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PubMed1989

X-ray-induced specific-locus mutations in the ad-3 region of two-component heterokaryons of Neurospora crassa. IV. Irreparable mutants of genotype ad-3A and ad-3B result from multilocus deletion and an unexpectedly high frequency of multiple-locus mutations.

The induction of specific-locus mutations in the ad-3 region of Neurospora crassa after X-irradiation was studied in a two-component heterokaryon to determine: (1) the ratio of reparable ad-3 mutants (presumed gene/point mutations, designated ad-3R) to irreparable ad-3 mutants (presumed multilocus deletions, designated ad-3IR), and (2) the induction kinetics of each class (Webber and de Serres, 1965). More extensive genetic tests made subsequently (de Serres, 1989a) on the 832 X-ray-induced specific-locus mutations recovered in those experiments showed that unexpected high frequencies of reparable and irreparable ad-3 mutants are actually multiple-locus mutants that have additional, but separate, sites of recessive lethal (RLCL) damage in the immediately adjacent genetic regions (designated ad-3R + RLCL or ad-3IR + RLCL). The frequencies of these X-ray-induced multiple-locus mutants in the ad-3 region are orders of magnitude higher than expected on the basis of target theory (where the frequency of the double mutant is expected to be the product of the frequencies of each single mutant) and classical models of chromosome structure during interphase (de Serres, 1989a). In the present paper, a random sample of 832 X-ray-induced ad-3 mutants of genotype ad-3A or ad-3B that are irreparable have been subjected to more extensive genetic fine-structure analysis. These experiments were designed to determine the extent of the functional inactivation in individual mutants in the ad-3 and immediately adjacent genetic regions in mutants classified as presumptive multilocus deletions or multiple-locus mutations. These experiments have shown that in Neurospora crassa most X-ray-induced irreparable mutants of genotype ad-3A or ad-3B map as a series of overlapping multilocus deletions. Among the 29 irreparable mutants of genotype ad-3A, there are 16 different subgroups of complementation patterns; and among the 63 irreparable mutants of genotype ad-3B, there are also 16 different subgroups. In addition, mutants classified as presumptive multiple-locus mutants result from a variety of separate, but closely linked, sites of genetic damage.(ABSTRACT TRUNCATED AT 400 WORDS)

Alleles↗
PubMed2002

Phenotypic correction of primary Fanconi anemia T cells with retroviral vectors as a diagnostic tool.

OBJECTIVE: The aim of this study was to develop a rapid laboratory procedure that is capable of subtyping Fanconi anemia (FA) complementation groups FA-A, FA-C, FA-G, and FA-nonACG patients from a small amount of peripheral blood. MATERIALS AND METHODS: For this test, primary peripheral blood-derived FA T cells were transduced with oncoretroviral vectors that expressed FANCA, FANCC, or FANCG cDNA. We achieved a high efficiency of gene transfer into primary FA T cells by using the fibronectin fragment CH296 during transduction. Transduced cells were analyzed for correction of the characteristic DNA cross-linker hypersensitivity by cell survival or by metaphase analyses. RESULTS: Retroviral vectors containing the cDNA for FA-A, FA-C, and FA-G, the most frequent complementation groups in North America, allowed rapid identification of the defective gene by complementation of primary T cells from 12 FA patients. CONCLUSION: Phenotypic correction of FA T cells using retroviral vectors can be used successfully to determine the FA complementation group immediately after diagnosis of the disease.

3T3 Cells↗
PubMed1988

Use of transmissible plasmids as cloning vectors in Caulobacter crescentus.

Cloning vectors for studies of Caulobacter crescentus genes should be transferrable between Escherichia coli and C. crescentus since a transformation system has not been developed for C. crescentus. We have tested a large number of vectors containing IncP or IncQ replicons and found that many of the vectors containing IncQ replicons, and all but one of the vectors containing IncP replicons, are readily transferred by conjugation into C. crescentus. All of the plasmids tested were maintained in C. crescentus at 1 to 5 copies per cell, but plasmids containing IncP replicons were more stable than plasmids containing IncQ replicons. Further studies with a derivative of the IncQ plasmid R300B showed that when a promoterless kanamycin (Km)-resistance gene (npt2) was inserted into the intercistronic region of the sul-aphC (SuR-SmR) operon, Km resistance was expressed only when the npt2 gene was inserted such that it would be transcribed from the sul promoter. These data indicate that R300B does not contain sequences which would provide promoter function in C. crescentus in the orientation opposite to that of the sul operon and that any genes cloned in this orientation would require native promoters for expression. To provide greater versatility for cloning into R300B, additional vectors were constructed by the addition of multiple cloning sites in the intercistronic region of the sul-aphC operon. In addition, chromosomal DNA libraries were constructed in R300B and in the cosmid vector pLAFR1-7. Specific clones from these libraries containing genes of interest were identified by complementation of the appropriate C. crescentus mutants.

Cloning, Molecular↗
PubMedDate not supplied

Correction of Fanconi anemia type C phenotypic abnormalities using a clinically suitable retroviral vector infection protocol.

Fanconi anemia (FA) is a complex autosomal recessive disease with hematologic manifestations characterized by a progressive hypoplastic anemia, hypersensitivity to clastogenic agents, and an increased incidence of acute myelogenous leukemia. The cDNA that corrects one of four FA complementation subtypes, named Fanconi anemia Type C (FAC) has recently been identified. We constructed a a simplified recombinant retrovirus (vMFGFAC) encoding only the FAC cDNA, and tested its ability to correct the FAC defect in a lymphocytic cell line and primary mobilized blood progenitor cells. In addition, the gene transfer efficiency using a clinically applicable gene transfer protocol into normal primitive hematopoietic progenitor cells, high proliferating potential colony forming cells (HPP-CFC), derived from CD34+ purified cord blood cells was examined. The gene transfer efficiency was significantly enhanced when cells were transduced with supernatant while adherent to a 30/35 KD fragment of fibronectin, FN30/35, and was similar to efficiency obtained by coculture with retrovirus packaging cells. Transduction of an FAC deficient lymphoid cell line with vMFGFAC supernatant resulted in an enhanced cell viability, and G-CSF mobilized peripheral blood cells from an FAC-deficient patient transduced with the vMFGFAC virus demonstrated enhanced progenitor cell colony formation. These data indicate that the vMFGFAC virus allows functional complementation of FAC in lymphoblasts and primary hematopoietic progenitors, and that primitive cord blood hematopoietic stem/progenitor cells can be transduced at an efficiency comparable to protocols using cocultivation if adherent to FN 30/35 fragment.

Base Sequence↗
PubMed1996

The mutation rate of human immunodeficiency virus type 1 is influenced by the vpr gene.

A system has been designed to study the in vivo forward rate of mutation of human immunodeficiency virus type 1 (HIV-1) during one round of replication. A HIV-1 shuttle vector was used that contained the lacZ alpha peptide gene as a reporter for mutations. The forward mutation rate of HIV-1 was found to be 3 x 10(-5) mutations per target base pair per cycle, or about 20-fold lower than the error rates reported for purified HIV-1 reverse transcriptase with sense-strand RNA and DNA templates of the lacZ alpha peptide gene in a cell-free system. To test the hypothesis that the vpr gene product might, at least in part, account for the lower mutation rate observed in vivo, a HIV-1 vector was replicated to determine if the mutation rate was higher in the absence of the wild-type vpr gene product. A vpr- shuttle vector had an overall mutation rate as much as 4-fold higher than that of the parental vector. A shuttle vector with an amino acid substitution in Vpr that prevents efficient incorporation of Vpr into virus particles was found to have a mutation frequency similar to that of the vpr- vector, and was interpreted to indicate a requirement for Vpr incorporation into the virus particle in order to observe the influence of vpr on the mutation rate. Replication of a vpr- shuttle vector in the presence of a wild-type vpr expression plasmid led to a mutation frequency similar to that of the parental vector, suggesting that the vpr mutation could be complemented in trans. Immunoprecipitation analysis indicated that Vpr virion incorporation coincided with the influence of vpr on the mutation rate.

Animals↗
PubMed1992

The conserved helicase motifs of the herpes simplex virus type 1 origin-binding protein UL9 are important for function.

The UL9 gene of herpes simplex virus encodes a protein that specifically recognizes sequences within the viral origins of replication and exhibits helicase and DNA-dependent ATPase activities. The specific DNA binding domain of the UL9 protein was localized to the carboxy-terminal one-third of the molecule (H. M. Weir, J. M. Calder, and N. D. Stow, Nucleic Acids Res. 17:1409-1425, 1989). The N-terminal two-thirds of the UL9 gene contains six sequence motifs found in all members of a superfamily of DNA and RNA helicases, suggesting that this region may be important for helicase activity of UL9. In this report, we examined the functional significance of these six motifs for the UL9 protein through the introduction of site-specific mutations resulting in single amino acid substitutions of the most highly conserved residues within each motif. An in vivo complementation test was used to study the effect of each mutation on the function of the UL9 protein in viral DNA replication. In this assay, a mutant UL9 protein expressed from a transfected plasmid is used to complement a replication-deficient null mutant in the UL9 gene for the amplification of herpes simplex virus origin-containing plasmids. Mutations in five of the six conserved motifs inactivated the function of the UL9 protein in viral DNA replication, providing direct evidence for the importance of these conserved motifs. Insertion mutants resulting in the introduction of two alanines at 100-residue intervals in regions outside the conserved motifs were also constructed. Three of the insertion mutations were tolerated, whereas the other five abolished UL9 function. These data indicate that other regions of the protein, in addition to the helicase motifs, are important for function in vivo. Several mutations result in instability of the mutant products, presumably because of conformational changes in the protein. Taken together, these results suggest that UL9 is very sensitive to mutations with respect to both structure and function, perhaps reflecting its multifunctional character.

Amino Acid Sequence↗
PubMed1971

Genetics of sulfate transport by Salmonella typhimurium.

Sixty-four mutants were isolated from the LT-2 wild-type strain of Salmonella typhimurium by selecting for chromate resistance. The majority of lesions were shown to lie in the cysA gene. (i) The mutants cannot take up sulfate, a finding which verifies the role of cysA in sulfate transport. In addition, 52 sulfate-transport mutants isolated without chromate selection were defective in the cysA gene. (ii) Most had less than 25% of the binding activity of the wild-type strain. (iii) Most had normal sulfite reductase (H(2)S-nicotinamide adenine dinucleotide phosphate oxidoreductase, EC 1.8.1.2) activity. (iv) Their sulfate-binding protein (binder) appears electrophoretically and immunologically normal. (v) Amber cysA mutants also make apparently normal binder in small amounts. (vi) All classical cysA mutants tested, including two with long deletions, had normal binding activity. From these observations, it is suggested that the cysA gene does not code for the binder. But many mutations in this gene reduce the binding activity in some unknown way. Other mutants, identified as cysB mutants, had neither binding nor uptake activities and their sulfite reductase activities were similarly reduced, thus confirming the regulatory role of the cysB gene. When binder was detectable, it had wild-type properties. No mutations in the binder gene were found among more than 100 mutants examined. Thus, sulfate binding has not been established as a part of sulfate transport. However, the production of binder is intimately connected with cysA, the established sulfate transport gene, and is regulated by the same mechanism that regulates both transport and the rest of the cysteine biosynthetic pathway.

Acrylates↗
PubMed2002

A system for deletion and complementation of Candida glabrata genes amenable to high-throughput application.

We describe a method for deleting or modifying genes from the pathogenic fungus Candida glabrata as well as a companion vector for complementation or ectopic expression experiments. A linear deletion fragment generated by polymerase chain reaction was used to replace a gene of interest with the C. glabrata gene encoding imidazoleglycerol-phosphate dehydratase (HIS3). As test cases, the chromosomal loci of the C. glabrata genes encoding aminoimidazole ribonucleotide carboxylase (ADE2) and encoding isopropylmalate dehydrogenase (LEU2) were deleted. To facilitate application of the deletion technique to essential genes, we also constructed vectors to allow expression of a complementing copy of the wildtype gene under control of the copper-inducible C. glabrata metallothionein I (MT-1) promoter. One version of the vector carried the Saccharomyces cerevisiae centromere (CEN) and autonomously-replicating sequence (ARS) regions. The C. glabrata ADE2 and LEU2 genes and a transposon-derived neomycin/kanamycin resistance gene were successfully expressed from this vector, with expression of the ADE2 and LEU2 genes complementing the ADE2 and LEU2 deletion mutations, respectively. However, this vector showed regulated expression only for the ADE2 gene. A second version of the vector, which carried an additional C. glabrata CEN and ARS region for stable plasmid maintenance, did show regulated expression for the LEU2 and neomycin/kanamycin resistance genes. This deletion and expression system is potentially applicable to any C. glabrata gene and is amenable to high-throughput application. We anticipate that these tools will have broad utility in deletion or modification of specific C. glabrata genes. This approach is also applicable to other yeast fungi.

3-Isopropylmalate Dehydrogenase↗
PubMed1996

Increased transcomplementation properties of plasmids carrying HSV-1 origin of replication and packaging signals.

This report describes a very simple method that facilitates the analysis of the functional activity of essential genes of herpes simplex virus type 1 (HSV-1) in cell culture. The method, which depends on the use of complementing plasmids containing a virus origin of replication (ori s) and a packaging signal (amplicon plasmids), was tested for its ability to detect complementation of the defective HSV-1 Cgal delta 42 virus, a UL42 null mutant, by amplicon plasmids carrying either wild-type (pA-UL42) or mutated alleles (pA-delta UL42) of the HSV-1 UL42 gene. In nonpermissive Vero cells transfected with amplicon plasmid pA-UL42 and superinfected with the defective Cgal delta 42 virus, both the plasmid and the helper genomes were amplified and packaged, giving rise to a self-complementing amplicon/helper virus population able to disseminate and to form lytic plaques in cell culture. These plaques were due to complementation and not to recombination between the defective virus and the amplicon plasmid as confirmed by Southern blot analysis of individual plaques. No complementation was observed by superinfection of cells transfected with the noncomplementing pA-delta UL42 amplicon plasmid. Instead, small foci were observed in cells transfected with plasmids which express wild-type UL42 but were unable to amplify or to become packaged, and the few true lytic plaques that were observed in these systems resulted from the spread of competent recombinant viruses. Results presented in this work indicate that (i) self-complementation between a defective virus and an amplicon plasmid provides a strong and very sensitive method to assess function activity of an HSV-1 essential gene in a one-step experiment and (ii) oris-carrying plasmids could be instrumental in the production and selection of recombinant HSV-1 vectors.

Animals↗
PubMed2000

Functional analysis of heterologous holin proteins in a lambdaDeltaS genetic background.

Holins are small hydrophobic proteins causing non-specific membrane lesions at the end of bacteriophage multiplication, to promote access of the murein hydrolase to their substrate. We have established a lambdaDeltaS genetic system, which enables functional expression of holins from various phages in an isogenic phage lambda background, and allows qualitative evaluation of their ability to support lysis of Escherichia coli cells. Synthesis of Holins is under control of native lambda transcription and translation initiation signals, and the temperature-sensitive CIts857 repressor. A number of different holins were tested in this study. The opposing action of phage lambda S105 and S107 holin variants in lysis timing could be confirmed, whereas we found evidence for a functionally non-homologous dual translational start motif in the Listeria phage Hol500 holin, i.e., the Hol500-96 polypeptide starting at Met-1 revealed a more distinct lytic activity as compared to the shorter product Hol500-93. The largest holin known, HolTW from a Staphylococcus aureus phage, revealed an early lysis phenotype in the lambdaDeltaSthf background, which conferred a plaque forming defect due to premature lysis. Mutant analysis revealed that an altered C-terminus and/or a V52L substitution were sufficient to delay lysis and enable plaque formation. These results suggest that the extensively charged HolTW C-terminus may be important in regulation of lysis timing. The gene 17.5 product of E. coli phage T7 was found to support sudden, saltatory cell lysis in the lambdaDeltaSthf background, which clearly confirms its holin character. In conclusion, lambdaDeltaSthf offers a useful genetic tool for studying the structure-function relationship of the extremely heterogeneous group of holin protein orthologs.

Amino Acid Sequence↗
PubMed1988

Spectra of molecular changes induced in DNA of Drosophila spermatozoa by 1-ethyl-1-nitrosourea and X-rays.

Mutations induced in Drosophila spermatozoa at the alcohol dehydrogenase Adh locus by 1-ethyl-1-nitrosourea (ENU) were compared to X-ray-induced mutations using genetic tests for complementation, southern blotting, western blotting and northern blotting. 8 of 10 ENU-induced mutations complemented all known adjacent loci and were presumed to be intragenic. In contrast, 8 of 30 X-ray-induced mutations were intragenic. Southern blot analysis showed that 2 of 7 intragenic mutations induced by X-rays were altered at the Adh locus, whereas all 8 intragenic ENU mutants appeared normal. Western blot analysis showed 4 of 7 intragenic mutants induced by X-rays produced a detectable polypeptide; 1 of the 4 had normal molecular weight and charge. In contrast, 7 of the 8 intragenic mutants induced by ENU produced a polypeptide of normal molecular weight and charge. One ENU and two X-ray-induced mutants, which had normal southern blots and no detectable polypeptide, produced normal molecular weight mRNA by northern blots. The interpretation of these results is that in spermatozoa X-rays induce primarily deletions that either produce deficiencies of the Adh locus or nonsense mutations within the locus, whereas ENU induces primarily missense mutations. This forward mutation assay based on loss of enzymatic activity efficiently recovered a broad spectrum of mutations ranging from missense to intragenic deletions and multi-locus deficiencies. Only 3 of these 40 mutations produced a polypeptide detectable as an electrophoretic variant.

Alcohol Dehydrogenase↗
PubMed2004

An efficient method for creation and functional analysis of libraries of hybrid type I polyketide synthases.

Bacterial type I polyketide synthases (PKSs) generate a structurally diverse group of natural products with a wide range of biological activities. Hybrid type I PKSs in which domains of one multifunctional polypeptide are replaced with components from heterologous systems have generated significant interest over the past decade. Almost invariably only one or several specific hybrids are made at a time and tested for functionality. This approach is slow, dependent upon a fortuitous choice of specific fusions points, and often leads to inactive or minimally active hybrid systems. We describe herein a method for generating and screening a library of hybrid pikAI complementation plasmids (encoding the loading domain and the first two extension domains of pikromycin PKS) able to restore pikromycin in a BB138 Streptomyces venezuelae pikAI-deletion mutant. In the first step the plasmid sequence encoding the loading domain AT(0)-ACP(0) was replaced by a counter selectable marker, sacB. DNA family shuffling was then used to generate a diverse library of chimeric AT(0)-ACP(0) fragments, which were used to replace sacB by lambda-Red-mediated in vivo recombination in an Escherichia coli host. This method resulted in the rapid and efficient generation of a large number of hybrid pikAI complementation plasmids, which were used to transform S.venezuelae BB138. A bioassay of over 4000 of these transformants successfully revealed three different PikAI hybrids which were able to lead to pikromycin production. The study suggests that most of the hybrids are not detectably functional, and underscores the need to generate and screen large and diverse libraries in which different fusion points are tried. The methodologies applied in this study address this need and can be used for directed evolution of any component of the PikPKS, and potentially other type I PKS systems.

Acyltransferases↗
PubMed1992

Evolution of the mouse H-2K region: a hot spot of mutation associated with genes transcribed in embryos and/or germ cells.

Active gene transcription is known to promote genetic change in neighboring DNA. We reasoned that the change would be readily heritable if transcription was occurring in germ cells or early embryonic cells before the germ cells are set aside. The H-2K region of the major histocompatibility complex (MHC) provides a good vehicle for testing this hypothesis because it is replete with such genes. We have compared the amount of polymorphism in 240 kb of DNA contiguous with H-2K and 150 kb of DNA flanking a homologous duplicated region in t-haplotypes and inbred strains. Using 90 probes and three restriction enzymes, we find a staggering difference in the amount of polymorphism in the H-2K region vs. the duplicated region (26% vs. 0%) of t-haplotypes. The disparity in the rate of divergence between the two regions indicates that the spatial distribution of genes and their expression pattern might be important factors in sequence evolution. Since t-haplotypes normally show extremely limited variability among themselves due to their recent divergence from a single ancestor, these results imply that the mutation rate in the H-2K region is unusually high. This is in apparent contradiction to the current view that the MHC loci have evolved at the same rate as other loci. The implications for the evolution of the H-2K gene are discussed.

Animals↗
PubMed2004

A large-scale screen for mutagen-sensitive loci in Drosophila.

In a screen for new DNA repair mutants, we tested 6275 Drosophila strains bearing homozygous mutagenized autosomes (obtained from C. Zuker) for hypersensitivity to methyl methanesulfonate (MMS) and nitrogen mustard (HN2). Testing of 2585 second-chromosome lines resulted in the recovery of 18 mutants, 8 of which were alleles of known genes. The remaining 10 second-chromosome mutants were solely sensitive to MMS and define 8 new mutagen-sensitive genes (mus212-mus219). Testing of 3690 third chromosomes led to the identification of 60 third-chromosome mutants, 44 of which were alleles of known genes. The remaining 16 mutants define 14 new mutagen-sensitive genes (mus314-mus327). We have initiated efforts to identify these genes at the molecular level and report here the first two identified. The HN2-sensitive mus322 mutant defines the Drosophila ortholog of the yeast snm1 gene, and the MMS- and HN2-sensitive mus301 mutant defines the Drosophila ortholog of the human HEL308 gene. We have also identified a second-chromosome mutant, mus215(ZIII-2059), that uniformly reduces the frequency of meiotic recombination to <3% of that observed in wild type and thus defines a function required for both DNA repair and meiotic recombination. At least one allele of each new gene identified in this study is available at the Bloomington Stock Center.

Alleles↗
PubMed1992

Discordant segregation of Na+,K(+)-adenosine triphosphatase alleles and essential hypertension.

OBJECTIVES: To determine whether the alpha 2 and or beta 1 isoforms of the Na+,K(+)-adenosine triphosphatase (Na+,K(+)-ATPase) are involved in the pathogenesis of essential hypertension. DESIGN: Segregation analysis of polymorphic DNA markers was used to test the involvement of Na+,K(+)-ATPase in essential hypertension. PARTICIPANTS: Children with persistent hypertension having one parent with essential hypertension were included in the study. Criteria for persistent hypertension were blood pressure readings with systolic and/or diastolic levels exceeding the 95th percentile based upon age and sex. The diagnosis of hypertension for adults, including parents and older siblings, was confirmed using criteria recommended in the 1988 report of the Joint National Committee on Detection, Evaluation, and Treatment of High Blood Pressure. RESULTS: In three essential hypertensive families consisting of 18 members including 11 hypertensives, several obligate recombinants between the Na+,K(+)-ATPase alpha 2 isoform marker and the hypertension phenotype were observed. Similarly, in one hypertension family consisting of four members, obligate recombinants between the beta 1 isoform marker and the disease were observed. CONCLUSIONS: The discordant segregation of the alpha 2 and beta 1 isoform markers and essential hypertension suggests that neither the Na+,K(+)-ATPase alpha 2 nor beta 1 isoform genes play a primary role in the pathogenesis of hypertension in the families studied.

Adult↗
PubMed1994

Drosophila P element transposase induces male recombination additively and without a requirement for P element excision or insertion.

P element dysgenesis-associated male recombination in Drosophila was examined with a selective system focused upon a section of the third chromosome divided into eight recombination segments. Tests compared crossing over in the presence of none, one and two doses of P(delta 2-3)(99B), a non-mobile transposase source, in the absence of a mobilizable P element target in the genome. In the presence of the P transposase source, and without a P element target, significant male recombination occurred in genomic regions physically separated from the P(delta 2-3) site. Using two doses of P(delta 2-3) without a P element target, the male recombination rate doubled, and 90% of the crossovers occurred in the pericentric region. The distribution of recombination events, in the absence of P element targets approximates that seen in studies of radiation induced mitotic crossing over and the metaphase chromosome map. Another experiment examined the effects of one dose of P(delta 2-3) on a genome with a single P element target, P(lArB)(87C9), in the third recombination segment. Crossovers increased 58-fold in the immediate region of the P element target.

Animals↗
PubMed1986

Genetic analysis of DNA repair in Aspergillus: evidence for different types of MMS-sensitive hyperrec mutants.

To identify genes which affect DNA repair and possibly recombination in Aspergillus nidulans, mutants hypersensitive to methyl methanesulphonate (MMS) were induced with ultraviolet light (UV) or gamma-rays. About half of them contained associated translocations and many were hypersensitive to UV and/or defective in meiosis. Two are alleles of the known uvsB gene while most others define new genes. In addition, among available uvs mutants many were found to be MMS-sensitive. Some of the various uncharacterized ones were identified as alleles of known uvs, but 5 of them were mapped in 2 new genes, uvsH and uvsJ. To identify functional and epistatic groups, mutants from each uvs gene were tested for effects on recombination and mutation, and double mutant uvs strains were compared for UV survival to their component single mutant strains. 3 epistatic pairs were identified, (1) uvsF and H, (2) uvsB and D, and (3) uvsC and E. Conclusive interpair tests were difficult, because such double mutant combinations were frequently lethal or nearly so. The first pair, uvsF and H, shared some of the properties of excision-defective mutants, both uvs being very highly sensitive to UV for mutation as well as survival. But unlike such mutants, uvsH was also sensitive to gamma-rays and defective in meiosis. Both uvs showed normal levels of meiotic recombination, but greatly increased spontaneous mitotic crossing-over, being the most "hyperrec" types among all uvs. The second pair, uvsB and uvsC, which was similarly hyperrec showed only slight increases of UV-induced mutation (less than 2-fold). As a main effect, these uvs caused very high frequencies of unbalanced, unstable segregants from diploid conidia (30 X), but few of these were recognizable aneuploids. The third pair, uvsC and E, which are known to be rec- for gene conversion, caused reduced mitotic crossing-over in diploids and increased levels of haploid segregants. These mutants are spontaneous mutators, but showed less UV-induced mutation than wild-type controls.

Alleles↗
PubMed1990

Genetic organization of the cellulose synthase operon in Acetobacter xylinum.

An operon encoding four proteins required for bacterial cellulose biosynthesis (bcs) in Acetobacter xylinum was isolated via genetic complementation with strains lacking cellulose synthase activity. Nucleotide sequence analysis indicated that the cellulose synthase operon is 9217 base pairs long and consists of four genes. The four genes--bcsA, bcsB, bcsC, and bcsD--appear to be translationally coupled and transcribed as a polycistronic mRNA with an initiation site 97 bases upstream of the coding region of the first gene (bcsA) in the operon. Results from genetic complementation tests and gene disruption analyses demonstrate that all four genes in the operon are required for maximal bacterial cellulose synthesis in A. xylinum. The calculated molecular masses of the proteins encoded by bcsA, bcsB, bcsC, and bcsD are 84.4, 85.3, 141.0, and 17.3 kDa, respectively. The second gene in the operon (bcsB) encodes the catalytic subunit of cellulose synthase. The functions of the bcsA, bcsC, and bcsD gene products are unknown. Bacterial strains mutated in the bcsA locus were found to be deficient in cellulose synthesis due to the lack of cellulose synthase and diguanylate cyclase activities. Mutants in the bcsC and bcsD genes were impaired in cellulose production in vivo, even though they had the capacity to make all the necessary metabolic precursors and cyclic diguanylic acid, the activator of cellulose synthase, and exhibit cellulose synthase activity in vitro. When the entire operon was present on a multicopy plasmid in the bacterial cell, both cellulose synthase activity and cellulose biosynthesis increased. When the promoter of the cellulose synthase operon was replaced on the chromosome by E. coli tac or lac promoters, cellulose production was reduced in parallel with decreased cellulose synthase activity. These observations suggest that the expression of the bcs operon is rate-limiting for cellulose synthesis in A. xylinum.

Arabidopsis Proteins↗