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H V Colot

Publications and source records attributed to H V Colot.

16 recordsLinked to original sources
PubMed2001

Analysis of expressed sequence tags from two starvation, time-of-day-specific libraries of Neurospora crassa reveals novel clock-controlled genes.

In an effort to determine genes that are expressed in mycelial cultures of Neurospora crassa over the course of the circadian day, we have sequenced 13,000 cDNA clones from two time-of-day-specific libraries (morning and evening library) generating approximately 20,000 sequences. Contig analysis allowed the identification of 445 unique expressed sequence tags (ESTs) and 986 ESTs present in multiple cDNA clones. For approximately 50% of the sequences (710 of 1431), significant matches to sequences in the National Center for Biotechnology Information database (of known or unknown function) were detected. About 50% of the ESTs (721 of 1431) showed no similarity to previously identified genes. We hybridized Northern blots with probes derived from 26 clones chosen from contigs identified by multiple cDNA clones and EST sequences. Using these sequences, the representation of genes among the morning and evening sequences, respectively, in most cases does not reflect their expression patterns over the course of the day. Nevertheless, we were able to identify four new clock-controlled genes. On the basis of these data we predict that a significant proportion of the expressed Neurospora genes may be regulated by the circadian clock. The mRNA levels of all four genes peak in the subjective morning as is the case with previously identified ccgs.

Blotting, Northern↗
PubMed1999

Rat8p/Dbp5p is a shuttling transport factor that interacts with Rat7p/Nup159p and Gle1p and suppresses the mRNA export defect of xpo1-1 cells.

In a screen for temperature-sensitive mutants of Saccharomyces cerevisiae defective for mRNA export, we previously identified the essential DEAD-box protein Dbp5p/Rat8p and the nucleoporin Rat7p/Nup159p. Both are essential for mRNA export. Here we report that Dbp5p and Rat7p interact through their Nterminal domains. Deletion of this portion of Rat7p (Rat7pDeltaN) results in strong defects in mRNA export and eliminates association of Dbp5p with nuclear pores. Overexpression of Dbp5p completely suppressed the growth and mRNA export defects of rat7DeltaN cells and resulted in weaker suppression in cells carrying rat7-1 or the rss1-37 allele of GLE1. Dbp5p interacts with Gle1p independently of the N-terminus of Dbp5p. Dbp5p shuttles between nucleus and cytoplasm in an Xpo1p-dependent manner. It accumulates in nuclei of xpo1-1 cells and in cells with mutations affecting Mex67p (mex67-5), Gsp1p (Ran) or Ran effectors. Overexpression of Dbp5p prevents nuclear accumulation of mRNA in xpo1-1 cells, but does not restore growth, suggesting that the RNA export defect of xpo1-1 cells may be indirect. In a screen for high-copy suppressors of the rat8-2 allele of DBP5, we identified YMR255w, now called GFD1. Gfd1p is not essential, interacts with Gle1p and Rip1p/Nup42p, and is found in the cytoplasm and at the nuclear rim.

Adenosine Triphosphatases↗
PubMed1998

Dbp5p/Rat8p is a yeast nuclear pore-associated DEAD-box protein essential for RNA export.

To identify Saccharomyces cerevisiae genes important for nucleocytoplasmic export of messenger RNA, we screened mutant strains to identify those in which poly(A)+ RNA accumulated in nuclei under nonpermissive conditions. We describe the identification of DBP5 as the gene defective in the strain carrying the rat8-1 allele (RAT = ribonucleic acid trafficking). Dbp5p/Rat8p, a previously uncharacterized member of the DEAD-box family of proteins, is closely related to eukaryotic initiation factor 4A(eIF4A) an RNA helicase essential for protein synthesis initiation. Analysis of protein databases suggests most eukaryotic genomes encode a DEAD-box protein that is probably a homolog of yeast Dbp5p/Rat8p. Temperature-sensitive alleles of DBP5/RAT8 were prepared. In rat8 mutant strains, cells displayed rapid, synchronous accumulation of poly(A)+ RNA in nuclei when shifted to the non-permissive temperature. Dbp5p/Rat8p is located within the cytoplasm and concentrated in the perinuclear region. Analysis of the distribution of Dbp5p/Rat8p in yeast strains where nuclear pore complexes are tightly clustered indicated that a fraction of this protein associates with nuclear pore complexes (NPCs). The strong mutant phenotype, association of the protein with NPCs and genetic interaction with factors involved in RNA export provide strong evidence that Dbp5p/Rat8p plays a direct role in RNA export.

Alleles↗
PubMed1998

A comprehensive biochemical and genetic analysis of the yeast U1 snRNP reveals five novel proteins.

The U1 snRNP is essential for recognition of the pre-mRNA 5'-splice site and the subsequent assembly of the spliceosome. Yeast U1 snRNP is considerably more complex than its metazoan counterpart, which suggests possible differences between yeast and metazoa in early splicing events. We have comprehensively analyzed the composition of yeast U1 snRNPs using a combination of biochemical, mass spectrometric, and genetic methods. We demonstrate the specific association of four novel U1 snRNP proteins, Snu71p, Snu65p, Nam8p, and Snu56p, that have no known metazoan homologues. A fifth protein, Npl3p, is an abundant cellular component that reproducibly co-purifies with the U1 snRNP, but its association is salt-sensitive. Therefore, we are unable to establish conclusively whether it binds specifically to the U1 snRNP. Interestingly, Nam8p and Npl3p were previously assigned functions in (pre-m)RNA-metabolism; however, so far, no association with U1 snRNP has been demonstrated or proposed. We also show that the yeast SmB protein is a U1 snRNP component. Yeast U1 snRNP therefore contains 16 different proteins, including seven snRNP core proteins, three homologues of the metazoan U1 snRNP-specific proteins, and six yeast-specific U1 snRNP proteins. We have simultaneously continued the characterization of additional mutants isolated in a synthetic lethal (MUD) screen for genes that functionally cooperate with U1 snRNA. Consistent with the biochemical results, mud10, mud15, and mud16 are alleles of SNU56, NAM8, and SNU65, respectively. mud10 and mud15 affect the in vivo splicing efficiency of noncanonical introns. Moreover, mud10p strongly affects the in vitro formation of splicing complexes, and extracts from the mud15 strain contain a U1 snRNP that migrates aberrantly on native gels. Finally, we show that Nam8p/Mud15p contributes to the stability of U1 snRNP.

Amino Acid Sequence↗
PubMed1996

The yeast splicing factor Mud13p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex.

The mechanism by which pre-mRNAs are initially recognized by the splicing machinery is not well understood. In the yeast system, commitment complexes are the earliest identified splicing complexes. They contain pre-mRNA, U1 snRNP, and the splicing factor Mud2p and probably correspond to the mammalian E complexes, which contain pre-mRNA, U1 snRNP, and the splicing factor U2AF. To identify other yeast commitment complex components, we have characterized mutant strains that are synthetic lethal with viable U1 snRNA mutations. We report here that MUD13 is a nonessential gene that encodes the yeast homolog of CBP20, the small subunit of the vertebrate nuclear cap-binding complex (CBC). Characterization of splicing in the delta-MUD13 strain and extract indicates that Mud13p is a yeast splicing factor and is the second identified non-snRNP commitment complex component. The observations also suggest that CBC interacts with other commitment complex components as well as with the substrate cap. Taken together with the accompanying results for a mammalian system, our data indicate that cap-binding proteins as well as the pre-mRNA cap contribute to early steps in spliceosome assembly.

Amino Acid Sequence↗
PubMed1992

Requirements for U2 snRNP addition to yeast pre-mRNA.

The in vitro spliceosome assembly pathway is conserved between yeast and mammals as U1 and U2 snRNPs associate with the pre-mRNA prior to U5 and U4/U6 snRNPs. In yeast, U1 snRNP-pre-mRNA complexes are the first splicing complexes visualized on native gels, and association with U1 snRNP apparently commits pre-mRNA to the spliceosome assembly pathway. The current study addresses U2 snRNP addition to commitment complexes. We show that commitment complex formation is relatively slow and does not require ATP, whereas U2 snRNP adds to the U1 snRNP complexes in a reaction that is relatively fast and requires ATP or hydrolyzable ATP analogs. In vitro spliceosome assembly was assayed in extracts derived from strains containing several U1 sRNA mutations. The results were consistent with a critical role for U1 snRNP in early complex formation. A mutation that disrupts the base-pairing between the 5' end of U1 snRNA and the 5' splice site allows some U2 snRNP addition to bypass the ATP requirement, suggesting that ATP may be used to destabilize certain U1 snRNP:pre-mRNA interactions to allow subsequent U2 snRNP addition.

Adenosine Triphosphate↗
PubMed1989

A new mutation at the period locus of Drosophila melanogaster with some novel effects on circadian rhythms.

A new period mutation has been induced and characterized in D. melanogaster. It causes flies to be apparently arrhythmic in tests of locomotor activity and thus is superficially similar to the original per01 mutant. Yet, the new "zero" allele, per04, has some novel properties and effects: Behaviorally, per04 adults often exhibit weak, long-period rhythms of locomotor activity in constant darkness; this low-frequency rhythmicity usually was not obvious in the analog behavioral records but was readily revealed by spectral analyses. These treatments of the data also extracted hidden high-frequency (ultradian) rhythms in many of the behavioral records, of the type associated with per01 and other per-nulls. The wide range of periodicities exhibited by different per04-expressing flies implies the expression of multiple oscillatory modes by this mutant. The new mutation also leads to a tendency for flies to be hyperactive during activity monitoring and is thus dissimilar to the other arrhythmic variants in the per gene but similar to the effects of a deletion of the locus. During light:dark cycling, per04 adults once more behave differently from other per0's and in fact tend to resemble wild-type flies in these conditions. The new mutation is not caused by the same nucleotide substitution that created a stop codon in the original arrhythmic per mutant and, as it turns out, per02 and per03 as well. per04 is also not a null variant at the transcriptional level; but it leads to an anomalous form of per mRNA, which is smaller than the normal 4.5 kb species encoded by this clock gene.

Alleles↗
PubMed1988

Interspecific comparison of the period gene of Drosophila reveals large blocks of non-conserved coding DNA.

We have cloned and sequenced the coding region of the period (per) gene from Drosophila pseudoobscura and D. virilis. A comparison with that of D. melanogaster reveals that the conceptual translation products consist of interspersed blocks of conserved and non-conserved amino acid sequence. The non-conserved portion, comprising approximately 33% of the protein sequence, includes the perfect Thr-Gly repeat of D. melanogaster, which is absent from the D. pseudoobscura and D. virilis proteins. Based on these observations and cross-species transformation experiments, we suggest that the interspecific variability in the per primary amino acid sequence contributes to the control of species-specific behaviors.

Amino Acid Sequence↗
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↗
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↗
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↗
PubMedDate not supplied

A family of unusually spliced biologically active transcripts encoded by a Drosophila clock gene.

Complementary DNA cloning of the transcripts of the Drosophila clock gene period reveals three distinct transcripts. These result from unusual splicing pathways, one involving a CG 3' splice site and one resulting in the use of two different reading frames in one exon, and they predict three separate proteins. Two of the cloned cDNAs can restore clock function to mutant arrhythmic flies.

Animals↗
PubMedDate not supplied

Behaviour modification by in vitro mutagenesis of a variable region within the period gene of Drosophila.

The period gene of Drosophila melanogaster, implicated in the control of both the circadian and male courtship song rhythms, is found to be polymorphic. Alleles differ in the length of a region of the gene encoding a series of threonine-glycine repeat units. The phenotypes of transformed fruit flies, in which the only functional period gene lacks the entire perfect threonine-glycine repeat region, show that the effects of the period gene on the circadian and male courtship song rhythms can be dissociated.

Alleles↗