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R Maxson

Publications and source records attributed to R Maxson.

At least 37 records · Page 2Linked to original sources
PubMed1994

Promoter analysis meets pattern formation: transcriptional regulatory genes in sea urchin embryogenesis.

Analyses of spatial and temporal gene control mechanisms in the sea urchin embryo have identified several important trans-regulatory factors, including some that are related to known developmental control genes of the fly and mouse. Recent advances in gene perturbation technologies, including the use of antisense oligonucleotides to target mRNAs in early-stage embryos, as well as the injection of mRNAs into zygotes to express genes ectopically, have made it possible to test the functions of such factors directly.

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PubMed1994

A POU gene required for early cleavage and protein accumulation in the sea urchin embryo.

SpOct is a POU gene expressed during oogenesis and early embryogenesis of the sea urchin, Strongylocentrotus purpuratus. In the first use of antisense technology in the sea urchin embryo, we report that disruption of SpOct gene function in 1-cell zygotes by the injection of antisense oligodeoxynucleotides arrests development prior to the first cell division. We show that single-stranded antisense oligodeoxynucleotides specifically block cleavage, and that injection of SpOct mRNA overcomes this block. The accumulation of [35S]methionine into zygotically synthesized protein is significantly reduced in antisense-injected embryos. DNA synthesis is also reduced by the antisense regimen as expected from the antisense inhibition of protein accumulation. That protein accumulation prior to the first cleavage is retarded by antisense targeting of a transcription factor is very surprising in light of classical work showing that the activation of protein synthesis does not require zygotic transcription. We conclude that either some new transcription is obligate for the accumulation of new protein, or that the SpOct gene plays a novel, non-transcriptional role in this process.

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PubMed1993

Direct induction of DNA hypermethylation in sea urchin embryos by microinjection of 5-methyl dCTP stimulates early histone gene expression and leads to developmental arrest.

The role of DNA methylation in gene inactivation has been studied extensively in vertebrates but it is not clear whether it serves similar functions in other organisms. We devised a novel approach to induce hypermethylation of both endogenous and injected DNA in the sea urchin Lytechinus pictus in order to study the effect of DNA methylation on gene expression in this invertebrate. By injecting 5-methyl dCTP either alone or together with a cloned DNA construct into fertilized sea urchin eggs, replicating DNA became hypermethylated from the random incorporation of the methylated nucleotide in place of cytosine during DNA synthesis. During subsequent rounds of replication, the injected 5-methyl dCTP became depleted but methylation at CpG sites was still elevated presumably due to the action of a methyltransferase enzyme. Using this approach, we studied the effect of hypermethylation on two members of the sea urchin multigene family, the early H2B and the late H2B genes. De novo methylation was shown to occur at known cis-regulatory regions of the genes. The effect of methylation on gene activity was probed using RNase protection assay. Methylation resulted in increased early H2B histone gene expression but had no effect on late H2B histone gene expression. These results demonstrate that methylation does not necessarily inactivate genes in the sea urchins as previously thought. Interestingly, the development of embryos injected with 5-methyl dCTP typically was arrested at the blastula stage, and analysis of the genomic DNA extracted from injected embryos showed a significant increase in the endogenous methylation content. These data suggest that perturbation of methylation patterns in developmental sea urchin embryos may be responsible for the developmental arrest through altering the gene expression pattern.

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PubMed1993

SpOct, a gene encoding the major octamer-binding protein in sea urchin embryos: expression profile, evolutionary relationships, and DNA binding of expressed protein.

We have characterized a sea urchin gene, SpOct, that encodes a 78-kDa POU-domain protein related to mammalian Oct-1 and Oct-2. The SpOct protein binds octamer elements in the promoters of the alpha H2B (Bell et al., 1992, Dev. Biol. 150, 363-371) and CyIIIa actin genes, and it closely resembles the major octamer-binding activity obtained from sea urchin blastula nuclear lysates in the size of its DNase I footprint on a canonical octamer element and in its relative binding affinity (Kr) for the octamer element versus poly(dAT) (1.4 x 10(4)). Moreover, partial protein sequences obtained from affinity-purified octamer-binding protein match sequences present in SpOct. These data suggest that SpOct is closely related to, if not identical with, the major octamer-binding activity in blastula nuclear extracts. RNA gel blots reveal four forms of SpOct mRNA, ranging in size from 4 to 12 kb. They are regulated coordinately in the embryo: all are present in the unfertilized egg, increase 28-fold in amount by the 8-hr blastula stage, and decline 6-fold by the 12-hr blastula stage. The same four size classes of SpOct mRNAs are present in several adult tissues, although their relative amounts vary. The temporal profile of SpOct mRNA expression in embryos closely resembles that of the alpha histone H2B gene. Our previous work (Bell et al., 1992) showed that expression of the alpha H2B gene in blastula-stage embryos was entirely dependent on an octamer element. Together, these data strongly suggest that SpOct may be the key regulator of the alpha H2B gene.

Amino Acid Sequence↗
PubMed1992

An octamer element is required for the expression of the alpha H2B histone gene during the early development of the sea urchin.

Early (alpha) histone genes are one of several histone gene families in the sea urchin genome. They are expressed at high levels in blastula-stage embryos and are inactivated by the early gastrula stage. By microinjecting mutant early H2B genes into sea urchin zygotes and monitoring their transcriptional activity in blastula- and gastrula-stage embryos, we sought to identify the cis-regulatory elements responsible for this dramatic change in early H2B gene activity. We found that deletion of DNA 5' of -71 and 3' of +591 did not affect the timing or magnitude of early H2B gene expression. Neither was early H2B gene expression affected by the replacement of sequences downstream of -36 with the corresponding region of the L1 late H2B gene, expressed after the peak transcription of the early H2B gene. Further deletion of early H2B promoter sequences from -71 to -56, removing a conserved octamer element, resulted in near-complete inactivation of the early H2B gene in both blastula- and gastrula-stage embryos. Also inactivating early H2B gene expression were an internal deletion of the octamer element and a base substitution mutation that altered its sequence. This base substitution mutation also caused a parallel reduction in the ability of the octamer element to bind a factor present in nuclear extracts of sea urchin blastulae. These data strongly suggest that the proper expression of the early H2B gene in cleavage- and blastula-stage embryos depends on the octamer element and a factor with which it interacts.

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PubMed1991

Activation of the L1 late H2B histone gene in blastula-stage sea urchin embryos by Antennapedia-class homeoprotein.

The L1 late H2B histone gene of the sea urchin Strongylocentrotus purpuratus is transcriptionally activated in late blastula stage embryos by a mechanism that depends on an enhancer element located 3' of the gene (Zhao et al., 1990). A protein factor, designated H2B abp 1, binds this element at a site that resembles the consensus recognition sequence of Antennapedia-class homeodomain proteins. We demonstrate here that Antennapedia (Antp) and Hbox4 proteins, members of the Antennapedia class of homeoproteins from Drosophila and sea urchin respectively, bind the L1 H2B abp 1 site, and that the Drosophila Antp protein acts through this site to trans-activate the L1 H2B gene, in vivo. In addition, RNA gel blot analysis demonstrated that Hbox4 transcripts accumulate in developing embryos with a time course that closely resembles that of H2B adp 1 DNA binding activity and the activity and the transcription rate of the L1 late H2B gene. Finally, we show that antibody prepared against the sea urchin Hbox4 protein, a member of the Abd-B subclass of the Antennapedia class, specifically inhibits binding of the H2B abp 1 factor to the L1 H2B enhancer, suggesting that H2B abp 1 is encoded by Hbox4 or a closely related gene.

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PubMed1990

Activation of a late H2B histone gene in blastula-stage sea urchin embryos by an unusual enhancer element located 3' of the gene.

In the sea urchin embryo, late histone genes are transcribed at low levels during cleavage and blastula formation and at substantially higher levels in later stages of embryogenesis. To investigate the molecular basis of the stage-specific expression of a late H2B histone gene, we injected mutant genes lacking portions of 5'- and 3'-flanking regions into Lytechinus pictus embryos and monitored their expression by RNase protection. A 200-bp region located 489 bp downstream of the mRNA 3' terminus was necessary for the increase in transcription of the late H2B gene at the mid-blastula stage of development. DNase I and methylation interference footprint analyses located only one factor-binding site in this region, and gel mobility shift experiments showed that the DNA-binding activity of this factor (designated H2B abp 1) paralleled the transcriptional activity of the L1 H2B gene. Additional mutagenesis and microinjection experiments located the activator element to a 32-bp DNA segment that includes the H2B abp 1-binding site. These experiments also showed that the 32-bp fragment functions independently of position and orientation and therefore has the hallmarks of an enhancer. That this fragment contains most or all of the L1 H2B gene transcription-stimulatory activity makes it unusual among enhancerlike elements, which generally consist of several clustered factor-binding sites that act additively or cooperatively to affect transcription. The nucleotide sequence of the L1 H2B enhancer element suggests that the trans-acting factor that interacts with it is a member of the antennapedia or engrailed class of homeodomain proteins.

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PubMed1989

Cell cycle regulation of H2b histone octamer DNA-binding activity in Chinese hamster lung fibroblasts.

The promoter regions of H2b histone genes contain a 14-base-pair element which includes the octamer ATTTGCAT. Mutational analysis has implicated the octamer element in the cell cycle-dependent expression of H2b histone genes. In this report, we address the question of whether the DNA-binding activity of the octamer transcription factor is itself cell cycle regulated. By using a gel mobility shift assay, we measured the relative amounts of octamer-binding activity during various phases of the cell cycle in serum-synchronized Chinese hamster fibroblasts. We found that the activity increased approximately fivefold between late G1 phase and early S phase and then decreased threefold between late S phase and G2 phase. These cell cycle-dependent changes in octamer DNA-binding activity may in part account for the selective transcription of H2b histone genes in late G1 and S phases.

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PubMed1988

Synthesis and turnover of late H2B histone mRNA in developing embryos of the sea urchin, Strongylocentrotus purpuratus.

In sea urchins, "early" histone proteins are synthesized during cleavage and blastula formation, "late" histone proteins in subsequent stages of development. To understand the molecular mechanisms responsible for this ontogenic switch in histone subtype synthesis, we determined the absolute amounts, rates of synthesis, and rates of turnover of late H2b histone mRNAs during development. We showed previously that late H2b mRNA comprises several mRNA isotypes. In this study, we used both a class-specific DNA probe to measure the amounts of the late H2b mRNA isotypes collectively, and a gene-specific probe to measure amounts of a particular late H2b mRNA encoded by a gene known as L1. We found that the amount of late H2b mRNA increased dramatically from 85,000 molecules per embryo in the 16-hr blastula to a peak of 670,000 molecules per embryo in the 24-hr mesenchyme blastula, and fell to 380,000 molecules per embryo in the 72-hr pluteus larva. The L1 late H2b mRNA achieved its maximum abundance earlier than the late H2b mRNA class as a whole, reaching a peak of 34% of total late H2b in the 14-hr blastula and declining to 7% in the pluteus larva. Measurements of the rate of incorporation of [3H]uridine into late class H2b mRNA, performed by a novel in vivo isotope incorporation method, enabled us to calculate both synthesis rates and half-lives of late H2b mRNA during development. These calculations showed (1) that the increase in late H2b mRNA level between 16 and 24 hr postfertilization is regulated primarily if not entirely at the level of mRNA synthesis; and (2) that the half-life of late H2b mRNA is comparatively short, around 20 min, at all stages examined.

Animals↗
PubMed1988

Closely linked early and late histone H2B genes are differentially expressed after microinjection into sea urchin zygotes.

An early and a late histone H2B gene from the sea urchin Stronglyocentrotus purpuratus were linked in a single plasmid and injected into the eggs of the sea urchin Lytechinus pictus. The levels of transcripts of injected early and late genes and of endogenous early genes were monitored during development by a ribonuclease protection assay. Transcripts of both the injected and endogenous early genes peaked during the blastula stage and decreased severalfold by the mesenchyme blastula stage. Transcripts of the injected late gene became detectable at the blastula stage and increased in amount subsequently, until at least the early gastrula stage, 28 hr after fertilization. Thus, the pattern of expression of the injected early and late H2B genes is similar to that of their endogenous counterparts. These results show that DNA sequences regulating the temporal pattern of early and late H2B gene expression must lie within the cloned DNA segments; i.e., within 600 base pairs of the early H2B gene and 3 kilobases of the late H2B gene.

Animals↗
PubMed1988

Both basal and ontogenic promoter elements affect the timing and level of expression of a sea urchin H1 gene during early embryogenesis.

Late histone H1-beta mRNA accumulates with the correct ontogenic pattern following microinjection of the cloned gene into fertilized sea urchin eggs. Sequences upstream of the gene encoding the sea urchin H1-beta protein contain both basal and developmentally regulated elements. One late H1-specific activator sequence (USE IV) is required for the accumulation of mRNA following the blastula stage of development. All late H1 genes also contain a highly conserved GC-rich sequence resembling a low-affinity binding site for the mammalian transcription factor Sp1 that is required for basal expression of the H1-beta gene at all stages of embryogenesis. When this GC-rich sequence (GGGCTG) is converted to a perfect core Sp1 sequence (GGGCGG), the H1-beta transcripts accumulate to much greater levels and their peak accumulation is shifted to the early blastula stage rather than late blastula and gastrula stages of development. Coincidently, early H1 genes, whose peak expression is also at the early blastula stage, all contain the same core consensus sequence (GGGCGG). Thus, both gene-specific activator sequences, as well as sequences that resemble sites for general transcription factors, may play a major role in determining the temporal patterns of gene expression during early embryogenesis.

Animals↗
PubMed1988

Differential stimulation of sea urchin early and late H2B histone gene expression by a gastrula nuclear extract after injection into Xenopus laevis oocytes.

Sea urchin early histone genes are active in preblastula embryos; late histone genes are maximally expressed during subsequent stages of embryogenesis. We used the Xenopus laevis oocyte to assay for trans-acting factors involved in this differential regulation. Sea urchin nuclear proteins were prepared by extracting gastrula-stage chromatin successively with 0.45, 1, and 2 M NaCl. We injected three fractions into oocytes along with plasmids bearing sea urchin early and late H2b histone genes. While neither the 0 to 0.45 M nor the 1 to 2 M salt fraction affected H2b gene expression, the 0.45 to 1 M salt fraction stimulated early and late H2b mRNA levels significantly. Late H2b gene expression was stimulated preferentially when the early and late genes were coinjected into the same oocytes. This extract did not stimulate the accumulation of transcripts of injected herpesvirus thymidine kinase genes or of the sea urchin Spec 1 gene, suggesting that the stimulatory activity is not a general transcription factor. We localized the DNA sequence required for the stimulatory effect to a region of the late H2b gene located between -43 and +62 relative to the transcription start site. A component of the 0.45 to 1 M salt wash fraction specifically bound to the 105-base-pair late gene DNA sequence and to the corresponding early gene fragment. The abundance of this binding activity decreased on a per genome basis during early development of the sea urchin.

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PubMed1987

Evolution of late H2A, H2B, and H4 histone genes of the sea urchin, Strongylocentrotus purpuratus.

Sea urchins possess several distinct sets of histone genes, including "early" genes, maximally active in cleavage and blastula stages, and "late" genes, active from the late blastula stage onwards. We determined the nucleotide sequences of six sea urchin (Strongylocentrotus purpuratus) late histone genes located on four genomic segments. Comparative analysis of these sequences identified several conserved elements in 5' flanking regions, including the sequences ATGPyATANTATA shared by all late genes and GGCGGGAAATTGAAAA shared by two late H4s. Comparisons of protein-coding sequences of late H4 and H2B genes with their early counterparts showed that silent sites have diverged to the theoretical maximum, indicating that early and late histone gene classes diverged at least 200 million years ago. Since extant echinoderms evolved from a common ancestor at about that time, it is likely that early and late histone gene sets are characteristic of all echinoderm groups. Amino acid sequences derived from nucleotide sequences of late H2A and H2B gistone genes differ substantially from amino acid sequences of their late counterparts. Most such differences are in highly mutable positions. A few, however, occur in positions that do not mutate frequently and thus may reflect functional differences between the early and late forms of the H2A and H2B proteins.

Amino Acid Sequence↗
PubMed1987

Differential expression of early and late embryonic histone genes in adult tissues of the sea urchin Strongylocentrotus purpuratus.

The sea urchin synthesizes distinct classes of histone mRNAs at different stages of development. "Early" embryonic histone mRNAs are synthesized in large amount in cleavage and blastula stage embryos. "Late" embryonic histone mRNAs are the predominant forms in postblastula embryos. To learn more about how early and late histone genes are regulated during the life cycle of the sea urchin and to search for additional classes of developmentally regulated histone mRNAs, we examined histone mRNAs in sea urchin adult tissues. Using methods of primer extension and S1 nuclease protection, we found that tube foot, intestine, testis, and ovary contain a subset of the several H2b mRNA species synthesized by the embryo. We detected early H2b mRNA in ovary, but not in other tissues. Three late H2b mRNA species were present in all tissues tested, while a fourth late H2b was not detected. Using a probe that hybridized specifically with transcripts of a single-copy late H2b gene, we found that this gene was transcribed in both embryos and adults. Interestingly, its level of expression relative to other late H2b genes varied among tissues. Finally, we identified two H2b mRNA species that were distinct from early and late embryonic forms and were synthesized only in adult tissues.

Animals↗
PubMed1985

Simultaneous expression of early and late histone messenger RNAs in individual cells during development of the sea urchin embryo.

The transition from early (E) to late (L) histone gene expression in developing sea urchin (Strongylocentrotus purpuratus) embryos was examined for H2B, H3, and H4 mRNAs by in situ hybridization of class-specific probes. Hybridization patterns indicate that the shift from E to L mRNAs occurs gradually and simultaneously in all blastomeres. Thus, during the transition the ratio of L to E mRNAs is similar in most cells. This suggests that no sudden changes in histone composition occur in individual cells which might be related to alterations in gene expression associated with differentiation of cell lineages. Around the midpoint of the transition, clusters of cells progressively appear which contain little, if any, E or L histone mRNA. This modulation of expression is coordinated for the three late genes examined because most individual cells contain either high or low levels of all three mRNAs. At blastula stage these clusters of unlabeled cells appear to be randomly distributed throughout the embryo. Subsequently the unlabeled regions expand and are found predominantly in aboral ectoderm as these cells cease to divide. Thus, the L/E histone mRNA ratio is not differentially regulated in diverse cell lineages, and the major differences in total histone mRNA content among individual cells may be related to cell cycle and/or the cessation of division.

Animals↗
PubMed1983

Distinct organizations and patterns of expression of early and late histone gene sets in the sea urchin.

The set of histone genes that are active late in embryogenesis of the sea urchin (Strongylocentrotus purpuratus) are present in 5-12 copies per genome and, unlike the clustered, tandemly arrayed early histone genes, are dispersed and irregularly arranged. Late H2B gene expression is activated by events accompanying fertilization and its mRNAs a, first detectable by as early as 6 h of development (16 cells) and increase only slightly in amount during the period of rapid cleavage between 6 and 14 h. However, during the short interval between 14 and 16 h, while the amount of early histone mRNA is declining, there is a greater than 15-fold burst in the rate of late H2B mRNA accumulation.

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