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E H Davidson

Publications and source records attributed to E H Davidson.

At least 127 records · Page 7Linked to original sources
PubMed1988

An in vivo titration of regulatory factors required for expression of a fusion gene in transgenic sea urchin embryos.

We report that endogenous regulatory factors mediating expression of a lineage-specific sea urchin embryo gene can be titrated in vivo by introduction of a sufficient molar excess of DNA-binding sites. Thus we obtain an estimate of the quantity of limiting factor(s) required for developmental activation and transcriptional expression, which can be compared with estimates of factor prevalence obtained by measurements in vitro carried out under equilibrium conditions. A fusion construct in which the bacterial gene for chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2.3.1.28) is controlled by cis-regulatory elements of the CyIIIa cytoskeletal actin gene (CyIIIa-CAT) was introduced in varying numbers of copies into sea urchin eggs. The activity of the CyIIIa-CAT fusion gene in 24-hr blastula-stage embryos was shown to saturate as the number of exogenous genes was increased. The mean number of CyIIIa-CAT fusion genes per nucleus at which half saturation was obtained was 105 +/- 40 (mean +/- SD). This result suggests that equilibrium parameters measured earlier in vitro may apply, at least approximately, within the embryo nuclei.

Actins↗
PubMed1988

Spatially deranged though temporally correct expression of Strongylocentrotus purpuratus actin gene fusion in transgenic embryos of a different sea urchin family.

We report the unexpected observation that cis-regulatory sequences of a Strongylocentrotus purpuratus actin gene, which direct a particular, lineage-specific pattern of embryonic expression, confer a completely different spatial pattern of expression when introduced into embryos of another sea urchin species. We utilized a fusion gene construct in which the bacterial chloramphenicol acetyl transferase (CAT) reporter gene is driven by CyIIIa actin regulatory sequences. We previously showed that the regulatory region that is included suffices to promote the accumulation of CAT mRNA in transgenic S. purpuratus embryos, on the same developmental schedule and in the same embryonic region, the aboral ectoderm, in which the CyIIIa actin gene is normally expressed (Flytzanis et al. 1987; Hough-Evans et al. 1987). When injected into zygotes of Lytechinus variegatus, which belongs to a different echinoid family, the expected temporal pattern of expression of CAT enzyme was observed. Thus, in both S. purpuratus and L. variegatus embryos, expression is activated at the early blastula stage, although this stage is attained several hours sooner in L. variegatus embryo cultures. Similar kinetics of CAT enzyme accumulation were obtained whether the gene was introduced directly into the L. variegatus zygote nucleus or into the cytoplasm. However, when examined by in situ hybridization, the transgenic L. variegatus embryos were found to display a totally new pattern of CAT mRNA accumulation. Copious CAT transcripts were detected not only in aboral ectoderm cells, but also in skeletogenic mesenchyme cells, gut cells, and oral ectoderm, all cell types that in the transgenic S. purpuratus controls are invariably devoid of detectable CAT transcripts.

Acetyltransferases↗
PubMed1988

A regulatory domain that directs lineage-specific expression of a skeletal matrix protein gene in the sea urchin embryo.

DNA sequences derived from the 5' region of a gene coding for the 50-kD skeletal matrix protein (SM50) of sea urchin embryo spicules were linked to the CAT reporter gene and injected into unfertilized eggs. CAT mRNA and enzyme were synthesized from these fusion constructs in embryos derived from these eggs, and in situ hybridization with a CAT antisense RNA probe demonstrated that expression is confined to skeletogenic mesenchyme cells. A mean of 5.5 of the 32-blastula-stage skeletogenic mesenchyme cells displayed CAT mRNA (range 1-15), a result consistent with earlier measurements indicating that incorporation of the exogenous injected DNA probably occurs in a single blastomere during early cleavage. In vitro mutagenesis and deletion experiments showed that CAT enzyme activity in the transgenic embryos is enhanced 34-fold by decreasing the number of SM50 amino acids at the amino-terminus of the fusion protein from 43 to 4. cis-regulatory sequences that are sufficient to promote lineage-specific spatial expression in the embryo are located between -440 and +120 with respect to the transcriptional initiation site.

Amino Acid Sequence↗
PubMed1988

A long, nontranslatable poly(A) RNA stored in the egg of the sea urchin Strongylocentrotus purpuratus.

Nontranslatable transcripts containing interspersed repetitive sequence elements constitute a major fraction of the poly(A) RNA stored in the cytoplasm of both the sea urchin egg and the amphibian oocyte. We report the first complete sequence of a representative interspersed maternal RNA transcript, called ISp1. The transcript is about 3.7 kb in length [including poly(A) tail]; and the 5' half consists of a cluster of repetitive sequences, whereas the 3' half is single copy. Other repetitive sequences occur in the 5' and 3' regions flanking the transcription unit. In several cloned alleles, the flanking repetitive and single-copy sequences differ, indicating a high degree of insertional and deletional rearrangement around, as well as within, the transcription unit. No significant open reading frames exist in any region of the ISp1 transcript, nor is it spliced to give rise to translatable mRNA in egg or embryo. A 620-nucleotide repetitive sequence element at the 5' end of the ISp1 transcript is also represented in a large number of other long interspersed maternal poly(A) RNAs. In addition, this sequence appears in a prevalent set of small polyadenylated RNAs about 600-nucleotides in length, which disappear almost completely by the gastrula stage of development. The structural features of the ISp1 RNA uncovered in this work exclude several hypotheses of interspersed maternal poly(A) RNA origin and function.

Animals↗
PubMed1988

Developmental appearance of factors that bind specifically to cis-regulatory sequences of a gene expressed in the sea urchin embryo.

Previous gene-transfer experiments have identified a 2500-nucleotide 5' domain of the CyIIIa cytoskeletal actin gene, which contains cis-regulatory sequences that are necessary and sufficient for spatial and temporal control of CyIIIa gene expression during embryogenesis. This gene is activated in late cleavage, exclusively in aboral ectoderm cell lineages. In this study, we focus on interactions demonstrated in vitro between sequences of the regulatory domain and proteins present in crude extracts derived from sea urchin embryo nuclei and from unfertilized eggs. Quantitative gel-shift measurements are utilized to estimate minimum numbers of factor molecules per embryo at 24 hr postfertilization, when the CyIIIa gene is active, at 7 hr, when it is still silent, and in the unfertilized egg. We also estimate the binding affinity preferences (Kr) of the various factors for their respective sites, relative to their affinity for synthetic DNA competitors. At least 14 different specific interactions occur within the regulatory regions, some of which produce multiple DNA-protein complexes. Values of Kr range from approximately 2 x 10(4) to approximately 2 x 10(6) for these factors under the conditions applied. With one exception, the minimum factor prevalences that we measured in the 400-cell 24-hr embryo nuclear extracts fell within the range of 2 x 10(5) to 2 x 10(6) molecules per embryo, i.e., a few hundred to a few thousand molecules per nucleus. Three developmental patterns were observed with respect to factor prevalence: Factors reacting at one site were found in unfertilized egg cytoplasm at about the same level per egg or embryo as in 24-hr embryo nuclei; factors reacting with five other regions of the regulatory domain are not detectable in egg cytoplasm but in 7-hr mid-cleavage-stage embryo, nuclei are already at or close to their concentrations in the 24-hr embryo nuclei; and factors reacting with five additional regions are not detectable in egg cytoplasm and are low in 7-hr embryo nuclei, i.e., less than or equal to 10% per embryo of the level they attain in 24-hr embryo nuclei. The rise in concentration of factors of the latter class could provide the proximal cause for the temporal activation of the CyIIIa gene at the early blastula stage.

Animals↗
PubMed1988

Direct introduction of cloned DNA into the sea urchin zygote nucleus, and fate of injected DNA.

A method is described for microinjection of cloned DNA into the zygote nucleus of Lytechinus variegatus. Eggs of this species are unusually transparent, facilitating visual monitoring of the injection process. The initial fate of injected DNA fragments appears similar to that observed earlier for exogenous DNA injected into unfertilized egg cytoplasm. Thus after end-to-end ligation, it is replicated after a lag of several hours to an extent indicating that it probably participates in most of the later rounds of DNA synthesis undergone by the host cell genomes during cleavage. The different consequences of nuclear versus cytoplasmic injection are evident at advanced larval stages. Larvae descendant from eggs in which exogenous DNA was injected into the nuclei are four times more likely (32% versus 8%) to retain this DNA in cell lineages that replicate very extensively during larval growth, i.e. the lineages contributing to the imaginal rudiment, and thus to display greatly enhanced contents of the exogenous DNA. Similarly, 36% of postmetamorphic juveniles from a nuclear injection sample retained the exogenous DNA sequences, compared to 12% of juveniles from a cytoplasmic injection sample. However, the number of copies of the exogenous DNA sequences retained per average genome in postmetamorphic juveniles was usually less than 0.1 (range 0.05-50), and genome blot hybridizations indicate that these sequences are organized as integrated, randomly oriented, end-to-end molecular concatenates. It follows that only a small fraction of the cells of the average juvenile usually retains the exogenous sequences. Thus, even when introduced by nuclear microinjection, the stable incorporation of exogenous DNA in the embryo occurs in a mosaic fashion, although in many recipients the DNA enters a wider range of cell lineages than is typical after cytoplasmic injection. Nuclear injection would probably be the route of choice for studies of exogenous DNA function in the postembryonic larval rudiment.

Actins↗
PubMed1987

Structure and organization of the CyIII actin gene subfamily of the sea urchin, Strongylocentrotus purpuratus.

We describe here the organization of the CyIII subfamily of cytoskeletal actin genes in the sea urchin Strongylocentrotus purpuratus. The functional genes CyIIIa and CyIIIb are linked at a 6 X 10(3)-base distance. Gene CyIIIc appears to be a pseudogene that lacks 5' exons and displays unselected mutational changes. Gene CyIIIa codes for a protein that differs at only nine out of 376 residues from that coded by another cytoskeletal actin gene, CyI. However, five of these nine changes occur within an 11-amino acid region that could represent a functional specialization of the CyIIIa actin protein. The CyIIIa gene possesses three introns, located, respectively, 25 nucleotides upstream from the translation start site, between the codons for amino acids 121 and 122, and within the codon for amino acid 204. These intron positions have also been observed in other cytoskeletal sea urchin actin genes. Comparison of both intron and 3'-terminal sequences shows that the CyIIIa and CyIIIb genes are closely related, while no homology in these untranslated sequences is observed between the CyIII genes and the other cytoskeletal actin genes of the S. purpuratus genome. The CyIII genes probably arose by duplication events at least 40 X 10(6) years ago, prior to radiation of the genus Strongylocentrotus. Consideration of the biological role of the embryo and larval aboral ectoderm cells to which CyIIIa and CyIIIb transcripts are confined suggests that these actins might contribute to cytoskeletal elements that endow the larval body wall with its rigid structure.

Actins↗
PubMed1987

Correct cell-type-specific expression of a fusion gene injected into sea urchin eggs.

A fusion gene construct containing the bacterial chloramphenicol acetyltransferase (CAT) gene under the control of CyIIIa actin gene regulatory sequences was injected into unfertilized eggs of the urchin Strongylocentrotus purpuratus, and early pluteus stage embryos that developed from these eggs were fixed and sectioned for analysis by in situ hybridization. A [3H]RNA antisense probe for CAT mRNA was hybridized to 5-micron embryo sections. Autoradiographic signal denoting the presence of CAT mRNA was detected only over aboral ectoderm cells, in which the CyIIIa gene is normally expressed, and not over any recognizable regions of gut or oral ectoderm included in the same sections.

Acetyltransferases↗
PubMed1987

A lineage-specific gene encoding a major matrix protein of the sea urchin embryo spicule. II. Structure of the gene and derived sequence of the protein.

A lambda gt11 cDNA clone isolated by use of a polyclonal antispicule matrix protein antiserum is shown in the accompanying paper [S. C. Benson, H. M. Sucov, L. Stephens, E. H. Davidson, and F. Wilt (1987) Dev. Biol. 120, 499-506] to encode a prominent 50-kDa spicule matrix protein (SM50). This clone was used to select homologous genomic recombinants, and the structure of the gene was determined. The SM50 gene occurs once per haploid genome. It contains a single intron located within the 35th codon. A unique transcription initiation site 110 nucleotide pairs prior to the translation start signal was mapped by primer extension. The mRNA is 1895 nucleotides in length, excluding the 3' poly(A) sequence, and contains a single open reading frame 450 codons in length. Though rare in whole embryo RNA the prevalence of the SM50 mRNA is calculated to be about 1% of the total mRNA in skeletogenic mesenchyme cells. The derived peptide sequence indicates a typical N-terminal signal peptide, and an N-linked glycosylation site near the C terminus. About 45% of the length of the protein is included in a domain composed of consecutive approximate repetitions of a 13-amino-acid element, the consensus sequence of which is Trp-Val-Gly-Asp-Asn-Gln-Ala-LeuTrp-Val-IleAsp-Asn-GlnPro+ ++-ValGlu. The protein also contains an internal domain unusually rich in proline residues and a very basic C-terminal region.

Amino Acid Sequence↗
PubMed1987

Sea urchin actin gene linkages determined by genetic segregation.

Genetic linkage between the actin genes of Strongylocentrotus purpuratus was investigated by observing the segregation of restriction fragment length polymorphisms (RFLPs). Specific RFLPs of actin gene pairs CyI/CyIIa and CyIIIa/CyIIIb always cosegregated, confirming the linkage groups CyI-CyIIa-CyIIb previously previously determined by molecular cloning. In contrast, RFLPs of actin genes CyI/CyIIa, CyIIIa/CyIIIb, and M all segregated at random with respect to one another. This demonstrates that the known actin gene clusters CyI-CyIIa-CyIIb, CyIIIa-CyIIIb, and the M actin gene are not closely linked.

Actins↗
PubMed1987

Expression of myosin heavy chain gene in the sea urchin: coregulation with muscle actin transcription in early development.

A fragment of a Strongylocentrotus purpuratus (Sp) myosin heavy chain (MHC) gene was isolated from a genomic recombinant DNA library by cross-reaction with a cloned Drosophila melanogaster (Dm) MHC probe. A portion of a 227-nucleotide Sp coding sequence that is included in this fragment predicts a peptide very closely homologous with a region of the Dm sequence. The MHC gene sequence is present in a single copy per haploid Sp genome, and the gene is utilized in adult as well as embryonic muscle. The quantity of MHC transcript was measured in embryos of various stages by single-strand RNA probe excess titration. Transcripts are not observed until postgastrular stages, after which they accumulate rapidly. The time course of accumulation closely parallels that measured earlier for muscle actin message.

Actins↗
PubMed1987

Ontogenic activation of a fusion gene introduced into sea urchin eggs.

Regulatory sequences of a sea urchin cytoskeletal actin gene (CyIIIa) were ligated to the bacterial gene coding for chloramphenicol acetyltransferase (CAT; acetyl-CoA:chloramphenicol O3-acetyltransferase, EC 2.3.1.28) and the construct was injected into unfertilized sea urchin eggs. CAT activity is detected at the early blastula stage, when transcripts of the endogenous CyIIIa gene normally appear. Our measurements show that during activation the amount of CAT enzyme increases at least 100-fold; that there are present in late blastula stage embryos about 5 X 10(5) molecules of CAT mRNA (i.e., approximately 6 times the number of endogenous CyIIIa mRNAs); and that within the range studied the amount of CAT enzyme produced is independent of the number of CyIIIa-CAT genes incorporated per embryo, probably because the genes are present in excess of factors required for their activation. Activation of the CyIIIa-CAT construct is seriously inhibited, or abolished, by successive deletions of upstream CyIIIa sequences.

Acetyltransferases↗
PubMed1987

Lineage and fate of each blastomere of the eight-cell sea urchin embryo.

A fluoresceinated lineage tracer was injected into individual blastomeres of eight-cell sea urchin (Strongylocentrotus purpuratus) embryos, and the location of the progeny of each blastomere was determined in the fully developed pluteus. Each blastomere gives rise to a unique portion of the advanced embryo. We confirm many of the classical assignments of cell fate along the animal-vegetal axis of the cleavage-stage embryo, and demonstrate that one blastomere of the animal quartet at the eight-cell stage lies nearest the future oral pole and the opposite one nearest the future aboral pole of the embryo. Clones of cells deriving from ectodermal founder cells always remain contiguous, while clones of cells descendant from the vegetal plate (i.e., gut, secondary mesenchyme) do not. The locations of ectodermal clones contributed by specific blastomeres require that the larval plane of bilateral symmetry lie approximately equidistant (i.e., at a 45 degree angle) from each of the first two cleavage planes. These results underscore the conclusion that many of the early spatial patterns of differential gene expression observed at the molecular level are specified in a clonal manner early in embryonic sea urchin development, and are each confined to cell lineages established during cleavage.

Animals↗
PubMed1987

Ontogenic expression of a CyI actin fusion gene injected into sea urchin eggs.

The 5' terminus of the CyI actin gene transcription unit of Strongylocentrotus purpuratus was located by primer extension and other procedures, and the flanking upstream region was partially sequenced and mapped. A fusion gene was constructed containing about 2.5 kb of 5' flanking sequence, the transcribed leader sequence, and the first few codons of the CyI gene ligated to the bacterial gene coding for chloramphenicol acetyl transferase (CAT). This was micro-injected into the cytoplasm of S. purpuratus eggs, and CAT enzyme activity was measured at various stages of embryonic development. CAT synthesis was activated between 10 and 14 h postfertilization, the same time at which newly synthesized transcripts of the endogenous CyI gene first appear. The exogenous CyI.CAT fusion DNA replicated actively during cleavage, as observed previously for other DNAs injected into sea urchin egg cytoplasm. Thus the absence of CAT activity prior to 10 h postfertilization could not be due to insufficient CyI.CAT genes. The amounts of CAT enzyme produced by embryos bearing CyI.CAT deletions that lack various regions of the CyI sequence were measured. As little as 254 nucleotides of upstream CyI sequence suffice for correct temporal activation of the fusion construct, although the level of CAT enzyme produced in embryos bearing any deletion retaining less than 850 nucleotides of upstream sequence was significantly lowered compared to controls bearing the complete CyI.CAT fusion construct.

Actins↗
PubMed1986

Cell lineage-specific programs of expression of multiple actin genes during sea urchin embryogenesis.

We have determined spatial patterns of expression of individual actin genes in embryos of the sea urchin Strongylocentrotus purpuratus. Radioactively labeled probes specific for each of five cytoplasmic-type (Cy) and the single muscle-type (M) mRNAs were hybridized in situ to sections of fixed embryos. M actin mRNA appears only late in development and is confined to a few cells associated with the coelomic rudiments. The five Cy mRNAs fall into three sets, whose times and sites of expression during development are highly distinctive. Different cell lineages express messages of one or more of these sets, but never all three. Although all Cy actin mRNAs exhibit monophasic accumulation in the RNA of whole embryos during the course of development, such accumulation in many cases results from the summation of both increases and decreases in abundance within individual sets of cells. Within the genomic linkage group CyI-CyIIa-CyIIb, expression of CyI and CyIIb appears to be co-ordinate, and quite distinct from that of CyIIa. CyI and CyIIb are expressed in all lineages at some point in embryogenesis, but confined mainly to oral ectoderm and portions of the gut of the pluteus larva. CyIIa mRNAs are restricted to mesenchyme lineages throughout late gastrula stage, and subsequently accumulate in parts of the gut. The CyIIIa and CyIIIb genes, which form a separate linkage group, are expressed only in aboral ectoderm and its precursors. Furthermore, CyIII messages are the only detectable actin mRNAs in this cell lineage after late blastula stage.

Actins↗
PubMed1986

Activation of sea urchin actin genes during embryogenesis. Measurement of transcript accumulation from five different genes in Strongylocentrotus purpuratus.

The number of molecules of mRNA transcribed from each of five different actin genes are reported for developing embryos of the sea urchin Strongylocentrotus purpuratus. Transcripts of the cytoskeletal actin genes CyI, CyIIa, CyIIb and CyIIIa, and of the muscle actin gene M, were measured in unfertilized egg and embryo RNAs of cleavage, blastula, gastrula and pluteus stages. The measurements were obtained by probe excess titrations of these RNAs, using a set of single-stranded RNA probes each identifying the mRNA transcripts of a specific actin gene. These mRNAs can be identified by their distinct 3' non-translated trailer sequences. We confirm prior observations that the prevalence of actin mRNA in the unfertilized egg is low. Cytoskeletal actin genes CyI and CyIIIa each contribute 1 X 10(3) to 2 X 10(3) maternal mRNA molecules, and CyIIb contributes less than 2 X 10(2) mRNA molecules, while no detectable maternal mRNAs derive from cytoskeletal actin gene CyIIa or the muscle actin gene M. During certain periods of development, transcripts derived from the individual cytoskeletal actin genes accumulate rapidly, with kinetics specific to each mRNA. Transcripts of the muscle actin gene are absent until after gastrulation, when the initial muscle progenitor cells are formed. At late stages of development, each of the five genes studied is represented by 10(4) to 10(5) mRNA molecules per embryo. The present measurements permit calculation of the levels of each actin mRNA species in the particular cell types in which each gene functions in the fully differentiated embryo.

Actins↗