Biomedical subjects

R J Britten

Publications and source records attributed to R J Britten.

At least 73 records · Page 4Linked to original sources

PubMed1990

Negative spatial regulation of the lineage specific CyIIIa actin gene in the sea urchin embryo.

The CyIIIa.CAT fusion gene was injected into Strongylocentrotus purpuratus eggs, together with excess ligated competitor sequences representing subregions of the CyIIIa regulatory domain. In this construct, the chloramphenicol acetyltransferase (CAT) reporter gene is placed under the control of the 2300 nucleotide upstream regulatory domain of the lineage-specific CyIIIa cytoskeletal actin gene. CAT mRNA was detected by in situ hybridization in serial sections of pluteus stage embryos derived from the injected eggs. When carrier DNA lacking competitor CyIIIa fragments was coinjected with CyIIIa.CAT, CAT mRNA was observed exclusively in aboral ectoderm cells, i.e. the territory in which the CyIIIa gene itself is normally expressed (as also reported by us previously). The same result was obtained when five of seven different competitor subfragments bearing sites of DNA-protein interaction were coinjected. However, coinjection of excess quantities of either of two widely separated, nonhomologous fragments of the CyIIIa regulatory domain produced a dramatic ectopic expression of CAT mRNA in the recipient embryos. CAT mRNA was observed in gut, mesenchyme cells and oral ectoderm in these embryos. We conclude that these fragments contain regulatory sites that negatively control spatial expression of the CyIIIa gene.

Actins↗

PubMed1989

In vivo competition identifies positive cis-regulatory elements required for lineage-specific gene expression in the sea urchin embryo.

Several cis-regulatory elements within the 5' regulatory region of the lineage-specific CyIIIa actin gene have been identified by in vivo competition. Sea urchin eggs were coinjected with a fusion construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by the CyIIIa regulatory domain, together with molar excesses of various DNA subfragments that are derived from this region. Each subfragment studied includes one or several known sites where highly specific interactions occur in vitro with nuclear DNA-binding proteins. Coinjection of excess molecules of some of these subregions results in a decrease in the activity of the CyIIIa-CAT fusion gene, as a function of the molar subfragment: CyIIIa-CAT ratio. This result implies that these sites complete with cis sequences linked to the CAT reporter gene for limited factors that positively regulate CyIIIa transcription in the embryo, and demonstrates the functional importance of a number of the DNA-protein interactions that have been observed in vitro.

Actins↗

PubMed1989

Expression of two actin genes during larval development in the sea urchin Strongylocentrotus purpuratus.

We report the first measurements of cell number, total RNA, and transcript accumulations for two actin genes during larval development of the sea urchin Strongylocentrotus purpuratus. At 5 weeks of feeding, when development of laboratory-raised larvae is completed, the cell number has increased about 100-fold with respect to the pluteus-stage embryo to about 150,000 +/- 50,000, and the total RNA has increased 46-fold to about 130 ng per larva. The transcripts of the Cylla cytoskeletal actin gene, which is expressed in adult tissues, continue to accumulate throughout larval development. A contrasting pattern of transcript accumulation is observed for Cyllla, a different cytoskeletal actin gene that in the embryo is expressed only in aboral ectoderm. These transcripts increase in number early in larval development, when the larval epidermis is differentiating, and then decline in quantity. It is known that at metamorphosis the larval epidermis is largely histolyzed and that the Cyllla gene is not expressed in the juvenile or adult.

Actins↗

PubMed1989

The current source of human Alu retroposons is a conserved gene shared with Old World monkey.

A significant fraction of human Alu repeated sequences are members of the precise, recently inserted class. A cloned member of this class has been used as a probe for interspecies hybridization and thermal stability determination. The probe was reassociated with human, mandrill, and spider monkey DNA under conditions such that only almost perfectly matching duplexes could form. Equally precise hybrids were formed with human and mandrill DNA (Old World monkey) but not with spider monkey DNA (New World). These measurements as well as reassociation kinetics show the presence in mandrill DNA of many precise class Alu sequences that are very similar or identical in quantity and sequence to those in human DNA. Human and mandrill are moderately distant species with a single-copy DNA divergence of about 6%. Nevertheless, their recently inserted Alu sequences arise by retroposition of transcripts of source genes with nearly identical sequences. Apparently a gene present in our common ancestor at the time of branching was inherited and highly conserved in sequence in both the lineage of Old World monkeys and the lineage of apes and man.

Animals↗

PubMed1989

The oral-aboral axis of a sea urchin embryo is specified by first cleavage.

Several lines of evidence suggest that the oral-aboral axis in Strongylocentrotus purpuratus embryos is specified at or before the 8-cell stage. Were the oral-aboral axis specified independently of the first cleavage plane, then a random association of this plane with the blastomeres of the four embryo quadrants in the oral-aboral plane (viz. oral, aboral, right and left) would be expected. Lineage tracer dye injection into one blastomere at the 2-cell stage and observation of the resultant labeling patterns demonstrates instead a strongly nonrandom association. In at least ninety percent of cases, the progeny of the aboral blastomeres are associated with those of the left lateral blastomeres and the progeny of the oral blastomeres with the right lateral ones, respectively. Thus, ninety percent of the time the oral pole of the future oral-aboral axis lies 45 degrees clockwise from the first cleavage plane as viewed from the animal pole. The nonrandom association of blastomeres after labeling of the 2-cell stage implies that there is a mechanistic relation between axis specification and the positioning of the first cleavage plane.

Animals↗

PubMed1988

Mosaic incorporation and regulated expression of an exogenous gene in the sea urchin embryo.

A fusion gene construct in which the bacterial chloramphenicol acetyltransferase (CAT) gene is controlled by CyIIIa actin gene cis-regulatory sequences was injected into unfertilized eggs of the sea urchin Strongylocentrotus purpuratus. The distribution of CAT DNA sequences was measured directly by in situ hybridization in squashed 24-hr blastula preparations derived from these eggs. Earlier studies had shown that stable mosaic incorporation of the exogenous DNA occurs during cleavage, after which the exogenous sequences replicate at approximately the pace of the host cell genomes. The fractions of embryonic cells observed in this study to include CAT DNA sequences imply that their stable incorporation into a replicating nuclear form occurs most often in a single cell at the 3rd or 4th cleavage stages, though it may occur as early as 2nd cleavage, or as late as 7th cleavage. Corroborative measurements were carried out by the same method on squashed preparations of embryos at earlier stages, and by in situ hybridizations of CAT mRNA, both in dissociated embryos and in cytological sections of 72-hr pluteus-stage embryos. Hybridizations to CAT mRNA and to CAT DNA were carried out on alternate sections of several embryos. The results confirm unequivocally that although CAT mRNA appears only in the aboral ectoderm in embryos derived from eggs injected with the CyIIIa.CAT fusion gene, the exogenous sequences are indeed present, though silent, in the various other cell types of the late embryo.

Acetyltransferases↗

PubMed1988

Sources and evolution of human Alu repeated sequences.

Alu repeated sequences arising in DNA of the human lineage during about the last 30 million years are closely similar to a modern consensus. Alu repeats arising at earlier times share correlated blocks of differences from the current consensus at diagnostic positions in the sequence. Using these 26 positions, we can recognize four subfamilies and the older ones are each successively closer to the 7SL sequence. It appears that there has existed a series of conserved genes that are the primary sources of the Alu repeat family, presumably through retroposition. These genes have probably replaced each other in overlapping relays during the evolution of primates.

Animals↗

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↗