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The genes for the glucose-regulated protein GRP94 and GRP78 are co-ordinately regulated by common trans-acting factors

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The genes for the glucose-regulated protein GRP94 and GRP78 are co-ordinately regulated by common trans-acting factors

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Content THE GENES FOR THE GLUCOSE- REGULATED PROTEIN GRP94 AND GRP7 8 ARE CO-ORDINATELY REGULATED BY COMMON TRANS-ACTING FACTORS by Shin C. Chang A D i s s e r ta ti o n Presented to the FACULTY OF THE GRADUATE SCHOOL UNIVERSITY OF SOUTHERN CALIFORNIA I n Pa rt i al F ul fil l ment of the Requirements for the Degree DOCTOR OF PHILOSOPHY ( Bi ochemi stry) May 1989 Copyright 1989 Shin C. Chang UNIVERSITY OF SOUTHERN CALIFORNIA THE GRADUATE SCHOOL UNIVERSITY PARK P t h LOS ANGELES, CALIFORNIA 90089 p . > “ 1 This dissertation, written by ...................................................... under the direction of h.& r . Dissertation Committee, and approved by all its members, has been presented to and accepted by The Graduate School, in partial fulfillm ent of re quirements for the degree of D O CTO R OF PHILOSOPH Y Dean of Graduate Studies p )aie Febrjja r*y 13, 1989 DISSERTATION COMMITTEE Chairperson ACKNOWLEDGMENTS I would like to thank my research adviser, Dr. Amy Lee, for her guidance and advice during my graduate s t ud y. I sincerely appreciate and am thankful for the advice and encouragement from Drs. Michael Lai, Robert Maxon, Pradip Roy-Burman, and Robert Stellwagen who served as the members in my d i s s e r t a t i o n and guidance c ommi 1 1 e e s . I thank my labo r ato r y part ners for th eir helpful di scussio n s and techni cal a ss is tan ce s. These include Elpidio Resendez, J er ry Ting, Augustine Lin, Takafumi Nakaki , Ajay Sharma, Adrienne Day, Scott Wooden, Alex Artis hevsky, Steven Wells, Aulikki Flagan, Janet A tt e n el lo , Yong Kim, and Kyu Kim. Fi na lly , special thanks go to my parent s, Ching-Tzer and Ying-Hsia Chang and my husband, Ming-Fu for the love and support they have always given me. INTRODUCTION TABLE OF CONTENTS Page 1 Chapter 1 ISOLATION AND SEQUENCE ANALYSIS OF THE HUMAN GENOMIC CLONES ENCODING THE 9 4KD GLUCOSE-REGULATED PROTEIN (GRP94) 7 1.1 INTRODUCTION 7 1 .2 MATERIALS AND METHODS 9 1.2.1. Cell Lines and Culture Conditions 9 1.2.2. I solatio n of Cytoplasmic RNA and RNA Blot Hybri di zat i on 10 1.2.3. DNA Sequence An a 1y s i s 11 1.2.4. Isolatio n of Human GRP94 Genes 1 1 1.2.5. Phage DNA Pr eparation 13 1.2.6. Southern Blot Analysis 15 1.2.7. SI Nuclease P rotection 15 1.2.8. Primer Extension 17 1.3 RESULTS 18 1.3.1. Analysis of Hamster GRP94 cDNA Clone p4A3 18 1.3.2. Southern Blot Analysis of GRP94 Sequences in Human Genomic DNA 19 1.3.3. Iso lati on and Sequence Analysis of the Human GRP94 Pseudogene 20 1.3.4. Isolati on and Sequence An alys is of the Human GRP94 5 ’ Region 23 1.4 DISCUSSION 27 Chapter 2 FUNCTIONAL STUDIES OF THE HUMAN GRP94 AND THE RAT GRP7 8 PROMOTERS 76 2 .1 INTRODUCTION 7 6 2 . 2 MATERIALS AND METHODS 81 2.2.1. Cell Lines and Culture Conditions 81 2. 2.2. PI a smi d s 81 2.2.3. Conditions for Transient Tra n sfectio n 83 2. 2. 4. Assays for the CAT A c tiv ity 84 2.2.5. Isola tio n of Cytoplasmic RNA and RNA Blot Hybridiza ti on 85 2.3 RESULTS 8 6 2. 3.1. Promoter A c ti vi ty of the Human GRP94 Gene 8 6 i i i Page 2.3.2. L ocal i zat i on of the Regulatory Regions Important for High Level Expression of GRP94 8 8 2.3.3. Effect of Progesterone on the Transc ri pt io n of GRP94 Gene 89 2.3.4. Regulatory Sequence Contained within the 5 ’-Flanking Sequence of the Rat GRP78 Gene 90 2.3.5. Deletion Analysis of the 5 ’-Flanking Sequence of the GRP78 Gene 91 2.3.6. The Sma/Stu Fragment Can Compete for Trans-act ing Regulatory Factors 93 2.4 DISCUSSION 95 Chapter 3 STUDIES OF THE INTERACTIONS BETWEEN PROTEIN FACTORS AND 5 ’ -FLANKING SEQUENCES OF THE GRP94 AND GRP78 GENES 118 3.1 INTRODUCTION 118 3 .2 MATERIALS AND METHODS 123 3.2.1. DNA Sequence Analysis 123 3.2.2. Cell Lines and Culture Conditions 123 3.2.3. Isolation of Cytoplasmic RNA and RNA Blot Hybr i di zat i on 123 3.2.4. P1 a smi d s 123 3.2.5. Pr eparat i on of HeLa Nuclear Extracts 124 3.2.6. Gel Retar dat i on Assay 124 3.2.7. DN ase I Footprint An al ysi s 125 3.2.8. Transient Tran sfection and Assay for CAT A c tiv ity 126 3.3 RESULTS 127 3. 3.1. Sequence Conservation between the GRP P r omo ters 12 7 3.3.2. GRP94 and GRP78 Are Regulated Similar ly during Stress 128 3 .3.3. GRP94 and GRP78 Promoters Can Com pete for Nuclear Factors La Vi t r o 128 3 .3.4. Binding Sit es of the Human GRP94 Promoter to Prot ein Factors 130 3.3.5. GRP94 and GRP78 Promoters Can Com pete for Cel lul ar Factors La Vivo 131 3.4 DISCUSSION 134 REFERENCES 154 i v LIST OF FIGURES 1 . 2 . 3 . 4 . 5 . 6 . 7 . s . 9 . 10 . 1 1 . 12 . 13 . 14 . 15 . A r e s t r i c t i o n map of the p4A3 cDNA clone Hybr i di zat i on of pa rt ia l cDNA insert s of the p4A3 with hamster K12 cytoplasmic RNA P a rti al nucleot ide sequence of the cDNA clone (p4A3) encoding the hamster GRP94 Southern blot anal ysi s of GRP94 sequences R e s tr i c ti o n map of a human GRP94 gene from the phage clones hu94-24 and hu94-7 Southern blot anal ysi s of the phage clone hu94 - 24 The sequence of 5 ’ region of the human GRP94 gene from phage clone hu94-24 Nucleotide sequences of the human GRP9 4 genes and their comparison with the chicken, hamster and murine cDNA sequences encoding the GRP94 Determination of the tra n s cr ip ti o n a l i n i t i a t i o n site of the human GRP94 tr a n s cr ip ts in HeLa and 293 cel ls P a r t i a l r e s t r i c t i o n map of the human GRP94 gene from the phage clone hu94-19 Sequence of the human GRP94 5 ’ region isolated from the phage clone hu94-19 and its comparison with the chicken HSP108/GRP94 genomic sequence Comparison of the 5 ’ -UTR sequences of the human and murine GRP94 genes Amino acid sequence comparison among GRP94, H S P 8 3 , and HSP90 CAT a c t i v i t i e s of GRP94-CAT fusion genes containing 5 ’ regions from the human phage clone hu94-24 Promoter a c t i v i t i e s of GRP94-CAT fusion genes contai ning 5 ’ sequences of the human phage clone hu94- 19 Page 32 34 36 38 40 42 44 4 6 61 65 67 69 71 100 102 v age 105 107 109 1 1 1 1 1 3 1 16 138 140 142 144 146 148 150 152 age 1 15 v i Pormot e r a c t i v i t i e s of GRP9 4-CAT fusion genes in He pG2 cel ls Northern blot analysis of the GRP94 mRNA on pr og est er on e-treate d cell s In du c ib ili ty of pIlO by calcium ionophore A231 87, g 1 u c o s e - s t a r va t i on and K12 t_s_ mu t a t i on Promoter a c t i v i t i e s of the rat GRP78 5 ’ deletion CAT constructs Regions of the rat GRP78 promoter important for the induction by A231 87 and K12 t_s mu t a t i on C omp e t i t i o n for t r a n s regulatory factors by the Sma/Stu fragment Sequence conservation of the GRP promoters Tr anscript level of GRPs under st ress conditions Binding of HeLa nuclear factors to the human GRP94 p r omo ter Similar binding pat t er ns formed between the human GRP94 promoter and HeLa nuclear factors i sol ated from induced and non-induced conditions Competition for nuclear factor Ln vitro DNase I foo tprint analysis of the human GRP94 p r omo ter Competition for c el l ul ar f act or s Ln v i vo by the rat GRP78 Sma / St u(- 375/- 8 8 ) fragment Competition for c el lu la r factors i_n vivo by the rat GRP78 common doma in (nt -170 to -135) TABLE Effect of 5 ’ del etions on CAT a c t i v i t y ABSTRACT The goal of th is study is to dissect regul at or y domains of the genes encoding the gl ucose-reg u lated proteins (GRPsi and to look for the possible mechanisms by which GRPs are co- ord inately regulated. Both the 94 k ilo da lto n (GRP94) and 78 kilodalto n (GRP78) GRPs are lo calized in the endoplasmic ret i cul um (ER). Their syntheses are gre atly enhanced at the t r a n s c r i p t i o n a l level under a va ri e ty of stress conditions which block g 1ycosy1 ation or prote in processing in the ER. In e a r l i e r work, the gene encoding GRP78 has been isolated from the r at. In this study, the promoter of the human gene encoding the GRP94 w as isolated. The 5 ’- f l anki ng regions important for the expression of GRPs were id e n t i f i e d by delet ion analysis. Comparison of the GRP78 and GRP94 promoters derived from hum an, r at, and chicken reveals a common d o m ain o f 2 8 nt w ith in the put ative regul atory regions of both genes. Since the GRP94 and GRP78 genes are t r a n s c r i p t i o n a l l y regulated wi t h si mi l ar k i n e ti c s under induced condi t i ons, possible mechanisms for their co- or di nat e expression were tested through La. v i t r o and La vi V Q competition assays. Results i ndi cat e that the protein factors which in te ra c t with the GRP94 promoter also h a v e a f f i n i t y for the conserved d o m ain of th e GRP78 promoter. These v i i suggest that GRP94 and GRP78 are co-o r din ately r egulated through common tra ns -a ct in g f act or s which recognize the common r egulatory domains of GRP p r omo t e r s . INTRODUCTION To understand the m e c h a n is m s by which cel ls respond to environmental changes, p a r t i c u l a r l y s t r es s c o n d i t i o n s , is a fundamental questi on in studying the c o n tr o l of gene e x p r e s s i o n in eukaryotes. Cells accommodate s tre ss by synthesizing a set of pro teins, known as s tr e s s - i n d u c i b l e protei ns . It is assu m ed that these p r ot ei ns are synthesized to p r ot ect ce lls from the effe ct s of s tr e ss . One family of s tr e s s - i n d u c i b l e pr ot eins that has been extensively i nves tig ated are the heat shock protein s (HSPs). The heat shock response was o r i g i n a l l y discovered in Drosophila (Ri t os sa, 1 964) as the co- or di nat e a c t i v a t i o n of a small num ber of cytogenetic loci in response to heat. Further studies h a v e shown that heat shock genes are expressed in response to a wide range of p h y s io lo g ic al ly and chemically induced st re ss conditions and is an e v o lu ti o n a ri ly conserved response in al l li vi ng species ( L i ndqui st, 1986). The HSPs are localize d in the cytoplasm, but accumulate in large a m o u n ts in the n u cl eoli during the s tre ss (W elch and Feramisco, 1984). The glucose-regulated prote in s (GRPs) represent another class of s t r e s s - i n d u c i b l e p rot ei ns . H o w ev e r, GRPs are lo calized in the endoplasmic reti culum (ER). Th e y we re f i r s t i d e n t i f i e d as prote in s wh i c h are 1 s p e c i f i c a l l y synthesized wh en eukaryotic cell s are depleted of glucose (Shiu et a 1 . , 1977). Subsequently, i t wa s found that a var ie ty of reagents wh i c h block pr ot ei n glycosylat i on (Pouyssegur et a 1 . , 1977; Olden et a 1 . , 1978) or deple te i n t r a c e l l u l a r calci um stores (Wu et a 1 . , 1981; Lee et a 1 . , 1984; Resendez et a 1 . , 1985; Drummond et a 1 . , 1987) also enhanced the syntheses of GRPs. In addition, in a Chinese hamster t emp e r a t u r e - s e n s i t i v e ( t_s ) mutant cell line, k! 2 , which is blocked in prot ei n glycosy lat i on, the GRPs are s p e c i f i c a l l y induced at non-permissive temperature (Lee et a 1 . , 1981; Melero, 1981; Lee et a 1 . , 1983). In a recent study, these stress gene inducers were c l a s s i f i e d into three groups (Watcwich and Morimoto, 1 988 ). The f i r s t one induces the expression of a 78 kilodalton GRP (GRP78), and includes in hi bitors of glycoprotein processing. The second class ac tivates the synthesis of both the GRP 7 8 and a 70 kil odalt on HSP (HSP70). This includes am ino acid analogs and heavy m eta ls. Calcium ionophore A23187 and the glucose analog 2 -deoxyglucose belong to the third class wh ich induces GRP78 but represses the expression of HSP70. The 94 kilod alto n GRP (GRP94) is the most abundant glycoprotein in the ER, commonly observed in the chicken, r at , hamster and human (Shiu et a 1 . , 1977; Welch et a l . , 1983; Lee et a l . , 1984; Lee, 1981; McCormick et a l . , 1979). It is also referr ed to as GP100 (Koch et a l . , 1985 ) , ERp99 (Lewis et a l . , 1985) or endoplasmin (Koch et a l . , 1986) and was found to be s u b s t a n t i a l l y overexpressed in t issues or ce lls that are rich in ER (Lewis et a l . , 1985). The locat ion of GRP94 wit hi n the ER remains cont rov er si al. Studies on the protease s e n s i t i v i t y in iso l ated microsomes demonstrated that GRP94 is an ER transmembrane glycoprotein (Lewis et a l . , 1985; Mazzarella and Green, 1987). In contrast, independent studies by im m unoe1 ec t ron microscopy and biochemical analyses indicated that GRP94 is a soluble prot ei n which resides wit h i n the lumen of ER (Koch, 1987; Koch et a l . , 1988). In order to understand the physiological functions and regul at i on of the GRPs, GRP94 was purified and its amino-terminal sequence determined (Lee et a l . , 1984). It was hypothesized that the acidic ami n o - t e rmi nu s of GRP9 4 may provide binding sites for calcium ions (Lee et a l . , 1984). cDNA clones for the hamster and murine GR P94 have been i sol ated and sequenced (Lee et a l. , 1983; Sorger and Pelham, 1987; Smith and Koch, 1987; Mazzarella and Green, 1987). Analysis of the predicted amino acid sequence also revealed highly acidic residues at the carboxy1 - 1 erm i nus (Sorger and Pelham, 1987 ). Ca1cium-binding studies showed that GRP 9 4 contains 1 ow a f f i n i t y but high capacity binding sites 3 (Koch et a l . , 1986), suggesting that GRP94 may serve as an important source for i n t r a c e l l u l a r calcium se qu est ra ti on. During the course of this work, cDNA and genomic clones for chicken HSP108 were also obtained and studied (Kulomaa et a l . , 1986; Kleinsek et a l . , 1986). I t wa s i n i t i a l l y identified as a heat-shock inducible protein in chicken. However, its predicted amino acid sequence is almost i dent i cal to that reported for hamster GRP94 (Lee et a l., 1984; Sorger and Pelham, 1987). In te re s ti n g ly , GRP94 shares about 50% amino acid ide ntity with that of cytoplasmic yeast HSP90 and Drosophila HSP83 (F arrelly and F i nke1 stein, 1984; Blackman and Me se Ison, 1986; Sorger and Pelham, 1 987; Mazzarella and Green, 1987). Thus, GRP94 and HSP90/HSP83 may have evolved from the same protein family. HSP90 was shown to complex with ster oid recept or s. It wa s speculated that a highly negat ivel y charged amino acid domain and the helical conformation of HSP90 can mimic a DNA binding domain of hormone regul at or y elements (HRE) that complex with ster oid r e c e p t o r s . Th is prevents receptors fr om binding to the HRE and increasing t ra n s c r ip ti o n of regulated genes (Baulieu, 1987; C at e 11i et a l . , 1985; Sanchez et a l., 1985; C a t e 11i et a l . , 1988). GRP94 has a native molecular weight of 192 kDa (Lee et a l. , 1984), 4 suggesting that it may form a dimer or it may be coval ent l y linked with receptors or other pro teins. Another abundant protein in the ER is the GRP78. This pr otein which has been l ocal ized to the lumen of ER (Zala et a l . , 1980; Munro and Pelham, 1986) is a phosphoprotein but is not glycosylated (Welch et a l . , 1983; Lee et a l . , 1984). GRP78 w as recently ide ntified as the immunoglobulin heavy chain binding prot ei n BiP (Munro and Pelham, 1986) which binds immunoglobulin molecules in B cells (Bole et a l . , 1986). This in te ra ct io n may prevent the formation of heavy chain aggregates and premature secretion of prot ei ns before assembly is complete. Further, GRP78/BiP was found to as sociat e with c el lu lar prot ei ns in a hamster fib ro bl as t cell line (Hendershot et a l . , 1988). Therefore, it is possible that GRP78 may serve a similar function to that postulated for HSP70; that is to bind to and s ta b i li z e abnormal pro teins. (Munro and Pelham, 1986; Ananthan et a l . , 1986). The gene encoding the GRP78 has been isolated from the rat ( At t enell o and Lee, 1984) and human gen om es (Ting and Lee, 1988). Studies have sh own that the rat GRP 7 8 promoter is highly active and a 288-nucleotide (nt -375 to - 8 8 ) fragment was identified as having enhancer-like p rop erti es (Lin et a l . , 1986; Resendez et a l . , 1988). 5 Using cDNA clones encoding hamster GRP94 and GRP78 as probes, it was shown that both GRP94 and GRP78 are c o n st i t u t i v e 1y expressed at low level s in eukaryotic c e l l s , and that both GRP genes are regulated simil arl y at the t r a n s c r i p t i o n a l level in d i f f e r e n t species and t i s s u e s under conditions which block gl ycosylation or deplete i n t r a c e l l u l a r calcium stores (Lee, 1987; Kim and Lee, 1987). This suggests that these two unlinked genes coding for two di s si m il ar proteins are capable of responding to some common stimuli generated by diverse physiological stre ss condi t i ons. Thus, the GRP genes r epresent an useful model system for studying co- o r d i n ate gene regul ation in m am m alian c e l l s . To study the possi b l e m e c h a n is m s by which GRPs are c o- ord ina te ly regul ated, the GRP78 and GRP94 genes were iso la ted and thei r promoters char act er iz ed. The aim of my d i s s e r t a t i o n wa s three fold: f i r s t , the is o l a t i o n and c h a r a c t e r i z a t i o n of the human GRP94 gene; second, the d etermination of the 5 ’- flanki ng regions that are important for the expression of the human GRP94 and th e r a t GRP78 genes; and thir d, studies on the possi bl e co-o rdinate regu la tio n mechanism of the GRP94 and GRP78 genes using both La v i vo and La v i t r o systems. 6 Ch aptcr 1 I s o l a t i o n and sequence a n a l y s i s of the human gen om ic c lo n es encoding the 94 kD g l u c o s e - r e g u l a t e d p r o t e i n (GRP94) 1.1 INTRODUCTION The t emp e r a t u r e - s e n s i t i ve ( t_s) mutant cell line K12, derived from the Chinese hamster fib ro bl as t WglA, has been shown to overproduce GRP94 and GRP78 when th e ce lls are grown at the non -permi ssive temperature (Lee, 1981). As a f i r s t step to understand the stru cture and r egu l ati o n of the GRP genes, a cDNA li brary was constructed using poly(A+) RNA extracted from the hamster K12 cel ls incubated at 4 0.5°C. Two cDNA clones, p3C5 and p 4 A 3 , were isolated from this libr ary by hyb rid -s el ect tr a n s l a t i o n (Lee et a l . , 1981). The p3C5 contained sequence coding for the GRP78, while the p4A3 clone contained sequence coding for the GRP94 (Lee et a l . , 1981; Lee et a l . , 1983). Northern blot analysis demonstrated that p3C5 hybridized to an RNA species of 2700 nucleo tid es (nt) and p4A3 hybridized to RNA of 3 2 00 nt. These agree with the sizes predicted from the molecular masses of GRP78 and G R P94. However, the sizes of the inse rts for p3C5 and p4A3 were estimated to be 2500 n t and 1400 nt, respec tive ly (Lee et a l . , 1983). 7 The p4A3 was therefore concluded to be a p a rt ia l cDNA clone . Using these cDNA clones as probes, it was estab li shed that the two GRP genes are regulated sim ila rly at the t r a n s c r ip ti o n a l level under phys iological stres s conditions (Lee et a l . , 1983; Lin and Lee, 1984; Resendez et a l . , 1985; Kim and Lee, 1987). In order to determine whether 5 ’ -flanking sequences of the GRP genes contain signals required for the regulati on, it wa s i mp ortant to isola te their genomic clones. Using p3C5 as a probe, genomic clones for GRP 7 8 have been isolated from the rat and human ( Att enell o and Lee, 1984; Ting and Lee, 1988). In this chapter, the t r a n s c r ip ti o n a l or ie nta tio n and pa rti al nucleot ide sequence of the p r o t e i n - coding porti on of the p4A3 cDNA clone were determined (chapter 1 . 3 . 1 . ) . Sequence analysis suggested that p4A3 contains the cDNA encoding the carboxyl-terminal half of GRP9 4. With this information and the ami n o - t e rmi n a 1 sequences known for hamster GRP94 (Lee et a l . , 1984), the s tr u c tu ra l gene encoding GRP94 was i solated from a h uma n gen om i c 1 i b r a r y . 1 . 2 MATERIALS AND METHODS 1 . 2 . 1 . C e ll l i n e s and c u lt u r e c o n d i t i o n s . The I_S . mutant c e l l line K12, de riv ed from Chinese hamster lung f i b r o b l a s t line Wg 1A, has been d e scr ib e d (Roscoe et a l . , 1973; Lee 1981; Melero 1981). It was r o u t i n e l y m a in ta in ed at 3 5°C in Dulbecco* s m o d ifie d E a g l e ’ s medium (DMEM) c o n ta in in g 4 .5 m g/m l g lu c o s e and supplemented w i t h 10% cadet c a l f serum. The HeLa D98 AH2 monolayer c e l l s were obtained from R. E. K. Fournier and were m ain tain ed in DMEM supplemented w i t h 10% f e t a l c a l f serum. The 293 human embryonic kidney c e l l s were obtained from M. Karin (UCSD) and were m a in ta in e d in DMEM supplemented w i t h 10% f e t a l c a l f s e rum. For i s o l a t i o n of cy to p la sm ic RNA, c e l l s were grown to about 8 0% c o n f lu e n c y . Fresh me d i um wa s then added and the c e l l s were e i t h e r t r e a t e d w i t h 7 ;uM of calciu m ionophore A23187 at 35°C, s h i f t to the non- pe r m issiv e temperature ( 3 9 . 5 ° C ) , or m a in ta in ed at 35°C for 16 hrs pr io r to the e x t r a c t i o n , except for HeLa c e l l s and 293 c e l l s which were exposed to ca lciu m ionophore for 5 hrs p r io r to the e x t r a c t i o n . Whenever g 1u c o s e - s t a r v a t i o n c o n d i t i o n was used, the c e l l s were c u lt u r e d in g l u c o s e - f r e e medium for 16 hrs p r io r to the e x t r a c t i o n . G l u c o s e - f r e e medium was prepared by supplementing 9 regular EMEM ( g l u c o s e om itted) w i t h 10% cadet c a l f s e r urn wh ich had been d ia l y z e d overn ight a g a in st phosphat e - buff e red s a l i n e (PBS) (Lee, 1981). 1 . 2 . 2 . I s o l a t i o n of cytoplasm ic RNA and RNA b lot h y b r i d i z a t i o n . Total cytoplasm ic RNA was i s o l a t e d from the c e l l s as p r e v i o u s l y d e s cr ib e d (Lee et a l . , 1983). B r i e f l y , cu ltu re d c e l l s were washed twice w i t h cold PBS and then lyzed w i t h 0.5% NP-40 in 0 .7 ml of cold Isoh igh b u ffer (140 nM NaCl , 10 mM Tr i s (pH 8 . 4 ) , 1.5 mM MgC^)* The supernatant was mixed w i t h 0 .3 ml NETS b u ffer (100 mM NaCl, 1 nM EDTA, 10 nM Tris (pH 8 . 4 ) , 1% SDS), e x t r a c t e d twice w i t h pheno1/ c h i o r o f o r m and then ethanol p r e c i p i t a t e d . Ten /ig o f RNA f r om each s amp 1e wa s e l e c t r o p h o r e s e d on a fo rmamide- fo rma1dehyde denaturing agarose gel and b l o t t e d onto n i t r o c e l l u l o s e paper (Lee et a l . , 1983). In h y b r i d i z a t i o n r e a c t i o n s , the f i l t e r s c o n t a in in g t r a n s f e r r e d RNA were p r e t r e a t e d w i t h b u ffe r c o n t a in in g 10X Denhardt ’ s s o l u t i o n (IX D e n h a r d t’ s s o l u t i o n : 0.02% each of bovine serum albumin, p o l y v i n y l p y r r o l i d o n e , F i c o l l and SDS), 50 nM sodium phosphate b u ffer (pH 6 . 8 ) , and 5X SSC ( IX SSC: 150 nM NaCl, 15 nM sodium c i t r a t e ) for 3 hrs at 42°C, p r e h y b r id iz e d w i t h a s o l u t i o n c o n t a i n i n g 50% d e i o n i z e d formamide, IX D e n h a r d t ’ s s o l u t i o n , 20 nM sodium phosphate (pH 6. 8) , 10 5X SSC, 0.1% SDS, and 50 Aig/ml of denatured salm on sperm DNA at 4 2°C for 1 hr, fo llo w ed by h y b r i d i z a t i o n in the same buffer c o n t a i n i n g denatured la b e le d probe for o v e r n ig h t at 42°C. The f i l t e r s we re then wa shed once in 4X SSC, 0.1% SDS, 25 mM sodium phosphate (pH 6 . 8 ) , and 0.1% sodium pyrophosphate at room temperature for 40 min, three times in IX SSC, 0.1% SDS, 25 mM sodium phosphate, and 0.1% sodium pyrophosphate at 50°C for 45 min each, and twice in 0. 3X SSC, 0.1% SDS, 25 mM sodium phosphate, and 0.1% sodium pyrophosphate at room temperature for 30 min each. 1 . 2 . 3 . DNA sequence a n a l y s i s . The DNA contain ed w i t h i n the p4A3 cDNA or phage c lo n e s was i s o l a t e d and sequenced by the d id eoxy c h a i n - t e rmination method (Sanger et al . , 1977) u sin g e i t h e r the s i n g l e stranded Ml3 or double stranded pTZ18U DNA as template (Messing et a l . , 1981; Y an isch-P erron et a l . , 1985; Guo et a l . , 1983; United S t a t e s B iochem ical Corp., C le v e la n d , OH). A bou t 90% of the sequence was determined on both s t r a n d s . The sequence was analyzed u sin g the I n t e 11i - G e n e t i c s Bionet computer SEQ:SEARCH program. 1 . 2 . 4 . I s o l a t i o n of hum an GRP94 g e n e s. The human f e t a l l i v e r genomic l i b r a r y (Lawn e t a l . , 1978) was screened 11 using as probes, a p a r t i a l cDNA plasm id, p4A3 encoding the hamster GRP94 (Lee et a l . , 1983; Sorger and Pelham, 1987 ) and a synt hetic 4 1 -me r ( 5 ’ - AP PTCQTCQTCNACNGTNCCP TCNACPTCNACQTCPTCPTC-3 ’ ; P: A /G, Q: T/C, N: I/C) d eri v ed from the unique ami no - termi na 1 sequence ( r e s i d u e s 1 through 14, Lee et a l . , 1984) of the hamster GRP9 4 . A pseudogene was obtained (s e e below, chapter 1 . 3 . 3 . and 2 . 3 . 1 . ) . To i s o l a t e the f u n c t i o n a l gene, the s ame 1 i b r a r y wa s screened u sin g two probes , the 415 nt fragment d erived from Bam HI and Eco RV d i g e s t i o n of the human pseudogene i s o l a t e d from the phage clone hu94-24 (as i n d i c a t e d in F ig . 5) and a s y n t h e t i c 18 -me r ( 5 ’ - TTCCACATCAACTTCATC- 3 ’ ) corresp ondin g to a part of the hamster 4 1 -me r sequence, o btained from the human phage clo n e s hu94-7 and hu94-24 (as i n d i c a t e d in F ig . 8). F i l t e r s r e p r e s e n t i n g about 10^ recombinant phage were prepared for the primary s cr e en . H y b r i d i z a t i o n was performe d usin g e i t h e r hex ame r - l a b e l e d DNA fra gme n t s (F ein b erg and V o g e l s t e i n , 1983) or 5 ’ - end - 1 a be 1ed s y n t h e t i c oligom ers as probes. Whenever DNA fragments were used as p robes, the f i l t e r s were p r e h y b r id i z e d in 50% formamide, 5X SSC (IX SSC; 150 mM NaCl, 15 mM sodium c i t r a t e ) , 5X D e n h a r d t’ s s o l u t i o n (IX D e n h a r d t’ s s o l u t i o n : 0.02% each of bovine serum albumin, p o l y v i n y l p y r r o l i d o n e , F i c o l l and SDS), 50 mM sodium 12 phosphate buffer (pH 6 . 8 ), 1% glycine and 50 >ug/ml of denatured salmon sperm DNA at 4 2°C for 1 hr, followed by hybridization in the same buffer overnight at 42°C wi t h denatured labeled probe. Th e f i l t e r s were then washed three times in 5X Denhar dt ’s soluti on , 3X SSC, 0 . 1% SDS and 0 . 1% sodium pyrophosphate at 50°C for 60 min each, then twice in IX SSC, 0 . 1 % SDS and 0 . 1% sodium pyrophosphate at 5 0 °C for 60 min each. When oligomers were used as probes, the synt hetic oligomers were end-labeled with [ ^ P] ATP using the T4 polynucleotide kinase. F i l t e r s were pr ehybridized in 6 X SSC, 1 OX Denhard t’s solut i on, 50 /Ug/ml of denatured salmon sperm DNA and 30 jugml of denatured E. coli DNA at room temperature for 15 min. Hyb r i dizat i o n was then • 2 y carri ed out in the same buffer with the labeled oligomer overnight at room temperature. After hybri di zat ion , the f i l t e r s were washed twice in 6 X SSC at room temperature for 10 min each, then twice .at 42°C for 15 min each. 1.2.5. Phage DNA preparatio n. A single KH802 colony grow n on a NZYCM plate ( 1 % NZ- amine, 0.5% NaCl, 0.5% yeast ex tract, 0.1% casamino acid, 10 mM Mg SO4 and 1.2 5% bactoagar) was inoculated into 15 ml of overnight broth (2% t rypt one, 1% NaCl, 1 % yeast extrac t and 0.1% maltos e) at 37°C without shaking overnight. T w elv e ml 1 3 of the overnight culture (OD^qq = 1.2 to 1.4) was mixed with (2-5) X 10 t it e r e d phage in lambda dilu ent (10 mM T r i s (pH 8 ) and 2 mM Mg CI 2 ) and adsorbed 15 min at 37°C , then dil uted into 500 ml prewarmed S i n sheimer’s broth (45 mM Na2 HP04 , 20 mM KH2 P 04 , 7.5 mM NaCl, 0.1% NH4 C1, 0.36% glucose, 0.4% maltose, 1 % casamino acids, 5 mM M gCl2 and 0.1 mM CaCl2 ). This cul t ure was incubated at 3 7°C with shaking for 6 - 8 hrs. When white threads appeared, an indi cat i on of lysis of ba cte ria l c e l l s , one ml of chloroform wa s added and the culture incubated for 10 min more. After c en tr if u g a ti on at 10 k r pm in a Sorva 1 1 GSA rotor for 20 min at 4°C , the supernatant was obtained which could be stored as phage stock or used to prepare phage DNA. To prepare phage DNA, t h i r t y gm NaCl wa s added and dissolved at 4°C , then 35 gm of PEG6000 was added and di ssol ved. The mixture was held at 4°C for 1 hr, and the PEG p r e c ip it a te d phage col l ect ed by c en tr ifu ga tio n at 7.5 krpm in a Sorva 11 GSA rotor for 30 min. The pe ll e t was resuspended in 5 ml of TM10 (10 mM Tris (pH 7.4) and 10 mM MgCl2 ) and incubated with 20 jul of DNase sol ut i on (5 mg /ml ) at room temperature for 30 min. The PEG was rem oved by chloroform ex tra cti on. The phage p a r t i c l e s in TM10 were incubated with 20 mM EDTA, p rot ei nase K (0.5 mg/ml) and 0.5% SDS at 3 7°C for 1 hr. The DNA was purified by phenol-chloroform 1 4 extraction followed by d ia ly s is at 4°C against TE (10 mM Tris, 1 mM EDTA). 1 . 2 . 6 . Southern b lot a n a l y s i s . After r e s t r i c t i o n enzyme digestion, DNA fragments were s iz e - f r a c ti o n a te d by agarose gel electrophoresis. The gel was denatured by two incubations in 0.4 M NaOH and 0 . 8 M NaCl for 30 min each, rinsed several times w7 i t h d i s t i l l e d water and neut ra liz ed by three wa shes with a solution containing 0.5 M Tris (pH 7.4) and 1.5 M NaCl for 20 min each. The gel wa s then blotted onto n i t r o c e l l u l o s e paper and subjected to hybr id iza ti on. The hy bridi zation and washing conditions were the same as described above in ’’is ola tio n of human GRP94 genes” (chapter 1 . 2 . 4 . ) . 1.2.7. SI n u cle a s e p r o t e c t i o n . RNA samples for SI protection assays were prepared from the HeLa D9 8 AH2 monolayer ce lls and the 293 human embryonic kidney cells as described in the chapters 1 . 2 . 1 . and 1 . 2 . 2 .. To generate probes, a 1.8 kb Hind III fragment was isolated from the phage clone hu94-24. This Hind III fragment was shown to hybridize w7 i t h the s ynthet ic 41-mer corresponding to the ami no- termi na 1 sequence of the hamster GRP94 , but not wi t h the p4A3 cDNA probe (see chapter 1.3.3. and Figs. 5 and 6 ). The DNA fragment was 5 ’ -end-labeled wri th [ f '- ' ^ P ] ATP using the 15 T4 polynucleotide kinase and digested with Eco R1 to generate the 1.75 kb Eco RI/Hind III fragment labeled at the Hind III s it e . This Eco RI/Hind III fragment was used as a probe. To prepare a second probe, the 1.8 kb Hind III fragment was subcloned into the unique Hind III site of the pTJC8 . With the a v a i l a b i l i t y of a Sal I sit e in the poly li nker of pUC8 , a 1.35 kb Hind II I/ A sp718 fragment was prepared from a 5 ’-end-labeled Sal I/Asp718 fragment which contains the 5 ’-half of the Hind III fragment (see Fig. 5). About 5 X 10^ cpm of the heat denatured probe was hybridized with 30 >ug of the RNA samples f r om either the HeLa cell s or the 293 cells. The hy bridization was carried out in 8 09c formamide, 0.4 M NaCl, 4 0 mM PIPES [piperazine-N,N’ -bis(2-etha ne sul fo nic acid)] ( pH 6.4), and 1 m\l EDTA at 5 8 °C for 16 hrs. The DNA-RNA hybrid was then digested wT ith 20 units of SI nuclease (PL B i ochemica 1s ) in 200 >u 1 of r eact i on mixture containing 0.28 M NaCl, 50 mM s o d i um acet at e (pH 4.6), 4.5 mM ZnSC>4 and 50 ;ug/ml of denatured salmon sperm DNA at 2 5°C for 30 min. Undigested DNA was p r e c i p i t a t e d . The p e l l e t was resuspended in a loading buff er containing 0.1 N NaOH and 95% formamide, and el ectr ophoresed on a 4% polyacrylamide sequencing gel a ft e r denaturing at 9 5 °C for 2 min. 16 1.2.8. Primer extension. The synthet ic o l i g o n u cleot i d e (18-me r ) with sequence 5 ’ - TTCCACATCAACTTCATC- 3 ’ as described in the chapter 1.2.4. was used as the primer. The primer was 5 ’-end-labeled with ATP using the T4 pol ynucleotide kinase to a specific a c t i v i t y of 10^ c pm/jug DNA. About 1.5 X 10^ cpm of the primer was hybridized with 50 jog of cytoplasmic RNA prepared from the HeLa D 98 AH2 monolayer ce lls or the 293 c e l l s . The h yb rid iz at ion was car ried out at 2 5°C for 5 hrs in a sol ut i on containing 8 0 % formamide, 0.4 M NaCl, 40 m\l PIPES [piperazine-N-N’-bis(2- eth an esu lf oni c acid)] (pH 6 . 4) , and 1 mM EDTA. After p r e c i p i t a t i o n , 40 uni t s of a v i an my e lo b la st os is virus reverse tr a n s c r ip ta s e (Life Science) was added to the RNA/DNA hybrid in a 100 jul r eaction mixture containing 10 mM MgC12 » 12 0 mM KC1 , 50 mM Tris (pH 8.3), 30 mM ^-m er cap toe tha no l, and 1 mM each of unlabeled deoxyribonucleos ide t r iph osphates. The r eacti o n mixture was incubated at 37°C for 90 min. Sodium hydroxide was then added to a final concentration of 0 . 2 N and incubated at 4 5 °C for 60 min. Aft er subsequent n e u t r a l i z a t i o n writh HC1 , the pr imer- extended products were p r e c ip it a te d and elect r ophoresed on a 6 % polyacrylamide sequencing gel. 1 7 1.3 RESULTS 1.3. 1. Analysis of h a m s t e r GRP94 cDNA clone p 4 A 3 . Based on a Northern blot analysis and the i nsert size estimated for the p4A3 cDNA clone (Lee et a l . , 1981; Lee et a l . , 1 983; as described in the chapter 1. 1. ), the p4A3 was concluded to be a p a rt ia l cDNA clone. It was important to carry out a more de ta il ed anal ysi s of this clone before it could be used for further stud ies. As a f i r s t step, the r e s t r i c t i o n map of p4A3 was determined and is shown in Fig. 1. To determine the or ie n ta ti o n ( 5 ’ to 3 ’ ) of the cloned DNA, RNA - DNA hybridi zat ion studies were car ried out. A 980 nt fragment generated by Bam HI and Eco RY dig es tio n of p4A3 as indicated in Fig. 1 was isola ted . The fragment wa s 5 ’ - end - labeled wi t h [ Jf' - ^ ^ P ] ATP using the T4 pol ynucleotide kinase, and then digest ed with Hind III r e s t r i c t i o n enzyme. The resul tan t asymmetrically labeled fragments were resolved on a low-melting agarose gel. Fragments of sizes 290 and 690 nt were isolated and used as the probes to hybr i dize wit h RNA prepared from the hamster t_s mutant cell line K12. As shown in Fig. 2, only the 690 nt Hind I I I / Eco RV fragment hybridi zed to the RNA to give specific signal s corresponding to the GRP94 t r a n s c r i p t s , ind i cat i n g that the Bam HI site is located 5 ’ to the 1 8 Eco RV s i t e in th e cDNA clone p 4 A 3 . Two n o n - s p e c i f i c h y b r i d i z a t i o n s i g n a l s matched to the p o s i t i o n s of 28S and 18S ribosomal RNAs were seen for a l l the samples. F u r t h e r , and in agreement w i t h previous s t u d i e s (Lee et al., 1983; Resendez et a l . , 1985), s i g n a l s were stro n g er for RNA s amp le s i s o l a t e d fr om c e l l s t r e a t e d w i t h A23187 or grown at the non - pe rmi s s i ve temperature ( F i g . 1A, lanes 2 and 3) than from c e l l s m a in ta in e d at 35°C in EMEM ( F ig . 1A, lane l ) . To a s c e r t a i n whether the p a r t i a l cDNA clone p4A3 c o n ta in s the poly(A) t a i l and t r a n s l a t i o n a l stop codon, sequence downstream of the Eco RV r e s t r i c t i o n enzym e s i t e was determined ( F i g . 3 ) . Sequence a n a l y s i s show ed that a t r a n s l a t i o n a l stop codon (TAA) is lo c a t e d 230 nt downstream of the Eco RV s i t e and an open reading frame was found from Eco RV s i t e to the stop codon. Later on, a n e a r l y complete sequence a n a l y s i s of the p4A3 i n s e r t fu r t h e r confirmed that p4A3 c o n ta in s only sequences encoding the carb o x y 1 - termi n a 1 h a l f of the hamster GRP94 (Sorger and Pelham, 1987; F ig . 8). 1 . 3 . 2 . Southern b l o t a n a l y s i s of GRP94 seq uences in hum an genomic DNA. As a p r e l i m i n a r y to the s c r e e n in g of a genomic l i b r a r y to o b t a in the GRP94 gene, a Southern b lo t of human genomic DNA was probed w i t h the p4A3 cDNA c l o n e . A simple h y b r i d i z a t i o n p a t t e r n was o bta in ed 19 (Fig. 4A) , implying that GRP94 does not e x i s t as a mutiple gene family in the human genome. 1 . 3 . 3 . I s o l a t i o n and sequence a n a l y s i s of the human GRP94 pseudogene. In order to obtain genomic clones contai ning the 5 ’ - flanking sequences of the GRP94 gene, both the p a r t i a l cDNA clone (p4A 3) and the syn th et i c 41-mer derived from the unique ami no - termi n a 1 sequence (r e si d u e s 1 through 14; Lee et al., 1984) of the hamster GRP 9 4 were used to screen a human genomic 1 i b r a r y . For the primary screen, the p4A3 w as used as a probe. Three p o s i t i v e plaques, designa ted hu94-7, hu94-23, and hu94-24, were i s o l a t e d . Further h y b r i d i z a t i o n using the 41-mer as a probe revealed that hu94-7 and hu94-24 but not the hu94-23 contain the sequence coding for the ami no- termi nus of GRP94. DNA w as i s o l a t e d from the phage clones hu94-7 and hu94-24 and r e s t r i c t i o n maps determined (Fig. 5). R esu l ts in d ic ate d that these two phage clones are derived from d i f f e r e n t regions of the same GRP94 gene in the human genome. S u r p r i s i n g l y , Southern blot a n a l y s i s showed that a 2.5 kb Bam HI fragment contained w i t h i n these two clones could hybridize to both the s y n t h e t i c 41-mer corresponding to the ami n o - termi n a 1 sequence of the hamster GRP94 and th e p4A3 cDNA clone 20 encoding for the carboxyl-terminal half of the hamster GRP94 (Fig. 6; Lee et a l . , 1983; Sorger and Pelham, 1987). It is very u n l i kel y that the 2.5 kb B am HI fragment could contain an entire GRP94 st ru c tu ra l gene since the size of GRP94 tr a n s cr ip t is about 3.2 kb (Lee et a l . , 1983). To f ur t her characterize thi s human GRP94 gene, a 1.8 kb Hind III fragment containing the 5*-half of the 2.5 kb Bam HI fragment was r e s t r i c t i o n mapped (Fig. 5B), and sequenced (Figs. 7 and 8). This 1.8 kb fragment hybridized wT i t h the synt hetic 4 1-mer, but not the p4A3 cDNA probe (Fig. 6). Sequence analysis indicated that the DNA domain corresponding to the hamster synthetic 4 1-mer was located 137 nt 3 ’ to the Bam HI site. Further, sequence d own s t r e am of the ATG t r a n s la t io n a l i n i t i a t i o n codon wa s quite conserved (Fig. 8) wT h e n it wra s c omp ared to that of the chicken GRP94 (Kulomaa et a l . , 1986; referred to as HSP1-0 8) and the murine GRP94 (Mazza r e l 1 a and Green, 1987; r eferred to as ERp99). Tr ans criptional i n i t i a t i o n site was mapped by SI nuclease prote ction assay and primer extension (Fig. 9). For this purpose, total cy topi a sm i c RNA wT a s isolated from HeLa D98 AH2 monolayer cel ls and 293 cell s grown under induced or non-induced condition. The RNA samples were hybridized with a 1.75 kb Eco RI/ 2 1 Hind III fragment or a 1.35 kb Hind III/Asp718 fragment contained with in the 1.8 kb Hind III fragment of the phage clone hu94-24 (as indicated in Fig. 5). Protected fragments 1.15 kb and 0.59 kb, re sp ectively, were detected following SI nuclease d i gestio n (Figs. 9A and 9B). To determine the i n i t i a t i o n site more p re c is e ly , primer extension was car ried out. Primer used was a synt hetic 18 -me r ( 5 ’ - TTCCACATCAACTTCATC- 3 * ) corresponding to the antisense strand of the put ati v e amino terminal sequence of the human GRP94 isolated f r om the phage clone hu94-24 (as indicated in Fig. 8). A d is cr ete band of 188 nt wa s detected on the autoradiogram (Fig. 9C). Furthermore, in te n s it y of the s i g n a 1 wa s enhanced wh e n RNA s amp les prepared fr om cells g r O W 'D under induced conditions were used. These res ult s suggested that the tr a n s c r i p t i o n a l i n i t i a t i o n sit e is located 170 nt upstream from the put ati v e tra n s la t io n a l i n i t i a t i o n sit e ( GTG in the phage clone hu94-24) of the human GRP94. P r omo t e r - 1 i k e sequences such as, TATAA and CCAA.T were observed in this GRP9 4 gene (Fig. 7). However , several f ea tures observed fr om sequence anal ysis are inconsis tant wi t h this being a f unctional s tr u c tu ra l gene: i) the ATG codon was replaced by GTG; i i) no introns were found; i i i ) deletions were found , some of which have r es ul ted in frame shift mutations although 22 the mutations and deletio ns were not detected in the SI protection assay under the condition used; and iv) tr a n s la t io n a l te rm ination codon wa s found within a putati v e open reading frame. In lig h t of these observation and further functional studies (see chapter 2 . 3 . 1 . ) , it was concluded that these two human GRP94 clones, hu94-24 and hu94-7 are derived from a processed pseudogene. Unlike most pseudogene, neit her bounding r epeats, nor a st retch of adenosine residues, and polyadenyl ation signal ( AATAAA) at the 3 ’ end wa s observed in this GRP9 4 processed gene. 1.3.4. Isolation and sequence anal ysi s of the human GRP94 5 ’ region. Using the information obtained from studies of the human GRP94 pseudogene, the human gen om i c libr ary wa s rescreened to obtain a functional gene encoding the human GRP94. The probes used were a 415 nt fragment derived from Bam HI and Eco RV dig es tio n of the phage clone hu94-24 (as indicated in Fig. 5B) and a synthetic o l i gonu cle ot id e ( 1 8 -me r , 5 ’ - TTCCACATCAACTTCATC- 3 ’ ), corresponding to a part of the hamster 41-m er sequence, obtained from the human phage clones hu94-7 and hu94-24 (as indicated in Fig. 8). Nine positiv e plaques were isolated among 10^ phage screened. Based on r e s t r i c t i o n map anal ysi s and hyb ridi zation with both the synthet ic olig om er probe of 2 3 the human GRP94 ( 1 8 -me r ) and the cDNA probe of the hamster GRP94 (p4A3), these clones were found to represent two d if f e r e n t GRP94 genes. One is a processed pseudogene without introns as described in the chapter 1 .3 .3 . . The other contains introns as described for the chi cken HSP10 8 /GRP94 gene (Kleinsek et a l . , 1986); this was concluded based on a p a r t i a l r e s t r i c t i o n mapping and sequence analysis of the phage clone hu94-l9 (Figs. 8 and 10). The 415 nt Bam HI/Eco RV fragment was used as a 5 ’ -probe for Southern blot anal ys i s of human genomic DNA (Fig. 4B). As expected, the bands sh own on the autoradiogram matched to r e s t r i c t i o n maps of either the clones hu94 - 24 and hu94-7 (Hind II I, 1.8 kb; Bam HI, 2.5 kb; Eco RI, 10.5 kb; Fig. 5) or the clone hu94-19 (Eco RI, 3.1 kb; Fig. 10). It wa s not surp ri sing to see a difference wh en the hybr idization pa ttern in Fig. 4B was compared to that in the Fig. 4 A (chapter 1. 3 .2 .) since the two probes used in Southern blot anal y sis do not have any overlapping nucl eotide sequences. To determine whether the human GRP94 gene isolate d from phage clone h u 9 4 -1 9 contains a functi onal promoter, i t s 5 ’ region was id e n ti fi e d and sequenced (Fig. 11). The sequences of 5 *- u n t r a n s 1 ated region (UTR) are highly conserved b etw een this human GRP 9 4 gene and the human pseudogene described in chapter 24 1.3.3. (Fig. 12), wh i 1e sequences diverge upstre am o f the putati v e t ra n s c r ip ti o n a l i n i t i a t i o n sit e (Figs. 7 and 11). The f i r s t exon-intron junction was determined based on the sequence comparison with the pseudogene iso lated from phage clone hu94-24. This GRP94 promoter sequence contains both typical and atypical c h a r a c t e r i s t i c s for an eukaryotic gene. Several putative s ite s for eukaryotic t r a n s c r i p t i o n a l f act or s such as Spl, AP-2 and CCAAT elements commonly found in eukaryotic genes are found. Str iki ng ly, this GRP 9 4 promoter has six CCAAT sequences within 300 nt of its 5 ’- f lanking sequence. However, the human GRP94 promoter is unusual in that it lacks the typical TATA sequence. A comparison of the human and chicken GRP 9 4 5 ’ sequences (Fig. 11) revealed several in te res ti ng feat ure s: ( i ) both human and chicken promoters possess atyp ical TATA sequence, GTGAAA4. an d TTGATAA., re sp ect iv el y , around 30 nt upstream of the t r a n s c r i p t i o n a l i n i t i a t i o n s ite ; ( i i ) five CCAAT sequences are conserved in both promoters with four of them in the inverted o ri e n ta ti o n ATTGG; in addi ti on , the human promoter has a sixth inverted CCAAT sequence; ( i i i ) there is a region of high sequence conservation (65%) b etw een n t -195 and -72, although chicken and human have diverged from each other about 300 m ill i on years ago, suggesting that this domain may encompass 25 sequences important for GRP94 expression; and ( iv) the lengths of the 5 ’ - UTR and the f i r s t short exon wh i c h codes for part of the leader peptide are conserved. This promoter from human phage clone h u 9 4 -1 9 was further studied using i_H vivo sys t em to e s t a b l i s h its fu n c t i o n a l i t y (see chapter 2). 26 1 .4 DISCUSSION In this chapter, c h a r a c te ri z a ti o n of the p a rt ia l cDNA clone p4A3 which encodes the hamster GRP94 has been described. Based on the information obtained and the known ami no - termi na 1 sequence for the hamster GRP 94 (Lee et a l . , 1984), two GRP94 genes were isolated from a human genomic librar y. It is known that mammalian genomes contain fami l i es of related sequences. Further, in mo s t cases, only one or a few members in a family are f unctional genes. The others are nontran scribed pseudogenes arising from gene duplication through RNA int ermedi ates. The term ’’retroposon” was suggested for this class of RXA-derived sequence to stand for their RNA origin and their dispersed positions (Rogers, 1983). The retroposons g enerall y possess no promoter, no int r ons, retain a 3 ’ polyadenyla ted sequence and are flanked by short (7- to 21- base pair ) repeats at the ins er tio n s i t e s . These retropseudogenes are u sually inact ive even if the coding regions s t i l l remain i n tact (Weiner et a l . , 1986; Karin et a l . , 1984). Sequence anal ys i s of the two human GRP94 genes indicated that one (hu94-19) contains i ntron s, wh i 1e the other does not. I n t e r e s t i n g l y , the intr onless gene has acquired CCAAT elements and a typical TATA sequence 27 (Fig. 7) required for the t r n s c r i p t i o n in eukaryotes. Moreover, ne ith er the polyadenine t r a c t , nor the flanking repeats were found in this human GRP94 gene. These featu r es are similar to a few f u n c ti o n a l genes such as, the rat p r e p r o i n s u 1 i n I gene (Soares et al., 1985), the human p r o i n t e r 1 e uk i n 1 beta gene (Clark et al., 1986), and the phosphog1ycerate kinase gene, pgk-2 (Boer et al., 1987) wh ich have s ome of the c h a r a c t e r i s t i c s of retroposons. The processed genes may have occurred by i n t e g r a t i o n of reverse transcribed cDNA into regions where promoters are lo ca te d . A l t e r n a t i v e l y , a processed mRNA may acquire a promoter a f t e r r e t r o p o s i t i o n . Howe v e r , the mu t i p l e g e n e ti c l e s i o n s (Fig. 8) found in the i n t r o n l e s s human GRP94 gene make t r a n s l a t i o n of any tr a n s c r i p t into a fu n c t i o n a l p r o te in imp oss ib le. In a d d it io n , instead of AUG, i t s p u t a t iv e t r a n s l a t i o n i n i t i a t i o n codon is replaced by GUG. Although both GUG and UUG could also be em ployed as i n i t i a t i o n codons in prokaryotic system (yet at lo w er e x t e n t ) , only the codon AUG is used as a t r a n s l a t i o n a l i n i t i a t o r in eukaryotic cytoplasm. Further Ln v i vo st u d ie s (chapter 2) showed that the i n t r o n l e s s human GRP94 gene is a n o n - f u n c t i o n a 1 p se ud oge ne . The promoter of the i n t r o n - c o n t a i n i n g GRP94 gene p o s s e s s e s an uncommon TATA sequence, GTGAAAA, and 28 several other putative s ite s for eukaryotic t r a n s c r i p t i o n a l factors such as, Spl, AP-2 and CCAAT (Fig. l l ) . An a t y p t i c a l TATA sequence in w h i c h a G residue int e rr u p t s the A-T rich oligo nucleoti de is also found in the chicken HSP108/GRP94 gene ( TTGATAA, Kleinsek et a l . , 1986), beta-g lobin gene (GATAAAA, D ay e t a l . , 1981) and ty p e 5 actin gene (ATAGAAA, Bergsma et a l . , 1985). H ow ever, a l l of them r e t a i n a ATA sequence except the int ro n-containing human GRP94 gene isola ted in this study. A ssum ing that the GTGAAAA promoter sequence of the human GRP94 gene is acquired genomically and not generated by a cloning a be rr at ion , it probably i n t e r a c t s with a new class of promoter factors d i f f e r e n t fr om those wh ich recognize the typi cal TATA sequences. A comparison among GRP94 genes showed that the 5 ’ -UTR sequence is highly conserved b e tw e e n murine and human genes (Fig. 12), but not wit h the chicken HSP10 8 /G R P94. The conservat i on b e tw e e n the fun ctional and processed human genes is not surpr is ing as the retropseudogene often extend to the norm al t r a n s c r i p t i o n a l i n i t i a t i o n s i t e . Further, sequence c o m p a r is o n of the 5 ’-f l anki ng region of the fun ction al human GRP94 gene wi t h that of chicken reveals a region of high sequence conservat i on b e tw ee n n t -195 and -72, suggesting that this region may contai n sequences 29 important for the expression and regul ation of the GRP94 gene. A c omp ar i son of the predicted am ino acid sequence of chicken and murine GRP 9 4 showed 92% hom ology (Fig. 13; Sargan et al., 1 986; Mazzarel la and Green, 1987). A comparison of the p a r t i a l hamster and human predicted amino acid sequences to those of chicken and murine, also showed a high conservat i on (Fig. 13). Fu rther, GRP94 shares about 50% amino acid hom ology wi t h that of yeast HSP90 ( F a r r e l ly and F in k e l s t e i n , 1984) and P r o s oph i l a HSP83 (Blackm an and Meselson, 1986) (Fig. 13), suggesting that the genes coding for the GRP94 and HSP90/HSP83 may have arisen from a common ancestral gene (Sorger and P elham , 1987; Mazzarel l a and Green, 1987). H ow ever, in co n t r ast to GRP and m o st genes in higher eukaryotes, heat shock genes are conspicuously free of i nt ron s. The gene encoding the Dr o s oph i 1 a HSP83 is one exception. The HSP83 gene does contai n intron s. 11 wa s s h own that severe heat sh o ck blocks the processing of i nt ron (Y o st and L ind quist, 1 9 8 6 ). Some of the uns pliced t r a n s c r i p t s may leave the nucleus re s ul ti n g in the production of ab n o rm al p ro te in s. Most of the heat sh o c k genes lack i nt ron s, this ma y all o w th ei r t r a n s c r i p t s b y p a ss the block in RNA sp lic ing and consequent production of norm al funct ion al proteins p r e s u m a b l y needed for prot ec tin g cel ls from the effe ct s 30 of s tr e s s . The HSP83 gene possesses i n t ron s, but it is c o n s t i t u t i v e l y expressed at a substa nt ial quantit y even before heat shock. The fact that GRP genes do contain intr ons may also explain why GRPs are not h e a t- in d u c i b le . Although the preci s e functions of HSPs and GRPs are not clear, HSP90 has been shown to bind steroi d hormone receptor s and a v a ri e ty of viral tyrosine protein kinases p ri o r to the as sociation with plasma membrane (Baulieu, 1987; C a t e 11i et a l . , 1985; Schuh et a l . , 1985). It is po ssi ble that GRP94 is also capable of binding to recepto rs. Furthermore, as suggested by Pelham (1986), both HSPs and GRPs may somehow be involved in prot ein assembly that occurs in the cytoplasm as well as in the ER. The pr otein assembly is p a r t i c u l a r l y important when abnormal proteins are produced due to stress conditions such as, heat shock and glucose s ta r v a ti o n . The is o l a t i o n of the human gene encoding GRP94 provides us with an additio nal tool with which to study the molecular mechanism of i t s expression and the mechanisms by which GRPs are co- ordinately regul ated. Wi th the gene a va il a b le , it ma y also aid in understanding the bi ol ogical functions of GRPs through gene manipulation. 31 FIG. 1 A r e s t r i c t i o n map of the p4A 3 cDNA clone. The cDNA insert indi cat ed by an open bar, was introduced into the unique Bam HI r e s t r i c t i o n enzyme s ite of pBR3 2 2 (Lee et a l . , 1981). The Bam HI site was destroyed during cloning. The r e s t r i c t i o n map was const ruct ed on the basis of double enzyme dig e st io n and di ge st io n of r e s t r i c t i o n fragments isol at ed by SeaPlaque gel e le c tr o p h o r e s is . The 980 nt Bam H I/E c o RV fragment indi cat ed was is olated and used for determining the o r i e n t a t i o n of the insert contained w ith in cDNA clone p4A3 . 32 lO O nt 9 8 0 n t Bam HI EcoRV Bam HI) FIG. 2 Hy bridization of p a r t i a l cDNA ins erts of the p4A3 with hamster K12 cytoplasmic RNA. Total cytoplasmic RNA was ext r act ed from the hamster fi b r o b l a s t K12 ce lls w h ic h were maintained at 3 5°C in EMEM (lane l) , t r eat ed with 7 txM of A2 31 87 (lane 2), or shifted to the non- permissive temperature 3 9 .5 ° C (lane 3) for 16 hrs. RNA (10 jug) from each sample was used for Northern blot ana ly sis . In panel A, the 690 nt Hind III/Eco RV fragment of p4A3 inser t labeled at the Eco RV r e s t r i c t i o n enzyme si t e was used as a probe. In panel B, the 290 nt Bam HI/Hind III fragment of p4A3 i nsert labeled at the Bam HI r e s t r i c t i o n enzyme site was used as a probe. Their cooresponding h y b rid iz at ion pa tterns are sh own . Solid lines indicate the DNA probes, and broken lines i ndicate the cytoplasmic RNA. The r e s t r i c t i o n enzyme sites indicated are: Bam HI (B), Hind III (H), and Eco RV (E). Autoradiograms are shown and the positi on corresponding to the GRP94 t r a n s c r i p t s is indi cat ed. 34 A. B . 1 0 0 n t B H E B H E 5’1 ------------1 -----------------------------3’ 3’W---------1 ---------------------------- J 5 3 ’ 1 — 1 1 r~i—1 1 | { 1 1 | 5 -X - -X'5’j r-r | 1 j 1 3 i i j i i i j i i i i i 1 i i i i i i 3’ 3’ -*-' - - - --------------------------- 5’ m R N A mR N A 1 2 3 1 2 3 35 FIG. 3 P a r t i a l n ucleotide sequence of the cDNA clone (p4A3) encoding the hamster GRP94. S equence d o w n s t r e a m of the Eco RV r e s t r i c t i o n enzyme s ite of the p4A 3 insert and i t s p redi ct ed am ino acid sequence are sh own . The t er mi nat i on codon TAA is boxed. 36 Eco RV ▼ AT CTT Leu CTA Leu CCA Pro GAC As p ACC Thr AAG Lys AGA Arg ATG Me t CTT Leu CGC Arg CTC Leu AGC Ser TTA Leu GTA V al GAA G lu GAA G lu GAA G lu CCG Pro GAA G lu GAA G lu GAC As p ACA Thr GAA G lu CAA G in GAC As p GAG G lu GAG G lu GAA G lu GAA G lu GAA G lu GAA G lu GAA G lu GAG G lu GAA G lu ACA Thr GCA A l a GAA G lu AAA Lys GAT As p GAG G lu TTA Leu GCA TAT GCA GAT AGA ATA GAG A la Tyr A la As p Arg I 1 e Glu AAC ATT GAC CCT GAA GCA CAG Asn l i e As p Pro G lu Al a Gin GAG CCT GAA GAC ACC ACA GAA G lu Pro G lu As p Thr Thr G lu GAA GAA GTG GAT GCA GGC ACA G lu G lu V al Asp A l a G ly Thr CAG GAA ACA GCA AAG GAA TCT G in G lu Thr Al a Lys G lu Ser F taaI t t a t c c t c t c a c c a c a g a t c c t g TER 37 FIG. 4 Southern blot analys is of GR P94 sequences. Ten jag of HeLa gen om i c DNA wa s di gested with r e s t r i c t i o n enzymes as i ndicated. The re s ul ta nt fragments were el ectrophoresed on 1% agarose gel s, blott ed onto n i t r o c e l l u l o s e papers, and probed wi t h (A) the hamster cDNA clone p4A3 , (B) the 415 nt Bam HI/ Eco RV fragment of the human pseudogene isolated from phage clone hu94 - 24 (as i ndicated in Fig. 5). Nucleotide sequence of the 415 nt fragment and that of the p4A3 are not ov e rla p ed . Autoradiograms are shown. 38 B. (Kb) 2 3 .1 - 9.4- 6.6 4 .4 EcoRI Bam HI Hind III I 1 0 .5 — 2.3- 2j0- Hindlll Eco RI SamHI t 39 FIG. 5 R e s t r i c t i o n map of a human GRP94 gene from the phage clones hu94-24 and hu94-7. A) The phage clones hu94-24 and hu94-7, represen ti ng overlapping sequences of a human GRP94 gene are shown. I t s 5* to 3* o r i e n t a t i o n is in d i cat ed . W a v y l i n e s i ndi cat e the DNA arms of the lambda Char on4A in w h ic h th e human genomic l i b r a r y was constru cted. A 2.5 kb Bam HI f r a g m e n t w h ic h hybri dized to both the synt hetic o l i g o m e r probe and the cDNA probe from hamster GRP94 is indi cat ed by a hatched box. Two DNA fra gme nts used in the SI p ro t e c t i o n assay (Fig. 9) are shown. Ast er isk s indicat e the [JT- 3 2 P] ATP labeled 5 ’ -ends. B) A 1.8 kb Hind III fragment contai ning the 5 ’- half of the 2.5 kb Bam HI fragment was expanded. A DNA dom ain corresponding to the hamster synthetic 41-m er is indi cat ed by a black box. A 415 nt B am HI/ E c o RV fra gme n t wh i c h wa s used for a l i b r a r y screening and for a Southern blot anal ysi s (chapt er 1 .3.4. and Fig. 4B) is indicated. R e s t r i c t i o n e n z yme s it e s i ndicated are: Eco RI ( RI ) , Sst I (SS), Bam HI (B), Hind III (H) , Ava II ( All), Ava I (Al ) , Sma I (SM), Eco RV ( R V ), and Kpn I (K). 40 A. B. 103 nt \ ■ i -If-----------------------------------------------------------------1 M v v w hu 94-24 hu 94-7 w m ----------------------------------------------------------------- —ih I-------------1* I 1* i i i i i i . . Rl SS Rl B SS HRI B H H B SS B Rl O 1 — 1 - - - - - - - - - - - - 1 - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - * ---- 1 - - - - - - - - - - - - - - - - “ - - - - - M iiiim iinitm iliiiTim nm - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - FIG. 6 Southern blot anal ys i s of the phage clone h u 9 4 - 2 4. DNA isolated fr om the phage clone h u 9 4 -2 4 wa s r e s t r i c t i o n digested with Eco Rl (lanes 1 and 4), Bam HI (lanes 2 and 5) or Hind III (lanes 3 and 6). The re s u lt an t fragments were resolved on an agrose gel and blotted onto a n i t r o c e l l u l o s e paper. Hybridization probes used were the synth etic 4 1 -me r corresponding to the ami no - te r m i n a 1 sequence of the h a m s t e r GRP94 (lanes 1 to 3) and the p4A3 cDNA clone encoding the carboxy1 - termi n a 1 half of h a m s t e r GRP94 (lanes 4 to 6). Autoradiograms are shown. Hy bridization si gnals corresponding to the DNA f r a g m e n t of sizes 2.5 kb and 1.8 kb are i ndicated. 42 h u 94-24 — . m R P9nH RRp^"vll 4 4 _o xa xc in C N oo rH FIG. 7 The sequence of 5 ’ region of the human GRP94 gene from phage clone hu94-24. The 5' region of human GRP 94 gene isol at ed from phage clone h u 9 4 -2 4 was sequenced. The putative tr a n s c r i p t i o n a l i n i t i a t i o n site ( r — ^ ) determined by Si pr ot e c tio n assay and primer extension (shown in Fig. 9) is ind icated. The putat ive TATA an d CCAAT sequences are underl ined. The Bam HI s ite contained w ith in the 1.8 kb H ind III fragment, sh o w n in Fig. 5B, is underlined with a wavyline. The puta tive t r a n s l a t i o n a l i n i t i a t i o n codon GTG is boxed and the amino acid sequence t r a n s l a t e d . 44 CTGACTTCGATCGAAAT CTGATTTGACTATAGCAAT CCTTTTTAAACTCGATCATCCCTGCTATGAA ACCATATTACTGACCTTTTTAACCCTTTAACNACAATGGTATAAGCAATA TTTTGACAAGGATTATAGCCTAGATTTTCCCATGTTTGTTCTTTATGTAT AAAGGACAATTTGTGATTGGATTACTTATTAACTATCTGCCATAGCCAAT GGGGTAGACTTTGTTCAGAGAATTGACCTTTGCTTATAAAAGTTAAATTT r— . . . . . GTGGCGGACCACGCGGCTGGAGGTGTGAGGATCCAACCCGGGGGTGAGAG V W W V W V W GGTGGAGGTGGCTCTTGCGATCGAAAGGGACTTGAGACTCACCAGCCACA a g c t I g t^ a a g g c c c t g t g g t g c t g g g g c c t c t g c t g c g t c c t g c t g a c c V a 1LysA laL euT r pC ysT rpG lyL euC ysC ysV a1LeuLeuTh r 45 FIG. 8 Nucleotide sequences of the human GRP 9 4 genes and their comparison wi t h the chicken, hamster and murine cDNA sequences encoding the GRP94 (Kulomaa et a l . f 1986; Sorger and Pelham, 1 9 87; Mazzare 1 la and Green, 1987). The human sequence shown was determined f r om the phage clone hu94-19, wh ile the sequence of human pseudogene was determined from the phage clone hu94-24. Nucleotides are numbered with t r a n s c r i p t i o n a l i n i t i a t i o n site of the chicken HSP10 8/GRP9 4 gene designated as +1. Solid tr i an g le s (▼) separate the chicken cDNA sequence into 18 domains corresponding to the 18 exons of the chicken H SP 108/GRP94 gene (Kleinsek et a 1 . , 1986). The t r a n s l a t i o n a l star t codons (ATG) and sequences corresponding to am i no - termini of the mature proteins are sh own in bold. A d ownwa r d a r r ow i n d i cates the cleavage si t e for the matured p r ot ein of chicken HSP1 0 8 /GRP94. Undetermined sequences are bracketed. Nucleotides identical between two human GRP94 genes or between two species that are aligned next to each other are indicated by v e r t i c a l dots. The sequence corresponding to the sense strand of the synthetic 18 -me r ( 5 ’ - TTCCACATCAACTTCATC- 3 ’ ) used for primer extension (Fig. 9) and lib r ar y screening is double-underl i ned. Hamster cDNA sequence d o w n s t r e a m of the E c o RV site indicated with an underline at position 2278 has been sh own in Fig. 3. 46 CHICKEN HAMSTER MURINE HUMAN H U M A N (pseudo) CHICKEN HAMSTER MURINE HUM AN HUMAN( pseudo) CHICKEN HAMSTER MURINE HUM AN HUMAN( pseudo) CHICKEN HAMSTER MURINE HUM AN HUMAN( pseudo) CHICKEN HAMSTER MURINE HUM AN MET 140 ATGAAGTCAGCGTGGGCGCTGGCTCTGGCATGCACGCTT ( ) ATGAGGGTCCTGTGGGTGTTGGGCCTCTGCTGTGTCCTG ATGAGGGCCCTGTGGGTGCTGGGCCTGGCATGCACGCTT GTGAAGGCCCTGTGGTGCTGGGGCCTCTGCTGCGTCCTG t v ASPGLU 176 CTCCTGGCCGCATCGGTGACCGCT GAGGAGGTGGAT ( ) CTGACCTTCGGGTTCGTCAGAGCTGATGATGAAGTCGAC CTGACCTTCGG ( ) CTGACCTTCGGGTCGGTCAGAGCTGTTGATGAAGTTGAT 215 GTGGATGCGACCGTGGAAGAAGATCTGGGTAAAAGTAGA ( ) GTGGATGGCACAGTGGAAGAGGACCTGGGTAAAAGTCGA ( ) GTGGAAGGTACAGTAGAAGAGGATCTGGGTAAAAGTAGA y 254 GAAGGGTCCCGAACTGATGATGAAGTTGTTCAGAGAGAG ( ) GAAGGCTCAAGGACAGATGAAGTTGTTCAGCAGAGAGAG ( ) CAAGGCTCAAGGATGGAT - AT - AAGTAGT ACAGAG AG AG 293 GAAGAAGCTATCCAGCTGGATGGCCTAAATGCATCCCAG ( ) GAAG AAGCTATTC AGTTGGATGGGTTAAACGCATCACAG ( ) HUM AN (pseudo) GAAGAAGCTATTCAGTTGGATAAATTAAATGCATCACAA 47 FIG. 8 ( c o n t i n u e d ) CHICKEN HAMSTER MURINE HU M A N HUMAN(pseudo) 332 ATCAAAGAAATCAGAGAAAAATCTGAGAAGTTTGCCTTT ( ) ATAAGAGAACTTAGAGAAAAATCTGAAAAGTTCGCCTTC ( ) ATAAGAGAACTTAGAGAGAAGTTGGAAAAGTTCGCCTTC 371 CHICKEN CAAGCAGAAGTTAACAGAATGATGAAGCTTATTATTAAC HAMSTER ( ) MUR INE CAAGCTGAAGTGAACAGGATGATGAAACTTATCATCAAT HUM AN ( ) HUMAN(pseudo) CAAGCTGAAGTTAACAGAATGATGAAACTTATCATCAAT y 410 CHICKEN TCTTTATATAAGAACAAAGAGATTTTCCTGAGAGAGCTT HAMSTER ( ) MURINE TCTTTGTATAAAAATAAAGAGATTTTCCTGAGAGAACTG HUM AN ( ) HUM AN (pseudo) TCATTATATGAAAATAAAGAGATTTTCCTTAGAGAACTG CHICKEN HAMSTER MURINE HUM AN 449 ATTTCAAATGCTTCAGATGCTTTGGATAAGATACGCTTA ( ) ATTTCAAATGCTTCTGATGCTTTAGACAAGAT AAGGCTC ( ) HUM AN (pseudo) AT------------------ TCTGACGCTTTAGTTAAGATAAGGCTG 488 CHICKEN ATATCCTTGACTGATGAAAATGCTCTTGCTGGT AATGAG HAMSTER ( ) M UR I NE ATCTCCCTAACTGATGAAAATGCACTCGCTGGAAATGAG HUM AN ( ) HUM AN (pseudo) ATATCACTGACTAATGAAAATGCTTGCTCTGGAAATGAG 48 f i g . 8 ( c o n t i n u e d ) C H I C K E N H A M S T E R M U R IN E HUMAN HUMAN ( p s e u d o ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) f 527 GAGCTCACTGTTAAAATCAAGTGTGATAAAGAGAAGAAC ( ) GAGTTAACGGTCAAGATTAAGTGTGACAAAGAGAAAAAC ( ) GAACTAACAGTCAAAATTAAGTGTGATAAGGTGAAGAAC 566 ATGCTTCATGTTACAGATACGGGTATTGGCATGACAAAA ( ) CTGCTGCATGTCACAGACACGGGTGTAGGAATGACTAGA ( ) CTACTGCATGTCACAGACACTGGTGTAGGAATGACCAGA 605 GAGGAGTTGATTAAAAACCTTGGTACCATTGCAAAGTCT ( ) GAGGAGTTGGTTAAAAATCTCGGCACCATAGCCAAATCT ( ) GAAGAGTTGGTTAAAAACCTC( ) 644 GGTACAAGTGAATTCTTAAACAAGATGACTGAAATGCAG ( ) GGAACAAGCGAGTTTTTAAACAAAATGACAGAAGCTCAA ( ) ( ) C H I C K E N H A M S T E R MURINE HUMAN HUMAN( p s e u d o) 683 GATGATAGCCAGTCGACATCTGAGTTAATTGGCCAGTTT ( ) GAAGATGGTCAGTCAACCTCTGAACTGATTGGCCAGTTT ( ) ( ) 49 FIG. 8 (c o n t i n u e d ) C H IC K E N H A M S T E R MURINE HUMAN H U M A N (p se u d o ) 722 GGTGTTGGCTTTTATTCTGCTTT CTTGGTAGCAGACAGA C ) GGTGTCGGTTTTTATTCTGCCTTCCTTGTAGCAGAT AAG ( ) ( ) C H IC K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) 761 GTTATTGTCACATCAAAGCACAACAATGATACCCAACAT ( ) GTCATTGTCACATCGAAACACAACAATGATACCCAGCAC ( ) ( ) C H I C K E N H A M S T E R M U R IN E HUM AN HUMAN( p s e u d o ) 800 ATTTGGGAGTCAGATTCAAATGAGTTCTCTGTGATTGAT ( ) ATCTGGGAATCAGACTCCAATGAATTCTCTGTAATTGCT ( ) ( ) C H I C K E N HAMSTER MURINE HUM AN HUMAN(ps eudo) 839 GATCCAAGAGGAAACACTCTGGGACGTGGCACAACCATA ( ) GACCCAAGAGGAAACACACTAGGTCGTGGAACAACAATT ( ) ( ) C H I C K E N H A M S T E R M U R IN E HUMAN HUMAN(ps eu d o) « 878 ACCCTTGTCTTGAAGGAAGAAGCCTCTGATTATCTTGAG ( ) ACTCTTGTCTTAAAAGAAGAAGCAT CTGATTACCTTGAA ( ) CCTTGTCTTAAAAGAAGAAGCATCTGATTACCTTGAA ( ) 50 FIG. 8 ( c o n t i n u e d ) C H I C K E N H A M S T E R MURINE HUMAN HUMAN ( p se u d o ) 917 TTGGACACTGTTAAAAATCTAGTCAAGAAATATTCACAG ( ) TTGGACACAATTAAAAATCTTGTCAGGAAGTACTCTCAG TTGGATACAATTAAAAATCTCGTCAGAAAATATTCACAG ( ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) -956 TTCATAAACTTCCCCATATATGTGTGGAGCAGCAAGACA ( ) TTCATCAACTTTCCCATCTACGTGTGGAGTAGCAAGACA TTCATAAACTTTCCTATTTATGTATGGAGCAGCAAGC ) ( ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) 992 GAGACTGTTGAAGAGCCAGTTGAAGAGGAGGAAGCA- - - ( ) GAGACTGTTGAGGAGCCCTTGGAAGAAGATGAAGCAGCA ( ) ( ) C H I C K E N H A M S T E R M U R IN E HUM AN HUMAN( p s e u d o ) 1031 AAGGAGGAGAAAGAAGAAACAGATGAT AATGAAGCAGCG ( ) AAAGAAGAGAAAGAAGAATCTGATGAT GAAGCTGCA ( ) ( ) C H IC K E N H A M S T E R MURINE HUMAN HUMAN( pseudo) 1070 GTTGAAGAGGAGGAGGAAGAGAAGAAACCAAAAACTAAG ( ) GT AGAGGAGGAAGAAGAAGAAAAGAAACCAAAAACT AAG ( ) ( ) 51 FIG. 8 (c o n t i n u e d ) C H IC K E N HAMSTER MUR IN E HUMAN HUMAN (p se u d o ) w 1109 AAGGTTGAAAAGACTGTCTGGGATTGGGAGCTCATGAAT ( ) AAAGTTGAAAAAACTGTGTGGGATTGGGAACTTATGAAT ( ) ( ) C H IC K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) 1148 GACATAAAACCAATCTGGCAGAGACCATCTAAAGAAGTT ( ) GATATCAAACCAATATGGCAGAGACCATCCAAAGAAGTA ( ) ( ) C H IC K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) 1187 GAAGAGGATGAATATAAAGCTTTTTACAAAACCTTTT CC ( ) GAAGAAGACGAATACAAAGCTTT CTACAAATCATTTT CA ( ) ( ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) « 1226 AAGGAACACGATGATCCAATGGCTTACATCCATTTTACT ( ) AAGGAAAGTGATGACCCCATGGCTTATATCCACTTCACT ( ) ( ) C H I C K E N H A M S T E R MURINE HUMAN HUMAN(ps eudo) 1265 GCTGAAGGGGAAGTAACTTTCAAATCAATCTTGTTTGTT ( ) GCAGAAGGGGAGGTCACCTTCAAGTCGATTTTGTTTGTA ( ) ( ) 52 FIG 8 ( c o n t i n u e d ) C H I C K E N H A M S T E R M U R IN E HUMAN H U M A N ( p s e u d o ) 1304 C C T A A T T C T G C T C C A C G T G G C T T G T T T G A T G A G T A T G G A ( ) C C C A C A T C T G C A C C T C G A G G T C T G T T T G A T G A A T A T G G A ( ) ( ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) « 1343 T C C A A A A A A A G T G A T T T C A T T A A G C T G T A T G T T A G A A G A ( ) A A G A A G A G C G A T T A T A T T A A G C T C T A T G T G C G C C G A T C T A A G A A G A G T G A T T A T A T T A A G C T G T A T G T A C G C C G C ( ) ( ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) 1382 G T G T T C A T C A C T G A T G A C T T C C A T G A C A T G A T G C C C A A A G T A T T C A T C A C C G A T G A C T T C C A T G A T A T G A T G C C C A A G G T A T T C A T C A C A G A T G A C T T C C A T G A T A T G A T G C C C A A A ( ) ( ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) y 1421 T A T C T T A A C T T C G T T A A A G G T G T T G T G G A T T C T G A T G A T T A C C T T A A T T T T G T C A A A G G T G T T G T G G A T T C A G A T G A T T A C C T T A A T T T T G T C A A A G G T G T T G T G G A T T C C G A T G A T ( ) ( ) C H I C K E N H A M S T E R MURINE HUMAN HUMAN( p s e u d o) 1460 C T T C C T T T G A A T G T A T C T C G T G A A A C A C T T C A G C A G C A T C T C C C C C T T A A T G T T T C C C G T G A G A C T C T T C A G C A A C A T C T C C C C C T C A A T G T T T C C C G T G A G A C T C T T C A G C A A C A T ( ) ( ) 53 FIG. 8 ( c o n t i n u e d ) C H IC K E N H A M S T E R M URIN E HUMAN H U M A N ( p se u d o ) C H IC K E N H A M S T E R M U R IN E HUMAN H U M A N ( p s e u d o ) f 1499 A A G T T A C T A A A G G T G A T C A G A A A G A A G C T T G T T C G T A A A A A A C T G C T C A A G G T G A T A A G G A A G A A G C T C G T C C G T A A A A A A T T G C T C A A G G T G A T T A G G A A G A A G C T T G T C C G A A A A ( ) ( ) 1538 A C C C T C G A T A T G A T C A A G A A A A T T G C A G A A G A A A A A T A C A C T C T G G A C A T G A T C A A G A A G A T T G C A G A T G A G A A G T A C A C T C T G G A C A T G A T C A A G A A G A T T G C T G A T G A G A A G T A T ( ) ( ) C H IC K E N H A M S T E R M U R IN E HUMAN H U M A N ( p s e u d o ) 1577 A A T G A C A C A T T C T G G A A A G A G T T T G G T A C T A A T G T A A A G A A T G A T A C T T T C T G G A A A G A G T T T G G C A C C A A C A T C A A G A A C G A C A C T T T C T G G A A G G A G T T C G G C A C G A A C A T C A A G ( ) ( )A G 1616 C H I C K E N C T T G G A G T T A T T G A A G A T C A C T C C A A T C G C A C A C G A C T G H AM S T E R C T T G G T G T G A T T G A G G A C C A C T C A A A T C G G A C G C G G C T T M U R IN E C T T G G T G T G A T T G A A G A C C A C T C A A A T C G A A C A C G G C T T HUM AN ( ) HUM AN ( p s e u d o ) C T T G G T G T A A T T G A A G A C C A C T C G A A T C G A A C A T G T C T T 54 FIG. 8 ( c o n t i n u e d ) C H IC K E N H A M S T E R MURINE HUMAN H U M A N (p se u d o ) 1655 G C T A A A C T G C T T C G C T T C C A G T C T T C C C A T C A T G A A A G T G C T A A A C T T C T T A G G T T C C A G T C T T C T C A T C A C T C A A C T G C T A A A C T T C T T A G G T T C C A G T C T T C T C A C C A T T C A A C T ( ) G C T A A A C T T C T T A G G T T C C A G T C T T C T C A T C A T C C A G C T C H IC K E N H A M S T E R M U R IN E HUM AN 1694 A A C C T T A C A A G C C T T G A C C A A T A T G T G G A A A G A A T G A A A G A C A T T A C T A G T T T A G A C C A G T A T G T G G A A A G A A T G A A G G A C A T T A C T A G T T T A G A C C A G T A T G T G G A A A G A A T G A A G ( ) HUM AN ( p s e u d o ) G A C A T T A C T A G C C T A C A C C A G G A T G T T G A A A G A A T G A A G 1733 C H I C K E N G A G A A A C A A G A C A A A A T T T A T T T C A T G G C A G G T G C C A G C H A M S T E R G A G A A G C A G G A C A A A A T C T A C T T C A T G G C T G G G T C A A G C M U R I N E G A A A A A C A G G A C A A A A T C T A C T T C A T G G C T G G G T C A A G C HUM AN ( ) HUMAN ( p s e u d o ) G A A A A A C A A G A C A A A A T C T G C C T C A T G G C T A G G T C C A G C y 1772 C H I C K E N A G A A A G G A G G C T G A G T C T T C A C C A T T T G T T G A A C G C C T T H A M S T E R A G G A A A G A G G C C G A A T C T T C T C C A T T T G T T G A G A G G C T T M U R IN E A G A A A G G A G G C G G A A T C T T C T C C A T T T G T T G A G A G A C T T HUM AN ( ) H U M A N (pseudo) AGAAAAGAGGCTGAATCTTCTCCATTTGTTTAGCGACTT 55 FIG. 8 ( c o n t i n u e d ) 1811 C H I C K E N C T G A A A A A G G G C T A T G A A G T G A T C T A C C T G A C T G A A C C T H A M S T E R C T G A A G A A G G G C T A T G A G G T T A T T T A C C T C A C A G A G C C T M UR IN E C T G A A G A A G G G C T A T G A A G T C A T T T A T C T C A C A G A G C C T HUMAN ( ) HUMAN ( p s e u d o ) C T G A A A A G G G G C T A T G A A G T T A T T T A C C T C A C A G A A C C T 1850 C H I C K E N G T A G A T G A A T A C T G C A T T C A G G C T C T G C C A G A G T T T G A T H A M S T E R G T G G A T G A A T A C T G C A T T C A G G C T C T T C C C G A G T T T G A T M U R I N E G T G G A T G A A T A C T G C A T T C A G G C T C T T C C C G A G T T T G A T HUM AN ( ) HUM AN ( p s e u d o ) G T G G T T G A A T A C T G C A T T C A G G C C C T T C C C G A A T T T G A T 1889 C H I C K E N G G C A A G A G G T T T C A G A A T G T A G C A A A G G A G G G A G T T A A G H AM S T E R G G G A A G A G G T T C C A G A A T G T T G C C A A A G A A G G A G T G A A G M U R IN E G G G A A G A G G T T T C A G A A T G T T G C C A A A G A A G G G G T G A A G HUM AN ( ) H UM A N ( p s e u d o ) G G G A A G A G G T T C C A G A A T G T T G C C A A A G G A G G A G T G A A G C H I C K E N H A M S T E R M U R IN E HUM AN 1928 T T T G A G G A A A G T G A A A A G T C C A A G G A G A G T C G T G A A G C C T T T G A T G A G A G C G A G A A A A C G A A G G A A A A C C G G G A A G C A T T T G A T G A G A G T G A G A A A A C T A A A G A A A G T C G G G A A G C A ( ) HUMAN(pseudo) TTTGATGACAGTGAGAAAACTAAGGAGAGTCATGAAGCA 56 FIG. 8 ( c o n t i n u e d ) C H I C K E N H A M S T E R MURINE HUMAN 1967 T T G G A G A A G G A A T T T G A A C C A C T C T T G A A C T G G A T G A A A A C A G A G A A G G A G T T T G A G C C C C T G C T C A A C T G G A T G A A A A C A G A G A A G G A G T T T G A A C C T C T G C T G A A C T G G A T G A A A ( ) HUMAN ( p s e u d o ) G T T G A G A A A G A A T T T G A G C C T C T G C C C A A T T G G G T G A A A v 2006 C H I C K E N G A C A A A G C T C T A A A G G A C A A G A T T G A A A A G G C T G T G C T G H A M S T E R G A C A A G G C C C T T A A G G A C A A G A T T G A A A A G G C T G T G G T A M U R IN E G A T A A G G C C C T C A A G G A C A A G A T A G A A A A G G C T G T G G T G H UM AN ( ) H U M A N ( p s e u d o ) G A T A A A G C C A T T A A G G A C A A G A T T G A A A A G G C T A T G G T A 2045 C H I C K E N T C T C A A C G T C T A A C A C A G T C T C C A T G T G C A C T C G T G G C T H A M S T E R T C T C A G C G T C T C A C G G A A T C T C C T T G T G C T C T T G T G G C C M U R IN E T C G C A G C G C C T C A C A G A G T C T C C C T G T G C T C T T G T G G C C HUM AN ( ) H U M A N ( p s e u d o ) T C T C A G T G C C T G A C A G A A T C T C C G T G T G C T T T G G T G G C C 2084 C H I C K E N A G T C A G T A C G G A T G G T C T G G T A A C A T G G A A A G A A T C A T G H A M S T E R A G C C A G T A T G G A T G G T C T G G C A A C A T G G A G A G G A T T A T G M U R IN E A G T C A G T A T G G A T G G T C T G G C A A C A T G G A G A G G A T C A T G HUM AN ( ) HUMAN ( p s e u d o ) AGCCAGTACGGGTGGTCTGGCAACATGGAGAGAATCATG 57 FIG. 8 ( c o n t i n u e d ) y 2123 C H I C K E N A A G G C T C A A G C T T A C C A A A C T G G G A A G G A C A T A T C T A C A H A M S T E R A A G G C A C A A G C A T A C C A A A C G G G C A A G G A C A T C T C T A C A M UR I N E A A G G C A C A A G C A T A C C A G A C G G G C A A G G A C A T C T C T A C A HUMAN ( ) H U M A N ( p s e u d o ) A A A G ( ) C H I C K E N H A M S T E R M U R IN E HUM AN HUMAN( p s e u d o ) 2162 A A T T A C T A T G C T A G C C A G A A G A A G A C A T T T G A A A T A A A T A A T T A C T A T G C A A G T C A G A A G A A A A C A T T T G A A A T T A A T A A T T A C T A T G C C A G T C A A A A G A A A A C G T T C G A A A T C A A T ( ) ( ) C H I C K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) 2201 C C C A G A C A T C C A C T G A T C A A A G A C A T G C T G A G G C G A G T T C C C A G A C A T C C G C T G A T C A G A G A C A T G C T T A G G A G G G T T C C T A G A C A C C C A C T G A T C A G A G A C A T G T T G C G G C G G A T T ( ) ( ) C H IC K E N H A M S T E R M U R IN E HUM AN H U M A N ( p s e u d o ) y 2240 A A G G A A A A T G A A G A T G A C A A A A C A G T T T C A G A T C T T G C A A A G G A A G A T G A A G A T G A C A A G A C T G T C T T G G A T C T T G C T A A G G A A G A T G A A G A T G A C A A G A C A G T C A T G G A T C T T G C T ( ) ( ) C H I C K E N H A M S T E R M U R IN E HUM AN HUMAN( p s e u d o ) 2279 G T G G T G T T G T T T G A A A C T G C G A C T T T G A G A T C A G G A T A T g t a g t t t t g t t t g a a a c a g c a a c a c t t c g g t c a g g a t a t G T A G T T T T G T T T G A A A C G G C A A C A C T T C G G T C A G G A T A T ( ) ( ) 58 FIG. 8 ( c o n t i n u e d ) C H I C K E N HAMSTER MURINE HUMAN H U M A N ( p s e u d o ) 2318 A T G T T A C C A G A C A C A A A G G A A T A T G G A G A C A G A A T A G A A C T T C T A C C A G A C A C C A A G G C A T A T G C A G A T A G A A T A G A G C T T C T A C C A G A C A C C A A G G C G T A T G G A G A T A G A A T A G A A ( ) ( ) 2357 C H I C K E N A G G A T G C T T C G T T T G A G T T T A A A C A T T G A C C T G G A T G C A H AM S T E R A G A A T G C T T C G C C T C A G C T T A A A C A T T G A C C C T G A A G C A M U R IN E A G A A T G C T T C G C C T C A G T T T A A A C A T T G A C C C T G A A G C A HUM AN ( ) H U M A N ( p s e u d o ) ( ) T C A G T T T G A A C A - - C A T C C T G A T G C A C H I C K E N H A M S T E R M U R IN E HUM AN « 2393 A A G G T G G A G G A G G A A C C T G A A G A G C C T G A A G A T G C A C A G G T A G A A G A A G A A C C G G A A G A A G A G C C T G A A G A C A C C C A G G T G G A G G A A G A A C C A G A A G A A G A G C C T G A A G A C A C C ( ) HUM AN ( p s e u d o ) A A G G T G G A A G A A C C C G A C G A A G A A C C T G A G G A G A C A C H IC K E N H A M S T E R M U R IN E HUM AN 2429 G C T G A G G A G G C A G A G C A A G A T G A A G A A G A G G T G G A T A C A G A A G A C A C A G A A C A A G A C G A G G A G G A A G A A G T G G A T T C A G A A G A C G C A G A A G A C T C A G A G C A G G A T G A G G G A G A A ( ) HUMAN ( p s e u d o ) GCAG AAGACAAAGAGCAAGACAAAGAC AAA---------- A ---- 59 FIG. 8 ( c o n t i n u e d ) C H IC K E N H A M S T E R M U R INE HUMAN HUMAN ( p se u d o ) 2453 G C T G A T G C T ------------------------------ G A A G A C A G T G A A A C A G C A G G C A C A G A A G A A G A A G A A G A A G A G G A A C A G G A A A C A G A G A T G G A T G C A G G G A C A G A A G A A G A A G A G G A G G A A A C A ( ) - - -A T G G A T G T G G G A A C A G A T G A A G A A A A A C A A G A A A C A C H I C K E N H A M S T E R M U R IN E HUM AN v 2486 * C A G A A G G A A T C C A C A G A T G T A A A A G A C G A A C T G T A A G C A A A G G A A T C T A C A G C A G A A A A A G A T G A G T T A T A A G A A A A G G A A T C T A C A G A G A A G G A T G A A T T G T A A ( ) HUM AN ( p s e u d o ) G C A A A G G A A T C T A C A G C T G A A A A G A C T G A A T T G T A A 6 0 FIG. 9 Determination of the t r a n s c r i p t i o n a l i n i t i a t i o n site of the human GRP94 tr a n s c r i p t s in HeLa and 293 c e l l s . A) SI prot ec tio n assay using a 1.75 kb Eco Rl' Hind III fragment as the probe. RNA samples were prepared from either the HeLa D9S AH2 monolayer cel ls (lanes 1-3) or the 293 cel ls (lanes 4, 5, and 7) maintained at 3 5°C in DMEVl (lanes 1 and 4), tr eated with 7 juM of A231 87 at 3 5 °C for 5 hrs (lanes 2, 5, and 7), or g r own in glu cose-f ree medium at 35°C (lane 3) for 16 hrs pri or to RNA ext ra cti on . As controls, yeast tRNA was used in lane 6, and no SI nuclease was added in the r eaction mixture in lane 7. Autoradiogr am i s shown. The lane marked M r efers to a size marker generated fr om Hae III r e s t r i c t i o n enz ym e dig estion of the X17 4 DNA. Th e prot ect ed fra gme nt about 1.15 kb in size is i ndicated. B) SI p ro te c tio n assay using a 1.35 kb Hind III/Asp718 fra gme nt as the probe. RNA samples were prepared from ei th e r the HeLa D98 AH2 monolayer cells (lanes 1 and 2) or the 293 ce lls (lanes 3 and 4) maintained at 35°C in DMEM (lanes 1 and 3), t reated with 7 >uM of A23187 at 3 5°C for 5 hrs (lane 4) or grown in glucose -free medium at 3 5 °C (lane 2) for 16 hrs prior to RNA e xt ra ct i on . Yeast t RNA wa s used in lane 5. The protected fragment about 590 nt in size is indicated . C) Primer extension using a syn thetic 18-mer as the probe. The RNA samples used were yeast tRNA 6 1 (lane 1 ) or RNA prepared from either the HeLa cell s (lanes 2 to 4) or 293 cel ls (lanes 5 and 6) maintained at 3 5 °C in DMEM (lanes 2 and 5), tr eated with 7 >uM of A231 87 at 35°C for 5 hrs (lanes 3 and 6), or grown in gl ucos e-free medium at 3 5 °C (lane 4) for 16 hrs prior t o RNA extract io n. Th e RNA wa s reannealed to the syn t h etic 1 8 -me r ( 5 ’ - TTCCACATCAACTTCATC- 3 ’ , indi cat ed in Fig. 8). The p r ime r - e x t e nd e d GRP94 t r a n s c r i p t was el ect r ophor es ed on a 6% polyacrylamide sequencing gel along with DNA size marker (M) . The autoradiogram is shown. The position of primer-extended products is i ndi cat ed . 62 A B. 63 c. 1 2 3 4 5 6 M - 1 0 7 8 * iV f* K- ; « ? 64 FIG. 10 P a r t i a l r e s t r i c t i o n map of the human GRP94 gene from the phage clone h u 9 4 - 1 9 . DNA was isol at ed from the phage genomic clone h u 9 4 -1 9 and subjected to Eco Rl d ig e st io n. Two Eco Rl fragments of 3.1 kb and 6.1 kb in size, sh own as open bars, we re fu rt her subcloned into the unique Eco Rl r e s t r i c t i o n enzyme site of pUC8, designated p h u 9 4 -1 9 P and p h u 9 4 -1 9 C , r e s p e c ti v e ly . The wavy lines i n di cat e the DNA arms of th e lambda phage C haron4A and the crossin g-o ver double li nes ind icate the pUC8 vector sequences. The t r a n s c r i p t i o n a l i n i t i a t i o n s it e (*“►) i s also indic ated. R e s t r i c t i o n enzyme s it e s m ark ed are: Eco Rl (Rl), Bam HI (B), H ind III (H) , Pst I (PS), Pvu II (PV), Sma I (S), and Ava I (A). 65 hu94-19 Rl — ► Rl Rl wm — i i — i i — 1 . A M , A A HR.' ? H H B Rl phu94-19C — • • 1 i i ooc m i o o FIG. 11 Sequence of the human GRP94 5 ’ region isolated from the phage clone h u 9 4 -1 9 and its comparison wi t h the chicken HSP108/GRP94 genomic sequence. Nucleoti des are numbered with t r a n s c r i p t i o n a l i n i t i a t i o n si te ( n^ ) of the human GRP94 gene desi gnat ed as +1. Bases d o w n s t r e a m a r e num bered p o s i t i v e l y and base upstream are num bered negati vel y. R e s t r i c t i o n s it e s used for subcloning and for constru ctin g GRP94-CAT fusion plasmids in the chapter 2 are in di cat ed. The sym bols m ark ed a re ( ) CCAAT, ( ) CCAAT in vert ed, ( /S. ) putative Spl binding s i t e , and ( ) AP- 2 binding s i t e . The Go 1dberg-Hogness sequences and the t r a n s l a t i o n a l s t a r t codon ATG are boxed. The f i r s t exon of the GRP94 pr ote in sequence is t r a n s la t e d . The f i r s t exon- i nt r on junct i on is in d icated and intr on sequence is w r i t t e n in low er case. Nucleoti des identi cal b e tw e e n the human GRP94 and chicken HSP108/GRP94 sequences are i ndicated by v e r t i c a l dots . The conserved d o m ain s shared with GRP78 (see chapter 3) are bracketed with solid l i ne. The fo o tp r in te d regions (see chapter 3) are bracketed wi th dashed line s. 67 HUMAN C ltlC K - 50 I A TCTG A A A G G G TTCTAGGGG ATTTGCAACCTCTCTCGTGTG T T T C T T C T T T C C G CGCCGAGTGAUGACTACAGCTCCCAGCAGCCACTGCGGCACCACACGCAAGCCT HUMAN AGAAGCGCCGCCACACGAGAAAGCTGGCCGCGAAAGTCOTGCTGGAATCACTTCCAACGAAACCCCCAGGCA'I AGATGGGAAAGGGTGAAGAACACGTTC CHICK GCCACTTGCGCTTAGCCGCCGTACTTTATGTCAGCCCCGTCCGTCACTGGGGCTTGCGGGCGACGCGACCGGAAACAGTTCCACGGCAACAGGGTCCCCG BimHI(- 3 5 7 ) - 30 1 y HUMAN G CA TG G CTA CCGTTTCCCCG GTCACG GAATA AACGCTCTCTA GGATCCGG AAG TAGTTCCGCCG CGA CCTCTCTAAAA GGATG GATGTG TTCTCTG CTTA CHICK G TTA A CA TCCCCAGTGCTCCCGG AAGCAGTCGA GCCrrGGCGTG CCCATGTGG AGCTTGTCCTTAAAG CTG CTG ACTGCAGCCA CG GTAGCTCGACTAOCC S s t I I ( - 2 3 1 ) - 2 0 1 y r--------- 3 T ” HUMAN CATTCjVrTOgACOTTTTCCCTTAGAGGCCAAGGCUCCAGOAAAUOGCGTCCCACGTGTGAGGGGCCCGCGGAGCCATTTGATTGGAGAAAAUCTGCAAAC CHICK TCICCGCAUCCTTCGAGCTOACOOTGCGCCCTGAAAAAAACAACATTCGCCGTATCGTTAAACCCCCCCGCGGATACCCGATTGOACCGAGCCGOTCCTC C H IC K H st lil K |64 ) ^ _ -101 T ' HUMAN CCTGACCAATCGGAAGGAGCCACGCTTCGGGCAirGGTCACCGCACCrOGACAGCTCCGATTGGTGGACTTCCGCCCCCCCTCACGAATCCTCATTGGGT CH I CK C C - GGCCAATCGACGCCGGCCACGC3 C C G ^G C A - - - GAAACCGCACATG GAAAG CCACGATTGGTAG TGTAt’C G C C C C CC C TC CC G C G TTC TC A TTG G CT |_________________________________ TC I '— ~ ’ GGCC "AA A T*"------' J t I 1 ( - 4 2 ) - 1 IIUMAN G CCG TGGG TGCGTGOTG CG GCG CGATTGGTG G G TTC A TG TTTC C CG TCC C c c g c c c o c o c g a a g t g g g g E t g a a a a Jg c g g c c c g a c c t g c t t g c g g t g t a CT11 CK G CA TG T TTG G CG TGG TGCGCTCCCATTGGCAG GCGCG GAGA ACCCCCCCG CCCTCCA TCG G G - GC - GCGfTTGATA^GGCGGAGGGGGAGGGTGCGGCGTC ' ' ----- " " C 'c 6 'c QemH I ( *30) +100 r~ . . r . . . . . IRJMAN GTGGCGGACCGCGCGGCTGGAGGTOTGAGGATCCGAACCCAGGGGTGGGG GGTGGAOGCGGCTCC TG C G A T CG AAGGGG A C rT G A G ACTCACCGGCCGCA CH I CK AGCGGGTTCGGCGGCGG rOCGGGAGGCGTTGCGGIXjGGG IT C T A C G G C l C GGG GCAGGGCGTTGGGCCG TTTTTCTCTCA O CA TCA G CA G G CCG CGGCA M ET A r g A l a l.eu T rp Va I l.eu Gly l.eu Cy« Cyi Va I Leu Leu T h r Phe HUMAN CO CC |A T 0| AGO GCC CTG TOG G I'G CTG GGC C T C T G C TG C G TC CTG CTG ACC T T C G g t g a g t g a t t c t g g a g g a c g c a g a c g i - - TC IATGI AAG TCA GCG TUG GCG crTG G C r CTG G CA TG C ACG C T T C T C CTO GCC G g l g a g t g c t g t a g c c t t t c g g a a a c g t c g MET Lyi Ser Ala Tr p Ala Leu Ala Leu Ala Cy« Thr Leu Leu Leu Ala rt 68 FIG. 12 C o m p a ris o n of the 5 ’ -UTR sequences of the human and murine GRP94 genes. The 5 ’ -UTR sequence of the functi o n al human GRP94 gene is o la te d from phage clone hu94-l9 is shown. Nucleotides are num bered with i ts t r a n s c r i p t i o n a l i n i t i a t i o n s ite 0—► ) desi gnat ed as + 1 . Sequence from the analogous region of the human GRP94 pseudogene isol at ed from phage clone h u 9 4 -2 4 and the 5 * -UTR sequence of the murine GRP94 gene w e re com pared to that of the funct i onal human GRP 9 4 gene. Identical nucl eot ide s are shown as dot s. The ATG t r a n s l a t i o n a l i n i t i a t i o n codon is shown in bold. 69 Huma n (funct ion (pseudo) Mu rine ,------------► 39 1) GTGGCGGACCGCGCGGCTGGAGGTGTGAGGATCCGA- ACC ......................... A ............................................................... .....................................G . T T . G . . T 78 CAGGGGTG - GGGGGTGGAGGCGGCTCCTGCGATCGAAGGG .G .............. A .A ........................T .............T ............................A . . .G . .AT. .G .......................................................A. .C. . . .AA. 104 GACTTGAGACTCACCGGCCGCACGCCATG .........................................A . . .A . .A . .TG. . ..............C .............G ................A . G . A ............ 70 FIG. 13 Amino acid sequence c o m p a r is o n among GRP94, HSP83, and H S P 90. Predi cted amino acid sequences derived from the human genomic clone and the cDNA clones of hamster, murine, and chicken GRP94 , yeast HSP90 ( F a r r e l ly and Fi nk e 1s t ein, 1984), and Dr o s oph i 1 a HSP83 (B lackm an and Meselson, 1986) were co m p ared . Undetermined sequences are bracketed. Amino acids are num bered wit h t r a n s l a t i o n a l s t a r t codon (ATG) of the chicken HSP108/GRP94 designated as +1. Amino acids id e n ti c al b e tw e e n two species that are aligned next to each other are i n d i cat ed by v e r t i c a l dots. 71 HAMG94 MURG94 CHKG9 4 YHSP9 0 DHSP8 3 HAMG94 MURG94 CHKG94 YHSP90 DHSP8 3 HAMG94 MURG94 CHKG94 YHSP90 DHSP8 3 HAMG94 MURG94 CHKG94 YHSP90 DHSP8 3 70 DDEVDVDGTVEEDL ?K( ) DD EVDVDGTVE EDLGK S R EG S RTDDEWQRE E EAIQLDGLNAS QIRELRE - EEVDVDATVEEDLGKSREGSRTDDEVVQREEEAIQLDGLNASQIKEI RE ( )MA- ( )MPE 120 ( ) KSEKFAFQAEVNRNMKLI INSLYKNKEIFLRELI SNASDALDKI RL I SLT KSEKFAFQAEVNRMMKLI INSLYKNKEIFLRELISNASDALDKIRLISLT - SETFEFQAEITQLMSLIINTVYSNKEIFLRELISNASDALDKIRYKSLS EAETFAFQAEIAQLMSLI INTFYSNKEI FLREL I SNASDALDK I RYE SLT 170 ( ) DENALAGNEELTVKIKCDKEKNLLHVTDTGVGMTREELVKNLGTIAKSGT DENALAGNEELTVKIKCDKEKNMLHVTDTGIGMTKEELIKNLGTI AKSGT DPKQLETEPDLFIRITPKPEQKVLEIRDSGIGMTKAELINNLGTIAKSGT DP SKLDSGKELYIKLIPMKTAGTLT11DGTIGMTKSDLVNNLGTIAKSGT 220 ( ) SEFLNKMTEAQEDGQSTSELIGQF GVGF Y S AFLVADKVIVT SKHNNDTQH SEFLNKMTEMQDDSQSTSELIGQFGVGFYSAFLVADRVIVTSKHNNDTQH KAFMEALSAGADVS-------- MIGQFGVGFYSLFLVADRVQVI SKSNDDEQY KAFMEALQAGAD I S -------- MI GQFGVGFY SAYLVADKVTVTSKNNDDEQY 72 FIG. 13 ( c on t i nued ) 269 HAMG94 ( ) HUMG94 ( ) LVLKEEASDYLELDTIKNLVRK MURG9 4 IWE SDSNEF SVIADPRGNT- LGRGTTITLVLKEEASDYLELDTIKNLVRK CHKG94 FWESDSNEF SVIDDPRGNT-LGRGTTITLVLKEEASDYLELDTVKNLVKK YHSP90 IWE S NAGG S F TVT LDEVN E RIGRGTILRLFLKDDQLEYLEEKRIKEVIKR DHSP8 3 VWE S SAGG S FTVRADN SEP-LGRGTKIVLYIKEDQTDYLEE SKIKEIVNK 309 HAMG94 ( ) HUMG94 YSQFINFPIYWSSKC ) MURG94 YSQFINFPIYVWS SKTETVEEPLEEDEAAK----------------- EEKEESDD-EA CHKG9 4 YSQF INFPI YVWS SKTETVEEPVEEEEA-K----------------- EEKEETDDNEA YHS P 9 0 HSEFVAYPIQLWTKEVEKEVPIPEEEKK DEEKKDEEKKDEDDKKP DHS P 8 3 HSQFI GYP IKLLVEKEREKEVSDDEADDEKKEGDEKKEMETDEPKIEDVG 357 HAMG94 ( ) MURG94 AVEE- - EEEEKKPKTKKVEKTVWCWELMNDIKPIVQRPSKEVEEDEYKAF CHKG94 AVEE- - EEEEKKPKTKKVEKTVWCWEIJMNDIKPIVQRPSKEVEEDEYKAF YHS P 9 0 KLEEVDEEEEKKPKTKKVKEEVQEIEELNKTKPLWTRNP SDITQEEYNAF DHS P 8 3 EDEDADKKDKDAKKKKTIKEKYTEDEELNKTKPIWTRNPDDI SQEEYGEF 407 HAMG94 ( )KKSDY MURG94 YKS F SKE SDDPMAYIHFTAEGEVTFKSILFVPTSAPRGLFDEYGSKKSDY CHKG94 YKTF SKEHDDPMAYI HFTAEGEVTFKS ILFVPNSAPRGLFDEYGSKKSDF YHSP90 YKS I SNEKVEDPLYVKHFSVEGQLEFRAILF IPKRAPFDLFESK- -KKKNN DHSP8 3 YKSLTNDWEDHLAVKHFSVEGQLEFRALLFIPRRTPFDLFENQ--KKRNN 73 PIG. 13 ( c o n t i n u e d ) HAMG94 MURG94 CHKG94 YHSP9 0 DHSP8 3 HAMG94 MURG94 CHKG94 YHSP90 DHSP8 3 HAMG94 MURG94 CHKG94 YHSP90 DHSP8 3 HAMG94 MURG94 CHKG94 YHSP90 DHSP8 3 457 IKLYVRRVFI TDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLK IKLYVRRVF ITDDFHDMMPKYLNFVKGVVDSDDLPLNVSRETLQQHKLLK IKLYVRRVF ITDDFHDMMPKYLNFVKGWDSDDLPLNVSRETLQQHKLLK I KLYVRRVF ITDEAEDLI PEWLSFVKGWDSEDLPLNLSREMLQQNKIMK IKLYVRRVF IMDNCEDLI PEYLNFMKGWDSEDLPLNI SREMLQQNKVLK 506 VIRKKLVRKTLDMIKKIAD-EKYNDTFWKEFGTNIKLGVIEDHSNRTRLA VI RKKLVRKTLDM IKKI AD - EKYNDTFWKE FGTN I KLGV I EDHSNRTRLA VI RKKLVRKTLDM I KK IAE - EKYNDTFWKEFGTNVKLGVI EDHSNRTRLA VIRKNIVKKLI EAFNEIAEDSEQFEKFYSAFSKNIKLGVHEDTQNRAALA VIRKNLVKKTMELIEELTEDKENYKKFYDQF SKNLKLGVHEDSNNRAKLA 556 KLLRFQS SHHSTDIT S LDQYVE RMKEKQDKIYFMAGS SRKEAE S S PFVER KLLRFQS SHHSTDITSLDQYVERMKEKQDKIYFMAGS SRKEAE S S PFVER KLLRFQS SHHESNLTSLDQYVERMKEKQDKIYFMAGASRKEAE S S PFVER KLLRYNSTKSVDELTSLTDYVTRMPEHQKNIYYITGESLKAVEKSPFLDA DFLRFHTSASGDDFCSLADYVSRMKDNQKHVYFITGESKDQVSNSAFVER 606 LLKKGYEVIYLTEPVDEYCIQAL P E FDGKR FQNVAKEGVKFDE S EKTKEN LLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFDESEKTKES LLKKGYEVIYLTEPVDEYCIQALPEFDGKRFQNVAKEGVKFEE SEKSKE S LKAKNFEVLFLTDPIDEYAETQLKEFEGKTLVDITKDF - ELEETDEEKAE VKAJRGFEWYMTEPIDE YVIQHLKE YKGKQLVS VTKEGLEL P EDE S EKKK 74 FIG. 1 HAMG94 MURG94 CHKG9 4 YHSP90 DHSP8 3 HAMG94 MURG94 CHKG94 YHSP90 DHSP8 3 HAMG94 MURG94 CHKG94 YHSP90 DHSP8 3 HAMG94 MURG94 CHKG94 YHSP90 DHSP8 3 3 ( c o n t i n u e d ) 656 REATEKEF E PLLNWMKDKALKDK IEKAW SQRLTE S PCALVASQVGWSGN REATEKEFE PLLNVA4KDKALKDKIEKAWSQRLTE S PCALVASQYGWSGN REALEKE F E PLLN\\MKDKALKDK IEKAVL SQRLTQS PCALVASQYGWSGN REKE IKEVEPLTKALKE - ILGDQVEKVWSYKLLDAPAAIRTGQFGWSAN REEDKAKFESLCKLMKS - ILDNKVEKVWSNRLVDS PCCIVTSQFGWSAN 705 MERIMKAQAYQTGKDI STNYYASQKKTFEINPRHPLIRDMLRRVKED-ED MERIMKAQAYQTGKDI STNYYASQKKTFE I NPRHPL IRDMLRRIKED - ED MER I MKAQAY QTGKD I STNYYASQKKTFE I NPRHPL IKEMLRRVKEN - ED MER IMKAQAL RDS SMS SYMS SKKTFE SPKSP I IKELKKRVDEGGAQ MER IMKAQAL RDTATMGYMAGKKQLEINPDHPIVETLRQKADADK - N 755 DKTVLDLAWLFETATLR SGYLLPDTKAYADRIERMLRLSLNIDPEAQVE DKTVMDLAWLFETATLRSGYLLPDTKAYGDRI ERMLRLSLN IDPEAQVE DKTVSDLAWLFETATLRSGYMLPDTKEYGDRI ERMLRLSLN IDLDAKVE DKTVKDLTKLLYETALLTSGFSLDEPTSFASRINRLISLGLNIDEDEETE DKAVKDLVILLFETSLLSSGFSLDSPQVHASRIYRMIKLGLGIDEDEPMT 795 EEPEEEPEDTTED TEQDEE- - EEVDAGTEEEEEEEQETAKESTAEKDEL EEPEEEPEDTSEDAEDSEQDEG-- EEMDAGT EEEEEETEKEST-EKDEL EEPEE - PEDAAEEAE QDE EEVDADA--------- EDS ETQKE STDVKDEL TAPEASTAAPVEEVPADTE MEEVD TDDAQS AGDAP SLVE - DTEDASHMEEVD 75 C hap tcr 2 F u n c t i o n a l s t u d i e s of the human GRP94 and the Rat GRP7 8 p rom oters 2 .1 INTRODUCTION T h e regu la tio n of eukar yotic gene expression has been found to involve the i n t e r a c t i o n of s pe c if ic t r a n s - a c t i n g factors wi t h c i s - a c t i n g re gula tory elements. Two such re gu la tor y elements commonly found in the 5 ’ -flan king region of eukaryotic genes are termed the promoter and enhancer elements. The promoter element acts in a p o s i t i on - d e p e nd e n t manner. This can be subdivided into proximal elements, including the t r a n s c r i p t i o n a l i n i t i a t i o n s ite and the TATA element which is required for i n i t i a t i o n of t r a n s c r i p t i o n , and d i s t a l elements, w h ic h can be locat ed several hundred n ucleot ides upstream. The enhancer e le m e n t appears to increase t r a n s c r i p t i o n a l e ff i c i e n c y in a m a n n er r e l a t i v e l y independent of its p o s it io n and o r i e n t a t i o n with respect to a nearby gene. Enhancers have been found w i t h i n a number of DNA viruses and re t r o v i r u s e s (K houry and Gruss, 1983). The SV40 enhancer is the best c ha r ac te ri z ed . It acts in a wide v a r i e t y of hosts and t i s s u e s . In r e t r o v i r u s e s , enhancers play a crucia l role in oncogenesis (Cullen et 76 a l . , 1985; W eber and Schaffner, 1985). Certain c e l l u l a r genes such as, immunoglobulin, in te rf e ro n , and m e ta ll o th io n e in genes also possess their own enhancers ( G il l ie s et a l . , 1983; Goodbourn et a l . , 1985; Serfling et a l . , 1985; Haslinger and Karin, 1985). Although enhancer a c t i v i t y is often found upstream of the t r a n s c r i p t i o n uni t (Gruss et a l . , 1981), they are also found d o w n s t r e a m of a t r a n s c r i p t i o n uni t (L u sk y et a l . , 1983), w it h in an intron ( G i l l i e s et a l . , 1983), or w i th in a coding sequence i t s e l f (Osborne et a l . , 1984). Vi r al enhancers often reside in higher DNase I s e n s i t i v i t y domains. A working hypothesi s was thus suggested that the increase of t r a n s c r i p t i o n a l a c t i v i t y wa s regu l ated by a l t e r i n g the chr oma tin s tr u c tu re and superh e1i c i t y (S aragost i et a l . , 1980; Jongstr a et a l . , 19 84; K houry and Gruss, 1983). H ow ever, studi es have demonstrated that enhancers and promoter elements overlap both ph y si ca lly and fu n c t i o n a l l y . E f f i c i e n t t r a n s c r i p t i o n requi r es the presence of both elements. They consist of a modular arrangement of short DNA mo ti fs to confer the t issu e s p e c i f i c i t y , i n d u c i b i l i t y , or a general en h an c e m en t of t r a n s c r i p t i o n . T h ese short mo t i f s are pres uma bly binding s i t e s for a v a r i e t y of t r a n s c r i p t i o n fac tor s (Dynan and Tjian, 1985). T h ro u g h analyses of d e le t io n and base - s u b s t i t u t i o n m ut ations, regu latory elements other than the TATA 77 sequence have been id e n t i f i e d . In the case of S V 4 0 early promoter, both upstream 21-nt repeats and the 72-nt enhancer element are required for e f f i c i e n t t r a n s c r i p t i o n (Baty et a l . , 1983; Gruss et a l . , 1981; Benoist and Chambon, 1981). Herpes simplex vi ru s (HSV) t h ym idine kinase ( t_k) gene contains t r a n s c r i p t i o n a l regulato r y domain located approximately 50 to 100 nt upstream of the tr a n s c r i p t i o n a l i n i t i a t i o n si t e required for f ul l promoter a c t i v i t y i_& vivo (Mcknight, 1982; Mcknight et a l . , 1984). Th i s HSV tk, r egul atory domain contains repeats of a hexanuc1eotide sequence, GGGCGG, known as a GC box. The GC box was l a t e r shown to be the binding site for the tr a n s c r i p t i o n factor Spl (Gidoni et a l . , 1984). Th e Spl and CAAT-binding tr a n s c r i p t i o n f act or (CTF) act in conjunction at the upstream region of the HSV tk, gene to mediate RNA polymerase II recogniti on. The me t a 1 1 o t h i on e i n genes of man and mouse 'are one of the w e ll -s tu d ie d inducible gene fa mi lie s . Many d i s t i n c t DNA m oti fs involved in t r a n s c r i p t i o n regul ation of these genes are id e n ti fi e d w it h in the upstream regions. These include metal-Tes ponsive elements, g 1u c o c o r t i c o i d - responsive element and the Spl, AP - 1 and AP - 2 binding s it e s (Karin et a l . , 1984; Angel et a l . , 1987; Lee et a l . , 1987). Tr an s c ri p ti o n factor AP-1 recognizes a TPA-responsive element (TRE) 78 with conserved mo t i f as TGACTCA, wT h ile the reco g n it ion mot i f of AP - 2 is (T /C )C (C /G )C C (A /C )N (G /C )(C /G )(G /C ). AP-1 a c t i v i t y can be modulated by TPA-induced c e l l u l a r changes, while AP - 2 appears to mediate tr a n s c r i p t i o n a l a c t i v a ti o n in response to both TPA-activated pro tein kinase C and cAMP-dependent pr otein kinase A (Imagawa et a l . , 1987; Chiu et a l . , 1987). The SV4 0 early promoter also possesses Spl and AP-2 binding site s within the 21-nt repeats, and AP-1 and AP-2 binding si te s with in the 72-nt enhancer fragment (Dynan and Tjian, 1983; Lee et a l . , 1987; Mitchell et a l . , 1987). The AP-2 binds to the 21-nt repeats with a lower a f f i n i t y than to the 72-nt fragment. In the case of heat shock response, a 5 ’-f lanking region (nt -66 to -10) of HSP70 wa s id e n ti fi e d to be s u f f i c i e n t in conferring heat i n d u c i b i l i t y , and nt -66 to -47 were necessary (Pelham, 19 82 ). More r ec entl y, the heat shock element (HSE) res ponsible for the heat shock i n d u c i b i l i t y was defined as a dimer of a 10-nt consensus sequence, NTTCNNGAAN (Xiao and Lis, 1988). As the examples described above, common or promoter - spec i f i c c i s - a c t i n g reg u lator y elements are pres ent to i n i t i a t e the t r a n s c r i p t i o n , to confer the i n d u c i b i l i t y or to provide a general enhancement of t r a n s c r i p t i o n . To id e n ti fy and localize these common or specif ic c is - a c ti n g r egu l ato r y elements, is an 79 e ss e n ti a l step towards the understanding of DNA-protein i n te ra c t io n and the elucidati on of the mechanism by wh ich genes are r egulated. In the previous chapter, two human GRP94 genes were isolated and the sequence of their 5 ’ regions determined. The gene i solated from clone hu94-24 does not have a typical ATG tr a n s l a t i o n a l i n i t i a t i o n codon, nor does it contain int r ons. However, sequence analysis indi cat ed that both genes possess promoter-1 ike sequences. Therefore, it is of i n t e r e s t to determine f i r s t l y , whether these two human genes contain funct i onal prom oters and secondly, to ide ntify regions e sse n tia l for the expression and induction of GRP94. In the case of GRP7 8, preli mi n ary s tudies using the techniques of i_n v it r o mutagenesis and DNA-mediated gene tra n s fe r had id e n ti fi e d the promoter and an enhancer element located within the 5 ’-f l anki ng region of this gene (At t ene 1 1 o and Lee, 1984; Lin et a l . , 1986). To further define the sequences required for the expression and induction of GRP78, a series of hybrid genes contai ning the 5 ’ - flanking region of GRP78 gene were const ruct ed and their promoter a c t i v i t i e s determined. The re su lts of these studi es are described in the following chapter. 80 2.2 MATERIALS AND METHODS 2 .2 .1 . Cell lines and culture condition s. The Chinese hamster f i b r o b la s t c e l l s , WglA, the t_§_ mutant cell line K12, and their cul t ur e conditions have been described (chapteT 1 . 2 . 1 .; Lee, 1981). The human hepatoma cell l ine, HepG2 was obtained from R. E. K. Fournier and was maint ai ned in DMEM supplemented with 10% feta l calf serum. The T4 7D human breast cancer cell line was obtained from M. Phal (La Jolla Cancer Research Foundation) and was maintained in RPMI16 40 medium supplemented with 5% fet al bovine serum, 1% non es sen ti al amino acids and 0.2 l.U./ml of insul i n. 2.2. 2. Plasmids, (i ) pSV2CAT. Plasmid pSV2CAT contains the SY4 0 early promoter and ori gin of r e p l i c a t i o n fused to the ba ct e ria l chloramphenicol a c e t y l t r a n s f e r a s e (CAT) gene (Gorman et a l . , 1982) and was obtained from B. Howard (National I n s t i t u t e of Health). ( i i ) p94( 0 . 7HB)GAT, p94( 1 . 4PA)CAT and p94( 1 . 5PB)CAT. PIasmi ds p 9 4( 0. 7HB)CAT, p94( 1 . 4PA)CAT, and p94(1.5PB)CAT contain 0.7, 1.4, and 1.5 kb, r e s p e c ti v e ly , of the human GRP94 5* region isolated f r om the phage clone hu94-24, fused 5 ’ to the CAT gene in the p 1 a smi d p SVOCAT (Go rma n e t a l . , 1982). Pla smi d s contai ning the 5* sequence and CAT gene fused in the 81 same t r a n s c r i p t i o n a l or ie nt a ti on were ide nt if ie d by r e s t r i c t i o n mapping. ( i i i ) pGRP94(- 1170)CAT, pGRP94(-357)CAT, and pGRP94(- 164)CAT. CAT fusion constru cts pGRP94(- 357 )CAT, and pGRP94(- 164)CAT contain Bam HI/Bam HIC-357/+29) and Bst EII/Bam Hl(-164/+29) fragments, re sp ect iv el y, of the 5 ’ region of the human GRP94 gene isolated from the phage clone hu94-19, subcloned into the unique Hind III site of the pSVOCAT. Plasmid pGRP94(-1 170)CAT was const ruct ed by inse rting the Bst EII/Bst Eli (- 1 1 70/- 1 64) fragment into the Bst El I (-164) r e s t r i c t i o n enzyme site of the pGRP94 (- 357 )CAT. The plasmid pGRP94 ( - 1 1 70)CAT t herefore contains an extra Bam HI/Bst E I I ( - 35 7 / - 1 64 ) 5 ’ to the Bst EII/Bam HI (-1170/+29) fragment. ( i v) pIlO, pK, pA2 , pE4 3 , pGRP7 8 ( - 4 80 )CAT, p3K, p2A, and p3H. Constr uct ion of these CAT fusion plasmids contai ning the rat GRP78 promoter has been described (Resendez et a l . , 1985; Chang et a l . , 1987). These CAT fusion plasmids have a same 3 ’ endpoint, the Bssh II site which is located 37 nt upstream from the major t r a n s c r i p t i o n a l i n i t i a t i o n site of the rat GRP78 gene, but various 5 ’ endpoints (Chang et a l . , 1987 ). The 5 ’ endpoints of these CAT constru cts are pI10(-1290), pK(-1215) , pA2(-780), pE43(-480), p3K(-355) and p2A(-265) determined by r e s t r i c t i o n mapping. The p3H 82 used in tra ns ien t tra n sf e ct io n assay does not r eta in any of the rat GRP78 5 ’-flanking sequence. The plasmid pE43 is also ref erred to as pGRP78(- 480)CAT in chapter 3 . (v) pGRP7 8C-375/-88) . Plasmid pGRP78(- 375/- 88 ) contains the rat GRP78 Sma / Stu(- 375/ - 88 ) promoter/ enhancer fragment subcloned into the Sma I s ite of pUC8 with the Sma I si te (-3 7 5 ) proximal to the Eco Rl si t e on the polvli n k er of pUC8 . It is also referr ed to as pUC2 9 1R. 2.2 .3. Conditions for tr an si ent t r a n s f e c t i o n . The conditions for DN A tr a n s fe ct io n have been described (Att ene ll o and Lee, 1984; Wang and Lee, 1983). Briefly , 3 to 5 ;u g t ra n s fe ct in g DNA was mixed w i t h high- mo 1 ecu1 ar-we i ght HeLa cell c a r ri e r DNA to a tot al of 10 >ug in the t r a n s f e c t i o n buffer (0.275 M NaC1 , 10 m\l KC1 , 1.5 mM Na 2 HP0 4 , 0.22% glucose, and 4 5 m\l HEPES (pH 7.4)) and added to the K12 cel ls or HepG2 cells grown to about 30-4 0% confluency. Cells we re incubated at 35°C for 20 min before the additi o n of 6 ml of fresh EMEM. The incubation was continued for 4 hrs at 35°C before glycer ol shock. Fresh me d i um wa s then added and cel ls were harvested wi thout se le c ti o n 48 hrs afte r t ra n s f e c t i o n . To test the i n d u c i b i l i t y of the promoters contained withi n the tr a n s fe ct in g plasmids, ce lls were 83 eith er treated with 7 >uM calcium ionophore A23187 at 35°C, changed to gl uco se-free medium at 35°C, shift ed to the non-permi ssive temperature (39.5°C), or maintained in EMEM at 3 5°C for 16 hrs pri or to harvest. 2 . 2 . 4 . A s s a y s for th e CAT a c t i v i t y . The level of CAT a c t i v i t y was determined by the procedure of Gorman et a l . (1982) with the following m o d if ic a ti o n s . The trans fe cte d ce lls were washed three times with PBS and then incubated with 1 ml buffer containing 40 mM Tris (pH 7.4), 1 mM EDTA and 150 mM NaCl at room temperature for 5 min. Cells were then pe lle te d and resuspended in 5 0 Ail of 0.2 5 M Tris (pH 7.8). The ce lls were disrupt ed by three f r e e z e - th aw cycles and the supernatant was assayed for pro tein concent rat i on using the Bio-Rad protein assay. For CAT a c t i v i t y analys is , equal amount of protein extracts from each sample was incubated with 0.2 5 mC i of [ ^ C ] - cbl orampheci col (57.8 mCi /mmol ; New England Nuclear Corp.) in a 75 Ail re action mixture containing 35 Aimo 1 Tris (pH 7.8) and 0.04 auuo 1 acetyl CoA for 30 min at 3 7°C. The samples were extr ac ted wi t h ethyl a ce ta te , and spotted on s i l i c a gel t h in - la y e r chromatography p l a te s . After developing in chloroform- met h an ol ( 95:5) , the th in - la y e r chromatography p late wT a s exposed to XAR - 2 Kodak film. For q u a n t i t a t i o n , 84 spots corresponding to the un ac etylated and ace tyl at ed forms of the chloramphenicol were cut out, and counted in a l i quid s c i n t i l l a t i o n counter. The percentage of conversion to the acety lated fo rm wa s calc ula te d by d i vi di ng the counts per minute in the ac e ty la te d forms by the total counts per minute. The f o 1 d - i ndu c t i on of CAT a c t i v i t y was calcula ted as the r a t i o of conversion between the induced and the non-induced conditio ns. 2 . 2 . 5 . I s o l a t i o n of c y t o p l a s m i c RNA and RNA blot h y b r i d i z a t i o n . The i s o l a t i o n of cytoplasmic RNA and RNA blot h y b r id i z a ti o n have been descr i bed (chapter 1. 2 . 2 .). 85 2 . 3 RESULTS 2. 3 .1 . Promoter a c t i v i t y of the human GRP94 genes. To t est whether the GRP 9 4 genes is olated from the human gen om ic l i b r a r y contain f unctional pr omo t e r s , th e ir 5 ’ regions were fused to the b a c t e r i a l chloramphenicol a c e t y l t r a n s f e r a s e (CAT) gene contained w it h in the plasmid pSVOCAT (Gorman et a l . , 1982). As shown in Fig. 14, less than 1% CAT a c t i v i t y wa s d etected for each of the three GRP94-CAT hybrid genes p94( 0 . 7HB)CAT, p94( 1 . 4PA)CAT, and p94( 1 . 5PB)CAT constructed from the 5 ’ -fla nk in g region of phage clone hu94-24. These r e s u l t s t ogether with the presence of frame s h if t mu ta tio ns and absence of introns, i n dicat e that the GRP94 gene r epr esent ed by the phage clone hu94-24 is a p rocessed, i nactive pseudogene in the human genome. In c o n tr a s t, the phage clone hu94-19 does contai n a funct ion al promoter, since CAT a c t i v i t y s i g n i f i c a n t l y higher than background was detected when cel ls were tr a n s fe c te d with a CAT fusion const ruct containi ng promoter sequences derived from the phage clone hu94-19. This plasmid, re fe rr e d to as pGRP94(- 35 7)CAT, contai ns a 386 nt Bam HI fragment (-357/+29) from the 5* region of hu94-19. As a comparison, pGRP78(-480)CAT cont aini ng the rat GRP 7 8 promoter, and pSV2CAT containi ng the SV40 early promoter were also 86 tr an sf ect ed into the K12 c el ls . The GRP 7 8 genes have been is olated from both the human and rat (At tenel 1 o and Lee, 1984; Ting and Lee, 1988). Their promoter sequences are about 80% conserved wit hin 340 nt region upstream from the t r a n s c r i p t i o n a l i n i t i a t i o n site of the human GRP78 gene. The rat GRP78 promoter contained with in pGRP78(- 480)CAT was shown to be act ive and is inducible by calci um ionophore and the K12 L§_ mu t a t i o n (see chapter 2 . 3 . 5 . ) . As shown in Fig. 15 A and summarized in 15C, the 386 nt Bam HI fragment of the phage clone hu94-19 contains the regulator y elements required for basal level expression and i n d u c i b i l i t y of the GRP9 4 . Five- and four-fold increase in CAT a c t i v i t y wa s detected for A23187 tr eated ce lls and for K12 cel ls grown at 3 9.5°C, re s p ec tiv e ly . In co ntr ast , only about 1.5 fold increase in CAT a c t i v i t y was detected under both induced conditions in the case of pSV2CAT. A comparison of the pGRP94(- 357 )CAT a c t i v i t y to that of pGRP78(- 480)CAT demonstrates that the CAT a c t i v i t y under the d ir e c ti o n of GRP78 promoter is about 4- to 5-fold higher than that of GRP94, although GRP94 and GRP78 CAT fusion genes are s i m i l a r l y regulated under induced condi t i ons. These observat i ons are consistent wi t h previous t r a n s c r i p t i o n a l measurements showing that the GRP78 gene is expressed at higher level than the GRP94 87 gene (Lee et a l . , 1983; Lin and Lee, 1984; Resendez et al . , 1 985). 2. 3 .2 . Local iz ati on of the regul atory regions important for high level expression of GRP94. To lo ca lize the r egulatory regions in the GRP94 promoter which are important for expression, two other CAT fusion plasmids were cons t ruct ed. These plasmids, pGRP94(- 1 170)CAT and pGRP94(- 164)CAT, contain 1170 nt and 164 nt re s p ec tiv e ly of the GRP94 5 ’ -f lanking sequences (Fig. 15C). Their promoter a c t i v i t i e s were t ested (Fig. 15B) and the re su lts are summarized in Fig. 15C. A sli ght decrease ( l ess than 2-fold) in the basal a c t i v i t y was observed when the 5 ’-f lanking sequence was reduced fr om 1170 nt to 357 nt, and a large decrease (about 7-f ol d) when the sequence was reduced to 164 nt. Both pGRP94(- 1170)CAT and pGRP94(- 357)CAT were highly inducible by A23 1 87 and the K12 t_s mutation , while p a r t i a l A231 87 and K12 t_s i n d u c i b i l i t y were observed wit h pGRP94(- 164)CAT. When HepG2 cells were transfected wi t h these GRP94-CAT fusion genes, simi lar res ult s were observed (Fig. 16). These combined re s u l t s i ndi cat ed that an important domain required for high basal level expression of the human GRP94 gene is located between nt -357 and -165. Sequence anal ysi s (Fig. 11) revealed three CCAAT elements reside within 88 thi s domain, which may p a r t i a l l y con t r i b u t e to the high basal level expression. In add i t i on, at least part of the region important for i n d u c i b i l i t y appears to be located in sequences fu rther downstream. Within the region, three CCAAT elements, one AP-2, and two Spl binding s ite s were found. 2 . 3 . 3 . E f f e c t of progesterone on the t r a n s c r i p t i o n of GRP 9 4 gene. Progesterone was reported to induce the tr a n s c r i p t i o n of HSP10 8/GRP94 in chicken oviduct (Baez et a l . , 1987). Therefore, an attempt was made to lo calize a p o te nt ia l progesterone responsible domain of the human GRP9 4 gene. As a f i r s t step, Northern blot analysis wa s carried out. Th e eff ect of progesterone was tested on the T47D human breast cancer cell l i ne, wh ich has acquired high content of progesterone receptor (Horwitz et a l . , 1982). The fi b r o b la s t cell line s, K12 and WglA, were also t ested in paralleT (Fig. 17). As expected, progesterone treatment has no effe ct on the accumulation of GRP94 t r a n s c r i p t in f i b r o b l a s t cell lines (Figs. 17B and 17C). In the T47D cell l i ne, when trea ted wi t h 5 or 20 nM of progesterone for 24 hrs, about 2- to 3-fold increase in the GRP94 t r a n s c r i p t level was observed (Fig. 17A). This increase in the level of GRP94 mRNA is relat ively smal 1 compared 89 to that observed for the chicken HSP108/GRP94 gene to wh ich a 20- to 50-fold increase wa s detected 16 hrs a ft e r a secondary stim ul ation wi t h estrogen or proges ter one (Baez et a l . , 1987). In the 5 ’-flanking region of the chicken HSP108/ GRP94 gene, consensus sequences responsible for steroi d i n d u c i b i l i t y have been id e n ti fi e d (Kleinsek et a l . , 1986). Sequence anal ysi s withi n the 550 nt region upstream of the human GRP94 tr a n s c r i p t i o n a l i n i t i a t i o n site (chapter 1; Fig. 11) d i d n ’t reveal any sequence homology to eith er the steroi d responsible element or the heat shock consensus sequences. The progesterone eff ect could t herefore be a t i s s u e - s p e c i f i c or a s p e c i e s - speci fi c response due to the presence of d i f f e r e n t c e l l u l a r f act or s in the chicken oviduct or due to the specifi c regu l atory elements l ocalized wit hin the chicken promoter sequence that has been changed during evolution. 2 . 3 . 4 . Regulatory sequence contained w i t h i n the 5 ' - f l a n k i n g sequence of the rat GRP78 gene. Previous s t u d i e s have shown that a hybrid gene containing the rat GRP78 5 ’ -f la nk in g sequences fused to the b a c t e r i a l n e omy cin r e s i s t a n c e ( n e o ) gene is a c ti v e and can confer i n d u c i b i l i t y by g l u c o s e - s t a r v a t i o n , calcium ionophore A2 3187 treatment, or by temperature when introduced 90 into the K12 t_s_ mutant cell line (At t enell o and Lee, 1984; Resendez et a l ., 1985). To lo calize the regul atory region important for the expression of GRP78 gene, a 1.25 kb fragment of i t s 5 ’- flanking sequence was subcloned into the unique Hind III site of pSVOCAT. This plasmid, designated pIlO was transfected into the K12 cells and CAT a c t i v i t y dete rm ined (Fig. 18). Results showed that the 1.25 kb 5 ’-f lanking sequence could di re c t basal level a c t i v i t y and contained a funct i onal regulator y sequence for the i n d u c i b i l i t y by calcium i onophore A23 1 87 and by the t_s_ mutation. Comparing with other c e ll u la r promoters by transient t r a n s f e c ti o n assay, the rat GRP78 promoter is highly acti ve (Lin et a l . , 1986). 2.3.5. Del et ion anal ysis of the 5 ’-flanking sequence of the GRP 7 8 gene. To id e n tif y the DNA region that is important for the regulati o n of response to calcium ionophore and t_s_ mutation, plasmids of 5 ’ deletions from the GRP78 promoter region were constr ucted by BAL-31 nuclease treatment. The 3 ’ endpoint of all the deleted fragments was the Bssh II s i t e , which is about 20 nt downstream from the TATA sequence (Chang et a l . , 1987). Fusion of these 5 ’ regions of various lengths to the b a c te ri a l CAT gene yielded a series of rec omb i n a n t s containing from 1250 nt to none of the 5 ’ -flanking 91 sequence upstream of the Bssh II site of the rat GRP78 gene. These recombinants were tr ansfected into the hamster K12 c e ll s. Cell extracts were prepared and assayed for CAT enzyme a c t i v i t y (Fig. 19). As the 5 ’-fl anki ng sequences were dele ted, both the basal and the A23187 induced l evel s decreased. When the hamster f i b r o b la s t t_s mutant cell l i ne, K12 was incubated at the non- permi ssive temperature (39.5°C), the CAT a c t i v i t y also decreased foil owT i ng the deletion of 5 ’ - flanking sequences. Th e percent conversion of [ ^ C ] - chi oramphen i col to i t s acetylated forms was q ua n ti ta te d (Fig. 20), and fold of induction cal cul ated for each of the de letion const ruct s (Table l). Results in d i cat e that a region, about 500 nt upstream of the Bssh II s i t e , was most important for A23187 induction: del et ion mu tants within this region resulted in a dramatic decrease in the A23187 inducible CAT a c t i v i t y . The DNA domain important for the induction of K12 t s mutation was also analyzed to determine whether the t s induction and the calcium i onophore treatment involved the same regulatory mechanisms. By comparing the increase over the basal l evel, a region i mp o r t a n t for both calcium ionophore and K12 i_s inductions was i d e n t i f i e d within 480 nt upstream from the major t r a n s c r i p t i o n a l i n i t i a t i o n site of the rat GRP78 gene (Table l). The r e s u lt s also demonstrated that deletions 92 into the 5 ’ region reduced the basal level a c t i v i t y of the GRP78 promoter. This reduction is unli kely to be due to the pBR322 sequence being brought closer to the CAT gene, since the reduction effect was not observed in d e le t io n studies of the histone promoter when the length of 5 ’ - flanking sequence contained w ith in the CAT fusion con s t ruct wa s reduced fr om 1000 nt to about 4 00 nt (Wells and Lee, unpublished r e s u l t s ) . 2.3.6. The Sma/Stu fragment can compete for t r a n s acting re gu latory fa c to rs . The Sma/ St u(- 375/- 88 ) fragment of the rat GRP78 5 ’ sequence was found to have enhancer - l i ke a c t i v i t i e s (Lin et a l . , 1986). It contains tandem-repeat domains and shares sequence homology wi t h SY4 0 early promoter and other c el l u la r and v ira l enhancers such as, El A core enhancer and immunoglobulin heavy-chain enhancer (Lin et a l . , 1986; L i n and Lee, 1986; Wu et a l . , 1986). It acts in c i s t o enhance expression of the neomycin phosphot ransf erase ( n e o) gene driven by the herpes simplex virus thymidine kinase promoter in an o r i e n t a t i o n - independent manner (Lin et a l . , 1986). It is i n t e r e s t i n g that this enhancer fragment resi des w ith in the 480 nt domain s h own to be i mp or t ant for both calci um ionophore and K12 t_s induct i ons. As enhancer and promoter element often overlap, both physically and fu n c ti o n a ll y , this 93 enhancer fragment was test ed for its a b i l i t y to confer the i n d u c i b i l i t y to A23 1 87 and t_s mutation in K12 c e l l s . Competition experiment was performed using the pi 10 CAT const ruct as a test plasmid and the Sma/Stu enhancer fragment as a competitor. In trans ient tr a n s fe c ti o n assay, it wa s s h own (Fig. 18) that pIlO can be stimulated by A2 31 87 and temperature in K12 cells to produce high levels of CAT a c t i v i t y . Th u s , addit i o n of increasing amount of the Sma/Stu fragment (contained in pGRP78(- 375/- 88)) should compete away the factors binding to pIlO, thereby diminishing the CAT a c t i v i t y f r om this fusion pla sm id in a dosage-dependent manner. The re su lts are shown in Fig- 21. Ulien increasing amount of the Sma/Stu fragment was added to c o -t r a n s f e c t i o n mi xtures, the CAT a c t i v i t y in response to A2318 7 and temperature, decreased correspondi ngly while the basal level was r e l a t i v e l y unaf f ected . These re s u l t s suggest that the Sma/Stu fragment contains sequences capable of i n te ra ct in g with d i f f u s i b l e factors invloved in the induction by A23187 and the K12 t s mut at i on. These re s u lt s also suggest that the reduction in both basal and inducible promoter a c t i v i t y fo il o w ing the d e le t io n of the 5 ’ - flanking sequences (descri bed in the chapter 2 . 3 . 5 .) ma y be p a r t i a l l y due to the removal of this element. 94 2. 4 DISCUSSION With the technique of mol ecul ar cloning, it has been p o s s i b l e to study the reg ula tio n of gene e x p r e s s i o n . One important control of gene express ion is at the level of t r a n s c r i p t i o n . Using cDNA clones encoding hamster GRP94 and GRP78 as h y b ri d i z a ti o n probes (Lee et a l . , 1983), it has been sh own that the syn thesis of both GRPs are g r e a t l y enhanced at the t r a n s c r i p t i o n a l level in mammalian cel ls under a v a r i e t y of st re ss conditions (Lee, 1987; Kim and Lee, 1987). RNA polymerase II requi res a TATA element in the promoter as well as other control elements for proper i n i t i a t i o n and regu la tio n of the t r a n s c r i p t i o n . To i d e n t i f y the regulatory elements that control the expression of GRP genes, both GRP78 (A ttenello and Lee, 1984; Ting and Lee, 1988) and GRP94 (chapter 1) genes were isol at ed and the 5 ’ sequences determined. Although sequence analy sis has revealed some putat ive consensus domains shown to be recognized by common t r a n s c r i p t i o n facto rs such as, binding s it e s for the C TF/N F-1 (C A A T -b in d in g t r a n s c r i p t i o n fa c to r, nucl ear factor l ) , Spl and AP-2, the regulatory domain of GRP genes needed to be loc alized through La vivo assay system. Using b a c t e r i a l chloramphenicol a c e t y l t r a n s f e r a s e ( CAT) gene as a reporter gene, fusion 95 plasmids containing d if f e r e n t lengths of the 5 ’-flanking region of eith er GRP94 or GRP78 gene were const ruct ed and promoter a c t i v i t i e s t ested . Delet ion analysis ide nt if ie d a cruci al funct i onal domain, be tween nt -357 and -164 of the human GRP94 gene required for the basal level expression (Fig. 15). In ad dition, the region important for induci b i 1 i t y appears to be located with in this domain and extended fu r th er downstream. The promoter region of the rat GRP78 gene has enhancer - 1 ike pro perties (Lin et a l . , 19 86). When the Sma/Stu (- 375/-88) subfragment from the promoter is fused to a heterologous marker gene, it s u b s t a n t i a l l y increased the tr a n s c r ip t l evel s of the marker gene. Del et ion anal ysis of the promoter provides evidence that the most important control regions for the induction of GRP78 gene reside within the Sma/ St u (- 375/- 88 ) fragment (Fig*. 19 and 20). The str ength of the GRP 7 8 promoter was about 5- f old higher than that of GRP 9 4 promoter (Fig. 15A), as demonstrated by CAT a c t i v i t y driven by the GRP promoters contained withi n the CAT fusion plasmids pGRP78(- 480 )CAT and pGRP94(- 357 )CAT, re sp ect iv el y . These o b se rv ation s are consistent with the re s u l t s from Northern blot analy sis wh ich also sh owe d that the magnitudes of response is always 2- to 5- fol d higher for GRP78 than GRP94. However, the k i n e t i c s of 96 induction for GRP94 and GRP78 are similar (Lee et a l . , 1983; Lin and Lee, 1984; Resendez et a l . , 1985; Kim and Lee, 1987). To understand how the di fference of promoter strengths occurred, a careful anal ysis on the promoter sequence was carried out. F i r s t , sequence comparison be tw een GRP94 and GRP78 genes (chapter 3 . 3 . 1 . ) revealed a 28 nt domain that is highly conserved between the two promoters. Moreover, the GRP94 promoter contains only half of the palindromic sequence observed in the GRP 7 8 (Fig. 22). Palindromic sequences are often targets for sp eci fic DNA-binding p r oteins in prokaryotes (Pabo and Sauer, 1984). All the prokaryot i c repr essor - and a c t i v a to r - b in d in g s ite s that occur as dyad sequences are recognized by pr o tein dimers that exhibit twofold r o ta ti o n a l s ymmetry (Takeda et a l . , 1983). This is important for giving funct i onal c o o p er ativ it y in terms of i n t e r a c t i o n between DNA-binding proteins. Tr an s c ri p ti o n fa ctors which act as monomers have g r e a tl y reduced DNA-binding a f f i n i t y and t r a n s c r i p t i o n a l e ff ic ie nc y. Such a concept may bring relevance to the eukaryotic syst em. Th e nuclear p ro te in , CREB which binds to the cAMP response element (CRE) of the rat somatost at i n gene as an example was shown to stimulate t r a n s c r i p t i o n as a dimer. CREB is r e l a t i v e l y i nactive as a monomer and becomes act ive as 97 dimer upon phos phoryl at i on (Yamamoto et a l . , 1988). In the case of heat shock gene, it wa s s h own that heat - shock t r a n s c r i p t i o n factor (HSTF) binds to the HSE in a cooper ative manner for maximal induction both i n v i t r o (Topol et a l . , 1985) and i_£. vivo (Dudler and Travers, 1984). Through al ky la tio n int erference and p ro te c tio n experiments, the residues which contact with the HSTF were determined. The contacts exhibited ro ta ti o n a l symmetry, suggestive of a multimer ic HSTF (Shuey and Parker, 1986a). This type of cooperative i n t e r a c t i o n may also p a r t i a l l y explain why the GRP 7 8 promoter is stronger than that of GRP94 since the GRP94 gene contains only half of the palindr om ic sequence observed in the GRP78 gene. In addition, it is i n te r e s t in g to note that both human and chicken GRP94 genes possess an unusual TATA sequence, GTGAAAA and TTGATAA, re sp ect ive ly (chapter 1; k le in s ek et a l . , 1986). It is now clear that many promoters, p a r t i c u l a r l y those of ’’housekeeping” genes, lack TATA elements, and instead consist of GC-rich elements located within me thy 1 a t i o n - f ree islands (Bird 1986). However, unlike the GRP94 gene, those ’’housekeeping” genes are often t r ans cribed at mu ltiple i n i t i a t i o n s i t e s . Since it was shown that a T-to-G, or T-to-A tr a ns ver sio n, and an A-to-G t r a n s i t i o n reduced the level s of t r a n s c r i p t i o n Ln. v it r o and i_n vivo 98 (Wa sylyk et. a l . , 1980; Wa sylyk et a l . , 1981; Zarucki- Schulz et a l . , 1982; Dierks et a l . , 1983), the uncommon TATA sequence of the GRP94 gene may p a r t i a l l y account for i t s lower promoter a c t i v i t y compared to that of GRP78. With the information obtained from sequence comparison as well as deletion mutation analysis, we have made a step towards understanding the regulati o n mechanisms of the GRP genes. 99 FIG - 14 CAT a c t i v i t i e s of GRP94-CAT fusion genes cont aini ng 5 ’ regions from the human p h a g e clone hu94-24. K12 ce lls were tra ns fe ct e d with 5 /.lg each of p 9 4 ( 0 . 7 HB)CAT (lanes 1 , 5, and 8), p94( 1 . 4PA)CAT (lanes 2 and 9), p94 ( 1 . 5 PB)CAT (lanes 3, 6, and 10) and, as a contr ol , pSV2CAT (lanes 4, 7, and 11). Aft er 28 hrs, cel ls were eithe r treated with calcium ionophore (A 2 3 1 8 7 ) a t 35°C (lanes 8 - l l ) , sh ifted to the non - permi ssive temperature 3 9 .5 ° C (lanes 5-7), or mai nt ai ned at 35°C (lanes 1-4) for 16 hrs. Protein e xtract was prepared and equal am ount (1 2 0 yug) from each s amp 1 e wa s assayed for CAT a c t i v i t y . Th e autoradiogram is show n. The p o s iti on s of chloramphenicol (CM) and its acet yla te d forms ( 3Ac and l A c ) are in di cat ed. 100 101 0 1 C O > > o o T Y - • ro • ^ 0 0 • • o n • £ It ? * o > . § - J * £ • i 0 0 * ^ o o • # • s 9 = 1 •• FIG. 15 P rom oter a c t i v i t i e s of GRP94-CAT f u s i o n genes contai n ing 5* sequences of the human phage clone hu94-19. A) K12 cell s were tran sf ect ed with 3 /ig each of pGRP94 ( - 357 ) CAT , pGRP7 8 ( - 480 )CAT and pSV2CAT. Af t er 2 8 hrs, the ce lls we re e ith er ma int ai ned at 35 °C (lane l) , trea ted wi t h 7 juM of A2 3187 a t 3 5 °C (lane 2) or sh ift e d to the non-pe rmissive temperature, 3 9 .5 ° C (lane 3). Aft er 16 hr s, prote in extract was prepared from the t r a n s f e c t a n t s and equal amo unt ( 12 ju g ) fr om each s amp 1e was assayed for CAT a c t i v i t y . The autoradiograms are show n. The p o si tio ns of chloramphenicol (CM) and it s a ce tyl at ed forms ( 3Ac and l A c ) are ind icated. B) K12 ce ll s were tra ns fe ct e d with CAT fusion genes contai ning vari ous lengths ( 1 1 7 0 , 357 or 164 n t ) of th e human GRP94 promoter. Conditions of treatment are as descri bed above. Sixty jig of cell extra ct fr om each sample was used for CAT assay a na ly s is . The autoradiograms are show n. C) The fea tur es of GRP promoters contained w it h in the CAT fusion genes are sch emati ca ll y presen t ed. The s y m b o ls marked are ( I ) Go 1 dbe r g -H o g n e s s sequence, ( ) CCAAT, ( ) CCAAT i nverted, ( A ) pu tative Spl binding s i t e , ( ) p u ta tiv e A P-2 binding s i t e , ( ES2) th e h o m o lo g o u s d o m a in between GRP94 and GRP78 promoters, and ( I ^ ) the t r a n s c r i p t i o n a l i n i t i a t i o n s i t e . The Sma/ S t u (- 375/- 88) enhancer fragment of the rat GRP78 promoter is 102 indic ated. Th e GRP-CAT fusion r e l a t i v e pr omo ter a c t i v i t i e s of the constru cts are summarized. four 103 104 A pGRP78(-480)CAT pGRP94(-357)CAT PSV2CAT B . pGRP94(-1170)CAT pGRP94{-357)CAT pGRP94(-!64)CAT 3A e^ • • • • • • • • • • • • • • • m + 1 Ac** • • • - * • # • * • • • • CM ► • • • i « • • • • < h '§ Y # • * ♦ * % C . hV/ h - 5 0 0 pGRP94(-U?0)CAT \ - / h pGRP94(-35/)CAT pGRP94(-164)CAT pGRP76(-480)CAT - 4 0 0 -300 -200 -100 + 1 Bit EH I 1 j t ™i a 4. ■ r "o — er"— u u — ™ ----1 — B o m H l) CAT « j C T r r i 4 — — u -------c r - CAT A . i r [ cat t S tul M —[ "cat" Relative CAT Activity control A23I87 39.5*C 100 550 4 70 60 3 0 0 260 1 5 50 40 290 1370 1080 FIG. 16 Promoter a c t i v i t i e s of GRP94-CAT fusion genes in HepG2 c e l l s . The HepG2 ce lls were trans fecte d with 3 ng each of pSV2CAT (lanes 1 and 2), pGRP94 ( - 1 170 )CAT (lanes 3 and 4), pGRP94 ( - 3 5 7 )CAT (lanes 5 and 6), and pGRP94 ( - 164 )CAT (lanes 7 and 8). Six hrs pri or to the pre paratio n of cell e x t r a c t s , cel ls were changed to fr esh DMEM in the absence (-) or presence ( + ) of 7 aM A231 87. Equal amount (200 Aig) of cell e xtr act s from each sample was assayed for CAT a c t i v i t y . The autoradiogram is show n. The positions of chloramphenicol (CM) and i t s ace tylated forms ( lAc , 3Ac. and 1,3Ac) are indicated . The assay performed with 0.08 unit of CAT (P-L Biochemi ca 1s ) is also showm (lane 9). The percent conversion of chloramphenicol to its ac etvlated forms are i ndicated . 105 C D 00 C D LO 00 c\ j 106 FIG. 17 Northern blot a n a l y s i s of the GRP94 mRNA on p r o g e s t e r o n e - t r e a t e d c e l l s . A) human breast cancer cell line T47D, B) hamster f i b r o b l a s t cell line WglA, and C) t s mutant cell lin e, K12 derived from WglA. Cells were treated wi t h various concentr atio ns (20, 5, or 0.5 nM) of progester one for d i f f e r e n t periods of time (24, 7, or 1 hr) prior to tota l cytoplasmic RNA i s o l a t i o n . As a c o n t r o 1 , RNA s amp 1 e s we re also prepared fr om cel ls maintained at normal cul t ur e condition (35°C), incubated with 7 juM of A231 87, or sh ift ed to the non-permi ssive temperature (39.5°C) for 16 hrs prio r to RNA e xt ra cti on . The s ynt hesi s of GRP94 tr a n s cr ip t should be induced in both the K12 cel ls and WglA cell s wh en A23187 wa s added in the cul t ur e medi um, wh i 1 e only K12 ce lls would respond to the induction of t_s mutation. Rel at i ve masses of the RNA samples are i ndicated. The RNAs were probed for GRP94 tr a n s cr ip t using a hexamer labeled 980 nt Bam HI/Eco RY fragment isolated from the p a r t i a l cDNA clone p4A3 as described in the chapter 1. 107 A . relative m ass 1.3 12 11 07 1 07 1 09 09 0 8 11 09 24 hr 7 hr 1 hr 35 c 3 9 5 c A231&7 co n e (nM) 20 5 C.5 2C 5 0.S 20 5 05 0 0 0 B . relative m ass o & 13 1 1 0.9 09 os o& o .s 12 1 11 24 hr 7 hr 1 hr 35cc 395°c A23L&7 c o n e . { n M ) 20 5 0.5 20 s 0.5 20 5 as 0 c 0 C. relative m ass 09 1 1 o s o s 1 1 1 1 1 2 4 hr 7 hr 1 hr 35°c 39 5"c c o n e . (nM) 5 05 20 5 05 2 0 5 05 0 0 FIG. 18 In d u e i b i 1it y of pIlO by calcium ionophore A23187, g 1 uc o s e - s t a r va t i on and K12 l_s. mutation. pIlO (3 >ug) was used to t ra n s fe c t K12 c e l l s . At 28 hrs a ft e r t r a n s f e c t i o n , ce lls were either treated with 7 ;uM of calcium ionophore (A23187) at 35°C, changed to glucose -f ree (GF) medium at 35°C, sh ift e d to the non-pe rmissive temperature (39.5°C) or main tained at 35°C ( c o n tr ol ) for 16 hrs. Total cell e xt ra ct s were prepared and assayed for protein content . Various amount of prote in was used for the CAT assay. The autoradiogram is shown. The amount of p ro te in (in micrograms) used in each enzyme assay and the percentage of chloramphenicol (CM) converted to it s lAc and 3Ac acet yla te d forms are in dicated. The assay performed wit h 0.05 unit of CAT (P-L B i o chemi c a 1 s ) is also s h own . 109 n o 3 Ac 1 Ac I > C M I P ro te in (jig), 100 200 25 50 100 % c o n v e rsio n 4 6 23 36 71 L - t — J 1 r-------------- 1 C ontrol A23187 m 100 200 100 200 0.0511 CAT 50 \ 3 9 . 5°C 71 7 __J I _____ 11 GF 95 FIG. 19 Promoter a c t i v i t i e s of the rat GRP78 5* d e l e t i o n CAT c o nst ru cts . K12 cell s were tr an sf ect ed wit h 3 jug of pIlO, pK, pA2 , pE43, p3K, or p2A, which contained various lengths of the 5 ’-fl an k i n g sequence of the rat GRP 7 8 gene fused 5 ’ to the CAT gene. The plasmid p3H does not r e ta in any of the GRP78 5 ’ -flanking sequence. A) Cells were cont i nuousl y incubated at 35°C in regular DMEM. B) Twenty-eight hrs a f t e r t r a n s f e c t i o n , EMEM supplemented with 7 ;uM A23187 was added to the cell s and incubat ion was continued at 35°C for 16 hrs . C) Twenty-eight hrs a f t e r t r a n s f e c t i o n , the cell s were sh ift ed to 39.5°C and incubated for 16 hrs. Cell extr act s were prepared 48 hrs a f t e r t r a n s f e c t i o n . Equal amount of prote in (75 jug) f r om each s amp 1 e wa s assayed for CAT a c t i v i t y . The autoradiograms are shown. The assay performed with 0.08 unit of CAT (P-L Bioch em ical s) is also sh own. Positi on s of chloramphenicol (CM) and it s ace tyl at ed forms ( 3Ac and lAc) are i ndicated . 111 1 1 2 Std CAT 110 (-1290) K (-1215) A2 (-780) E43 (-480) 3K (-355) 2A (-265) 3H • n • • • • • K • f t •ft • I I f t f t • ft 1 I I f t » ft • • f t f t • . • ft » f t f t • • ft I f t • - f t • f t 0 * FIG. 20 Regions of the rat GRP78 p r o m o t e r important for the i nduction by A23187 and K12 UL mu tatio n. Percent conversion of [ ^C ]-chloramphenicol to i t s acetylate d forms in various 5 ’ delet ion constructs pres ented in Fig. 19 was q u a n ti ta te d . These values were pl ot te d agai nst the p o s it io n of each of the 5 ’ de le ti o n constru cts along the promoter sequence. X, incubation at 35°C; • , i ncubat ion in EMEM cont aini ng A23187; A , incubat ion at 39.5°C. Results are summarized in Table 1. The Sma/Stu fragment previo usly i d e n t i f i e d as an enhancer element with two repeat domains (Lin et a l . , 1986) is r epr esent ed by two hatched arr ows b e 1 ow the r e s t r i c t i o n map. The region important for A23187 and K12 t_s_ mu ta ti o n induction is bracketed (480 nt upstre am f r om the t r a n s c r i p t i o n a l i n i t i a t i o n s i t e ) . 113 % conversion Position, nt IOOfT-1200 -1000 - 6 0 0 -8 0 0 4 0 0 200 [TATA CCAAT A23187 V / W / S / l V / 777?> -400 4 0 ° C 3 5 ° C [10 A2 E43 3K 2 A \ 3H 114 Table 1. Effect of 5' deletions on CAT activity Plasmid Deletion endpoint Fold increase over basal level A23187 39.5°C pIlO -1290 8.6 6.4 pK -1215 8.2 6.3 pA2 -7 8 0 7.9 5.2 pE43 -4 8 0 7.8 4.0 p3K -3 5 5 5.2 4.5 p2A -2 6 5 2.2 2.4 p3H 0.78 0.78 115 FIG. 21 Competition for trans r eg u la to ry f a c t o r s by the Sma/Stu fragment. K12 cells were co tra nsf ect ed wi t h 1 jug of t est plasmid ( p 110; 5.7 kb in si ze) and incr easing amount of competitor plasmid ( pGR P 7 8 ( - 3 7 5 - 8 8 ) ; 3.0 kb in s iz e ). To ma i n t a i n a constant amount of DNA in each experiment, control p 1 a sm id ( pUC 8; 2.7 kb in si ze) wa s added to adjust the tot a l plasmid DNA to 11 jug • At 48 hrs a f t e r t r a n s f e c t i o n , cell e xt rac ts were prepared from the tr a n s fe c te d ce lls treated for 16 hrs wi t h 7 juM A23187 a t 35°C (A), from tr a n s fe ct e d cel ls tr ea ted for 16 hrs at 3 9.5°C ( • ) , or from control cel ls maint ai ned in normal medium at 3 5°C (o). Equal amount of pr ot e in (75 /i g ) fr om each s amp 1 e wa s assayed for CAT a c t i v i t y . Th e r e l a t i v e CAT a c t i v i t y , expressed as fold increase over that of the control c e l l s , wa s plott ed agai ns t the mo l ar r a t i o of the c omp e t i t o r to the test DNA. 116 TEST COMPETITOR CONTROL *> o co h- < O <D > jC O o QC \ \ A X W T / P i 10 (PGRP78 M C A \(— 375/— I p u c a 7 2 15 5 molar ratio: Competitor/ Test 117 Chap ter 3 Studies of the i n t e r a c t i o n s between protei n f a c t o r s and 5 * -f la n k in g sequences of the GRP94 and GRP78 genes 3.1 INTRODUCTION In order to respond quickly to enviro nm e n t a 1 changes, bact er ia have developed a f le x ib le and economical gene or g anization . Bacterial genes are grouped into c lu st er s so that genes r esponsible to make e n z ym es needed for a p a r t i c u l a r metabolic pat hwa v are adjacent to one another. This allows the expression of all the genes in the unit to be co -ordin ately regulated by co ntrolling the t r a n s c r i p t i o n via in te ra ct io ns between r egu l atory protein s and control domains lie close to the promoter. The lact ose ( l a c ) operon contains three genes, 1 a cZ , .1 acY and 1 a cA wh i c h are tran sc rib ed into a single mRNA from a promoter just upstream from the 1 a c Z. The t r a n s c r i p t i o n of these genes is c o -o rdi na te ly regulated and the r e la ti v e amounts of the three enzymes always remain the same under various induced condi t i o ns. In eukaryotic syst em, co- ord i n ate regulation has been observed in many systems although eukaryotic genes are tr anscribed as monocistronic mRNAs. HSP family is a 11 8 good example. The heat shock response was o r i g i n a l l y discovered in Dr o soph i 1 a ( Ritoss a, 1964) as the coordinate a c t i v a t i o n of a small number of cyt ogenet i c loci in response to heat. Later, it wa s s h own that the induction of these loci coincided wi t h the s ynt hesis of a set of pro teins cal led HSPs (heat shock p r o t e i n s ) . Fur t her stu d i es have sh own that heat shock genes are expressed in response to a wide range of p h y s i o l o g i c a l l y and chemically induced stre ss condition s, and that the response is evo1u t i o n a r i 1y conserved in al l li vi ng species (Lindqu ist, 1986). The co nser vat i on of HSP is not only in the p r o t e i n - c od i ng sequences, but also in the re gula tory sequences (Pelham, 1982). When a Pros oph i 1 a heat shock gene and 5 ’-flanki ng region were introduced into mammalian c e l l s , the introduced HSP gene was ac ti v at e d . A common reg ula tory mechanism appears to control the t r a n s c r i p t i o n of heat shock genes in Dr o s oph il a and m am m alian c e l l s . Intensive stu d i es on the genes encoding the HSP70 provide us an exam ple of gene famil ies that are c o- or di na te ly regulated through sp eci fic protein-DNA i n t e r a c t i o n s . Chromatin m a p p in g observed mu lt ip l e DNase I h y p er sen si tiv e s i t e s at the 5' region of Pros oph i 1 a HSP70 and HSP83 genes. The p o s it io n and number of the h yp er sen si tiv e s it e s changed upon heat shock (Wu, 1980; 119 Wu , 19 84). The 5 ’ s e n s i t i v i t y may re f l e c t a s t r u c t u a l l y mo di fi ed or a specia lly exposed st re tc h of DNA for the rec ognit io n of t r a n s c r i p t i o n fac tor s. Del et ion anal ysis and s i t e - s p e c i f i c mutagenesis have i d e n ti f ie d a short DNA domain required for tr a n s c r i p t i o n a l a ct iv at io n of the HSP70 gene (Pelham, 1982). This heat shock element ( HSE) is now defined as a dimer of a 10-nt consensus sequence NTTCNNGAAN (Xiao and Lis, 1988). I n t e r e s t i n g l y , it was shown that the sequence around HSEs of the non-induced genes have hy persensitive site s (Wu , 1984), and acti vat ing facto rs bind to the HSEs in a cooperative manner for maximal induction (Topol et a 1 . , 1 9 8 5; Dudler and Travers, 1984). Heat shock tr a n s c r i p t i o n a l f act or (HSTF) and heat shock a ct i v at o r p r o t ein (RAP) we re isol at ed fr om heat-shocked Dr o s oph ila ce lls (Parker and Topol, 1 984 ) and Schneider emb ryo cell line (Wu , 19 8 4; Wu e t a 1 . , 1987), res p ec tiv e ly . The HAP p r e - e x i s t s in normal ce lls in a non-binding form. Only a ft e r heat shock a c t i v a t i o n , it is converted to a high a f f i n i t y , sequence-spec ific DNA binding pro tein po stulated by a p o s t - t r a n s l a t ional mo d i f i c a t i o n (Wu , 19 84; Wu e t a 1 . , 19 8 7; Z i ma r i n o and Wu , 1987). Similar observation wa s obtained in human cells (Kingston et a l . , 1987; Sorger et a l . , 1987). However, heat shock f act or (HSF) isol at ed fr om the yeast . ce revi siae acts d i f f e r e n t l y . 1 20 The HSF binds to DNA even in unstr essed cel ls (Sorger et a l . , 1987; Jakobsen and Pelham, 1988), and the binding is not s u f f i c i e n t for t r a n s c r ip ti o n a l a c t i v a t i o n . Phosphorylation is involved in the re gulation of i t s heat shock response. GRPs are a set of ER pro teins that are cons t i tut ively expressed and can be co-or dinately enhanced under diverse physiological stre ss conditions in mammalian cell s (Lee, 1987). Extensive studies have establ is he d that the expression of the GRP genes is regul at ed at the tr a n s c r i p t i o n a l level in the hamster t s mu tant cell line K12 (Lee et a l . , 1983; Lin and Lee, 1984; Resendez et a l . , 1985; Kim and Lee, 1987). These obser vat i ons raise the p o s s i b i l i t y that the r egu l ati o n of both genes may involve the binding of c omm o n tr a ns -a ct in g factors to a putati v e conserved regul at or y DNA d oma in as the HSP genes a c t i v a t i o n , wh ich involves the in te ra ct io ns between heat shock facto rs and the consensus HSE found in all heat shock genes from d i f f e r e n t organisms analyzed to date. In l i ght of t h i s , it was important to id e n tif y both c is-elements and t ra n s -a c ti n g facto rs that control or influence the co-o rdinate regula tion of the GRP genes. In the chapter 2, functi onal regions of the GRP94 and GRP78 promoters were defined. To f a c i l i t a t e the search for pr otein fa ctors that in te ra ct with the 121 DNA regu l atory domains of the GRP genes, the HeLa cell nuclear extr ac ts were obtained and used for i_n vi t r o studies. Both GRPs were shown to be regulated si mi la rly in the HeLa and the K12 cel ls under the same conditions (chapter 3 . 3 . 2 . ) . Furthermore, La. v i t r o tr a n s c r i p t i o n assay showed that the nuclear extracts isolated from HeLa cel ls tr anscribed GRP78 promoter e f f i c i e n t l y (Resendez et a l . , 1988). Using both i_n vivo and j_n vi t r o assay systems, the hypothesi s that the co-ordinate regulati o n of GRP genes acts through common trans -actin g facto r binding to a pu tative conserved regul at or y DNA d oma i n , wa s tested and is described in the foilow ing chapter. 122 3.2 MATERIALS AND METHODS 3.2.1 . DNA sequence a n a l y s i s . The sequence was analyzed using the Int e 1 1 i-Genet i cs Bionet SEQ:SEARCH program. 3.2.2. Cell l i n e s and culture c o n d i t i o n s . The Chinese hamster fi b r o b la s t t_s. mutant cell line K1 2 , the HeLa D9 8 AH2 monolayer c el ls , and their cul t ur e conditions have been described (chapter 1 . 2 . 1 . ) . The culture condition for HeLa S3 suspension ce lls is the same as described for the HeLa D9 8 AH2 monolayer cell s except that the calcium ionophore A231 87 treatment was at a concent r ati on of 2 >uM for 6 hrs. 3 . 2 . 3 . Iso lation of cytoplasmic RNA and RNA blot hy b ri di za ti on . The isolation of cytoplasmic RNA and RNA blot hy b rid iz a tio n have been described (chapter 1 . 2 . 2 .). 3.2. 4. Plasmids. ( i ) Plasmids pGRP94( - 2 3 1 / - 165) and p G R P 9 4 ( - 2 3 1 / - 4 3 ) . These plasmids are subclones of the human GRP94 promoter. Plasmid pGRP94 (- 2 3 1 /- 165) contains the Sst II/Bst EI I (- 231 /- 165 ) fragment subcloned into the SmaI site of pTZ18U. Plasmid pGRP94(-231/-43) contains the Sst II /S s t II (-2 31/-43) fragment subcloned into the SmaI site of pUC8. 123 ( i i ) Plasmids pGRP78 ( - 3 7 5 / - 8 8 ) and p G R P 7 8 ( - 1 7 0 / - 1 3 5 ) . The plasmid pGRP78(- 375/- 88 ) has been described (chapter 2 . 2 . 2 . ) . Plasmid pGRP78 (- 1 70/- 1 35) contains the synthetic rat GRP78 promoter sequence from nt -170 to -135 (with flanking li nker s it e s , Xho I and Sal I) cloned into the Sma I s ite of pUC8. ( i i i ) Plasmid pGRP94(- 3 5 7 )CAT. The constr ucti on of pGRP94(- 357 )CAT has been described (chapter 2 . 2 . 2 . ) . 3.2. 5. P reparati on of HeLa nuclear e x tr a ct s . Nuclear prot ein ext ract was prepared from the HeLa S3 cells growTi in suspension to a densi ty of 1 X 10^ cel ls/ ml in DMEM supplemented by 10% fetal calf serum as described (Shapiro et a l . , 1988; Resendez et a l . , 1988). To prepare nuclear extr act fr om induced HeLa S3 c e l l s , the cel ls were cultured in the gl ucos e-free medium for 16 hrs or in the medium containing 2 >uM of calcium ionophore A23187 for 6 hrs. 3. 2.6. Gel r e t a r d a t i o n assay. To prepare a probe for the gel re ta rd a ti o n assay, the 118 nt Eco Rl/Hind III fragment of the pGRP94( - 2 3 1 / - 165) contai ning 67 nt of the human GRP94 promoter, was labeled wi t h [ -*^P ] dATP using the Klenow fragment of DNA polymerase I. The binding r eact i on was performed in a 20 j a 1 volume 124 containing 10 mM Tris (pH 7.5), 50 mM NaCl, 1 mM EDTA, 10 mM b e t a -me r c a p t o e t h an o 1 , indicated amount of HeLa nuclear e xt ra ct , poly(dl -dC), and 1 ng (45,000 cpm) of the labeled fragment. Indicated amount of various c omp et i tors wa s added to the react i on mixtur es for the competition experiments. The reacti on mixtur es were incubated at room temperature for 25 min and elect r ophoresed on 59c low s a l t , non-denaturing polyacrylamide gels (a weight r a ti o of acrylamide/ b i s a c r y 1 ami d e was 80) containing 6.7 mM Tris (pH 7.5), 3.3 mM sodium acet at e and 1 mM EDTA. 3.2.7. DNase I fo otp ri nt analysis. The 118 nt Eco RI/ Hind III fragment labeled as in the gel re ta rd a ti o n assay was digest ed with Asp718 r e s t r i c t i o n enzyme and resolved on a non-denaturing polyac rylami de gel to obtain the 111 nt A sp 7 1 8 /H in d III fragment with labeled non-coding strand. To prepare the labeled coding strand, a 207 nt Eco Rl/Pst I fragment containing the Sst II /Sst Il (- 23 1/- 43 ) promoter sequence of human GRP94 was isolated from the plasmid pGRP94 ( - 2 3 1 /-43 ) , and its Eco RI end was labeled with [© £-^P] dATP using the K lenow fragment of DNA polymerase I. Binding reacti ons were carried out as in the gel r e ta rd a ti o n assay, except that the react i ons contained 0.4-0.7 ng end-labeled DNA, 1 >ug poly(dl-dC) and varying amounts 125 of nuclear e x tra ct . After 2 5 min incubation at room temperature, 2 jul of 20 mM MgCl 2 and 20 ill of 5 m\l C a C ^ / l mM EDTA were added. The reacti on mixtur es were subjected to DNase I dig e st io n (400 ng and 4 ng for samples with or without e x tr a c t, res p ec ti v e ly ) at room temperature for 60 sec. The reacti on was terminated by adding 50 ul of STOP buffer contai ning 200 mM NaCl , 2 0 mM EDTA, 1% SDS and 250 *ig tRNA/ml , followed by phenol / chl or of or m ex traction and ethanol p r e c i p i t a t i o n . The DNA was then resuspended in formamide dye mix and electr oph o resed on an 8 or 6% polyacrylamide sequencing gel. To localize the p r otect ed regions, the end-labeled DNA was subjected to Ma x am-G i 1 b e r t G sequencing reac ti on (Maxam and G i l b e r t , 1980) and used as a ma rke r . 3. 2.8. T ransi ent t r a n s f e c t i o n and assay for CAT a c t i v i t y . Tran sfection of DNA into K12 c e l l s , pre paration of the cell e xt ra ct , measurement of the p rot ei n concent r ati o n and assays for the CAT a c t i v i t y have been described (chapter s 2.2. 3. and 2 . 2 . 4 . ) . 126 3. 3 RESULTS 3.3.1. Sequence conservation b e tw ee n the GRP promoters. Since both GRP94 and GRP78 are regulated sim ila rly , it is possible that r egul ation of both genes involves common tr a ns -a ct in g f act or s which recognize si mi l ar DNA mot i fs in the GRP78 and GRP94 promoters. To ide ntify the put ati v e common regul at or y domains of the GRP genes, the human GRP94 promoter sequence was compared with that of chicken GRP94 and 'th e human and r a t GRP78 genes. The GRP94 and GRP78 promoter sequences are d is si m il ar with the exception of a short domain located from nt -193 to -168 of the human GRP94 gene (Fig. 22). This highly conserved region lie s with in the Sma/ St u(- 375/- 88 ) fragment implicated in high level expression of GRP78 gene by deletion analysis and i n vivo competition (chapter 2; Chang et a l . , 1987; Lin et al., 1986). Furt h er, this domain li es with in the r e g i on wh ich is highly conserved be t we en the chicken and human GRP94 promoters (nt -195 to -72). The delet ion analysis described in chapter 2 indi cat es that thi s domain is cruci al for the expression of GRP94 gene. In v i t r o studies have demonstrated that nuclear fa ctors s p e c i f i c a l l y bound to the highly similar region in both the rat and human GRP78 genes (Resendez et a 1 . , 1988). 127 3.3.2. GRP94 and GRP78 are regulated s im ila rly during s t r e s s . In order to study the r egulati o n of GRPs using HeLa nuclear ex tr a ct s , we f i r s t determined whether the GRP94 and GRP78 genes are regulated in a similar fashion in the HeLa D98AH2 monolayer c e l l s . Cytoplasmic RNA was ext r act ed from HeLa cel ls subjected to eit h er glucose s tar vat io n or calcium ionophore A23187 treatment. The level of GRP 7 8 and GRP 9 4 mRNA was monitored by RNA blot hybridi zat ion using hamster cDNA probes of similar spec ifi c a c t i v i t y . The mRNA level was also compared to that of hamster K12 c el l s. As shown in Fig. 23, the mRNA l evel s of GRP94 and GRP78 are enhanced both in human and hamster ce lls under the same induced condition. Furthermore, hy bri di zat ion signals of the hamster K12 ce lls are stronger than those of HeLa cel ls . The divergence between human and hamster coding sequences may account for this d if f er en c e. When a human GRP94 probe was used for hy br id i za ti o n , the human RNA samples gave higher signal s than those of h ams ter. 3.3.3. GRP94 and GRP78 p r o m o t e r s can com pete for n u clear fac tor s i_fi v i t r o . To t est the h 3rpot hesis that GRP94 and GRP78 share common tra ns -a ct in g fa ctors recognizing the 28 nt conserved DNA domain, speci fi c i n te ra c t io n s b etw een the human GRP94 promoter and 1 28 nuclear f act or s we re f i r s t establi sh ed by gel r e ta rd a ti o n assays (Fried and Crothers, 1981). A 118 nt Eco RI/Hind III fragment containing the GRP common domain was isolated from pGRP94(- 2 3 1 / - 165 ) , end- label ed, and mixed with nuclear ext r act prepared from HeLa S3 cel ls grown under non-induced condition. The DNA-protein complexes were resolved on polyacrylamide gels and detected by autoradiography. As shoun in Fig. 24, a major complex (l abel ed as ”C ” ) was det ected. Furthermore, the binding pa ttern betw een GRP94 promoter and nuclear facto rs did not change wh en nuclear ext r act prepared from the HeLa S3 ce lls treat ed with calcium ionophore A2 3187 or grown under glucose starv ation condition was used (Fig. 25). This may not be surprising because the DNA fragment used for the gel re ta rd a ti o n assays only contains the GRP94 promoter sequence nt -231 to -165. This sequence located with in the region sh own to be i mp ortant for basal level expression but have only p a r t i a l effect on the i n d u c i b i l i t y (Fig. 15). The s p e c i f i c i t y of the binding a c t i v i t y for the human GRP94 promoter sequence was confirmed by competition assays. The competition experiments were carried out in the presence of eith er hom ologous or heterologous DNA compet itors. The re su lts indicated that the complex formed is spec if ic for the GRP94 and 129 binds with high a f f i n i t y and s t a b i l i t y because a 120- fold molar excess of a non-homo1ogous fragment (a Pvul I fragment of pTZ18U) was unable to compete away the complex, whereas a 40-fold molar excess of the homologous unlabeled DNA completely eliminated the complex (Fig. 2 6A). A Sma/ Stu(- 375/- 88 ) fragment imnediately 5 ’ to the TATA element of rat GRP78 gene has been id e n t i f i e d as an enhancer (Lin et a l . , 1986). In add i t i o n , it was s h own to c omp ete for c e l l u l a r factors in te ra ct in g with the GRP78 promoter in cot ra nsf ect ion assays (Fig. 21). To investigate whether GRP94 and GRP78 share common tra n s -a c ti n g factors, the GRP78 Sm a/ S t u ( - 3 1 5 / -88) fragment was used as a competitor in the gel r e ta rd a ti o n assay. As sh own in Fig. 26B, the Sma , S tu(- 375/ - 88 ) fragment competed away the complex at a 40-fold molar ratio of unlabeled GRP78/1 ab e1ed GRP94 DNA. These competition re su lts suggest that nuclear fa ctors which bind to the GRP 9 4 promoter also have a f f i n i t y for the GRP78 Sma / Stu fragment. The res ult s also support the hypothesi s that GRP94 and GRP78 share common tr a n s -a ct in g fa ctors which may be involved in the co- or di nat e regulatio n of GRP genes. 3.3 .4 . Binding s it e s of the human GRP94 p r o m o t e r to pr o te in f a c to r s . In the gel re ta rd a ti o n assays, a major 1 30 complex was formed between the HeLa S3 nuclear extract and the human GRP94 Sst II/Bst EI I (- 231 /- 165 ) promoter fragment. To define the binding site s more preci sel y, the labeled non-coding strand wa s subjected to DN a s e I fo o tp r in t assay. Results i ndicated a region spanning f r om nt -165 to -190 wa s protect ed fr om DN a s e I di gestion (Fig. 27A). This domain resides within the region required for high basal level expression of GRP 9 4 (Fig. 15), and the domain is conserved between the GRP94 and GRP78 genes (Fig. 22). A bigger domain spanning from nt -161 to -203 was pr ot ect ed when the coding strand of a 207 nt Eco R1 Psi I fragment containing the Sst II / Sst I I ( - 231/-43) sequence of GRP9 4 promoter was end-labeled and used for foo tp r in ti ng analysis (Fig. 27B). Other minor footprinted regions were also seen. The f o otpri nt ed regions are indicated in the Fig. 11. Addition of the unlabeled GRP78 consensus domain to the binding r eact i on reduced the protec tion (Fig. 27B). This confirms the r esult from gel re ta rd a ti o n assay that nuclear factors which bind to the GRP94 promoter also have a f f i n i t y for the GRP78 promoter and the DNA domain conserved b etw ee n GRP94 and GRP78 genes. 3.3.5. GRP94 and GRP78 promoters can com pete for c e l l u l a r facto rs i_n v i v o . Pr eviously, plasmid 131 pGRP7 8(- 375/- 88 ) containing the GRP78 Sma/Stu promoter/enhancer fragment has been shown to compete for c e l l u l a r factors binding to the GRP78 promoter i n vivo (chapter 2; Fig. 21). To corr el at e the i_n v it r o c omp e t i t i o n r e s u lt s (sh own in the chapters 3 .3.3. and 3 . 3 . 4 . ) with biolo g ical functions Li v i v o , this GRP 7 8 Sma / S t u fra gm e n t wa s used to test i t s a b i l i t y to compete for the f act or s in te rac ti ng with the GRP94 promoter i_n vivo . Various amount of the pGRP7 8 (- 3 7 5 /- 88 ) was co -tr ansfected with the test plasmid pGRP94(- 357 )CAT into the K12 c e l l s . To maintain a constant amount of exogenous DNA in each tr a n s f e c t i o n , pUCS was added. As shown in Fig. 28, ad d i t i on of increasing amount of the competitor plasmid d imi n i shed CAT a c t i v i t y driven by the GR P 9 4 p r omo ter under both induced and non-induced cond i t i ons, suggesting that the GRP78 promoter fragment can compete away c e ll u la r facto rs int e ra ct in g wi t h the- GRP94 promoter. This provides an evidence that common f act or s * are important for the co- or di nat e expression of GRP94 and GRP 7 8 genes. To t es t fu rther whether the common facto rs recognize the common GRP regulatory domain, the competition experiments were perfomed with the synthetic GRP78 sequence (nt -170 to -135) subcloned into pUC8. The r e s u l t s , as shown in Fig* 29, 132 demonstrated that this 36 nt region e ff e c t iv e ly c omp etes for c e l l u l a r f act or s involved in both basal level and induced expression of GRP94 by A23187 and the K12 t_s mutation. At a com peti t o r / t e s t plasmid molar ratio of 6 to 1, the CAT a c t i v i t y of the test pla sm i d pGRP94 (- 357 )CAT was reduced by h alf. In contr ast, the promoter a c t i v i t y of pSV2CAT was not af f ected in p a r a l l e l competition experiments (Fig. 29). These combined res ult s indicate that the facto rs necessary for the expression of GRP94 recognize the common GRP domain. Since this domain is unique among the GRP genes and is devoid of binding sites for general tr a n s c r i p t i o n f actor s such as, Spl, AP-2 or CAA.T, i t is likely that novel nuclear factors sp ecifi c for the GRP genes may be involved. 1 3 3 3.4. DISCUSSION In eukaryotic system, mRNA is synthesized by RNA polymerase II. However, extens ively p u rif ie d RNA polymerase II can not recognized its cognate promoters i n v i t r o (We il et a l . , 1979). Au x i l i a r y factors are required to i n i t i a t e tr a n s c r i p t i o n accur ate ly as well as to optimize the RNA synthesis through in te ra ct io n s between promoter/enhancer elements and pr otein f act or s (Ma tsui et a l . , 1980; Samu e l s et a l . , 1982). C e l l- fr e e systems for RNA polymerase II that ac curately and s p e c i f i c a l l y tr ans cribe exogenous DNA t emplates have been est ablished (Man le y et a l . , 1980; Shapiro et a l . , 1988). With the c e l l - f r e e systems, gel re ta rd a tio n assay (Fried and Cr ot her s, 1981), and foo tp ri nt in g analy si s, many pr otein factors have been id e n t i f i e d . S om e of the facto rs have been pu ri f ie d to homogeneity and the corresponding genes cloned. Of the k n own t r a n s c r i p t i o n fac tors, it is clear that s om e are important for the expression of a wide v a r i e t y of genes such as, CTF/NF-1, Spl, AP-1, and AP-2, wh i 1 e others appear to regulate a r ather s pe ci fic gene such as, HSTF which s p e c i f i c a l l y regul at es the expression of the heat shock gene family. I d enti fy ing the common regu latory domains and pro tein fac to rs involved in the co- or di nat e reg u l ati o n 1 3 4 of a speci fic set of genes is p a r t i c u l a r l y i n t e r e s t i n g . The GRP genes encode a set of ER pr ot eins whose syntheses are gr e a tl y enhanced under s tre ss conditions that s p e c i f i c a l l y a ffe ct s the ER a c t i v i t y (Lee, 1987). Therefore, GRP genes represent an unique m odel for studying the regulation of genes encoding ER lo ca lized prot ei ns . In this chapter, I have att emp ted to localized DNA sequences w ith in the GRP genes that may serve as sig nals for the regulation and to determine w h e t h e r prote in fa ctors in te ra ct wi th these DNA dom ains to r egul at e the expression of the GRP genes. Di r ect sequence c omp arison sh owe d that there is a DNA d oma i n consisting of 28 nt that is highly conserved b e tw ee n GRP94 and GRP78 (Fig. 22). This dom ain lie s w ith in the crucial region for basal level expression of GRP94 as well as GRP7 8. The consensus sequences in both GRP genes we re pro tec ted fr om DN ase I dige st ion (Fig. 27; Resendez et a l . , 1988). Furthermore, both La vivo and i n v i t r o c omp e t i t i o n assays sh owe d that the facto rs w h ic h bind to the GRP94 promoter also have a f f i n i t y for the GRP78 Sma / St u(- 3 7 5 / - 88 ) f r a g m e n t and the conserved dom ain ( n t -170 to - 1 3 5 ) of GRP 7 8 p r o m o t e r (Figs. 26, 28, and 29). These com bined re s ul ts suggest that GRP 9 4 and GRP78 share common t r a n s c r i p t i o n fa c to rs . D if f e r e n t m e ch a n is m s that are involved in the 135 r e g u l a t i o n of developmental process and i n d u c i b i l i t y have been obser ved. A c t i v a t i o n of t r a n s c r i p t i o n could be due to a d d i t i o n a l DNA-factor i n t e r a c t i o n s th at do not occur in the oth er developmental s t a t u s or non- ind uced c e l l s . A l t e r n a t i v e l y , a c t i v a t i o n may r e g u l a t e d by the same f a c t o r s w i t h d i f f e r e n t m o d i f i c a t i o n s . P h o s p h o r y l a t i o n of t r a n s c r i p t i o n f a c t o r s is one type of m o d i f i c a t i o n which a c t i v a t e s t r a n s c r i p t i o n . Examples have been seen w i t h the cAMP response element binding p r o t e i n (CREB) under hormone induced c o n d i t i o n (Yamamoto et a l . , 1988) and the ye as t HSF under s t r e s s c o n d i t i o n s (Sorger and Pel ham, 1 988 ). In order to examine wh ether d i f f e r e n t binding p a t t e r n s e x i s t between the GRP94 promoter and n u c l e a r f a c t o r s upon a c t i v a t i o n and under normal c o n d i t i o n , n u c l e a r e x t r a c t s fr om HeLa S3 c e l l s gr own under induced and non-induced c o n d i t i o n s were used for the gel r e t a r d a t i o n ass ay. No s i g n i f i c a n t changes ‘in the bi ndi ng p a t t e r n were observed (Fig. 25). It is p o s s i b l e t h a t the same f a c t o r s r e g u l a t e the t r a n s c r i p t i o n under both induced and non- ind uced c o n d i t i o n s , su g g e s t i n g th a t the f a c t o r s ma y be mo d i f i e d under one c o n d i t i o n but not the o t h e r . Moreover, the s i m i l a r binding p a t t e r n observed may simply imply t h a t the DNA fragment used in the gel r e t a r d a t i o n assays only c o n ta i n ed the DNA domain which is important for the GRP94 basal level 136 e x p r e s s i o n . The in d u c i b l e element may p r e s en t somewhere f u r t h e r downstream as suggested by the La vivo s t u d i e s (F ig . 15). A l t e r n a t i v e l y , f u l l i n d u c i b i l i t y may r e q u i r e more than one DNA domain i n t e r a c t i n g w i t h m u l t i p l e f a c t o r s . I d e n t i f i c a t i o n of the minimal DNA sequences involved in GRP e x p r e s s i o n under induced and non-induced c o n d i t i o n s , and p u r i f i c a t i o n of the t r a n s - a c t i n g f a c t o r s involved in the r e g u l a t i o n w i l l f u r t h e r our u n d e r s t a n d i n g of the c o - o r d i n a t e r e g u l a t i o n mechanism of the GRP genes. 137 FIG. 22 Sequence i s o 1 a t e d l o c a t i o n t r a n s c r i i n v e r t e d genes (R Sequence c o n s e r v a t i o n of the GRP pr om ot er s, s conserved between GRP94 and GRP78 promoters from human, chicken and rat are shown. The of each sequence is numbered w i t h i t s own p t i o n a l i n i t i a t i o n s i t e d e s i g n a t e d as +1 . An repeat found in both human and rat GRP78 esendez et a l . , 1988) is i n d i c a t e d . 138 139 Chick GRP94 Human GRP94 Human GRP78 Rat GRP78 202 AATCGACGCCGGCCACGCTCCGTCCGCA 19 3 AATCGGAAGGAGCCACGCTTCG - - GGCA 1 3 5 GGGCCGCTTCGAAT CGGCGGCGGCCA - GCTTGGT - GGCC 1 7 2 AGGCCGCTTCGAATCGGCAGCG(^CA - GCTTGGT - GGCA g T 7 Z / J l g f t » ^ ZT777'//771 I____________________________________________ I -175 -168 - 99 - 1 36 FIG. 23 T r a n s c r i p t level of GRPs under s t r e s s c o n d i t i o n s . Total cyt oplasmic RNA was e x t r a c t e d from the hamster t_§_ mutant K12 c e l l s (l a n e s 1 to 3) and the human HeLa D98 AH2 monolayer c e l l s ( l a n e s 4 to 6) m a i n t a i n e d at 35°C in DMEM ( l a n e s 1 and 4), t r e a t e d w i t h 7 juM of A231 87 at 35°C ( l a n e s 2 and 5), s h i f t e d to the non-permi ss ive temp erature 39.5°C ( l a n e 3) or grown in g l u c o s e - f r e e medium at 3 5°C (la ne 6). Except for lane 5 wh e r e the c e l l s were t r e a t e d for 5 h r s , o t h e r s were a l l t r e a t e d for 16 h r s . Equal amount (10 >ug) of RNA f r om each s amp 1e wa s used for N o rt h e rn blot a n a l y s i s . The f i l t e r s were probed w i t h a l a b e l e d 1500 nt fragment g e n e r a t e d by Eco RI and Pst I d i g e s t i o n of p3C5, a cDNA clone encoding the hamster GRP 7 8 (Lee et a l . , 1983; Ting et a l . , 1987), or a l a b e l e d 980 nt fragment ge n er ate d by Bam HI and Eco RV d i g e s t i o n of p4A3, a cDNA clone encoding the hamster GRP94 ( c h a p t e r 1, Lee et a l . , 1983; Sorger and Pelham 1987). The au to rad iog rams are shown. 140 hamster human i---------------------------------- 1 i----------------------------- 1 2 3 4 5 6 -< GRP78 GRP94 141 FIG. 24 Binding of HeLa n u c le a r f a c t o r s to the human GRP94 promoter. Gel r e t a r d a t i o n assays were c a r r i e d out using the l a b el ed 118 nt Eco RI/Hind III fragment c o n t a i n i n g the 67 nt Sst II/'Bst E I I ( - 2 3 1 / - 1 6 5 ) region of the human GRP94 promoter. The n u c l e a r e x t r a c t was prepared f om HeLa S3 suspension c e l l s gr own under non-induced c o n d i t i o n . Reactio ns c onta i ne d e i t h e r 1 jug ( l a n e s 2 to 7) or 4 jug ( l a n e s 8 to 12) of n u c l e a r e x t r a c t except lane 1 to wh ich only 1 ng (45,000 c pm) of the l a b el ed 118 nt fragment was added. The t i t r a t i o n p r o f i l e s w i t h i n c r e a s i n g amount of p o l y ( d l - d C ) are s h own . Lanes 2 to 12 contained 0, 0 . 5 , 1, 1.5, 2, 3, 1, 2.5, 5, 7.5, and 10 jug of p o l v ( d l - d C ) , r e s p e c t i v e l y . The au to ra dio gra m s are shown. The p o s i t i o n s of unbound DNA (F) and the major DNA - f a c t o r complex (C) are i ndi cat ed. 142 c - F - 143 FIG. 25 Sim ilar binding p a t t e r n s formed between the human GRP94 promoter and HeLa n u c l e a r f a c t o r s i s o l a t e d f r om induced and non-induced c o n d i t i o n s . DNA binding r e a c t i o n was c a r r i e d out w i t h the la b el ed 118 bp fragment co n t ai n i n g the GRP94 Sst I I / B s t EI 1 (-231 /- 165) promoter sequence and n u c l e a r e x t r a c t s pre pared from the HeLa S3 suspension c e l l s gr own under non-induced c o n d i t i o n ( l a n e s 1, 3, 5, 7, 9, and 11), c al c i um ionophore tr eatm ent for 5 hrs (l a n e s 2, 6, and 10) or g l u c o s e - s t a r v a t i on for 16 hrs (l a n e s 4, 8, and 12) before the e x t r a c t i o n . The aut oradio gra ms are shown. Lanes 1 to 4 contain ed 1 u g p o l y ( d l - d C ) , and 1 ju g of c el l e x t r a c t . Lanes 5 to 8 contain ed 3 jug p o l y ( d l - d C ) and 1 jug of c e l l e x t r a c t , and lanes 9 to 12 c o n t a i n e d . 1 jug p o l v ( d l - d C ) and 2 jug of c e l l e x t r a c t . The p o s i t i o n s of unbound DNA (F) and the major DNA-fact or complex (C) are i n d i c a t e d . 1 4 4 1 2 3 4 5 6 7 8 9 10 11 12 4 « ■ m ~ V c - 145 FIG. 2 6 Com petit ion for n u c l e a r f a c t o r i_s. v i t r o . Nuclear e x t r a c t wa s prepared fr om HeLa S3 suspension c e l l s g r own under non-induced c o n d i t i o n . Each r e a c t i o n co n ta i n ed 4 jug of the n u c l e a r e x t r a c t except the f i r s t lane in panel A, to wh ich only the l a b e l e d 118 nt Eco RI/Hind III fragment c o n t a i n i n g the 67 nt Sst I I / B s t Eli ( - 2 3 1 / - 1 6 5 ) region of the human GRP94 promoter was added. Varying amount of u n l a b e l e d co m p et it o r was added t o g e th e r w i t h 1 ng (45,000 cpm) of the probe. The autor adiog rams are shown. The molar r a t i o of u n la b el ed c omp e t i t o r / l a b e l e d GRP94 promoter fra gm ent, and the p o s i t i o n s of unbound DNA (F) and the ma j o r DNA-factor c omplex (C) are indi c a t e d . Th e c omp e t i t o r s used were: A) a PvuI I fragment ( n t 5 3 t 0 398) of pTZl8U and the u nla bel ed 118 nt Eco RI/Hind I I 1 fra gme nt c o n t a i n i n g GRP94 promoter sequence n t - 2 3 1 t 0 -165; B) the 298 nt Eco Rl/Bam HI fragment c o n t a i n i n g GRP78 promoter sequence nt -375 to -88. 146 147 A. co m p e tito r. pTZ18U (5 3 /3 9 8 ) G R P94 (-2 31/- 1 6 5 ) m o la r ra tio : 0 H 4 12 4 0 120, r i 4 12 4 0 120* B. G R P 78(-375/-88) 0 ' I 4 12 4 0 1 FIG. 2 7 DNase I f o o t p r i n t a n a l y s i s of the human GRP94 p r omo t e r . Th e DNA probe wa s incubated w i t h n u c l e a r e x t r a c t prepared fr om the HeLa S3 c e l l s gr owm under non-ind uced c o n d i t i o n and su b je c te d to DN a s e I d i g e s t i o n . The auto radio gra ms are shown. A) Non-coding s t r a n d f o o t p r i n t . Lanes 1 and 4 are DN ase I t r e a t e d probes w i t h o u t p r o t e i n e x t r a c t . Lane 2 and lane 3 are DN ase I t r e a t e d probes w ith 63 and 42 ^ig of p r o t e i n e x t r a c t , r e s p e c t i v e l y . The box ( - 1 9 0 / - 1 6 5 ) r e p r e s e n t s the f o o t p r i n t re g io n . B) Coding stran d f o o t p r i n t . Lanes 1 and 4 are DN ase I t r e a t e d probes wi thout p r o t e i n e x t r a c t . Lanes 2 and 3 are DN ase I t r e a t e d probes c o n t a i n i n g 72 ;ug of p r o t e i n e x t r a c t w i t h o u t (l a n e 2) or wit h (lane 3) a 5 5 -f o ld molar excess of a co m p et it o r fra gme n t . Th e c omp e t i t o r used wa s an Eco R I / S a 1 1 fragment i s o l a t e d from pGRP78 (- 1 70/- 1 35 ) which c o n t a i n s the s y n t h e t i c rat GRP78 consensus sequence (nt -170 to -135). The major f o o t p r i n t e d region ( - 2 0 3 / - 1 6 1 ) is boxed. Lanes M are the Maxam-Gi 1 be r t G sequencing r e a c t i o n of the l a b e l e d s t r a n d . 148 A . N o n * * C o d in g 1 2 3 4 M rl65 H 9 0 -231 231 -2 0 3 -161 C o d in g 149 FIG. 2 8 C omp e t i t i o n for c e l l u l a r f a c t o r s La vivo b y the rat GRP 7 8 Sma/ S t u (- 375/- 88 ) fragment. K12 c e l l s were c o - t r a n s f e c t e d w i t h 5 jug of the t e s t p la sm id , pGRP94(- 357)CAT, and i n c r e a s i n g amount of the co m p et it o r plasmid, pGRP78 (- 375/- 88 ). To m a i n t a i n a c o n s t a n t amount of DNA for each t r a n s f e c t i o n , pUC8 was added to a d j u s t the t o t a l plasmid DNA to 20 jug. P r o t e i n e x t r a c t s we re pre pared fr om the t r a n s f e c t e d c e l l s m a i n t a i n e d in DMEM at 3 5°C ( O ), t r e a t e d wT it h 7 juM of A231 87 ( A ), or s h i f t e d to 39.5°C ( # ) . The CAT a c t i v i t y wT a s d e t e rm ined using 60 jug of p r o t e i n fr om each cell e x t r a c t . Th e CAT a c t i v i t y , exp ressed as pe rc en ta g e conver sion of ^ C - chioTampheni col to i t s a c e t v l a t e d fo rm s , wa s p l o t t e d a g a i n s t the mo 1 a r r a t i o of com pet ito r to the t e s t plasmid. 150 TEST COMPETITOR CONTROL A A pGRP94 (-3 5 7 ) CAT p U C 8 CAT 80 70 60 50 o 40 O 30 20 10 4 3 5 molar ratio: Competitor/ Test 151 FIG. 2 9 Competition for c e l l u l a r f a c t o r s Ln vivo by the rat GRP78 common domain (nt -170 to -135). K12 c e l l s were c o - t r a n s f e c t e d w it h 3 jug of t e s t plasmid, pGRP94(- 357 )CAT or pSV2CAT, and i n c r e a s i n g amount of the c om pe ti to r plasmid, pGRP78(- 170/- 135). To m a i n t a i n a c o n st a n t amount of DNA for each t r a n s f e c t i o n , pUC8 was added to a d j u s t the t o t a l plasmid DNA to 20 jag . P r o t e i n e x t r a c t s were pre pared from the t r a n s f e c t e d c e l l s m a i n t a i n e d in DMEM at 35°C ( c o n t r o l ) , t r e a t e d w i t h 7 juM of calcium ionophore (A23187), or s h i f t e d to the n o n - permi ssive tempe rat ure (39.5°C) for 16 h r s . The CAT a c t i v i t y was determined using 120 jag of p r o t e i n from each cell e x t r a c t . The auto rad iog rams are shown. The molar r a t i o of c o m p e t i t o r / t e s t plasmid in each t r a n s f e c t i o n and the CAT a c t i v i t y , expressed as 1 4 p e r c en ta g e conv ersio n of C-chlorampheni col to i t s a c e t v l a t e d forms, are i n d i c a t e d . 152 153 Test plasmid Treatment: control 1 Ac — C M - C om petitor/T est molar ratio: */• CAT conversion : 2.4 pGRP94(-357)CAT _________ I __________ A23187 pSV2CAT 39.5* C control m m • • * m m • • • • • • 0 2 6 10.3 9.7 5.1 0 2 6 5.9 6.5 7.7 REFERENCES An an than, J . , A. L. Goldberg and R. Vo e limy. 1 986. 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The metabolism of the two di-deuterobutyric acids as indicated by the deuterium content of the excreted betahydroxbutyric acid

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A study of the effect of glycogen on the oxidation of butyrate by rat liver slices

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Calculations of electrostatic interactions in proteins

Asset Metadata

Creator Chang, Shin C. (author)

Core Title The genes for the glucose-regulated protein GRP94 and GRP78 are co-ordinately regulated by common trans-acting factors

School Graduate School

Degree Doctor of Philosophy

Degree Program Biochemistry

Degree Conferral Date 1989-05

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag chemistry, biochemistry,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-6325

Unique identifier UC11347662

Identifier DP21628.pdf (filename),usctheses-c17-6325 (legacy record id)

Legacy Identifier DP21628.pdf

Dmrecord 6325

Document Type Dissertation

Rights Chang, Shin C.

Type texts

Source University of Southern California (contributing entity), University of Southern California Dissertations and Theses (collection)

Access Conditions The author retains rights to his/her dissertation, thesis or other graduate work according to U.S. copyright law. Electronic access is being provided by the USC Libraries in agreement with the au...

Repository Name University of Southern California Digital Library

Repository Location USC Digital Library, University of Southern California, University Park Campus, Los Angeles, California 90089, USA