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J L Woolford

Publications and source records attributed to J L Woolford.

46 records · Page 3Linked to original sources
PubMed1983

Molecular cloning and analysis of the CRY1 gene: a yeast ribosomal protein gene.

Using cloned DNA from the vicinity of the yeast mating type locus (MAT) as a probe, the wild type allele of the cryptopleurine resistance gene CRY1 has been isolated by the technique of chromosome walking and has been shown to be identical to the gene for ribosomal protein 59. A recessive cryR1 allele has also been cloned, using the integration excision method. The genetic distance from MAT to CRY1 is 2.2 cM, while the physical distance is 21 kb, giving a ratio of about 10 kb/cM for this interval. The phenotypic expression of both plasmid borne alleles of the gene can be detected in vivo. The use of this gene as a hybridization probe to examine RNA processing defects in the rna 2, rna 3, rna 4, rna 8, and rna 11 mutants is also discussed.

Alleles↗
PubMed1981

Ribosomal protein genes rp 39(10 - 78), rp 39(11 - 40), rp 51, and rp 52 are not contiguous to other ribosomal protein genes in the Saccharomyces cerevisiae genome.

A library of recombinant phage containing EcoR1 fragments of Saccharomyces cerevisiae DNA has been constructed. This library was screened with four different recombinant plasmids, each containing a different yeast ribosomal protein gene, in order to isolate chromosomal fragments extending in both directions from these genes. These chromosomal fragments were assayed for the presence of additional ribosomal protein genes by hybridization selection and cell free translation, and none were found. These four regions are not closely linked to each other, since DNA from one domain does not cross hybridize with DNA from any of the other three, except for the sequences within the homologous ribosomal protein 39 gene pair. Northern blots demonstrate that although the concentrations of ribosomal protein mRNAs are diminished significantly in a strain containing the ts mutation rna2, transcripts from genes in these flanking segments are relatively unaffected.

DNA Restriction Enzymes↗
PubMed1981

The effect of temperature-sensitive RNA mutants on the transcription products from cloned ribosomal protein genes of yeast.

The levels of four ribosomal protein (rp) mRNAs in different mutant strains were determined by hydridization of radiolabeled cloned genes to RNA fractionated on CH3HgOH gels and transferred to DBM paper. Two ribosomal protein genes (rp 51 and rp 52) controlled by the locus RNA2 have dramatically decreased mRNA levels after a shift-up to the nonpermissive temperature in a strain carrying the rna2 mutation (ts368). Two ribosomal protein genes not controlled by the RNA2 locus and several control nonribosomal protein genes are relatively unaffected by the temperature shift in this strain. Other genes in the vicinity of one of the rna2-sensitive ribosomal protein genes (th rp 51 gene) are insensitive to the rna2 gene product, suggesting that all ribosomal protein genes do not occur in clusters and that the RNA2 gene product does not affect a large region of chromatin. In ts368 at the nonpermissive temperature, the concentration of higher molecular weight transcripts complementary to the rp 51 and the rp 52 plasmids is increased. Analysis of the rp 51 plasmid transcripts reveals that the temperature-induced higher molecular weight transcripts differ from the mature rp 51 mRNA by the presence of an intron. This observation and the kinetics with which the concentration of the various rp 51 transcripts change after a temperature shift suggest that the effect of rna2 may be at the level of processing of rp mRNA.

Cloning, Molecular↗
PubMed1979

The use of R-looping for structural gene identification and mRNA purification.

A method is presented for the purification of mRNAs and the identification of structural gene sequences in recombinant DNA molecules. RNA is hybridized to double-stranded linear DNA such that R-loops are formed between most DNAs and their complementary RNA sequences. These R-loops are purified from unhybridized RNAs by gel filtration chromatography in the presence of a high concentration of salt. The complementary RNAs are released from the R-loops by heating, and are assayed by gel electrophoresis or cell free translation to determine their purity and to identify the proteins for which they code. We have demonstrated that recombinant DNAs containing sequences for abundant or moderately abundant mRNAs of Saccharomyces cerevisiae can be identified by this means.

Base Sequence↗
PubMed1979

Isolation of cloned DNA sequences containing ribosomal protein genes from Saccharomyces cerevisiae.

Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribosomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMB9 hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for 35S-methionine-labeled polypeptides which co-migrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the five clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for "tight" linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal protein gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA.

DNA, Fungal↗
PubMed1977

Adsorption protein of bacteriophage fl: solubilization in deoxycholate and localization in the fl virion.

A complex containing the minor coat protein or adsorptionprotein (A protein) of bacteriophage fl has been solubilized from the fl virion, using the detergent deoxycholate. This complex was resolved from the fl DNA and from the fl major coat protein, or B protein, by gel filtration in the presence of deoxycholate. The A protein complex migrated as a single band on sodium dodecyl sulfate-urea-polyacrylamide gels corresponding to a molecular weight of 60 000. Analysis of the amino acid composition and amino terminal residues of this preparation indicates that the preparation contains a 20% contamination of additional protein species. Antibody against purified fd A protein is cross-reactive with deoxycholate-purified fl A protein and with fl phage. Electron microscopic observation of negatively stained complexes of fl phage with this anti-fd A protein antibody and ferritin conjugated goat anti-rabbit IgG antibody revealed phages with ferritin particles at their termini or complexes of two or more phages joined together at one end by ferritin, indicating that the complex of A protein molecules is located at one end of the filamentous fl virion.

Adsorption↗
PubMed1975

Interaction of deoxycholate and of detergents with the coat protein of bacteriophage f1.

The major coat protein of bacteriophage f1, which is localized in the host membrane during phage maturation, has a hydrophobic binding site capable of binding deoxycholate and a variety of detergents to form a soluble particle, and in that respect, resembles many membrane proteins. The soluble particle has properties that suggest it is formed by simple insertion of protein into a deoxycholate or detergent micelle, but molecular weight measurements show that the protein is present as a dimer, even in sodium dodecyl sulfate, indicating the existence of unusually strong forces for self-association. A by-product of the investigation has been to show that detergents can be very helpful in the fractionation of the constituent molecules of the virus: deoxycholate-solubilized virus is readily fractionated by gel chromatography into DNA, A protein, and B protein, with virtually no cross-contamination.

Binding Sites↗
PubMed1975

Proteolytic digestion of the micellar complex of f1 coat protein and deoxycholate.

The major coat protein of bacteriophage f1 radioactively labeled with specific amino acids was solubilized with deoxycholate and digested with trypsin or alpha-chymotrypsin. The degree of proteolysis of the coat protein was assayed by gel filtration chromatography of the digest in the presence of deoxycholate. Hydrolysis occurred at residues in the hydrophilic termini of the coat, releasing peptides containing proline, lysine, and phenylalanine. No cleavage occurred at the tyrosine or methionine residues in the hydrophobic core. However, chymotrypsin could cleave somewhat at these residues in the absence of deoxycholate. A model for the topography of the micellar complex of coat protein and deoxycholate is presented in which the hydrophobic sequence of the coat is bound to deoxycholate within a micelle, while the hydrophilic termini of the coat project from the micelle.

Amino Acid Sequence↗