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Biomedical subjects

W R Folk

Publications and source records attributed to W R Folk.

At least 55 records · Page 3Linked to original sources
PubMed1983

An assay for the rates of cleavage of specific sites in DNA by restriction endonucleases: its use to study the cleavage of phage lambda DNA by EcoRI and phage P22 DNA containing thymine or 5-bromouracil by HindIII.

A method to measure the rates of cleavage of specific sites in DNAs by restriction endonucleases is described. Partial digests are prepared by incubating DNAs with limiting amounts of endonuclease. The termini generated by cleavage are labeled with 32P by the polynucleotide kinase-exchange reaction. The labeled termini are then identified by completing the digestion with the same endonuclease and separating the products by gel electrophoresis. As the products of complete digestion of DNA are often easily separated and can be unequivocally identified, this procedure permits comparison of the rates of cleavage of specific sites in DNAs; furthermore, because detection of the products of cleavage utilizes radioautography and does not depend upon their size, or amount, only small amounts of DNA need to be utilized. This method has been used to examine the cleavage of phage lambda DNA by EcoRI endonuclease, and to demonstrate that 5-bromouracil substitution in phage P22 DNA reduces the rate of cleavage of most sites by HindIII endonuclease approximately threefold and the rate of cleavage of one site approximately tenfold.

Bacteriophage lambda↗
PubMed1983

A detailed mutational analysis of the eucaryotic tRNAmet1 gene promoter.

We have isolated phage M13 clones containing the X. laevis trnamet1 gene, each having one or a few C leads to T transitions in the tRNA coding sequence. Nearly every G-C and C-G base pair in the tDNA has been mutagenized. The importance of these altered nucleotides in transcription by RNA polymerase III has been assessed by injecting the cloned DNAs into frog oocyte nuclei together with alpha-32P-GTP and measuring the synthesis of labeled tRNAmet1. Several G-C and C-G base pairs in the structural gene appear to be major promoter determinants, because when mutated, transcription is reduced 3-fold to 20-fold. Most of these determinants occur between nucleotides 7 to 19 and 49 to 61 in sequence regions highly conserved among eucaryotic as well as procaryotic tRNAs. Several additional G-C and C-G base pairs between these two regions also contribute to promoter activity; their location suggests that a stem-loop structure in the DNA encoding the tRNA's anticodon arm is important for RNA polymerase III promoter function.

Animals↗
PubMed1983

Comparison of the DNA sequence of the Crawford small-plaque variant of polyomavirus with those of polyomaviruses A2 and strain 3.

The DNA sequence of two wild-type strains of polyomavirus (A2 and strain 3) are known. We have determined the majority of the DNA sequence of a third strain, the Crawford small-plaque virus. This virus has been noted for its capacity to induce readily detected tumor-specific transplantation antigen in hamster cells, a property that is most likely attributable to an altered middle T-antigen. A comparison of its DNA sequence with those of the A2 and strain 3 viruses reveals numerous nucleotide substitutions, insertions, and deletions throughout the genome. Most sequence changes in coding regions are silent mutations; however, variability in proteins can be predicted from these sequence data at 5 locations in middle T-antigen, 10 in large T-antigen, and 10 in VP1. The Crawford small-plaque virus noncoding regulatory region contains, in addition to nucleotide substitutions, a 44-base-pair tandem repeat of sequences on the late side of the origin of DNA replication.

Antigens, Viral, Tumor↗
PubMed1982

Structure and organization of a mammalian 5 S gene cluster.

We have cloned and characterized a G+C-rich 2.2-kilobase pair Bam HI fragment which contains a single Syrian hamster 5 S rRNA gene. The cloned fragment directs the synthesis in vitro of 5 S RNA catalyzed by RNA polymerase III. Southern hybridization analysis of genomic hamster DNA indicates that the sequences present in the clone are tandemly repeated with a redundancy of 2700 copies per diploid cell.

Animals↗
PubMed1982

Structure of the Syrian hamster ribosomal DNA repeat and identification of homologous and nonhomologous regions shared by human and hamster ribosomal DNAs.

We have cloned and partially characterized Bam HI fragments of Syrian hamster DNA containing most of the ribosomal RNA-coding region. Several restriction site polymorphisms within the transcribed portions of the hamster rDNA repeats have been noted. Approximately three-fifths of the repeats contain a Bam HI site upstream of the 18 S coding sequences. Approximately four-fifths of the repeats contain a Bam HI site very close to the 5' end of the 28 S coding sequences. This microheterogeneity has been maintained in the DNA of baby hamster kidney (BHK)-21 cells, a cell line established nearly 20 years ago. R-loop analysis with homologous hamster rRNAs has established the size of the coding regions and the internal transcribed spacer. Heterologous R-loop analyses with cloned hamster rDNAs and human rRNAs reveal several well-defined regions within the 28 S gene where the homology between human and hamster RNAs is greatly reduced. These regions are not detectable in heteroduplexes of hamster and human rDNAs. Sequences encoding the 18 S gene do not exhibit such reduced homology.

Animals↗
PubMed1981

Nucleotide sequence changes in polyoma virus A gene mutants.

The mutational alterations in polyoma virus mutants ts-a and ts-25E which cause their large T-antigens to be thermolabile have been identified. In ts-a, a G leads to A transition at nucleotide 2193 causes the replacement of Ala (GCT) by Thr (ACT). In ts-25E, a G leads to T transversion at nucleotide 2883 causes the replacement of Gly (GGC) by Cys (TGC). Revertants of both mutants have been isolated and shown to have the original nucleotides restored at these positions.

Amino Acid Sequence↗
PubMed1979

Quantitation of the various termini generated by type II restriction endonucleases using the polynucleotide kinase exchange reaction.

Parameters of the polynucleotide kinase-catalyzed exchange reaction between [gamma-32P]ATP and 5'-phosphoryl DNAs have been measured with the termini generated by the following endonucleases: EcoRI (Berkner, K. L., and Folk, W. R. (1977) J. Biol. Chem. 252, 3176-3184), Hpa II, BamHI, and HindIII (external termini); HindII and Hpa I (blunt terminal); Hae II and Hha I (internal termini). In every case, exchange is reproducible and proportional to the number of termini. However, in most cases, the exchange reaction does not proceed to the theoretical maximum. External termini and single-stranded DNAs are labeled more rapidly and to approximately 5-fold higher levels than blunt or internal termini. Concentrations of 100 to 200 microns ADP and 12 microns ATP are optimal for labeling all types of termini with the exchange reaction.

Coliphages↗
PubMed1979

The effects of substituted pyrimidines in DNAs on cleavage by sequence-specific endonucleases.

The rates of cleavage of DNAs containing substituents at position 5 of thymine or cytosine have been measured for a variety of sequence-specific endonucleases, so as to determine which features in the DNA sequence are being probed. Phage phi e DNA fully substituted with 5-hydroxymethyluracil is cleaved more slowly by enzymes whose recognition sequences contain A-T base pairs than are DNAs containing thymine, but both types of DNA are cleaved at similar rates by enzymes recognizing sequences composed only of G-C base pairs. Phage PBS2 DNA with uracil completely substituted for thymine is cleaved slowly by several enzymes which recognize sequences containing A-T base pairs (endonucleases Hpa I, HindII, and HindIII), while the rates of cleavage by other enzymes (endonucleases EcoRI and BamHI) are not affected. Phage lambda- and P22 DNAs containing 5-bromouracil are cleaved more slowly by several enzymes (endonucleases HindIII, Hpa I, BamHI) than are thymine-containing DNAs. Enzymes that recognize sequence isomers with the composition G:C:2A:2T (endonucleases EcoRI, Hpa I, HindIII) are not equally affected by substitution at position 5 of thymine, suggesting that they differ in their contacts with A-T base pairs. DNA containing glucosylated 5-hydroxymethylcytosine in place of cytosine is resistant to cleavage by all the endonucleases examined.

Base Sequence↗
PubMed1979

Polyoma viruses with mutations at endonuclease HindII site 1: alterations at the COOH terminus of VP1.

Four mutants of polyoma virus lacking endonuclease HindII site 1 were isolated and characterized with respect to the VP1 coding sequence. Three of these mutants had deletions that removed 0.2 to 0.3% of the genome. All three deletion mutants encoded VP1 proteins that were smaller than wild type and that lacked one or more tryptic peptides normally found in the wild-type VP1 protein. Our results suggest the HindII site 1 is at, or very near, the carboxy terminal end of the coding sequence for VP1. A model for the peptide organization in that region is presented.

Capsid↗
PubMed1979

Deletion mutants of polyoma virus defining a nonessential region between the origin of replication and the initiation codon for early proteins.

Mutants of polyoma virus with deletions as large as 90 base pairs were isolated by selecting spontaneously arising genomes resistant to endonuclease HaeII or by treating HaeII- or BglI- cleaved linear DNAs with S1 nuclease and exonuclease III. All of the mutants were viable and, therefore, defined a nonessential region in the polyoma genome between the origin of DNA replication and the initiation codon for translation of early proteins. Several mutants with large deletions had altered growth properties, giving smaller plaques and lower virus yields than the parental wild-type virus. These viruses may lack sites that are important for DNA replication or for transcription and translation of early mRNA's. All of the mutants tested could transform BHK-21 cells to anchorage independence.

Animals↗