Chromosomal deletions in Streptomyces griseus that remove the afsA locus

We have recently constructed a physical map of the Streptomyces griseus 2247 genome using the restriction enzymes AseI and DraI, which revealed that this strain carries a 7.8 Mb linear chromosome. Based on this map, precise macrorestriction fragment and cosmid maps were constructed for both ends of the chromosome, which localized the afsA gene 150 Kb from the left end. Two afsA ^? mutants were found to have suffered chromosomal deletions that removed the afsA locus. The sizes of the deletions were 20 and 130 Kb at the right end and 180 and 350 kb at the left end, respectively. Hybridization experiments using cosmids carrying a deletion endpoint indicated that the ends of the chromosome in the mutants were fused to form a circular chromosome.     

Genome structures in a lysogenic Rhizobium meliloti system

Summary The genome structure of the temperateRhizobium meliloti phage and the attachment site of this phage on the host chromosome were examined by genetic means. The heat-sensitive mutants used in 2 and 3 point crosses gave a linear chromosome map. There was no evidence for map circularity. The immunity region has a distal position on the phage chromosome. The functional grouping of the used 23 phage mutants was made byin vivo andin vitro complementation tests and 20 cistrons were obtained. The cistrons, near to the immunity region, were identified as early genes, the remaining ones as morphogenetic cistrons. The latter inin vitro complementation tests gave two complementing groups, presumably as head and tail donors. The attachment site of the prophage on the host chromosome was localized by pulse mutagen treatments in synchronously replicating cultures. The sequence of markers are O-str — hs — att ¹⁶⁻³ — T.     

Aluminum-sensitive mutants of Saccharomyces cerevisiae

We are developing budding yeast, Saccharomyces cerevisiae, as a genetic system for the study of tolerance to the trivalent aluminum cation (Al³⁺). We have isolated eight mutants that are more sensitive to Al³⁺ than the wild type. Each mutant represented a different complementation group. A number of the mutants were pleiotropic, and showed defects in other stress responses, changes in tolerance to other metal cations, or abnormal morphology. Two mutants also showed increased dependence on supplemental Mg²⁺ and Ca²⁺. One mutant with a relatively specific sensitivity to Al³⁺ was chosen for molecular complementation. Normal Al³⁺ tolerance was restored by expression of the MAP kinase gene SLT2. Strains carrying deletions of the SLT2 gene, or of the gene for the corresponding MAP kinase–kinase SLK1, showed sensitivity to Al³⁺. These results indicate that the SLT2 MAP kinase signal transduction pathway is required for yeast to sense and respond to Al³⁺ stress. Received: 17 April 1996 / Accepted: 21 October 1996     

Restriction mapping of DNA of temperate Rhizobium meliloti phage 16-3: comparison of genetic and physical maps indicates a long, genetically silent chromosomal arm.

Summary The complete restriction map of DNA (61.57 Kb) of temperate Rhizobium meliloti phage 16-3 has been constructed for enzymes BglII, HindIII, HpaI, KpnI, and a partial map for EcoRI. The strategy employed for mapping included the analysis of double, triple, and partial digests; comparison of wild type and deletion mutants; and detailed analysis of subfragments, exploiting the presence of cohesive ends of the phage. Comparison of the genetic and physical maps indicates that one arm of the chromosome is genetically silent and/or contains nonessential genes.     

Preferential mutagenesis of lacZ integrated at unique sites in the Escherichia coli chromosome

To study the variation in spontaneous mutation frequencies in different chromosomal domains, a mini-Mu-kan-lacZ ⁻transposable element was constructed to insert the lacZ ⁻(Trp570 → Opal) allele into many different loci in the Escherichia coli chromosome. Papillation on MacConkey lactose plates was used to screen for mini-Mu insertion mutants with elevated levels of spontaneous mutagenesis of lacZop → LacZ⁺ candidates were then screened for normal mutation frequencies in other genes. Two different insertion mutants with this enhanced mutagenesis phenotype were isolated from 14 000 colonies, and named plm-1 (preferential lacZmutagenesis) and plm-2. The frequency of LacZ⁻→ LacZ⁺ mutations in these plm mutants was over 400-fold higher than that in isogenic strains containing mini-Mu-kan-lacZop insertions at other loci. Six Lac⁺ reversion (or suppression) mutations obtained from each of the two plm mutants were mapped by P1 transduction and all were found to be linked to the Kan^r gene in the mini-Mu-kan-lacZop, suggesting that a localized mutagenic event is responsible for the preferential mutagenesis. Furthermore, both the LacZ⁺→ LacZ⁻and Kan^r→ Kan^s mutant frequencies of these Lac⁺ revertants were in the range of 10⁻³ to 10⁻², indicating that this putative localized mutagenesis is neither allele nor gene specific. To identify the plm loci, the chromosomal regions flanking the mini-Mu insertion sites were cloned and sequenced. A computer-assisted database search of homologous sequences revealed that the plm-1 locus is identical to the mutS gene; the mini-Mu insertion most probably results in the production of a truncated MutS protein. We suggest that the enhanced lacZ mutation frequency in plm-1 may be associated with an active process involving the putative truncated MutS protein. The DNA sequence of the plm-2 locus matched a putative malate oxidoreductase gene located at 55.5 min of the E. coli chromosome. Received: 1 August 1996 / Accepted: 3 April 1997     

Artificial circularization of the chromosome with concomitant deletion of its terminal inverted repeats enhances genetic instability and genome rearrangement in Streptomyces lividans

The unstable linear chromosome of Streptomyces lividans was circularized by homologous recombination and its terminal inverted repeats deleted. Strains with circularized chromosomes showed no obvious phenotypic disadvantages compared to the wild type. However, they segregated about 20 times more chloramphenicol-sensitive mutants than the wild type (24.3% vs. 1.4%), due to a higher incidence of large deletions. In addition, in all circularized chromosomes amplification of 30–60 kb fragments was observed at the new chromosomal junction, to levels of approximately 10 copies per chromosome. Arginine auxotrophs that arose spontaneously among the progeny of strains with a circularized chromosome showed high-copy-number amplification of the DNA element AUD1, as also seen in mutants of the wild type. These observations demonstrate that the circular form of the Streptomyces chromosome is more unstable than the linear one. Received: 28 July 1996 / Accepted: 18 November 1996     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ⁻ cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon. Received: 8 October 1996 / Accepted: 27 November 1996     

Cloning, mapping and functional characterization of the hemB gene of Pseudomonas aeruginosa , which encodes a magnesium-dependent 5-aminolevulinic acid dehydratase

During tetrapyrrole biosynthesis 5-aminolevulinic acid dehydratase (ALAD) catalyzes the condensation of two molecules of 5-aminolevulinic acid (ALA) to form one molecule of the pyrrole derivative porphobilinogen. In Escherichia coli, the enzyme is encoded by the gene hemB. The hemB gene was cloned from Pseudomonas aeruginosa by functional complementation of an E. coli hemB mutant. An open reading frame of 1011 bp encoding a protein of 336 amino acids (M_r = 37 008) was identified. The gene was mapped to SpeI fragment G and DpnI fragment G of the P. aeruginosa chromosome, corresponding to the 10 to 12 min region of the new map or 19 to 22 min interval of the old map. The 5′ end of the hemB mRNA was determined and the −10 and −35 regions of a potential σ⁷⁰-dependent promoter were localized. No obvious regulation of the hemB gene by oxygen, nitrate, heme or iron was detected. Alignment of the amino acid sequences deduced from hemB revealed a potential metal-binding site and indicated that the enzyme is Mg²⁺-dependent. P. aeruginosa hemB was overexpressed in an E. coli hemB mutant using the phage T7 RNA polymerase system and its Mg²⁺-dependent activity was directly demonstrated. Received: 11 July 1997 / Accepted: 9 October 1997     

Nicotiana chloroplast genome

Summary N. accuminata has a chloroplast genome of 171 Kb which is larger than 160 Kb reported for N. tabacum. A physical map of the former has been constructed by SalI, BglI, and PvuII enzymes in comparison with N. tabacum. They both share identical restriction map except in the extra segment of N. accuminata. This extra segment is located on the right-hand border of an inverted repeat in the large-single copy region of N. accuminata. It contains the following restriction sites: two for BglI and SmaI; three for SalI and XhoI. The size of inverted repeat measured by electron microscope is 22.67±0.78 Kb and 19.28±0.61 Kb for N. accuminata and N. tabacum, respectively. This is the first case where a larger inverted repeat region accompanied by a larger genome size was found among over 30 Nicotiana species studied thus far.     

The Pik m gene, conferring stable resistance to isolates of Magnaporthe oryzae , was finely mapped in a crossover-cold region on rice chromosome 11

The Pik ^m gene in rice confers a high and stable resistance to many isolates of Magnaporthe oryzae collected from southern China. This gene locus was roughly mapped to the long arm of rice chromosome 11 with restriction fragment length polymorphic (RFLP) markers in the previous study. To effectively utilize the resistance, a linkage analysis was performed in a mapping population consisting of 659 highly susceptible plants collected from four F₂ populations using the publicly available simple sequence repeat (SSR) markers. The result showed that the locus was linked to the six SSR markers and defined by RM254 and RM144 with ≈13.4 and ≈1.2 cM, respectively. To fine map this locus, additional 10 PCR-based markers were developed in a region flanked by RM254 and RM144 through bioinformatics analysis (BIA) using the reference sequence of cv. Nipponbare. The linkage analysis with these 10 markers showed that the locus was further delimited to a 0.3-cM region flanked by K34 and K10, in which three markers, K27, K28, and K33, completely co-segregated with the locus. To physically map the locus, the Pik ^m -linked markers were anchored to bacterial artificial chromosome clones of the reference cv. Nipponbare by BIA. A physical map spanning ≈278 kb in length was constructed by alignment of sequences of the clones anchored by BIA, in which only six candidate genes having the R gene conserved structure, protein kinase, were further identified in an 84-kb segment.     

The structure of the chloroplast genome in members of the genus Asparagus

 In a previous study we constructed a physical map of the chloroplast DNA (ctDNA) of garden asparagus (Asparagus officinalis L. cv ‘Mary Washington 500W’; Lee et al. 1996). In the present study we have constructed and compared HindIII and XhoI restriction maps of the ctDNAs of eight species of Asparagus: namely, A. officinalis, A. schoberioides, A. cochinchinensis, A. plumosus, A. falcatus, A. sprengeri, A. virgatus and A. asparagoides. The ctDNA of A. officinalis has 32 and 23 sites that are recognized by HindIII and XhoI, respectively. Taking the physical map of the ctDNA of A. officinalis as a standard, we found that the ctDNAs of A. falcatus, A. sprengeri, and A. asparagoides each had one additional HindIII site and lacked one XhoI site. We also detected two relatively large deletions of nucleotides in the ctDNA from A. cochinchinensis by sequencing analysis. Both of these deletions were located in a non-coding region between the ndhC and trnV genes and were 95 bp and 347 bp in length, respectively. The regions around the deletions exhibited strong homology, and short direct-repeat sequences were detected at the borders of the deletions, an indication that these deletions were the result of intramolecular recombination mediated by the direct repeats. Received: 16 June 1997 / Accepted: 17 July 1997     

Genetic and physical mapping of an hydrogenase gene cluster from Rhodobacter capsulatus

Summary An hydrogenase-deficient (Hup^–) mutant of Rhodobacter capsulatus was obtained by adventitious insertion of IS21 DNA into an hydrogenase structural gene (hup) of the wild-type strain 1310. The resulting Hup^– mutant, strain JP91, selected by its inability to grow autotrophically (Aut^– phenotype) together with other Hup^– mutant strains obtained by classical ethyl methane sulphonate mutagenesis were used in R plasmid-mediated conjugation experiments to map the hup/aut loci on the chromosome of R. capsulatus. The hup genes tested in this study were found to cluster on the chromosome in the proximity of the his-1 marker. A cluster of hup genes comprising the structural genes was isolated from a gene bank constructed in the cosmid vector pHC79 with 40 kb insert DNA. The clustered hup genes, characterized by hybridization studies and complementation analyses of the R. capsulatus Hup^– mutants, span 15–20 kb of DNA.     

A further characterization of the cinnamon gene in Drosophila melanogaster

Summary Two mutants of Saccharomyces cerevisiae lacking pyruvate kinase (EC.2.7.1.40) are described. The mutations are recessive, segregate 2⁺:2^- in tetrads and do not complement each other. Single-step spontaneous revertants, isolated on glucose plates, get back pyruvate kinase activity. The enzymes from various revertants display a wide spectrum of specific activity, thermolability and altered affinity for ligands such as P-enol pyruvate, ADP and fructose 1,6-diphosphate. The mutants produce materials crossreacting to the rabbit antibody raised against purified pyruvate kinase from the wild type yeast. These mutations thus define the structural gene of pyruvate kinase.The mutations map on the leaft arm of chromosome I and form a single complementation group with five other pyruvate kinase mutations in the pyk1 gene that was earlier suggested to be a regulatory locus controlling the synthesis of this enzyme. A comparative study of these mutants has been made with the structural mutants described here.     

Sesqui-Ds , the chromosome-breaking insertion at bz-m1 , links double Ds to the original Ds element

The bz-m1 mutation in maize was one of the first to arise by direct transposition of the chromosome-breaking Ds element from its original or `standard' location in chromosome 9S to a known locus in the same chromosome arm. Thus, elucidation of its structure should shed light on the nature of the original Ds element described by McClintock in 1948. The Ds insertion in bz-m1 has been reported to be only 1.2 kb long – much shorter than other chromosome-breaking Ds elements that have been described. We have characterized here the Ds element in our bz-m1 stocks and have confirmed by genetic and molecular tests that, in the presence of Ac, it acts as a chromosome breaker. The Ds insertion at bz-m1 is 1260 bp long. Besides its normal 5′ and 3′ ends, it contains an internal 3′ end at the same junction as the chromosome-breaking double Ds element that has been found in several sh mutations. Thus, it appears to have arisen from the 4.1-kb double Ds by internal deletion of 2.9 kb. Because the element has lost one internal 5′ end, but retains the chromosome-breaking properties of double Ds, we have named it sesqui-Ds (sDs). The origin, structure and properties of sDs vis-à-vis double Ds support the hypothesis that double Ds corresponds to the chromosome-breaking Ds element at the `standard' location in 9S. Received: 10 March 1997 / Accepted: 2 May 1997     

The mapping of phytochrome genes and photomorphogenic mutants of tomato

The map positions of five previously described phytochrome genes have been determined in tomato (Lycopersicon esculentum Mill.) The position of the yg-2 gene on chromosome 12 has been confirmed and the classical map revised. The position of the phytochrome A (phy A)-deficient fri mutants has been refined by revising the classical map of chromosome 10. The position of the PhyA gene is indistinguishable from that of the fri locus. The putative phyB1-deficient tri mutants were mapped by classical and RFLP analysis to chromosome 1. The PhyB1 gene, as predicted, was located at the same position. Several mutants with the high pigment (hp) phenotype, which exaggerates phytochrome responses, have been reported. Allelism tests confirmed that the hp-2 mutant is not allelic to other previously described hp (proposed here to be called hp-1) mutants and a second stronger hp-2 allele (hp-2 ^j ) was identified. The hp-2 gene was mapped to the classical, as well as the RFLP, map of chromosome 1. Received: 24 May 1996 / Accepted: 14 June 1996     

Chromosome mapping in Alealigenes eutrophus CH34

Mutants and mobilizing plasmids were developed as genetic tools in Alcaligenes eutrophus CH34. In order to map the chromosome, spontaneous and ethyl methane sulphonate (EMS)-induced mutants (mostly auxotrophs) were isolated. Another source of mutants was provided by the phenomenon of temperature-induced mortality and mutagenesis that is observed at 37° C and is characteristic of many metallotolerant strains of A. eutrophus. Plasmid pULB113 (RP4::miniMu) was used to map the available mutations. Twenty-five loci were ordered in a circular map. pMOL50, a rearranged derivative of plasmid pMOL28, which was obtained in a survivor at 37° C and displayed chromosome mobilizing activity (Cma+), was also used to mobilize chromosomal markers: resulting linkages were stronger than with pULB113, allowing confirmation of the circularity of the A. eutrophus CH34 chromosome with a small number of crosses.     

Isolation of novel pre-mRNA splicing mutants of Schizosaccharomyces pombe

 New prp (pre-mRNA processing) mutants of the fission yeast Schizosaccharomyces pombe were isolated from a bank of 700 mutants that were either temperature sensitive (ts^-) or cold sensitive (cs^-) for growth. The bank was screened by Northern blot analysis with probes complementary to S. pombe U6 small nuclear RNA (sn RNA), the gene for which has a splicesomal (mRNA-type) intron. We identified 12 prp mutants that accumulated the U6 snRNA precursor at the nonpermissive temperature. All such mutants were also found to have defects in an early step of TFIID pre-mRNA splicing at the nonpermissive temperature. Complementation analyses showed that seven of the mutants belong to six new complementation groups designated as prp8 and prp10-prp14, whereas the five other mutants were classified into the known complementation groups prp1, prp2 and prp3. Interestingly, some of the isolated prp mutants produced elongated cells at the nonpermissive temperature, which is a phenotype typical of cell division cycle (cdc) mutants. Based on these findings, we propose that some of the wild-type products from these prp ⁺ genes play important roles in the cellular processes of pre-mRNA splicing and cell cycle progression. Received: 15 April 1996/Accepted: 9 July 1996