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E H Davidson

Publications and source records attributed to E H Davidson.

At least 19 recordsLinked to original sources
PubMed1992

Complexity of sea urchin embryo nuclear proteins that contain basic domains.

We describe a quantitative two-dimensional gel electrophoretic analysis of nuclear extract from 24-hr sea urchin embryos. The extract was fractionated by using a weak cation-exchange resin, and eight known DNA-binding proteins were shown to be entirely included in a salt eluate that releases proteins containing basic domains. This fraction and a lower-salt fraction containing the majority of the protein species were mapped two-dimensionally by using new algorithms that permit reproducible spot identification, storage of intensity and map-position data, and subtractive comparison of one pattern with respect to another. By reference to a previously characterized DNA-binding factor, spot intensity could be interpreted in terms of the number of molecules per embryo nucleus. A map was constructed displaying all nuclear proteins containing basic domains that are present within the concentration range per nucleus of a set of known DNA-binding factors of the sea urchin embryo. The map includes 265 spots that fulfill both of these criteria, probably representing about 100 different protein species.

Animals↗
PubMed1992

Calculation of sequence divergence from the thermal stability of DNA heteroduplexes.

Measurements are reported of the thermal stability of DNA heteroduplexes between clones of the eta-globin pseudogene from a variety of primates. The known sequences of this 7.1-kb region differ from each over a range from 1.6% for human versus chimp to nearly 12% for human versus spider monkey. Thermal stability was determined by standard hydroxyapatite thermal elution, and the results show a precisely linear decrease in thermal stability with divergence. The slope of the regression line is 1.18% sequence divergence per degree centigrade reduction in thermal stability.

Animals↗
PubMed1992

Territorial expression of three different trans-genes in early sea urchin embryos detected by a whole-mount fluorescence procedure.

We have developed a new procedure for detection of the protein product of chloramphenicol acetyltransferase (CAT) reporter genes in whole mounted sea urchin embryos. The position of a commercially available anti-CAT antibody is visualized by video or confocal microscopy, and thus the spatial domains of exogenous reporter gene expression can be determined with regard to the intact three-dimensional structures of the embryo. We show that in pluteus stage embryos CAT protein expression patterns for SM50 . CAT or CyIIIa . CAT reporter genes are similar to those previously obtained by in situ hybridizations with radioactive probes. Taking advantage of the superior resolution of cellular CAT expression patterns using the antibody visualization method, we found for the first time that, in addition to the expression in aboral ectoderm, some cells in the ciliated band of the pluteus express CyIIIa . CAT. The expression of a new fusion construct, CyIIa . CAT, was also examined. As expected from the localization of endogenous CyIIa mRNA, CAT protein was expressed under control of the CyIIa promoter in gut and skeletogenic mesenchyme cells.

Animals↗
PubMed1992

Modulation of sea urchin actin mRNA prevalence during embryogenesis: nuclear synthesis and decay rate measurements of transcripts from five different genes.

The parameters determining the prevalence of the five actin gene transcripts that are differentially expressed during embryogenesis in the sea urchin Strongylocentrotus purpuratus were measured in vivo. These results and previous studies show that the developmental appearance of the cytoskeletal actin mRNA, CyI, CyIIa, CyIIb, and CyIIIa, and the muscle-specific actin message M, is transcriptionally regulated. The cytoskeletal actin genes are activated at the 64-cell stage or shortly thereafter. At this stage the specification of the early embryonic lineages has just completed. M gene transcription was detected only after muscle cells appear in the late embryo. The CyI, CyIIa, and CyIIb genes are transcribed at a moderate rate that does not vary significantly during development. In contrast, during late cleavage CyIIIa transcripts are produced at the maximum rate observed for structural genes in this embryo. In later stages, CyIIIa transcription is reduced at least 30-fold. The rate at which new actin transcripts enter the cytoplasm was also measured. The data show that essentially all primary actin gene transcripts are processed into mature messages. Actin message stability does not change during development. The mRNA half-life of the various messages was found to range from 4 hr to greater than 14 hr.

Actins↗
PubMed1992

Expression of spatially regulated genes in the sea urchin embryo.

Spatially controlled genes expressed in the early sea urchin embryo have been characterized, and the patterns of expression in terms of the mechanisms by which this embryo accomplishes its initial set of founder cell specifications are the subject of current discussion. Sea urchin transcription factors that have been cloned are classified with respect to their target sites and the genes they regulate. Among the best known of the sea urchin cis-regulatory systems is that controlling expression of the Cyllla gene, which encodes an aboral ectoderm-specific cytoskeletal actin. The Cyllla regulatory domain includes approximately 20 sites of DNA-protein interaction, serviced by about ten different factors. Certain of these factors are known to negatively control spatial expression, while others positively regulate temporal activation and the level of Cyllla gene expression. Differential, lineage-specific gene expression is instituted in the sea urchin embryo by mid-late cleavage, prior to any cell migration or overt differentiation, and shortly following lineage segregation.

Animals↗
PubMed1992

SpCoel1: a sea urchin profilin gene expressed specifically in coelomocytes in response to injury.

SpCoel1 is a single copy gene that is specifically expressed in most of the coelomocytes of the adult purple sea urchin, Strongylocentrotus purpuratus. The 4-kb transcript from this gene has a relatively short (426 nucleotide) open reading frame (ORF) with long 3' and 5' untranslated regions. The ORF encodes a protein that has strong amino acid sequence similarity to profilins from yeast to mammals. Transcript titrations of SpCoel1 show significant increases per coelomocyte in animals that have been physiologically challenged. Increases in transcript levels are of similar magnitudes between animals receiving different treatments, such as injuries from needle punctures or from injections of foreign cells. The evidence presented here implies a molecular mechanism by which this lower deuterostome defense system responds to external insult, viz that an external "injury signal" activates a signal transduction system, which in turn mediates the alterations in cytoskeletal state that are required for coelomocyte activation.

Amino Acid Sequence↗
PubMed1992

Automated sequential affinity chromatography of sea urchin embryo DNA binding proteins.

An automated method of running a tandem sequence of oligonucleotide affinity columns was used to purify factors that interact specifically with cis-regulatory sites of the CyIIIa cytoskeletal actin gene of the sea urchin embryo (Strongylocentrotus purpuratus). The method allows quantitative enrichment in a single chromatographic run of up to 12 different sequence-specific DNA binding proteins, each of which may then be readily purified to homogeneity by methods such as preparative gel electrophoresis. The affinity chromatography and identification of six different CyIIIa-regulatory factors is described, and the general utility of the method is discussed.

Actins↗
PubMed1991

Retroviral-like element in a marine invertebrate.

Retroviral-like elements (RL elements) include retroviruses and long terminal repeat (LTR)-containing retrotransposons. We report the presence of sea urchin RL elements (termed SURL) in eight species of sea urchins and find that these RL elements belong to several subfamilies. The complete DNA sequence of one SURL element in Tripneustes gratilla is 5266 base pairs long, including 254-nucleotide-long identical long terminal repeats (LTRs). It contains a single open reading frame nearly 4 kilobases long including the gag and pol genes. Comparison of conserved DNA sequences of RL elements from different sea urchin species indicates that active elements have been inserting copies into echinoid genomes for at least 200 million years.

Animals↗
PubMed1991

Cell type specification during sea urchin development.

Recent discoveries indicate that cell lineages and fates play a key role in the establishment of spatially restricted gene expression during sea urchin development. Unique sets of founder cells generate five territories of gene expression by means of an invariant pattern of complete cleavage. Cell lineage analysis demonstrates that the second embryonic axis, the oral-aboral axis, is specified with reference to the first cleavage plane. In the undisturbed embryo, clones that contribute to one territory or another begin to appear at the third cleavage, and founder cell segregation to all five territories is completed by the sixth cleavage. Founder cell segregation is a key feature of mechanisms that establish the spatially defined gene activity of sea urchin embryogenesis.

Animals↗
PubMed1991

Comparison of the bindin proteins of Strongylocentrotus franciscanus, S. purpuratus, and Lytechinus variegatus: sequences involved in the species specificity of fertilization.

Bindin is the sea urchin sperm acrosomal protein that is responsible for the species-specific adhesion of the sperm to the egg. Two new bindin cDNA sequences that contain the entire open reading frame for the binding precursor are reported: one for Strongylocentrotus franciscanus and one for Lytechinus variegatus. Both contain inverted repetitive sequences in their 3' untranslated regions, and the S. franciscanus cDNA contains an inverted repetitive sequence match between the 5' untranslated region and the coding region. The middle third of the mature bindin sequence is highly conserved in all three species, and the flanking sequences share short repeated sequences that vary in number between the species. Cross-fertilization data are reported for the species S. purpuratus, S. franciscanus, L. variegatus, and L. pictus. A barrier to cross-fertilization exists between the sympatric Strongylocentrotus species, but there is no barrier between the allopatric Lytechinus species.

Amino Acid Sequence↗
PubMed1991

Gene regulatory factors of the sea urchin embryo. I. Purification by affinity chromatography and cloning of P3A2, a novel DNA-binding protein.

The P3A2 regulatory protein interacts with specific sites in the control region of the CyIIIa actin gene. Previous studies showed that this interaction is required to confine expression of a CyIIIa.CAT fusion to the aboral ectoderm, the embryonic territory in which CyIIIa is normally utilized. P3A2 also binds specifically to similar target sites located in the regulatory region of the SM50 gene, which is expressed only in skeletogenic mesenchyme lineages. The P3A2 factor was purified by affinity chromatography from nuclear extracts of 24 h sea urchin embryos, and partial peptide sequences were used to isolate a cDNA clone encoding the complete protein. There are no significant similarities between P3A2 and any other protein in existing sequence data bases. P3A2 thus includes a novel type of DNA-binding domain. To examine the differential utilization of P3A2 in CyIIIa and SM50 genes, we measured the specific affinity of this protein for the various target sites in the regulatory DNAs of each gene, and identified the core target site sequences. The stability of P3A2 complexes formed with SM50 target sites is 50-100 times greater than that of the complexes formed with CyIIIa target sites, though the factor binds to very similar core sequence elements. P3A2 is one of at least twelve different proteins whose interaction with CyIIIa regulatory DNA is required for correct developmental expression. The results reported demonstrate that it might be possible to purify most of these regulatory proteins, or any other specific DNA-binding proteins of the sea urchin embryo, by using the simple procedures described for P3A2.

Amino Acid Sequence↗
PubMed1991

Gene regulatory factors of the sea urchin embryo. II. Two dissimilar proteins, P3A1 and P3A2, bind to the same target sites that are required for early territorial gene expression.

Previous work demonstrated that a negative regulatory interaction mediated by factor(s) termed 'P3A' is required for correct territory-specific gene expression in the sea urchin embryo. A probe derived from a P3A target site in the skeletogenic SM50 gene of Strongylocentrotus purpuratus was used to isolate a cDNA clone coding for a factor that binds specifically to this site. This factor, called P3A1, contains two sequence elements that belong to the Zn finger class of DNA-binding motifs, and in these regions is most closely similar to the Drosophila hunchback factor. The P3A1 factor also binds to a similar target sequence in a second gene, CyIIIa, expressed in embryonic aboral ectoderm. Another sea urchin embryo protein factor, P3A2, has been isolated by affinity chromatography and cloned, as described in Calzone et al. Development 112, 335-350 (1991). P3A2 footprints the same target sites in the SM50 and CyIIIa genes as does P3A1, but lacks the Zn finger sequence motifs and in amino acid sequence is almost entirely dissimilar to P3A1. A deletion analysis of P3A2 delimited the DNA-binding region, revealing that five specific amino acids in the first P3A1 finger region and four in the second P3A1 finger region are also present in equivalent positions in P3A2. The P3A1 and P3A2 factors could function as regulatory antagonists, having evolved similar target specificities from dissimilar DNA-binding domains.

Amino Acid Sequence↗
PubMed1991

Spatial mechanisms of gene regulation in metazoan embryos.

The basic characteristics of embryonic process throughout Metazoa are considered with focus on those aspects that provide insight into how cell specification occurs in the initial stages of development. There appear to be three major types of embryogenesis: Type 1, a general form characteristic of most invertebrate taxa of today, in which lineage plays an important role in the spatial organization of the early embryo, and cell specification occurs in situ, by both autonomous and conditional mechanisms; Type 2, the vertebrate form of embryogenesis, which proceeds by mechanisms that are essentially independent of cell lineage, in which diffusible morphogens and extensive early cell migration are particularly important; Type 3, the form exemplified by long germ band insects in which several different regulatory mechanisms are used to generate precise patterns of nuclear gene expression prior to cellularization. Evolutionary implications of the phylogenetic distribution of these types of embryogenesis are considered. Regionally expressed homeodomain regulators are utilized in all three types of embryo, in similar ways in later and postembryonic development, but in different ways in early embryonic development. A specific downstream molecular function for this class of regulator is proposed, based on evidence obtained in vertebrate systems. This provides a route by which to approach the comparative regulatory strategies underlying the three major types of embryogenesis.

Animals↗
PubMed1991

Differential stability of expression of similarly specified endogenous and exogenous genes in the sea urchin embryo.

The object of these experiments was to determine whether competitive titration in vivo of factors required for expression of the CyIIIa.CAT fusion gene would affect expression of the endogenous CyIIIa gene in the same embryos. Earlier work showed that expression of this fusion gene after injection into sea urchin eggs is stoichiometrically reduced when low molar excesses of DNA fragments containing only its regulatory domain are coinjected. In order to compare endogenous (i.e. CyIIIa) and exogenous (i.e. CyIIIa.CAT) expression simultaneously in embryos bearing excess competitor regulatory DNA, we developed, and here describe, a new procedure for generating transgenic sea urchin embryos in which all of the cells in many embryos, and most in others, bear the exogenous DNA. Such large reduction of mosaicism can be achieved by multiple injection of the exogenous DNA fragments into fertilized eggs. Using this method, we demonstrate that at a level of competitor DNA incorporation which reduces CyIIIa.CAT expression by 85%, endogenous CyIIIa mRNA levels are wholly unaffected. Nor is spatial expression of the endogenous CyIIIa gene disturbed. Since the CyIIIa.CAT genes are properly expressed under control of the CyIIIa regulatory sequences, they must participate in the same set of necessary DNA-protein interactions. However, we infer from the results that we report here that the regulatory complexes in the endogenous CyIIIa gene are greatly stabilized relative to those of the exogenous CyIIIa.CAT genes.

Animals↗
PubMed1991

Macromere cell fates during sea urchin development.

This paper examines the cell lineage relationships and cell fates in embryos of the sea urchin Strongylocentrotus purpuratus leading to the various cell types derived from the definitive vegetal plate territory or the veg2 tier of cells. These cell types are gut, pigment cells, basal cells and coelomic pouches. They are cell types that constitute embryonic structures through cellular migration or rearrangement unlike the relatively non-motile ectoderm cell types. For this analysis, we use previous knowledge of lineage to assign macromeres to one of four types: VOM, the oral macromere; VAM, the aboral macromere, right and left VLM, the lateral macromeres. Each of the four macromeres contributes progeny to all of the cell types that descend from the definitive vegetal plate. Thus in the gut each macromere contributes to the esophagus, stomach and intestine, and the stripe of labeled cells descendant from a macromere reflects the re-arrangement of cells that occurs during archenteron elongation. Pigment cell contributions exhibit no consistent pattern among the four macromeres, and are haphazardly distributed throughout the ectoderm. Gut and pigment cell contributions are thus radially symmetrical. In contrast, the VOM blastomere contributes to both of the coelomic pouches while the other three macromeres contribute to only one or the other pouch. The total of the macromere contribution amounts to 60% of the cells constituting the coelomic pouches.

Animals↗
PubMed1990

Sequences of the CyIIIa actin gene regulatory domain bound specifically by sea urchin embryo nuclear proteins.

Expression of the CyIIIa cytoskeletal actin gene is a marker of differential gene activation in the aboral ectoderm of the early sea urchin embryo. Gene transfer experiments have defined a 2,300 nucleotide cis-regulatory domain required for the correct spatial and temporal control of this gene. This domain includes at least 20 sites at which relatively stable DNA--protein complexes form in vitro on reaction with embryo nuclear extracts. We report the nucleotide sequence of the whole regulatory domain and map the sites at which high-specificity DNA--protein interactions occur. These were located initially by gel shift assays carried out on progressive restriction digests of given subfragments of the large regulatory domain and were located more exactly by oligonucleotide gel shift competitions. Eight of the sites of specific interaction are unique within the CyIIIa regulatory domain, and the remainder consist of five different sites that occur more than once. We observe some well known sequences also found in regulatory regions of other genes, e.g., "CCAAT" and "octamer" elements. The various sites have been classified regarding putative biological function in other work, and the present studies permit an assessment of the number and complexity of interactions constituting each functional class and of the relative locations of sites of each class.

Actins↗
PubMed1990

Rapid evolution in a fraction of the Drosophila nuclear genome.

Previous observations have indicated that Drosophila DNA contains a component that evolves so rapidly that it fails to hybridize between the DNAs of sibling species. To establish the reality of this component and study its properties, the fraction (about 20%) of Drosophila simulans (Dsim) DNA that fails to hybridize to Drosophila melanogaster (Dmel) DNA has been isolated. The majority of the hybridizable part of this isolated fraction (based on control tests on Dsim DNA) fails to hybridize with Dmel DNA under the conditions used for the initial fractionation. Clones of this fraction do hybridize with Dmel DNA at open criterion producing duplexes with greatly reduced thermal stability, indicating that the underlying process is rapid sequence divergence rather than loss of the homologous sequences by relatively large deletions. Cloned fragments from the nonhybridizing fraction from Dsim are more than 15% divergent from the Dmel homologues, whereas the fraction that does hybridize is only 3-5% divergent. In comparison, synonymous substitutions in the coding regions of five genes show a 9% average divergence between Dsim and Dmel. They appear to be intermediate in their degree of divergence between the hybridizing and nonhybridizing components.

Animals↗