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Biomedical subjects

M Rosbash

Publications and source records attributed to M Rosbash.

At least 181 records · Page 10Linked to original sources
PubMed1982

Open reading frame cloning: identification, cloning, and expression of open reading frame DNA.

A plasmid was constructed that facilitates the cloning and expression of open reading frame DNA. A DNA fragment containing a bacterial promoter and the amino terminus of the cI gene of bacteriophage lambda was fused to an amino-terminally deleted version of the lacZ gene. An appropriate cloning site was inserted between these two fragments such that a frameshift mutation was introduced upstream of the lacZ-encoding DNA. This cloning vehicle produces a relatively low level of beta-galactosidase activity when introduced into Escherichia coli. The insertion of foreign DNA at the cloning site can reverse the frameshift mutation and generate plasmids that produce a relatively high level of beta-galactosidase activity. A large fraction of these plasmids produce a fusion protein that has a portion of the lambda cI protein at the amino terminus, the foreign protein segment in the middle, and the lacZ polypeptide at the carboxyl terminus. The production of a high level of beta-galactosidase and a large fusion polypeptide guarantees the cloning of a DNA fragment with at least one open reading frame that traverses the entirety of the fragment. Hence, the method can identify, clone, and express (as part of a larger fusion polypeptide) open reading frame DNA from among a large collection of DNA fragments.

Base Sequence↗
PubMed1982

Sporulation and rna2 lower ribosomal protein mRNA levels by different mechanisms in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, the levels of ribosomal protein mRNAs are regulated coordinately. Vegetative strains carrying the temperature-sensitive rna2 mutation exhibit a dramatic decrease in the levels of most ribosomal protein mRNAs at the restrictive temperature. Similarly, in wild-type cells induced to sporulate by nitrogen starvation, there is a fivefold reduction in the relative synthesis rate of ribosomal proteins. Using Northern gel analysis and cloned ribosomal protein genes, we compared the way in which ribosomal protein mRNA is affected under these two conditions. In vegetative rna2 cells, incubation at 34 degrees C led to the disappearance of ribosomal protein mRNAs and the accumulation of higher-molecular-weight precursor RNAs. A different phenotype was observed during sporulation. Although sporulating conditions led to a significant reduction in the relative abundance of ribosomal protein mRNA, there was no detectable accumulation of precursor RNAs even in rna2/rna2 diploids at 34 degrees C. A suppressor of rna2 and of other rna mutations, SRN1, at least partially relieved the block in the splicing of the ribosomal protein 51 intron in vegetative rna2 cells but did not detectably affect the level of ribosomal protein mRNA in sporulating cells. We concluded that the rna2 mutation and sporulation conditions affected ribosomal protein mRNA metabolism in two quite different ways. In vegetative cells the mutant rna2 effected a block which occurred primarily in post-transcriptional processing, whereas in sporulating cells the ribosomal protein mRNA levels were decreased by some other mechanism, presumably a change in the relative rate of transcription or mRNA turnover. Furthermore, the data suggest that the mutation rna2 has no additional effect on ribosomal protein mRNA metabolism in sporulating cells.

Gene Expression Regulation↗
PubMed1982

Further evidence that the rna2 mutation of Saccharomyces cerevisiae affects mRNA processing.

The relative rate at which ribosomal protein 51 (rp51) mRNA is synthesized was measured by pulse-labeling cells in vivo with [3H]adenine. Two strains of Saccharomyces cerevisiae were compared: A364A (wild type) and ts368 (rna2), a temperature-sensitive strain in which the level of rp51 mRNA decreases and an intron-containing rp51 precursor RNA increases. When cells were shifted up to the nonpermissive temperature (36 degrees C), the rate of rp51 RNA synthesis was only marginally affected (75% of wild type) by the presence of the rna2 mutation. The precursor RNA was the predominant transcription product at 36 degrees C. This precursor could be converted into RNA equal in size to mature mRNA by further incubation at either 36 or 23 degrees C in the presence of unlabeled adenine. The relative half-life of the rp51 transcripts at 36 degrees C also decreased approximately twofold in ts368 as compared with A364A. All of these data imply that the precursor (intron-containing) RNA is processed inefficiently to mature mRNA and that the rp51 precursor RNA is continuously synthesized and degraded in the mutant strain at 36 degrees C.

Gene Expression Regulation↗
PubMed1981

DNase I hypersensitive sites of the chromatin for Drosophila melanogaster ribosomal protein 49 gene.

By using an indirect end-labelling technique for mapping, five DNase I hypersensitive sites have been located in Drosophila melanogaster chromatin at the 5'-end of the gene coding for ribosomal protein 49. These sites typically span about 100-600 base pairs and are approximately the length of a nucleosome apart (center to center distance ca 245 bp). This is the first analysis of the chromatin structure of a constitutive house-keeping gene. The results support the hypothesis that the presence of such a DNase 1 hypersensitive site in chromatin is necessary for transcription in vivo. The presence of such sites may reflect some local changes in the conformation of the chromatin in the presumptive regulatory region.

Animals↗
PubMed1981

Ribosomal protein genes rp 39(10 - 78), rp 39(11 - 40), rp 51, and rp 52 are not contiguous to other ribosomal protein genes in the Saccharomyces cerevisiae genome.

A library of recombinant phage containing EcoR1 fragments of Saccharomyces cerevisiae DNA has been constructed. This library was screened with four different recombinant plasmids, each containing a different yeast ribosomal protein gene, in order to isolate chromosomal fragments extending in both directions from these genes. These chromosomal fragments were assayed for the presence of additional ribosomal protein genes by hybridization selection and cell free translation, and none were found. These four regions are not closely linked to each other, since DNA from one domain does not cross hybridize with DNA from any of the other three, except for the sequences within the homologous ribosomal protein 39 gene pair. Northern blots demonstrate that although the concentrations of ribosomal protein mRNAs are diminished significantly in a strain containing the ts mutation rna2, transcripts from genes in these flanking segments are relatively unaffected.

DNA Restriction Enzymes↗
PubMed1981

The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast.

The levels of four ribosomal protein (rp) mRNAs in different mutant strains were determined by hydridization of radiolabeled cloned genes to RNA fractionated on CH3HgOH gels and transferred to DBM paper. Two ribosomal protein genes (rp 51 and rp 52) controlled by the locus RNA2 have dramatically decreased mRNA levels after a shift-up to the nonpermissive temperature in a strain carrying the rna2 mutation (ts368). Two ribosomal protein genes not controlled by the RNA2 locus and several control nonribosomal protein genes are relatively unaffected by the temperature shift in this strain. Other genes in the vicinity of one of the rna2-sensitive ribosomal protein genes (th rp 51 gene) are insensitive to the rna2 gene product, suggesting that all ribosomal protein genes do not occur in clusters and that the RNA2 gene product does not affect a large region of chromatin. In ts368 at the nonpermissive temperature, the concentration of higher molecular weight transcripts complementary to the rp 51 and the rp 52 plasmids is increased. Analysis of the rp 51 plasmid transcripts reveals that the temperature-induced higher molecular weight transcripts differ from the mature rp 51 mRNA by the presence of an intron. This observation and the kinetics with which the concentration of the various rp 51 transcripts change after a temperature shift suggest that the effect of rna2 may be at the level of processing of rp mRNA.

Cloning, Molecular↗
PubMed1981

Determination of cellular RNA concentrations by electron microscopy of R loop-containing DNA.

R loop hybridizations and electron microscopy have been used to determine cellular RNA concentrations for cloned genes. In plasmid DNA sequence excess, all the complementary RNA is driven into R loop structures that can be assayed by electron microscopy. To determine the concentration of a particular poly(A)+ RNA, plasmid DNA crosslinked once every 2000-5000 base pairs with trioxsalen and UV light is hybridized in DNA sequence excess to various known amounts of total poly(A)+ RNA, and the R loops are stabilized by treatment with glyoxal. If necessary, excess nonhybridized RNA is removed by Sepharose 2B chromatography, which enables the visualization of less abundant transcripts. Reconstruction experiments demonstrated that electron microscopic determination of the fraction of plasmid DNA molecules containing specific RNA loops gives accurate values of specific RNA weight fractions or concentrations in the total poly(A)+ RNA populations. These methods were also used to determine the concentrations of five RNA species complementary to sequences on TRT3, a recombinant DNA plasmid containing yeast histone 2A and 2B genes and three other nonhistone genes. The methods described allow one to visualize the R loop structures for both abundant and nonabundant transcripts and to estimate concentrations of these RNA species simply by determining the fraction of DNA containing R loops.

Animals↗
PubMed1980

Isolation and mapping of a cloned ribosomal protein gene of Drosophila melanogaster.

Molecular cloning techniques are particularly well suited to the study of gene organization in Drasophila melanogaster because recombinant DNA can easily be localized in the genome by in situ hybridization to salivary gland polytene chromosomes. We report here the isolation and preliminary characterization of a recombinant phage, designated C25, containing a bona fide D. melanogaster ribosomal protein gene. In situ hybridization demonstrates that this sequence maps to region 99D on chromosome 3.

Animals↗
PubMed1980

Accumulation of individual pA+ RNAs during oogenesis of Xenopus laevis.

RNA metabolism during amphibian oogenesis has been investigated by the analysis of individual cDNA cloned sequences. Two cDNA clone libraries were constructed from the pA+ RNA of Xenopus ovary and of tadpole. The accumulation of RNAs complementary to individual clones was examined during oogenesis by isolating RNA from oocytes of different stages. There are only two patterns of accumulation during oogenesis. Mitochondrial mRNA sequences accumulate throughout all the stages of oogenesis, whereas all other sequences detected cease accumulation early in oogenesis and remain at a constant steady state level for the remainder of oocyte development. In the earliest stages examined, the individual RNAs are already present at approximately 1/4 their final level, and the subsequent increase of all sequences appears to be coordinant. These basic observations were confirmed by colony screening approximately 500 clones. The pattern of RNA accumulation observed is significant, because it had been determined that the total steady state pA+ RNA levels in the oocyte also ceased to increase at the same stage in oogenesis in which each individual sequenced reached a plateau level. In addition, lampbrush chromosomes, which have traditionally been thought to be the site of oocyte pA+ RNA synthesis, are maximally active after the stage in which all pA+ RNAs seem to have reached their final level of accumulation.

Animals↗
PubMed1979

The use of R-looping for structural gene identification and mRNA purification.

A method is presented for the purification of mRNAs and the identification of structural gene sequences in recombinant DNA molecules. RNA is hybridized to double-stranded linear DNA such that R-loops are formed between most DNAs and their complementary RNA sequences. These R-loops are purified from unhybridized RNAs by gel filtration chromatography in the presence of a high concentration of salt. The complementary RNAs are released from the R-loops by heating, and are assayed by gel electrophoresis or cell free translation to determine their purity and to identify the proteins for which they code. We have demonstrated that recombinant DNAs containing sequences for abundant or moderately abundant mRNAs of Saccharomyces cerevisiae can be identified by this means.

Base Sequence↗
PubMed1979

Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae.

Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.

DNA, Fungal↗
PubMed1979

Isolation of yeast histone genes H2A and H2B.

Analysis of cloned sequences for yeast histone genes H2A and H2B reveals that there are only two copies of this pair of genes within the haploid yeast genome. Within each copy, the genes for H2A and H2B are separated by approximately 700 bp of spacer DNA. The two copies are separated from one another in the yeast genome by a minimum distance of 35-60 kb. Sequence homology between the two copies is restricted to the genes for H2A and H2B; the spacer DNA between the genes is nonhomologous. In both copies, the genes for H2A and H2B are divergently transcribed. In addition, both plasmids code for other nonhistone proteins. Sequences coding for histones H3 and H4 have not been detected in the immediate vicinity of the genes for H2A and H2B.

Base Sequence↗