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J R Roth

Publications and source records attributed to J R Roth.

At least 91 records · Page 5Linked to original sources
PubMed1986

Structural and functional studies of insertion element IS200.

The nucleotide sequence of the insertion element IS200 has been determined partially, including the junctions between the element and the host chromosome at the insertion site. At most, two bases (A-A) are found repeated at the junctions and could be duplications of host sequences generated by the insertion of the element. No obvious sequence repeats, either direct or inverted, have been detected between the sequences just within the two ends of the element. The element is an extremely strong block to host transcription across the insertion site. A sequence similar to known transcription termination signals was found just within the element near the right end. Removal of less than 50 base-pairs at the right end of the element abolishes the transcription block. The putative terminator sequence is located within this 50 base-pair region. Genetic studies suggest that the element contains a promoter located more than 93 base-pairs from its left end. The proposed promoter and terminator are in proper orientation to form an internal transcription unit.

Autoradiography↗
PubMed1986

Target sequences for mutagenesis in Salmonella histidine-requiring mutants.

Nucleotide target sequences involved in reversion to the wild type phenotype are diagrammed for Salmonella frameshift histidine-requiring mutants hisD3052, hisD3018, hisD6610, and hisD6580 and for base-substitution mutants hisG46 and hisG428. Frameshift strain hisC3076 probably reverts by nucleotide changes similar to those that occur during reversion of hisD3018 and hisD6610. Multiple modes of reversion characterize each strain. Each strain also has a particularly diagnostic mutagen-susceptible sequence. These highly mutagen-susceptible stretches are the hisD3052 GCGCGCGC sequence, the hisD6610 CCCCCC sequence, the hisD6580 AAAAA sequence, and the A/T containing codon of hisG428 and G/C containing codon of hisG46, respectively. Between them, hisG46 and hisG428 are reverted by all of the six possible base substitution transition and transversion mutations.

Base Sequence↗
PubMed1985

Directed formation of deletions and duplications using Mud(Ap, lac).

A genetic procedure is directed for the isolation of chromosomal deletions and duplications with predetermined endpoints. These rearrangements are generated in transduction crosses using a mixture of P22-transducing phage lysates grown on two strains, each carrying a Mud-lac insertion. The formation of duplications and deletions was demonstrated in the his operon using insertions of Mud 1-8 (a transposition-defective Mud-lac phage). This technique was also used to make larger chromosomal duplications between Mud 1-8 insertions in the thr and leu biosynthetic operons and between Mud insertions in the thr and pyrB operons. Genetic evidence is presented that strongly suggests that inheritance of a single Mud prophage by P22-mediated crosses requires two transduced fragments; each carrying part of the Mud prophage. The two fragments must be involved in three recombinational exchanges; one exchange joins the donor Mud fragments and two exchanges occur between the composite fragment and the recipient chromosome, one on either side of the complete donor Mud element. Since duplications only occur between Mud insertions in the same orientation on the chromosome, the method of duplication formation provides a simple means of determining the orientation of Mud 1-8 on the chromosome and, therefore, the direction of transcription of the gene into which Mud is inserted. This method was also used to construct recombinants between a Mud 1-8 prophage and Casadaban's protein fusion vector Mud2 and, thereby, isolate Mud2-8, a Mud derivative containing the protein fusion ability of Mud2 and the defective transposition functions of Mud1-8.

Bacterial Proteins↗
PubMed1985

Mutations in the spoT gene of Salmonella typhimurium: effects on his operon expression.

The spoT gene of Salmonella typhimurium has been identified. Mutations in spoT map between gltC and pyrE at 79 min. The spoT1 mutant has elevated levels of guanosine 5'-diphosphate-3'-diphosphate (ppGpp) during steady-state growth and exhibits a slower than normal decay of ppGpp after reversal of amino acid starvation. The spoT1 mutation elevates his operon expression but is distinct from known his regulatory mutations. Elevated his operon expression in spoT mutants causes resistance to the histidine analogs, 1,2,4-triazole-3-alanine and 3-amino-1,2,4-triazole. These properties of spoT mutants allowed us to identify and characterize additional spoT mutants. Approximately 40% of these mutants are temperature sensitive for growth on minimal medium, suggesting that the spoT function is essential or that excessive accumulation of ppGpp is lethal.

Alcohol Oxidoreductases↗
PubMed1984

Conditionally transposition-defective derivative of Mu d1(Amp Lac).

A Mu d1 derivative is described which is useful for genetic manipulation of Mu-lac fusion insertions. A double mutant of the specialized transducing phage Mu d1(Amp Lac c62ts) was isolated which is conditionally defective in transposition ability. The Mu d1 derivative, designated Mu d1-8(Tpn[Am] Amp Lac c62ts), carries mutations which virtually eliminate transposition in strains lacking an amber suppressor. In such strains, the Mu d1-8 prophage behaves like a standard transposon. It can be moved from one strain of Salmonella typhimurium to another by the general transducing phage P22 with almost 100% inheritance of the donor insertion mutation. When introduced into a recipient carrying supD, supE, or supF, 89 to 94% of the Ampr transductants were transpositions of the donor Mu d1-8, from the transduced fragment into new sites. The stability of Mu d1-8 in a wild-type, suppressor-free background was sufficient to permit use of the fusion to select constitutive mutations without prior isolation of deletions to stabilize the fusion. Fusion strains could be grown at elevated temperature without induction of the Mu d prophage. The transposition defect of Mu d1-8 was corrected by a plasmid carrying the Mu A and B genes.

DNA Restriction Enzymes↗
PubMed1984

Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic growth conditions.

In this paper, we report that the enteric bacterium Salmonella typhimurium synthesized cobalamin de novo under anaerobic culture conditions. Aerobically, metE mutants of S. typhimurium need either methionine or cobalamin as a nutritional supplement for growth. The growth response to cobalamin depends upon a cobalamin-requiring enzyme, encoded by the gene metH, that catalyzes the same reaction as the metE enzyme. Anaerobically, metE mutants grew without any nutritional supplements; the metH enzyme functioned under these conditions due to the endogenous biosynthesis of cobalamin. This conclusion was confirmed by using a radiochemical assay to measure cobalamin production. Insertion mutants defective in cobalamin biosynthesis (designated cob) were isolated in the three major branches of the cobalamin biosynthetic pathway. Type I mutations blocked the synthesis of cobinamide, type II mutations blocked the synthesis of 5,6-dimethylbenzimidazole, and type III mutations blocked the synthesis of cobalamin from cobinamide and 5,6-dimethylbanzimidazole. Mutants that did not synthesize siroheme (cysG) were blocked in cobalamin synthesis. Genetic mapping experiments showed that the cob mutations are clustered in the region of the S. typhimurium chromosome between supD (40 map units) and his (42 map units). The discovery that S. typhimurium synthesizes cobalamin de novo only under anaerobic conditions raises the possibility that anaerobically grown cells possess a variety of enzymes which are dependent upon cobalamin as a cofactor.

Anaerobiosis↗
PubMed1983

IS200: a Salmonella-specific insertion sequence.

A new IS element (IS200) has been identified in Salmonella. The sequence was identified as an IS element by the following criteria: its insertion caused the mutation hisD984; six copies of the sequence are present in strain LT2 of S. typhimurium; and transposition of the sequence has been observed on several occasions. IS200 is found in almost all Salmonella species examined but is absent from most other enteric bacteria. The specificity of this element for Salmonella (and the absence of IS1-IS4 from Salmonella) suggest that transfer of insertion sequences between bacterial groups may be less extensive than is commonly believed. Alternatively, the distribution may suggest that these elements play a selectively important role in bacteria.

Base Sequence↗
PubMed1983

New suppressors of frameshift mutations in Salmonella typhimurium.

Several new types of suppressor mutants have been isolated. These were identified among revertants of mutants originally generated by mutagens other than the acridine-derived ICR191. The new suppressors correct mutations other than those with runs of C or G which are recognized by the previously described suppressors. Several frameshift mutations are corrected by more than one suppressor type. Apparently, the DNA base sequence near these mutant sites includes sites of action for several distinct suppressor types.

Base Sequence↗
PubMed1983

Genetic characterization of the sufj frameshift suppressor in Salmonella typhimurium.

A new suppressor of +1 frameshift mutations has been isolated in Salmonella typhimurium. This suppressor, sufJ, maps at minute 89 on the Salmonella genetic map between the argH and rpo(rif) loci, closely linked to the gene for the ochre suppressor tyrU(supM). The suppressor mutation is dominant to its wild-type allele, consistent with the suppressor phenotype being caused by an altered tRNA species. The sufJ map position coincides with that of a threonine tRNA(ACC/U) gene; the suppressor has been shown to read the related fourbase codons ACCU, ACCC, ACCA.--The ability of sufJ to correct one particular mutation depends on the presence of a hisT mutation which causes a defect in tRNA modification. This requirement is allele specific, since other frameshift mutations can be corrected by sufJ regardless of the state of the hisT locus.--Strains carrying both a sufJ and a hisT mutation are acutely sensitive to growth inhibition by uracil; the inhibition is reversed by arginine. This behavior is characteristic of strains with mutations affecting the arginine-uracil biosynthetic enzyme carbamyl phosphate synthetase. The combination of two mutations affecting tRNA structure may reduce expression of the structural gene for this enzyme (pyrA).

Base Sequence↗
PubMed1983

Genetic methods for analysis and manipulation of inversion mutations in bacteria.

A number of genetic methods for the isolation, characterization and manipulation of large chromosomal inversions in Salmonella typhimurium are described. One inversion-carrying mutant is characterized in detail and used to demonstrate a number of unique genetic properties of bacterial inversions. --Contrary to expectation, it was found that large inversion mutations can be repaired by generalized transduction. The repair results from the simultaneous introduction of two wild-type transduced fragments into a single recipient cell. Homologous recombination between the two transduced fragments and the two inversion breakpoints causes the inverted segment to be reinverted. This results in regeneration of the wild-type orientation of this chromosome segment. Similar recombination events allow a large inversion mutation to be introduced into a wild-type strain; two transduced fragments from an inversion strain cause recombination events resulting in inversion of a large chromosome segment. --Genetic methods for mapping the extent of a large inversion mutation by generalized transduction are described and tested. The methods are operationally simple and allow good resolution of the two inversion breakpoints.

Chromosome Mapping↗
PubMed1983

Selection and endpoint distribution of bacterial inversion mutations.

This paper describes the isolation and characterization of spontaneous inversion mutants of Salmonella typhimurium. The mutants are selected by demanding that an unexpressed hisD gene acquire a new promoter. Chromosome rearrangements that juxtapose the hisD gene and a foreign promoter are obtained by this selection. Although a number of inversions are found, the frequency was lower than expected. The breakpoint of these inversions are not distributed randomly either in the his operon or on the chromosome. The his breakpoint lies in the hisG-hisD intercistronic region, a sequence known to occur at several places on the bacterial chromosome. In most of the inversions, the 'non-his' breakpoint lies across the chromosome, so that the inverted region includes the origin or terminus of DNA replication. The significance of these results is discussed.

Chromosome Mapping↗
PubMed1983

Genetic mapping of IS200 copies in Salmonella typhimurim strain LT2.

The wild-type Salmonella typhimurium strain LT2 contains six copies of the insertion sequence element IS200 which is unique to Salmonella. We have determined the chromosomal locations of all six copies of IS200 in strain LT2. This was done by mapping the positions of Tn10 elements inserted near each copy of IS200. Such Tn10 insertions were detected by Southern hybridization as IS200-containing restriction fragments with altered electrophoretic mobility. The copies are located at quite evenly spaced sites in the chromosome. Some are found in regions with many known genes; others are in regions with few known functions. There is no indication of a possible function for IS200. The method described here should be applicable to the mapping of IS elements in general.

Chromosome Mapping↗
PubMed1983

Internal promoters of the his operon in Salmonella typhimurium.

Two internal promoters in the his operon of Salmonella typhimurium have been precisely mapped genetically. The internal promoters are found in, or very close to, gene border regions in the his operon. The his operon was examined for the presence of additional internal promoters whose transcripts were sensitive to rho-mediated transcription termination and therefore had escaped detection. No new internal promoters were found. It is argued that the internal promoters described here are not likely to be fortuitous message start sites, but may play a physiologically important role in operon expression.

Chromosome Mapping↗
PubMed1983

Regulation of proline utilization in Salmonella typhimurium: characterization of put::Mu d(Ap, lac) operon fusions.

The genes for proline utilization were fused to the structural genes of the lac operon by use of the hybrid Mu phage derivative Mu d(Ap lac). Stable deletion derivatives of these fusions were selected and used to study the transcriptional regulation of the put genes. Analysis of these fusions showed that the putA gene product, a bifunctional oxidase-dehydrogenase, also serves to negatively control transcription of the putA and putP genes. Transcription of the put genes is repressed only in putA+ strains; this repression is lifted when exogenous proline is supplied. Transcription of the put genes is stimulated by cyclic AMP in putA+ and putA strains. Maximal induction of the put genes in putA+ strains requires oxygen or an alternative electron acceptor. This oxygen effect is mediated by the putA protein since putA mutants show maximal transcription even without an electron acceptor. The orientation of the Mu d(Ap lac) insertions was determined by formation of Hfr's via the lac homology on F'ts114 lac+. The direction of chromosome mobilization by these Mu d(Ap lac)-directed Hfr's demonstrated that the putP and putA genes are divergently transcribed from a central regulatory region lying between them.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1983

6-Aminonicotinamide-resistant mutants of Salmonella typhimurium.

Resistance to the nicotinamide analog 6-aminonicotinamide has been used to identify the following three new classes of mutants in pyridine nucleotide metabolism. (i) pncX mutants have Tn10 insertion mutations near the pncA locus which reduce but do not eliminate the pncA product, nicotinamide deamidase. (ii) nadB (6-aminonicotinamide-resistant) mutants have dominant alleles of the nadB gene, which we propose are altered in feedback inhibition of the nadB enzyme, L-aspartate oxidase. Many of these mutants also exhibit a temperature-sensitive nicotinamide requirement phenotype. (iii) nadD mutants have mutations that affect a new gene involved in pyridine nucleotide metabolism. Since a high proportion of nadD mutations are temperature-sensitive lethal mutations, this appears to be an essential gene for NAD and NADP biosynthesis. In vivo labeling experiments indicate that in all the above cases, resistance is gained by increasing the ratio of NAD to 6-aminonicotinamide adenine dinucleotide. 6-Aminonicotinamide adenine dinucleotide turns over significantly more slowly in vivo than does normal NAD.

6-Aminonicotinamide↗
PubMed1983

An indispensable gene for NAD biosynthesis in Salmonella typhimurium.

We have located the nadD locus between lip and leuS at 14 min on the Salmonella typhimurium chromosome, and we have shown it to be the structural gene for nicotinic acid mononucleotide adenylyltransferase. This is the first indispensable gene of pyridine nucleotide metabolism that has been identified. Mutants altered at this locus, isolated by their 6-aminonicotinamide resistance phenotype, accumulate abnormally large pools of nicotinic acid mononucleotide in vivo; many exhibit a temperature-sensitive lethal phenotype. Enzyme assays reveal markedly lower transferase activity in mutant extracts than in nadD+ extracts. The partial dominance of nadD mutants when placed in a nadD+/nadD diploid suggests that nicotinic acid mononucleotide adenylyltransferase is a multimeric enzyme.

Amide Synthases↗