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M Rosbash

Publications and source records attributed to M Rosbash.

At least 145 records · Page 8Linked to original sources
PubMed1988

Spatial and temporal expression of the period gene in Drosophila melanogaster.

The temporal and spatial expression of the period gene of Drosophila melanogaster has been analyzed by examining the expression of a per beta-galactosidase fusion gene in transformants and by in situ hybridization experiments with wild-type flies. Several strains of Drosophila melanogaster, transformed with the fusion gene, have been generated. The gene is active in mid-late embryos in the midline of the nervous system. Thereafter, beta-galactosidase activity is undetectable until the pupal stage when the prothoracic gland-corpora allata and the optic lobes are beta-galactosidase positive. In adults a surprisingly large number of tissues stain positively, including antennae, proboscis, eyes, optic lobes, cells of the central brain, cells of the thoracic ganglia, gut, Malpighian tubules, and ovarian follicle cells. The temporal pattern of expression agrees well with previous estimates made from developmental Northern blots with RNA extracted from wild-type animals. We suggest that many of the tissues that express the per gene contain their own intrinsic oscillator activity.

Animals↗
PubMed1988

A chemical modification/interference study of yeast pre-mRNA spliceosome assembly and splicing.

A chemical modification/interference assay was used to determine the yeast pre-mRNA sequence requirements for in vitro spliceosome assembly and splicing. Modifications of any of the nucleotides within the 5' splice site and branch point (TACTAAC box) consensus sequences as well as less conserved intron and exon positions were found to inhibit assembly and/or splicing. The interference pattern of the 5' splice site and TACTAAC box lesions increased as spliceosome assembly proceeded (complex III----complex I----complex II) and as splicing proceeded, suggesting that these sequence elements play multiple roles in the assembly of yeast spliceosomes and in the removal of intervening sequences. Furthermore, modification (or mutation) of a TACTAAC-like sequence upstream of the branch point was found to inhibit the rate of spliceosome assembly, implying a possible role for degenerate branch point sequences in modulating the efficiency of spliceosome assembly.

Base Sequence↗
PubMed1988

Early commitment of yeast pre-mRNA to the spliceosome pathway.

Pre-mRNA splicing in vitro is preceded by complex formation (spliceosome assembly). U2 small nuclear RNA (snRNA) is found in the earliest form of the spliceosome detected by native gel electrophoresis, both in Saccharomyces cerevisiae and in metazoan extracts. To examine the requirements for the formation of this early complex (band III) in yeast extracts, we cleaved the U2 snRNA by oligonucleotide-directed RNase H digestion. U2 snRNA depletion by this means inhibits both splicing and band III formation. Using this depleted extract, we were able to design a chase experiment which shows that a pre-mRNA substrate is committed to the spliceosome assembly pathway in the absence of functional U2 snRNP. Interactions occurring during the commitment step are highly resistant to the addition of an excess of unlabeled substrate and require little or no ATP. Sequence requirements for this commitment step have been analyzed by competition experiments with deletion mutants: both the 5' splice site consensus sequence and the branch point TACTAAC box sequence are necessary. These experiments strongly suggest that the initial assembly process requires a trans-acting factor(s) (RNA and/or proteins) that recognizes and stably binds to the two consensus sequences of the pre-mRNA prior to U2 snRNP binding.

Chromosome Deletion↗
PubMed1987

S. cerevisiae U1 RNA is large and has limited primary sequence homology to metazoan U1 snRNA.

We have cloned and sequenced the yeast SNR19 gene and show here that snR19 is the yeast homolog of metazoan U1 snRNA. sn R19 is 569 nucleotides long, strikingly larger than its metazoan counterpart. The two molecules resemble each other closely in the predicted secondary structure of their first 50 nucleotides. Primary sequence homology is restricted to some of their single-stranded regions, including 11 consecutive nucleotides at the 5' end of the two molecules, the region that interacts with pre-mRNA 5' splice junctions. snR19 is spliceosome-associated and required for in vitro pre-mRNA splicing. We also note that 8 sequences in snR19 have extensive complementarity to snR20, the large yeast U2 RNA, suggesting that yeast U1 may interact with yeast U2 by base-pairing.

Animal Population Groups↗
PubMed1987

Molecular mapping of point mutations in the period gene that stop or speed up biological clocks in Drosophila melanogaster.

The pero1 and the pers mutations in Drosophila melanogaster, which seem to eliminate or speed up, respectively, the clocks underlying biological rhythmicity, were mapped to single nucleotides. Chimeric DNA fragments consisting of well-defined wild-type plus mutant DNA subsegments were constructed, introduced into flies by germ-line transformation, and assayed for biological activity. These experiments localized both pero1 and pers to a 1.7-kilobase DNA fragment that is mostly coding DNA. Sequencing of this subsegment from each mutant showed that pero1 is completely accounted for by a nonsense mutation in the third coding exon of a 4.5-kilobase RNA transcribed from this locus. The pers mutation is also a single nucleotide substitution, in the fourth coding exon, which results in a serine-to-asparagine substitution in the per gene protein product. The functional significance of these changes is discussed with reference to the phenotypes of the two mutations.

Amino Acid Sequence↗
PubMed1987

A novel role for the 3' region of introns in pre-mRNA splicing of Saccharomyces cerevisiae.

To investigate the importance of sequences between the yeast (Saccharomyces cerevisiae) branch point (TACTAAC box) and 3' splice site (AG), we generated a series of pre-mRNA substrates that differed in the length of RNA retained on the 3' side of the TACTAAC box. These pre-mRNAs were compared as substrates for the first step of in vitro splicing (5' cleavage and lariat formation) and in vitro spliceosome assembly (complex formation) in a whole-cell yeast extract. The results indicate that for rp51A pre-mRNA at least 29 nucleotides of RNA on the 3' side of the TACTAAC box are required for 5' cleavage and lariat formation, as smaller substrates fail to manifest any detectable cleavage or ligation events. Analysis of splicing complex assembly indicates that these smaller substrates undergo efficient yet incomplete complex formation; they are blocked at a late stage of spliceosome assembly, the complex I to complex II transition (Pikielny et al. 1986), a result which suggests that the failure to form lariats is due to a specific assembly defect. The lariat formation block (and assembly defect) can be relieved by the addition of ribohomopolymer "tails" to the 3' end of the shortened rp51A pre-mRNAs, and similar results were obtained with shortened actin pre-mRNAs. The results of this study indicate that this region of the pre-mRNA serves a specific function late in in vitro spliceosome assembly.

Base Sequence↗
PubMed1986

Differential nuclease sensitivity identifies tight contacts between yeast pre-mRNA and spliceosomes.

The oligonucleotide-directed RNase H sensitivity of a yeast (Saccharomyces cerevisiae) pre-mRNA was determined in an in vitro splicing reaction. While most of the pre-mRNA was sensitive to cleavage, the regions of the 5' splice site and TACTAAC box were found to be highly resistant. The biochemical requirements for protection against nuclease attack parallel those of both spliceosome formation and splicing. Most of the uncleaved pre-mRNA remaining after RNase H challenge was found associated with two forms of the yeast spliceosome. Differences in the RNase H sensitivity of pre-mRNA found in the two spliceosome forms indicate an increased association of splicing factors with the 5' splice site during spliceosome assembly.

Base Sequence↗
PubMed1986

Efficient trans-splicing of a yeast mitochondrial RNA group II intron implicates a strong 5' exon-intron interaction.

The reaction mechanism for self-splicing introns requires the existence of a 5' exon binding site on the intron. Experimental evidence is now presented consistent with the existence of such a binding site by demonstrating efficient and accurate trans-self-splicing of a yeast mitochondrial group II intron. Partial and complete trans-splicing reactions take place in the absence of branch formation, part of the usual pathway of nuclear splicing and group II self-splicing. In addition to indicating the existence of a 5' exon binding site on the intron, the results have mechanistic implications for group II self-splicing and perhaps for nuclear splicing as well.

Exons↗
PubMed1986

The period clock locus of D. melanogaster codes for a proteoglycan.

The period (per) gene of D. melanogaster is involved in the generation of biological rhythms. The most striking feature of the predicted coding sequence, corresponding to the key 4.5 kb transcript from this locus, is an extensive run of alternating Gly-Thr residues. This is homologous to a series of Gly-Ser repeats in a chondroitin sulfate proteoglycan. To determine whether the per transcript codes for a proteoglycan, a region of its coding sequence was expressed (in bacteria) as part of a fusion protein, which was used to immunize rabbits. When the resultant immune sera were used to probe fly protein preparations, they detected an antigen that is present in wild-type flies and absent in a per- mutant. Biochemical characterization of this antigen indicated that it is indeed a proteoglycan.

Amino Acid Sequence↗
PubMed1986

Specific small nuclear RNAs are associated with yeast spliceosomes.

Two different methods have been devised for the analysis and purification of spliceosomes formed in a yeast in vitro splicing system. The first method relies on the electrophoretic separation of ribonucleoprotein particles in composite acrylamide-agarose gels. A large fraction of added substrate is located in spliceosomes, the formation of which can be shown to be dependent on the presence of both a yeast 5' splice junction and a TACTAAC box on the RNA substrate. The second method relies on oligo(dT)-cellulose chromatography of spliceosomes formed with a polyadenylated substrate. Purification of spliceosomes by either method indicates that at least three small nuclear RNAs, approximately 160, 185, and 215 nucleotides in length, are specifically associated with yeast spliceosomes.

Chromatography↗
PubMed1986

Embryonic expression of the period clock gene in the central nervous system of Drosophila melanogaster.

We have examined the temporal and spatial expression of the 4.5-kb mRNA that is transcribed from the period locus of Drosophila melanogaster and is the best candidate for the per gene product. Both Northern blot analyses and hybridizations in situ to tissue sections reveal significant expression of the 4.5-kb mRNA in embryos. This expression is limited to the central nervous system of the developing embryo and is localized within the brain and ventral ganglia. The 4.5-kb mRNA is enriched in adult heads (by Northern blotting) although we were not able to detect specific localization (in situ). In addition to the physiological role the 4.5-kb mRNA might have in maintaining biological rhythms, we now suggest that it has a developmental role for establishing mechanisms that are necessary for eventual expression of clock functions.

Animals↗
PubMed1986

Blastoderm-specific and read-through transcription of the sry alpha gene transformed into the Drosophila genome.

The serendipity (sry) locus contains three tightly clustered genes: beta, alpha, and delta. The alpha gene lies between beta and delta and is expressed predominantly at the blastoderm stage of embryogenesis. Additional features of this locus include read-through transcription and the fact that the predicted beta and delta polypeptides show remarkable homology to the Xenopus RNA polymerase III transcription factor TFIIIA. To identify cis-acting elements controlling the expression of the alpha and beta transcripts, we created transformed lines containing modified versions of these genes. In lines containing derivatives of both the alpha and beta genes, the expected modified mRNAs are transcribed. An alpha gene variant containing only 798 bp of upstream DNA is also transcribed, and at the proper time; thus blastoderm-specific alpha gene transcription is independent of expression of the adjacent upstream beta gene. Analysis of transformed lines confirmed the beta-alpha read-through transcription, which was eliminated by the insertion of a different polyadenylation site within the coding region of the beta gene. We conclude that the transcription of the beta and alpha genes is independently regulated.

Animals↗
PubMed1986

RNA splicing and intron turnover are greatly diminished by a mutant yeast branch point.

Two mutant genes, both of which contain an A----C transversion at the absolutely conserved branch point of the yeast "TACTAAC box" (TACTAAC----TACTACC), were constructed and introduced into yeast cells. Splicing and gene expression are almost completely eliminated by this mutation, but a low level (approximately equal to 0.1%) of proper splicing is detectable. Branch point mapping indicates that the mutant branch is formed at the normal location--i.e., to cytidine rather than adenosine. The mutant branch is also a very poor substrate for the HeLa cell debranching enzyme. Although splicing of the mutant transcripts is very poor, the cells contain a high level of mutant intron because these excised introns are remarkably stable. The results imply that the normal branch point is important not only for branch formation and splicing but also for intron turnover.

Base Sequence↗
PubMed1986

Alternative branch points are selected during splicing of a yeast pre-mRNA in mammalian and yeast extracts.

Pre-mRNA splicing in yeast and higher eukaryotes proceeds by similar pathways, in which a probable splicing intermediate and the excised intron are in a lariat configuration. To compare the pre-mRNA splicing mechanisms in yeast and higher eukaryotes, we have analyzed the RNA products resulting from in vitro processing of a yeast intron-containing pre-mRNA in HeLa cell and yeast extracts. In yeast, the RNA branch (2'-5' phosphodiester bond) of the RNA lariat forms at the third adenosine of the TACTAAC box in vivo and in vitro. In contrast, in the HeLa cell extract, the yeast pre-mRNA is accurately spliced, but the RNA lariats contain RNA branches located significantly closer to the 3' splice site than the TACTAAC box. In yeast, mutant pre-mRNAs that lack the TACTAAC box are not spliced in vivo or in vitro. However, these same mutant pre-mRNAs are accurately spliced in the HeLa cell extract. Therefore, although pre-mRNA splicing in yeast and higher eukaryotes proceeds by the same basic pathway, there are substantial differences in the specificity of the biochemical components that mediate the formation of the RNA processing products.

Base Sequence↗