Sulfonamide Resistance Gene in a Transferable R Plasmid of Pasteurella piscicida

The sulfonamide resistance (SA^r) determinant was cloned from a transferable R plasmid of Pasteurella piscicida, pSP9351, and the sequence was determined. The resistance gene (pp-sul) was localized to an approximately 1-kb region that includes the PstI-EcoRI site in the restriction map. An open reading frame coding a sul II-type gene composed of 810 nucleotides was identified. A direct repeat sequence was shown in the 5′ flanking region of pp-sul, and a plasmid recombinational event may have occurred during the construction of pSP9351. In the 3′ flanking region of the gene, a sequence homologous to the 5′ noncoding sequence of the trimethoprim resistance gene, dhfr IX was found.     

Fine Mapping of Xa2, a Bacterial Blight Resistance Gene in Rice

The rice bacterial blight resistance gene, Xa2, confers resistance to T7147 of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae. It is located on the long arm of chromosome 4. Here, we report the fine mapping of Xa2 by genetic recombination analysis with simple sequence repeat (SSR) markers according to the genome sequence. Two F₂ populations are constructed to localize Xa2. In a primary analysis with 136 random F₂ plants of Zhenzhuai/IRBB2, it was found that Xa2 was located in approximately 20 cM region. To accurately determine the locus of Xa2, 120 new SSR markers were developed in this region by screening the sequence. Twelve new SSR markers were successfully used in genetic recombination analysis in IR24/IRBB2 population, while 20 in ZZA/IRBB2 population. We found that the nearest SSR markers to Xa2 are HZR950-5 and HZR970-4, which cover approximately 190-kb region. The sequence analysis of this 190-kb region revealed the presence of a homologous sequence of leucine rich repeat (LRR)-kinase. These results are very useful for transferring or pyramiding Xa2 by molecular marker-assistant selection in rice breeding programs and for cloning Xa2 by map-based cloning in combination with a long-range PCR strategy. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users.     

Novel Tetracycline Resistance Determinant Isolated from an Environmental Strain of Serratia marcescens

Resistances to tetracycline and mercury were identified in an environmental strain of Serratia marcescens isolated from a stream highly contaminated with heavy metals. As a step toward addressing the mechanisms of coselection of heavy metal and antibiotic resistances, the tetracycline resistance determinant was cloned in Escherichia coli. Within the cloned 13-kb segment, the tetracycline resistance locus was localized by deletion analysis and transposon mutagenesis. DNA sequence analysis of an 8.0-kb region revealed a novel gene [tetA(41)] that was predicted to encode a tetracycline efflux pump. Phylogenetic analysis showed that the TetA(41) protein was most closely related to the Tet(39) efflux protein of Acinetobacter spp. yet had less than 80% amino acid identity with known tetracycline efflux pumps. Adjacent to the tetA(41) gene was a divergently transcribed gene [tetR(41)] predicted to encode a tetracycline-responsive repressor protein. The tetA(41)-tetR(41) intergenic region contained putative operators for TetR(41) binding. The tetA(41) and tetR(41) promoters were analyzed using lacZ fusions, which showed that the expression of both the tetA(41) and tetR(41) genes exhibited TetR(41)-dependent regulation by subinhibitory concentrations of tetracycline. The apparent lack of plasmids in this S. marcescens strain, as well as the presence of metabolic genes adjacent to the tetracycline resistance locus, suggested that the genes were located on the S. marcescens chromosome and may have been acquired by transduction. The cloned Tet 41 determinant did not confer mercury resistance to E. coli, confirming that Tet 41 is a tetracycline-specific efflux pump rather than a multidrug transporter.     

A new family of polymorphic metallothionein-encoding genes MTH1 (CUP1) and MTH2 in Saccharomyces cerevisiae.

By pulsed-field gel electrophoresis of chromosomal DNA and hybridization with a cloned MTH1 (CUP1) gene, we determined the locations of metallothionein-encoding gene sequences on chromosomes in monosporic cultures of 76 natural strains of Saccharomyces cerevisiae. Most of the strains (68) exhibited a previously known location for the MTH sequence on chromosome (chr.) VIII. Seven strains (resistant or sensitive to Cu2+) showed a MTH sequence in a new locus, MTH2, on chr. XVI. One strain carried an MTH locus on both chromosomes VIII and XVI. Restriction fragment and Southern blot analyses showed that the two MTH loci were very closely related. The strains displayed heterogeneity in the size and structure of their MTH2 locus. The length of the repeat unit of MTH2 varied: a 1.9-kb or 1.7-kb unit was found, instead of the 2.0-kb unit of the MTH1 locus. The most resistant strain (resistant to 1.2 mM CuSO4) contained a 0.9-kb repeat unit in addition to those of 1.9 kb and 1.7 kb. All three sensitive (to over 0.3 mM CuSO4) strains with an mth2 locus had a repeat unit of 1.9 kb or 1.7 kb, suggesting the presence of at least two copies of the MTH2 gene, with one always being in the junction area outside of the repeat unit. A monogenic tetrad segregation of 2:2 was usually found in crosses of resistant MTH2 and sensitive mth2 strains. Hybrids between strains with different MTH loci in all combinations showed low ascospore viability, suggesting that the complete lack of an MTH locus may lead to the death of segregants on YPD medium. The MTH1 and MTH2 loci were exchangeable. Strains with a high level of Cu2+ resistance were also resistant to Cd2+. However, these two properties did not cosegregate in heterozygotic hybrids.     

Chromosomal heteromorphism linked to the mating type locus of the oomycetePhytophthora infestans

The mating type locus of the oomycete,Phytophthora infestans, is embedded in a region of DNA that displays distorted and non-Mendelian segregation. By using DNA probes linked to the mating type locus to genetically and physically characterize that region, a large zone of chromosomal heteromorphism was detected. LocusS1 was shown to represent a tandemly repeated array of DNA that was typically present in a hemizygous state in A1 isolates while being absent from A2 isolates. The analysis of the parents and progeny of seven crosses indicated that the tandem array was linked in cis to the A1-determining allele of the mating type locus. A worldwide survey of genotypically diverse field isolates ofP. infestans indicated thatS1 was present in each of 48 isolates of the A1 mating type that were tested, but was absent in 46 of 47 A2 strains. Physical analysis ofS1 indicated that the tandemly repeated DNA sequence spanned about 300 kb and had evolved from a 1.35-kb monomer. Internal deletions occurred withinS1 during sexual propagation. This and other mutations apparently contributed to a high degree of polymorphism within theS1 array.     

Cloning and characterization of the glutamate 1-semialdehyde aminomutase gene from Xanthomonas campestris pv. phaseoli

The gene from Xanthomonas campestris pv. phaseoli for glutamate 1-semialdehyde (GSA) aminomutase, which is involved in the C5 pathway for synthesis of -aminolevulinic acid (ALA), was cloned onto a multicopy plasmid, pUC18, by the complementation of an ALA-deficient mutant (hemL) of Escherichia coli. Subcloning of deletion fragments from the initial 3.5-kb chromosomal fragment allowed the isolation of a 1.7-kb fragment which could complement the hemL mutation. Nucleotide sequence analysis of the 1.7-kb DNA fragment revealed an open reading frame (ORF) that is located downstream from a potential promoter sequence and a ribosome-binding site. The ORF encodes a polypeptide of 429 amino acid residues, and the deduced molecular mass of this polypeptide is 45,043 Da. The amino acid sequence shows a high degree of homology to the HemL proteins from other organisms, and a putative binding site for pyridoxal 5-phosphate is conserved. Correspondence to: Y. Murooka     

Molecular cloning and characterization of the genetic determinants that express the complete Shigella serotype D (Shigella sonnei) lipopolysaccharide in heterologous live attenuated vaccine strains

The genetic determinants for the complete Shigella sonnei lipopolysaccharide (LPS) have been cloned, characterized by restriction mapping, and expressed in heterologous genetic backgrounds, including Salmonella typhi and Vibrio cholerae live attenuated vaccine strains. The rfb/rfc locus encoding the polymerized serotype-specific O polysaccharide was mapped within 23 kb of DNA isolated from S. sonnei virulence plasmid pWR105. A highly similar chromosomal DNA sequence was identified by Southern hybridization analysis in Plesiomonas shigelloides known to have the same O serotype specificity as S. sonnei. Expression studies of the rfb/rfc locus have shown that S. sonnei. O polysaccharide is covalently bound to LPS cores of both the K-12 and RI types, but neither to Salmonella (Ra-type) nor to V. cholerae O1 cores. In order to express a compatible core structure in the latter organisms, chromosomal rfa loci encoding R1-type LPS were isolated from both an Escherichia coli R1 strain (rfaR1) and from S. sonnei (rfd_sonnei). Restriction mapping and functional analysis of cloned DNA allowed us to localize the rfaR1 locus and to orient it with respect to the neighbouring cysE chromosomal marker. A high degree of sequence similarity was found at the DNA level between rfa loci of enterobacterial species characterized by Ri-type LPS. Co-expression studies involving S. sonnei rfb/rfc and rfa loci propagated on compatible plasmids have shown that, at most, 13 to 14 kb of r/api DNA are required for the expression of complete phase-l-like S. sonnei LPS in E. coli K-12 and S. typhi, whereas an adjacent region of about 3.5 kb is needed in the more stringent host, V. cholerae, S. sonnei O antigen expressed in a V. eholerae recombinant vaccine strain is present on the cell surface in a form suitable for the induction of a specific antibody response in vaccinated rabbits.     

Fine physical mapping of the rice stripe resistance gene locus, Stvb-i

The Stvb-i gene confers stripe disease resistance to rice. For positional cloning, we constructed a physical map spanning 1.8-cM distance between flanking markers, consisting of 18 bacterial artificial chromosome (BAC) clones, around the Stvb-i locus on rice chromosome 11. The 18 clones were isolated by screening a BAC library derived from a japonica cultivar, Shimokita, with three Stvb-i-linked RFLP markers and DraI-digested DNAs of a yeast artificial chromosome (YAC) clone. The results of Southern hybridization and restriction enzyme analyses indicated that these BAC clones are contiguous and cover about a 700-kb region containing the Stvb-i allele. Utilizing end and internal fragments of the BAC insert DNAs, 33 molecular markers were generated within a small chromosomal region including the Stvb-i locus. Genotyping analysis with these markers for a resistant cultivar and four nearby recombinants selected from 120 F₂ individuals indicated that Stvb-i is contained within an approximately 286-kb region covered with two overlapping BAC clones. Received: 25 August 1999 / Accepted: 16 November 1999     

Analysis of a histone H2A variant from fission yeast: evidence for a role in chromosome stability

We have isolated and characterised the pht1 gene from the fission yeast Schizosaccharomyces pombe. The sequence of the predicted translation product has revealed a striking similarity to the family of H2A.F/Z histone variant proteins, which have been found in a variety of different organisms. Cells deleted for the pht1 gene locus grow slowly, exhibit an altered colony morphology, increased resistance to heat shock and show a significant decrease in the fidelity of segregation of an S. pombe minichromosome. We propose that the histone H2A variant encoded by the pht1 gene is important for chromosomal structure and function, possibly including a role in controlling the fidelity of chromosomal segregation during mitosis.     

Fertility restorer locus Rf1 of sorghum (Sorghum bicolor L.) encodes a pentatricopeptide repeat protein not present in the colinear region of rice chromosome 12

With an aim to clone the sorghum fertility restorer gene Rf1, a high-resolution genetic and physical map of the locus was constructed. The Rf1 locus was resolved to a 32-kb region spanning four open reading frames: a plasma membrane Ca²⁺-ATPase, a cyclin D-1, an unknown protein, and a pentatricopeptide repeat (PPR13) gene family member. An ~19-kb region spanning the cyclin D-1 and unknown protein genes was completely conserved between sterile and fertile plants as was the sequence spanning the coding region of the Ca²⁺-ATPase. In contrast, 19 sequence polymorphisms were located in an ~7-kb region spanning PPR13, and all markers cosegregated with the fertility restoration phenotype. PPR13 was predicted to encode a mitochondrial-targeted protein containing a single exon with 14 PPR repeats, and the protein is classified as an E-type PPR subfamily member. To permit sequence-based comparison of the sorghum and rice genomes in the Rf1 region, 0.53 Mb of sorghum chromosome 8 was sequenced and compared to the colinear region of rice chromosome 12. Genome comparison revealed a mosaic pattern of colinearity with an ~275-kb gene-poor region with little gene conservation and an adjacent, ~245-kb gene-rice region that is more highly conserved between rice and sorghum. Despite being located in a region of high gene conservation, sorghum PPR13 was not located in a colinear position on rice chromosome 12. The present results suggest that sorghum PPR13 represents a potential candidate for the sorghum Rf1 gene, and its presence in the sorghum genome indicates a single gene transposition event subsequent to the divergence of rice and sorghum ancestors.An erratum to this article can be found at     

Investigation into the nature of a Bacillus promoter cloned into a promoter-probe plasmid

The alpha-amylase-coding gene (amy) of Bacillus amyloliquefaciens NCP1 was cloned into the Bacillus subtilis promoter probe vector pPL603b.1, using a BglII digest of chromosomal DNA. The resulting plasmid, pVC102, was shown to have a BglII site within the insert. It was determined that this was the result of the fortuitous co-cloning of 2.88-kb and 0.92-kb BglII fragments separated in NCP1 DNA by approx. 3 kb. Unexpectedly, this co-cloning was readily repeated. Subcloning showed that while the 2.88-kb amy-bearing fragment was sufficient for amylase production, it might not have been capable of promoting sufficient levels of chloramphenicol resistance under the conditions used in the cloning experiments. The promoter on the 0.92-kb BglII fragment was more efficient, although its sequence differed from the canonical promoter sequence recognised by B. subtilis RNA polymerase E.sigma 43. As other promoter-bearing fragments from NCP1 DNA operated equally efficiently when cloned into pPL603b.1, the reason for the repeated co-cloning of the 2.88-kb and 0.92-kb NCPI BglII fragments may well be due to structural parameters, whereby certain nucleotide sequences are more readily cloned than others.     

Genetic and physical mapping of <Emphasis Type="Italic">Pi36</Emphasis>(t), a novel rice blast resistance gene located on rice chromosome 8

Blast resistance in the indica cultivar (cv.) Q61 was inherited as a single dominant gene in two F₂ populations, F₂-1 and F₂-2, derived from crosses between the donor cv. and two susceptible japonica cvs. Aichi Asahi and Lijiangxintuanheigu (LTH), respectively. To rapidly determine the chromosomal location of the resistance (R) gene detected in Q61, random amplified polymorphic DNA (RAPD) analysis was performed in the F₂-1 population using bulked-segregant analysis (BSA) in combination with recessive-class analysis (RCA). One of the three linked markers identified, BA1126₅₅₀, was cloned and sequenced. The R gene locus was roughly mapped on rice chromosome 8 by comparison of the BA1126₅₅₀ sequence with rice sequences in the databases (chromosome landing). To confirm this finding, seven known markers, including four sequence-tagged-site (STS) markers and three simple-sequence repeat (SSR) markers flanking BA1126₅₅₀ on chromosome 8, were subjected to linkage analysis in the two F₂ populations. The locus was mapped to a 5.8 cM interval bounded by RM5647 and RM8018 on the short arm of chromosome 8. This novel R gene is therefore tentatively designated as Pi36(t). For fine mapping of the Pi36(t) locus, five additional markers including one STS marker and four candidate resistance gene (CRG) markers were developed in the target region, based on the genomic sequence of the corresponding region of the reference japonica cv. Nipponbare. The Pi36(t) locus was finally localized to an interval of about 0.6 cM flanked by the markers RM5647 and CRG2, and co-segregated with the markers CRG3 and CRG4. To physically map this locus, the Pi36(t)-linked markers were mapped by electronic hybridization to bacterial artificial chromosome (BAC) or P1 artificial chromosome (PAC) clones of Nipponbare, and a contig map was constructed in silico through Pairwise BLAST analysis. The Pi36(t) locus was physically delimited to an interval of about 17.0 kb, based on the genomic sequence of Nipponbare.     

Genetic control of translational fidelity in yeast: molecular cloning and analysis of the allosuppressor gene SAL3

Summary The fidelity of translation in the yeast Saccharomyces cerevisiae is controlled by a number of gene products. We have begun a molecular analysis of such genes and here describe the cloning and analysis of one of these genes, SAL3. Mutations at this locus, and at least four other unlinked loci (designated SAL1-SAL5), increase the efficiency of the tRNA ochre suppressor SUQ5, and are thus termed allosuppressors. We have cloned the SAL3 gene from a yeast genomic library by complementation of a sal3 mutation. Integration of the cloned sequence into the yeast chromosome was used to confirm that the SAL3 gene had been cloned. SAL3 gene is present in a single copy in the yeast genome, is transcribed into a 2.3-kb polyadenylated mRNA and encodes a protein of M_r 80 000. The size of the SAL3 gene product strongly suggests that it is not a ribosomal protein.     

Identification and characterization of a chlorate-resistant mutant of Arabidopsis thaliana with mutations in both nitrate reductase structural genes NIA1 and NIA2

Mutant plants defective in the assimilation of nitrate can be selected by their resistance to the herbicide chlorate. In Arabidopsis thaliana, mutations at any one of nine distinct loci confer chlorate resistance. Only one of the CHL genes, CHL3, has been shown genetically to be a nitrate reductase (NR) structural gene (NIA2) even though two NR genes (NIA1 and NIA2) have been cloned from the Arabidopsis genome. Plants in which the NIA2 gene has been deleted retain only 10% of the wildtype shoot NR activity and grow normally with nitrate as the sole nitrogen source. Using mutagenized seeds from the NIA2 deletion mutant and a modified chlorate selection protocol, we have identified the first mutation in the NIA1 NR structural gene. nia1, nia2 double mutants have only 0.5% of wild-type shoot NR activity and display very poor growth on media with nitrate as the only form of nitrogen. The nial-1 mutation is a single nucleotide substitution that converts an alanine to a threonine in a highly conserved region of the molybdenum cofactor-binding domain of the NR protein. These results show that the NIA1 gene encodes a functional NR protein that contributes to the assimilation of nitrate in Arabidopsis.     

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.     

Characterization of an autonomously replicating region from the Streptomyces lividans chromosome.

The chromosomal replication origin of the plasmidless derivative (TK21) from Streptomyces lividans 66 has been cloned as an autonomously replicating minichromosome (pSOR1) by using the thiostrepton resistance gene as a selectable marker. pSOR1 could be recovered as a closed circular plasmid which shows high segregational instability. pSOR1 was shown to replicate in Streptomyces coelicolor A3(2) and in S. lividans 66 and hybridized with DNA from several different Streptomyces strains. Physical mapping revealed that oriC is located on a 330-kb AseI fragment of the S. coelicolor A3(2) chromosome. DNA sequence analyses showed that the cloned chromosomal oriC region contains numerous DnaA boxes which are arranged in two clusters. The preferred sequence identified in the oriC region of Escherichia coli and several other bacteria is TTATCCACA. In contrast, in S. lividans, which has a high GC content, the preferred sequence for DnaA boxes appears to be TTGTCCACA.     

Fine mapping and identification of tightly linked DNA markers of blast resistance gene <Emphasis Type="Italic">Pia</Emphasis> by using an introgression line

An introgression line (INL) for a major rice blast resistance gene, Pia, was developed, with the genetic background of a blast susceptible variety, US-2. The reaction pattern of the INL was characterized by using 20 standard blast isolates from the Philippines. The introgression of the Pia gene was confirmed by DNA markers on the short arm of chromosome 11 where Pia was previously mapped. A genome-wide DNA marker survey revealed that most of the chromosomal regions were US-2 type. By using an F2 population derived from a cross between the INL and US-2, the chromosomal location of the Pia locus was mapped between RM26281 and RM3701. For fine mapping of the Pia locus, five additional markers were developed based on the genomic sequence of the corresponding region of a japonica-type variety, Nipponbare. The candidate region of Pia was delimited between two DNA markers, RM26281 and 82N19365, corresponding to a 140 kb region on the Nipponbare genome sequence. We obtained three DNA markers within this region. The developed INL, information on the map position of Pia, and DNA markers developed in the candidate region of the Pia locus are useful tools for blast resistance studies and a marker-aided breeding strategy.