Genetic characterization of the folding domains of the catalytic chains in aspartate transcarbamoylase

In Salmonella typhimurium strains which produce high constitutive levels of aspartate transcarbamoylase due to the pyrH700 mutation, the bulk of the carbamoyl phosphate of the cell is consumed for the biosynthesis of pyrimidines. As a consequence, there is little substrate available for arginine synthesis and the cell growth is impeded. Suppression of arginine auxotrophy by mutations which block aspartate transcarbamoylase activity provides a positive selection technique for mutant strains defective in this enzyme activity. A genetic analysis was performed on 29 mutant strains harboring defects in the structural gene pyrB, encoding the catalytic chains of aspartate transcarbamoylase of Escherichia coli. Extracts from 15 strains contained intact, inactive enzyme-like molecules of the same size as the purified wild type enzyme. These same extracts contained a predominant polypeptide chain which migrated electrophoretically at the same rate as catalytic chains from wild type enzyme. In addition to these 15 different missense mutants, 14 others (presumably chain-terminating mutants) were isolated; no polypeptides corresponding to full length catalytic chains were detected in these strains. Based on their reversion and suppression properties, seven were designated as frameshift and two as amber nonsense. A fine structure recombination map of the pyrB locus was constructed from a series of three-factor transductional crosses. Mutational sites were correlated with regions in the polypeptide sequence by relating their map positions to that of mutation pyrB231 which results in an amino acid replacement at position 128. Moreover, since recent crystallographic studies indicate that residue 128 is located near the junction between the NH2- and COOH-terminal folding domains, the mutational sites can be placed within either of these two regions of tertiary structure. Interallelic complementation experiments showed four units of complementation. Those defining the alpha and beta units were missense mutants with their mutational sites in the NH2- and COOH-terminal domains, respectively. The mutants determining the delta and gamma units involved premature polypeptide chain termination and their mutational sites were correlated with distal regions of the two respective domains. Several mutants of the chain-terminating type failed to complement members of more than one unit. Possible effects of the various mutations and their implications for mechanisms of complementation and enzyme activity are presented.     

Nonsense Motility Mutants in SALMONELLA TYPHIMURIUM

Of 313 motility-deficient mutants isolated from an LT2 his(amber) strain fixed in phase 1 by gene vh2(-), 25 regained motility when amber or ochre suppressors were introduced, in F' factors or by transduction. The fla mutants (23 amber, 1 ochre) fell in complementation groups A, B, C, F, K, a new group, M, and at least one further new group; the hypothesis of a fla gene which specifies only an RNA structural component of a flagellum-synthesizing basal apparatus is disproven for the corresponding genes. Hfr and transductional crosses confirmed gene assignments from complementation and indicated that flaM and another new fla locus map near H1. A small minority of motile bacteria were detectable in many of the amber fla mutants. In groups A and F some pairs of amber fla mutants complemented each other, and perhaps each of these groups corresponds to more than one structural gene. The suppressed derivatives of a mutant with an amber mutation in H1 made flagella morphologically and serologically indistinguishable from wild-type flagella. A slow-spreading but flagellate mutant showed mainly non-translational motility in broth, and in a viscous medium the bacteria reversed very frequently; its amber mutation, probably near H1, is inferred to cause a defect in chemotaxis, so that the bacteria give the avoidance reaction continuously.     

Regulator mutants for the synthesis of a 2d purine nucleoside phosphorylase in Escherichia coli K-12. II. Mapping and study of the dominance of pndR mutations

The synthesis of a second purine nucleoside phosphorylase (PNPII) in the wild type strains of Escherichia coli K-12 is induced by xanthosine. Three types of pndR mutants were studied, which are altered in regulation of PNPII synthesis: 1) constitutive, 2) inducible by nucleosides of hypoxantine and adenine as much as by xanthosine and 3) defective in synthesis of PNPII. All pndR mutations are located in transductional crosses on 51 min of E. coli genetic map. The order of genes established is as follows: pndR-ptsH-cysA. Mutations of the first and second type are dominant, while pndR21 mutation of the third type is recessive to the pndR+ allele on F' episome. The data obtained support the suggestion that the product of pndR regulatory gene is an activator protein necessary for the expression of the PNPII structural gene.     

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.     

A Deletion Map of cyc1 Mutants and Its Correspondence to Mutationally Altered Iso-1-Cytochromes c of Yeast

Mutants arising spontaneously from sporulated cultures of certain strains of yeast, Saccharomyces cerevisiae, contained deletions of the CYC1 gene which controls the primary structure of iso-1-cytochrome c. At least 60 different kinds of deletions were uncovered among the 104 deletions examined and these ranged in length from those encompassing only two adjacent point mutants to those encompassing at least the entire CYC1 gene. X-ray-induced recombination rates of crosses involving these deletions and cyc1 point mutants resulted in the assignment of 211 point mutants to 47 mutational sites and made it possible to unambiguously order 40 of these 47 sites. Except for one mutant, cyc1-15, there was a strict colinear relationship between the deletion map and the positions of 13 sites that were previously determined by amino acid alterations in iso-1-cytochromes c from intragenic revertants.     

The gua operon of Escherichia coli K-12: evidence for polarity from guaB to guaA

Guanine auxotrophs of Escherichia coli K-12 were isolated after mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine, ethyl methane sulfonate, or the acridine mustard ICR 372. guaA (xanthosine 5'-monophosphate [XMP] aminase-less) mutants were distinguished from guaB (inosine 5'-monophosphate [IMP] dehydrogenase-less) mutants by their growth response to xanthine and by enzyme assay. Mutations were classified as base substitutions or frameshift on the basis of mutagen-induced reversion patterns. All guaA strains, including three frameshift mutants, produced derepressed levels of IMP dehydrogenase when cultured with a growth-limiting concentration of guanine. The guaB strains were of two types: (i) those producing derepressed levels of XMP aminase, and (ii) those producing basal levels of XMP aminase when grown under conditions of guanine starvation. In the guaB strains of the second type, the expression of the adjacent guaA gene is reduced. It is proposed that this pleiotropic effect of some guaB mutations is a result of polarity. The orientation of polarity suggests the gene order "operator"-guaB-guaA. Gel diffusion studies with IMP dehydrogenase antiserum showed that strains carrying polar guaB mutations do not produce cross-reacting material (CRM). The remaining guaB mutants were either CRM(+) or CRM(-). Mapping the mutations by three-factor crosses showed that polar and nonpolar guaB sites are clustered in a small genetic region cotransducible with guaA. The relative positions of the guaB mutational sites established that the polar mutations lie within the structural gene for IMP dehydrogenase.     

Point Mutants in the D2a Region of Bacteriophage T4 Fail to Induce T4 Endonuclease IV

We have studied the properties of presumptive point mutants in the D2a region of bacteriophage T4. Dominance tests showed that the D2a mutation was recessive to the wild-type allele. The mutations were shown to map in the D2a region by complementation against rII deletions. The D2a mutations were also located between gene 52 and rIIB by two- and three-factor crosses. The mutants are located at at least two distinct loci in the D2a region. The point mutants grow normally on all hosts tested and none of the mutants makes T4 endonuclease IV. We propose the name "denB" for the D2a locus.     

Transfer of the delta (argF-lac)U169 mutation between Escherichia coli strains by selection for a closely linked Tn10 insertion

To facilitate construction of mutants harboring delta lac for use in gene fusion studies, strains were constructed that carry the transposon Tn10 next to the well defined lac deletion U169. This deletion can now be moved to other Escherichia coli strains in transductional or conjugational crosses by selecting resistance to tetracycline.     

Regions of Salmonella typhimurium flagellin essential for its polymerization and excretion.

Immunological methods were used to examine the flagellin production of Salmonella typhimurium strains that carried a mutation in one of the two possible genes for flagellin (H1 or H2) and also were incapable of expressing the other gene. Some mutants produced flagellin that was excreted into the culture medium; others accumulated flagellin intracellularly. These two phenotypes were detected in both H1 and H2 mutants. The mutation sites were mapped on the corresponding deletion map (consisting of 21 segments in the case of H1 and 31 segments in the case of H2). H1 and H2 mutations causing excretion of flagellin were clustered mainly in segment 12 and segment 6 from the proximal end, respectively, suggesting that the corresponding segments of the flagellins play a role in polymerization. Mutations causing accumulation in the cytoplasm were clustered in segments 19 to 21 of the H1 map and in segments 25 to 29 of the H2 map, suggesting that an essential region for flagellin transport exists toward the C terminus of flagellin.     

On the role of the single-stranded DNA binding protein of bacteriophage T4 in DNA metabolism. I. Isolation and genetic characterization of new mutations in gene 32 of bacteriophage T4

Summary The product of gene 32 of bacteriophage T4 is a single-stranded DNA binding protein involved in T4 DNA replication, recombination and repair. Functionally differentiated regions of the gene 32 protein have been described by protein chemistry. As a preliminary step in a genetic dissection of these functional domains, we have isolated a large number of missense mutants of gene 32. Mutant isolation was facilitated by directed mutagenesis and a mutant bacterial host which is unusually restrictive for missense mutations in gene 32. We have isolated over 100 mutants and identified 22 mutational sites. A physical map of these sites has been constructed and has shown that mutations are clustered within gene 32. The possible functional significance of this clustering is considered.     

Regulation of fatty acid degradation in Escherichia coli: isolation and characterization of strains bearing insertion and temperature-sensitive mutations in gene fadR.

Transposon Tn10 was used to mutagenize the fadR gene in Escherichia coli. Mutants bearing fadR:Tn10 insertion mutations were found to (i) utilize the noninducing fatty acid decanoate as sole carbon source, (ii) beta-oxidize fatty acids at constitutive rates, and (iii) contain constitutive levels of the five key beta-oxidative enzymes. These characteristics were identical to those observed in spontaneous fadR mutants. The constitutive phenotype presented by the fadR:Tn10 mutants was shown to be genetically linked to the associated transposon-encoded drug resistance. These results suggest that the fadR gene product exerts negative control over the fatty acid degradative regulon. The fadR gene of E. coli has been mapped through the use of transposon-mediated fadR insertion mutations. The fadR locus is at 25.5 min on the revised map and cotransduces with purB, hemA, and trp. Three-factor conjugational and transductional crosses indicate that the order of loci in this region of the chromosome is purB-fadR-hemA-trp. Spontaneous fadR mutants were found to map at the same location. Strains that exhibit alterations in the control of the fad regulon in response to changes in temperature were also isolated and characterized. These fadR(Ts) mutants were constitutive for the fad enzymes at elevated temperatures and inducible for these activities at low temperatures. The fadR(Ts) mutations also map at the fadR locus. These results strongly suggest that the fadR gene product is a repressor protein.     

Genetic analysis of Escherichia coli K-12 mutants defective for the structural and regulatory genes for second purine nucleoside phosphorylase

Two types of mutants lacking the second purine nucleoside phosphorylase (PNPase 2) activity were isolated using the Escherichia coli K-12 pndR strains with constitutive or inosine-inducible synthesis of the PNPase 2. The mutations of the first type are recessive to the pndR+ allele on the F' episome. They are closely linked to the original pndR+ mutations and therefore affect the pndR gene encoding the activator protein. The mutations of the second type affect the PNPase 2 structural gene (pndA) and are recessive to the pndA+ allele on the F' episome. The nupC-pndR-pndA-ptsH-cysA gene order was established by means of four- and five-factorial transductional crosses.     

An osmosensing histidine kinase mediates dicarboximide fungicide resistance in Botryotinia fuckeliana (Botrytis cinerea)

A two-component histidine protein kinase gene, homologous to os-1 from Neurospora crassa, was cloned and sequenced from a single ascospore isolate of Botryotinia fuckeliana. A series of nine spontaneous mutants resistant to dicarboximide fungicides was selected from this strain and characterized with respect to fungicide resistance and osmotic sensitivity. Genetic crosses of the mutants with an authentic Daf1 strain showed that the phenotypes mapped to this locus. Single point mutations (seven transitions, one transversion, and one short deletion) were detected in the alleles of the nine mutants sequenced. The mutational changes were shown to cosegregate with the dicarboximide resistance and osmotic sensitivity phenotypes in progeny obtained from crossing selected resistant strains with a sensitive strain. All mutations detected are predicted to result in amino acid changes in the coiled-coil region of the putative Daf1 histidine kinase, and it is proposed that dicarboximide fungicides target this domain.     

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.     

Genetic analysis of the Escherichia coli K-12 srl region.

Specialized transducing lambda derivatives, deletion mapping, and Plkc transductional crosses have been used to analyze the genetic organization and regulation of the srl genes. Transducing phages obtained from a secondary site lambda insertion in srlA are of two types: lambdapsrlC1 and lambdaprecA are substituted in the b2 region of the lambda chromosome (galtype) and carry the srlC gene but not srlD; lambdapsrlD is substituted in the early region of the phage deoxyribonucleic acid (biotype) and carries the srlD gene but not srlC. The lambdapsrlC1 phage, which lysogenizes at attlambda, complements srlC mutants in trans, indicating that this gene codes for a diffusable positive regulatory element. The srl genes have been ordered relative to the cysC, recA, and alaS genes by two- and three-factor P1kc crosses. The order, cysC...srlD-srlA-srlC-recA-alaS, has been obtained. The srlA and srlD genes comprise an operon with srlD operator distal. From the secondary site lysogen, it has been possible to obtain deletion mutants of this region that are sensitive to ultraviolet light and are recombination deficient. Genetic evidence suggests that these deletions extend from srl into the recA gene.     

Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QH2-cytochrome c reductase.

1. Three methods are described for the genetic analysis of yeast cytoplasmic mutants (mit- mutants) lacking cytochrome oxidase or coenzyme QH2-cytochrome c reductase. The procedures permit mutations in mitochondrial DNA to be mapped relative to each other and with respect to drug-resistant markers. The first method is based upon the finding that crosses of mit- mutants with some but not other isonuclear q- mutants lead to the restoration of respiratory functions. Thus a segment of mitochondrial DNA corresponding to a given mit- mutation or to a set of mutations can be delineated. The second method is based on the appearance of wild-type progeny in mit- X mit- crosses. The third one is based on the analysis of various recombinant classes issued from crosses between mit-, drug-sensitive and mit+, drug-resistant mutants. Representative genetic markers of the RIBI, OLII, OLI2 and PAR1 loci were used for this purpose. 2. The three methods when applied to the study of 48 mit- mutants gave coherent results. At least three distinct regions on mitochondrial DNA in which mutations cause loss of functional cytochrome oxidase have been established. A fourth region represented by closely clustered mutants lacking coenzyme QH2-cytochrome c reductase and spectrally detectable cytochrome b has also been studied. 3. The three genetic regions of cytochrome oxidase and the cytochrome b region were localized by the third method on the circular map, in spans of mitochondrial DNA defined by the drug-resistant markers. The results obtained by this method were confirmed by analysis of the crosses between selected mit- mutants and a large number of q- clones whose retained segments of mitochondrial DNA contained various combinations of drug-resistant markers. 4. All the genetic data indicate that the various regions studied are dispersed on the mitochondrial genome and in some instances regions or clusters of closely linked mutations involved in the same respiratory function (cytochrome oxidase) are separated by other regions which code for entirely different functions such as ribosomal RNA.     

Genetic fine structure of the glucosyltransferase gene of bacteriophage T2

14 mutants of T2, which carried mutations in the gene coding for glucosyltransferase, were isolated. Although ambers were not selected for, six mutants appeared to be of the amber type. These mutants, as well as another twelve, 5 missense and 7 amber, were located and a genetic map was constructed. The amber and non-amber mutations were not equally distributed over the gt gene. The part transcribed first carried mainly amber mutations; the tail part contained only non-amber mutations. A possible relation between the non-random location of the two kinds of mutation and functional differences within the enzyme is discussed. No intragenic complementation could be demonstrated. The recombination frequencies of amgt mutants are reduced to about two-thirds if crosses are performed under conditions where the DNA of the mutants remains unglucosylated.     

Sequence analysis of lacZ- mutations induced by ion beam irradiation in double-stranded M13mp18DNA

While M13mp18 double-stranded DNA was irradiated with ion beam, and transfected intoE. coli JM103, a decrease of transfecting activity was discovered. The lacZ^- mutation frequency at 20% survival could reach (3.6–16.8) × 10⁴, about 2, 3–10 times that of unirradiated M13DNA. Altogether, 27 IacZ^-mutants were selected, 10 of which were used for sequencing. 7 of the sequenced mutants show base changes in 250-bp region examined (the remaining 3 mutants probably have base changes outside the regions sequenced). 5 of the base-changed mutants contain more than one mutational base sites (some of them even have 5–6 mutational base sites in 250-bp region examined); this dense distribution of base changes in polysites has seldom been seen in X-rays, Y-rays or UV induced DNA mutations. Our experiments also showed that the types of base changes include transitions(50%), transversions (45%) and deletion (5%); no addition or duplication was observed. The transitions were mainly C→T and A→G; the transversions were mainly C→A and C→G. The mutations involving cytosine residue (in the template strand) constitute about 60% of all the base changes observed. In comparison with the surrounding sequences of mutational base sites, the base located between TG and CT is found to be easily substituted.     

Distribution and characterization of mutations induced by nitrous acid or hydroxylamine in the intron-containing thymidylate synthase gene of bacteriophage T4

The detailed distribution and characterization of 51 hydroxylamine (HA)-induced and 59 nitrous acid (NA)-induced mutations in the intron-containing bacteriophage T4 thymidylate synthase (td) gene is reported here. Mutations were mapped in 10 regions of thetd gene by recombinational marker rescue using plasmid or M13 subclones of thetd gene. Phage crosses using deletion mutants with known breakpoints in the 3′ end of thetd intron subdivided HA and NA mutations which mapped in this region. At least 31 of the mutations map within the 1-kb group I self-splicing intron. Intron mutations mapped only in the 5′ and 3′ ends of the intron sequence, in accordance with the hypothesis that the 5′ and 3′ domains of the T4td intron are essential for correct RNA splicing. RNA sequence analysis of a number of mappedtd mutations has identified two intron nucleotides and one exon nucleotide where both HA- and NA-induced mutations commonly occur. These three loci are characterized by a GC dinucleotide, with the mutations occurring at the cytosine residue. Thus, these data indicate at least three potential sites of both HA- and NA-induced mutagenic hotspot activity within thetd gene.