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S G Clarkson

Publications and source records attributed to S G Clarkson.

At least 37 records · Page 2Linked to original sources
PubMed1987

Structure and transcription termination of a lysine tRNA gene from Xenopus laevis.

Termination of RNA polymerase III transcripts commonly occurs at clusters of T residues. A T4 tract located 72 base-pairs beyond a lysine tRNA gene from Xenopus laevis serves as an efficient termination site for the tRNA(Lys) precursors synthesized from this gene in homologous cell-free extracts. Nucleotides following this T tract influence the extent of read-through transcription in vitro, but in a way that differs from Xenopus 5 S RNA termination. Only approximately 50% of the transcripts initiated in vitro extend as far as this downstream T cluster. The remainder prematurely terminate at a second T4 tract located within the gene itself. The contrasting behaviour of these two T tracts in injected oocytes indicates that termination can be influenced by more than just RNA polymerase III alone, and that different components may contribute to, or hinder, termination at these sites. Prematurely terminated tRNA(Lys) transcripts are detectable in RNA from ovary tissue but not from a kidney cell line, suggesting that read-through transcription beyond intragenic T clusters can be modulated in vivo.

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PubMed1986

A dispersed tyrosine tRNA gene from Xenopus laevis with high transcriptional activity in vitro.

Several kinds of tRNA genes of Xenopus laevis are clustered together within tandemly repeated 3.18-kilobase DNA fragments. Other members of these reiterated tRNA gene families are dispersed and irregularly arranged in the genome. Here we report the isolation and some characteristics of one such dispersed gene that codes for a tyrosine tRNA. It is located within a low copy number 9.4-kilobase restriction fragment that contains no other RNA polymerase III gene functional in vitro. The dispersed gene differs from the clustered tyrosine tRNA gene by a single purine transition within the coding region, by extensive sequence differences within the intervening sequence and 5' and 3' flanking regions, and by its approximately 6-fold higher transcriptional activity in homologous S-100 extracts. Analyses of hybrid genes and deletion mutants demonstrate that this differential transcription is due to DNA in the 5' flanking regions.

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PubMed1984

Structure and in vitro transcription of a glycine tRNA gene from Bombyx mori.

We report the sequences of a Bombyx mori tRNA1Gly gene, its flanking regions, and its in vitro transcription products. The 5' flanking DNA contains the sequences TATAC, TATTTT and TTC located 30, 18 and 4 nucleotides, respectively, in front of the transcription initiation site. These resemble, in both position and composition, sequences preceding other RNA polymerase III genes of B. mori. A deletion mutant retaining these conserved sequences and an additional 8 bp of flanking DNA is transcribed better than the wild-type gene in cell-free extracts from Xenopus laevis and B. mori. A mutant lacking the conserved sequences is expressed in the frog extract, but is inactive in the homologous system.

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PubMed1984

Chromosomal location of a major tRNA gene cluster of Xenopus laevis.

In Xenopus laevis, genes encoding tRNAPhe, tRNATyr, tRNAMet1, tRNAAsn, tRNAAla, tRNALeu, and tRNALys are clustered within a 3.18-kb (kilobase) fragment of DNA. This fragment is tandemly repeated some 150 times in the haploid genome and its components are found outside the repeat only to a limited extent. The fragment hybridizes in situ to a single site very near the telomere on the long arm of one of the acrocentric chromosomes of the group comprising chromosomes 13-18. All the chromosomes of this group also hybridize with DNA coding for oocyte-specific 5S RNA. The tRNA gene cluster is slightly proximal to the cluster of 5S RNA genes.

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PubMed1983

5'-flanking sequences that inhibit in vitro transcription of a xenopus laevis tRNA gene.

Two series of deletion mutants have been constructed in the 5'-flanking DNA of a variant methionine tRNA gene of X. laevis. When assayed in homologous cell-free extracts, mutants of both series are expressed at very different levels and they initiate transcription at either or both of the two major sites used by the wild-type gene. No similarities are evident in the 5'-flanking regions of well expressed mutants, but two DNA segments are identified that inhibit transcription. The most inhibitory one, between residues -20 and -12, consists of 9 bp of alternating purines and pyrimidines: TGCGCGTGC. The second has only a weak inhibitory effect when in its normal location from -43 to -32, but it can reduce transcription severely when brought closer to the gene; it is composed of 12 bp of alternating purines and pyrimidines with one residue out of alternation: ATGCACAGCGCA. The composition of these tracts has some possible implications for their conformation, which may account for their inhibition of gene expression.

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PubMed1982

Synthesis and maturation of Xenopus laevis methionine tRNA gene transcripts in homologous cell-free extracts.

A cloned initiator methionine tRNA gene from Xenopus laevis has been transcribed in cell-free extracts (S-100) prepared from cultured X. laevis kidney cells. RNA polymerase III produces two primary transcripts of this gene which initiate with a pppG and a pppA located seven and four nucleotides, respectively, in front of the mature tRNAMet1 5' end. Both terminate with a dT5 tract just beyond the nucleotides encoding the mature tRNA. The tRNA precursors are readily processed in vitro to mature length tRNA which contains six of the seven modified nucleotides found in the in vivo tRNAMet1. Many of these ribonucleotide modifications (m1G, m2G, m7G, D, and m1A) are introduced into the primary transcripts. The single exception is t6A which is found in the tRNAMet1 anticodon loop only after maturation of the 5' and 3' termini.

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PubMed1980

Mutations of the yeast SUP4 tRNATyr locus: transcription of the mutant genes in vitro.

Twenty-nine different SUP4-o tRNATyr genes with second-site mutations were transcribed in X. laevis cell-free RNA polymerase III transcription reactions, and the in vitro transcripts were analyzed by polyacrylamide gel electrophoresis. Nineteen mutant genes yield normal amounts of RNA that co-electrophorese with SUP4-o gene transcripts. RNA synthesized from a mutant gene lacing a single base pair migrated slightly faster in gels, as expected. The still shorter transcripts made from seven other mutant genes suggest that several mutations alter transcription starting or stopping points. Fingerprint analyses of transcripts from the two most extreme cases showed that premature termination occurred at new tracts of T residues resulting from the mutations. Two mutations significantly enhance transcription, and two mutations which alter the invariant C within the T psi CG sequence dramatically reduce SUP4-o gene transcription. The regions of the SUP4-o gene that surround these mutations are partially homologous to intragenic sequences in many other eucaryotic tRNA and 5S RNA genes. We hypothesize that these homologous sequences are recognized as promoter regions during RNA polymerase III transcription initiation.

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PubMed1980

Nucleotide sequence of genes coding for tRNAPhe and tRNATyr from a repeating unit of X. laevis DNA.

A cloned 3.18 kb repeating unit of Xenopus laevis DNA, known to contain two tRNA1Met genes, has been partially sequenced. The sequenced region could potentially code for two more tRNA species. One is a phenylalanine tRNA with the same primary sequence as that found in a variety of mammals. The other is a tyrosine tRNA with a 13 bp intervening sequence located immediately adjacent or very close to the 3' nucleotide of the anticodon. The two putative genes have opposite polarity and the sequences encoding the 5' nucleotides of the mature tRNAs are separated by only 72 bp of DNA. Homologous GC-rich sequences of dyad symmetry precede each gene. Neither gene codes for the 3' terminal CCA expected in the nature tRNAs. A short T cluster is present in the noncoding strand 3 bp away from the 3' end of the tRNATyr gene. A similar short T cluster is located 26 bp downstream of the tRNAPhe sequence. The nucleotides in between are very GC-rich and are capable of forming a stable RNA hairpin. The possible significance of these features for tRNA gene evolution, RNA splicing and transcription is discussed.

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PubMed1979

Delimitation of a promoter for RNA polymerase III by means of a functional test.

A Xenopus laevis DNA segment containing the structural gene for tRNA1Met and 22 base pairs at the 5' side of the gene is active in tRNA production. The DNA segment was ligated to sea urchin histone DNA and was also inserted into plasmid pCR1. Both recombinant DNAs were shown to produce mature tRNA1Met at a high rate when injected into centrifuged Xenopus oocytes.

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PubMed1978

Sequence organization of a cloned tDNA met fragment from Xenopus laevis.

3.18 kb fragments of X. laevis DNA coding for tRNA 1 met have been inserted into a lambda vector via Hind III termini and cloned in E. coli. The organization of one cloned fragment has been analyzed by restriction endonuclease digestion and RNA-DNA hybridization. From the distribution of sites for three enzymes, this fragment appears to be typical of the majority of X. laevis tandem tDNA 1 met repeat units. Evidence is presented to suggest that it contains two genes coding for tRNA 1 met and at least one gene coding for a second as yet unidentified 4S RNA species. The two tRNA 1 met genes are located on the same DNA strand 0.96 and 1.38 kb from one end of the repeat unit. A detailed restriction map for 19 enzymes reveals that the spacers between these genes are not identical, and it provides no indication of short repetitive sequence elements within the spacers.

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PubMed1978

Transcription of cloned tRNA gene fragments and subfragments injected into the oocyte nucleus of Xenopus laevis.

Cloned 3.18 kilobase fragments of Xenopus laevis DNA containing genes for tRNAMet1 and for at least one other 4S RNA species are transcribed rapidly after their injection into the nucleus of X. laevis oocytes. The newly synthesized RNA can be resolved by gel electrophoresis into a few predominant 4S RNA species and a series of slower migrating components. One of the 4S RNA species appears to be identical, by fingerprint analysis, to the tRNAMet1 isolated by hybridization of somatic cell RNA to this cloned tRNA gene fragment (tDNA). Thus, the tRNAMet1 produced after injection can be both fully processed and modified. Its rate of synthesis is estimated to be about 6 x 10(9) molecules/hr in each oocyte injected with 2 ng of tDNA. When the tDNA fragment is cleaved into two halves with restriction endonuclease Sst I, each injected half gives rise to a subset of the RNAs produced after injection of the intact fragment. This experiment thus suggests the presence of at least two transcriptional units on this cloned tDNA. This simple way of biologically testing defined restriction fragments may be of value for analyzing the functional organization of other cloned eukaryotic DNA units.

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