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W B Dempsey

Publications and source records attributed to W B Dempsey.

At least 19 recordsLinked to original sources
PubMed1994

traJ sense RNA initiates at two different promoters in R100-1 and forms two stable hybrids with antisense finP RNA.

RNase protection experiments show that the sizes of the two R100 finP molecules are 74 and 135 nucleotides. In an RNase III mutant, finP transcripts form stable double-stranded hybrids of 108 bp and 68 bp with traJ transcripts. RNase protection experiments also show that most R100-1 transcripts originating in traM cross the traM-traJ intergenic region and end inside the untranslated leader region of traJ. Some extend into the traJ open reading frame. These findings mean that the antisense finP RNA, thought to regulate traJ translation, must regulate traJ transcripts from both J and M promoters.

Bacterial Outer Membrane Proteins↗
PubMed1994

Regulation of R100 conjugation requires traM in cis to traJ.

Deletion mutants of R100-1 were constructed by classical methods to remove various segments of the traM open reading frame, pTraM-binding sites and the traM promoters. Complementation tests showed that traM was efficiently complemented only when the trans-acting fragment contained both the complete traM gene and the adjacent traJ promoter and leader sequences. The conclusion is that traM and traJ constitute a complex operon. A deletion mutant lacking all of the traJ gene, and one containing a frameshifting traM deletion, retained the ability to transfer at a low level, thereby showing that neither pTraM nor pTraJ is absolutely essential for transfer.

Bacterial Outer Membrane Proteins↗
PubMed1992

Suppression of insertions in the complex pdxJ operon of Escherichia coli K-12 by lon and other mutations.

Complementation analyses using minimal recombinant clones showed that all known pdx point mutations, which cause pyridoxine (vitamin B6) or pyridoxal auxotrophy, are located in the pdxA, pdxB, serC, pdxJ, and pdxH genes. Antibiotic enrichments for chromosomal transposon mutants that require pyridoxine (vitamin B6) or pyridoxal led to the isolation of insertions in pdxA, pdxB, and pdxH but not in pdxJ. This observation suggested that pdxJ, like pdxA, pdxB, and serC, might be in a complex operon. To test this hypothesis, we constructed stable insertion mutations in and around pdxJ in plasmids and forced them into the bacterial chromosome. Physiological properties of the resulting insertion mutants were characterized, and the DNA sequence of pdxJ and adjacent regions was determined. These combined approaches led to the following conclusions: (i) pdxJ is the first gene in a two-gene operon that contains a gene, temporarily designated dpj, essential for Escherichia coli growth; (ii) expression of the rnc-era-recO and pdxJ-dpj operons can occur independently, although the pdxJ-dpj promoter may lie within recO; (iii) pdxJ encodes a 26,384-Da polypeptide whose coding region is preceded by a PDX box, and dpj probably encodes a basic, 14,052-Da polypeptide; (iv) mini-Mud insertions in dpj and pdxJ, which are polar on dpj, severely limit E. coli growth; and (v) three classes of suppressors, including mutations in lon and suppressors of lon, that allow faster growth of pdxJ::mini-Mud mutants can be isolated. A model to account for the action of dpj suppressors is presented, and aspects of this genetic analysis are related to the pyridoxal 5'-phosphate biosynthetic pathway.

ATP-Dependent Proteases↗
PubMed1990

Integration host factor affects expression of two genes at the conjugal transfer origin of plasmid R100.

Integration host factor (IHF) binds to two sites near the origin of transfer of the conjugative antibiotic resistance plasmid, R100. DNase I footprinting shows that one site is immediately adjacent to oriT and the gene X promoter, and another is adjacent to the traM promoter. A third site, known only from retardation gels, is near the traJ promoter. The relative promoter activities of genes X, traJ and traM are reduced in himA mutants (IHF-), as measured by chloramphenicol-resistance assays. Transcript analyses by Northern blots showed a reduction in size of the principal gene X and traJ transcripts in the absence of IHF.

Bacterial Proteins↗
PubMed1989

Sense and antisense transcripts of traM, a conjugal transfer gene of the antibiotic resistance plasmid R100.

The region of the antibiotic resistance plasmid R100 that encodes the plasmid-specific transfer gene traM has two tandemly aligned promoters separated by 145 nucleotides. The principal transcripts are 705 and 562 nucleotides long. Minor transcripts are 1550 and 1700 nucleotides long. The 705-base transcript appears to encode an 11 kD traM protein. The 562-base transcript does not encode a detectable protein. When subcloned on short fragments, the promoter for the 562-base transcript initiates efficiently but that for the 705 site does not. The 3' ends of the 705 and 562 base transcripts end inside the traJ ORF. Thus they provide additional sense RNA to compete with traJ for finP, the antisense translational regulator of traJ. A model is proposed for the participation of these sense and antisense transcripts in the control of expression of the traJ gene.

Bacterial Proteins↗
PubMed1987

Fertility inhibition gene of plasmid R100.

The fin0 gene of R100 was isolated from the Fin0+ transducing phage VA lambda 57. The limits of the gene were determined by BAL31 digestions and by analysis of deletion mutations derived from an internal restriction site. The DNA sequence contained an open reading frame of 558 nucleotides that would encode a protein of 21,268 daltons. Synthesis of such a protein was observed only when the fragment was cloned in front of the TAC promoter. Deletions entering the large open reading frame from either end were Fin0-, while internal frame shift mutations retained high Fin0 activity. One such strain had a 13 bp internal deletion that would produce a protein of 63 amino acid residues of which 21 were basic. We were consequently unable to rigorously establish that the 558 base orf encoded a fin0 product. The strand opposite the large open reading frame contained several transcription termination signals, and it is possible that the active gene product is one or two small RNAs from this strand.

Bacteriophage lambda↗
PubMed1987

Transcript analysis of the plasmid R100 traJ and finP genes.

Single-stranded RNA probes were used to study the regulation of plasmid transfer in the infectious antibiotic resistance plasmid R100. Transcription of the positive transfer control gene traJ of R100 appears to be initiated continuously. In the presence of finO, the traJ transcript is 235 bases long, and in the absence of finO it is 1050. These sizes are strain specific. finO increases four-to tenfold the amount of the transcript from the finP gene that is detectable in cells containing R100, R136, or the sex factor F. The size of the principal finP transcript from R100 as determined on Northern blots is 105 bases. A secondary transcript with a size of 180 bases was detected in small amounts in R100 extracts. The finP transcript size was also determined by nuclease protection experiments. In this case the size was 74 bases. The 5' ends of the finP and traJ transcripts were located by primer extension experiments. A new model of FinO/P control is proposed.

Base Sequence↗
PubMed1987

oriT sequence of the antibiotic resistance plasmid R100.

We present the nucleotide sequence of the oriT region from plasmid R100. Comparison to other IncF plasmids revealed homology around the proposed nick sites as well as conservation of inverted repeated sequences in the nonhomologous region. Three areas showed strong homology (eight of nine nucleotides) to the consensus sequence for binding of integration host factor, suggesting a role for this DNA-binding protein in nicking at oriT.

Anti-Bacterial Agents↗
PubMed1986

Cloning, mapping, and sequencing of plasmid R100 traM and finP genes.

The fertility control gene finP, the transfer gene traM, and the transfer origin, oriT, of plasmid R100 were isolated on a single 1.2-kilobase EcoRV fragment and were then subcloned as HaeIII fragments. The sequence of the 754-base-pair finP-containing fragment is reported here. In addition to the finP gene, the sequence includes all but two bases of the R100 traM open reading frame and apparently all of the leader mRNA sequence and amino end of the traJ gene of R100. The sequence contains two open reading frames which encode small proteins on the opposite strand from the traM and traJ genes. It also shows two sets of inverted repeats that have the characteristics of transcription terminators. One set is positioned as if it was the traM terminator, and the other set, which is downstream from the first, sits in the middle of the leader mRNA sequence for traJ. On the bottom strand, this inverted repeat has the structure of a rho-independent terminator. Other less-stable inverted repeats overlap this second terminator in the same way as is seen in attenuation sequences, and the two separate small open reading frames on the bottom strand also totally overlap the stem of the rho-independent terminator, suggesting that their translation would cause shifting of termination to the bottom strand homolog of the putative traM terminator. The finP gene product was not identified, but the gene was mapped to the sequence which contains the traJ gene. It either overlaps traJ or is antisense to it.

Base Sequence↗
PubMed1983

The finO gene of antibiotic resistance plasmid R100.

Lambda phages carrying the R100 finO gene have been isolated from an R100:: lambda cointegrate in which lambda was inserted into the R100 traD gene at kb coordinate 72.1. Physical analyses of these phages place the finO gene within R100 SalI fragment D, near kb coordinate 82.0. Analysis of proteins synthesized by the phages did not identify the finO gene product, although a constitutive protein of m.w. 30,100 was encoded by R100 DNA between kb coordinates 78.7 and 81.2.

Bacterial Proteins↗
PubMed1983

Low muscle levels of pyridoxine in McArdle's syndrome.

Pyridoxal phosphate is a covalently bound cofactor of glycogen phosphorylase. Phosphorylase is a major muscle protein and therefore represents a significant pool of pyridoxal phosphate. Muscle pyridoxine content was measured in three patients with myophosphorylase deficiency (McArdle's syndrome) in whom there was a marked diminution or absence of phosphorylase protein as determined by acrylamide gel electrophoresis. Total muscle pyridoxine in the patients with McArdle's syndrome (0.55 +/- 0.08 microgram/g wet weight, mean +/- SD) was markedly reduced compared with 11 human control subjects who had normal levels of muscle phosphorylase (total muscle B6 = 2.49 +/- 0.47). Despite such drastically low levels of muscle pyridoxine, these patients had no evidence of pyridoxine "deficiency." These results suggest that low muscle B6 in McArdle's syndrome represents the specific loss of pyridoxal phosphate normally bound to phosphorylase apoenzyme and imply that phosphorylase pyridoxal phosphate accounts for 75 to 80 percent of the total pyridoxine in normal human muscle.

Adolescent↗
PubMed1982

Threonine prevents derepression of pyridoxine synthesis in Escherichia coli B.

After 40 min of pyridoxal starvation, pyridoxine phosphate oxidase-less mutants of Escherichia coli B derepressed pyridoxine biosynthesis 13-fold to a rate of 1.7 X 10(-9) mol/h per mg of cells. Threonine at 100 mg/liter prevented this derepression but did not affect the continued synthesis of pyridoxine. Neither serine nor branched-chain amino acids altered the threonine effect.

Amino Acids, Branched-Chain↗
PubMed1979

Lambda transducing phages derived from a FinO- R100::lambda cointegrate plasmid: proteins encoded by the R100 replication/incompatibility region and the antibiotic resistance determinant.

Three lambda transducing phages have been isolated from pEDR20, an R100::lambda cointegrate plasmid in which the lambda insertion inactivated the R100 finO gene. Physical analysis of the three phages showed that the lambda is inserted at kilobase coordinate 81.3 of R100. All three phages carry different amounts of R100 DNA in the left arm of lambda. Each pahge contains ISlb, the mer genes and the region between coordinate 81.3 and 88.6; thus, all contain the genes necessary for R100 replication. One phage, VA lambda 73, contains the entire r-determination of R100 in addition to the above DNA. Five proteins coded by the region between 81.3 and 88.6 were detected. These had subunit molecular weights of 10,400; 12,200; 16,200; 19,600; and 38,300. The first was made constitutively and the other four only from a lambda promoter. Other constitutive proteins were one from the cml fus region with a molecular weight of 22,400 (cml) and two from the str sul region with molecular weights of 31,500 (str?) and 30,100 (sul?). Mercuric ion induced synthesis of at least 10 proteins. Six of these were known from earlier work. The total size of the proteins which appear to derive from the mer genes exceeds by a factor of 1.5, the coding capacity of this region without overlapping genes. Some, or all of these extra proteins may be chromosomal in origin, possibly derepressed in response to mercury gene products.

Bacteriophage lambda↗
PubMed1978

Pyridoxine-requiring mutants of Escherichia coli: glycolaldehyde dehydrogenase is not coded for by the pdxB gene.

Twenty-seven independent pyridoxineless mutants belonging to genetic linkage group I were assayed for glycolaldehyde dehydrogenase. Some mutants lacked enzyme activity entirely, and others showed activity ranging from very low to wild-type levels. Reversion to pyridoxine independence usually had no effect upon this activity. Transfer of the pyridoxine genes to a common host that had wild-type levels of enzyme activity made the recipient pyridoxineless without affecting the activity. These results negate the idea of an obligatory role for glycolaldehyde dehydrogenase in pyridoxine biosynthesis.

Acetaldehyde↗