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W M Holmes

Publications and source records attributed to W M Holmes.

At least 37 records · Page 2Linked to original sources
PubMed1988

Sequence determinants for promoter strength in the leuV operon of Escherichia coli.

The promoter for the leuV tRNA operon of Escherichia coli has been studied. Derivatives of this promoter were examined in vivo, fused to the cat gene or to the lacZ gene. When compared to other promoters, the leuV promoter was found to be at least three times stronger than the tyrT promoter (for the tyrT tRNA operon), or the lac promoter (trp::lac promoter fusion) and as strong as the P1,P2 promoter of the rrnB operon (a ribosomal RNA operon). Deletion analysis revealed that, while removal of sequences downstream from +11 (relative to the transcription start point) did not affect activity, removal of sequences upstream from -39 resulted in a ten-fold reduction in expression. Unlike rRNA operons which also display upstream activation, sequences responsible for this effect in the leuV promoter are separated into two regions, one between -76 and -47, and the other between -45 and -39. DNA fragments carrying the leuV promoter migrate aberrantly on polyacrylamide gels, a phenomenon usually associated with DNA bending. One sequence thought to be involved in bending is a TTTTT run centered around -71. Point mutations engineered at this T5 region resulted in a loss of activation but had no apparent effect on migration rate. Transcription efficiency of promoter derivatives was examined in vitro using supercoiled, relaxed, or linearized plasmids as templates. Upstream activation was observed only when using relaxed templates, although maximum activity was obtained using supercoiled forms. Insertion of the very efficient 16S transcription terminator between the leuV promoter and the cat gene resulted in barely detectable activities, indicating that no antitermination mechanism was present.

Base Sequence↗
PubMed1988

Development of a new shuttle plasmid system for Escherichia coli and Clostridium perfringens.

We constructed a 7.9-kilobase-pair recombinant shuttle plasmid, designated pHR106, by combining desired segments of three plasmids: an Escherichia coli plasmid (pSL100) which provides a multiple cloning site, a Clostridium perfringens plasmid (pJU122) which provides a clostridial origin of replication, and an E. coli plasmid (pJIR62) which provides an E. coli origin of replication, an ampicillin resistance gene, and a chloramphenicol resistance gene of clostridial origin. The shuttle plasmid transformed E. coli HB101 with a frequency of 1 transformant per 10(4) viable cells and C. perfringens L-phase strain L-13 with a frequency of approximately 1 transformant per 10(6) viable cells. Because of the set of unique cloning sites and the chloramphenicol resistance marker, this shuttle plasmid should be particularly useful for studies of gene regulation and for enzyme production with C. perfringens.

Chloramphenicol Resistance↗
PubMed1988

Isolation of promoters from two anaerobic bacteria.

Promoters which function in Gram-positive organisms show, with few exceptions, remarkable conservation of sequences identical with those in Escherichia coli. An E. coli system was tested to select putative promoters of two anaerobes, the Gram-positive Clostridium absonum and the Gram-negative Bacteroides thetaiotaomicron. Random restriction fragments of chromosomal DNA from these organisms were fused to the galactokinase (galK) gene of E. coli within a plasmid vector. Approximately 10% of these fragments functioned as promoters in E. coli, and a broad range of activities was evident. A single 88 base pair (bp) C. absonum DNA fragment yielded, in the E. coli plasmid vector, approximately the same high activity as that provided by the E. coli galK promoter. Sequence analysis of this fragment showed typical -35 and -10 sequences, with about five -10-like sequences closely flanking each other, some overlapping, and this appears to result in multiple start sites for transcription. The transcriptions of E. coli plasmid fragments in vitro with both E. coli RNA polymerase and C. absonum RNA polymerase showed pairs of transcripts corresponding to two start sites. By colony hybridization with the 88 bp fragment, radioactively labelled, as a probe, a 4.2 kilobase segment of C. absonum chromosomal DNA containing the 88 bp fragment was isolated. About 375 bp of this fragment was sequenced. A putative Shine-Dalgarno sequence and ATG start site were detected, followed by an opening reading frame. Using a sequence about 100 bp downstream from the 88 bp sequence, a 17-base oligonucleotide was synthesized to serve as a primer. With C. absonum RNA as a template, a reverse transcriptase primer extension assay located a pair of transcription start sites just downstream from the 88 bp sequence, proving that the 88 bp sequence functions as a promoter in C. absonum.

Bacteroides↗
PubMed1987

A human tRNA(iMet) gene produces multiple transcripts.

A third nonallelic locus of the human methionyl-tRNA multigene family (tRNA(iMet-3) was isolated. This gene, unlike two other tRNA(iMet) loci, lacks a remarkable run of T and C residues which functions as a termination of transcription signal. Instead, three tandem termination signals, each containing no more than four thymidylate residues, function as relatively inefficient termination signals. As a result, polymerase readthrough generates at least three transcripts in vitro. The efficiency of apparent termination varies significantly at these sites. All resulting transcripts appear to be processed in vitro.

Base Sequence↗
PubMed1983

Stain intensity of human nucleolus organizer region reflects incorporation of uridine into mature ribosomal RNA.

The stain intensity of the nucleolus organizer regions (NORs) of acrocentric chromosomes was correlated positively with incorporation of [3H]uridine into 18S rRNA and 28S rRNA from cultured diploid human skin fibroblasts. An analysis of these data from twins by a path model indicated that no other common genetic or environmental parameters were required to explain the relationship between NOR scores and uptake of [3H]uridine into mature rRNA species.

Adolescent↗
PubMed1982

Fusion of the Escherichia coli tRNALeu1 promoter to the galK gene: analysis of sequences necessary for growth-rate-dependent regulation.

We have fused DNA fragments derived from an Escherichia coli tRNALeu1 operon to the galK gene of E. coli to identify sequences necessary for the in vivo initiation of transcription and growth-rate-dependent regulation. Promoter sequences consisting of residues from -50 to +56 or -50 to +5 with respect to the in vivo site for initiation of transcription were introduced into chimeric plasmids upstream from the galK gene. Cells bearing these chimeric plasmids exhibited much higher levels of galactokinase than did cells bearing plasmids wherein the galactose promoter was fused to galK. This indicates that the tRNALeu1 promoter is substantially more efficient than the gal promoter. The tRNALeu1 promoter-galK chimeras exhibited marked growth-dependent regulation in a manner consistent with that reported for tRNA regulation. Since tRNALeu1 DNA spanning residues -50 to +5 was sufficient to provide growth rate regulation of galK, an inverted repeat centered at position +17 is not, under the conditions we used, required for this type of regulation.

Base Sequence↗
PubMed1981

Nucleotide sequence of an Escherichia coli tRNA (Leu 1) operon and identification of the transcription promoter signal.

Fourteen different DNA fragments containing Escherichia coli tRNA genes have been cloned using the vector pBR322. We report the methods of cloning, the identification of specific tRNA genes, and the presence or absence of rRNA genes on these cloned DNA fragments. In particular, one chimeric plasmid contains a 17.0 kilobase pair EcoRI fragment bearing tRNA(Leu 1) sequences. Using nucleotide sequence analysis we have identified a cluster of three tandem tRNA(Leu 1) genes separated by intergenic spacers of 27 and 34 base pairs, respectively. The nucleotide sequence upstream of the first gene contains a transcription promoter site. A G-C rich sequence (5'-CGCCTCC-3') found between the Pribnow box and initiation site is very similar to the corresponding sequence found in other genes which are under stringent control.

Base Sequence↗
PubMed1980

The distal end of the ribosomal RNA operon rrnD of Escherichia coli contains a tRNA1thr gene, two 5s rRNA genes and a transcription terminator.

The distal region of the Escherichia coli ribosomal RNA operon rrnD has been sequenced to identify transfer RNA genes which might be contained within this transcriptional unit, and to define a possible transcription termination site. A nucleotide sequence corresponding to the gene for tRNA1thr is located between two genes for 5S rRNA. The spacer region between the proximal 5S rRNA gene and the tRNA1thr gene is 12 base pairs long, whereas the spacer region between the distal 5S rRNA gene and the tRNA1thr gene is 37 base pairs. Possible sites for RNA processing are discussed, and the sequence of the tRNA1thr gene is found to possess the same anticodon as the tRNA3thr gene. A putative transcription termination site for rrnD was located about 25 base pairs distal to the 3' end of the second 5S rRNA gene. The rrnD and rrnC terminators are compared with each other and with other prokaryotic terminators. Located at exactly the same site on the complementary strand is another putative transcription termination site for a gene being transcribed towards rrnD. The possibility of overlapping terminators is discussed.

Base Composition↗
PubMed1980

Organization of transfer ribonucleic acid genes in the Escherichia coli chromosome.

The arrangement of transfer ribonucleic acid (RNA) genes in the chromosome of Escherichia coli K-12 (C600) was examined with the techniques of restriction endonuclease digestion and Southern blotting. The number and size of restriction fragments containing transfer or ribosomal RNA sequences or both were estimated by a variety of restriction endonucleases, including EcoRI, BglI, SmaI, SalI, BamHI, and PstI. EcoRI liberated a minimum of 27 fragments which hybridized to transfer RNA and 16 which hybridized to ribosomal RNA. Enzymes which did not cut within the ribosomal RNA operons (PstI and BamHI) liberated 16 and 13 fragments, respectively, which hybridized to transfer RNA. Five PstI and six BamHi fragments also hybridized to ribosomal RNA, suggesting that there may be at least 11 chromosomal locations distinct from ribosomal RNA operons which encode transfer RNA genes. In addition, our data indicated that several transfer RNA genes may be very close to the 5' proximal ends of certain ribosomal RNA operons and close to the 3' distal ends of all seven ribosomal RNA operons. Similar studies have been carried out with 22 purified species of transfer RNA, and we report here the number and size of EcoRI restriction fragments which hybridize to these transfer RNA species.

Base Sequence↗
PubMed1979

Synthesis of guanosine tetra- and pentaphosphates by the obligately anaerobic bacterium Bacteroides thetaiotaomicron in response to molecular oxygen.

Guanosine 5'-diphosphate 3'-diphosphate (ppGpp) and guanosine 5'-triphosphate 3'-diphosphate (pppGpp) were detected in formic acid extracts of air-exposed culutres of Bacteroides thetaiotaomicron. The identification of ppGpp and pppGpp in B. thetaiotaomicron was based on the following results: (i) cochromatography of 32P-labeled hyperphosphorylated nucleotides in two different two-dimensional solvent systems with authentic ppGpp and pppGpp; (ii) incorporation of [3H]guanosine into the putative ppGpp and pppGpp; (iii) alkaline lability; and (iv) resistance, to periodate oxidation. There was a marked increase in the concentration of ppGpp and pppGpp after shift from anaerobic to aerobic conditions, and accumulation of both ppGpp and pppGpp was blocked under these conditions by pretreatment of the culture with rifampin or tetracycline. Growth and incorporation of [3H]guanosine, [3H]tymidine, [14C]succinate, and L-[35S]methionine into macromolecules were inhibited immediately upon exposure to air. The accumulation of ppGpp and pppGpp in B. thetaiotaomicron upon exposure to air may represent a novel signal for synthesis of these compounds.

Anaerobiosis↗
PubMed1977

Differential utilization of leucyl-tRNAs by Escherichia coli.

The utilization of the isoaccepting species of leucyl-tRNA in protein synthesis has been examined in E. coli. Two minor leucyl-tRNA species, isoacceptors tRNA3Leu and tRNA4Leu, are the predominant species found bound to ribosomes during exponential growth in minimal medium. In rich medium, an increased proportion of tRNA1Leu is found bound to ribosomes. One species, tRNA5Leu, is always absent from ribosomes. In a mutant strain in which normal tRNA3Leu and tRNA5Leu are reduced or absent, tRNA1Leu is a major ribosome-bound species in minimal medium. Protein synthesis in vitro with RNA from E. coli further suggests that tRNA5Leu is rarely used for total protein synthesis; however, this species is active with RNA from MS2 phage. We propose that tRNA1Leu can substitute for tRNA3Leu under rapid growth conditions, and that tRNA5Leu is used minimally in total protein synthesis.

Bacterial Proteins↗
PubMed1975

Anthranilate synthase from Bacillus subtilis. The role of a reduced subunit X in aggregate formation and amidotransferase activity.

With respect to its sulfhydryl groups, subunit X can exist in at least two forms, oxidized (Xox) and reduced (Xre). The importance of the Xre form for the formation of an EX complex and for amidotransferase activity has been examined. Subunit Xre is rapidly inactivated by p-chloromercuribenzoate and bromopyruvate, whereas subunit Xox, which is not catalytically functional in amidotransferase activity, is not affected. The glutamine analogue 6-diazo-5-oxo-L-norleucine (DON) has no effect on Xre alone but rapidly inactivates the EXre complex. DON-inactivated subunit X cannot be reactivated by 2-mercaptoethanol but can be readily displaced from subunit E by free subunit Xre. The integrity of the EXre complex is maintained following gel filtration on Sephadex G-100 in the presence of glutamine and 2-mercaptoethanol, thus the binding of glutamine to the complex does not require the binding of other substrates. Subunit Xox, however, does not aggregate with subunit E since no EXox complex is found following gel filtration on Sephadex G-100 in the presence of glutamine and in the absence of 2-mercaptoethanol. Thus, a reduced sulfhydryl group(s) is not only essential for amidotransferase activity but also for the formation of the aggregate as well. The following model is proposed to explain these results. Free subunit Xre does not bind DON or glutamine to the catalytically functional sulfhydryl group. Upon aggregation with subunit E, however, the glutamine or DON binds to the glutamine catalytic site on subunit Xre and amidotransfer or alkylation occurs. An EX complex which has been alkylated by DON can be readily dissociated and it is suggested that following catalysis the EX complex may also dissociate.

Amides↗
PubMed1975

Separation of transfer ribonucleic acid by sepharose chromatography using reverse salt gradients.

The transfer ribonucleic acids of Escherichia coli bind to unsubstituted Sepharose in the presence of high concentrations of ammonium sulfate at pH 4.5. Transfer RNA species are eluted individually from the Sepharose by a gradient from high to low concentrations of ammonium sulfate; leucine tRNA is fractionated into five isoaccepting species. The order of elution of these isoaccepting species differs from that seen with reverse phase chromatography. By means of only these two procedures, one isoaccepting species of leucine tRNA can be purified to apparent homogeneity. Isoaccepting tRNA species for 9 amino acids have been resolved. This established the general utility of this chromatographic system for the separation and purification of specific isoaccepting transfer RNAs.

Chromatography, Gel↗