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Biomedical subjects

R J Britten

Publications and source records attributed to R J Britten.

At least 127 records · Page 7Linked to original sources
PubMed1980

Evolutionary conservation of repetitive sequence expression in sea urchin egg RNA's.

Cloned repetitive DNA sequences were used to determine the number of homologous RNA transcripts in the eggs of two sea urchin species, Strongylocentrotus purpuratus and S. franciscanus. The eggs of these species contain different amounts of RNA, and their genomes contain different numbers of copies of the cloned repeats. The specific pattern of repetitive sequence representation in the two egg RNA's is nonetheless quantitatively similar. The evolutionary conservation of this pattern suggests the functional importance of repeat sequence expression.

Animals↗
PubMed1980

Evolution of sea urchin non-repetitive DNA.

New methods have been applied to the determination of single copy DNA sequence differences between the sea urchin species Strongylocentrotus purpuratus, S. franciscanus, S. drobachiensis, and Lytechinus pictus. The thermal stability of interspecies DNA duplexes was measured in a solvent (2.4 M tetraethylammonium chloride) that suppresses the effect of base composition on melting temperature. The lengths of duplexes were measured after digestion with S1 nuclease and correction made for the effect of length on thermal stability. The degree of base substitution that has occurred in the single copy DNA during sea urchin evolution is significantly larger than indicated by earlier measurements. We estimate that 19% of the nucleotides of the single copy DNA are different in the genomes of the two sea urchin congeners, S. purpuratus, and S. franciscanus, which apparently diverged only 15 to 20 million years ago.

Animals↗
PubMed1980

Four sizes of transcript produced by a single sea urchin gene expressed in early embryos.

This report concerns a set of sea urchin egg and embryo transcripts complementary to a single-copy region of a cloned DNA fragment (Sp88). Three distinct 16-cell embryo polysomal RNA species were found to hybridize with this fragment. These RNAs are about 1700, 3000, and 4000 nucleotides (nt) in length, and the same species were identified in unfertilized eggs. A significant fraction of all three species of the egg and early embryo transcripts is polyadenylylated. At gastrula stage Sp88 transcripts are almost completely confined to the nucleus [Lev, Z., Thomas, T. L., Lee, A. S., Angerer, R. C., Britten, R. J. & Davidson, E. H. (1980) Dev. Biol, 75, in press]. The Sp88 transcripts of gastrulae are present as a fourth RNA species approximately 5800 nt in length. The four species share a sequence element of cloned DNA fragment that is about 1000 nt long. These RNAs constitute a set of alternative partially overlapping transcripts from the same genomic region.

Animals↗
PubMed1980

Messenger RNA prevalence in sea urchin embryos measured with cloned cDNAs.

mRNA prevalence during sea urchin development was measured by treating cDNA clone colonies with labeled cDNAs transcribed from unfertilized egg and embryo poly(A)-RNAs. The number of cytoplasmic transcripts per embryo complementary to several clones was determined independently by titration with poly(A)-RNA in solution, and the amount of cDNA bound to these clones in colony hybridizations was shown to be proportional to the concentration of the respective poly(A)-RNAs in the embryo cytoplasm. At the gastrula stage, the most prevalent mRNA species occur in about 10(6) molecules per embryo. If all cells were equivalent, this would be a few hundred molecules per cell. By pluteus stage, the prevalence of some sequences has increased more than 10-fold. Most, though not all, sequences prevalent in later embryos are also present in the maternal RNA of the unfertilized egg. For most poly(A)-RNA sequences, the prevalence levels determined during oogenesis are maintained through the pluteus stage, whereas a minority of sequences display sharp stage-specific changes in representation during development.

Animals↗
PubMed1980

Complexity of RNA in eggs of Drosophila melanogaster and Musca domestica.

Comparative measurements are presented of the sequence complexity of the RNA stored in the eggs of two dipteran flies, Musca domestica and Drosophila melanogaster. The genome of Musca is about five times the size of the Drosophila genome and contains about 3.6 times as much single-copy sequence. As shown earlier, the interspersion of repetitive and single-copy sequence is of the short-period form in Musca, and is of the long-period form in Drosophila. The egg RNA complexities were determined by hybridization of excess RNA with radioactively labeled single-copy DNA. Complexity is expressed as the length (in nucleotides) of diverse single-copy sequence represented in the RNA. The complexity of the RNA of the Musca egg is about 2.4 X 10(7) nucleotides, and that of the Drosophila egg is about 1.2 X 10(7) nucleotides. The RNA of the Musca egg is similar to or very slightly lower in complexity than that of other egg RNAs, e.g., those of Xenopus and sea urchin. Compared to all previously measured egg RNAs, Drosophila egg RNA is low in sequence complexity.

Animals↗
PubMed1979

Regulation of gene expression: possible role of repetitive sequences.

Large contrasts are observed between the messenger RNA populations of different tissues and of embryos at different stages of development. Nevertheless, coding sequences for genes not expressed in a cell appear to be present in its nuclear RNA. Though many nuclear RNA transcripts of single copy DNA sequences are held in common between tissues, an additional set, probably consisting of non-message sequences, is not shared. Nuclear RNA also contains transcripts of repetitive DNA sequences. Certain repeat families are represented at high levels in the nuclear RNA of particular tissues and much lower levels in others. It is surprising that both complements of most repeat sequences are present in nuclear RNA. These observations lead to model for regulation of gene expression in which the formation of repetitive RNA-RNA duplexes controls the production of messenger RNA.

Animals↗
PubMed1979

Distinct single-copy sequence sets in sea urchin nuclear RNAs.

The purpose of this study was to determine whether nuclear RNAs (nRNA) of sea urchin embryos and adult tissues contain identical or partially distinct sets of single-copy sequence transcripts. A DNA tracer was prepared consisting mainly of sequences absent from gastrula nRNA; 3.6% of this tracer reacted with adult intestine nRNA but not with gastrula nRNA. The existence of a differentially transcribed DNA fraction was verified by its partial purification and rehybridization to intestine and gastrula nRNAs. About one-third of the genomic single-copy sequence is represented in both nRNAs, or about 2 x 10(8) nucleotides. The differentially transcribed portion of the single-copy genome identified in this work includes about 3.5 x 10(7) nucleotides.

Animals↗
PubMed1978

A large-scale laboratory maintenance system for gravid purple sea urchins (Strongylocentrotus purpuratus).

A large-scale laboratory facility for the maintenance of several thousand gravid sea urchins (S. purpuratus) is described. Gametes of this species provide an important research resource for studies of animal development. Methods are described for culturing adult S. purpuratus for periods up to two years or more with almost no mortality after the first three to four weeks following collection. Adult females can be maintained in a fertile state in the culture system for four to six months. When spawned, gravid females living in the culture facility will routinely regenerate normal sized populations of fertile oocytes at 1 to 2-month intervals. Such females can be used repeatedly as a source of mature gametes for laboratory research. The reproductive performance of these females can be predicted approximately by the number of late vitellogenic oocytes present in their ovaries. After several months the pool of these oocytes is exhausted and no further mature oocytes can be found for a long period. We show, however, that such females are able to carry out a complete annual cycle of oogenesis if held for a long enough period of time in the culture system.

Animals↗
PubMed1978

Evolutionary change in the repetition frequency of sea urchin DNA sequences.

The frequency of occurrence of particular repetitive sequence families has been estimated in the DNA of the three sea urchin species Strongylocentrotus purpuratus. Strongylocentrotus franciscanus and Lytechinus pictus using individual cloned S. purpuratus repetitive sequence elements. Cloned repetitive sequence elements as described by Scheller et al. (1977a) were prepared by reassociation of S. purpuratus DNA fragments to repetitive Cot, digestion with single-strand-specific nuclease S1 and ligation of synthetic restriction sites to their ends. The sequences were cloned by insertion at the Eco RI site of plasmid RSF2124, labeled, strand-separated and reassociated with 800--900 nucleotide long unlabeled DNA. Both kinetic (genomic DNA excess) and saturation (cloned DNA excess) estimates of frequencies were made. For nine cloned fragments, the ratio of the repetition frequency in S. purpuratus DNA to that in S. franciscanus DNA ranges from about 20 to about 1. In the four cases examined, only a few copies were detected in the DNA of L. pictus. Estimates have also been made of frequency changes in many repetitive families by measuring the reassociation of labeled repetitive DNA fractions of each species with total DNA from other species. In each reciprocal comparison, the labeled repetitive sequences reassociate more slowly with DNA of other species than with DNA of the species from which they were prepared. Thus it appears that the dominant repetitive sequence families in the DNA of each species are present at lower frequencies in the DNA of closely related species. Measurements of thermal stability have been made of S. purpuratus cloned repetitive sequences reassociated with S. franciscanus DNA or S. purpuratus DNA. Most families have changed both in frequency and sequence, although some have changed little in sequence but show great changes in frequency.

Animals↗
PubMed1978

The single-copy DNA sequence polymorphism of the sea urchin Strongylocentrotus purpuratus.

The single-copy DNA sequence difference between individual sea urchins of the species Strongylocentrotus purpuratus has been estimated by comparing the thermal stability of reassociated DNA duplexes from two individuals with that for DNA from an individual. Thermal stability was measured by hydroxyapatite thermal chromatography, S1 nuclease resistance after heating in a solvent which neutralizes the effect of DNA base composition, and spectrophotometric melting. One pair of individuals appear to differ from each other in about 4% of the nucleotide pairs of their single-copy DNA sequence. The differences in DNA sequence among individuals in local populations are not distinguishably smaller than those among populations as far apart as 2000 kilometers along the Pacific coast of North America.

Animals↗
PubMed1978

Repetitive sequence transcripts in the mature sea urchin oocyte.

The expression of interspersed repetitive sequences in the RNA of mature sea urchin oocytes was investigated. 3H-DNA tracers representing short interspersed repetitive sequences a few hundred nucleotides long, and long repetitive sequences approximately 2000 nucleotides long, were prepared from genomic DNA of the sea urchin, Strongylocentrotus purpuratus. These tracers were reacted with excess RNA from the mature oocyte. About 80% of the reactable short repeat tracer and 35% of the long repeat tracer hybridized. Thus most of the repetitive sequence families in the short repeat tracer are represented in oocyte RNA, and transcripts complementary to both strands of many repeat sequences are present. The kinetics of the reaction show that some transcripts are highly prevalent (greater than 10(5) copies per oocyte), while others are rare (approximately 10(3) copies per oocyte). Nine cloned repetitive sequences were labeled, strand-separated and reacted with the oocyte RNA. Transcripts of both strands of all nine repeats were found in the RNA. The prevalence of transcripts of the cloned repeat families varied from approximately 3000 to 100,000 copies per oocyte. Studies with both cloned and genomic tracers show that transcript prevalence is independent of the genomic reiteration frequency of the transcribed repetitive sequences. Most of the families represented by prevalent transcripts have fewer than 200 copies per haploid genome. The RNA molecules with which the cloned repeats react are at least 1000-2000 nucleotides in length. Other experiments show that a majority of the members of repeat families represented by prevalent transcripts in the oocyte RNA are interspersed among single-copy sequence elements in the genome.

Animals↗
PubMed1978

Specific representation of cloned repetitive DNA sequences in sea urchin RNAs.

Nine cloned repetitive sequences were labeled, strands-separated and individually hybridized with RNA extracted from the nuclei of gastrula stage sea urchin embryos and of adult sea urchin intestine cells. The concentration of transcripts complementary to each cloned sequence was measured by RNA excess hybridization kinetics and by a DNA excess titration method. Transcripts of certain of the repeat families are present at over 100 times the concentration of transcripts of other families in each RNA. The set of repetitive sequence families highly represented in intestine nuclear RNA is different from that highly represented in gastrula nuclear RNA. Together with the results obtained with mature oocyte RNA and presented in the accompanying paper by Costantini et al. (1978), these findings show that quantitative patterns of repetitive sequence representation in RNA are specific to each cell type. Both strands of all of the nine cloned repeats are represented at some level in all the RNAs studied. Usually, though not always, the concentration of transcripts complementary to the two strands of each repeat do not differ by more than a factor of two. The cloned tracers do not react with polysomal messenger RNA, and the nuclear RNA molecules with which they hybridize are many times larger than the repetitive sequences themselves.

Animals↗
PubMed1978

Characteristics of individual repetitive sequence families in the sea urchin genome studied with cloned repeats.

Cloned repetitive sequences from the S. purpuratus genome a few hundred to approximately 1000 nucleotides long were used to investigate the characteristics of individual repetitive sequence families. They wer terminally labeled by the kinase procedure and reacted with sheared S. purpuratus DNA. Repetition frequencies were measured for 26 individual families and were found to vary from a few to several thousand copies per genome. Estimates of sequence divergence were made for 18 cloned repeat families by measuring thermal stability of the heteroduplexes formed between the genomic DNA and the cloned fragments, compared with that of the renatured cloned fragments. The difference was less than 4 degrees C for three of the 18 families, and less than 10 degress C for 13 of the 18 families. These 13 repetitive sequence families lack any detectable highly divergent sequence relatives, and the results reported are shown not to change when the renaturation criterion is lowered below 55 degrees C in 0.18 M Na+. Five of the 18 cloned families displayed greater sequence divergence. The average sequence divergence of the total short repetitive sequence fraction of S. purpuratus DNA was found to match closely the average of the divergences of the cloned repeat sequences.

Base Sequence↗