Genetic studies with a phosphoglucose isomerase mutant of Saccharomyces cerevisiae.

Summary A mutation pgi1 in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2⁺:2^- in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: tsm134-lys2-pgi1-tyr1 approximately 15 map units from tyr1. The mutation pgi1 defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgi1 locus.     

Ribosomal abnormality in recA mutants of Escherichia coli.

Summary The tif-1 mutation has been shown to affect protein synthesis in vitro by increasing translational ambiguity (Ephrati-Elizur, Luther-Davies and Hayes, 1976). It is demonstrated here that some recA mutations confer similar abnormality. By comparing suitable combinations of ribosomes and soluble proteins from recA ⁺ and recA cells the defect is shown to be associated with ribosomes. The recA mutation, which suppresses most phenotypic characteristics of the tif-1 mutation (Castellazzi, George and Buttin, 1972(b)) does not suppress the ribosomal abnormality. Sience the closely linked tif-1 and recA mutations lead to the expression of a common property they may be in the same gene.     

Induction of recA+-protein synthesis in Escherichia coli.

Summary Escherichia coli was infected with precA ⁺to determine the genetic and physiological factors controlling recA ⁺gene expression. When precA ⁺replication was prevented by superinfection immunity, recA ⁺protein synthesis was induced by UV radiation. The recA ⁺gene is negatively controlled by the lexA ⁺gene product because i) a dominant lexA mutation, lexA3, prevented induction of recA ⁺protein synthesis ii) a recessive lexA mutation, tsl-1, caused induction of recA ⁺protein synthesis. Conversely positive control of recA ⁺gene expression requires recA ⁺protein because i) a co-dominant tif-1 mutation (a recA mutation) caused induction of recA ⁺protein synthesis ii) a recessive mutation, recA1, prevented cis-induction of recA protein synthesis. recA ⁺protein and Protein X of UV irradiated bacteria co-migrated and were subject to the same physiological and genetic controls. It is concluded that Protein X is recA ⁺protein. lysogenic induction was prevented by TPCK, a protease inhibitor. However TPCK did not prevent induction of recA ⁺protein synthesis, indicating that induction of the two processes occurs in different ways. It is suggested that the lexA ⁺and recA ⁺proteins normally combine to repress the recA ⁺gene. Derepression might occur after DNA damaging treatments because the amount of this complex would be reduced by recA ⁺protein i) binding to single-stranded DNA and/or ii) being activated to function proteolytically towards regulatory molecules such as repressor.     

Prophage induction and cell division in E. coli. II. Linked (recA, zab) and unlinked (lex) suppressors of tif-1-mediated induction and filamentation

Summary The pleiotropy of tif-1, a mutation in E. coli K12 causing, among other effects, cellular filamentation at 42° and thermal induction of lysogenic derivatives, can be explained by the participation of the tif ⁺ gene product in more than one reaction pathway. Pathways that involve the tif ⁺ product may be analyzed by selection of secondary mutations that reverse both tif-1-mediated prophage induction and cell filamentation. Among revertants of a tif-1 lysogen among 20% are recombination deficient. These appear to carry a recA mutation. In addition to this class is a rarer (7%) phenotypically distinguishable class of revertants, called zab, first described here. Markers tif-1, recA and zab are closely linked. Mutations lex which are dominant and located near malB also appear (3%) among tif-1 revertants. The lex ⁺ function is needed for normal UV, X-ray and mitomycin C induction of prophage .The zab mutation resembles recA in causing (1) high sensitivity to UV, X-rays and mitomycin C, (2) drastic DNA degradation following UV irradiation but normal capacity to repair UV-damaged infecting phage (Hcr⁺), (3) failure to carry out UV reactivation and UV mutagenesis of UV-irradiated bacteriophage , (4) a markedly reduced level of spontaneous induction of . In contrast, other capacities, strikingly diminished by recA, are affected less, if at all by zab. Thus zab (1) permits 30–60% normal recombination proficiency, (2) shows real, although very low inducibility of by UV or mitomycin C, (3) permits 100% efficiency of plating of red gam, and (4) does not degrade DNA spontaneously.The hypothesis is proposed that the tif-1 mutation is a regulatory mutation controlling the activity, or more likely the synthesis of repair enzyme(s). The level of these repair enzyme(s), rather than DNA lesions, may govern the stability of the prophage repressor and the capacity of the bacteria to form septa.     

Genetic analysis of two new mutations resulting in herbicide resistance in the cyanobacterium Synechcoccus sp. PCC 7002

Two herbicide-resistant strains of the cyanobacterium Synechococcus sp. PCC 7002 are compared to the wild-type with respect to the DNA changes which result in herbicide resstance. The mutations have previously been mapped to a region of the cyanobacterial genome which encodes oneof three copies of psbA, the gene which encodes the 32 kDa Q_b-binding protein also known as D1 (Buzby et al. 1987). The DNA sequence of the wild-type gene was first determined and used as a comparison to that of the mutant alleles. A point mutation at codon 211 in the psbA1 coding locus (TTC) to TCC) results in an amino acid change from phenylalanine to serine in the D1 protein. This mutation confers resistance to atrazine and diuron at seven times and at two times the minimal inhibitory concentration (MIC) for the wild-type, respectively. A mutation at codon 211 resulting in herbicide resistance has not previously been described in the literature. A second point mutation at codon 219 in the psbA1 coding locus (GTA to ATA) results in an amino acid change from valine to isoleucine in the D1 protein. This mutation confers resistance to diuron and atrazine at ten times and at two times the MIC for the wild-type, respectively. An identical codon change conferring similar herbicide resistance patterns has previously been described in Chlamydomonas reinhardtii. The atrazine-resistance phenotype in Synechococcus sp. PCC 7002 was shown to be dominant by plasmid segregation analysis.Abbreviations At ^r atrazine resistance - Du ^r diuron resistance - Km ^r kanamycin resistance - Ap ^r ampicillin resistance - MIC Minimum inhibitory concentration     

Isolation and characterization of an operator-constitutive mutation in the recA gene of E. coli K-12

Summary The recA gene of E. coli is regulated by a specific repressor, the lexA protein, which binds to an operator in the recA regulatory region. We describe in this paper the isolation and characterization of a mutant thought to carry an operator-constitutive mutation in the recA gene. This mutation has the following properties: 1) It partially supresses the UV sensitivity of lexA ^– strains. 20 It maps near the recA gene. 3) It allows constitutive high-level synthesis of recA protein in both lexA ^– and lexA ⁺ backgrounds. 4) It allows constitutive synthesis of the recA messenger RNA. 5) It is cis–acting. The mutation does not restore induced cellular mutagenesis in a lexA ^– background. The expression of induced repair and mutagenesis of UV irradiated phage lambda or the regulation of the lexA gene is not affected by the presence of the mutation in either a lexA ⁺ or lexA ^– strain. These observations confirm other findings that high levels of recA protein synthesis per se is not sufficient for the expression of UV inducible functions and that the lexA protein represses other genes besides the recA gene.Abbreviations UV ultraviolet - Kd kilodalton - PAGE polyacrylamide gel electrophoresis     

Pathways for repair of DNA damaged by alkylating agent in Escherichia coli.

Summary A strain with both the polA12 and the alk-1 mutation is only slightly more sensitive to methyl methane sulfonate (MMS) than isogenic strains with only one of the mutations. On the other hand, alk-1 recA1 double mutant is much more sensitive to MMS than are strains carrying either one of alk or recA mutation. It was suggested that the alk and the polA gene products are involved in the same DNA repair process whereas the recA function is independent from the process. The yield of MMS-induced mutation (Arg^- (argE) to Arg⁺ reversion) in alk mutant is considerably higher than that in wild type strain. Thus, the repair process in which the alk gene product is involved is relatively accurate. When MMS-treated phages were plated on MMS-treated bacteria, there were considerable increases in survival of treated phage even in recA alk double mutant. It seems that a new repair pathway, which is specific for alkylating agent-induced damages and is not dependent on the RecA function, may be induced on exposure of bacteria to the alkylating agent.     

Identification of protein X of Escherichia coli as the recA+/tif+ gene product.

Summary Protein X, molecular weight 40,000, has been separated from the other proteins of E. coliby a two-dimensional gel electrophoretic technique which separates proteins according to isoelectric point (pI) in the firstdimension and according to molecular weight in the second. When protein X is induced in wild-type cells by mitomycin C treatmentit has a pI6.0. However, when protein X is induced in a tif-1 mutant, either by temperatureshift-up to 42° or by mitomycin C treatment at 30°, it has a pI6.2. The low level of protein X which is present inuninduced tif mutants at 30° also has a pI6.2. These results suggest thattif-1 is a mis-sense mutation in the gene coding for protein X. Since transduction andcomplementation studies indicate that tif-1 is a mutation of therecA ⁺ gene (Castellazzi, Morand, George and Buttin, 1977) it follows that protein X is the recA ⁺ gene product.A model has been formulated to account for the relationship between protein X synthesis and the recA ⁺ and lexA ⁺ genes. In this model, a repressor coded by lexA ⁺ binds to the operator of the recA ⁺ gene from whence it can normally only be removed by the combined action of an inducer and protein X, the recA ⁺ product. Thus, protein X controls its own synthesis. The tif-1 mutation leads to a temperature sensitive form of protein X which, at 42°, can spontaneously remove the repressor without the intervention of the inducer.     

Suppressor mutation of position-effect variegation in Drosophila melanogaster affecting chromatin properties

The dominant mutation Su-var(2)1 ⁰¹ which suppresses position-effect variegation and displays recessive butyrate sensitivity was found to result in significant hyperacetylation of histone H4. This biochemical finding, as well as the genetic properties of this mutation, strongly suggest that the wild-type product of the corresponding locus is involved in histone H4 deacetylation. In larvae containing the suppressor mutation the accessibility of chromatin to endogenous nucleases is significantly increased which might be causally connected with histone H4 hyperacetylation. The suppressor mutation Su-var(2)1 ⁰¹ has, therefore, to be classified as a chromatin condensation mutation.     

A new RNA synthesis mutant of E. coli

A temperature-sensitive mutant of E. coli is described. At the nonpermissive temperature, the capacity for RNA and protein synthesis decreases logarithmically in the mutant. The mutant is unable to support the growth of f2 or T7 virus, even at the permissive temperature. The temperature-sensitive mutation maps approximately 1 away from rif ^r in E. coli and therefore affects a gene previously undescribed. The temperature sensitivity is suppressed by sublethal concentrations of rifampicin. Moreover, in rif ^r T^s double mutants, the T ^s mutation suppresses rif ^r and vice versa. The partially purified RNA polymerases from mutant and wild-type cells have different temperature and salt optima.This research was supported by Public Health Service grant GM-14368 from the National Institute of General Medical Sciences and by grant IN-29 from the American Cancer Society. One of us (D.P.) is a predoctoral trainee, supported by a National Science Foundation Graduate Traineeship Program and by a National Institutes of Health Predoctoral Research Fellowship. S. Marshall is supported by LASBAU.     

A dnaB analog ban, specified by bacteriophage P1: genetic and physiological evidence for functional analogy and interactions between the two products.

Summary Bacteriophage P1 has been shown previously to determine a product ban than can substitute in DNA replication for the protein specified by cistron dnaB of Escherichia coli. However, ban product furnished by P1 bac prophage (ban constitutive) substitutes only poorly for DNA replication in the absence of dnaB product in a strain bearing an unsuppressed amber mutation, dnaB266, as shown by the cryosensitivity of the dnaB266 (P1 bac) lysogen and its unability to support growth. An additional mutation (termed crr) in the P1 bac prophage has been obtained which confers cryoresistance to the sup ⁺ dnaB266 (P1 bac crr) lysogen and restores its ability to support growth. ban product produced in P1 bac crr lysogen fulfills all dnaB roles in vivo, especially in the various instances in which ban product expressed in P1 bac lysogens does not. The ban product is expressed constitutively in P1 crr prophage. The crr-1 mutation is tightly linked to the bac-1 and ban-1 mutations and is dominant over crr ⁺. The nature of the crr mutation is discussed: two hypotheses are considered, that of a mutation in the ban gene rendering the ban product more active or that of a site mutation in the ban operon increasing the level of ban expression. Expression of ban product (wild type or altered) leads to interactions with the variously altered dnaB product. Both positive and negative interactions are described. Genetic results presented here suggest that ban and dnaB subunits interact to form hybrid dnaB-like molecules; the average composition of which depends on the relative quantities of ban and dnaB subunits in the cell.     

Genetic interaction between the nip1 mutation and genes affecting integration and excision in phage P2

Summary The excision of prophage P2 is controlled by two genes, int and cox. (The cox gene discussed in this report is defined by the cox class II mutants, defined by Six and Lindqvist, 1978). The combined activity of these two genes is rather inefficient, however, since only about 1% of the lysogens carrying an int ⁺ cox ⁺ prophage actually produce phage when derepressed. The efficiency of phage production (and presumably excision) can be increased 100-fold by an additional mutation called nip1 (Calendar et al., 1972), which is dominant and is located in or near the int gene.The nip1 mutation was mapped between c5, a mutation in the C gene, and an amber int mutation, int150. Phages carrying nip1 and either int150 or a cox mutation, cox3, were prepared by recombination. The nip1 mutation was found to increase excision only when it was located on the same chromosome as an active int ⁺ gene and only if cox ⁺ gene product was also available. The cox gene, known to be located between genes B and C (Lindahl and Sunshine, 1972), was further localized to a region between 77.2 to 78.1% from the conventional left end of the P2 chromosome by comparing the ability of phages with overlapping deletions to promote excision of the prophage in a P2 nip1 c5 cox3 lysogen.Other features of the integration-excision system in P2 are discussed.     

Isolation of a formamidopyrimidine-DNA glycosylase (fpg) mutant of Escherichia coli K12

Summary The fpg ⁺ gene of Escherichia coli coding for formamidopyrimidine-DNA glycosylase was previously cloned on a multicopy plasmid. The plasmid copy of the fpg ⁺ gene was inactivated by cloning a kanamycin resistance gene into the open reading frame, yielding the fpg-1:: Kn^r mutation. This mutation was transferred to the chromosome in the following steps: (i) linearization of the plasmid bearing the fpg-1::Kn^r mutation and transformation of competent bacteria (recB recC sbcB); (ii) selection for chromosomal integration of the fpg-1::Kn^r mutation; (iii) phage P1 mediated transduction of the fpg-1::Kn^r mutation in the AB1157 background. The resulting fpg ^- mutant exhibited no detectable Fapy-DNA glycosylase activity in crude lysates. The insertion mutation was localized by means of genetic crosses between mtl and pyrE, at 81.7 min on the E. coli linkage map. Sequence analysis confirmed this mapping and further showed that fpg is adjacent to rpmBG in the order fpg, rpmGB, pyrE. The formamidopyrimidine-DNA glycosylase defective strain does not show unusual sensitivity to the following DNA damaging treatments: (i) methylmethanesulfonate, (ii) N-methyl-N-nitro-N-nitrosoguanidine, (iii) ultraviolet light, (iv) -radiation. The fpg gene is neither part of the SOS regulon nor the adaptive response to alkylating agents.     

The regulation of urea amidolyase of Saccharomyces cerevisiae: mating type influence on a constitutivity mutation acting in cis.

Summary Constitutivity for the synthesis of the urea amidolyase bienzymatic complex is obtained by dur0^hmutations located in the regulatory genetic region adjacent to the dur1, dur2 gene cluster. The dur0^hmutations act only in cis and are a new case of cis effect strongly cancelled in /a diploid, similar to cargA ⁺0^hmutation shown previously to lead to arginase constitutivity. Illegitimate diploids do not show such a cancellation of constitutivity.The constitutivity produced by dur0^hmutation comprises the process of induction and the release of the glutamine effect. It does not impair the NH ₄ ⁺ effect.     

Genetic mapping of the lexC-113 mutation.

Summary A mutation, previously designated lex-113 and suspected to be situated in the lexA locus, has been positioned by transductional studies to a unique site on the chromosome of E. coli B separate from the lexA102 and uvrA155 mutations. The order of genes in this chromosomal region was demonstrated to be malB-lexA-uvr A-lex-113. The allele designation lexC-113 is suggested for this mutation in a new gene functional in the regulation of inducible lex ⁺ - and rec ⁺ — dependent SOS activities.     

A Ds-mutation of the Adh1 gene in Zea mays L.

Summary An unstable spontaneous mutation in the maize Adh1 gene, coding for alcohol dehydrogenase, was selected by allyl alcohol poisoning of wild type Adh1 pollen from a maize line carrying Ds at the Bz2 locus and one copy of Ac in an unknown position. The mutant has a null phenotype. No wild type pollen grains were detected in strains devoid of Ac, but in the presence of Ac, wild type pollen grains were detected with a frequency of between 10^-4 and 10^-3. In addition, events have been identified in the aleurone in which reversions of both bz2-m and the unstable adh1 mutation occurred in the same patch of tissue, presumably in response to an alteration of Ac. By these criteria, the Adh1 mutant is caused by Ds. DNA blotting experiments have shown the presence of a 1.3 kb insertion in the Adh1 gene. All or part of this Ds insertion is transcribed, and is detected as an insertion within the ADH1-mRNA. The longer mRNA hybridizes to an authentic Ds probe.This Ds element differs in size from other known Ds insertions.     

Scd1 ab-Xyk : a new asebia allele characterized by a CCC trinucleotide insertion in exon 5 of the stearoyl-CoA desaturase 1 gene in mouse

We describe here a spontaneous, autosomal recessive mutant mouse suffering from skin and hair defects, which arose in the outbred Kunming strain. By haplotype analysis and direct sequencing of PCR products, we show that this mutation is a new allele of the asebia locus with a naturally occurring mutation in the Scd1 gene (a CCC insertion at nucleotide position 835 in exon 5), which codes for stearoyl-CoA desaturase 1. This mutation introduces an extra proline residue at position 279 in the Scd1 protein. The mutant mice, originally designated km/km but now assigned the name Scd1 ^ab-Xyk (hereafter abbreviated as ab ^Xyk / ab ^Xyk ), have a similar gross and histological phenotype to that reported for previously characterized allelic asebia mutations ( Scd1 ^ab , Scd1 ^abJ , Scd1 ^ab2J , and Scd1 ^tm1Ntam ). Histological analysis showed they were also characterized by hypoplasic sebaceous glands and abnormal hair follicles. In a cross between Kunming- ab ^Xyk / ab ^Xyk and ABJ/Le- ab ^J / ab ^J mice, all the progeny showed the same phenotype, indicating that the two mutations were non-complementing and therefore allelic. Comparisons with the other four allelic mutants indicate that the Scd1 ^ab-Xyk mutation causes the mildest change in Scd1 function. This new mouse mutant is a good model not only for the study of scarring alopecias in humans, which are characterized by hypoplasic sebaceous glands, but also for studying the structure and function of the Scd1 protein.Communicated by G. ReuterThe first two authors contribute equally to this work     

Lop12, a Mutation in Mouse Crygd Causing Lens Opacity Similar to Human Coppock Cataract

A new cataract mutation was discovered in an ongoing program to identify new mouse models of hereditary eye disease. Lens opacity 12 (Lop12) is a semidominant mutation that results in an irregular nuclear lens opacity similar to the human Coppock cataract. Lop12 is associated with a small nonrecombining segment that maps to mouse Chromosome 1 close to the eye lens obsolescence mutation (Cryge^Cat2-Elo), a member of the γ-crystallin gene cluster (Cryg). Using a systemic candidate gene approach to analyze the entire Cryg cluster, a G to A transition was found in exon 3 of Crygd associated with the Lop12 mutation and has been designated Crygd^Lop12. The mutation Crygd^Lop12 leads to the formation of an in-frame stop codon that produces a truncated protein of 156 amino acids. It is predicted that the defective gene product alters protein folding of the γ-crystallin(s) and results in lens opacity.     

The paromomycin resistance mutation (parr-454) in the 15 S rRNA gene of the yeast Saccharomyces cerevisiae is involved in ribosomal frameshifting

Summary The leaky expression of the yeast mitochondrial geneoxi1, containing a frameshift mutation (+1), is caused by natural frameshift suppression, as shown previously (Fox and Weiss-Brummer 1980). A drastic decrease in the natural level of frameshifting is found in the presence of thepar ^r-454 mutation, localized at the 3′ end of the 15 S rRNA gene. This mutation causes resistance to the antibiotic paronomycin in the yeast strains D273-10B and KL14-4A (Li et al. 1982; Tabak et al. 1982). The results of this study imply that in the yeast strain 777-3A this mutation alone is sufficient for restriction of the level of natural frameshifting but is insufficient to confer resistance to paromomycin. A second mutation, arising spontaneously with a frequency of 10⁻⁴ leads, in combination with thepar ^r-454 mutation, to full paromomycin resistance in strain 777-3A.