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J L Woolford

Publications and source records attributed to J L Woolford.

At least 37 records · Page 2Linked to original sources
PubMed1993

A protein containing conserved RNA-recognition motifs is associated with ribosomal subunits in Saccharomyces cerevisiae.

Using PCR cloning techniques, we have isolated a Saccharomyces cerevisiae gene encoding a protein that contains two highly conserved RNA-recognition motifs. This gene, designated RNP1, encodes an acidic protein that is similar in sequence to a variety of previously isolated RNA binding proteins, including nucleolin, poly (A) binding protein, and small nuclear ribonucleoproteins. The RNP1 gene maps to the left arm of chromosome XIV centromere distal to SUF10. Haploid yeast containing a null allele of RNP1 are viable, indicating that RNP1 is dispensible for mitotic growth. However genomic Southern blot analysis indicated that several other loci in the S. cerevisiae genome appear to contain sequences similar to those in the RNP1 gene. The majority of the Rnp1 protein is cytoplasmic. Extra copies of RNP1 cause a decrease in levels of 80S monoribosomes. A fraction of Rnp1 protein cosediments on sucrose gradients with 40S and 60S ribosomal subunits and 80S monosomes, but not with polyribosomes.

Amino Acid Sequence↗
PubMed1993

Molecular genetics of cryptopleurine resistance in Saccharomyces cerevisiae: expression of a ribosomal protein gene family.

The Saccharomyces cerevisiae CRY1 gene encodes the 40S ribosomal subunit protein rp59 and confers sensitivity to the protein synthesis inhibitor cryptopleurine. A yeast strain containing the cry1-delta 1::URA3 null allele is viable, cryptopleurine sensitive (CryS), and expresses rp59 mRNA, suggesting that there is a second functional CRY gene. The CRY2 gene has been isolated from a yeast genomic library cloned in bacteriophage lambda, using a CRY1 DNA probe. The DNA sequence of the CRY2 gene contains an open reading frame encoding ribosomal protein 59 that differs at five residues from rp59 encoded by the CRY1 gene. The CRY2 gene was mapped to the left arm of chromosome X, centromere-proximal to cdc6 and immediately adjacent to ribosomal protein genes RPS24A and RPL46. Ribosomal protein 59 is an essential protein; upon sporulation of a diploid doubly heterozygous for cry1-delta 2::TRP1 cry2-delta 1::LEU2 null alleles, no spore clones containing both null alleles were recovered. Several results indicate that CRY2 is expressed, but at lower levels than CRY1: (1) Introduction of CRY2 on high copy plasmids into CryR yeast of genotype cry1 CRY2 confers a CryS phenotype. Transformation of these CryR yeast with CRY2 on a low copy CEN plasmid does not confer a CryS phenotype. (2) Haploids containing the cry1-delta 2::TRP1 null allele have a deficit of 40S ribosomal subunits, but cry2-delta 1::LEU2 strains have wild-type amounts of 40S ribosomal subunits. (3) CRY2 mRNA is present at lower levels than CRY1 mRNA. (4) Higher levels of beta-galactosidase are expressed from a CRY1-lacZ gene fusion than from a CRY2-lacZ gene fusion. Mutations that alter or eliminate the last amino acid of rp59 encoded by either CRY1 or CRY2 result in resistance to cryptopleurine. Because CRY2 (and cry2) is expressed at lower levels than CRY1 (and cry1), the CryR phenotype of cry2 mutants is only expressed in strains containing a cry1-delta null allele.

Alkaloids↗
PubMed1993

DRS1 to DRS7, novel genes required for ribosome assembly and function in Saccharomyces cerevisiae.

To identify Saccharomyces cerevisiae mutants defective in assembly or function of ribosomes, a collection of cold-sensitive strains generated by treatment with ethyl methanesulfonate was screened by sucrose gradient analysis for altered ratios of free 40S to 60S ribosomal subunits or qualitative changes in polyribosome profiles. Mutations defining seven complementation groups deficient in ribosomal subunits, drs1 to drs7, were identified. We have previously shown that DRS1 encodes a putative ATP-dependent RNA helicase necessary for assembly of 60S ribosomal subunits (T. L. Ripmaster, G. P. Vaughn, and J. L. Woolford, Jr., Proc. Natl. Acad. Sci. USA 89:11131-11135, 1992). Strains bearing the drs2 mutation process the 20S precursor of the mature 18S rRNA slowly and are deficient in 40S ribosomal subunits. Cloning and sequencing of the DRS2 gene revealed that it encodes a protein similar to membrane-spanning Ca2+ ATPases. The predicted amino acid sequence encoded by DRS2 contains seven transmembrane domains, a phosphate-binding loop found in ATP- or GTP-binding proteins, and a seven-amino-acid sequence detected in all classes of P-type ATPases. The cold-sensitive phenotype of drs2 is suppressed by extra copies of the TEF3 gene, which encodes a yeast homolog of eukaryotic translation elongation factor EF-1 gamma. Identification of gene products affecting ribosome assembly and function among the DNAs complementing the drs mutations validates the feasibility of this approach.

Adenosine Triphosphatases↗
PubMed1993

Yeast ribosomal protein L1 is required for the stability of newly synthesized 5S rRNA and the assembly of 60S ribosomal subunits.

Ribosomal protein L1 from Saccharomyces cerevisiae binds 5S rRNA and can be released from intact 60S ribosomal subunits as an L1-5S ribonucleoprotein (RNP) particle. To understand the nature of the interaction between L1 and 5S rRNA and to assess the role of L1 in ribosome assembly and function, we cloned the RPL1 gene encoding L1. We have shown that RPL1 is an essential single-copy gene. A conditional null mutant in which the only copy of RPL1 is under control of the repressible GAL1 promoter was constructed. Depletion of L1 causes instability of newly synthesized 5S rRNA in vivo. Cells depleted of L1 no longer assemble 60S ribosomal subunits, indicating that L1 is required for assembly of stable 60S ribosomal subunits but not 40S ribosomal subunits. An L1-5S RNP particle not associated with ribosomal particles was detected by coimmunoprecipitation of L1 and 5S rRNA. This pool of L1-5S RNP remained stable even upon cessation of 60S ribosomal subunit assembly by depletion of another ribosomal protein, L16. Preliminary results suggest that transcription of RPL1 is not autogenously regulated by L1.

Alleles↗
PubMed1993

TIF4631 and TIF4632: two yeast genes encoding the high-molecular-weight subunits of the cap-binding protein complex (eukaryotic initiation factor 4F) contain an RNA recognition motif-like sequence and carry out an essential function.

The 5' ends of eukaryotic mRNAs are blocked by a cap structure, m7GpppX (where X is any nucleotide). The interaction of the cap structure with a cap-binding protein complex is required for efficient ribosome binding to the mRNA. In Saccharomyces cerevisiae, the cap-binding protein complex is a heterodimer composed of two subunits with molecular masses of 24 (eIF-4E, CDC33) and 150 (p150) kDa. p150 is presumed to be the yeast homolog of the p220 component of mammalian eIF-4F. In this report, we describe the isolation of yeast gene TIF4631, which encodes p150, and a closely related gene, TIF4632. TIF4631 and TIF4632 are 53% identical overall and 80% identical over a 320-amino-acid stretch in their carboxy-terminal halves. Both proteins contain sequences resembling the RNA recognition motif and auxiliary domains that are characteristic of a large family of RNA-binding proteins. tif4631-disrupted strains exhibited a slow-growth, cold-sensitive phenotype, while disruption of TIF4632 failed to show any phenotype under the conditions assayed. Double gene disruption engendered lethality, suggesting that the two genes are functionally homologous and demonstrating that at least one of them is essential for viability. These data are consistent with a critical role for the high-molecular-weight subunit of putative yeast eIF-4F in translation. Sequence comparison of TIF4631, TIF4632, and the human eIF-4F p220 subunit revealed significant stretches of homology. We have thus cloned two yeast homologs of mammalian p220.

Amino Acid Sequence↗
PubMed1992

A putative ATP-dependent RNA helicase involved in Saccharomyces cerevisiae ribosome assembly.

We have isolated a cold-sensitive mutant of Saccharomyces cerevisiae in which there is a deficit of 60S ribosomal subunits. Cold sensitivity and the assembly defect are recessive and cosegregate, defining a single essential gene that we designated DRS1 (deficiency of ribosomal subunits). The wild-type DRS1 gene was cloned by complementation of the cold-sensitive phenotype of drs1. Sequence analysis reveals a high degree of similarity to a family of proteins that are thought to function as ATP-dependent RNA helicases. Pulse-chase analysis of ribosomal RNA synthesis and processing indicates that the drs1 mutant accumulates the 27S precursor of the mature 25S rRNA. These results suggest that, as in pre-mRNA splicing, RNA helicase activities are involved in ribosomal RNA processing.

Amino Acid Sequence↗
PubMed1992

RNA splicing in lower eukaryotes.

Recently, cis-acting elements and trans-acting RNA and protein factors necessary for splicing nuclear pre-mRNAs, group II introns or group III introns, have been discovered, and new roles for the splicing factors have been elucidated. Parallels among the pathways for splicing these different classes of introns have been identified.

Animals↗
PubMed1991

Assembly of 60S ribosomal subunits is perturbed in temperature-sensitive yeast mutants defective in ribosomal protein L16.

Temperature-sensitive mutants defective in 60S ribosomal subunit protein L16 of Saccharomyces cerevisiae were isolated through hydroxylamine mutagenesis of the RPL16B gene and plasmid shuffling. Two heat-sensitive and two cold-sensitive isolates were characterized. The growth of the four mutants is inhibited at their restrictive temperatures. However, many of the cells remain viable if returned to their permissive temperatures. All of the mutants are deficient in 60S ribosomal subunits and therefore accumulate translational preinitiation complexes. Three of the mutants exhibit a shortage of mature 25S rRNA, and one accumulates rRNA precursors. The accumulation of rRNA precursors suggests that ribosome assembly may be slowed in this mutant. These phenotypes lead us to propose that mutants containing the rpl16b alleles are defective for 60S subunit assembly rather than function. In the mutant carrying the rpl16b-1 allele, ribosomes initiate translation at the noncanonical codon AUA, at least on the rpl16b-1 mRNA, bringing to light a possible connection between the rate and the fidelity of translation initiation.

Amino Acid Sequence↗
PubMed1990

Depletion of yeast ribosomal proteins L16 or rp59 disrupts ribosome assembly.

Two strains of Saccharomyces cerevisiae were constructed that are conditional for synthesis of the 60S ribosomal subunit protein, L16, or the 40S ribosomal subunit protein, rp59. These strains were used to determine the effects of depriving cells of either of these ribosomal proteins on ribosome assembly and on the synthesis and stability of other ribosomal proteins and ribosomal RNAs. Termination of synthesis of either protein leads to diminished accumulation of the subunit into which it normally assembles. Depletion of L16 or rp59 has no effect on synthesis of most other ribosomal proteins or ribosomal RNAs. However, most ribosomal proteins and ribosomal RNAs that are components of the same subunit as L16 or rp59 are rapidly degraded upon depletion of L16 or rp59, presumably resulting from abortive assembly of the subunit. Depletion of L16 has no effect on the stability of most components of the 40S subunit. Conversely, termination of synthesis of rp59 has no effect on the stability of most 60S subunit components. The implications of these findings for control of ribosome assembly and the order of assembly of ribosomal proteins into the ribosome are discussed.

Galactose↗
PubMed1988

Sequence and genetic analysis of a dispensible 189 nucleotide snRNA from Saccharomyces cerevisiae.

The structure of a Saccharomyces cerevisiae gene that encodes a small nuclear RNA (snRNA) of 189 nucleotides is described. This gene, designated SNR189, is located 400 base pairs upstream of the CRY1 gene on yeast chromosome III. Gene replacement analysis revealed the SNR189 gene to be dispensable for growth under a variety of culture conditions. The snR189 sequence lacks homology with other sequenced yeast or metazoan snRNAs.

Base Sequence↗
PubMed1988

Depletion of Saccharomyces cerevisiae ribosomal protein L16 causes a decrease in 60S ribosomal subunits and formation of half-mer polyribosomes.

We constructed yeast strains containing deletion-insertion null alleles of the RPL16A or RPL16B genes encoding the 60S ribosomal subunit protein L16 to determine the role of L16 in the synthesis and function of ribosomes. Strains lacking a functional RPL16A gene grow as rapidly as wild type, whereas those containing a null allele of RPL16B grow more slowly than wild type. RNA analysis using RPL16 probes revealed that both RPL16 genes are transcribed and that RPL16B transcripts accumulate to twice the level of RPL16A transcripts. No evidence was obtained for the occurrence of dosage compensation at the level of RPL16 mRNA accumulation in either mutant. Strains lacking both RPL16 genes are apparently inviable, demonstrating that L16 is an essential yeast ribosomal protein. Introduction of an extra copy of either RPL16 gene into rpl16b mutants restored wild-type growth rates, indicating that the two forms of the L16 protein are interchangeable. rpl16 mutants are deficient in 60S ribosomal subunits relative to 40S subunits. 43S preinitiation complexes accumulate in half-mer polyribosomes in the absence of sufficient 60S subunits. We postulate that the slow-growth phenotype of rpl16 mutants results from the perturbation of initiation of protein synthesis.

Fungal Proteins↗
PubMed1988

Ribosomal protein synthesis is not regulated at the translational level in Saccharomyces cerevisiae: balanced accumulation of ribosomal proteins L16 and rp59 is mediated by turnover of excess protein.

We have investigated the mechanisms whereby equimolar quantities of ribosomal proteins accumulate and assemble into ribosomes of the yeast Saccharomyces cerevisiae. Extra copies of the cry1 or RPL16 genes encoding ribosomal proteins rp59 or L16 were introduced into yeast by transformation. Excess cry1 or RPL16 mRNA accumulated in polyribosomes in these cells and was translated at wild-type rates into rp59 or L16 proteins. These excess proteins were degraded until their levels reached those of other ribosomal proteins. Identical results were obtained when the transcription of RPL16A was rapidly induced using GAL1-RPL16A promoter fusions, including a construct in which the entire RPL16A 5'-noncoding region was replaced with the GAL1 leader sequence. Our results indicate that posttranscriptional expression of the cry1 and RPL16 genes is regulated by turnover of excess proteins rather than autogenous regulation of mRNA splicing or translation. The turnover of excess rp59 or L16 is not affected directly by mutations that inactivate vacuolar hydrolases.

Gene Expression Regulation↗
PubMed1987

Evidence for related functions of the RNA genes of Saccharomyces cerevisiae.

The yeast genes RNA2-RNA11 are necessary for splicing of nuclear intron-containing pre-mRNAs. We investigated the relationships among these genes by asking whether increased expression of one RNA gene leads to suppression of the temperature-sensitive lethality of a mutation in any other RNA gene. The presence of extra plasmid-borne copies of the RNA3 gene relieves the lethality of temperature-sensitive rna4 mutations. A region of the yeast genome (SRN2) is described that suppresses temperature-sensitive rna2 mutations when it is present on either medium or high-copy number plasmids. Neither suppression occurs via a bypass of RNA gene function since null alleles of rna2 and rna4 are not suppressed by elevated dosage of SRN2 and RNA3, respectively. These results suggest that the SRN2 and RNA2 gene products have related functions, as do the RNA3 and RNA4 gene products.

Genes, Fungal↗
PubMed1987

Structure and expression of the Saccharomyces cerevisiae CRY1 gene: a highly conserved ribosomal protein gene.

The Saccharomyces cerevisiae CRY1 gene encodes ribosomal protein rp59, a component of the 40S ribosomal subunit. Mutations in CRY1 can confer resistance to the alkaloid cryptopleurine, an inhibitor of the elongation step of translation. The nucleotide sequence of the cloned CRY1 gene was determined. The predicted amino acid sequence shows that CRY1 encodes a 14,561-dalton polypeptide that has 88% amino acid sequence homology to the hamster or human S14 ribosomal protein responsible for emetine resistance and 45% homology to Escherichia coli ribosomal protein S11. Analysis of the DNA sequences upstream from CRY1 revealed the presence of three sequences, HOMOL1 (consensus, A/TACATCC/TG/ATA/GCA), RPG (consensus, ACCCA/GTACATT/CT/A), and a thymine-rich sequence, found upstream of more than 20 other cloned yeast genes encoding components of the translational apparatus. We exploited the ability to assay the expression of CRY1 in vivo by using the cryptopleurine resistance phenotype to demonstrate that these three consensus sequences are necessary for the transcription of CRY1. We previously showed that the upstream promoter element of the yeast RP39A gene consists of these identical sequence motifs. Therefore, we suggest that these three sequences define a consensus promoter element for the genes encoding the yeast translational apparatus. CRY1 is one of several hundred yeast genes, including ribosomal protein genes, whose expression is transiently decreased 10-fold upon heat shock. We found that the HOMOL1 and RPG consensus sequences are not necessary for the heat shock response of CRY1.

Amino Acid Sequence↗
PubMed1986

Identification and nuclear localization of yeast pre-messenger RNA processing components: RNA2 and RNA3 proteins.

Temperature-sensitive mutations in the RNA2 through RNA11 genes of yeast prevent the processing of nuclear pre-mRNAs. We have raised antisera that detect the RNA2 and RNA3 proteins in immunoblots of extracts of yeast containing high copy number RNA2 and RNA3 plasmids. Subcellular fractionation of yeast cells that overproduce the RNA2 and RNA3 proteins has revealed that these proteins are enriched in nuclear fractions. Indirect immunofluorescence results have indicated that these proteins are localized in yeast nuclei. These localization results are consistent with the fact that these genes have a role in processing yeast pre-mRNA.

Cell Nucleus↗
PubMed1986

Tripartite upstream promoter element essential for expression of Saccharomyces cerevisiae ribosomal protein genes.

To initiate a genetic analysis of yeast ribosomal protein gene promoters, we have constructed a gene fusion between the yeast ribosomal protein gene RP39A and the Escherichia coli lacZ gene. This gene fusion contains approximately 1,030 nucleotides of the 5' flanking region and the first 49 1/3 codons of RP39A fused in frame to a large 3' end fragment of lacZ. Whether it is introduced into yeast cells on a moderately high-copy-number plasmid, or integrated into the yeast genome at the RP39A locus, this RP39A-lacZ gene directs the synthesis of a hybrid transcript which encodes beta-galactosidase activity. Deletions in the 5' flanking region of RP39A-lacZ were constructed by linker insertion and BAL 31 mutagenesis. The expression of the mutant genes in yeast cells was assayed by measuring RP39A-lacZ mRNA and beta-galactosidase levels. By these means we have shown that the sequences between nucleotides -256 and -170 upstream of RP39A are essential for expression of this gene. Three sequence motifs, HOMOL1, RPG, and a T-rich region, which were found in that order 5'----3' upstream of most yeast ribosomal protein genes, were present within this interval. We found that substitution of the CYC1-lacZ upstream activation site with the fragment from nucleotides -298 to -172 upstream of RP39A, containing the HOMOL1-RPG-T-rich motif in that 5'----3' orientation, fully restored expression of the CYC1-lacZ gene. The essentially of HOMOL1, the RPG sequence, and the T-rich region for wild-type levels of expression of RP39A, the conserved location and order of these sequence motifs in yeast ribosomal protein genes, and the ability of a DNA fragment carrying these three sequence elements to substitute for the upstream activation site regions of CYC1 indicate that these three oligonucleotides may be essential to the transcription of yeast ribosomal protein genes.

Base Sequence↗
PubMed1984

Isolation and characterization of the RNA2, RNA3, and RNA11 genes of Saccharomyces cerevisiae.

Temperature-sensitive mutations in the genes RNA2 through RNA11 cause accumulation of intervening sequence containing precursor mRNAs in Saccharomyces cerevisiae. Three different plasmids have been isolated which complement both the temperature-sensitive lethality and precursor mRNA accumulation when introduced into rna2, rna3, and rna11 mutant strains. The yeast sequences on these plasmids have been shown by Southern transfer hybridization and genetic mapping to be derived from the RNA2, RNA3, and RNA11 genomic loci. Part of the RNA2 gene is homologous to more than one region of the yeast genome, whereas the RNA3 and RNA11 genes are single copy. RNAs homologous to these loci have been identified by RNA transfer hybridization, and the specific RNAs which are associated with the Rna+ phenotype have been mapped. This was done by a combination of transcript mapping, subcloning, and in vitro mutagenesis. The transcripts are found to be enriched in polyadenylated RNA and are of very low abundance (0.01-0.001% polyadenylated RNA).

Base Sequence↗