A Genetic Characterization of the Nadc Gene of Salmonella Typhimurium

The nadC gene of Salmonella encodes the pyridine biosynthetic enzyme PRPP-quinolinate phosphoribosyltransferase. Using a combination of genetic techniques, a deletion map for the Salmonella nadC gene has been generated which includes over 100 point mutants and 18 deletion intervals. The nadC alleles obtained by hydroxylamine mutagenesis include those suppressed by either amber, ochre, or UGA nonsense suppressors as well as alleles suppressed by the missense suppressor, sumA. Deletions were obtained by three separate protocols including spontaneous selection for loss of the nearby aroP gene, recombination between aroP::MudA and nadC::MudA insertion alleles, and selection for spontaneous loss of tetracycline resistance in a nearby guaC::Tn10dTc insertion mutant allele. The nadC mutants comprise one complementation group and the nadC+ allele is dominant to simple, nadC auxotrophic mutant alleles. Intragenic complementation of two nadC alleles, nadC493 and nadC494, mapping to deletion intervals 17 and 18, respectively, suggests that nadC encodes a multimeric enzyme. Both nadC and the nearby aroP locus are transcribed counterclockwise on the standard genetic map of Salmonella, in opposite orientation to the direction of chromosome replication.     

Histidine Mutants Requiring Adenine: Selection of Mutants with Reduced hisG Expression in SALMONELLA TYPHIMURIUM

A method is described for the selection of Salmonella typhimurium mutants with reduced levels of hisG enzyme activity. This method is based on the fact that the hisG enzyme catalyzes the consumption of ATP in the first step of histidine biosynthesis. Normally, this reaction is closely regulated, both by feedback inhibition and by repression of the operon. However, conditions can be set up that result in the uncontrolled use of adenine in histidine biosynthesis. Cells grown under these conditions become phenotypic adenine auxotrophs. Some revertant clones that no longer require adenine contain mutations in hisG, hisE, or the his-control region. The hisG mutations are of all types (nonsense, frameshift, missense, deletion and leaky types), and they map throughout the hisG gene.     

Temperature-Sensitive Mutants Blocked in the Folding or Subunit Assmbly of the Bacteriophage P22 Tailspike Protein. I. Fine-Structure Mapping

As part of a study of protein folding, we have constructed a fine-structure map of 9 existing and 29 newly isolated UV- and hydroxylamine-induced temperature-sensitive (ts) mutations in gene 9 of Salmonella bacteriophage P22. Gene 9 specifies the polypeptide chain of the multimeric tail spikes, six of which form the cell attachment organelle of the phage. The 38 ts mutants were mapped against deletion lysogens with endpoints in gene 9. They mapped in 10 of the 15 deletion intervals. Two- and three-factor crosses between mutants within each interval indicated that at least 31 ts sites are represented among the 38 mutants. To determine the distribution of ts sites within the physical map, we identified the protein fragments from infection of su^- hosts with 10 gene 9 amber mutants. Their molecular weights, ranging from 13,900 to 55,000 daltons, were combined with the genetic data to yield a composite map of gene 9. The 31 ts sites were distributed through most of the gene, but were most densely clustered in the central third.—None of the ts mutant pairs tested exhibited intragenic complementation. Studies of the defective phenotypes of the ts mutants (Goldenberg and King 1981; Smith and King 1981) revealed that most do not affect the thermostability of the mature protein, but instead prevent the folding or subunit assembly of the mutant chains synthesized at restrictive temperature. Thus, many of thes ts mutations identify sites in the polypeptide chain that are critical for the folding or maturation of the tail-spike protein.     

A deletion map of the WAGR region on chromosome 11.

The WAGR (Wilms tumor, aniridia, genitourinary anomalies, and mental retardation) region has been assigned to chromosome 11p13 on the basis of overlapping constitutional deletions found in affected individuals. We have utilized 31 DNA probes which map to the WAGR deletion region, together with six reference loci and 13 WAGR-related deletions, to subdivide this area into 16 intervals. Specific intervals have been correlated with phenotypic features, leading to the identification of individual subregions for the aniridia and Wilms tumor loci. Delineation, by specific probes, of multiple intervals above and below the critical region and of five intervals within the overlap area provides a framework map for molecular characterization of WAGR gene loci and of deletion boundary regions.     

Defining regions and rearrangements of the Silene latifolia Y chromosome

We combine data from published marker genotyping of three sets of S. latifolia Y chromosome deletion mutants with changed sex phenotypes and add genotypes for several new genic markers to refine the deletion map of the Y chromosome and compare it with the X chromosome genetic map. We conclude that the Y chromosome of this species has been derived through multiple rearrangements of the ancestral gene arrangement and that none of the rearrangements so far detected was involved in stopping X-Y recombination. Different Y genotypes may also differ in their gene content and possibly arrangements, suggesting that mapping the Y-linked sex-determining genes will be difficult, even if many further genic markers are obtained. Even in determining the map of Y chromosome markers to discover all the rearrangements, physical mapping by FISH or other experiments will be essential. Future deletion mapping work should ensure that markers are studied in the parents of deletion mutants and should probably include additional deletions that were not ascertained by causing mutant sex phenotypes.     

Segregation of the mutator property of plasmid R46 from its ultraviolet-protecting property

Summary Plasmid R46 (an R factor conferring resistance to ampicillin, sulfonamides, streptomycin and tetracycline) reduces the bactericidal effect of UV irradiation but increases its mutagenic effect (reversion of hisG46), and raises the frequency of spontaneous reversion (mutator effect). Putative deletion mutants of R46 were obtained by transduction of the plasmid, then two successive conjugal transfers. Plasmids of five of six deletion classes, each with a different combination of drug resistance traits, retained conjugative ability and the UV-protecting, mutagenesis-enhancing and mutator effects of R46. (pKM101, used in the Ames system to enhance responsiveness to chemical mutagens, is one such mutant of R46.) Plasmids of a sixth class, represented by pKM115, conferred resistance only to streptomycin and were non-conjugative. All of several such plasmids (of independent origin) had a much stronger mutator effect than did R46, but lacked UV-protecting ability and did not enhance the mutagenic effect of UV irradiation. We infer that R46 possesses: (i) a gene, uvp, which increases capacity for error-prone repair of UV-damaged DNA, and thus causes both UV protection and enhancement of UV mutagenesis; (ii) gene(s) whose action in the absence of gene uvp greatly increases the frequency of spontaneous reversion of hisG46. A plasmid of another incompatibility group, pLS51, has UV-protecting and mutagenesis-enhancing effect but lacks the mutator property; introduction of pLS51 into a clone of hisG46 carrying a pKM115-type plasmid greatly reduced its spontaneous reversion rate, as expected if pLS51 also has a uvp gene able to modulate the mutator effect of R46-derived gene(s) in the pKM115-type plasmid.     

Polarity Effects in the Hisg Gene of Salmonella Require a Site within the Coding Sequence

A single site in the middle of the coding sequence of the hisG gene of Salmonella is required for most of the polar effect of mutations in this gene. Nonsense and insertion mutations mapping upstream of this point in the hisG gene all have strong polar effects on expression of downstream genes in the operon; mutations mapping promotor distal to this site have little or no polar effect. Two previously known hisG mutations, mapping in the region of the polarity site, abolish the polarity effect of insertion mutations mapping upstream of this region. New polarity site mutations have been selected which have lost the polar effect of upstream nonsense mutations. All mutations abolishing the function of the site are small deletions; three are identical, 28-bp deletions which have arisen independently. A fourth mutation is a deletion of 16 base pairs internal to the larger deletion. Several point mutations within this 16-bp region have no effect on the function of the polarity site. We believe that a small number of polarity sites of this type are responsible for polarity in all genes. The site in the hisG gene is more easily detected than most because it appears to be the only such site in the hisG gene and because it maps in the center of the coding sequence.     

Genetic mapping and characteristics of the actinophage phi C31 deletion mutants of Streptomyces coelicolor A3(2) incapable of lysogenization

Actinophage phi C31 deletion c mutants with impaired ability to make repressor were genetically studied. Genetic crosses indicate that the c28 deletion mutant is situated with the c-region of the phi C31 genetic map. Based on the results of a qualitive test for recombination between several c mutants, a scheme of their order relative to deletion mutants was presented. The approximate distances between eight c mutants have been represented in units of the physical DNA map estimation. Genetic studies of actinophage lyg deletion mutants which cannot lysogenize sensitive cultures were carried out. Mutants failed to lysogenize upon mixed infection with lyg+ phages. The absence of the effect of lyg+ gene in trans suggests that lyg deletions cause a structural defect in an integration site of the phage. Preliminary data on alignment of lyg positions on physical and genetic maps of phi C31 phage have been obtained. According to evidence from genetic crosses, lyg mutation has been located in the right half of the phi C31 genome.     

Genetic characterization of the glutamate dehydrogenase gene (gdhA) of Salmonella typhimurium.

Salmonella typhimurium mutants, either devoid or glutamate dehydrogenase activity or having a thermolabile glutamate dehydrogenase protein, were used to identify the structural gene (gdhA) for this enzyme. Transductions showed that the mutations producing these phenotypes were linked to both the pncA and nit genes, placing the gdhA locus between 23 and 30 U on the S. typhimurium chromosome. Additional transductions with several Tn10 insertions established the gene order as pncA-gdhA-nit. Since few genetic markers exist in this region of the chromosome, Hfr strains were constructed to orient the pncA-gdhA-nit cluster with outside genes. Conjugation experiments provided evidence for the gene order pyrD-pncA-gdhA-nit-trp. To further characterize gdhA, we used Mu cts d1 (Apr lac) insertions in this gene to select numerous strains containing deletions with various endpoints. Transductions of these deletions with strains containing different gdh mutations and with a mutant having a thermolabile glutamate dehydrogenase protein permitted us to construct a deletion map of the gdhA region.     

Physical mapping and complete nucleotide sequence of the denV gene of bacteriophage T4.

Phage T4 deletion mutants that are folate analog resistant (far) and contain deletions in the region of the T4 genome near denV have been isolated previously. We showed that one of these mutants (T4farP12) expressed normal denV gene activity, whereas another mutant (T4farP13) was defective in the denV gene. The rII-distal (right) physical endpoints of these deletions defined the limits of the interval in which the rII-proximal (left) endpoint of the denV gene should be located. The deletion endpoints were identified by restriction and Southern hybridization analyses of phage derivatives containing deoxycytidine instead of hydroxymethyldeoxycytidine in their DNAs. The results of these analyses localized the rII-proximal (left) end of the denV gene to a region between 62.4 and 64.3 kilobases on the T4 physical map. denV+ phage resulted from marker rescue with two of five denV- alleles tested, using plasmids containing a 1.8-kilobase fragment from this region or a 179-base-pair terminal fragment derived from it. Sequencing of the 179-base-pair fragment from wild-type DNA showed a 130-base-pair open reading frame with its termination codon at the rII-proximal end. Confirmation that this open reading frame is part of the denV coding sequence was obtained by identifying a TAG amber codon in the homologous DNA derived from a denV amber mutant strain. This mutant strain rescued the denV+ allele from plasmids containing the wild-type sequence. An adjacent overlapping restriction fragment was also cloned, permitting determination of the remaining denV gene sequence. Based on these results, the 3' end of the coding region of the denV locus was mapped to kilobase position 64.07 on the T4 physical map, and the 5' end was mapped to position 64.48.     

trans-Recessive mutation in the first structural gene of the histidine operon that results in constitutive expression of the operon.

The first enzyme for histidine biosynthesis, encoded in the hisG gene, is involved in regulation of expression of the histidine operon in Salmonella typhimurium. The studies reported here concern the question of how expression of the histidine operon is affected by a mutation in the hisG gene that alters the allosteric site of the first enzyme for histidine biosynthesis, rendering the enzyme completely resistant to inhibition by histidine. The intracellular concentrations of the enzymes encoded in the histidine operon in a strain carrying such a mutation on an episome and missing the chromosomal hisG gene are three- to fourfold higher than in a strain carrying a wild-type hisG gene on the episome. The histidine operon on such a strain fails to derepress in response to histidine limitation and fails to repress in response to excess histidine. Furthermore, utilizing other merodiploid strains, we demonstrate that the wild-type hisG gene is trans dominant to the mutant allele with respect to this regulatory phenomenon. Examination of the regulation of the histidine operon in strains carrying the feedback-resistant mutation in an episome and hisT and hisW mutations in the chromosome showed that the hisG regulatory mutation is epistatic to the hisT and hisW mutations. These data provide additional evidence that the first enzyme for histidine biosynthesis is involved in autogenous regulation of expression of the histidine operon.     

Characterization of a yeast mitochondrial locus necessary for tRNA biosynthesis. Deletion mapping and restriction mapping studies

Yeast mitochondrial DNA codes for a complete set of tRNAs. Although most components necessary for the biosynthesis of mitochondrial tRNA are coded by nuclear genes, there is one genetic locus on mitochondrial DNA necessary for the synthesis of mitochondrial tRNAs other than the mitochondrial tRNA genes themselves. Characterization of mutants by deletion mapping and restriction enzyme mapping studies has provided a precise location of this yeast mitochondrial tRNA synthesis locus. Deletion mutants retaining various segments of mitochondrial DNA were examined for their ability to synthesize tRNAs from the genes they retain. A subset of these strains was also tested for the ability to provide the tRNA synthesis function in complementation tests with deletion mutants unable to synthesize mature mitochondrial tRNAs. By correlating the tRNA synthetic ability with the presence or absence of certain wild-type restriction fragments, we have confined the locus to within 780 base pairs of DNA located between the tRNAMetf gene and tRNAPro gene, at 29 units on the wild-type map. Heretofore, no genetic function or gene product had been localized in this area of the yeast mitochondrial genome.     

Deletion map of the Escherichia coli structural gene for alkaline phosphatase, phoA.

Lambda transducing phages containing portions of the phoA gene have been isolated and used to construct a deletion map of the phoA gene. The isolation of a plaque-forming lambda transducing phage carrying the entire phoA gene is also described. Two new methods for screening or selection of mutants that have altered levels of alkaline phosphatase activity are reported.     

Cluster of genes controlling proline degradation in Salmonella typhimurium.

A cluster of genes essential for degradation of proline to glutamate (put) is located between the pyrC and pyrD loci at min 22 of the Salmonella chromosome. A series of 25 deletion mutants of this region have been isolated and used to construct a fine-structure map of the put genes. The map includes mutations affecting the proline degradative activities, proline oxidase and pyrroline-5-carboxylic dehydrogenase. Also included are mutations affecting the major proline permease and a regulatory mutation that affects both enzyme and permease production. The two enzymatic activities appear to be encoded by a single gene (putA). The regulatory mutation maps between the putA gene and the proline permease gene (putP).