The location of a temperature-sensitive trans-dominant mutation and its effect on restriction and modification in Escherichia coli K12

An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4 alpha gt57 beta gt14 and lambda vir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage lambda carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdXts+d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the lambda phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the Xts+d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of Xts+d mutation was studied. The results of complementation experiments, using F'-merodiploids or plasmid pBR322 with an inserted Xts+d mutation, support the idea that the HsdSts+d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.     

Cloning and genetic mapping of SNF1, a gene required for expression of glucose-repressible genes in Saccharomyces cerevisiae.

A functional SNF1 gene product is required to derepress expression of many glucose-repressible genes in Saccharomyces cerevisiae. Strains carrying a snf1 mutation are unable to grow on sucrose, galactose, maltose, melibiose, or nonfermentable carbon sources; utilization of these carbon sources is regulated by glucose repression. The inability of snf1 mutants to utilize sucrose results from failure to derepress expression of the structural gene for invertase at the RNA level. We isolated recombinant plasmids carrying the SNF1 gene by complementation of the snf1 defect in S. cerevisiae. A 3.5-kilobase region is common to the DNA segments cloned in five different plasmids. Transformation of S. cerevisiae with an integrating vector carrying a segment of the cloned DNA resulted in integration of the plasmid at the SNF1 locus. This result indicates that the cloned DNA is homologous to sequences at the SNF1 locus. By mapping a plasmid marker linked to SNF1 in this transformant, we showed that the SNF1 gene is located on chromosome IV. We then mapped snf1 to a position 5.6 centimorgans distal to rna3 on the right arm; snf1 is not extremely closely linked to any previously mapped mutation.     

Structure and molecular analysis of RGR1, a gene required for glucose repression of Saccharomyces cerevisiae.

An RGR1 gene product is required to repress expression of glucose-regulated genes in Saccharomyces cerevisiae. The abnormal morphology of rgr1 cells was studied. Scanning and transmission electron microscopic observations revealed that the cell wall of the daughter cell remained attached to that of mother cell. We cloned the RGR1 gene by complementation and showed that the cloned DNA was tightly linked to the chromosomal RGR1 locus. The cloned RGR1 gene suppressed all of the phenotypes caused by the mutation and encoded a 3.6-kilobase poly(A)+ RNA. The RGR1 gene is located on chromosome XII, as determined by pulsed-field gel electrophoresis, and we mapped rgr1 between gal2 and pep3 by genetic analysis. rgr1 was shown to be a new locus. We also determined the nucleotide sequence of RGR1, which was predicted to encode a 123-kilodalton protein. The null mutation resulted in lethality, indicating that the RGR1 gene is essential for growth. On the other hand, a carboxy-terminal deletion of the gene caused phenotypes similar to but more severe than those caused by the original mutation. The amount of reserve carbohydrates was reduced in rgr1 cells. Possible functions of the RGR1 product are discussed.     

Cloning, mapping and gene product identification of rhaT from Escherichia coli K12

Rhamnose utilization requires the function of a specific rhamnose transport system. Rhamnose transport mutants have been isolated and characterized. The structural gene, rhaT, encoding the rhamnose permease has been cloned from Escherichia coli. rhaT has been mapped in the rha locus (87.7 min) by analysis of cotransduction with glpK and other rha markers. The precise location of the gene has been determined by complementation analysis of rhamnose transport mutants transformed with recombinant plasmids containing different fragments of the cloned region. Gene order (counterclockwise) is established as glpK . . . rhaT-rhaR-rhaS-rhaB-rhaA-rhaD. The gene product has been identified by expression of rhaT in a T7 RNA polymerase/promoter system. This 23 kDa protein has been assigned to the rhaT product and has been shown to be located in the cell membrane.     

Genetic complementation by cloned bacteriophage T4 late genes.

Bacteriophage T4 containing nonsense mutations in late genes was found to be genetically complemented by four conjugate T4 genes (7, 11, 23, or 24) located on plasmid or phage vectors. Complementation was at a very low level unless the infecting phage carried a denB mutation (which abolishes T4 DNA endonuclease IV activity). In most experiments, the infecting phage also had a denA mutation, which abolishes T4 DNA endonuclease II activity. Mutations in the alc/unf gene (which allow dCMP-containing T4 late genes to be expressed) further increased complementation efficiency. Most of the alc/unf mutant phage strains used for these experiments were constructed to incorporate a gene 56 mutation, which blocks dCTP breakdown and allows replication to generate dCMP-containing T4 DNA. Effects of the alc/unf:56 mutant combination on complementation efficiency varied among the different T4 late genes. Despite regions of homology, ranging from 2 to 14 kilobase pairs, between cloned T4 genes and infecting genomes, the rate of formation of recombinants after T4 den:alc phage infection was generally low (higher for two mutants in gene 23, lower for mutants in gene 7 and 11). More significantly, when gene 23 complementation had to be preceded by recombination, the complementation efficiency was drastically reduced. We conclude that high complementation efficiency of cloned T4 late genes need not depend on prior complete breakage-reunion events which transpose those genes from the resident plasmid to a late promoter on the infecting T4 genome. The presence of the intact gene 23 on plasmids reduced the yield of T4 phage. The magnitude of this negative complementation effect varied in different plasmids; in the extreme case (plasmid pLA3), an almost 10-fold reduction of yield was observed. The cells can thus be said to have been made partly nonpermissive for this lytic virus by incorporating a part of the viral genome.     

Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli.

A structural gene for isocitrate lyase was isolated from a cosmid containing an ace locus of the Escherichia coli chromosome. Cloning and expression under control of the tac promoter in a multicopy plasmid showed that a 1.7-kilobase-pair DNA segment was sufficient for complementation of an aceA deletion mutation and overproduction of isocitrate lyase. DNA sequence analysis of the cloned gene and N-terminal protein sequencing of the cloned and wild-type enzymes revealed an entire aceA gene which encodes a 429-amino-acid residue polypeptide whose C-terminus is histidine. The deduced amino acid sequence for the 47.2-kilodalton subunit of E. coli isocitrate lyase could be aligned with that for the 64.8-kilodalton subunit of the castor bean enzyme with 39% identity except for limited N- and C-terminal regions and a 103-residue stretch that was unique for the plant enzyme and started approximately in the middle of that peptide.     

Isolation and characterization of the Aspergillus niger trpC gene

The Aspergillus niger trpC gene was isolated by complementation experiments with an Escherichia coli trpC mutant. Plasmid DNA containing the A. niger trpC gene transforms an Aspergillus nidulans mutant strain, defective in all three enzymatic activities of the trpC gene, to Trp+, indicating the presence of a complete and functional trpC gene. Southern blot analysis of DNA from these Trp+ transformants showed that plasmid DNA was present but that this DNA was not integrated at the site of the chromosomal trpC locus. The A. niger trpC gene was localized on the cloned fragment by heterologous hybridization experiments and sequence analysis. These experiments suggest that the organization of the A. niger trpC gene is identical to that of the analogous A. nidulans trpC and the Neurospora crassa trp-1 genes.     

Genetic analysis of the flaA locus of Bacillus subtilis.

We isolated two clones of recombinant lambda bacteriophage with overlapping inserts of Bacillus subtilis chromosomal DNA corresponding to part of the flaA locus. The flaA4 and flaA15 mutations were localized on the physical map by marker rescue experiments. The flaA locus and the flaB (sigD) gene were mapped in transduction crosses, and the order glnA polC flaB flaA was determined. FlaB was linked to polC in transformation crosses.     

Mutagenic potency and specificity of procarbazine in the ad-3 forward-mutation test in growing cultures of heterokaryon 12 of Neurospora crassa.

Procarbazine (Natulan) was tested for its mutagenic potency and specificity in the ad-3 forward-mutation test in heterokaryon 12 (H-12) of Neurospora crassa. In these experiments, procarbazine was a weak mutagen when present in growing cultures but nonmutagenic when conidial suspensions (nongrowing conidia) were treated. A total of 208 ad-3 mutants recovered after exposure of growing cultures of H-12 to 1 mg of procarbazine/ml, and 2 ad-3 mutants of spontaneous origin, were characterized genetically. These tests distinguish among gene/point mutations (ad-3R) at the ad-3A or ad-3B locus, multilocus deletion mutations ([ad-3]IR) covering one or more loci in the ad-3 and immediately adjacent regions, and 3 different classes of multiple-locus mutations: gene/point ad-3 mutations with a recessive lethal mutation elsewhere in the genome (ad-3R + RL), gene/point mutations with a closely linked recessive lethal mutation (ad-3R + RLCL), and multilocus deletion mutations with a closely linked recessive lethal mutation ([ad-3]IR + RLCL). All of the procarbazine-induced ad-3 mutants resulted from gene/point mutations; 92.2% (200/217) resulted from gene/point mutations at the ad-3A or ad-3B locus, and 3.7% (8/217) resulted from gene/point mutations with a recessive lethal mutation elsewhere in the genome. Identical percentages (15.4% [20/130] and 15.4% [12/78]) of the sigma ad-3BR and sigma ad-3AR mutants were leaky, and a high percentage (71.5% [93/130]) of the sigma ad-3BR mutants had nonpolarized complementation patterns. These results indicate that procarbazine-induced ad-3 mutants of Neurospora crassa are composed solely of gene/point mutations (ad-3R) that resulted, predominantly or exclusively, from base-pair substitutions. The Neurospora specific-locus data on procarbazine-induced ad-3 mutants are compared with data from similar experiments with the mouse using the morphological specific-locus assay; marked similarities were found between the mutagenic effects of procarbazine in the 2 specific-locus assays.     

Lethal Mutations Flanking the 68c Glue Gene Cluster on Chromosome 3 of DROSOPHILA MELANOGASTER

We have conducted a genetic analysis of the region flanking the 68C glue gene cluster in Drosophila melanogaster by isolating lethal and semilethal mutations uncovered by deficiencies which span this region. Three different mutagens were used: ethyl methanesulfonate (EMS), ethyl nitrosourea (ENU) and diepoxybutane (DEB). In the region from 68A3 to 68C11, 64 lethal, semilethal, and visible mutations were recovered. These include alleles of 13 new lethal complementation groups, as well as new alleles of rotated, low xanthine dehydrogenase, lethal(3)517 and lethal(3)B76. Six new visible mutations from within this region were recovered on the basis of their reduced viability; all proved to be semiviable alleles of lethal complementation groups. No significant differences were observed in the distributions of lethals recovered using the three different mutagens. Each lethal was mapped on the basis of complementation with overlapping deficiencies; mutations that mapped within the same interval were tested for complementation, and the relative order of the lethal groups within each interval was determined by recombination. The cytological distribution of genes within the 68A3-68C11 region is not uniform: the region from 68A2,3 to 68B1,3 (seven to ten polytene chromosome bands) contains at least 13 lethal complementation groups and the mutation low xanthine dehydrogenase; the adjoining region from 68B1,3 to 68C5,6 (six to nine bands) includes the 68C glue gene cluster, but no known lethal or visible complementation groups; and the interval from 68C5,6 to 68C10,11 (three to five bands) contains at least three lethal complementation groups and the visible mutation rotated. The developmental stage at which lethality is observed was determined for a representative allele from each lethal complementation group.     

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12

Summary Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20°C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.     

Molecular Mapping of a Gene Cluster Flanking the Drosophila Dopa Decarboxylase Gene

Nine lethal complementation groups flanking the Drosophila Dopa decarboxylase (Ddc) gene, have been localized within 100 kb of cloned chromosomal DNA. Six of these complementation groups are within 23 kb of DNA, and all ten complementation groups, including Ddc, lie within 78-82 kb of DNA. The potential significance of this unusually high gene density is discussed.     

Molecular analysis of SNF2 and SNF5, genes required for expression of glucose-repressible genes in Saccharomyces cerevisiae.

The SNF2 and SNF5 genes are required for derepression of SUC2 and other glucose-repressible genes of Saccharomyces cerevisiae in response to glucose deprivation. Previous genetic evidence suggested that SNF2 and SNF5 have functionally related roles. We cloned both genes by complementation and showed that the cloned DNA was tightly linked to the corresponding chromosomal locus. Both genes in multiple copy complemented only the cognate snf mutation. The SNF2 gene encodes a 5.7-kilobase RNA, and the SNF5 gene encodes a 3-kilobase RNA. Both RNAs contained poly(A) and were present in low abundance. Neither was regulated by glucose repression, and the level of SNF2 RNA was not dependent on SNF5 function or vice versa. Disruption of either gene at its chromosomal locus still allowed low-level derepression of secreted invertase activity, suggesting that these genes are required for high-level expression but are not directly involved in regulation. Further evidence was the finding that snf2 and snf5 mutants failed to derepress acid phosphatase, which is not regulated by glucose repression. The SNF2 and SNF5 functions were required for derepression of SUC2 mRNA.     

PEP4 gene of Saccharomyces cerevisiae encodes proteinase A, a vacuolar enzyme required for processing of vacuolar precursors.

The proteinase A structural gene of Saccharomyces cerevisiae was cloned by using an immunological screening procedure that allows detection of yeast cells which are aberrantly secreting vacuolar proteins (J. H. Rothman, C. P. Hunter, L. A. Valls, and T. H. Stevens, Proc. Natl. Acad. Sci. USA, 83:3248-3252, 1986). A second cloned gene was obtained on a multicopy plasmid by complementation of a pep4-3 mutation. The nucleotide sequences of these two genes were determined independently and were found to be identical. The predicted amino acid sequence of the cloned gene suggests that proteinase A is synthesized as a 405-amino-acid precursor which is proteolytically converted to the 329-amino-acid mature enzyme. Proteinase A shows substantial homology to mammalian aspartyl proteases, such as pepsin, renin, and cathepsin D. The similarities may reflect not only analogous functions but also similar processing and intracellular targeting mechanisms for the two proteins. The cloned proteinase A structural gene, even when it is carried on a single-copy plasmid, complements the deficiency in several vacuolar hydrolase activities that is observed in a pep4 mutant. A strain carrying a deletion in the genomic copy of the gene fails to complement a pep4 mutant of the opposite mating type. Genetic linkage data demonstrate that integrated copies of the cloned proteinase A structural gene map to the PEP4 locus. Thus, the PEP4 gene encodes a vacuolar aspartyl protease, proteinase A, that is required for the in vivo processing of a number of vacuolar zymogens.     

High-resolution mapping of the S and Z loci of Phalaris coerulescens.

Self incompatibility (SI) in Phalaris coerulescens is gametophytically determined by two unlinked multi allelic loci (S and Z). Neither the S nor Z genes have yet been cloned. As part of a map-based cloning strategy, high-resolution maps of the S and Z regions were generated from distorted segregating populations using RFLP probes from wheat, barley, oat, and Phalaris. The S locus was delimited to 0.26 cM with two boundary markers (Xwg811 and Xpsr168) and cosegregated with Xbm2 and Xbcd762. Xbcd266 was the closest marker linked to Z (0.9 cM). A high level of colinearity in the S and Z regions was found in both self-incompatible and -compatible species. The S locus was localized to the subcentromere region of chromosome 1 and the Z locus to the long arm end of chromosome 2. Several rice BAC clones orthologous to the S and Z locus regions were identified. This opens the possibility of using the rice genome sequence data to generate more closely linked markers and identify SI candidate genes. These results add further support to the conservation of gene order in the S and Z regions of the grass genomes.