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A POU gene expressed in the early sea urchin embryo: Structure, evolution and requirement for embryonic development

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A POU gene expressed in the early sea urchin embryo: Structure, evolution and requirement for embryonic development

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Content A POU GENE EXPRESSED IN THE EARLY SEA URCHIN EMBRYO:
STRUCTURE, EVOLUTION AND REQUIREMENT FOR
EMBRYONIC DEVELOPMENT
by
Bharat Raghunath Char
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Biochemistry and Molecular Biology)
November 1992
Copyright 1992 Bharat Raghunath Char
UMI Number: DP21640
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UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCHOOL
UNIVERSITY PARK
LOS ANGELES. CALIFO RNIA 9 0 0 0 7
This dissertation, written by
Bharat R. Char
under the direction of h Dissertation Com
mittee, and approved by all its members, has
been presented to and accepted by The Graduate
School, in partial fulfillment of requirements of
the degree of
Ph.D.
v 2 m o
' 92 .
’ > 7 / - , p i
D O C T O R OF P H I L O S O P H Y
Dean
Date November 2X 1992
DISSERTATION COMMITTEE
Chairman
ACKNOWLEDGEMENTS
I thank my thesis adviser, Dr. Robert Maxson, for his encouragement,
support and guidance during my period of graduate study at USC. He has
taught me a great deal about science and the scientific method, for which I will
always be grateful. Without his insights and advice, I would not have been able
to accomplish a significant portion of this project. He encouraged me to think
independently and pursue my own ideas, which is very important for a
beginning scientist. I also sincerely thank my thesis committee members, Dr.
Pradip Roy-Burman and Dr. Stanley Tahara, for their advice and support.
I thank all my fellow graduate students for making my Ph.D. study a
rewarding experience. In particular, I would like to thank past and present
members of the Maxson lab, namely (in chronological order): Jeff Bell, Anita
Colin, Mike Ito, Allan Zhao, Joanne Kearney, Alex Noveen, Liang Ma, Sonia
Dobias, Juan Chen and Tina Tan.
Finally, I thank my family: my wife Madhavi, for her unconditional
support through the financial deprivation of graduate school; my parents, for
enabling me to follow my dreams; and my brother Deshik, for many things,
among them buying me Barron's guide to the GRE exam.
TABLE OF CONTENTS
INTRODUCTION
CHAPTER 1
DNA-PROTEIN INTERACTIONS AT AN OCTAMER MOTIF
REQUIRED FOR THE EXPRESSION OF THE EARLY HISTONE
H2B GENE IN SEA URCHIN EMBRYOS
CHAPTER 2
CLONING AND CHARACTERIZATION OF SPOCT, A GENE
ENCODING THE MAJOR OCTAMER-BINDING PROTEIN OF
THE SEA URCHIN EMBRYO
CHAPTER 3
FUNCTIONAL ROLE OF THE SPOCT GENE IN THE EARLY
SEA URCHIN EMBRYO
CHAPTER 4
CONCLUSIONS AND FUTURE DIRECTIONS
REFERENCES
LIST OF FIGURES
1.1. Binding of sea urchin nuclear protein to the octamer element. 24
1.2. Temporal profile of sea urchin octamer-binding activity. 27
1.3. Oct-1 can bind to the early H2B gene octamer element. 29
1.4. A cloned sea urchin nuclear protein binds to the early
H2B octamer element. 31
1.5. Comparison of the binding ability of octamer elements
in the early and late H2B promoters. 33
2.1. PCR-based strategy for cloning sea urchin POU genes. 61
2.2. SpOct cDNA sequence and comparision of the SpOct POU
domain with that of other POU II proteins. 63
2.3. DNAse I footprint assay comparing bacterially-expressed
SpOct and affinity purified oct proteins. 66
2.4. Measurement of the relative binding affinity of the SpOct
protein. 68
2.5. Probes used for gel blots and a DNA gel blot probed
or SpOct. 71
2.6. Expression profile of SpOct cDNA during development. 74
2.7. Expression of SpOct in adult tissues of the sea urchin. 78
2.8. A comparison of domain structures of POU II proteins
from various species. 80
3.1. Effect of antisense ODNs on embryonic development. 99
3.2. Rescue of antisense ODN-injected embryos. 103
3.3. Incorporation of ^H-thymidine into DNA in antisense
and control ODN-injected embryos. 106
3.4. Incorporation of ^^S-methionine into proteins in
antisense and control ODN-injected embryos. 108
3.5. Incorporation of 35s-methionine into Ca++ ionophore
A23187-activated eggs injected with antisense ODNs. I l l
iv
Introduction
For many years, the field of developmental biology was the almost
exclusive domain of experimental embryologists. Through physical
manipulation of the embryo, they gained valuable insights into phenomena
such as regulation, induction and the cell movements associated with
developmental processes such as gastrulation. In recent years, however,
molecular biology has emerged as the driving force in developmental biology.
The reason for this is not hard to find-after all, genes control development.
The multitude of structural and "housekeeping" genes in the genome all have
to be regulated correctly to form a viable embryo. Regulatory proteins are
now known to be responsible for controlling these genes, but knowing this
only brings up more questions: What regulates the regulators? What
coordinates the incredibly complex patterns of gene expression in the embryo?
One approach to answer these questions has been to identify regulatory
proteins and then attempt to discover the downstream genes that they regulate.
Using this approach, it is hoped, a transcriptional hierarchy or network of
factors can be identified, within the framework of which development (or
some part of it) can begin to be explained. Comparative analyses of
embryogenesis, based on information obtained through experimental
embryology and molecular biology, have yielded useful information on the
developmental strategies of different species.
Davidson (1991) has delineated three types of embryogenesis, based
on how cell specification occurs during early development. In type 1
embryos, characteristic of most present-day invertebrates, cell specification is
1
in situ, and lineage plays an important role in spatial organization of the
embryo. In type 2 embryos, the vertebrate form, diffusible morphogens acting
through cell-cell interactions play a major role in cell specification. In type 3
embryos, characteristic of long germ-band insects such as D rosophila,
morphogens determine the spatial organization of the embryo before
cellularization occurs. Despite these differences in developmental strategies,
the Drosophila embryo is a paradigm for understanding the molecular basis of
development.
In the last few years, the study of the development of the Drosophila
embryo has provided insights into the sequence of events at the molecular
level that transform the fertilized egg into a fly. Meinhardt (1986) first
proposed that pattern formation depends on the sequential expression of
segmentation genes. Maternal effect gene products, localized in the oocyte
cytoplasm, form gradients and determine the antero-posterior and dorso-
ventral axes of the embryo. These proteins regulate the expression of gap
genes, mutants of which lack segments of the body (Nusslein-Volhard and
Wieschaus, 1980). Next, the pair-rule genes are activated, and appear as
stripes along the length of the embryo, since they are expressed in alternate
segments of the embryo (Scott and O' Farrell, 1986). The pair-rule genes
regulate the expression of segment-polarity genes which are expressed in the
posterior compartment of every segment, and specify the polarity of each
segment. Once the expression of these genes has been established, homeotic
selector genes of the Antennapedia and Bithorax complexes specify the
characteristic structures of each segment (Wakimoto et al., 1984; Lewis,
1978).
2
Many of the segmentation and homeotic selector genes of Drosophila
belong to the homeobox family. The homeobox is a 180 bp DNA sequence
that encodes a DNA-binding domain (McGinnis et al., 1984; Scott and
Weiner, 1984). Many of the D rosophila homeobox genes regulate one
another. Genes having high degrees of sequence similarity with various
Drosophila homeobox genes have been isolated from other species as diverse
as worms, sea urchins and vertebrates, and they all function in determining
cell fate at different positions in the embryo. This suggests that even animals
as different as flies and mice develop according to the same basic principles.
What is even more amazing, however, is that the homeobox genes that
determine anterior and posterior characteristics in flies and mice are arranged
in the same linear order on the chromosome in their respective genomes
(Gaunt and Singh, 1990). This finding highlights the role evolution plays in
determining developmental strategies.
A family of genes that are related to homeobox genes are the POU
genes, which are a major focus in this thesis. These genes have DNA
sequences resembling the homeobox, encoding a protein sequence known as
the POU-homeodomain, and they also have an adjacent sequence that is
unique to this family known as the POU-specific domain (Sturm et al., 1988).
The protein products of these genes play diverse roles both in general
transcriptional regulation, as well as during development. Members of the
POU family have been identified in many species including flies, worms, sea
urchins and vertebrates. The first POU gene to be isolated was the ubiquitous
mammalian transcription factor, Oct-1, which is involved in the cell-cycle
regulation of mammalian histone H2B genes. Recently, many new members
of the POU family have been discovered. Many of them play a role during
development of the nervous system of the embryo by determining neuronal
cell fate.
Embryos have to regulate their genes through space and time. Small
changes in the spatial and temporal expression of genes can have profound
effects on development. This has been amply demonstrated by expressing
developmentally important genes at inappropriate times or ectopic positions in
embryos. The sea urchin embryo has been the subject of experimental
embryology for over a century. The ease with which embryos can be cultured
and manipulated, as well as their transparency, made them very popular with
early workers. Gastrulation in vertebrate embryos is a complicated affair,
with cell layers moving under or over each other in a manner that is not easily
discernible. The simpler sea urchin embryo, on the other hand, provided
embryologists with a clear view of cell movements during gastrulation, and
remains to this day the textbook description of the process. The sea urchin
embryo is also ideal for studying gene regulation during early development
since large numbers of embryos are easily obtainable for biochemical
experimentation. The histone genes of the early sea urchin embryo provide an
interesting example of coordinately regulated temporal gene expression, and
have been the subject of study for nearly two decades.
The histone genes are expressed in three consecutive and overlapping
sets during the early development of the sea urchin embryo. The alpha-variant
histones predominate in the egg and early embryo, and are the result of 300-
400 tandem arrays of early histone genes. A second set, the cleavage-stage
variants, are found in the embryo only during the initial cleavage stages. A
4
final set of genes synthesize the late or gamma variant histones, which are
expressed in later embryos. The late histones are encoded by a small number
of dispersed genes (Maxson et al., 1983).
The unfertilized egg contains a large accumulation of histone mRNA
present in the form of ribonucleoprotein (Skoultchi and Gross, 1973). This
store includes both alpha-variant and cleavage-stage histones. Histone
synthesis at this point is occurring at low levels. Upon fertilization, the
cleavage-stage histones are utilized. These genes have not been characterized,
and little is known about how their synthesis is regulated. During the first few
hours of development the sea urchin zygote undergoes four synchronous cell
divisions to form a 16-cell embryo. During this time, histone mRNA is
mobilized from the maternal store of ribonucleoprotein (Baker and Infante,
1982). From the fifth division onwards, the sea urchin embryo begins a period
of rapid asynchronous cell cleavage, and at this time a dramatic increase in
histone synthesis apparent. This increase is accounted for by a rapid increase
in the rate of alpha-variant histone mRNA synthesis, which peaks at about 10-
12 hr post-fertilization and subsequently decreases just as dramatically
(Maxson and Wilt, 1981; Weinberg et al., 1983). Once the levels of the early
histone gene transcription has fallen off, late histone gene mRNA synthesis
takes over, and continues during later stages of development.
What causes this switch from early to late histone gene expression?
Recent evidence has pointed to the role of regulatory proteins that bind to cis-
acting elements in the nontranslated flanking sequences of histone genes. In
the case of the LI late H2B gene, a enhancer element 0.5 kb 3' of the gene
activates expression of the gene in late blastula-stage embryos by binding an
5
Antennapedia-class homeodomain protein (Zhao et al., 1990; 1991). The
temporal control of the early H2B gene began as the focus of this Ph.D thesis
project. An octamer element in the proximal promoter of the early H2B gene
is required for the high level of expression of the gene in blastula-stage
embryos (Bell et al., 1992). My first experiments were concerned with
characterizing sea urchin nuclear proteins that bind this element. The octamer
motif is known to be the target of binding for mammalian POU domain
proteins such as Oct-1 and Oct-2 (Sturm et al., 1988; LeBowitz et al., 1988). I
showed that the human Oct-1 protein binds to the early H2B gene octamer site
with high affinity. Taking advantage of the high degree of sequence
conservation between species within the POU domain, I cloned cDNAs
encoding two sea urchin POU domain proteins. I obtained full-length cDNAs
encoding one of these POU genes, related to Oct-1 and Oct-2, designated as
SpOct. The SpOct protein, when synthesized in vitro, also bound the octamer
element avidly. The temporal profile of expression of the SpOct transcript is
very similar to that of the early H2B gene, supporting the role of the SpOct
protein in the activation of the early H2B gene. There was no evidence for the
presence of transcripts of other POU genes at any embryonic stage.
We were interested in understanding the function of the SpOct gene.
Oct-1 has been implicated in DNA replication as well as transcriptional
activation, and we wanted to know whether SpOct played a similar role in the
sea urchin embryo, and also whether early H2B expression would be affected.
I tested the function of SpOct in sea urchin zygotes by targeting expression of
the gene by injecting antisense oligonucleotides against SpOct. I found that
perturbation of SpOct gene function prevented the one-cell zygote from
6
cleaving. DNA replication in the embryo was not affected, but protein
synthesis was inhibited significantly. This, apparently, prevents the first cell
division from taking place. This finding is significant because it contradicts
present evidence that activation of protein synthesis in the sea urchin zygote is
independent of the nucleus. The data to this point cannot distinguish whether
SpOct is required as a transcription factor in the activation of protein
synthesis, or is involved in a non-nuclear role. Experiments performed with
enucleated zygotes can address the question of whether the antisense
phenotype requires the presence of a nucleus.
In this study, we have identified a regulatory protein that is required
for protein synthesis as well as early histone H2B gene activation. The SpOct
protein also binds to the promoter of the Cyllla actin gene, and probably plays
a role in its regulation (Thiebaud et al., 1990; Theze et al., 1990). These
observations are consistent with the multiple roles played by Oct-1 in
mammals, although in early mammalian embryos other POU genes are in
evidence that determine cell specification in the developing nervous system
(He et al., 1989). The identification of SpOct protein and elucidation of some
of its functions have provided us an entry into the regulatory network of the
sea urchin embryo. A more complete picture of sea urchin development will
no doubt emerge when the interactions between such proteins are studied, and
their downstream genes are identified.
From an evolutionary standpoint, SpOct is an interesting protein. Sea
urchins belong to Phylum Echinodermata, which branched off from the
deuterostome lineage (which gave rise to the vertebrates) about 600 million
years ago. D rosophila, a protostome, possesses two closely-related POU
7
genes, members of the Oct-1 subfamily (Billin et al., 1991; Lloyd and
Sakonju, 1991). Like the homeobox genes, therefore, the POU genes are
"living fossils", a successful innovation that arose before the split between
protostomes and deuterostomes, almost a billion years ago. Since POU genes
have been cloned from number of key species, we are now in a position to
compare the various domains of POU genes, and assign functions to them.
In closing this introduction, I would like to direct the reader's attention
to my opening remarks regarding the relation between molecular biology and
embryology. An elegant analogy was drawn by Gilbert (1988) wherein he
stated that molecular biology is to embryology as abstract formalism is to
naturalism in art. Molecular biology defines development as a series of gene
interactions and differential gene expression, which can be applied broadly to
all species, while embryology looks at the embryo as a whole, in relation to its
surroundings. Molecular biology seeks to find simple, unifying principles that
can explain the bases of development regardless of the organism, whereas
embryology considers the differences in the way each species develops as
unique and worthy of our attention. Although the reductionist approach (some
call it a delusion) will surely unravel many of the mysteries of the embryo, I
think it is important that molecular developmental biologists not lose sight of
the organism as a whole, and the broader biological questions that a molecular
approach can address. Anyone who has watched a sea urchin embryo develop
from egg to pluteus larva cannot help but marvel at the beauty of the
transformation and wonder how it happens.
8
Chapter 1
DNA-protein interactions at an octamer motif that is required for the
expression of the early histone H2B gene in sea urchin embryos
Introduction
The timing of expression of genes is crucial for normal development in
the early embryo. Genes must be activated or repressed at the right time in
order to execute the developmental program laid out for the organism. The
temporal regulation of gene expression has been studied for many years in a
number of systems. The "early" histone genes in the sea urchin embryo have
been the focus of study for nearly two decades. These genes are one of
several families of differentially regulated histone genes, and are present in
tandem arrays repeated several hundred times in the genome (Hentschel and
Bimsteil, 1981; Kedes, 1979; Maxson et al, 1983). They are expressed during
oogenesis and early embryogenesis only, showing a dramatic increase in
transcription during the cleavage-stage embryo when demand for histones is at
its peak (Maxson and Wilt, 1981, 1982; Weinberg et al., 1983). They are
sharply reduced in expression by the late blastula-stage stage, and are not
expressed again. Gene transfer methods have been used by several
investigators to determine the regulatory sequences responsible for the
temporal profile of histone gene expression (Colin, 1986; Lai et al., 1988;
McMahon et al., 1985; Vitelli et al., 1988). Earlier work in this laboratory
showed that 572 bp of 5' flanking sequence and 465 bp of 3' flanking sequence
is sufficient for the correct temporal profile of expression of the early H2B
9
gene (Colin et al., 1988). Work on the early H3 gene identified a number of
5' flanking DNA sequence elements required for its transcription (DiLiberto et
al., 1989).
Important cis-regulatory sequences governing the expression of the
early H2B gene were identified (Bell et al., 1992). This work provided the
foundation on which the in vitro DNA-protein interaction studies described in
this chapter were based. Dr. Jeffrey Bell carried out a deletion analysis of the
S. purpuratus 6.5 kb early histone repeat which bears the early H2B gene
(Cohn et al., 1976; Overton and Weinberg, 1978). The various deletion
constructs were injected into Lytechinus pictus zygotes and the amount of
early H2B mRNA at the blastula and gastrula stages measured by an RNase
protection assay (Colin et al., 1988; Zhao et al., 1990). This assay can
distinguish between the endogenous L. pictus transcripts, which protect
several fragments around 60 nt in length, and transcripts from the injected S.
purpuratus gene, which protect a 149 nt fragment (Colin et al., 1988). The
amounts of early H2B DNA injected was also measured by the slot blot
method (Flytzanis et al., 1987). In most cases, expression of injected early
H2B genes did not change very much as a function of the amount of DNA
injected. This is probably because the DNA constructs were injected at levels
that saturated the transcription machinery of the zygote. When this is the case,
the early H2B mRNA signal would indicate the transcriptional activity of
theinjected gene, independent of the amount of early H2B DNA.
When the entire histone repeat, pC02, was injected, the early H2B
gene was expressed at high levels in the blastula-stage embryo, and at low
levels in the gastrula-stage embryo. This pattern of expression resembles that
10
of the endogenous L. pictus early H2B gene. When the injected early H2B
gene had 3 kb of 5' flanking and 0.5 kb of 3' flanking sequence (pSpl02), a
similar pattern of expression was observed. Further deletions of 5' flanking
DNA to -2300, -1250, -500 and -78 did not significantly affect the pattern or
amounts of transcript generated from injected early H2B genes.
The proximal promoter of the early H2B gene contains three DNA
sequence elements within 80 bp of the TATA box that are conserved amongst
different species of sea urchin. These consist of a CCAAT element at -87, an
octamer site at -57, and the sequence CGCAGC at -47. The CCAAT element
is also present in the LI late H2B gene of S. purpuratus, although in the
reverse orientation. The CCAAT element in the early H2B gene does not
seem to be essential for expression of the gene since the construct having only
78 bp of 5' flanking sequence is expressed in the correct temporal manner.
To further define the role of the octamer and the sequence at -47, a
series of finer 5' deletion constructs was made. These deletions had 5'
breakpoints that ranged from -100 to -29. The transcriptional activity of these
constructs was assayed in L. pictus zygotes by microinjection. A construct
beginning at -87 showed high levels of expression at the blastula stage and
much lower levels in the gastrula embryo. This construct lacks the CCAAT
element, confirming the result with the A-78 construct. Further deletion to
-59 resulted in a dramatic decrease in the expression of the injected early H2B
gene. This construct lacks the octamer element, suggesting that this DNA
element is required for high level expression of the gene. An even further
deletion to -29 was also inactive.
11
These experiments focused our attention on the octamer element. We
made a deletion construct starting at -80, in which the octamer site was
mutated by base substitutions. The transcriptional activity of this mutant was
compared to a A-71 mutant, the 5' end of which is 5 bp upstream of the
octamer element. The RNAse protection assay revealed that the -71 construct
was expressed at high levels in the blastula-stage embryo, and at lower levels
in the gastrula embryo. The oct mut construct, however, was expressed at
very low levels in the blastula-stage embryo and virtually absent in the
gastrula-stage embryo. The decline in the level of expression in the gastrula
stage embryo suggests that even though the octamer element is required for
high level expression, other sequences are responsible for the inactivation of
the early H2B gene in gastrula-stage embryos. The next experiment addressed
the question whether the octamer element is required for early H2B expression
when 570 bp of 5' flanking sequence is present. An H2B gene with an internal
deletion of sequences -84 to -59 was constructed, having 572 bp of 5' flanking
sequence. This mutant H2B gene was transcribed at much lower levels than a
control gene bearing 500 bp of upstream sequence. The results indicated that
sequences between the octamer site and -500 are not sufficient to activate the
early H2B gene.
We then studied the DNA-protein interactions in the region of the
promoter that was responsible for the activation of the early H2B gene. I
found that the octamer motif strongly binds a sea urchin nuclear protein,
whose activity can be competed specifically with an excess of the octamer
binding site. Mutating the octamer binding site greatly reduced the affinity for
the octamer protein, supporting the micro injection data. I used methylation
12
protection footprinting to show that this factor protects a residue within the
octamer binding site of the early H2B promoter. Octamer-binding activity at
different stages of development was assayed, revealing that blastula-stage
embryos have a greater amount of specific binding activity than gastrula-stage
embryos. Human Oct-1, which recognizes the octamer m otif in the
mammalian H2B genes, avidly bound to the octamer site present in the sea
urchin early H2B promoter. Finally, a cloned sea urchin protein also bound to
the octamer element with a high degree of affinity and specificity.
13
Materials and Methods
Gel retardation and methylation interference footprint assays
Nuclear protein was extracted from S. purpuratus blastulae as
described by Calzone et al. (1988). A 58-bp Bam HI-Ddel (-99 to -41)
fragm ent containing the octam er-binding site was purified by gel
electrophoresis and used as a probe in gel retardation assays as well as in the
methylation interference footprint. When used in gel mobility shift assays, the
fragment was radiolabeled at its 3' ends by using T4 DNA polymerase and
32P-dATP. In Figures 1.1 A and 1.4 probes were generated by PCR. For wild-
type probe, a 5' amplimer spanning -71 to -55 (5-G G C TC A TT-
TGCATACGG-3') was used with the common 3' amplimer that spanned
+572-+591. In the case of the octamer mutant probe, the "oct mut" amplimer
(5'-CGAGACCGAGGAGACGGGTACGCAGGACCGCAGCATACGG-3')
was used in conjunction with the common 3' amplimer. The PCR fragments
were radiolabeled at their 5' termini by using polynucleotide kinase and 32p_
ATP, digested with BamHI, and the appropriate fragments purified by
polyacrylamide gel electrophoresis. To generate the late H2B promoter probe
used in Figure 1.8, a 5' amplimer spanning the first octamer site (5'-
GATCCTCATTTGCATACCGTG-31 ) was used in conjunction with the
reverse primer to PCR amplify the promoter and body of the late H2B gene.
The resulting product was labeled with 32P-ATP, digested with BamHI and
the octamer-bearing fragment gel-purified.
Gel retardation assays were performed essentially as described in Zhao
et al. (1990) and Fried and Crothers (1981) except that nuclear extracts were
14
preincubated for 10 min with 5 jLXg of sheared salmon sperm DNA, in addition
to the other components of the binding reaction, before the radiolabeled probe
was added. Competitions were performed as described in Zhao et al. (1990)
using the double-stranded oligonucleotides 5-GATCCTAAGACCAAT-
GAAAG-3' as the CCAAT competitor and 5'-GGCTCATTTGCATACGG-3'
as the Oct competitor. Methylation interference footprint assays were
performed as described in Hendrickson and Schlief (1985).
In vitro translation of human Oct-1 and SpOct
pBSoct-l+, a full-length human Oct-1 cDNA cloned into pBluescript,
was obtained from Dr. Winship Herr. As described in Sturm et al. (1988),
H indlll-linearized pB Soct-l + was transcribed in vitro with T3 RNA
polymerase and translated in a reticulocyte lysate system (Ambion).
Sea urchin SpOct protein was prepared by transcription/translation, in
vitro. A 3.5 kb cDNA bearing the entire SpOct protein-coding sequence was
introduced into pBluescript. T3 RNA polymerase was used to transcribe
linearized (Hindlll) pBluescript-SpOct, and the resultant SpOct mRNA was
capped and translated in vitro in a reticulocyte lysate following the
manufacturer's instructions (Ambion). DNA binding assays were carried out
as described in Bell et al. (1992).
15
Results
DNA-protein interactions at the octamer element in the early H2B
promoter
I studied nuclear protein-DNA interactions at the octamer element
(Bell et al., 1992). The octamer element has been well studied in mammalian
systems, and several proteins that bind to the octamer site have been
characterized. The octamer site in the early H2B gene is identical to the one
in the mammalian histone H2B promoter over the 8 bp core sequence. The
conservation of this binding site over the long evolutionary distance that
separates mammals and echinoderms indicated to us that a sea urchin octamer
protein would recognize this sequence. Calzone et al. (1988) first identified
an octamer-binding protein in nuclear extracts of sea urchin blastulae. We
surmised that if an octamer protein is responsible for the activation of the
early H2B gene, blastula-stage embryos would contain such an activity. I
used gel mobility shift and methylation interference assays (Fried and
Crothers, 1981; Hendrickson and Schlief, 1985)to study interactions between
embryonic nuclear proteins and the octamer element of the early H2B gene. I
incubated an extract of blastula stage nuclei with a 58 bp radiolabeled DNA
probe spanning nucleotides -99 to -41 of the early H2B promoter. The
mobility shift gels revealed a slow migrating complex that appeared to be
made up of several proteins (Fig. 1.1 A). That this complex was specific for
the octamer site was revealed by competition with homologous and non-
homologous oligonucleotides. A 17-bp oligonucleotide bearing the early H2B
octamer site greatly reduced the octamer binding activity, whereas a 20-bp
16
oligonucleotide bearing the early H2B CCAAT element did not do so. To
further confirm this observation, I tested the ability of a mutated octamer
element to bind nuclear proteins. The mutant binding site had a much lower
affinity for octamer-binding proteins, showing only a weak retarded complex.
Next, a methylation interference footprint showed that methylation of a G
residue at position -62 within the octamer element interfered with complex
formation (Fig. 1.1B). No other protein-DNA reaction was observed along
the rest of the 58 bp probe.
The temporal profile of sea urchin octamer-binding activity
To correlate the temporal profile of the early H2B gene to octamer
proteins in sea urchin embryos, I determined the temporal profile of octamer
binding activity during early development. Nuclear protein extracts were
made from embryos at different stages of development, and their octamer
binding acitvity was assayed by gel mobility shift. These experiments
revealed that binding activity was present from the early blastula stage
through the gastrula stage (Fig. 1.2). The retarded complexes observed in the
gel mobility shift assay appeared to be comprised of more than one protein. I
observed a fair degree of variation in protein extracts made at different times
from embryos at the same stage of development. This made it difficult to
interpret the correct temporal profile of octamer binding activity. However,
the general pattern of octamer binding activity during development was an
increase after fertilization up till the late blastula stage, followed by a
decrease in activity in late gastrula stage embryos. The observation that
multiple proteins bind to the octamer site suggested that a family of octamer
proteins may be present in sea urchins, or alternatively that several forms of
the same protein were present during development.
Oct-1 can specifically bind to the early H2B promoter
Since the DNA binding domain of octamer-binding proteins are well
conserved through evolution, I tested the ability of the human Oct-1 protein to
bind to the octamer element in early H2B gene promoter. A full-length Oct-1
cDNA (pBSoct-l+), a gift of Dr. Winship Herr, was transcribed with T3 RNA
polymerase, and translated in a reticulocyte lysate system (Sturm et al., 1988).
The Oct-1 protein bound avidly to the early H2B octamer element (Fig 1.3).
This binding activity was specifically competedby an oligonucleotide bearing
the early H2B octamer element, but not by a non-homologous competitor, a
fragment of the sea urchin Spec gene promoter. The in vitro translated Oct-1
protein did not bind to a probe bearing the 3’ enhancer of the LI late H2b
gene, which is recognized by a Antennapedia-class homeodomain protein
(Zhao et al., 1991). These results suggest that the octamer element in the early
H2B promoter is recognized by a protein belonging to the POU family of
transcription factors.
A cloned sea urchin protein related to human Oct-1 also binds to the
octamer element in the early H2B promoter
A cloned sea urchin protein designated SpOct, the characterization of
which will be described in detail in chapter 2, was also transcribed and
translated in vitro and tested for its ability to bind the early H2B octamer
element. As shown in Figure 1.4, reticulocyte lysate programmed with SpOct
18
mRNA contained a strong octamer-binding binding activity, while untreated
lysate did not. Furthermore, when a labeled probe bearing the mutated
octamer site (described in the Introduction) was used in the gel-shift assay, the
SpOct-programmed reticulocyte lysate bound poorly, indicating the presence
of an octamer-specific binding activity. These results indicate that SpOct can
specifically recognize the octamer element in the early H2B promoter.
Comparison of the ability of octamer elements of the early and late H2B
genes to bind nuclear proteins, human Oct-1 and SpOct
The LI late H2B proximal promoter contains two consensus octamer
elements as opposed to the single one in the early H2B promoter (Maxson et
al., 1987). The function of the two closely spaced octamer sites in the late
H2B promoter is a matter of speculation, but it is possible that two binding
sites would have more affinity for octamer proteins in late-stage embryos than
one, and because of this the late H2B gene would out-compete the early H2B
gene, resulting in the turning-off of the latter. To determine if there is a
qualitative difference in octamer binding ability of the early and late
promoters, I labeled PCR fragments of early and late promoters bearing the
octamer sites that lie 5' of the conserved BamHI site in the two promoters. I
used these probes in gel mobility shift experiments with sea urchin embyonic
nuclear extracts, human Oct-1 and the cloned sea urchin protein SpOct (Figure
1.5). The three different proteins bound to the two octamer sites with no
appreciable qualitative difference, except that Oct-1 bound slightly more
avidly to the early H2B octamer element than to the late H2B element. From
this result we can conclude that, in isolation, the octamer elements of the early
19
and late H2B genes probably have no intrinsic difference in binding
properties.
20
Discussion
The early H2B gene requires only 71 bp of 5' flanking sequence in
order to be regulated in the correct temporal manner during early
development. The 71 bp of sequence contains a canonical octamer binding
site and a sequence unique to the early H2B gene. Deletion of the octamer
site, or mutating it by base substitution resulted in a dramatic decrease in the
transcriptional activity of the early H2B gene. This is consistent with the
observation of Wu and Simpson (1985) that a 100 nt nuclease hypersensitivity
site is present in the early H2B promoter, with the octamer element at its
center. This site is present in chromatin from early blastulae, in which the
early H2B gene is active, but is not present in chromatin from late blastulae, in
which this gene is inactive.
Before the regulatory regions of the histone genes were sequenced, it
was thought that they might have a common mechanism for the activation of
these genes in the early blastula embryo and subsequent down-regulation in
late-blastula and gastrula embryos. However, sequence comparisons revealed
few similarities between, for example, the early H2B and early H3 genes.
This suggests that a coordinated regulatory mechanism probably does not
exist in the case of the early histone genes. One aspect of histone gene
regulation that has been revealed is that the entire histone repeat does not have
to be intact for the individual genes to be correctly regulated. In addition to
the evidence presented by Colin et al. (1988) and Bell et al. (1992) in the case
of the early H2B gene, DiLiberto et al. (1989) have shown that the early H3
gene requires only 97 bp of 5' flanking sequence to be faithfully regulated.
21
In vitro DNA-binding experiments showed that a sea urchin nuclear
factor bound with high affinity to the octamer element in the early H2B gene.
This binding activity was specific for the octamer element since competition
with an oligonucleotide bearing the octamer element competed for the
retarded complex efficiently, whereas an oligonucleotide bearing a CCAAT
binding site did not. The octamer element, ATTTGCAT, is involved in the
expression and regulation of a number of genes. In the introduction to chapter
2 , 1 will describe the role that the octamer element plays in the regulation of
various genes. The ubiquitous transcription factor, Oct-1, was the first protein
identified that recognized the octamer element. A cDNA clone encoding the
Oct-1 protein was used to generate the Oct-1 protein in a reticulocyte lysate
system. Oct-1 bound to the early H2B octamer site with the same degree of
specificity and affinity as the octamer-binding activity present in sea urchin
nuclear extracts as judgedby gel mobility shift assays. This suggested
strongly that a sea urchin nuclear protein related to Oct-1 would recognize the
octamer element in the early H2B gene. In the next chapter I will show that
this is indeed the case.
The temporal profile of octamer binding activity was first reported by
Calzone et al. (1988). They characterized the DNA-protein interactions
occurring on the Cyllla actin gene promoter. The Cyllla promoter, like the
early H2B promoter contains a canonical octamer element. They reported that
embryonic octamer-binding activity increased slightly from the early blastula
stage to the gastrula stage. Our experiments came to the general conclusion
that blastula-stage embryos have more octamer-binding activity as compared
to gastrula-stage embryos. This is consistent with the temporal profile of
22
expression of the early H2B gene, viz., high levels of transcription in the
blastula-stage embryo, followed by a sharp decrease in expression in late
blastula-stage embryos.
23
Figure 1.1 Binding of sea urchin nuclear protein to the octamer element.
(A) Autoradiogram of mobility shift gel. Sea urchin nuclear protein from
blastula-stage embryos was incubated with probes containing either a wild
type (wt) or mutant (mut.) octamer consensus element (Materials and
Methods). We incubated these fragments alone, with 2 |ig blastula-stage
nuclear extract but without competitor DNA or, in the case of the wild type
probe, with either a 50-fold molar excess of octamer-bearing (Oct) or a
CCAAT-bearing (Cat) competitor oligonucleotide. We electrophoresed these
mixtures on a non-denaturing 5% acrylamide gel, and visualized the free
probe and protein-DNA complexes by autoradiography. A broad band
representing octamer protein-DNA complexes is indicated by brackets. The
single band below the bracketed complex, present in the control without
nuclear extract, is an artifact of probe preparation. (B) M eth y latio n
interference footprint of octamer protein-DNA complex. A 58 bp DNA
fragment bearing the octamer element (-99 to -41; Methods) was end-labeled,
methylated with DMS and incubated with 10 pg of blastula-stage nuclear
protein (Methods). The resultant protein-DNA complexes were resolved by
electrophoresis on a non-denaturing polyacrylamide gel. Bands corresponding
to free probe ("Free") or to octamer protein-DNA complexes ("Complex")
were excised. The labeled DNA was eluted, cleaved with piperidine,
electrophoresed on a denaturing urea-8 % polyacrylamide gel, and visualized
by autoradiography. G and G+A reactions (Maxam and Gilbert, 1980),
performed on the labeled probe, are shown as markers. We show only the
sense strand footprint (indicated by the arrow); no footprint was evident on the
opposite strand. The position of the octamer element is indicated to the left.
24
A
Probe wt mut
Extract - + + + +
Competitor - - oct cat
Complex
25
Figure 1.1 (continued)
26
Figure 1.2 Temporal profile of sea urchin octamer-binding activity.
Gel m obility shift assay of octam er-binding activity in different
developmental stages of the sea urchin embryo. The octamer probe used in
this experiment was the 58 bp early H2B promoter fragment used in Fig.
1.1 A. 3 jig of nuclear extracts from unfertilized eggs, blastula-stage embryos
and gastrula-stage embryos were loaded in the lanes indicated. The arrow
indicates the major retarded complex.
27
to
oo
free probe
probe only
egg
blastula
gastrula
Figure 1.3 Oct-1 can bind to the early H2B gene octamer element.
Gel mobility shift assay testing the ability of in vitro translated human Oct-1
to bind to probes containing the early H2B octamer element and the LI late
H2B enhancer binding site. The octamer probe was the 58 bp early H2B
promoter fragment used in Fig. 1.1 A, and the LI late H2B enhancer probe is
described in Zhao et al. (1990). Nuc ext., 3 fig blastula nuclear extract; -Oct-
1, 2 jil unprogrammed reticulocyte lysate; +Oct-l, 2 fil reticulocyte lysate
programmed with pBSoct-1 mRNA. Molar excesses of competitor added to
the +Oct-l lysate are indicated: octa, octamer oligonucleotide (Materials and
Methods); Spec, a promoter fragment of the sea urchin Spec gene.
29
;^robe only
free probe
early H2B octamer late H2B enhancer
30
^
Figure 1.4 A cloned sea urchin nuclear protein binds to the early H2B
octamer element.
Autoradiogram of mobility shift gel showing binding of a cloned sea urchin
POU protein, SpOct, to wild type or mutant octamer DNA probes. The probes
were the same as in Figure 1.1 A. SpOct was prepared by in vitro transcription
and translation as described in the Materials and Methods. Two jil of the
lysate was incubated with wild type or mutant octamer probes (+mRNA) in
the same manner as in Figure 1.1 A.
31
Probe wt mut
mRNA + - +
Complex [
32
Figure 1.5 Comparison of the binding ability of octamer elements in the
early and late H2B promoters.
Gel mobility shift assay of sea urchin blastula nuclear extracts, human Oct-1
and SpOct using octamer elements from the early and late H2B promoters.
For probe construction, refer to Materials and Methods. E, early H2B probe;
L, late H2B probe; -mRNA, 2 jil of unprogrammed reticulocyte lysate;
+SpOct, 2 |il of SpOct-programmed reticulocyte lysate; +O ct-l, 2 |il of
human O ct-1-programmed lysate; Nuc ext, 3 \i\ blastula nuclear extract.
Specific complexes are indicated by arrows.
33
| ft
S T
o
C 7
(D
oo
4 ^
probe only
-mRNA
+SpOct
+Oct-1
nuc ext
Chapter 2
Cloning and characterization of SpOct, a gene encoding the major
octamer-binding protein of the sea urchin embryo
Introduction
The octamer motif, ATGCAAAT, is a cis-regulatory element that has
been implicated in a variety of transcriptional regulatory phenomena. Among
these are the cell-cycle dependent regulation of mammalian histone H2B
genes (La Bella et al., 1988), the lymphoid-specific expression of light and
heavy chain immunoglobulin genes (Sen and Baltimore, 1986) and the
activation of certain viral promoters (e.g. Mackem and Roizman, 1982). A
number of different proteins that interact with the canonical octamer site have
been identified. These include Oct-1 (Sturm et al., 1987), Oct-2 (Gerster et al.y
1990; LeBowitz et a l, 1988; Scheidereit, et a l, 1987; Staudt et al., 1986) and
Oct-3 (Okamoto et al., 1990; Rosner et al., 1990) and possibly rat Brn-1 and
Brn-2 (Scholer, 1991). All are members of the POU protein family,
characterized by a highly conserved DNA binding motif consisting of a 60-62
amino acid homeodomain and a 69-71 amino acid sequence known as the
POU-specific domain (Herr et al., 1988).
Members of the POU family have been grouped in five subfamilies on
the basis of the amino acid sequence of the POU-specific domain and POU
homeodomain (reviewed in Rosenfeld, 1991). The POU I subfamily includes
Pit-1 (GHF-1), a transcription factor that regulates the rat prolactin and growth
hormone (GH) genes (Bodner et al., 1988). The POU II class includes the Oct
35
family of transcription factors that bind the octamer element. Oct-1 and Oct-2,
both present in mammals, have very similar POU domains but differ
substantially elsewhere. Ubiquitously expressed, Oct-1 is involved in the cell
cycle regulation of histone gene expression in mammalian cells, and may also
have a role in DNA replication (O'Neill, et al., 1988; Verrijzer et al., 1990).
Oct-2 stimulates the transcription of lymphoid-specific genes (Clerc et al.,
1988). Recently, two POU II genes from Drosophila, pdm-1 and pdm-2, were
characterized (Billin et al., 1991; Dick et al., 1991; Lloyd and Sakonju, 1991).
The POU III class, the most conserved of the POU factors, comprises Brn-1,
Brn-2 (both expressed in the developing nervous system) and Tst-1 expressed
in the testis and brain (He et al., 1989). Other members of this class are cfla
from Drosophila (Johnson and Hirsh, 1990) and ceh-6 from C. elegans (also
neuronal in expression). POU IV includes unc-86, responsible for the
determination of cell lineage in C. elegans (Finney et al., 1988; Ruvkun and
Finney, 1991), and rat Brn-3. The POU V class was created to accommodate
Oct-3 (Rosenfeld, 1991).
A canonical octamer element is present in the regulatory regions of
several developmental-stage-specific expressed sea urchin genes including the
temporally regulated alpha-histone H2B (Bell et al., 1992), the LI late histone
H2B gene (Maxson et al., 1987), the testes-specific histone H2B (Barberis et
al., 1989), and lineage-specific Cyl and Cyllla genes (Thiebaud et al., 1990;
Theze et al., 1990). The alpha histone genes are expressed exclusively during
oogenesis and early embryogenesis, reaching a sharp peak of transcription in
the early blastula-stage embryo when cleavage is rapid and the requirement
for histones reaches its zenith (Maxson and Wilt, 1981; Maxson and Wilt,
36
1982; Weinberg et al., 1983). The C yllla actin gene is transcriptionally
activated at the 64-128 cell stage (Lee et al., 1992). It is expressed exclusively
in cell lineages that give rise to the aboral ectoderm of the embryo and later
on, the larva (Angerer and Davidson, 1984; Cox et al., 1986). As described in
Chapter 1, deletion mutations that remove the octamer element, as well as
base substitution mutations that alter its sequence, greatly reduce expression
of a microinjected early H2B transgene in blastula-stage embryos,
demonstrating that the octamer is essential for the high-level expression of the
alpha histone genes in sea urchin blastulae.
Since the octamer element is important for the expression of the early
H2B and Cyllla actin genes, and since POU II proteins bind octamer elements
in general, we surmised that the expression, and perhaps the regulation, of
these and other sea urchin genes depends on a POU protein. Consistent with
this view, Calzone et al. (1988) showed that octamer binding proteins are
present in sea urchin blastulae, and Bell et al., (1992) demonstrated that both
human Oct-1 and a cloned sea urchin POU protein bind the early H2B
octamer element, in vitro . As a further test of the hypothesis that a POU
protein is involved in early H2B gene expression, we sought to identify a sea
urchin POU gene (or genes) expressed in early embryonic development. In
parallel with this approach, we collaborated with other investigators to purify
and characterize octamer binding proteins from sea urchin embryos by DNA
affinity chromatography.
In this chapter, I report the nucleotide sequence, expression profile,
and phylogenetic relationships of a cDNA encoding a protein designated as
SpOct, a member of the POU II class. In collaboration with Dr. F. Calzone's
37
laboratory at the University of California at Irvine, the DNA binding
characteristics of SpOct were studied. Peptide sequences from proteolytic
fragments of the purified octamer binding proteins closely matched the
deduced amino acid sequence of SpOct were obtained in collaboration with
Drs. J. Coffman and M. Harrington at the California Institute of Technology.
I show that SpOct mRNA is present at a low level in the unfertilized egg,
increases several fold in amount by the early blastula stage, then declines.
This profile is indistinguishable from the pattern of early H2B gene
transcription; thus the cloned sea urchin POU II gene may indeed participate
in the expression or regulation of the early H2B gene.
38
Materials and Methods
POU homeodomain amplification
Degenerate primers were synthesized to two domains conserved
between human Oct-1 and Oct-2. The 5' primer consisted of a 30-mer
spanning residues 335-344. The 3' primer was a degenerate 36-mer spanning
residues 426-436 (Figure 1). Polymerase chain reaction (PCR) was perfomed
using S. purpuratus genomic DNA (100 ng), 25 pmols of each degenerate
primer, each dNTP at 2.5 mM, 5 units of Taq polymerase (Perkin-Elmer
Cetus) in IX Taq polymerase buffer, in 100 pi reactions. The reaction
mixtures were overlaid with mineral oil, and 30 cycles of amplification (94
°C, 1 min; 50 °C, 1 min; 72 °C, 1 min) were carried out. The sequences of the
degenerate oligonucleotides are as follows:
5' primer: 5'-AAGCTTYAARAAYATGTGYAANTNAARCC-3*
3' primer: 5 ’ - A AGCTT YTT YTC YTT YTGNC KRTTRC AR A ACC A- 3'
Cloning and sequencing of amplification products and larger cDNAs
The 300 bp PCR product was cloned into the Smal site of double
stranded M13mp9 and sequenced using Sequenase (United States
Biochemicals). The 2 classes of inserts which showed significant sequence
similarity to POU domains were reamplified by PCR from individual clones
and labeled with 32p by random-priming. A 4 hr 1ZAP embryonic S.
purpuratus cDNA library was screened with both probes to obtain larger
cDNAs. Twenty-eight positives were obtained, which were plaque-purified.
pBluescript (Stratagene) containing the inserts was then excised from the
39
phage DNA as per the protocol provided by the company. The longest insert,
18-8 (3499 bp), was treated with exonuclease III (Boehringer Mannheim) to
create nested deletions (Ausubel et al., 1989) which were sequenced on both
strands by the dideoxy termination method. Sequence assembly and alignment
was done with the Intelligenetics software package.
Purification of octamer-binding proteins by affinity chromatography
Coffman et al., (1992) showed previously that octamer-binding
proteins obtained after a single cycle of affinity chromatography were purified
approximately 80-fold relative to total proteins in the nuclear extract which by
themselves represent only about 0.25% of total embryo proteins. We
constructed a DNA affinity column using a double stranded oligonucleotide
bearing a consensus octamer motif as described in Calzone et al., (1991) for
the P3A2 protein. The sequence used for the colum n was
GATCATCTCATTTGCATATCCT. Proteins that bound to the column were
eluted in steps of 0.1 M KC1, and octamer-binding activity was assayed by the
southwestern blot procedure. The major fraction of octamer binding activity
eluted from the site-specific column at 0.6M KC1, although several distinct
minor proteins are detected in other fractions. The Oct proteins detected by
this assay ranged from about 30-70 kD in size. A gel shift assay also showed
the major octamer-binding activity in the 0.6M KC1 fraction. When the 0.3M
and 0.6M fractions were used in a DNAse I footprinting assay, only the latter
showed protection over an octamer element, demonstrating that the major
octamer-binding activity was in the higher salt fraction. The probe for the
footprint assay, a DNA fragment spanning nucleotides -243 to -114 of the
40
CylllA actin promoter, contained a single consensus octamer-binding site
(Calzone et al., 1991).
Partial amino acid sequence determination of affinity purified octamer-
binding proteins
Affinity column fractions containing octamer-binding activity from
approximately 1 x 10*0 24-hour embryos were pooled, concentrated by
precipitation with 15% trichloroacetic acid, and further purified by SDS-
PAGE, using an 8% polyacrylamide resolving gel (Laemmli, 1970). The gel
was stained with coomassie blue and individual bands excised and
electroeluted in a Centriluor (Amicon) essentially as described (LeGrande and
Matsudaira, 1989). Aproximately 5-10 mg of electroeluted protein was
digested with Achromobacter Protease I (Wako), which cleaves on the
carboxyl side of lysine residues. The resulting fragments were separated by
reverse phase HPLC, using a 35x2.1 cm DEAE precolumn (ABI) to extract
the SDS (Kawazaki and Suzuki, 1990), and a either a 150x2.0 mm C8 column
(Beckman) or a 250x2.1 mm cl8 column (Vydac) to resolve the peptides.
Peptide peaks detected at 215 nm were sequenced using either a Porton
LF3000 or an ABI 473 automated amino acid sequencing system.
Preparation of SpOct proteins
A T7 RNA polymerase based expression system in E. coli developed
by Studier and Moffat (1986) was used to prepare recombinant SpOct
proteins. Three different expression constructs were prepared in pRSET
vectors (Invitrogen). The host used for expression was BL21 containing plys
41
S. The methods used to prepare and measure the amount and activity of
recombinant protein will be described elsewhere (C. Wilson and F. J. Calzone,
in preparation). The POU domain construct was prepared in three steps. The
Sphl-Clal fragment (1107-1870) of pSpOct (X8-8 inserted into the Eco RI site
of pBluescript) was first cloned into pSp72 (Promega) and released with
PvuII and Clal. This insert was then cloned into the Smal-Clal site of an
expression construct called pNL2 provided by A. Cameron (Caltech). The
pNL2 constuct contains sequences for eukaryotic expression and nuclear
localization will be described elsewhere. The POU domain was transfered into
the pRSET vector using SacI and Kpnl. The resulting construct contained
about 20 amino acid residues at the N-terminus derived from the NL2 vector.
The SpOct(A)C construct contained an EcoRI-Clal fragment (1-1870) of
pSpOct. The fragment was first cloned into pNL2 digested with EcoRI and
Clal. In this cloning step the EcoRI overhangs were made flush by the end-fill
reaction using Klenow fragment E. coli. DNA polymerase (New England
Biolabs). A BamHI adapter was inserted at the Clal site. The sequence at this
junction is (ATCGgatcc). The SpOct(A)C Sac 1-BamHI insert in the pNL2
construct was then transferred to the appropriate pRSET vector using SacI and
Bglll. The pSpOct WT contruct which expresses the full length SpOct protein
was derived from SpOct(A)C. The pSpOct(A)C construct was digested with
SphI and H indlll to remove the POU-homeodomain. The fragment was
replaced with the Sphl-Hindlll fragment of pSpOct containing the POU
domain and 3'trailer. The Hind III sites in fragments were derived from
vector. To increase the level of protein expression the 3' trailer beyond
position 2387 was removed by digestion with AafU and Hind III.
42
DNA binding assays
Gel retardation assays and DNase 1 footprints were carried out
essentially as described by Calzone et al. (1991). The oligonucelotide probe
used for the gel retardation assays was GATCAGCACCGAATCTC-
ATTTGCATATCCTTTTCAAGACT. The probe for DNAse I footprint
experiments was a Hind III-Dde I fragment of the S. purpuratus Cyllla actin
gene cis-regulatory region (-114 to -234). The sequence contains a single Oct
(designated P3B/OTF in Calzone et al., 1991) binding site is identical at 13/14
positions to the consensus Oct-1 binding site in H2B genes of higher
eukayotes. For measurement of equilibrium parameters gel-shift binding
assays contained 10 mM HEPES (pH 7.9), 0.5 mM DTT, 1.75 ug poly(dAdT;
Pharmacia), 55 mM KC1), 0.013% xylene cyanol, 0.013% bromphenol blue,
1.25% ficol (400), 0.66 ng of probe and 4 ul of bacterial proteins in buffer C
(Buffer C is 20 mM HEPES (pH 7.9), 40 mM KC1, 0.1 mM EDTA, 20%
glycerol, 0.1% NP-40). The reactions were incubated for 15 min on ice and
separated on 10% acrylamide gels in Tris-borate buffer.
RNA and DNA extraction
RNA was extracted from embryos and adult tissues by the
guanidinium isothiocyanate (GITC) method (Ausubel et al., 1989). Briefly, 5
ml packed volume of embryos was pelleted and washed in Ca++-Mg++ free
sea water. The embryos were then lysed in 10X volumes of 4M GITC. The
lysate was centrifuged through a 5.7 M CsCl pad for 20 hr at 24,000 rpm in a
SW28 rotor to isolate RNA. The pelleted RNA was extracted with phenol and
43
chloroform and ethanol precipitated. Genomic DNA was extracted from
individual S. purpuratus sperm samples by lysis at 50 °C overnight in 100
mM Tris (pH 8.0), 10 mM EDTA, 10 mM NaCl, 0.5% Sarkosyl, 10 mM 2-
mercaptoethanol containing 100 Jig/ml proteinase K, phenol-chloroform
extracted and dialyzed against 50 mM NaCl in TE.
RNA and DNA gel blots
RNA and DNA gel blots were performed essentially as described in
Maniatis et al.(1982). For the embryonic RNA time-course, the probe used
was the entire SpOct antisense RNA transcribed with T7 RNA polymerase. In
the case of the adult tissue RNA blot and the Southern blot, the probe was a
1.1 kb antisense transcript obtained by truncating the SpOct cDNA with Aat II
and transcribing with T7 RNA polymerase (Figure 2.5A). Autoradiograms
were exposed for 1 to 4 days. For the control RNA blots probed for histone
messages (Figure 2.6A), antisense RNA probes were generated with SP6
RNA polymerase (Bell et al., 1992; Zhao et al., 1990).
RNAse protection assay
The probe used was generated by digesting pNL2 containing the POU
domain (see above) with Eco RV and then using T7 RNA polymerase to
transcribe a 248 bp antisense message labeled with 32p_uTP. 10 jig of whole
cell RNA from embryos at various stages of development was hybridized
overnight to 3 x 10^ cpm of probe at 45 °C. The RNase protection assay was
performed using the RPA II kit (Ambion). The protected bands were resolved
44
on a 8% denaturing polyacrylamide gel. Gels were dried and exposed to
Kodak XAR-5 X-ray film (Eastman Kodak) with enhancing screens.
45
Results
Isolation of sea urchin POU genes
We designed degenerate oligonucleotides that would hybridize with
DNA encoding Oct-1 or Oct-2 but not Pit-1 or Unc-86, thus providing a
means of amplifying by PCR an approximately 300 bp DNA segment bearing
the majority of the POU homeodomain of Oct-1 or Oct-2- related proteins
(Figure 2.1). I used these oligonucleotides in a PCR reaction with sea urchin
genomic DNA, obtaining a band of the predicted size. This PCR product was
cloned into M l3, 30 clones were isolated and their inserts sequenced. These
sequences fell into three categories: 9 were of a sea urchin Oct gene
(designated SpOct), 10 were of a sea urchin POU Ill-class gene (SpPOUIII-1,
Figure 2.1); and 11 were novel sequences unrelated to POU genes (data not
shown). I was unable to detect transcripts of SpPOUIII-1 at any stage of
embryonic development. In preliminary experiments, we detected SpOct
transcripts in early embryos; hence we proceeded to examine the structure and
expression of the SpOct gene.
Using the PCR-amplified POU Il-class gene as a probe, I screened a 1
ZAP cDNA library prepared from cleavage-stage embryos (4 hr post
fertilization) derived from several sea urchin individuals. Of 200,000 plaques
screened, 28 hybridized with the POU Il-class probe. Nucleotide sequence
analysis showed that all had the same POU homeodomain sequence.
Restriction enzyme mapping of DNA flanking the POU-homeodomain
divided these clones into three major classes. Nucleotide sequencing of
several representatives of each class showed that sequence heterogeneities
46
(third base changes) were responsible for the differences in restriction maps
among the three categories. Given that the cDNA library was made from
several S. purpuratus individuals, and that there is a high level of sequence
polymorphism in the S. purpuratus population (Britten et al., 1978), it is not
surprising that multiple forms of the sea urchin Oct cDNAs are present in the
library.
I selected the largest clones of the majority class for complete
sequencing, and determined the nucleotide sequence of the 3499 bp insert of
clone 18-8. Sequencing of the 5' ends of another SpOct clone (17-2) gave an
additional 70 bp. The longest open reading frame starting with a methionine
encodes a protein of 729 amino acids (Figure 2.2A) whose predicted
molecular weight is 78 kD, similar vertebrate POU II class proteins. These
two SpOct cDNAs thus probably contain all or most of the SpOct protein-
coding region. The 3.6 kb of cDNA sequence is, however, substantially
shorter than the 8-12 kb mRNAs detected on RNA gel blots (Figures 2.6 and
2.7). This disparity in length is probably due to a very long 5' untranslated
sequence in the SpOct mRNAs. The presence of long untranslated sequences
is known to be a feature of some POU II mRNAs, the largest Oct-2 mRNA
species being 7.5-8.0 kb (Scheidereit, et al., 1988; Cockerill and Klinken,
1990).
To determine how the SpOct gene relates to other members of the
POU II family, we aligned the amino acid sequences of the POU domains of
POU II family members from human (Oct-1 and Oct-2), chicken (chicken
Oct-1) , Xenopus (X10ct-1) and Drosophila (pdm-1 and pdm-2) (Figure
2.2B). We found, interestingly, that SpOct has a slightly higher degree of
47
sequence similarity to the Drosophila POU II members within the POU
domain. For example, in the POU-specific domain at position 4, SpOct
shares a Thr with pdm-1 and pdm-2 while the vertebrate genes have a Ser, at
position 68 SpOct and pdm-l/pdm-2 have Gin while the vertebrates have Glu,
and at position 72 SpOct and pdm -l/pdm -2 have Asp or Glu while the
vertebrate genes have Asn (Figure 2.2B, in bold type). Similarly, in the
homeodomain at position 35, SpOct and pdml/pdm-2 share Leu while the
vertebrate POU II members have lie.
A comparison of the amino acid sequences outside the POU
homeodomain revealed that SpOct, like other members of the POU II family,
has a glutamine-rich domain in the N-terminal half, and a serine-threonine rich
domain in the carboxy end. The glutamine-rich domain of SpOct has an
unusually high percentage of glutamines (50%) compared to similar proteins
such as Oct-1 (26%) (Sturm et al., 1988). The motif, AQDLQQLQQLQQQN
(residues 295-308), located in the glutamine-rich domain, is shared by the
vertebrate POU II members, but is absent in pdm-1 and pdm-2 from
Drosophila.
Comparison of the DNA-binding properties of SpOct with that o f affinity
purified octamer-binding proteins
In parallel with the PCR cloning strategy to obtain cDNAs encoding
POU proteins, Dr. J. Coffman purified octamer-binding proteins from sea
urchin blastulae essentially as described in Calzone et al., (1991). We wished
to know whether embryos have octamer-binding activities other than SpOct.
In order to do this, we (i) tested the DNAse I footprinting characteristics of
48
bacterially-expressed SpOct versus that of affinity-purified Oct proteins to
determine whether embryonic nuclear extracts contain octamer proteins that
show different protection patterns from SpOct, (ii) quantitated the binding
affinity of bacterially-expressed SpOct in order to compare it to previously
measured affinity constants for crude nuclear octamer-binding activity in
blastula embryos (Calzone et al., 1988), and (iii) obtained peptide sequences
from proteolytic fragments of affinity-purified octamer proteins.
A T7 RNA polymerase expression system was used to produce full
length SpOct protein in E. coli by Dr. F. Calzone's laboratory (Studier and
Moffat, 1986). A DNase I footprint assay was then used to compare the DNA
binding specificity of recombinant SpOct protein with octamer-binding
proteins purified from blastula-stage nuclei using oligonucleotide affinity
chromatography. Recombinant SpOct and affinity-purified octamer binding
activity (0.6M KC1 fraction) gave essentially identical footprints on the
respective strands of the probe (Figure 2.3). The 0.6M KC1 fraction showed a
number of octamer-binding species when tested in gel retardation and
southwestern assays. The significance of these multiple bands is not clear, and
will be discussed later. It can thus be concluded that the octamer-binding
activity in the 0.6M KC1 fraction has the same DNA recognition domain as
the bacterially-expressed SpOct protein.
We then measured the Kr of bacterially-expressed SpOct, a measure of
the preference of the SpOct protein for binding to a 41 bp octamer element-
bearing probe versus synthetic poly(dAT) that is present in excess in each
binding reaction (Figure 2.4). Kr is defined as the ratio of the equilibrium
constant for the specific SpOct binding to the constant for the interaction of
49
SpOct protein with nonspecific DNA (i.e. Kr = Ks/K n). The method of
measurement for this parameter has been described previously (Calzone et al.,
1988), and is based on the treatment of Emerson et al. (1985) given the
condition that at the concentrations of specific probe and nonspecific DNA
used, essentially no free SpOct protein is present in the system. The Kr value
obtained for the full-length SpOct protein was 1.4 x 10^, not significantly
different than the Kr value obtained under similar conditions for the Oct (P3B)
complex in nuclear extracts (Calzone et al., 1988) (Figure 2.4A). A similar
value for Kr was obtained for the POU domain alone (residues 364-619)
(Figure 2.4B).
At higher probe concentrations, complexes suggestive of SpOct dimers
and higher multimers were observed with the bacterial protein (data not
shown). The probe contains a single octamer site (see Materials and
Methods). Similar complexes were observed in gel shift assays with SpOct
protein from which the serine-proline rich C-terminus was removed. This
suggests that Oct protein interactions are promoted by the glutamine-rich N-
terminus, since such higher order complexes were not detected with the POU-
domain alone at similar protein concentrations.
Sequence comparison of SpOct with that of affinity purified octamer
binding proteins
Drs. J. Coffman and M. Harrington obtained amino acid sequences of
proteolytic fragments of two different protein species purified by SDS-PAGE
from the eluate of the octamer affinity column. A proteolytic fragment from
an approximately 80 kD protein (the largest octamer-binding species in the
50
eluate) gave the sequence QGGGVTXVAAQQ/P, which matches amino acids
422-433 in the SpOct cDNA (Figure 2.2A). Another fragment of the same
protein gave the sequence ly/FLMQmK, which matches amino acids 555-561
encoded in the SpOct cDNA (with two mismatches, indicated in lower case;
see discussion). Both of these sequences are immediately C-terminal to a
lysine residue encoded in the SpOct cDNA, as would be expected of
fragments produced by digestion with LysC. A fragment from a smaller
protein species (approximately 70 kD) found in the same octamer affinity
column eluate gave the sequence QGGGVTSAAQPIPDI1/PH1, which
overlaps with the longer of the two sequences obtained from the 80 kD
protein, and matches amino acids 422-440 in the SpOct cDNA (with 2
mismatches, indicated in lower case). The SpOct cDNA encodes a protein of
78 kD, close in size to the largest affinity-purified protein. It is significant that
the same amino acid sequence (corresponding to SpOct residues 422-433),
which lies outside of both the POU-specific and POU-homeodomain, was
obtained from both the largest and a smaller protein species in the affinity
column eluate. This, together with the fact that SpOct is a single copy gene
(see below), suggests that the multiple octamer binding proteins detected on
southwestern blots of octamer affinity column fractions is either due to
alternate splicing of the SpOct transcript or proteolysis of the 78 kd species.
Genomic organization and developmental expression of SpOct
To determine the number of SpOct genes in the sea urchin genome, I
hybridized a radiolabeled SpOct probe (Figure 2.5A) with a gel blot
containing restriction-enzyme digested genomic DNA from several S.
51
purpuratus individuals. An autoradiogram of this blot (Figure 2.5B) shows,
as expected for a single copy gene, a single band when DNA from individual
2 was digested with H indlll or EcoRI (5 kb and 6.5 kb fragments
respectively). Individual 3 gave a 5.2 kb fragment with H indlll and 2
fragments of 9.4 kb and 7 kb with EcoRI. Individual 1 gave a result similar to
individual 3 with EcoRI. The latter two results probably reflect the presence
of different alleles of SpOct in individuals 1 and 3. I conclude that the SpOct
gene is present in the haploid sea urchin genome in a single copy.
We showed previously that the octamer element is essential for
expression of the early H2B gene in early development, and that the SpOct
protein binds the octamer element of the early H2B promoter (Bell et al.,
1992). If the SpOct protein has a role in the expression or regulation of early
H2B histone genes, then its concentration, and that of its cognate mRNA,
should be highest in early embryos, paralleling early H2B gene activity. To
test this proposition, I performed an RNA gel blot analysis. Using the entire
SpOct cDNA as a probe under stringent conditions of hybridization, we
detected several RNA species ranging in size from 4 kb to 12 kb (Figure
2.6A). The amounts of these mRNA species changed coordinately during
development. All were low in the egg, increased substantially during cleavage
and blastula formation and declined thereafter. Hybridization of the same
RNA developmental series to a probe for the early H2B mRNA revealed a
similar temporal pattern of accumulation and decay. As a further test of the
validity of the RNA developmental series, this blot was rehybridized with a
probe for the LI late H2B mRNA. The observed profile of this transcript
52
through early development was in agreement with previous studies (Ito et al.,
1988).
To confirm the rather dramatic increase and decline in SpOct mRNA
levels, I repeated the RNA gel blot experiment with a different RNA
developmental series and the 3' UTR probe used for the genomic DNA blot
and obtained virtually identical results (data not shown). Moreover, an
RNAse protection analysis also corroborated the SpOct developmental profile
(Figure 2.6B). An antisense probe from the POU domain (Figure 5A) was
hybridized to RNA samples of the same developmental series used in Figure
6A. As expected from its nucleotide sequence, the POU domain probe
protected a 203 nt segment of SpOct mRNA (Figure 2.6B). This protected
product was present in the unfertilized egg, increased 28-fold by the 8 hr hour
blastula stage, and declined approximately 6-fold by the 12 hr blastula stage
(graphically represented in Figure 2.6C). We conclude that SpOct mRNA
levels peak in early blastula-stage embryos, coincident with the maximum rate
of early H2B gene transcription and the maximum rate of DNA synthesis.
Oct-1 and Oct-2, the mammalian POU II genes most closely related to
SpOct, have very different spatial domains of expression. Oct-1 is expressed
ubiquitously, whereas Oct-2 is expressed predominantly in lymphoid cells
(Clerc et al., 1988). To determine which of these expression patterns is more
similar to that of SpOct-and thus which pattern may be ancestral— I examined
the expression of the SpOct gene in several tissues of the adult sea urchin
(Figure 2.7). In tube foot, intestine, coelomocyte and gonad, four forms of
message are evident, migrating at 4 kb, 8 kb, 10 kb and 12 kb. The ratios of
these four forms vary somewhat among tissues, but the significance of this is
53
not known. Since the probe is specific for the SpOct gene (see Southern blot,
Figure 2.5B), these multiple mRNA species are likely alternatively-processed
transcripts of the same gene. Identical transcripts are seen in 12 hr blastula-
stage embryos, with the 8 kb form predominant (Figure 2.7, extreme right
lane). The expression pattern of the SpOct gene thus resembles that of Oct-1
more closely than Oct-2.
54
Discussion
In this chapter, I have described the isolation of cDNAs encoding a sea
urchin POU protein designated SpOct, and I provide evidence that SpOct is at
least closely related to the major octamer binding protein purified from sea
urchin blastula nuclear extracts by DNA affinity chromatography.
Comparisons of the predicted SpOct amino acid sequence show that the SpOct
gene is a member of the POU II class, which includes the Oct-1 and Oct-2
genes of mammals and the pdm-1 and pdm-2 genes of Drosophila. The
longest open reading frame of the SpOct cDNA (Figure 2.2A) encodes a
protein of 78 kD, similar in size to the Oct-1 proteins of vertebrates. SpOct
encodes four size classes of transcripts ranging from 8 to 12 kb, all of which
are present in embryos and in the adult. Since SpOct is a single copy gene,
and since we have been unable to identify additional POU II class genes, we
believe that these multiple transcripts are probably the result of alternative
initiation, splicing or polyadenylation of SpOct transcripts. RNA gel blot and
RNAse protection assays show that the four size classes of SpOct transcripts
are present at low levels in the egg, increase coordinately several-fold in
rapidly cleaving early blastulae, and subsequently decline through the
remainder of embryonic development. All four forms of SpOct mRNA are
found in tubefoot, intestine, coelomocyte and gonad tissues of the adult,
though in contrast to their constant ratios in embryos, their relative amounts
vary. We do not yet know whether SpOct transcripts are localized to specific
regions or cell types in embryos as are the pdm-1 and pdm-2 transcripts of
Drosophila (Lloyd and Sakonju, 1991).
55
In parallel with our efforts to clone sea urchin Oct genes, we sought to
identify and characterize octamer binding proteins in sea urchin embryos.
Using sequence-specific DNA affinity chromatography, we purified octamer
binding proteins from a blastula nuclear extract (Coffman et al., 1992). The
0.5-0.6M KC1 eluate of the affinity column contained a DNA binding activity
that footprinted the octamer element in a manner identical to bacterially-
expressed SpOct. Moreover, a comparison of the binding affinity of SpOct for
its target sequence with that of the octamer binding activity in blastula nuclear
extracts showed them to be identical within experimental error. Consistent
with this view, southwestern blots revealed that the affinity column eluate
contained an approximately 80 kd protein, and amino acid sequences from
two fragments of this protein matched amino acids 422-433 and 555-561 (with
two mismatches in the latter sequence). However, both southwestern blots and
two-dimensional gel mobility shift assays (Coffman et al., 1992) reveal
several additional octamer-binding protein species of molecular weights
ranging from 30 to 70 kd, indicating that the octamer binding activity might
consist of a family of proteins. While we have not examined all of these lower
molecular weight octamer binding proteins closely, we have obtained limited
amino acid sequence from one of them. This sequence matched SpOct
residues 422-440 (with two mismatches), and overlapped with the longer of
the two sequences obtained from the 80 kD protein. The 4 mismatches
between SpOct and the amino acid sequences obtained directly from affinity
purified proteins generally occur towards the C-terminus of the peptides,
wherein amino acid sequence data becomes more difficult to obtain. In
addition, 3/4 of the mismatches correspond to serine residues, which often
56
present difficulties in sequencing (Tarr, 1977); therefore, the mismatches are
probably due to amino acid sequencing error. Given that SpOct is a single
copy gene, we believe that the diversity of octamer binding proteins found in
the affinity column eluate is due either to alternate processing of the SpOct
primary transcript, giving rise to a family of proteins that share some domains
but perhaps not all, or alternatively, to proteolysis of the 78 kd SpOct protein.
We showed previously that the sea urchin early H2B gene requires an
octamer element for expression at high levels in blastula-stage embryos (Bell
et al., 1992). Several lines of evidence suggest that the SpOct protein is the
trans-regulator that functions through this element; (i) Oct-1, a mammalian
protein related to SpOct, activates the transcription of mammalian H2B
histone genes (La Bella et. al., 1988); (ii) the SpOct protein avidly binds the
early H2B octamer sequence (Bell et al., 1992); (iii) the expression profile of
SpOct mRNA closely matches that of the early H2B gene; (iv) exhaustive
screening of sea urchin genomic DNA and embryonic cDNA libraries for
POU Il-class sequences has yielded only SpOct; (v) amino acid sequences of
peptides derived from affinity-purified sea urchin octamer binding proteins
match the predicted amino acid sequence of SpOct. These data suggest that
SpOct is a key regulator of the early H2B gene, and lead to the prediction that
experimentally-induced perturbations in SpOct activity will have a direct
effect on early H2B gene expression.
That an octamer binding protein— likely SpOct— also plays a role in the
expression of sea urchin cytoskeletal actin genes is suggested by several
observations. First, both Cyllla and Cyla genes possess octamer elements in
regions that are critical for gene function. Second, artificial promoters
57
containing various combinations of putative Cyllla regulatory elements must
contain an octamer element to drive CAT expression efficiently in embryos
(B. Hough-Evans and E. Davidson, unpublished). Finally, the Cyllla and Cyla
genes are transcriptionally activated at approximately 8 hours after
fertilization, coinciding with the peak accumulation of SpOct mRNA. Since
the Cyllla and Cyla actin genes are expressed regionally in the developing
em bryo-in contrast to the early H2B gene which is expressed in all cells— we
imagine that an octamer binding protein(s) is involved in the stage specific
activation of the Cyllla gene, but does not play a causal role in the region-
specific expression of Cyllla, though it may be required for this latter process
in some capacity.
The amino acid sequence of the SpOct POU domain is not
significantly different from the POU domains of either Oct-1 or Oct-2 of
mammals, or those of pdm-1 or pdm-2 of Drosophila (Figure 2.2B). This
result, together with the apparent lack of additional Oct genes in the sea urchin
genome, supports the view that the duplication that produced Oct-1 and Oct-2
occurred after sea urchins diverged from the lineage that gave rise to
chordates, and that the duplication that gave rise to pdm-1 and pdm-2 took
place after the divergence of Protostomes and Deuterostomes. A comparison
of the overall domain structures of SpOct with Oct-1, Oct-2, pdm-1 and pdm-
2 reveals several features that are common to all of these proteins and thus are
likely to be the most primitive: (i) in addition to the highly conserved POU-
homeodomain, all possess an N-terminal glutamine domain; (ii) all contain a
domain rich in serine and threonine adjacent to the POU-homeodomain on the
C-terminal side. These properties, preserved over a span of at least 500
58
million years, were probably features of the ancestral Oct molecule. Other
features typify an Oct-1-like subgroup. Both SpOct and Oct-1 possess
glutamine-rich domains at their N-termini, and both possess a domain at their
carboxy termini rich in serine and threonine, but not in proline as is
characteristic of Oct-2 (Figure 2.8) . These domains have been implicated in
the activation of mRNA promoters (Tanaka and Herr, 1990). Along with these
structural features, SpOct apparently shares with Oct-1 an ability to activate
the H2B promoter, as well as ubiquitous expression in tissues of the adult.
One structural feature found in Oct-1, but not in SpOct, is a C-terminal
domain (Figure 2.8, S/T*) involved in the activation of snRNA promoters
(Tanaka et. al., 1992). This domain is also absent from the pdm-1 and pdm-2
genes of Drosophila (Lloyd and Sakonju, 1991). Although Herr and
coworkers have suggested that the ability to trans-activate snRNA promoters
is an ancient property of Oct molecules (Tanaka et. al., 1992), it appears that
this function was acquired after the divergence of sea urchins from the lineage
that gave rise to Chordates. That sea urchin snRNA genes do not have
functional octamer elements provides further support for this scenario
(Stefanovic and Marzluff, 1992).
Our analysis of the sea urchin SpOct gene and octamer binding
proteins has provided insight into possible developmental roles of oct
proteins— the activation of early histone and cytoskeletal actin genes-and has
helped to distinguish features of the Oct molecule that are conserved in a wide
range of species and thus primitive, from features that are characteristic of
subgroups of animals, and are thus derived. In the next chapter, I will test
59
directly the developmental function of Oct binding proteins by a strategy that
alters Oct activity in embryos.
60
Figure 2.1 PCR-based strategy for cloning sea urchin POU genes.
Degenerate oligonucleotide primers (FKNMCKLKP and the reverse
complement of VWFCNRRQKEK) were made to conserved domains in Oct-
1, Oct-2 and Pit-1 (shown in boldface) and used in the PCR. The sequence of
the degenerate oligonucleotides can be found in Materials and Methods.
Amino acids identical to those in Oct-1 are represented by stars. The predicted
amino acid sequences of the SpOct and SpPOU-1 partial cDNAs obtained by
PCR are shown below the original alignment.
61
O ct-1 FSOTTI SRFEAUJLSFIOBCKLWLLEKWLNDAENLSSDSSLSSPSAhNSPG - - 1EGL-SRRRKKRTSIETNIRVALEKSFLENQKPTSEEITMIADQLNMEKEV1RVIIFC1IRHQK8KRINP
P i t - 1 •» * •« * *S* * * EE* *QV--------------- G**YNEK--VGAN-E*K**R**T*SIAAKD***RH*G*HS**S*Q**MRM*EE**L****V*»*#«»#****#RVKT
SpOct * * * * * * * . * * * * * * * • • • • • • • • • * * ---------*TTV*N*AL*G-*H NSP**I -N **************IS******M QP*******VLLGE*MG*****V**#*********
SpPOU-1 • • • • • • • • • * . A***EE*-------- **TSG**TS*DK *MQ-G*K*****»**VT*KG***NA**KQP**SAQ**SRL*DG*QL****V*»»***«*»*«*
o\
to
Figure 2.2 SpOct cDNA sequence and comparision of the SpOct POU
domain with that of other POU II proteins.
(A) Sequence of an SpOct cDNA clone along with its deduced amino acid
sequence. The 5' borders of overlapping clones A/7-2 and ^,8-8 are indicated.
Other features marked are a glutamine-rich domain and a serine-threonine-rich
domain. The POU-specific and POU-homeo domains are boxed, and a
putative polyadenylation signal site is underlined. Nucleotide and amino acid
numbers are shown on the right. (B) Alignment of the amino acid sequences
of the POU domains of POU II genes from various species. The shaded areas
indicate identical amino acids. The residues that SpOct shares with pdm-1 and
pdm-2 but not with other members in the POU domain are shown in bold type.
The sequences were obtained from the following sources: Oct-1 (Sturm et al.,
1988); Oct-2 (Clerc et al., 1988); chicken Oct-1 (Petryniak et al., 1990);
X10ct-1 (Smith and Old, 1990); pdm-1 and pdm-2 (Lloyd and Sakonju, 1991).
63
A
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1 7 - 2 -» | N S T E T A E M C P A
CAGATGATTCACGTCATGAGTTCATCTXiGCCAGCCCAC G TTG G 'TTCAOCCGACCAACGTCATGACOGCTCAQOGCCTAQCTGCtaCAG U jl I'llH -A GlCTl'lLAACCOCIIOQ CM QCCCA
Q M r Q V M S S S O a P T L V H P T N V M T A Q C L A A A C F L S L P T A G S P
A P G H C R H L Q Q P Q Q F L . V N pg s gq vl l q tl pf aq ag at pi al
GCTAACTTCCAOQGTGCOCAACAQGTTQCACKXXTAC CTCTUCTCCTX J AGRTCCTCAKrrCACCCAOGAACCAAATCAAGATOQCCACCRTQCJICEflOQOOQOCCJOCIIflCTCCAflBCA
A M F Q G A Q Q V A L A T S A A Q M L K S P T M Q I K M A T M Q S A A Q Q L Q A
T ? QQ L Q LL 0 2Q S v 3 v I S P F A O I T R M « A M Q Q Q P Q Q Q Q Q Q Q H 251
|— Oln rictw
Q Q F Q Q Q N Q Q M Q Q Q O T L P P Q Q Q Q Q Q G V Q Q P K I A P Q L P G Q L Q 251
CPC ATC TC O G C A C A A G A C C TA C A A C A G T T G C A G C A A C TC C A G C A A C A G A A C C C C A A C C T G C A A C A C T A ' njrC'n'C TrACAAOCrCCtTrCCCAO G WCCCAC A A A CATO CPO CJUC O C CAA 1000
Q I S A Q D L Q Q L Q Q L Q Q Q N P H L Q Q Y V F L Q A P S Q V P T H N Q Q A Q 331
Q L L L 3 Q Q Q Q 0 Q Q Q Q Q Q M L A 0 Q Q G L L Q Q S M V G H L P Q Q 3 L I Q 3?1
gCTCAAQCCATC A A T C C A C A A C A A C A G ^TG C A G C A A C A A G C A C T C A C ^ !Y>:AOCAOGAATCt^JTC ^ fl»* r G ^,CULU' T TXtlGCJO O O G C TOCAACAACAAAroCOOfiRCICfCTACTQC^X^ 1320
A Q A I N A Q Q Q L Q Q C A L T A A G I Q S V S R A A A A A T T N A Q 3 V T A Q 411
<-Gtn rich—(
CAG CA G C TC CAfi<7I^A A gA A G C A C A G A A G C A A fiQ G G G C G C >C T G A rC TX irC G TC C C T G C C C A A C C C A TY X ?C C A G T A T T C CTC ACAG CCAKinCJUlGIOCAfi4Ctt3Arr^ rTM rtn*nrTC 1440
Q Q L Q L Q Q A Q K Q G G G V T S V A A Q P I PS
T A TTCC TCA CAO CCATG TTjA ^G AO CAfiA CO G A TCTCG 4Q O AO CTC
1 P H S H | V E E Q T D L 8 B L |
^ G AAf‘ -A£r!TCG CTA£CA£,CTTC AAflT A G A A ^ AAACyjGGCITCACACACOGTGATGTTCGTCTOGCCATCiQy A A G C*IT]TATnfirAAfIlA rTTC AQ CTC7AAAn«APr ATTTTTO BC 1540
Ie q F A R T F K Q R R I K L C F T Q C D V C L A H G K L Y G H D F S Q T T I S R I 451
CCC G AAgjCATCAACCC O C G C A G A A A A A A C O SCA CAAG CATCG AG AC CAACA TTJO G ATA TO CTrO CA AAAflAGL l'l! l.L'I ^ TOCJICCa3AA0CCAA035aaGAAflM3ATr o T0L TU L T i: 1900
P E G I N RI R R K X R T S I E T N I R I S L E K S F L H Q P * P T S B E I V L L f S71
GGGGAGCAGATryyryATTGACAVlGVyr?TyiTl !l.A G G 'j'l.'!'l‘.i< .» -V * 'I ’G T AAC-TrriT;A^AG AAGG AG AAQfTTTATC*AAf*ft~GC(!l!A.TC^Q AQ O CTGAATCTO g>GAA£^TCA50PO BgAg 1920
[g E 0 MG M E K E V V R V WP C N R R Q K E K R IN pI p V S S L M L Q M L M A H H I
11 |—Sor/Thr rieh-»
CACACCTCGCACTCCATATCATCGATGJ^^C,!l GTCACCAGGACTCACCCAGATCCAGCAGACCAACCC 'RAj» * rA CCCCTG?rCAOG C 'C C i'TCC A COCCCATCTCCTOCAAAATCATCJ>OC>OC 2040
H T $ H S I SS N N L « PG V ? q : qq ?MPGTPVTPST PIS SK IIS S 431
AOGCTAAGACCGTGGCrATACAGrCA'TCAGDGAGAACTATIOCmjTCAACTroOTCTCTOCAAOrAACn OTCAA 2140
A K T V A I C S S G R T I A V M L V S A T M G Q 471
»rC *G T T A C T C C G T , rCACATC T G G C C A O C C A C A G AT G TCTCTA ATCAA TPC A3CCAC CCAC AUULTAT l L ^ A U L TG 2200
7 T P 5 T S G Q P Q M S L I N S A T N R L S . 725
rT^rCGTCTCGCAAAAAQCAaGGArACGCA^ACACTAArACCAAC^ACAGCTAC^CAAL 'tVlGTrG CAAACTGCTAATCAACrgGCATCGTrATCAQGICTACOOAgTBBM CBOPO T 2400
■"A A A SO G C T G A A G T A G A C C C A A G T A L lT JG C TTTAAACTO G TCrAAO G G A G T A C T G A C G TL m T T C C TO G ACATATnTAAAl l'l 1U A ATAJOOCCArCTn w tnA.'fAT C TO CAPCTG AT 2520
A A G A C G A Llii ?TCAAGAGAGTTGAGAGAGACG^ACCGGTCAG7CAGTAAGCT^AGTCACTG*GOOCGACTGTCCAGTACCATCGTOCOG A C ATG n GA 1 ffAOCCOCTCtl>OCAATOOCO 2740
GTA TTACAGG I1 L ' !T l ISjLlO' IM TTI^G C TCATTCOGGTTT'IV^ I '77 ACATATCACrrCACTTCAAATTLTj TCCATPCAAAQO OC A C A A A < J l 11 U .IL 1 U 1 iVAAAAJCTACACAAtTPCA 2«00
r.'-TTGTATAAAATAATATTAATACTAAl'lT'^ .rAL H TT ATATAGCTCTTAATACATCTArGATG TCTCTAA I.il/1 111 A CAGATATAL TA TTAL.maATTCAlTG G A CTCAATAglTAC 3000
G«:>^A7tXTTTTTCAGGTrTATTATAAPCrr7rA?CGGrCAT,TrAA ATC G A AATTA AAATG C T .TGTTnT7rAGATCTCAMTCTATA rnV l'r i,lG^^CATTAJCAOaiA l'HVAlLVJUT 3240
rACAACAAATCAAATTCCGC CTTTC'TAl'l 'lGtUl 1 ' l'j .'j i ATATTrATTCATCAACTA'HTATCTACATCCACTATCCAGCCCCTGATACCATGTCATTgMCrTrAQOOCCM ilGTl 'ICA 3340
rcrCTATATGGAGAGTGJCATrtSCAATXAACTTrTC^TTVATCAGAAAAATAAGATAAACAAAATGCa^AAGrnGAAATAATCAGACCCAMaMP^AAJCrrroGAAArAWAG 3400
AT;ACTA7ACTTA?GrrAA7CTCAAAr?GGCAATT,?GAT T A O ' ^ A i r iV rVC^TCrACTCACTGACAAC T llU jr ri'AGAA A A A A A A A A 3549
B
- - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - P O U - s p e c i f i c
1 10 20 30 40 50 60 70
Oct -1 EEPSDLEELEQFAKTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLEKWLNDA- -ENLSSDSSLSS - - -
ChickOct-1 EEPSDLEELEQFAKTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLEKWLNDA--ENLSSDSTLSS---
XlOct-1 EE PS DLEELEQFAKTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLEKWLNDAVLENITSDSTLTN---
Oct - 2 EEPSDLEELEQFARTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLEKWLNDA- -ETMSVDSSLPS---
SpOct EEQTDLEELEQFARTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLQKWLDDA-------DTTVSN---
pdm-2 EETTDLEELEQFAKTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLQKWLDDA-------DRTIQAT-G
pdm-1 EETTDLEELEQFAKTFKQRRIKLGFTQGDVGLAMGKLYGNDFSQTTISRFEALNLSFKNMCKLKPLLQKWLEDA-------DSTVAKSGG
P O U - h o m e o d o m a i n
1 10 20 30 40 50 60
Oct-1 PSALNSPG---IEGL - S RRRKKRTSIETNIR VALEK S FLENQK PTSEEITMIADQ LNMEKEVIRVWFCNRRQKEKRIN P
ChickOct-1 ----PSALNSPGQG-IEGV-NRRRKKRTSIETNIRVALEKSFLENQKPTSEEITMIADQLNMEKEVIRVWFCNRRQKEKRINP
XlOct-1 ----QSVLNSPGHG-MEGL-NRRRKKRTSIETNIRVALEKSFLENQKPTSEEITMIADQLNMEKEVIRVWFCNRRQKEKRINP
Oct2 ----PNQLSSPSLG-FDGLPGRRRKKRTSIETNVRFALEKSFLANQKPTSEEILLIAEQLHMEKEVIRVWFCNRRQKEKRINP
SpOct ----PALLGPHN--SPEGI-NRRRKKRTSIETNIRISLEKSFLMQPKPTSEEIVLLGEQMGMEKEWRVWFCNRRQKEKRINP
pdm-2 GVFDPAALQATV-STPEII-GRRRKKRTSIETTIRGALEKAFLANQKPTSEEITQLADRLSMEKEWRVWFCNRRQKEKRINP
pdm-1 GVFNINTMTSTLSSTPESILGRRRKKRTSIETTVRTTLEKAFLMNCKPTSEEISQLSERLNMDKEVIRVWFCNRRQKEKRINP
L/\
Figure 2 .2 (continued)
Figure 2.3 DNAse I footprint assay comparing bacterially-expressed
SpOct and affinity purified oct proteins.
DNase I footprint assay comparing bacterially-expressed SpOct and affinity-
purified octamer-binding proteins. ^^P-labeled probes representing (A) the
coding and (B) the noncoding strand of the C yllla regulatory region
containing the octamer site (P3B) were prepared as described previously
(Calzone et al., 1991) and with increasing amounts of bacterially-expressed
SpOct (lanes 1-3) or affinity purified protein (lanes 4 and 5). Bacterially-
expressed SpOct was diluted 1:10 before the amounts indicated were used.
The regions protected from DNase I cleavage in each reaction are indicated by
open boxes.
66
B
Hindlll -243-,
-156
Oct (P3B)
-138
3
“■ Bact
o
1 2 3 4 5
A f f a. o. Sact
o o
1 2 3 4 5
o
Aff a.
o
a
V v j-
— -114 Dae
-138
Oct(P3B)
-156
□del -114
-243 Hindlll
Non-coaing Coding
67
Figure 2.4 Measurement of the relative binding affinity of the SpOct
protein.
Calculation of relative binding affinity of SpOct by quantitative gel shift
analysis. DNA binding reactions with SpOct proteins: (A) full-length protein,
(B) POU domain only (residues 364-619). Each binding reaction contained
41 bp probe (1.7 x 10" 9 M), poly(dAT) (3.45 x 10‘4 moles nucleotide/liter)
and unlabeled probe diluted by a factor of two starting with 3.2 x 10"7 M in
tube 1. The value of Kr was calculated using the following equation by linear
regression:
(PDs xD n)/D s = K r(P0-PDs)
where PDS is defined as the concentration of specific complexes in
moles/liter; Ds is the concentration of the free probe; Dn is the concentration
of free nonspecific competitor DNA (here taken to be the total amount of
poly(dAT) present in moles of nucleotide/liter; P0 is the number of molecules
of a given factor in the system. The complex which most likely represents the
intrinsic interaction of SpOct monomers with the single site octamer probe is
indicated by the arrow and referred to as a primary interaction. Secondary
interactions appeared at high SpOct protein and low probe concentrations. In
the case of full-length SpOct and the C-terminal deletion, the total amount of
complex in the reaction was taken as total amount of probe (labeled +
unlabeled) in the reaction minus the amount of free probe (i.e it was assumed
that SpOct monomers and multimers bound the probe with equal affinity).
While this assumption causes us to underestimate Po, the value of Kr is not
68
significantly affected. It should be noted that SpOct protein has a relatively
high affinity for poly(dAT). Kn is about 50-100 fold greater than reported
previously for the general Cyllla regulatory factor (Calzone et al., 1988).
69
S p O c t f u l l - l e n g t h
1 2 3 4 5 6 7
• i\ I
I vil li
7
Kr= 1.4 X 104
6
5
4
3
2
1
0
Free probe
B
S p O c t P Q U - d o m a i n
1 2 3 4 5 6 7
i
Free probe
1 0
8
6
4
2
0
70
Figure 2.5 Probes used for gel blots and a DNA gel blot probed for SpOct.
(A) The thin line represents the SpOct cDNA clone. The thick line shows the
extent of the coding region, and POU refers to the POU domain. All probes
were antisense RNA molecules generated from SpOct cDNA by T7 RNA
polymerase. For the DNA gel blot and adult tissue RNA blot, the probe was a
labeled 1.1 kb antisense RNA fragment ending at an Aat II site in the 3'UTR
(probe b). In the case of the developmental series RNA blot, the entire SpOct
cDNA was transcribed to make an antisense probe (probe c). For the RNAse
protection assay, a 248 bp probe was generated from the POU domain of
SpOct (see Materials and Methods) (probe a). (B) DNA gel blot of
S.purpuratus DNA probed for SpOct. 10 \ig of genomic DNA from individual
males was digested with Hindlll or EcoRI and then electrophoresed on a 0.8%
agarose gel. Urchin 1, 2 and 3 refers to individual S.purpuratus males from
whom sperm DNA was extracted. Size markers in kb are on the left.
71
A A A A
Figure 2.5 (continued)
B
Hind III
U rch in 2 3
23.1 k b -
9.4
6.6 -
4.3 -
2.3 _
2.0 -
EcoRI
1 2 3
73
Figure 2.6 Expression profile of SpOct cDNA during development.
(A) RNA developmental series probed for SpOct. The numbers above the
lanes indicate the time in hours post-fertilization at which RNA was extracted.
10 |ig of whole cell RNA was loaded in each lane. Ribosomal RNA is shown
below as a control for the amounts of RNA loaded. The same developmental
RNA series was probed for early H2B transcripts with an antisense RNA
probe (Bell et al., 1992). The blot was then stripped of probe and hybridized to
a LI late H2B antisense probe (Zhao et al., 1990). Ribosomal RNA amounts
are shown below. (B) RNAse protection assay on the same developmental
series used in (A). Probe b (Figure 2.5A) was used in this experiment. 10 fig
of whole cell RNA was loaded in each lane. yeast RNA treated with
RNAse. The sizes of the full-length probe and protected fragment are
indicated on the left. (C) Quantitation of relative amounts of SpOct mRNA
based on the RNAse protection in (B). The gel shown in (B) was scanned
with a laser densitometer, and the relative amounts of SpOct mRNA at the
different stages of development were normalized to the 8 hr blastula stage.
74
SpOct
Hr
0 8 12 21 36 60
12 Kb
8
rRNA
early H2B
late H2B
rRNA
75
Figure 2.6 (continued)
1
B
Hrs post-fert.
E 8 12 36 60
248 bp
203 bp
76
SpOct m R N A (arbitrary units)
Figure 2.6 (continued)
c
1.2
1.0
0.8
0.6
0.4
0.2
0 10 20 30 40
Hours post-fertilization
77
Figure 2.7 Expression of SpOct in adult tissues of the sea urchin.
5 pg of whole cell RNA from different tissues was loaded in each lane. Coel,
coelomocytes; Ov, ovary; Test, testis; Tbf, tubefoot. Whole cell RNA from 12
hr blastula embryos was also run to compare transcripts present in adult
tissues and embryos. A labeled 1.1 kb antisense RNA molecule was used to
probe this gel blot (Figure 2.5A and Materials and Methods).
78
VC
Blastula
Figure 2.8 A comparison of domain structures of POU II proteins from
different species.
P: proline-rich domain; Q : glutamine-rich domain; S/T: serine-threonine-rich
domain; S/T*: serine-threonine-rich domain unique to Oct-1; POU-sp.: POU-
specific domain; homeo: POU-homeodomain.
80
■POU domain —
SpOct Q S/T ►
POU-sp. homeo
O ct-1
Q Q S /T -S /T * -
O ct-2
Q S /T P
pdm -1
-S /T - Q S /T
pdm-2 Q Q S/T S/T
Chapter 3
Functional role of the SpOct gene in the early sea urchin embryo
Introduction
In the preceding chapters, I have presented evidence that the octamer
element plays an important role in the activation of the early H2B gene, and
that a DNA-binding protein that recognizes this element is expressed in a
temporal profile that closely matches that of the early H2B gene. This is
strong circumstantial evidence that the DNA-binding protein, SpOct, regulates
the expression of the early H2B gene. To directly prove the role of a factor
such as SpOct, however, one must demonstrate that ablation of function of the
regulatory molecule has a direct effect on the downstream gene of interest. In
a model system such as Drosophila, genetics provides a convenient tool for
investigating such developmental problems, by identifying mutants at
different loci (the regulator and the regulated) that display similar phenotypes.
In systems that are not genetically tractable, such as the frog and sea urchin,
the reverse genetics approach has to be adopted, in which modification of the
activity of a regulatory gene is used to understand its function. Examples of
this approach are expression of the regulatory molecule at abnormal positions
or times during development (resulting in a gain of function), and disruption
of gene activity (resulting in a loss of function). I utilized both these methods
in order to test whether SpOct directly activates early H2B expression.
Ectopic or abnormally-timed expression of the regulatory gene can
yield useful information on the role it plays. For example, if the SpOct
82
protein is indeed directly activating early histone H2B expression in the sea
urchin blastula, it should be possible to activate early H2B gene expression in
later embryos (when early H2B expression is normally turned off) by
expressing SpOct at later stages. I attempted to do this by placing the SpOct
sequence under the control of the LI late histone H2B promoter which is
expressed at its highest level in the late blastula embryo, and injecting this
construct along with the early H2B gene bearing its own promoter into
fertilized sea urchin eggs. The expectation was that the exogenously-
produced SpOct protein would activate the co-injected early H2B gene.
RNase protection assays revealed, however, that there was no effect on the
expression of the early H2B gene— it shut down in late blastula-stage embryos
as normal. The failure of this experiment could be attributed to a number of
causes, including an insufficient level of SpOct protein generated by the late
H2B promoter, and a mechanism of early H2B repression in late blastula
embryos that overrides any activating mechanism.
I then used the loss-of-function approach, in which the expression of a
gene is perturbed so that it is no longer functional. This can be done in the
germ-line of the animal, resulting in a transgenic organism which lacks the
gene, or transiently in the embryo by targeting the gene of interest by
antisense methodology. In the sea urchin, the many months required for a
pluteus larva to reach sexual maturity as an adult makes it impractical to
generate transgenic animals. Thus, transient perturbation of the SpOct gene
was the method of choice, although this approach had not been reported in the
literature as a means of targeting genes in the sea urchin embryo. It is known,
however, that injection of an excess of the octamer element into sea urchin
83
zygotes results in embryonic death at various stages upto the pluteus larva
(Franks et al., 1990) indicating the importance of octamer-binding proteins in
development. Since SpOct is expressed in the unfertilized sea urchin egg, we
thought it might be possible to affect its expression in the early embryo by
injecting antisense oligonucleotides.
In Xenopus, antisense targeting of gene expression has been
successfully used to disrupt the function of maternally-expressed genes (Vize
et al., 1991). Injection of antisense oligonucleotides to the coding regions of
various maternal messages, including cyclins and zinc-finger proteins, results
in the specific ablation of target gene expression as judged by RNA analysis
(Weeks et al., 1991; El-Baradi et al., 1991). An RNaseH-mediated degradation
of antisense oligonucleotide-mRNA hybrids in addition to a block in
translation of the target mRNA occurs in the Xenopus embryo, resulting in the
loss of expression of the gene (Vize et al., 1991).
As mentioned in the intrduction to Chapter 2, POU genes are
expressed during development in a number of species. Several POU III genes
are expressed in the developing nervous system of the mammalian embryo
(He et al., 1989), and Oct-3 is expressed in the germ-line of the mouse
(Rosner et al., 1990). These findings show the fundamental role POU proteins
play in early embrogenesis. POU II genes (the Oct subfamily) have been
extensively studied in tissue culture, but their role in early development has
not been directly addressed in any system. Since SpOct is expressed in a
temporally regulated manner in very early sea urchin embryos, we thought it
worthwhile to study its function. The binding site for Oct proteins,
At t t g c AT, is present in the regulatory regions of a number of
84
embryonically expressed genes in the sea urchin, and in order to gain an
understanding of the broader role SpOct plays in the sea urchin embryo, we
sought to disrupt its function in the one-cell zygote.
In this chapter, I will demonstrate that injection of antisense
oligodeoxynucleotides (ODNs) against SpOct prevents cleavage or retards
development when injected into fertilized sea urchin eggs. This cleavage
arrest is produced only by single-stranded antisense ODNs, and not by
antisense ODNs that have been annealed to their complementary strands prior
to injection. I also show that SpOct mRNA can overcome the cleavage-arrest
phenomenon. Antisense-injected embryos incorporated ^H-thymidine into
DNA similarly to noninjected embryos or embryos injected with contol
ODNs. Protein synthesis, however, was reduced in antisense-injected
embryos by 3-4 fold compared to controls. These data indicate that SpOct is
required in the activation of protein synthesis in the sea urchin zygote. When
eggs were activated with the Ca++ ionophore A23187 and then injected with
antisense ODNs, protein synthesis was reduced about 2-fold as compared to
controls. This is the first demonstration of an antisense-mediated gene
perturbation in the sea urchin embryo, and we believe that this method could
be sucessfully employed with other maternal genes or zygotic genes expressed
in the early embryo.
85
Materials and methods
Synthesis and purification of oligodeoxynucleotides
O ligodeoxynucleotides (ODNs) were synthesized on an Applied
Biosystems DNA synthesizer. The ODN pellets were resuspended in 1 ml of
glass-distilled water and purified on a C l8 column (Waters) acording to the
manufacturer's instructions. The ODNs were ethanol precipitated and
resuspended before further purification on Elutip columns (Schleicher and
Schuell). Following this step, the ODNs were ethanol precipitated once again,
before being resuspended in distilled water. For injection into sea urchin
zygotes, the ODNs were diluted to 1 mg/ml in a sterile 25% glycerol solution.
Double-stranded ODNs were generated by annealing complementary strands
of a given sequence in 10 mM KC1, heating to 95 °C and then gradually
cooling the ODN mixture to room temperature. The data represent injections
performed with antisense ODNs al (5'-TTCGGGGCTATTGTGAG3\ the
reverse complement of nucleotides 1666-1682 in the SpOct cDNA), a2 (5'-
CACCCTGGAAGTTAGCC-3', the reverse complement of nucleotides 479-
495 in the SpOct cDNA) and a3 (5-TCAGTGCTCATGATGAGGACA-3',
the reverse complement of -10— hi 1 in the SpOct cDNA), and control ODNs
cl (5-CAGCGCCAGCCGAGG-3', a fragment of 5' non-translated sequence
from the mouse Hox 8 gene), c2 (5'-ACCAACATTAGGATATCG-3\
nucleotides 1722-1739 in the SpOct cDNA), and c3 (5'-GCTGAGCAA-
GTGTAGATATCC-3', a randomized version of a3).
86
Injection of ODNs into sea urchin zygotes
ODNs were injected into fertilized eggs essentially as described in
Colin (1986) and McMahon et al., (1985). Briefly, eggs were collected and
dejellied after which they were attached to Falcon petridishes treated with a
1% solution of protamine sulfate. Usually about 50 embryos were injected per
dish; those left uninjected were removed. As far as possible, both antisense
and control ODNs were injected into the same batch of eggs on a given day.
After injection, embryos were cultured in sea water at 15 °C. At 2 hr
intervals, the dishes were examined and embryos were counted for stage of
development reached. Experiments in which there was greater than a 30%
incidence of mortality were discarded. Those embryos which were damaged
during the injection process were not counted. Each ODN was injected at
least on three different occasions, to control for variation in the batches of
eggs.
Generation of SpOct mRNA
To do the rescue experiment, SpOct mRNA was generated by using
the Megascript kit (Ambion Inc.). pBS-SpOct was linearized with Hindlll,
and T3 RNA polymerase was used to generate full-length SpOct transcripts
according to the manufacturer's protocol. The SpOct mRNA synthesized was
phenol-chloroform extracted and ethanol precipitated before being
resuspended in RNAse free water at a concentration of about 2 mg/ml. The
SpOct mRNA was then diluted 1:1 with sterile 50% glycerol, and was co
injected with antisense ODN which was at a concentration of 1 mg/ml.
87
Measurement of DNA synthesis in injected embryos
Following injection with antisense and control ODNs embryos were
incubated at 15 C in filtered sea water containing 50 mCi/ml of ^H-thymidine.
After 2.5 hrs, the embryos were collected and washed 3X in sea water, and
lysed in 0.5% SDS. 20 mg of yeast RNA was added to each sample, and
DNA precipitated with ice-cold TCA at a final concentration of 10%.
Samples were passed through a GF/C filter (Whatman) and washed 4X with 5
ml of 10% TCA, followed by washing 3X with cold 95% ethanol. The Filters
were air-dried and Protosol added to release the counts. In the experiments in
which DNA synthesis in embryos was blocked, aphidicolin was added at a
final concentration of 50 mg/ml.
Measurement of protein synthesis in injected embryos
Embryos were incubated at 15 °C in filtered sea water containing 50-
100 mCi/ml of 35S-methionine immediately after injection. Embryos were
collected by centrifugation, washed 3X with sea water, and lysed in 20 ml of
NETS-EGTA (Bell et al., 1992). 100% ice-cold TCA was added to a final
concentration of 10%, and embryo lysates placed on ice for 15 min.
Precipitated proteins were precipitated by centrifugation for 15 min in a
microcentrifuge, and the pellets thoroughly washed with ice-cold acetone.
The protein pellets were then resuspended in 2X SDS sample buffer and either
dried on a GF/C filter (Whatman) for counting, or loaded on 8.5% SDS gels.
SDS gels were treated with Enhance to intensify the signal.
88
Results
Effect of antisense ODNs on embryonic development
To interfere with SpOct gene function, I injected antisense
oligodeoxynucleotides (ODNs) against the SpOct cDNA into fertilized eggs.
To minimize the possibility that contaminants in the ODNs affected the
embryos, three different antisense ODNs synthesized at different times were
used (Figure 3.1 A). To control for any effect the injection procedure might
have on the rate of development of the embryos and any toxic effect due to the
ODNs themselves, I compared antisense-injected embryos to those injected
with control ODNs. Control ODNs consisted of a sense-strand ODN, a
randomized antisense ODN and an ODN made against an unrelated gene. The
data shown in Figure 3.IB is representative of the three antisense ODNs, al,
a2, and a3. First, two individual experiments are shown, in which a l and a2
were injected into fertilized eggs. ODNs c l and c2 were injected as controls.
Injection of al resulted in the cleavage arrest of 88% of the embryos, while
injection of a2 resulted in the cleavage arrest of 69%. The control-injected
zygotes generally cleaved at 100-110 min post-fertilization, while noninjected
ones cleaved by about 90 min (data not shown). Pooled data from
experiments done with al, a2 and a3, as well as control ODNs cl, c2 and c3
are also shown in Figure 3 .IB. These data show that upon injection of
antisense ODNs, 74% of the embryos failed to undergo the first embryonic
cleavage at the normal time. In contrast to this, only about 10% control ODN-
injected embryos remained uncleaved. I also found that antisense-injected
embryos that cleaved proceeded more slowly through development than
89
control-injected embryos. At 6-8 hr post-fertilization, the majority of control-
injected embryos had >16 cells whereas substantial numbers of antisense-
injected were still one-celled (Figure 3 .IB, pooled data). These results
indicate that antisense ODNs interfere with embryonic development, whereas
control ODNs did not have a significant effect on embryos.
Specificity of the antisense ODN effect
I performed additional tests for the specificity of the antisense ODNs.
To rule out the possibility that contaminants were causing the arrest in
development, I injected one-cell zygotes with double-stranded ODNs
composed of sense and antisense SpOct ODNs. The antisense ODN used in
these experiments produced the cleavage arrest phenotype. We expected that
injecting it annealed to its complementary strand would have little effect,
since the antisense ODN would not be available to bind to its target. If a
contaminant was responsible for the arrest in development, however, injection
of a double-stranded ODN would have the same effect as a single-stranded
antisense ODN. ODN al was annealed to its complementary strand and
injected into fertilized eggs (Figure 3.2A). As a control for DNA amounts
injected, al was mixed with cl and injected into embryos in parallel. In two
experiments, 55-77% of the embryos injected with the antisense+irrelevant
ODN mixture failed to cleave by 6 hr, whereas 94% of the double-stranded
ODN-injected embryos reached 16 or >16 cells. Thus the antisense phenotype
is a specific consequence of the antisense ODN, and not due to a teratogen
present in the ODN solution.
90
Rescue of antisense-injected zygotes with SpOct mRNA
If the single-stranded antisense ODN is causing the developmental
arrest, we reasoned that exogenously introduced SpOct mRNA should enable
the antisense ODN-injected embryos to overcome the block in development.
By the same token, an irrelevant RNA should not be able to rescue SpOct
antisense-injected embryos. I injected an antisense ODN against the 5' non
coding sequence (ODN a4, Figure 3 .IB) into one-cell zygotes along with
either SpOct mRNA or an irrelevant homeobox mRNA. The co-injected
SpOct mRNA was transcribed from a cDNA that is missing the 5' non-coding
sequence to which the ODN is complementary. Co-injection of SpOct mRNA
enabled 64% of the embryos to cleave by 2 hr, whereas about 12% of the
controls did so (Figure 3.2B). At 4 hr post-fertilization, the proportion of
cleaved embryos in the SpOct mRNA-injected and control-injected batches
was 87% and 36% respectively. These results indicate that SpOct mRNA can
rescue embryos in which the endogenous SpOct gene function has been
disrupted.
Assaying SpOct mRNA in uninjected embryos
In Xenopus, antisense-mediated disruption of gene expression occurs
through a RNaseH-like activity which causes degradation of RNA-DNA
hybrids. To determine whether a similar mechanism exists in sea urchins, I
injected embryos with antisense and control ODNs, and then assayed the
amount of SpOct mRNA in the embryos by an RNase protection assay.
Because the level of SpOct mRNA is very low in the one-cell zygote, it was
necessary to inject about 500 embryos with each ODN in order to get a strong
91
enough signal in the RNase protection assay to accurately quantitate the SpOct
transcript levels. The result of this experiment was that SpOct mRNA levels
were not significantly affected in antisense-injected embryos (data not shown).
From these results, I could not draw a conclusion as to whether an RNAse-H-
like mechanism is operative in these embryos. Values of AG of hybrids
formed by the various ODNs were calculated using the treatment of Saenger
(1983), and are as follows: al, -63.44 kcal/mol; a2, -62.34 kcal/mol; a3,
-88.01 kcal/mol; c2, -65.46 kcal/mol. These values indicate that the ODNs
used form stable hybrids (> -50 kcal/mol).
DNA synthesis is normal in antisense-injected embryos
Since Oct-1 (NF-IIIA) may have a role in regulating DNA replication
(O' Neill et al., 1988), and SpOct is closely related to Oct-1 in the DNA
recognition domain, we asked whether interfering with SpOct function affects
DNA replication. ^H-thymidine incorporation into embryonic DNA was
measured after one-cell zygotes were injected with antisense and control
ODNs. We used two sets of antisense and control ODNs in these
experiments. In fertilized eggs injected with antisense ODNs al and a3, 3H-
thymidine incorporation into newly synthesized DNA was 81% relative to
uninjected embryos (Figure 3.3). In the case of control ODNs c2 and c3,
^H-thymidine incorporation was 134% of that in uninjected embryos. To rule
out the possibility that the DNA synthesis observed in antisense-injected
embryos was due to mitochondrial DNA replication, I blocked nuclear DNA
synthesis in embryos with aphidicolin. Incorporation of ^H-thymidine into
the DNA of aphidicolin-treated embryos was about 9% relative to untreated
92
embryos, indicating that the counts due to mitochondrial DNA synthesis are
insignificant. These results suggest that DNA synthesis is occurring at normal
levels in embryos injected with antisense ODNs.
Protein synthesis is affected in antisense injected embryos
Since DNA replication was quantitatively normal in antisense-injected
embryos, but the embryos did not divide, it appeared that the one-cell zygotes
were blocked at a point in the cell cycle before mitosis began. A similar effect
is seen when embryos are subjected to a protein synthesis inhibitor. When
fertilized eggs are treated with emetine, DNA synthesis in the first cell cycle
occurs normally, but the embryos arrest during the early phases of mitosis,
and the first cell division fails to occur (Wagenaar, 1983). Because SpOct
antisense ODNs had the same effect on the zygote, we hypothesized that
protein synthesis was affected in some manner in the antisense-injected
embryos. Normally, protein synthesis is activated 5-10 min after the sperm
fuses with the egg membrane, following a change in membrane potential,
release of Ca++ from internal compartments and an increase in intracellular
pH.
To test whether protein synthesis was normal in antisense-injected
zygotes, I measured 35s_incorp0ration into newly synthesized proteins. In
experiments in which antisense ODNs a3 and al were injected, incorporation
of 35s_methionine was reduced to 17% and 37% as compared to untreated
embryos, while ODN c2-injected embryos incorporated 79% and 110% of the
counts incorporated in untreated controls (Figure 3.4A). Protein synthesis was
thus reduced 3-4 fold in antisense-injected embryos, while it was normal in
93
control-injected embryos. SDS-PAGE gels revealed that overall protein
synthesis was substantially reduced in antisense-injected embryos (Figure
3.4B). These results indicate that the injection of antisense ODNs reduces
general protein synthesis significantly, thereby preventing further
development in the one-cell zygote.
94
Measurement of protein synthesis in Ca++ ionophore A23187 activated
eggs
The Ca++ ionophore, A23187, is able to activate sea urchin eggs in the
absence of sperm. Eggs placed in sea water containing 20 p,M A23187
elevate fertilization membranes and physiologically mimic eggs fertilized by
sperm. In order to determine whether the inhibition of protein synthesis
requires the presence of a diploid nucleus, I injected antisense and control
ODNs into eggs activated with A23187 and then measured incorporation of
35S-methionine into newly synthesized proteins. I found that two antisense
ODNs, al and a3, reduced incorporation of ^^S-methionine approximately 2-
2.5-fold as compared to eggs injected with control ODN c2 (Figure 3.5).
95
Discussion
I have demonstrated that injection of antisense ODNs against the
SpOct gene specifically blocks protein synthesis in sea urchin embryos. This
apparently prevents the embryos from traversing the first cell cycle, and as a
result the embryos remain one-celled. In seeking to discover the reason for
the arrest in cleavage, I found that incorporation of ^H-thymidine into DNA
was normal through the first cell cycle, but that protein synthesis was
significantly reduced. As for the immediate reason for the inhibition of
cleavage in antisense-injected zygotes, we hypothesize that an insufficient
amount of proteins required for the process of cell division are made as a
result of the general inhibition of protein synthesis. These proteins, likely to
be the cyclins, are required by the cell to divide at the end of mitosis. Cyclins,
first discovered in sea urchin embryos, are encoded by maternal mRNAs
(Evans et al., 1983). Synthesis of these proteins increase linearly after
fertilization in parallel with general protein synthesis, but they are destroyed
during cell division. Cyclins are then resynthesized in the following cell cycle
from stored transcripts. As mentioned earlier, the SpOct antisensense-induced
effect on sea urchin zygotes resembles that of the protein inhibitory drug
emetine. When emetine is added 25 min post-fertilization, the embryos
divide, suggesting that by this time proteins required for the cleavage process
are synthesized (Wagenaar, 1983). The antisense-induced reduction of overall
protein synthesis in the one-cell sea urchin zygote probably prevents these
molecules from reaching the necessary levels for the cleavage process to be
initiated.
96
When eggs were activated with the Ca++ ionophore A23187, and then
injected with antisense ODNs, I found that protein synthesis was reduced
about 2-fold as compared to noninjected A23187 activated eggs. Protein
synthesis was thus not reduced as much as in sperm-fertilized eggs. The
significance of this is not clear at the present time, but could be ascribed to
subtle differences in the physiology of A23187- and sperm-activated eggs. In
sperm-fertilized eggs, there is first a change in membrane potential before
Ca++ is released from internal stores. This difference could result in some
signal transduction pathways that involve transcription factors like SpOct not
being activated in the case of A23187-treated eggs.
It has been known for a long time that chemically and physically
enucleated sea urchin eggs can activate protein synthesis when treated with
parthenogenic agents. Protein synthesis is activated in sea urchin zygotes in
the presence of actinomycin D, which blocks upto 94% of nuclear
transcription in the zygote (Gross and Cousineau, 1964), and experiments by
Brachet et al. (1963) and Denny and Tyler (1964) showed that enucleated egg
halves can also synthesize proteins. My results indicate the novel finding that
a transcription factor is required for the activation of protein synthesis. This
result suggests two possibilities, the first of these being that the SpOct gene
product as a trnscriptional activator at some stage in the activation of protein
synthesis. Since the SpOct transcript is present in the unfertilized egg, no new
nuclear transcription would be required for its own synthesis. SpOct could
thus be acting as a transcription factor to activate a downstream gene, even
though transcriptional activity in the one-cell zygote is hard to detect. The
second possibility is that SpOct is acting independent of the nucleus of the
97
zygote. For example, the SpOct gene product may be part of the signal
transduction mechanism set in motion when the sperm fuses with its receptor
on the egg membrane, or alternatively, the SpOct protein may play a direct
role in translation itself. These would be unusual roles for a transcription
factor, but if no transcription is indeed occurring in the zygote, it is a distinct
possibility. There is indirect evidence that octamer-binding protein(s) play
important roles in the early development of the sea urchin embryo. Injection
of an excess of the octamer binding site results in lethality, with embryos
dying at various times before the pluteus larva stage. My data is consistent
with this finding, and since I have been unable to obtain evidence for the
presence of octamer-binding proteins other than SpOct in the early embryo, I
believe SpOct is responsible for the regulation of a number of essential genes.
98
Figure 3.1 Effect of antisense ODNs on embryonic development
(A) Locations of antisense (al-a4) and control (cl-c3) ODNs on the SpOct
cDNA. POU refers to the POU domain. ODN sequences are given in
Materials and methods. (B) Effect of injection of antisense ODNs against
SpOct on sea urchin development. Individual experiments using ODNs al (n
= 38) compared to c2 (n = 60), and a2 (n = 34) compared to cl (n = 56). (C)
Pooled injection data for ODNs al, a2 and a3 (n = 197 except for the 8 hr time
point where n = 154) compared to ODNs c l, c2 and c3 (n = 216). The
columns refer to mean percent of embryos at each stage ± s.e.m.
99
Locations of antisense (a1-a4) and control (c1-c3) ODNs on the SpOct
cDNA
c1
c3
POU
c2
---------------------------------------------aaaa
a4 a3 a2 a1
o
o
% embryos % em bryos
Figure 3.1 (continued)
Effect of antisense ODNs on embryonic development
B
100 -
90-
80-
70-
60-
50-
40-
30-
2 0 -
1 0 -
0 -
2 hr 4 hr 6 hr 8 hr
100
80
60
2 hr 4 hr 6 hr 8 hr
100
a2
90
80
70
60
50
40
30
20
10
ta n
8 hr 2 hr 4 hr 6 hr
1 0 0
ct
90
80
70
60
50
40
30
20
1 0
8 hr 2 hr 6 hr 4 hr
g 1-ceii
0 2-cell
^ 4-cell
n 8-cell
g 16-cell
g >16-cell
101
^
% embryos
Figure 3.1 (continued)
Effect of antisense ODNs on embryonic development
110 -
100 -
90 -
80 -
70 -
60 ■
50 -
40 -
30 “
20 -
10 -
o-l*
pooled data
A C A C
2 hr
4 hr
6 hr 8 hr
Figure 3.2 Rescue of antisense ODN-injected embryos.
(A) Injection of double-stranded ODNs. In experiment 1, 48 embyros were
injected with antisense ODN al mixed with ODN cl at 0.8 mg/ml each (A),
while 17 embryos were injected with ODN al annealed to its complementary
strand at 1 mg/ml (D). In experiment 2, 40 and 17 embryos were used for the
same antisense and double-stranded ODNs respectively. Embryos were
followed for 6 hr after injection. The columns refer to mean percent of
embryos ± s.e.m. (B) Rescue of antisense ODN-injected embryos by in vitro
synthesized SpOct mRNA. Antisense ODN a4 (ATCTGGGCATCGGAAG-
ATTGT complementary to -56— 36 in the SpOct cDNA) at 1 mg/ml was co
injected with 1.5 mg/ml of capped SpOct transcript (a4+SpOct), n = 154.
Control embryos were injected with ODN a4 and 1.5 mg/ml of capped mRNA
transcribed from a genomic clone of the S. purpuratus SpHbox8 gene
(a4+SpHbox8), n = 280. The columns refer to mean percent of embryos ±
s.e.m.
103
% e m b r y o s
Rescue of antisense ODN-injected embryos
1 0 0 - /
9 0 - I
8 0 -
7 0 -
6 0 -
5 0 -
4 0 -
3 0 -
2 0 -
1 0 -
A D
2 h r 4 h r
E x p e r i m e n t 1
6 h r
2 h r 4 h r
E x p e r i m e n t 2
6 h r
■
1 - c e l l
6 3
2 - c e l l
m
4 - c e l l
a
8 - c e l l
□
1 6 - c e l l
□
> 1 6 - c e l l
% cleaved
Figure 3.2 (continued)
Rescue of antisense ODN-injected embryos
B
100
90
80
70
60
50
40
30
20
10
0
2 hr 4 hr
a4+SpOct a4+Hbox8 a4+SpOct a4+Hbox8
105
Figure 3.3 Incorporation of ^H-thymidine into DNA in antisense and
control ODN-injected embryos.
Approximately 160 embryos were injected with either antisense ODN al or
a3, and and an equal number with control ODNs cl or c3, in two separate
experiments. A sim ilar number received no treatment (uninjected).
Approximately 200 embryos were treated with aphidicolin. The columns
represent mean percent of embryos ± s.e.m.
106
I n c o r p o r a t i o n o f ^ H - t h y m i d i n e i n t o D N A i n a n t i s e n s e a n d c o n t r o l O D N -
i n j e c t e d e m b r y o s
200
1 8 0 -
1 6 0 -
1 4 0 -
a
120 -
100 -
8 0 -
6 0 -
4 0 -
2 0 -
a n t i s e n s e u n i n j e c t e d c o n t r o l -
i n j e c t e d - i n j e c t e d
107
Figure 3.4 Incorporation of ^^S-methionine into proteins in antisense
and control ODN-injected embryos.
(A) Relative amounts of counts incorporated into embryos after 2 hr of
developm ent. In experiment 1, antisense ODN al was compared to control
ODN c3. Approximately 60 embryos were injected with each ODN. An equal
number of uninjected embryos was used as a control for viability. In
experiment 2, antisense ODN al was compared with control ODN c2.
Approximately 40 embryos were injected with each ODN. (B) SDS-PAGE
gel of 35s-labeled proteins in 2hr- and 5hr-old embryos after injection of
antisense ODN al and control ODN c2. un, uninjected embryos. 80 embryos
were injected with each ODN; each lane contains proteins extracted from
about 40 embryos.
108
^corporation of 3 5 S-m ethionine into proteins in an tisen se and control
ODN-injected embryos
A
u n i n j e c t e d u n i n j e c t e d
% S-35 incorporation % S-35 incorporation
E x p e r i m e n t 1 E x p e r i m e n t 2
Figure 3.4 (continued)
Figure 3.5 Incorporation of ^^S-methionine into Ca++ ionophore A23I87-
activated eggs injected with antisense ODNs.
Relative incorporation of 35 S methionine into uninjected and antisense ODN-
injected eggs activated with A23187. The columns refer to the mean ± s.e.m.
of two experiments in which antisense ODNs al and a3 were injected into 25
and 40 embryos respectively, with an equal number of uninjected embryos as
controls.
Ill
I n c o r p o r a t i o n o f 35s-rnethionine i n t o C a + + i o n o p h o r e A 2 3 1 8 7 - a c t i v a t e d
e g g s i n j e c t e d w i t h a n t i s e n s e O D N s .
120
100
I 80
C O
k .
O
°r 6 0
o
o
c
4 0
CO
5 ?
20
0
u n i n j e c t e d a n t i s e n s e
i n j e c t e d
-
/ I
-
'
,
'
-
'
/ ...................................................
---- )
112
Chapter 4
Conclusions and future directions
Based on the data presented in the previous chapters, I can draw the
following conclusions:
(1) The octamer element in the proximal promoter of the early
histone H2B gene is required for the high level expression of the early H2B
gene. Sea urchin blastulae contain a nuclear factor which binds avidly to this
element. Since human Oct-1 also binds with high affinity to this element, we
conclude that the nuclear protein is probably related to Oct-1. These proteins
show no qualitative difference in binding to octamer elements either in the
early and late histone H2B promoter.
(2) Using a PCR-based strategy, I then cloned a gene encoding a POU-
domain one of which is expressed in early sea urchin blastulae, and was
designated SpOct. SpOct is related to mammalian Oct-1 and Oct-2, and is
expressed during oogenesis and early embryogenesis, peaking at about 8 hr
post-fertilization, after which levels of SpOct transcript decline. The gene is
still expressed in the adult, however, in several different tissues such as the
gonads, gut, tubefeet and coelomic fluid. In collaboration with other
investigators, octamer-binding proteins were purified from sea urchin nuclei
using site-specific DNA affinity chromatography. Peptide sequences obtained
from affinity purified proteins matched the deduced amino acid sequence of
SpOct. From this we conclude that in early blastulae, SpOct is probably the
major octamer-binding protein in the sea urchin.
113
(3) We then investigated the function of SpOct in the early sea urchin
embryo. When antisense ODNs were injected, the one-cell embryos failed to
divide, and embryonic development came to a standstill. Since this phenotype
could be rescued by the injection of SpOct mRNA but not by the injection of
an irrelevant RNA, we conclude that the antisense effect is specific for the
SpOct gene. When the SpOct message was assayed in antisense-injected
embryos by RNase protection, however, there was not a significant decrease
in the level of the transcript as compared to control-injected embryos. From
this result we conclude that the antisense ODNs exert their influence by
blocking translation of the SpOct message.
I then investigated the reason why the embryos did not cleave when
injected with antisense ODNs. I found that DNA replication in the antisense-
injected embryos was normal, but that protein synthesis was reduced
significantly. This reduction in protein synthesis is probably responsible for
the embryos arresting at the one cell stage. Proteins required for cell division,
such as the cyclins, are probably made in insufficient quantities in antisense-
injected embryos resulting in the cleavage arrest phenotype.
Future directions
That a transcription factor is required for protein synthesis in the early
sea urchin embryo is a novel finding. The present data do not differentiate
between SpOct acting as a transcriptional activator, or being involved in the
activation of protein synthesis post-transcriptionally (if we assume there is
virtually no transcriptional activity in the one-cell zygote). Protein synthesis is
activated in enucleated eggs, suggesting that the translation machinery is
114
independent of the nucleus. An important question to answer is whether the
antisense ODNs can block development in the absence of a nucleus. Eggs that
were activated with the Ca++ ionophore A23187 showed a lesser decrease in
protein synthesis than sperm activated eggs. The reason for this difference
may be that increasing intracellular Ca++ levels somehow partially bypasses
the requirement for SpOct-mediated activation of protein synthesis. To
answer the question of whether a nucleus is required for the SpOct antisense
effect to manifest itself, nucleated and enucleated egg halves can be injected
with antisense ODNs, and protein synthesis levels monitored. If both the
enucleated and nucleated halves fail to synthesize proteins normally, a post-
transcriptional role for SpOct would be indicated.
Now that an important regulatory protein has been identified in the sea
urchin embryo, it will be possible to ask many interesting questions. An entry
into the regulatory hierarchy of the early embryo can be gained by studying
the regulation of the SpOct gene itself. A sea urchin genomic library can be
screened with a probe made from the 5' end of the SpOct cDNA in order to
identify potential regulatory regions of the SpOct gene. A problem that might
be encountered with this approach, however, may be the extensive 5' non
translated region of the SpOct message, which is 4-8 kb in length. This could
make it extremely difficult to identify the regulatory sequences of the gene.
Another interesting question is the presence of at least four different mRNAs
apparent on RNA gel blots. This heterogeneity of transcripts could have a
regulatory function in the embryo, as is demonstrated by the alternate splicing
of the mammalian Oct-2 transcript.
115
Raising an antibody against SpOct will enable us to determine the
distribution of SpOct protein in the embryo. We would then know whether
SpOct is expressed in a subset of cells in the embryo, or if it ubiquitously
expressed. An interesting question that could be addressed would be the
localization of SpOct protein in antisense-injected embryos. Since the SpOct
transcript seems to be intact in these embryos as assayed by RNase protection,
antibody staining would tell us if the protein is indeed absent, as we suspect.
Immunoprecipitation methods would also enable us to attempt to identify
other genes in the embryo that are regulated by SpOct.
Lastly, the cloning of SpOct has enabled Dr. Frank Calzone at the
University of California, Irvine to investigate the role of protein-protein
interactions in the spatial control of gene expression. His work has revealed
that SpOct may play a role in the mechanism that restricts expression of the
Cyllla actin gene to the aboral ectoderm of the sea urchin embryo.
116
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Creator Char, Bharat Raghunath (author)

Core Title A POU gene expressed in the early sea urchin embryo: Structure, evolution and requirement for embryonic development

Degree Doctor of Philosophy

Degree Program Biochemistry and Molecular Biology

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

Tag health sciences, human development,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Maxson, Robert (committee chair), Roy-Burman, Pradip (committee member), Tahara, Stanly M. (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c17-13669

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Legacy Identifier DP21640.pdf

Dmrecord 13669

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Type texts

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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...

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