Search PubMed⌕ Search

Biomedical subjects

M Rosbash

Publications and source records attributed to M Rosbash.

At least 163 records · Page 9Linked to original sources
PubMed1986

Germ-line transformation involving DNA from the period locus in Drosophila melanogaster: overlapping genomic fragments that restore circadian and ultradian rhythmicity to per0 and per- mutants.

P-element-mediated transformations involving DNA fragments from the period (per) clock gene of Drosophila melanogaster have shown that several subsegments of the locus restore rhythmicity to per0 or per- mutants. Such fragments overlap in a genomic region complementary to one transcript, a 4.5-kb RNA which is probably the per message, in that it is necessary and (in terms of expression from this X-chromosomal locus) sufficient for the fly's circadian rhythms. It is also at least necessary for the high-frequency oscillations normally produced by courting males as they vibrate their wings. The entirety of the 4.5-kb transcript is not necessary for rather strong rhythmicity; nor does it seem to be sufficient, in transformants, for wild-type behavioral phenotypes. A 0.9-kb RNA, homologous to genomic region immediately adjacent to the source of the 4.5-kb species, oscillates in its abundance over the course of a day; but coverage of this transcript source in several transformants carrying a per0 mutation--which eliminates the 0.9-kb RNA's oscillation--does not restore rhythmicity. All of the independently isolated arrhythmic mutations tested were covered by the same array of overlapping per+-derived DNA fragments, implying that the only portion of the locus which has mutated to arrhythmicity is complementary to the 4.5-kb transcript.

Animals↗
PubMed1985

Sequence and structure of the serendipity locus of Drosophila melanogaster. A densely transcribed region including a blastoderm-specific gene.

The transcriptional organization of the Drosophila melanogaster serendipity (sry) locus (previously designated EH8) has been investigated by DNA sequencing, S1 nuclease mapping, and primer extension analysis. The data indicate that the five different (and partially overlapping) sry messenger RNAs detectable in early embryos are initiated at three separate sites, each directly upstream from one of the three protein-coding regions, designated (in 5' to 3' order) beta, alpha and delta. All of the sry mRNAs are transcribed in the same direction. The two blastoderm stage-specific sry mRNAs both include the alpha-coding region and have the same 5' terminus, only 183 nucleotides downstream from the 3' terminus of a beta region transcript (which is transcribed in ovaries). Similarly, only 331 nucleotides separate the 5' end of a delta region transcript from the major 3' end of the alpha region transcripts. One of the five sry embryonic mRNAs includes both the beta and alpha protein-coding segments and the spacer region in between, while another mRNA includes both the alpha and delta protein-coding regions as well as the spacer in between. The two read-through transcripts probably result from the failure to undergo a 3' cleavage and polyadenylation event rather than from differential splicing. As the three other sry embryo mRNAs are each transcribed from a single protein-coding region, it would appear that the alpha, beta and delta open reading frames correspond to three separate genes. Codon bias analysis reinforces the notion that these three genes code for bona fide proteins with translation starting at the first in-frame AUG codon. The predicted beta and delta polypeptides show partial amino acid sequence homology, suggesting a common evolutionary origin.

Amino Acid Sequence↗
PubMed1985

mRNA splicing efficiency in yeast and the contribution of nonconserved sequences.

A simple kinetic model for mRNA splicing predicts the way in which in vivo steady state precursor RNA levels (P) and messenger RNA levels (M) vary as a function of the rate constant of the splicing reaction (ksp). The model points to M/P as the best measure of ksp. The analysis of a set of intron mutations in a yeast gene supports the general features of the model and shows that the splicing efficiency of transcripts containing the wild-type intron is well in excess of what is necessary to generate normal mRNA levels. The data also suggest that regions of the intron, in addition to the well-conserved consensus sequences, contribute to efficient splicing.

Base Sequence↗
PubMed1985

A quantitative analysis of the effects of 5' junction and TACTAAC box mutants and mutant combinations on yeast mRNA splicing.

We have introduced four point mutations into the 5' junction (GTATGT) and TACTAAC box of a yeast intron-containing gene coding for beta-galactosidase. To analyze quantitatively mutant combinations, we deliberately avoided nucleotides adjacent to the sites of cleavage (GTATGT) and lariat formation (TACTAAC) and chose positions with expected weak effects on splicing. All four mutants do not affect, or only marginally affect, gene expression. The levels of precursor RNA and intermediates indicate, however, that splicing efficiency is affected in all cases; the first stage of splicing, 5' cleavage and lariat formation, is reduced two to 15-fold in these four mutants. The mutants were combined to generate compensatory and noncompensatory double mutant combinations. No evidence for a specific base-pairing interaction between the 5' junction and TACTAAC box could be obtained. The results suggest that all conserved positions in the 5' junction and TACTAAC box play a role in splicing efficiency.

Base Sequence↗
PubMed1985

Effect of RP51 gene dosage alterations on ribosome synthesis in Saccharomyces cerevisiae.

The Saccharomyces cerevisiae ribosomal protein rp51 is encoded by two interchangeable genes, RP51A and RP51B. We altered the RP51 gene dose by creating deletions of the RP51A or RP51B genes or both. Deletions of both genes led to spore inviability, indicating that rp51 is an essential ribosomal protein. From single deletion studies in haploid cells, we concluded that there was no intergenic dosage compensation at the level of mRNA abundance or mRNA utilization (translational efficiency), although phenotypic analysis had previously indicated a small compensation effect on growth rate. Similarly, deletions in diploid strains indicated that no strong mechanisms exist for intragenic dosage compensation; in all cases, a decreased dose of RP51 genes was characterized by a slow growth phenotype. A decreased dose of RP51 genes also led to insufficient amounts of 40S ribosomal subunits, as evidenced by a dramatic accumulation of excess 60S ribosomal subunits. We conclude that inhibition of 40S synthesis had little or no effect on the synthesis of the 60S subunit components. Addition of extra copies of rp51 genes led to extra rp51 protein synthesis. The additional rp51 protein was rapidly degraded. We propose that rp51 and perhaps many ribosomal proteins are normally oversynthesized, but the unassembled excess is degraded, and that the apparent compensation seen in haploids, i.e., the fact that the growth rate of mutant strains is less depressed than the actual reduction in mRNA, is a consequence of this excess which is spared from proteolysis under this circumstance.

Diploidy↗
PubMed1985

Posttranscriptional regulation and assembly into ribosomes of a Saccharomyces cerevisiae ribosomal protein-beta-galactosidase fusion.

To study the regulation of ribosomal protein genes, we constructed a 'lacZ fusion of the Saccharomyces cerevisiae RP51A gene, containing the first 64 codons of RP51A. In a strain lacking an intact RP51A gene (cells are viable due to the presence of an active RP51B gene), beta-galactosidase activity is 10-fold greater than in a strain containing RP51A. RP51A-lacZ mRNA levels are equal in the two strains, indicating that regulation is posttranscriptional. In the absence of the RP51A gene, the fusion protein is predominantly cytoplasmic and associated with polysomes, whereas in the presence of RP51A, the fusion protein is predominantly nuclear, and none is associated with polysomes. Deletions were made in the RP51A-coding portion of the fusion gene. The most extensively deleted gene, containing only the first seven RP51A codons fused to lacZ, produced a high level of beta-galactosidase activity in both the presence and the absence of the RP51A gene. In both cases, little or none of this shorter fusion protein was found associated with polysomes. Thus, a regulatory site (or sites) lies in the protein-coding region of RP51A. We suggest that posttranscriptional regulation of the rp51 fusion protein is related to assembly of the protein into ribosomes.

Escherichia coli↗
PubMed1984

Sequence, structure, and codon preference of the Drosophila ribosomal protein 49 gene.

In this communication, we describe several features of the D. melanogaster gene which codes for ribosomal protein 49 (rp49). Nucleotide sequence analysis in conjunction with primer extension and S1 nuclease protection experiments show that the structure of the rp49 gene consists of a 102 bp 5' exon, a single 59 bp intron, and a 420 bp 3' exon, encoding a total of 132 amino acids. The rp49 gene shares many features with other abundantly expressed Drosophila genes, including codon preference, which are discussed.

Amino Acid Sequence↗
PubMed1984

Drosophila maternal and embryo mRNAs transcribed from a single transcription unit use alternate combinations of exons.

We have investigated the organization and transcription of several genes in Drosophila melanogaster which are clustered on an 18-kb cloned DNA fragment (c25) that maps at 99D on the cytogenetic map. Multiple mRNAs, transcribed from genes which lie adjacent to a ribosomal protein ( rp49 ) gene, are present during oogenesis, embryogenesis, or both. At least five mRNAs are transcribed from one of these genes ( EH8 ); three are zygotic transcripts, of which two are blastoderm stage-specific, whereas two others accumulate during oogenesis and are therefore matenal mRNAs. The complex transcription pattern of this gene indicates that alternate usage of protein-coding exons results in the production of different mRNAs with different coding capabilities during oogenesis and embryogenesis. The EH8 transcription unit is framed by genes actively expressed in the adult male fly; thus, the blastoderm stage-specific promoter may be silent although within a region of transcriptionally active chromatin.

Amino Acid Sequence↗
PubMed1984

P-element transformation with period locus DNA restores rhythmicity to mutant, arrhythmic Drosophila melanogaster.

Mutations at the period (per) locus of Drosophila melanogaster disrupt several biological rhythms. Molecular cloning of DNA sequences encompassing the per+ locus has allowed germ-line transformation experiments to be carried out. Certain subsegments of the per region, transduced into the genome of arrhythmic pero flies, restore rhythmicity in circadian locomotor behavior and the male's courtship song.

Animals↗
PubMed1984

Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms.

We have isolated and analyzed DNA sequences encompassing the period (per) locus of Drosophila melanogaster. The location of this clock gene was delimited by the molecular mapping of chromosome aberrations at or very near the per locus. At least five RNAs are transcribed from this region. One of these transcripts, a 0.9 kb species, is strongly implicated in per's control of biological rhythms. Two independently isolated arrhythmic mutations at the per locus dramatically reduce the level of this transcript. Furthermore, the level of the 0.9 kb transcript is strongly modulated during a light/dark cycle. We discuss evidence, from previously reported genetic and phenotypic analysis of per's function, suggesting that this region may be complex and that several gene products from the per region, including this 0.9 kb transcript, may be involved in the different aspects of normal rhythmicity influenced by this clock gene.

Animals↗
PubMed1984

In vivo characterization of yeast mRNA processing intermediates.

Yeast mRNA introns contain a conserved sequence, TACTAAC, required for splicing. We previously identified a putative splicing intermediate characterized by a stop to reverse transcriptase at the TACTAAC box of the wild-type rp51A (ribosomal protein 51A gene) intron. We now show that this stop is due to a branch and occurs at the identical nucleotide in the actin intron TACTAAC box. We show further that the putative intermediate contains a complete intron and the 3' exon, but is missing the 5' exon. This RNA is largely in the form of a lariat. The lariat and the other putative splicing intermediates detected (two forms, of different molecular weights, of the excised intron and the free 5' exon) are compatible with the view that the cut at the 5' junction and lariat formation are early steps in yeast mRNA splicing and that substantial similarities exist between yeast and mammalian mRNA splicing.

Base Sequence↗
PubMed1984

Two genes for ribosomal protein 51 of Saccharomyces cerevisiae complement and contribute to the ribosomes.

We cloned and sequenced the second gene coding for yeast ribosomal protein 51 (RP51B). When the DNA sequence of this gene was compared with the DNA sequence of RP51A (J.L. Teem and M. Rosbash, Proc. Natl. Acad. Sci. U.S.A. 80:4403--4407, 1983), the following conclusions emerged: both genes code for a protein of 135 amino acids; both open reading frames are interrupted by a single intron which occurs directly after the initiating methionine; the open reading frames are 96% homologous and code for the same protein with the exception of the carboxy-terminal amino acid; DNA sequence homology outside of the coding region is extremely limited. The cloned genes, in combination with the one-step gene disruption techniques of Rothstein (R. J. Rothstein, Methods Enzymol. 101:202-211, 1983), were used to generate haploid strains containing mutations in the RP51A or RP51B genes or in both. Strains missing a normal RP51A gene grew poorly (180-min generation time versus 130 min for the wild type), whereas strains carrying a mutant RP51B were relatively normal. Strains carrying mutations in the two genes grew extremely poorly (6 to 9 h), which led us to conclude that RP51A and RP51B were both expressed. The results of Northern blot and primer extension experiments indicate that strains with a wild-type copy of the RP51B gene and a mutant (or deleted) RP51A gene grow slowly because of an insufficient amount of RP51 mRNA. The growth defect was completely rescued with additional copies of RP51B. The data suggest that RP51A contributes more RP51 mRNA (and more RP51 protein) than does RP51B and that intergenic dosage compensation, sufficient to rescue the growth defect of strains missing a wild-type RP51A gene, does not take place.

Base Sequence↗
PubMed1983

The rna2 mutation of yeast affects the processing of actin mRNA as well as ribosomal protein mRNAs.

The temperature sensitive rna2 mutation of Saccharomyces cerevisiae causes a rapid and dramatic decrease in the abundance of most ribosomal protein mRNAs We and others have recently shown that the processing of ribosomal protein mRNAs is defective at the nonpermissive temperature, suggesting that inefficient mRNA processing might be responsible for the decline in ribosomal protein mRNA levels. Actin is the only known intron-containing non-ribosomal protein yeast nuclear gene We show here that the processing of actin mRNA is also defective at the nonpermissive temperature in rna2-containing strains. The observation supports the notion that all intron-containing genes are affected in a similar fashion by the rna2 mutation.

Actins↗
PubMed1983

Evidence for the biochemical role of an internal sequence in yeast nuclear mRNA introns: implications for U1 RNA and metazoan mRNA splicing.

Sequence comparison of the introns of two yeast genes (rp51A and rp51B) coding for the same ribosomal protein shows homology only in the last 50 bases of the intron. This region of the intron contains an internal conserved sequence (ICS) present near the 3' end of all sequenced yeast nuclear mRNA introns. Removal of a 29 bp sequence containing the ICS prevents splicing of an intron-containing hybrid gene. In cells containing the wild-type gene, we have detected RNA molecules that we suggest are normal splicing intermediates, generated by an endonucleolytic cut in the primary transcript at the ICS. The homology of the ICS with a sequence near the 5' end of U1 snRNA suggests a model in which an interaction in cis between the ICS and the 5' splice junction in yeast is the counterpart of the interaction in trans between U1 and 5' splice junctions in higher eucaryotes.

Base Sequence↗
PubMed1983

Expression of a beta-galactosidase gene containing the ribosomal protein 51 intron is sensitive to the rna2 mutation of yeast.

The temperature-sensitive mutation rna2 causes the accumulation of higher molecular weight transcripts from the ribosomal protein 51 (rp51) gene of yeast and many other yeast ribosomal protein genes. We have determined the DNA sequence of the rp51 gene, confirming that it contains an intron and that the higher molecular weight transcript is an intron-containing precursor RNA. These data and other experiments suggest that the rna2 mutation affects mRNA processing (splicing) and that the presence of an intron is sufficient to render expression of a gene sensitive to the rna2 mutation. To test these hypotheses, we have inserted the rp51 intron into the coding region of a hybrid Escherichia coli beta-galactosidase gene, thereby interrupting the open reading frame subsequent to the initiating methionine codon. Despite the presence of the intron, the beta-galactosidase gene is expressed in yeast. Thus, the rp51 intron is properly excised from the normally intronless gene. The presence of the rp51 intron causes the beta-galactosidase activity to be sensitive to the rna2 mutation, consistent with the notion that this mutation affects gene expression at the level of splicing. The experiments suggest that an intron-containing beta-galactosidase gene can be used in a general way to study mRNA splicing.

Amino Acid Sequence↗