Separate regulation of purA and purB loci of Escherichia coli K-12.

We isolated a strain of Escherichia coli K-12 in which the lac structural genes are fused to the purB control region and used this strain to study the regulation of the purA and purB loci. The purA locus was derepressed in response to either limiting adenine or guanine growth conditions in the presence of excess guanine or adenine, respectively. The presence of hypoxanthine in the culture medium did not have any effect on the expression of the purA locus. The purB locus responded to limiting adenine growth conditions in the presence of either excess hypoxanthine or guanine alone but not when both hypoxanthine and guanine were present.     

Mapping of the nadB Locus Adjacent to a Previously Undescribed Purine Locus in Escherichia coli K-12

It is proposed that all mutants blocked in the de novo pathway of nicotinamide adenine dinucleotide biosynthesis be designated nad rather than nic. It is further suggested that mutants blocked in the pyridine nucleotide cycle be designated pnc. The nadB locus and a previously unidentified pur locus are cotransducible. These two loci have been mapped near minute 49 on the standard genetic map of Escherichia coli. The order of genes in that region is purC-guaB-purG-glyA-pur-nadB-tyrA-pheA.     

Isolation and characterization of a Saccharomyces cerevisiae mutant deficient in pyruvate kinase activity.

A mutant of the yeast Saccharomyces cerevisiae that is deficient in pyruvate kinase activity has been isolated. The mutant strain is capable of growth when supplied with lactate as the carbon source but not capable of growth when supplied with dextrose or other fermentable sugars or glycerol as the carbon source. Genetic analysis demonstrated that the phenotype of the pyruvate kinase-deficient strain was due to a single nuclear mutation, which was designated pyk1, and preliminary genetic mapping experiments located the pyk1 locus on chromosome I, 30 centimorgans from the ade1 locus. Adenine nucleotide levels in the mutant and parental strains were compared when the cells were subjected to various growth and starvation conditions. When carbon supply and energy production were dissociated by supplying the mutant strain with dextrose, adenine nucleotide levels fell dramatically. This result suggests that the initial reactions of glycolysis are not rate limiting, nor are they readily inhibited by feedback controls.     

Analysis of an esterase linked to a locus involved in the regulation of the melanoma oncogene and isolation of polymorphic marker sequences inXiphophorus

Melanoma formation in Xiphophorus hybrids is mediated by a growth factor receptor tyrosine kinase oncogene encoded by the Tu locus. In the wild-type parental fish no tumors occur due to the activity of a locus that regulates the activity of the melanoma oncogene. Molecular identification of this regulatory locus (R) requires a precise physical map of the chromosomal region. Therefore we studied esterase isozymes in Xiphophorus, two of which have been previously reported to be linked to locus R. We confirm that ES1 is a distant marker for R (approx. 30cM), and contrary to earlier studies, we show that this isozyme is present in all species of the genus and at similar activity levels in all organs tested. ES4, which has also been reported to be linked to R, was found to be a misclassification of liver ES1. In an attempt to identify markers that bridge the large distance between ES1 and R, we have generated DNA probes which are highly polymorphic. They will be useful in finding landmarks on a physical map of the R-containing chromosomal region.     

Properties of Escherichia coli mutants altered in calcium/proton antiport activity.

Mutants sensitive to growth inhibition by CaCl2 were found to have alterations in calcium uptake in everted membrane vesicles. These mutations map at different loci on the Escherichia coli chromosomes. A mutation at the calA locus results in vesicles which have two- to threefold higher levels of uptake activity than vesicles from wild-type cells. The calA mutation is phenotypically expressed as increased sensitivity to CaCl2 in a strain also harboring a mutation in the corA locus, which is involved in Mg2+ transport. The calA locus maps very close to purA and cycA at about min 97. The calB mutation results both in sensitivity to CaCl2 at pH 5.6 and in vesicles with diminished calcium transport capability. The CalB phenotype is also expressed only in a corA genetic background; the calB locus appears to map very near, yet separately from, the calA locus. When the cor+ allele is present, calA and calB mutations still result in a defect in calcium transport in vesicles. In addition, both calC and calD mutations result in vesicles with impaired calcium transport activity. calC is cotransducible with kdp and nagA, whereas calD is cotransducible with proC.     

Biochemical and regulatory properties of Escherichia coli K-12 hisT mutants.

Escherichia coli K-12 hisT mutants were isolated, and their properties were studied. These mutants are derepressed for the histidine operon, map close to the purF locus at about 49.5 min on the E. coli linkage map, and lack pseudouridylate synthetase activity. The defect in this enzyme leads to the absence of pseudouridines in the anticodon loop of several transfer ribonucleic acid species, as evidenced by the altered elution profile on reversed-phase chromatography and resistance to amino acid analogues. Finally, the hisT mutants studied have a reduced growth rate that appears to be linked to hisT, although it is not known whether it is due to the same mutation. The normal generation time can be restored by supplementing the medium with adenine, uracil, and isoleucine.     

Mutants of Escherichia coli with a growth requirement for either lysine or pyridoxine

Two distinct phenotypic classes of lysine requiring auxotrophs of Escherichia coli are described. Mutants of the LysA class produce little or no active diaminopimelic acid (DAP) decarboxylase and specifically require lysine for growth. Mutants of the LysB class produce a cryptic DAP decarboxylase which can be activated both in vivo and in vitro by higher than normal levels of its cofactor, pyridoxal 5'-phosphate. The LysB mutants have an alternate requirement for lysine or pyridoxine. Both LysA and LysB mutations map at 55 min, close to the thyA locus of E. coli. The association between pyridoxal phosphate and DAP decarboxylase appears to be much weaker in LysB mutants than in wild-type bacteria, and the mutant enzyme also sediments more slowly than wild-type enzyme in sucrose density gradients. The results suggest that the LysB mutations alter a specific region (or subunit) of the enzyme molecule which is needed to stabilize the binding of pyridoxal phosphate. These studies help to resolve certain contradictory observations on DAP decarboxylase reported earlier and may have relevance to pyridoxal phosphate enzymes in general. Prototrophic revertants of LysB mutants arise by second site mutations that result in increased availability of intracellular pyridoxal phosphate. These revertants appear to be derepressed for pyridoxine biosynthesis.     

Purine metabolism in Neisseria gonorrhoeae: the requirement for hypoxanthine

Strains isolated from disseminated gonococcal infections often require hypoxanthine for growth. The biochemical bases for the requirement for hypoxanthine in strains isolated from both disseminated (Ile-Val-Arg-Hyx-Ura-phenotype) and non-disseminated (Hyx-phenotype) infections were compared. The requirement for hypoxanthine was dependent upon the composition of the growth medium. In a complete defined medium, hypoxanthine was replaced by a mixture of adenine and guamine but not by either purine alone. The addition of adenine along inhibited gonococcal growth. This inhibition was reversed by the addition of guanine and most likely resulted from an inhibition of de novo purine biosynthesis. In a histidine-free medium, adenine replaced the hypoxanthine requirement in Ile-Val-Arg-Hyx-Ura-strains. Adenine did not replace the hypoxanthine requirement in Hyx- strains. The Ile-Val-Arg-Hyx-Ura- strains exhibited a markedly reduced rate of the novo purine biosynthesis while Hyx- strains were blocked in this pathway. In vivo concentrations of purines are important factors which may limit the intracellular or extracellular growth of these strains.     

The genetics of polyamine synthesis in Neurospora crassa

New mutations of the polyamine pathway of Neurospora crassa fell into three categories. The majority affected ornithine decarboxylase and lay at the previously defined spe-1 locus. One mutation, JP100, defining the new spe-2 locus, eliminated S-adenosyl-methionine decarboxylase and led to putrescine accumulation. Revertants of this mutation suggested that the locus encodes the enzyme. Two other mutations, LV105 and JP120, defined a third locus, spe-3. Strains with these mutations also accumulated putrescine and were presumed to lack spermidine synthase activity, which catalyzes the formation of spermidine from putrescine and decarboxylated S-adenosylmethionine. The three spe loci lay within about 20 map units of one another on the right arm of Linkage Group V in the order: centromere-spe-2-spe-1-spe-3. The requirement for spermidine for growth was much less in spe-2 and spe-3 mutants than in spe-1 mutants, which do not accumulate putrescine. This suggested that putrescine fulfills many, but not all, of the functions of spermidine, or that high levels of putrescine render spermidine more effective in its essential roles.     

Molecular genetic analysis of Saccharomyces cerevisiae C1-tetrahydrofolate synthase mutants reveals a noncatalytic function of the ADE3 gene product and an additional folate-dependent enzyme.

In eucaryotes, 10-formyltetrahydrofolate (formyl-THF) synthetase, 5,10-methenyl-THF cyclohydrolase, and NADP(+)-dependent 5,10-methylene-THF dehydrogenase activities are present on a single polypeptide termed C1-THF synthase. This trifunctional enzyme, encoded by the ADE3 gene in the yeast Saccharomyces cerevisiae, is thought to be responsible for the synthesis of the one-carbon donor 10-formyl-THF for de novo purine synthesis. Deletion of the ADE3 gene causes adenine auxotrophy, presumably as a result of the lack of cytoplasmic 10-formyl-THF. In this report, defined point mutations that affected one or more of the catalytic activities of yeast C1-THF synthase were generated in vitro and transferred to the chromosomal ADE3 locus by gene replacement. In contrast to ADE3 deletions, point mutations that inactivated all three activities of C1-THF synthase did not result in an adenine requirement. Heterologous expression of the Clostridium acidiurici gene encoding a monofunctional 10-formyl-THF synthetase in an ade3 deletion strain did not restore growth in the absence of adenine, even though the monofunctional synthetase was catalytically competent in vivo. These results indicate that adequate cytoplasmic 10-formyl-THF can be produced by an enzyme(s) other than C1-THF synthase, but efficient utilization of that 10-formyl-THF for purine synthesis requires a nonenzymatic function of C1-THF synthase. A monofunctional 5,10-methylene-THF dehydrogenase, dependent on NAD+ for catalysis, has been identified and purified from yeast cells (C. K. Barlowe and D. R. Appling, Biochemistry 29:7089-7094, 1990). We propose that the characteristics of strains expressing full-length but catalytically inactive C1-THF synthase could result from the formation of a purine-synthesizing multienzyme complex involving the structurally unchanged C1-THF synthase and that production of the necessary one-carbon units in these strains is accomplished by an NAD+ -dependent 5,10-methylene-THF dehydrogenase.     

TolF locus in Escherichia coli: chromosomal location and relationship to loci cmlB and tolD.

The tentative map position on the Escherichia coli chromosome of the tolF locus, determining tolerance to colicins A, E2, E3, K, and L, has been confirmed by three-point transduction. It lies between the aroA and pyrD loci at about 21 min on the linkage map of Bachmann et al. (1976). The cmlB locus, determining increased resistance to the antibiotics chloramphenicol and tetracycline, also lies in this region (Reeve, 1966). Phenotypic and genetic comparison of isogenic strains that carry a mutation in either the tolF or cmlB locus makes it likely that these loci are closely related or identical. The tolD locus determining tolerance to colicins E2 and E3 as well as increased resistance to antibiotics has been reported to be located close to the aroA locus as a result of conjugation experiments (Eriksson-Grennberg et al. 1965). However, tolD did not cotransduce with any of several loci in this region, indicating that the mutation is not located within the region of the genetic map corresponding to approximately 19 to 22.5 min.     

Genetic analysis of the components of winterhardiness in barley (Hordeum vulgare L.)

Winterhardiness in cereals is the consequence of a number of complex and interacting component characters: cold tolerance, vernalization requirement, and photoperiod sensitivity. An understanding of the genetic basis of these component traits should allow for more-effective selection. Genome map-based analyses hold considerable promise for dissecting complex phenotypes. A 74-point linkage map was developed from 100 doubled haploid lines derived from a winter x spring barley cross and used as the basis for quantitative trait locus (QTL) analyses to determine the chromosome location of genes controlling components of winterhardiness. Despite the greater genome coverage provided by the current map, a previously-reported interval on chromosome 7 remains the only region where significant QTL effects for winter survival were detected in this population. QTLs for growth habit and heading date, under 16 h and 24 h light, map to the same region. A QTL for heading date under these photoperiod regimes also maps to chromosome 2. Contrasting alleles at these loci interact in an epistatic fashion. A distinct set of QTLs mapping to chromosomes 1, 2, 3, and 5 determined heading date under 8 h of light. Under field conditions, all QTLs identified under controlled environment conditions were determinants of heading date. Patterns of differential QTL expression, coupled with additive and additive x additive QTL effects, underscore the complexity of winterhardiness. The presence of unique phenotype combinations in the mapping population suggests that coincident QTLs for heading date and winter survival represent the effects of linkage rather than pleiotropy.     

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.     

Potentiation by purines of the growth-inhibitory effects of sulphonamides on Escherichia coli K12 and the location of the gene which mediates this effect

The increased toxicity of sulphonamides for Escherichia coli in the presence of low concentrations (50-100 microM) of purines or purine nucleosides has been confirmed and investigated further. The potentiating effect of a purine was dependent upon the activity of the appropriate phosphoribosyl transferase: a gpt mutant strain was not potentiated by guanine but remained fully sensitive to the addition of adenine. Mutants resistant to the potentiating effect of all purines have been isolated and partially characterized. The site of these mutations has been located in the region between oriC and asnA at minute 83 on the E. coli chromosome map. It is suggested that this locus be temporarily designated psp (potentiation of sulphonamides by purines) because these mutants have unaltered sensitivities to sulphonamides acting alone. Mutations in PurA, purR and folB did not affect the potentiation of sulphonamides by purines. Hypoxanthine-insensitive strains harbouring lambda asn20 were as sensitive as the wild-type to the potentiating effect. This result suggests that these lysogens are heterozygous for psp and that the wild-type allele is dominant. It is probable that psp is a regulatory gene, affecting some rate-limiting step in the biosynthesis of methionine.     

Mapping and characterization of the nad genes in Salmonella typhimurium LT-2.

An ampicillin enrichment technique was used to isolate 39 nicotinic acid-requiring mutants of Salmonella typhimurium LT-2. Using interrupted-mating and transductional mapping procedures, three loci, designated nadA, nadB, and nadC, were identified. These loci mapped at 33, 82, and 6 min, respectively, on the S. typhimurium linkage map. The arrangement of the loci on the Salmonella linkage map corresponded closely to the nadA, nadB, and nadC loci on the Escherichia coli K-12 linkage map, indicating that the de novo pathway to nicotinamide adenine dinucleotide and the genes governing the enzymes involved in this pathway in S. typhimurium are very similar to those in E. coli. Evidence is also presented which indicates that the product of the nadC locus in S. typhimurium LT-2 is the enzyme quinolinic acid phosphoribosyltransferase. All nadC mutants of S. typhimurium secreted between 2 and 8 mumol of quinolinic acid per 100 ml of secretion medium. In addition, none of the nadC mutants isolated were able to grow in 10(-3) M quinolinic acid, whereas all nadA and nadB mutants of S. typhimurium grew well in the presence of quinolinic acid. Transductional crosses between nadB mutants provided evidence suggestive of more than one locus in the nadB region.     

A long-range restriction map of the interleukin-4 and interleukin-5 linkage group on chromosome 5

The genes for two of the hematopoietic growth factors, interleukin-4 and interleukin-5, are located on a small segment of chromosome 5 (q23-31), which is frequently deleted in myeloid disorders. Using pulsed-field gel electrophoresis, we demonstrate physical linkage of these two genes and present a long-range restriction map of the locus. The two genes are closely linked (maximum separation, 310 kb) and appear to be separated by an HTF island. We were unable to physically link these genes to two other closely related hematopoietic growth factor genes, interleukin-3 and granulocyte/macrophage colony-stimulating factor, which also map to this region of the genome. The clustering of these and other growth-related genes suggests that a higher order of genetic organization exists in this region of the chromosome.     

Construction of a 1.2-Mb BAC/PAC contig of the porcine gene RYR1 region on SSC 6q1.2 and comparative analysis with HSA 19q13.13

We screened a porcine bacterial artificial chromosome (BAC) and a P1 derived artificial chromosome (PAC) library to construct a sequence-ready approximately 1.2-Mb BAC/PAC contig of the ryanodine receptor-1 gene (RYR1) region on porcine chromosome (SSC) 6q1.2. This genomic segment is of special interest because it harbors the locus for stress susceptibility in pigs and a putative quantitative trait locus for muscle growth. Detailed physical mapping of this gene-rich region allowed us to assign to this contig 17 porcine genes orthologous to known human chromosome 19 genes. Apart from the relatively well-characterized porcine gene RYR1, the other 16 genes represent novel chromosomal assignments and 14 genes have been cloned for the first time in pig. Comparative analysis of the porcine BAC/PAC contig with the human chromosome (HSA) 19q13.13 map revealed a completely conserved gene order of this segment between pig and human. A detailed porcine-human-mouse comparative map of this region was constructed.     

Mapping of the fabA Locus for Unsaturated Fatty Acid Biosynthesis in Escherichia coli

fabA mutants of Escherichia coli require an appropriate unsaturated fatty acid for growth. The fabA locus has now been mapped at minute 21.5 of the linkage map of E. coli. The locus is cotransduced with pyrD and aroA but not with pyrC, purB, or pdxC. The clockwise order of markers in the region is pdxC, aroA, cmlB, pyrD, fabA, pyrC.