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M Rosbash

Publications and source records attributed to M Rosbash.

At least 109 records · Page 6Linked to original sources
PubMed1992

The analysis of new short-period circadian rhythm mutants suggests features of D. melanogaster period gene function.

A number of new period gene (per) mutants were generated by in vitro mutagenesis and germ line transformation. Missense mutations were made at amino acid 589, which is altered in the 19 h short-period (per(s)) mutant, and insertion mutations were generated with peptides commonly used for epitope tagging. Most of these new per mutants had short behavioral rhythms. Flies with heteroallelic combinations of these new mutant per genes were found to have "hybrid" periods, i.e., they had values that were usually in between those of the individual alleles. These findings suggest that short-period per mutants are not unusual gain-of-function mutants but rather more traditional loss-of-function mutants that are unable to influence the circadian pacemaker in a proper manner. The data also suggest that the per protein may engage in important intermolecular interactions.

Animals↗
PubMed1992

Expression of the period clock gene within different cell types in the brain of Drosophila adults and mosaic analysis of these cells' influence on circadian behavioral rhythms.

The product of the period (per) gene of Drosophila melanogaster is continuously required for the functioning of the circadian pacemaker of locomotor activity. We have used internally marked mosaics to determine the anatomical locations at which per expression is required for adult rhythmicity, and thus where the fly's circadian pacemaker is likely located in this holometabolous insect. We first provide a detailed description of the distribution and nature of per-expressing cells in the fly's CNS. Using an antibody to the per gene product, or to that of a reporter of per expression, in conjunction with an antibody to the embryonic lethal-abnormal visual system (elav) gene product--which is used as a marker of neuronal identity--we have experimentally confirmed previously proposed assignments of per-expressing cells to the neuronal and glial classes. Thus, we found that per expression and elav immunoreactivity colocalized in large cells located in the lateral cortex of the central brain, as well as in more dorsally located cells in the posterior central brain. In contrast, we found that cells located at the margins of the cortex and the neuropil, and within the neuropil, as well as smaller cortical cells found throughout the brain's cortex, were elav negative, supporting the notion that they are glial in nature. Using internally marked mosaics, we find that the pacemaker is located in brain but is not exclusive to the eyes, the ocelli, or the optic lobes, which is consistent with previous reports obtained in this and other insects of this class. Although the pacemaker may be a paired structure, we show that the functioning of one of them is sufficient for rhythmicity. Finally, we report that glial expression is sufficient for some behavioral rhythmicity to be manifest. However, the rhythmicities of animals for which per expression was confined to glia were weak, suggesting that neuronal per expression as well may be required for normal pacemaker function.

Animals↗
PubMed1992

The period gene encodes a predominantly nuclear protein in adult Drosophila.

The period gene of Drosophila melanogaster (per) is important for the generation and maintenance of biological rhythms. Previous light microscopic observations indicated that per is expressed in a variety of tissues and cell types and suggested that the per protein (PER) may be present in different subcellular compartments. To understand how PER influences circadian rhythms, it is important to define its subcellular location, especially in adult flies where inducible promoter experiments suggested that it is most relevant to circadian locomotor activity rhythms. To this end, we report the results of an immunoelectron microscopic analysis of wild-type flies and per-beta-galactosidase (beta-gal) fusion gene transgenics using a polyclonal anti-PER antibody or an anti-beta-gal antibody, respectively. Most of the PER antigen and the fusion gene product were located within nuclei, suggesting that PER acts in that subcellular compartment to affect circadian rhythms. The results are discussed in terms of per's possible biochemical functions.

Animals↗
PubMed1991

Genetic depletion indicates a late role for U5 snRNP during in vitro spliceosome assembly.

The pre-mRNA splicing pathway is highly conserved from yeast (S. cerevisiae) to mammals. Of the four snRNPs involved in splicing three (U1, U2 and U4/U6) have been shown to be essential for in vitro splicing. To examine the remaining snRNP, we utilized our previously described genetic procedures (Seraphin and Rosbash, 1989) to prepare yeast extracts depleted of U5 snRNP. The results show that U5 snRNP is necessary for both steps of pre- mRNA splicing and for proper spliceosome assembly, i.e., addition of the U4/U5/U6 triple snRNP. The prior steps of U1 and U2 snRNP addition occur normally in the absence of U5 snRNP.

Blotting, Northern↗
PubMed1991

A post-transcriptional mechanism contributes to circadian cycling of a per-beta-galactosidase fusion protein.

The period gene (per) of Drosophila melanogaster affects circadian rhythms. Circadian fluctuations in per mRNA levels are thought to contribute to circadian fluctuations in per protein levels in the heads of adult flies. To address the mechanisms underlying these oscillatory phenomena, we have analyzed RNA and protein cycling from two per-beta-galactosidase fusion genes. These studies demonstrate that 5' noncoding sequences from per are sufficient to cause the fusion mRNA levels to cycle in a wild-type (rhythmic) background. Protein cycling requires additional sequences derived from the per coding region. The data suggest that there is a per-dependent posttranscriptional mechanism that is under circadian clock control required for per protein levels to fluctuate in a rhythmic fashion.

Animals↗
PubMed1991

Molecular transfer of a species-specific behavior from Drosophila simulans to Drosophila melanogaster.

Drosophila males modulate the interpulse intervals produced during their courtship songs. These song cycles, which are altered by mutations in the clock gene period, exhibit a species-specific variation that facilitates mating. We have used chimeric period gene constructs from Drosophila melanogaster and Drosophila simulans in germline transformation experiments to map the genetic control of their song rhythm difference to a small segment of the amino acid encoding information within this gene.

Amino Acid Sequence↗
PubMed1991

The yeast branchpoint sequence is not required for the formation of a stable U1 snRNA-pre-mRNA complex and is recognized in the absence of U2 snRNA.

Commitment complexes contain U1 snRNP as well as pre-mRNA and are the earliest functional complexes that have been described during in vitro spliceosome assembly. We have used a gel retardation assay to analyze the role of the yeast pre-mRNA cis-acting sequences in commitment complex formation. The results suggest that only a proper 5' splice site sequence is required for efficient U1 snRNA-pre-mRNA complex formation. A role for the highly conserved UACUAAC branchpoint sequence is indicated, however, by competition experiments and by the direct analysis of branchpoint mutant substrates, which cannot form one of the two commitment complex species observed with wild-type substrates. The results suggest that the formation of a U1 snRNP-pre-mRNA complex is not dependent upon the presence of a branchpoint sequence but that the branchpoint sequence is recognized prior to U2 snRNP addition during in vitro spliceosome assembly.

Actins↗
PubMed1991

Cloning of the two essential yeast genes, PRP6 and PRP9, and their rapid mapping, disruption and partial sequencing using a linker insertion strategy.

In the yeast Saccharomyces cerevisiae, some thermosensitive (ts) mutants have been shown to be impaired in pre-mRNA splicing (prp mutants). From a yeast genomic library, we have isolated plasmids that complement prp6 or prp9 ts mutations. These plasmids also complement the ts growth defect of additional independent mutants identified as new prp6 and prp9 ts alleles, indicating that the cloned DNAs encode PRP6 and PRP9 genes, respectively. Here, we describe the restriction maps of these loci which are localized on chromosome II and IV, respectively. The limits of open reading frames (ORFs) within the cloned inserts have been determined using a linker insertion strategy combined with the ts complementation assay. Double-strand DNA sequencing was also performed directly on the yeast expression vector from the inserted linkers. Gene disruption experiments demonstrate that both genes are essential for viability.

Base Sequence↗
PubMed1991

The strength and periodicity of D. melanogaster circadian rhythms are differentially affected by alterations in period gene expression.

The per gene of D. melanogaster influences or participates in the generation of biological rhythms. Previous experiments have identified the head as the location from which per exerts its effect on circadian rhythms. To localize further this region and to examine the effects of altered levels and altered spatial expression patterns of the per gene on circadian rhythms of locomotor activity, we have characterized transformed lines containing per gene constructs missing substantial cis-acting regulatory information. The data suggest that wild-type levels of per gene expression are necessary in only a small fraction of the nervous system for near wild-type periods, whereas a larger fraction of per-expressing cells in the brain contributes to the strength of the circadian rhythms.

Animals↗
PubMed1991

Who's on first? The U1 snRNP-5' splice site interaction and splicing.

U1 small nuclear ribonucleoprotein (snRNP) is important for pre-mRNA splicing both in yeast (Saccharomyces cerevisiae) and mammalian systems. The RNA component of U1 snRNP, U1 snRNA, interacts by base pairing with pre-mRNA 5' splice sites. This article examines recent evidence suggesting that U1 snRNP is important for an early step in spliceosome assembly rather than a late step that contributes to the specificity of 5' splice-site cleavage.

Animals↗
PubMed1991

U1 snRNP can influence 3'-splice site selection as well as 5'-splice site selection.

To address the mechanisms that underlie splice site selection and splice site partner assignment, we analyzed the splicing of yeast (Saccharomyces cerevisiae) transcripts containing splice site region duplications. When the 5'-splice site region was duplicated, both sites were utilized to the same extent, indicating little or no influence of proximity on 5'-splice site choice. However, the effect of a 5'-mutant site was greatly enhanced by the presence of an adjacent wild-type site, and this effect was reversed by the restoration of base-pairing with U1 snRNA. 3'-Splice site choice was apparently influenced by proximity, as the site closest to the 5'-splice site was greatly preferred. Studies with strains carrying some U1 snRNA mutations showed an increase in the use of the distal 3'-splice site, indicating a role for U1 snRNP in 3'-splice site selection. The data are compared with those from mammalian splice site choice experiments and suggest mechanisms that influence differential splice site choice as well as exon skipping.

Base Sequence↗
PubMed1990

Exon mutations uncouple 5' splice site selection from U1 snRNA pairing.

It has previously been shown that a mutation of yeast 5' splice junctions at position 5 (GUAUGU) causes aberrant pre-mRNA cleavages near the correct 5' splice site. We show here that the addition of exon mutations to an aberrant cleavage site region transforms it into a functional 5' splice site both in vivo and in vitro. The aberrant mRNAs are translated in vivo. The results suggest that the highly conserved G at the 5' end of introns is necessary for the second step of splicing. Further analyses indicate that the location of the U1 snRNA-pre-mRNA pairing is not affected by the exon mutations and that the precise 5' splice site is selected independent of this pairing.

Base Sequence↗
PubMed1990

Feedback of the Drosophila period gene product on circadian cycling of its messenger RNA levels.

Mutations in the period (per) gene of Drosophila melanogaster affect both circadian and ultradian rhythms. Levels of per gene product undergo circadian oscillation, and it is now shown that there is an underlying oscillation in the level of per RNA. The observations indicate that the cycling of per-encoded protein could result from per RNA cycling, and that there is a feedback loop through which the activity of per-encoded protein causes cycling of its own RNA.

Animals↗
PubMed1990

Saccharomyces cerevisiae U1 small nuclear RNA secondary structure contains both universal and yeast-specific domains.

The five small nuclear RNAs (snRNAs) involved in mammalian pre-mRNA splicing (U1, U2, U4, U5, and U6) are well conserved in length, sequence, and especially secondary structure. These five snRNAs from Saccharomyces cerevisiae show notable size and sequence differences from their metazoan counterparts. This is most striking for the large S. cerevisiae U1 and U2 snRNAs, for which no secondary structure models currently exist. Because of the importance of U1 snRNA in the early steps of "spliceosome" assembly, we wanted to compare the highly conserved secondary structure of metazoan U1 snRNA (approximately 165 nucleotides) with that of S. cerevisiae U1 snRNA (568 nucleotides). To this end, we have cloned and sequenced the U1 gene from two other yeast species possessing large U1 RNAs. Using computer-derived structure predictions, phylogenetic comparisons, and structure probing, we have arrived at a secondary structure model for S. cerevisiae U1 snRNA. The results show that most elements of higher eukaryotic U1 snRNA secondary structure are conserved in S. cerevisiae. The hundreds of "extra" nucleotides of yeast U1 RNA, also highly structured, suggest that large insertions and/or deletions have occurred during the evolution of the U1 gene.

Base Sequence↗
PubMed1990

Phenotypic and genetic analysis of Clock, a new circadian rhythm mutant in Drosophila melanogaster.

Clock is a semidominant X-linked mutation that results in shortening the period of Drosophila melanogaster's free-running locomotor activity rhythm from ca. 24.0 to ca. 22.5 hr. This mutation similarly shortened the phase response curve, determined by resetting activity rhythms with light pulses. Eclosion peaks for Clk cultures were separated by only 22.5 hr instead of the normal 24 hr. Clk was mapped close to, but separable from, another rhythm mutation--period01--by recombination. The estimated distance between these two mutations was short enough to suggest that Clk could be a per allele. If this is the case, the new mutant is unique in that it, unlike other per variants, is associated with essentially normal 1-min courtship song rhythms when Clk is expressed in males. Also, the new rhythm variant could not, in contrast to a short-period per mutation, have its effects on free-running activity rhythms uncovered by deletions. This result, and the lack of coverage of Clk's effects by duplications, suggest that it is not a simple hypomorphic or amorphic mutation.

Animals↗