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K L Roy

Publications and source records attributed to K L Roy.

At least 37 records · Page 2Linked to original sources
PubMed1991

Isolation and molecular characterization of the ribosomal protein L6 homolog from Chlamydia trachomatis.

The cloning of a Chlamydia trachomatis eukaryotic cell-binding protein reported earlier from our laboratory (R. Kaul, K. L. Roy, and W. M. Wenman, J. Bacteriol. 169:5152-5156, 1987) represents an artifact generated by nonspecific recombination of chromosomal DNA fragments. However, the amino terminus of this plasmid-encoded fusion product demonstrated significant homology to Escherichia coli ribosomal protein L6. By using a 458-bp PstI-HindIII fragment of recombinant pCT161/18 (representing the 5' end of the cloned gene), we isolated and characterized a C. trachomatis homolog of the ribosomal protein L6 gene of E. coli. Sequence analysis of an 1,194-bp EcoRI-SacI fragment that encodes chlamydial L6 (designated CtaL6e) revealed a 552-bp open reading frame comprising 183 amino acids and encodes a protein with a molecular weight of 19,839. Interestingly, complete gene homology between C. trachomatis serovars L2 and J, each of which exists as a single copy per genome, was observed. Expression of a plasmid-encoded gene product is dependent on the lac promoter, since no product was obtained if the open reading frame was oriented in opposition to the lac promoter. Immunoblotting of purified ribosomes revealed functional, as well as antigenic, homology between the E. coli and C. trachomatis ribosomal L6 proteins.

Amino Acid Sequence↗
PubMed1990

Drosophila melanogaster tRNA(Ser) suppressor genes function with strict codon specificity when introduced into Saccharomyces cerevisiae.

The anticodon of the wild-type tRNA(7Ser) gene of Drosophila melanogaster was mutated using oligodeoxyribonucleotide-directed, site-specific mutagenesis, and all three nonsense suppressor derivatives of the gene were constructed. These constructs were cloned into an Escherichia coli-yeast shuttle vector (YRp7), and used to transform a Saccharomyces cerevisiae strain [JG 369-3B(alpha)] containing an array of nonsense alleles. When tested on appropriate omission media, the D. melanogaster suppressor genes were found to function in the yeast with strict codon specificity. Subsequent Northern hybridization analyses revealed that the D. melanogaster suppressor genes were transcribed and processed well, when in S. cerevisiae.

Animals↗
PubMed1990

Construction of an opal suppressor by oligonucleotide-directed mutagenesis of a Saccharomyces cerevisiae tRNA(Trp) gene.

In vitro mutagenesis was used to create putative opal suppressor alleles of a tRNA(Trp) gene of Saccharomyces cerevisiae. The construct with the requisite anticodon change did not result in an active suppressor, whereas when a second change was introduced into the portion of the gene encoding the intron, an active and specific opal suppressor was produced. We propose that the secondary structure of transcripts from the first mutant may prevent efficient pre-tRNA processing, whereas normal processing occurs with the double mutant.

Alleles↗
PubMed1988

Two human genes encoding tRNA(GCCGly).

Two human DNA fragments of 16.7 and 15.5 kb have been selected from a human lambda Charon-4A library by hybridization to an unfractionated tRNA probe. Restriction mapping and Southern and Northern hybridization analyses revealed the presence of a single tRNA-hybridizing region in each of the human DNA fragments. Nucleotide sequence analysis has identified two identical members of the tRNA(GCCGly) gene family. These tRNA(GCCGly) genes encode all of the conserved and semiconserved nucleotides of the tDNA split promoter sequences. Neither gene contains introns or encodes the CCA sequence present on the 3' terminus of mature tRNA. One of these identical tRNA(GCCGly) genes was found to be expressed at a substantially greater efficiency than the other in a HeLa cell lysate in vitro transcription system. No similarity was detected in the nucleotide sequences flanking these genes other than the characteristic, 3' oligo[dT] transcription termination signals and a TCTTT sequence located 7 to 10 bp upstream. These data are consistent with the hypothesis that as yet unidentified tDNA flanking sequences may have an important role in modulating human tRNA gene expression.

Bacteriophage lambda↗
PubMed1988

Isolation and characterization of a linear DNA plasmid from Streptomyces clavuligerus.

A linear DNA plasmid (pSCL) has been isolated from Streptomyces clavuligerus by a method employing high concentrations of protease. Rate-zonal sedimentation on sucrose gradients was used to purify the plasmid. The plasmid is 12 kb in length and appears to be linked to protein at its 5' termini. A restriction endonuclease map of the plasmid for ten enzymes has been determined. Evidence for terminally repeated sequences is provided by cross-hybridization analysis.

DNA Restriction Enzymes↗
PubMed1987

Analysis of a human gene cluster coding for tRNA(GAAPhe) and tRNA(UUULys).

A 13.8-kb fragment of human DNA isolated from a human lambda Charon-4A DNA library was found to contain four human tRNA genes. Nucleotide sequence analysis of approx. 3.7 kb of this segment of human DNA identified two lysine tRNA(UUU) genes identical in coding sequence to a previously reported human lysine tRNA gene [Roy et al., Nucl. Acids Res. 10 (1982) 7313-7322]. The other two tRNA genes were phenylalanine tRNA(GAA) genes, the first to be isolated from a mammalian source. These phenylalanine tRNA(GAA) genes were identical in sequence with the exception of a G/A polymorphism at coordinate 57. None of these tRNA genes contains introns. The tRNA(UUULys) and tRNA(GAAPhe) genes are organized in alternating order and are irregularly spaced, by intergenic regions of approx. 1.0, 2.6 and 5.0 kb, and randomly oriented. There was no evidence to indicate that any of these genes arose by gene duplication, since flanking sequence homology was limited to the putative RNA polymerase III termination signals in the 3'-flanking regions. A mature tRNA-sized product was identified following the transcription of each tRNA gene in a homologous in vitro transcription system. Interestingly, different levels of transcriptional activity of the three identical lysine tRNA genes were observed, suggesting modulation of tDNA expression by extragenic sequences. In addition, a minimum of eight regions of homology to Alu-type repetitive elements were detected in this human DNA fragment, one of which was located 53 bp upstream from a tRNA(GAAPhe) gene.

Base Sequence↗
PubMed1987

Plasmid transformation of Azotobacter vinelandii OP.

Azotobacter vinelandii OP which had been naturally induced to competence by growth in iron- and molybdenum-limited medium was transformed with the broad-host-range cloning vector pKT210. However, the transformation frequency at nearly saturating levels of DNA was 1000-fold lower for pKT210 than for a single chromosomal DNA marker (nif+). Plasmid- and chromosomal-DNA-mediated transformation events were competitive, magnesium-dependent, 42 degrees C-sensitive processes specific to double-stranded DNA, suggesting a common mechanism of DNA binding and uptake. The low frequency of plasmid transformation was not related to restriction of transforming DNA or to the growth period allowed for phenotypic expression. Covalently-closed-circular and open-circular forms of pKT210 transformed cells equally well whereas EcoRI- or HindIII-linearized pKT210 transformed cells with two to three times greater efficiency. Genetic transformation was enhanced 10- to 50-fold when pKT210 contained an insert fragment of A. vinelandii nif DNA, indicating that A. vinelandii possessed a homology-facilitated transformation system. However, all transformants failed to maintain the plasmid-encoded antibiotic resistance determinants, and extrachromosomal plasmid DNA was not recovered from these cells. Flush-ended pKT210 was not active in transformation; however, competent cells were transformed to Nif+ by HincII-digested plasmid DNA containing the cloned A. vinelandii nif-10 marker.

Azotobacter↗
PubMed1987

Cloning, expression, and primary structure of a Chlamydia trachomatis binding protein.

The gene encoding an 18,000-dalton eucaryotic cell-binding protein of Chlamydia trachomatis serovar L2 was cloned into Escherichia coli, and the nucleotide sequence of a 1,658-base-pair PstI restriction endonuclease fragment encoding this protein was determined. The recombinant chlamydial gene consists of a 486-base-pair open reading frame encoding a polypeptide of molecular weight 18,314. The resultant polypeptide, comprising 162 amino acids, possesses a highly charged carboxy-terminal end. The expression of this recombinant protein is under the control of a vector promoter. The recombinant 18,000-dalton protein possessed the same eucaryotic cell-binding characteristics as did the native chlamydial 18,000-dalton protein when electrophoresed and transferred to nitrocellulose. Polyclonal antibodies to the recombinant protein exhibited neutralizing activity.

Amino Acid Sequence↗
PubMed1986

Two human tyrosine tRNA genes contain introns.

A human lambda-phage recombinant which contains at least four tRNA genes, has been isolated. Two DNA fragments were subcloned to give the recombinant plasmids pM6 and pM6128. Nucleotide sequence analysis showed that each plasmid contained a different tyrosine acceptor tRNA (tRNATyr) gene. Both tRNATyr genes are interrupted by 21-bp introns. These recombinant plasmids have been shown to direct the in vitro synthesis of tRNA-sized products in a HeLa cell transcription system.

Base Sequence↗
PubMed1982

Nucleotide sequence of a segment of human DNA containing the three tRNA genes.

A 1.65 kb segment of DNA from a human-lambda recombinant, selected for human tRNA genes, was subcloned in the plasmid pAT 153 for sequence determination. Three human tRNA genes were found; one, a tRNAlys gene which appears to specify a tRNA identical in sequence to the previously described tRNA3lys from rat liver (AAA,G); another a tRNAgln gene, the product of which would be expected to recognize only the codon CAG; and a third which would direct synthesis of a tRNAleu specific for CUA and G. Intervening sequences are not found in any of these genes. The three tRNA genes are separated by about 0.5 kb segments of DNA containing no apparent informational sequences. Examination of flanking sequences shows some similarities at the 5'-ends of the three genes, but less similarity to 5' flanking sequences of tRNA genes in other eucaryotes. All three tRNA genes direct synthesis of appropriate sized products in a HeLa cell lysate in vitro transcription system.

Base Sequence↗
PubMed1982

Construction of a functional human suppressor tRNA gene: an approach to gene therapy for beta-thalassaemia.

A human tRNALys gene was converted to an amber suppressor by site-specific mutagenesis of the anticodon. The mutated tRNALys gene directed synthesis of a tRNA that suppressed the UAG amber nonsense mutation in beta O thalassemia mRNA. Such genes may be used to detect other nonsense mutations in mammalian cells and may provide an approach to gene therapy for beta O thalassaemia due to nonsense mutations.

Anticodon↗
PubMed1982

Dihydrouridine-deficient tRNAs in Saccharomyces cerevisiae.

A mutation in Saccharomyces cerevisiae, designated mia, is responsible for the production of isoaccepting tRNA molecules with reduced extents of nucleoside modifications. The mia isoacceptors of tRNAPhe and one of the mutant isoacceptors of tRNATyr were highly purified for nucleoside composition analyses. The data indicate that the mutant isoacceptors are lacking some of the dihydrouridine moieties. This is consistent with our previous hypothesis that the mutant isoacceptors were accumulated due to a defect in a modification process [Lo, R.Y.C. and Bell, J.B. (1981) Current Genetics 3, 73-82). Data from in vitro poly-U translation experiments also support the previous results, suggesting in vivo biological activity of these mutant tRNAs.

Base Composition↗
PubMed1978

Nucleotide sequence of rainbow trout (Salmo gairdneri) ribosomal 5.8 S ribonucleic acid.

The nucleotide sequence of rainbow trout (Salmo gairdneri) 5.8 S rRNA was determined to be (formula: see text). The major molecular species is 161 nucleotides long; the sequence differs in only eight nucleotides from that of Man. An estimate of the secondary structure retains essentially all of the features previously described in mammalian 5.8 S RNAs with several interesting variations. The very stable G-C-rich hairpin loop is extended by two additional hydrogen bond pairs; the A-U-rich loop is shorter; and a third 2'-O-methylation occurs at residue 8 close to the under methylated U(m) residue at position 14. Comparisons with other known sequences indicate that in eukaryotes 5.8 S rRNA is more highly conserved than 5 S RNA species.

Animals↗
PubMed1978

A proposed nucleotide sequence for the 5S ribosomal ribonucleic acid of rainbow trout (Salmo gairdneri).

Rainbow trout cell cultures have been exposed to 32P-labelled inorganic phosphate and the labelled RNA has been isolated. The 5S ribosomal ribonucleic acid (5S rRNA) was purified by polyacrylamide gel electrophoresis, then digested with RNase T1 or pancreatic RNase. The products of complete digestion were separated and their sequences determined. These analyses have allowed a sequence to be proposed which differs in eight positions from that of mammalian 5S rRNAs.

Animals↗