Physical map of a conditionally dispensable chromosome in Nectria haematococca mating population VI and location of chromosome breakpoints

Certain isolates of the plant pathogenic fungus Nectria haematococca mating population (MP) VI contain a 1.6-Mb conditionally dispensable (CD) chromosome carrying the phytoalexin detoxification genes MAK1 and PDA6-1. This chromosome is structurally unstable during sexual reproduction. As a first step in our analysis of the mechanisms underlying this chromosomal instability, hybridization between overlapping cosmid clones was used to construct a map of the MAK1 PDA6-1 chromosome. The map consists of 33 probes that are linked by 199 cosmid clones. The polymerase chain reaction and Southern analysis of N. haematococca MP VI DNA digested with infrequently cutting restriction enzymes were used to close gaps and order the hybridization-derived contigs. Hybridization to a probe extended from telomeric repeats was used to anchor the ends of the map to the actual chromosome ends. The resulting map is estimated to cover 95% of the MAK1 PDA6-1 chromosome and is composed of two ordered contigs. Thirty-eight percent of the clones in the minimal map are known to contain repeated DNA sequences. Three dispersed repeats were cloned during map construction; each is present in five to seven copies on the chromosome. The cosmid clones representing the map were probed with deleted forms of the CD chromosome and the results were integrated into the map. This allowed the identification of chromosome breakpoints and deletions.     

Construction of an Unstable Ring-X Chromosome Bearing the Autosomal Dopa Decarboxylase Gene in Drosophila melanogaster and Analysis of Ddc Mosaics

An unstable Ring-X chromosome, Ddc⁺- Ring-X carrying a cloned Dopa decarboxylase (Ddc) encoding segment was constructed. The construction involved a double recombination event between the unstable Ring-X, R(1)w^vC and a Rod-X chromosome which contained a P-element mediated Ddc ⁺ insert. The resulting Ddc⁺-Ring-X chromosome behaves similarly to the parent chromosome with respect to somatic instability. The Ddc⁺-Ring-X chromosome was used to generate Ddc mosaics. Analyses of Ddc mosaics revealed that while there was no absolute requirement for the Ddc ⁺ expression in either the epidermis or the nervous system, very large mutant clones did affect the viability of the mosaic.     

Genetic Analysis of the Role of Phytoalexin Detoxification in Virulence of the Fungus Nectria haematococca on Chickpea (Cicer arietinum)

Chickpea (Cicer arietium L.) produces the antimicrobial compounds (phytoalexins) medicarpin and maackiain in response to infection by microorganisms. Nectria haematococca mating population (MP) VI, a fungus pathogenic on chickpea, can metabolize maackiain and medicarpin to less toxic products. These reactions are thought to be detoxification mechanisms in N. haematococca MP VI and required for pathogenesis by this fungus on chickpea. In the present study, these hypotheses were tested by examining the phenotypes of progeny from crosses of the fungus that segregated for genes (Mak genes) controlling phytoalexin metabolism. Mak1 and Mak2, two genes that individually confer the ability to convert maackiain to its 1a-hydroxydienone derivative, were linked to higher tolerance of the phytoalexins and high virulence on chickpea. These results indicate that this metabolic reaction is a mechanism for increased phytoalexin tolerance in the fungus, which thereby allows a higher virulence on chickpea. Mak3, a gene conferring the ability to convert maackiain to its 6a-hydroxypterocarpan derivative, also increased tolerance to maackiain in strains which carried it; however, the contribution of Mak3 to the overall level of pathogenesis could not be evaluated because most progeny from the cross segregating for this gene were low in virulence. Thus, metabolic detoxification of phytoalexins appeared to be necessary, as demonstrated in the Mak1 and Mak2 crosses, but not sufficient by itself, as in the Mak3 cross, for high virulence of N. haematococca MP VI on chickpea.     

Map-based cloning of the tomato genomic region that spans the Sw-5 tospovirus resistance gene in tomato

Two yeast artificial chromosomes (YACs) containing genomic DNA from tomato have been isolated using CT220, an RFLP marker which is tightly linked to the tomato spotted wilt virus resistance gene, Sw-5. High-resolution mapping of the YAC ends and internal YAC probes demonstrated that one of the YAC clones, TY257 (400?kb), spans Sw-5. By chromosome walking in a cosmid library, the position of Sw-5 has been delimited within the YAC to a maximal chromosomal segment of 100?kb, spanned by nine overlapping cosmid clones.     

Three Genes for Metabolism of the Phytoalexin Maackiain in the Plant Pathogen Nectria haematococca: Meiotic Instability and Relationship to a New Gene for Pisatin Demethylase

Some isolates of the plant-pathogenic fungus Nectria haematococca mating population (MP) VI metabolize maackiain and medicarpin, two antimicrobial compounds (phytoalexins) synthesized by chickpea (Cicer arietinum L.). The enzymatic modifications by the fungus convert the phytoalexins to less toxic derivatives, and this detoxification has been proposed to be important for pathogenesis on chickpea. In the present study, loci controlling maackiain metabolism (Mak genes) were identified by crosses among isolates of N. haematococca MP VI that differed in their ability to metabolize the phytoalexin. Strains carrying Mak1 or Mak2 converted maackiain to 1a-hydroxymaackiain, while those with Mak3 converted it to 6a-hydroxymaackiain. Mak1 and Mak2 were unusual in that they often failed to be inherited by progeny. Mak1 was closely linked to Pda6, a new member in a family of genes in N. haematococca MP VI that encode enzymes for detoxification of pisatin, the phytoalexin synthesized by garden pea. Like Mak1, Pda6 was also transmitted irregularly to progeny. Although the unusual meiotic behaviors of some Mak genes complicate genetic analysis, identification of these genes should afford a more through evaluation of the role of phytoalexin detoxification in the pathogenesis of N. haematococca MP VI on chickpea.     

The genomic structure of human BTK,the defective gene in X-linked agammaglobulinemia

It has recently been demonstrated that mutations in the gene for Bruton's tyrosine kinase (BTK) are responsible for X-linked agammaglobulinemia. Southern blot analysis and sequencing of cDNA were used to document deletions, insertions, and single base pair substitutions. To facilitate analysis ofBTK regulation and to permit the development of assays that could be used to screen genomic DNA for mutations inBTK, we determined the genomic organization of this gene. Subcloning of a cosmid and a yeast artificial chromosome showed thatBTK is divided into 19 exons spanning 37 kilobases of genomic DNA. Analysis of the region 5' to the first untranslated exon revealed no consensus TATAA or CAAT boxes; however, three retinoic acid binding sites were identified in this region. Comparison of the structure ofBTK with that of other nonreceptor tyrosine kinases, includingSRC,FES, andCSK, demonstrated a lack of conservation of exon borders. Information obtained in this study will contribute to our understanding of the evolution of nonreceptor tyrosine kinases. It will also be useful in diagnostic studies, including carrier detection, and in studies directed towards gene therapy or gene replacement.     

Characterization of thelet-653 gene inCaenorhabditis elegans

A mutation in thelet-653 gene ofCaenorhabditis elegans results in larval death. The lethal arrest is concurrent with the appearance of a vacuole anterior to the lower pharyngeal bulb. The position of the vacuole is consistent with a dysfunction of the secretory/excretory apparatus. Germline transformation rescue experiments were able to position thelet-653 gene to two overlapping cosmid subclones. Sequence data generated from both cDNA and genomic DNA subclones indicated thatlet-653 encodes a mucin-like protein. Our characterization suggests that a mucin-like protein is essential for effective functioning of the secretory/excretory apparatus withinC. elegans.     

The Human Prohibitin (PHB) Gene Family and Its Somatic Mutations in Human Tumors

Five cosmid clones, isolated by procedures to screen genomic libraries for homologous variants of the human prohibitin gene (PHB), were analyzed to determine their genomic structures. Four of these (PHBP1-4) were found to be processed pseudogenes, each located on a different chromosome from their counterparts on chromosome 17q21. The DNA sequence of one clone (PHBP1, on chromosome 6q25) shared a 91.3% identity at the nucleotide level with the cDNA of functional prohibitin. A large number of human tumors of the breast, ovary, liver, and lung were examined for somatic mutations in the PHB gene. Although mutations were observed in a few sporadic breast cancers, none were identified in any of the other cancers.     

Plasmids controlling exclusion of the K2 killer double-stranded RNA plasmid of yeast

Saccharomyces strains of two types (K₁⁺R₁⁺ and K₂⁺R₂⁺) kill each other and K^?R^?-sensitive strains by secreting protein toxins. K₁ killer strains carry a 1.25 × 10⁶ dalton double-stranded RNA plasmid, [KIL-k₁], while K₂ killers have a 1.0 × 10⁶ dalton double-stranded RNA plasmid, [KIL-k₁]. Mating [KIL-k₁] haploids with [KIL-k₂] haploids yields only [KIL-k₁] diploids, that is, [KIL-k₁] excludes [KIL-k₂]. [EXL], a new non-Mendellan genetic element from a nonkiller strain, excludes [KIL-k₂] but does not exclude [KIL-k₂]. A second new non-Mendelian genetic element, called [NEX], when present prevents [EXL] from excluding [KIL-k₂]. [NEX] does not prevent [KIL-k₁] or [KIL-s₁] (a suppressive mutant of [KIL-k₁]) from excluding [KIL-k₂]. A chromosomal gene, called MKT1, is needed for maintenance of [KIL-k₂] if [NEX] is present. In the absence of [NEX], [KIL-k₂] does not need MKT1. [KIL-k₁] does not need MKT1 even if [NEX] is present. [EXL] replication depends on at least the products of MAK1, MAK3, MAK10and PET18. [NEX]replication depends on MAK3 but is independent of MAK4, MAKE, MAK27 and MKT1.     

Regulation of purine biosynthetic genes expression inSalmonella typhimurium IV Oc mutation site ofpurG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded bypurG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu- tamine and ATP for thede novo purine nucleotide biosynthesis.purG gene is negatively regulated by a repressor-operator system. The O⁺ purG and O^c purG were cloned respectivelyin vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O⁺) and pLBG-2 (O^c) were carried out. The hybrid plasmids pLB1933 (O⁺) and pLB1927 (O^c) containing 5′ control region ofpurG were constructed and the DNA sequences were determined respectively, DNA sequences data showed that O^c mutation ofpurG occurred at the 3rd position of 16 bp PUR box in the 5′ control region (G→A). Gel retardation experiment indicated that the repressor bound well with O⁺ PUR box, but not with O^c PUR box. The result strongly supported the idea that PUR box is the binding region of repressor protein and the 3rd position base G of PUR box is essential for the binding function with repressor protein.     

Mapping of mouse gamma crystallin genes on chromosome 1

Restriction fragments analysis of DNA from mouse-hamster somatic-cell hybrid clones revealed that a mouse gamma crystallin cDNA hybridized to genomic sequences located on mouse chromosome 1. Identification of restriction fragment length polymorphisms (RFLPs) in the gamma crystallin sequences of inbred strains of mice permitted the further localization of the gamma crystallin genes (Cryg) to the proximal region of chromosome 1 closely linked to the loci encoding isocitrate dehydrogenase (Idh-1), a low molecular weight (LM) crystallin protein polymorphism (Len-1), and fibronectin (Fn-1). A single recombinant was observed betweenLen-1 and an RFLP in the gamma crystallin gene family, consistent with the hypothesis thatLen-1 is one of the several structural loci encoding gamma crystallin genes.Len-1 is probably located on the centromeric end of theCryg gene family. Linkage ofIdh-1, Cryg, andFn-1 in mice extends the syntenic relationship of those loci to the human, bovine, and rodent genomes and may define a chromosomal region that is generally conserved among mammals. The map position ofCryg, near the eye lens obsolescence (Elo) locus, was confirmed by the discovery that the restriction fragment patterns of gamma crystallin sequences differed between strain C3H/HeJ and the congenic anophthalmic mutant strain, C3H.Elo. Therefore, the gamma crystallin genes were contransferred with the mutantElo gene in the derivation of C3H.Elo. The results establish that LEN-1 is a marker for the gamma crystallin gene family, position the gamma crystallin gene family relative to other markers on mouse chromosome 1, and provide additional evidence that theElo mutation is encoded at a locus closely linked to the gamma crystallin gene cluster. This study found no evidence of recombination hot spots within the gamma crystallin gene cluster.     

Ordered Cloned DNA Map of the Genome of Vibrio cholerae 569B and Localization of Genetic Markers

By using a low-resolution macrorestriction map as the foundation (R. Majumder et al., J. Bacteriol. 176:1105–1112, 1996), an ordered cloned DNA map of the 3.2-Mb chromosome of the hypertoxinogenic strain 569B of Vibrio cholerae has been constructed. A cosmid library the size of about 4,000 clones containing more than 120 Mb of V. cholerae genomic DNA (40-genome equivalent) was generated. By combining landmark analysis and chromosome walking, the cosmid clones were assembled into 13 contigs covering about 90% of the V. cholerae genome. A total of 92 cosmid clones were assigned to the genome and to regions defined by NotI, SfiI, and CeuI macrorestriction maps. Twenty-seven cloned genes, 9 rrn operons, and 10 copies of a repetitive DNA sequence (IS1004) have been positioned on the ordered cloned DNA map.     

Integration of hup Genes into the Genome of Chickpea-Rhizobium Through Site-Specific Recombination

The hup gene fragment of cosmid pHU52 was integrated into the genome of chickpea-Rhizobium Rcd301 via site-specific homologous recombination. Two small fragments of genomic DNA of strain Rcd301 itself were provided to flank cloned hup genes to facilitate the integration. The hup insert DNA of cosmid pHU52 was Isolated as an Intact 30.2 kb fragment using EcoRI, and cloned on partially restricted cosmid clone pSPSm3, which carries a DNA fragment of strain Rcd301 imparting streptomycin resistance. One of the recombinant cosmid clones, pBSL 12 thus obtained was conjugally transferred to the strain Rcd30₁. The integration of hup gene fragment into the genomic DNA through site-specific homologous recombination, was ensured by introducing an incompatible plasmid, pPH1 JI. The integration was confirmed by Southern hybridization. The integrated hup genes were found to express ex plants in two such constructs BSL 12–1 and BSL 12–3.     

Cloning and analysis of the glucosyl transferase gene encoding type I antigen in Shigella flexneri

The O-antigen of most Shigella flexneri serotypes contains an identical tetrasaccharide repeating unit. Apart from serotype Y, the O-antigen is modified by addition of a glucosyl and/or O-acetyl residue to a specific position in the O-unit. In this study the glucosyl transferase gene from a serotype 1a has been cloned and identified. The bacteriophage SfV integrase (int) gene was used to probe a S. flexneri Y53 (serotype 1a) cosmid library and 18 unique clones were identified. Southern hybridisation of these clones indicated two unlinked regions of the chromosome contained the int homologue. When expressed in a live candidate vaccine strain of S. flexneri serotype Y (SFL124), clones with one region produced type I antigen, whereas clones containing the other region produced mainly type Y antigen. One of the cosmid clones positive for type I antigen by agglutination and Western blotting was selected for further study. Genes involved in O-antigen glucosyl modification were mapped on a 5.8 kb fragment and subclones were produced which fully or partially expressed the type I antigen, depending on the extent of the clone. Fully and partially expressing clones may be useful vaccine candidate strains for protection against disease caused by two serotypes of S. flexneri.     

Fil1, a G-protein α-subunit that acts upstream of cAMP and is essential for dimorphic switching in haploid cells of Ustilago hordei

A constitutive mutation, fil1, that causes filamentous growth in the haplophase of the dimorphic smut fungus Ustilago hordei, was previously shown to be genetically associated with a 50-kb deletion within a 940-kb chromosome. Physiological studies suggested that a gene that functions upstream of adenylyl cyclase was deleted in the mutant. Representational difference analysis of isolated chromosomes was used to obtain deletion-specific DNA probes and corresponding genomic cosmid clones. Complementation analysis identified a cosmid clone and subsequently a 2.1-kb insert that converted transformants of the mutant strain10.1a(fil1) from the filamentous to the sporidial cell type. A single open reading frame of 354 codons that encodes a putative α-subunit of the heterotrimeric G-proteins was identified. Fil1 displayed a high degree of sequence identity to Gpa1 from the basidiomycete Cryptococcus neoformans and CPG-2 from the ascomycete Cryphonectria parasitica. FIL1, when introduced on a self-replicating vector, was found to suppress filamentous growth of starved haploid wild-type strains and restore normal mating response to the fil1 mutant, but did not suppress sexual dimorphism of either strain. Fil1 appears to function analogously to mammalian Gα proteins, which are coupled to cAMP production via adenylyl cyclase, to regulate dimorphic switching in ?U.?hordei.