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Genetic mapping of the compatibility between a lily isolate of Cucumber mosaic virus and a satellite RNA.

Five isolates of Cucumber mosaic virus (CMV) from Lilium sp. (lily), which were isolated from specimens in Japan, Korea and Taiwan, were unable to support satellite RNA (satRNA) accumulation. In order to map the CMV sequences that are involved in satRNA support, HL-CMV (Japanese lily isolate), Y-CMV (ordinary strain) and Y-satellite RNA (Y-sat) were used as the source material. The pseudorecombinants between Y-CMV and HL-CMV revealed that RNA1 was essential for satRNA replication in lily. The results of chimeric constructs and various mutations showed that two amino acid residues (at positions 876 and 891) in the 1a protein were the determinants for the inability of HL-CMV to support a satRNA. Specifically, Thr at position 876 had a more pronounced effect than Met at position 891. Specific changes in RNA sequence were also detected in the 3' terminus of Y-sat and these particular alterations allowed it to be supported by HL-CMV. It is believed that, through evolution, the adaptation of CMV to lily resulted in the introduction of amino acid changes in the 1a protein, changes that coincidentally affected the ability of lily CMV to support satRNAs.

Amino Acid Motifs↗

Mapping genetic and phenotypic diversity of Pseudomonas aeruginosa across clinical and environmental isolation sites.

Pseudomonas aeruginosa is a clinically significant opportunistic pathogen adept at thriving in both host-associated and environmental settings. To define the extent to which P. aeruginosa isolates specialize across niches and identify genotype-phenotype correlates, we performed whole genome sequencing and comprehensive phenotypic characterization of 125 P. aeruginosa isolates from diverse clinical and environmental sites, evaluating virulence-associated traits, including motility, cytotoxicity, biofilm formation, pyocyanin production, and antimicrobial susceptibility. We identify that genomic diversity does not correlate with isolation source or most virulence phenotypes. Instead, we find that the two major P. aeruginosa clades (Groups A and B) delineate phylogeny and cytotoxicity, with Group B strains showing significantly higher cytotoxicity than Group A. Sequence analysis revealed previously uncharacterized alleles of genes encoding type III secretion effector proteins. We observed high variability amongst strains and isolation sources in all four assayed virulence phenotypes. Antimicrobial resistance (AMR) is exclusively observed in clinical isolates, not environmental, reflecting antibiotic exposure-driven selection. Bacterial GWAS revealed a statistically significant association between cytotoxicity and exoU presence, and we identified a novel exoU allelic variant with decreased cytotoxicity, demonstrating that functional diversity within well-characterized virulence factors may still influence pathogenic outcomes. In summary, our analyses of 125 diverse isolates suggest that the ability of P. aeruginosa to thrive across diverse niches is driven by broadly conserved genetic repertoire rather than niche-specific accessory genes.

Journal Article↗

Tissue-specific expression, developmental regulation, and genetic mapping of the gene encoding CCAAT/enhancer binding protein.

This paper presents the results of experiments that determine the chromosomal location of the mouse gene encoding CCAAT/enhancer binding protein (C/EBP) and measure its expression as a function of tissue type and temporal period of development in mice and rats. Three alleles of the C/EBP gene were identified according to restriction fragment length polymorphisms. The strain distribution pattern of the three alleles was determined in recombinant inbred mouse strains and compared to that of other mouse genes. These results mapped the gene to a position within 2.5 centimorgans (cM) of the structural gene encoding glucose phosphate isomerase on chromosome 7 of the mouse. The expression pattern of the C/EBP gene was studied by a combination of nucleic acid hybridization and antibody staining assays. High levels of C/EBP mRNA were observed in tissues known to metabolize lipid and cholesterol-related compounds at uncommonly high rates. These included liver, fat, intestine, lung, adrenal gland, and placenta. More detailed analysis of two of these tissues, liver and fat, showed that C/EBP expression was limited to fully differentiated cells. Moreover, analysis of the temporal pattern of expression of C/EBP mRNA in two tissues, liver and intestine, revealed a coordinated induction just prior to birth. These observations raise the possibility that the synthesis of C/EBP may be responsive to humoral factors and that modulation in C/EBP expression might mediate coordinated changes in gene expression that facilitate adaptive challenges met during development or during the fluctuating physiological states of adult life.

Adipose Tissue↗

Bacterial artificial chromosome library of Finegoldia magna ATCC 29328 for genetic mapping and comparative genomics.

We constructed a bacterial artificial chromosome (BAC) library of Finegoldia magna ATCC 29328 DNA to facilitate further genome analysis of F. magna. The BAC library contained 385 clones with an average insert size of 55 kb, representing a 10.1-fold genomic coverage. Repeated DNA hybridization using primer sets designed on the basis of BAC-end sequences yielded nine contigs covering 95% of the chromosome and two contigs covering 98% of the plasmid. The contigs were localized on the physical map of F. magna ATCC 29328 DNA. A total of 121 BAC-end sequences revealed 103 unique genes, which had not been previously reported for F. magna. The homolog ORF of albumin-binding protein (urPAB), one of the known virulence factors from F. magna, was sequenced and localized on the physical map. Homology analysis of 121 BAC-end sequences revealed that F. magna is most closely related to clostridia, particularly Clostridium tetani. This close relationship is consistent with the recent classification of peptostreptococci based on 16S rRNA sequence analysis. The BAC library constructed here will be useful for the whole genome sequencing project and other postgenomic applications.

Chromosome Mapping↗

Genetic mapping of starch- and lambda-receptor sites in maltoporin: identification of substitutions causing direct and indirect effects on binding sites by cysteine mutagenesis.

Cysteine mutagenesis was used to test the proximity of 16 residues to protein-ligand interaction sites in maltoporin (LamB protein). LamB protein with additional cysteines was incorporated into the outer membrane of Escherichia coli except with a Ser-30----Cys substitution. Phage Lambda and starch binding was assayed before and after incubation of mutants with six thiol-specific reagents. Four categories of mutation were recognized on the basis of phenotype and modification for each of the Lambda- and starch binding sites. The thiol modification experiments helped to clarify whether the phenotype of a mutation was due to a substitution at the binding site or an indirect perturbation of the structure. This study suggests that the cysteine mutagenesis/thiol modification approach may be usefully applied to the operational mapping of surface-accessible binding sites or epitopes.

Bacterial Outer Membrane Proteins↗

Dipeptidyl aminopeptidase yspI mutants of Schizosaccharomyces pombe: genetic mapping of dpa1+ on chromosome III.

A mutant strain of Schizosaccharomyces pombe lacking dipeptidyl aminopeptidase yspI was isolated from a strain already defective in aminopeptidase activity by means of a staining technique with the chromogenic substrate ala-pro-4-methoxy-beta-naph-thylamide to screen colonies for the absence of the enzyme. The defect segregated 2+:2- in meiotic tetrads, indicating a single chromosomal gene mutation, which was shown to be recessive. Gene dosage experiments indicated that the mutation resides in the structural gene of dipeptidyl aminopeptidase yspI, dpa1+. The dpa1+ gene was located on chromosome III by using m-fluorophenylalanine-induced haploidization and mitotic analysis. dpa1 mutants did not show any obvious phenotype under a variety of conditions tested.

Chromosome Mapping↗

Physical and genetic map of the Bacteroides fragilis YCH46 chromosome.

The chromosome of Bacteroides fragilis strain YCH46 was shown to be a single circular DNA molecule of about 5.3 Mb having 16 NotI, seven AscI, and six I-CeuI sites. A physical map of the chromosome was constructed by four independent experimental approaches: linking clone analysis, cross-Southern hybridization, partial restriction digestion, and two-dimensional pulsed-field gel electrophoresis. Six rRNA operons and 10 known genes were localized on the physical map.

Bacteroides fragilis↗

Genetic mapping of the mouse proto-oncogene c-sis to chromosome 15.

The mouse homolog (c-sis) of the transforming gene of the simian sarcoma virus was mapped to chromosome 15 by the Southern blot analysis of DNA's from hamster-mouse somatic cell hybrids. Alterations in c-sis expression may thus play a role in the various murine neoplastic diseases characterized by rearrangements or duplications of chromosome 15.

Animals↗

Genetic Mapping in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium.

A method of meiotic segregation analysis based on recombinant selection in the homothallic basidiomycete Phanerochaete chrysosporium was developed. Using this method, we were able to reveal linkage relationships and to estimate recombination frequencies between seven mutations to auxotrophy. We detected two linkage groups, the first containing four and the second three of the seven mapped mutations.

Journal Article↗

Genetic map of the Actinobacillus pleuropneumoniae RTX-toxin (Apx) operons: characterization of the ApxIII operons.

Actinobacillus pleuropneumoniae RTX-toxin III (ApxIII) is implicated as an important virulence factor of A. pleuropneumoniae, the causative agent of porcine pleuropneumonia. Recently, the genes coding for ApxIII (apxIIICA) of serotype 8 were cloned and characterized. The toxin appeared to be a member of the RTX-toxin family, as are the other two secreted toxins of A. pleuropneumoniae, i.e., ApxI and ApxII. In this report, we describe the cloning and sequencing of the remaining part of the ApxIII operon of serotype 8. This sequence coded for the RTX secretion proteins ApxIIIB and ApxIIID, which showed 86 and 63% similarity to ApxIB and ApxID, respectively, and 83 and 63% similarity to HlyB and HlyD of Escherichia coli, respectively. Potential functional domains, such as eight transmembrane regions and an ATP-binding cassette, were present in ApxIIIB. We examined the presence of apxIIICABD sequences in the 12 serotypes of A. pleuropneumoniae and found that these sequences were present only in serotypes 2, 3, 4, 6, and 8, the serotypes that secrete ApxIII. Comparison of the apxIIICABD gene sequences of the serotypes revealed very few serotype-specific differences. Only the C terminus of ApxIIIA of serotype 2 differed from ApxIIIA of the other serotypes. The differences were located between the glycine-rich repeats and the secretion signal. The analysis of the apxIIICABD genes completed our efforts to characterize the ApxI, ApxII, and ApxIII operons of the reference strains of the 12 serotypes of A. pleuropneumoniae. We present a complete map of the ApxI, ApxII, and ApxIII operons and discuss this in terms of gene expression and complementation and the role of the toxins in pathogenesis.

Actinobacillus pleuropneumoniae↗

Comparative physical and genetic maps of the virulence plasmids of Salmonella enterica serovars typhimurium, enteritidis, choleraesuis, and dublin.

Using fragment profiling, PCR, and Southern hybridization, we found that Salmonella enterica serovar Choleraesuis harbored virulence plasmids of various sizes, whereas serovars Typhimurium, Enteritidis, and Dublin carried a plasmid of a unique size. Also, the virulence plasmid of Typhimurium contained genes in the same order detected in the other three plasmids, all of which contained deletions.

Base Sequence↗

Genetic mapping of the F plasmid gene that promotes degradation of stable ribonucleic acid in Escherichia coli.

F+ Escherichi coli cells that contain an srnA mutant allele degrade their stable ribonucleic acid (RNA) extensively after RNA synthesis is blocked at 42 degrees C. The relevant gene promoting degradation of stable RNA, srnB+, or its promoter was mapped between 1.7 and 2.8 kilobases on the F plasmid by using deleted F' plasmids and chimeric plasmids composed of pSC101 and fragments of F plasmid.

Chromosomes, Bacterial↗

Genetic mapping of bra genes affecting branched-chain amino acid transport in Pseudomonas aeruginosa.

Pseudomonas aeruginosa PAO mutants defective in the transport systems for branched-chain amino acids were isolated and characterized. Two mutations in strains selected for trifluoroleucine resistance, braA300 and braB307, were mapped in the met-9020-dcu-9108 and the nar-9011-puuC10 region, respectively. The mutation loci in strains selected for azaleucine resistance, braC310 and bra-311 through bra-314, were all located near the fla genes, with an order of region I fla-bra-region II fla. Strains with braA300 showed a marked reduction in the high-affinity branched-chain amino acid transport system (LIV-I) and a considerable decrease in the lower-affinity system (LIV-II). Strains with braB307 were found to be defective in the LIV-II system. Strains selected for azaleucine resistance were all defective only in the LIV-I system and fell into three phenotypically distinct classes. Strains with braC310 produced a binding protein for leucine, isoleucine, valine, alanine, and threonine (LIVAT-BP) altered in binding ability, indicating that the braC gene is the structural one for the LIVAT-BP. Strains with bra-311 or bra-312 showed a complete loss of production of the LIVAT-BP. Strains with bra-313 or bra-314 produced normal levels of functional LIVAT-BP, suggesting that these mutations are located in a gene(s) other than braC.

Amino Acids, Branched-Chain↗

Genetic mapping and characterization of Pseudomonas aeruginosa mutants defective in the formation of extracellular proteins.

We isolated 15 mutants of Pseudomonas aeruginosa PAO which were defective in the formation of certain extracellular proteins, such as elastase, staphylolytic enzyme, and lipase ( Xcp mutants). The mutations were mapped on the chromosome by conjugation and transduction. The locations were xcp -1 near 0', with the gene order cys-59- xcp -1- proB , and loci xcp -2, xcp -3, and xcp -31 at 35', with the gene order trpC , D- xcp -3/ xcp -31- xcp -2- argC . Loci xcp -4 and xcp -41 through xcp -44 were cotransducible with proA at 40'; loci xcp -5, xcp -51, xcp -52, and xcp53 were located at 55', with the gene order leu-10- trpF -met-9010- xcp -53- xcp -5/ xcp -51/ xcp+ ++-52, and xcp -6 was located at 65' to 70', between catA and mtu-9002. Nine mutations ( xcp -2, xcp -3, xcp -31, xcp -4, and xcp -41 through xcp -45) caused decreased production of extracellular enzymes. Six strains with mutations xcp -1, xcp -5, xcp -51, xcp -52, xcp -53, and xcp -6 produced cell-bound exoproteins and had defective release mechanisms. The regulation of production of alkaline phosphatase and phospholipase C is different from other exoproteins , such as elastase, but they all seem to share a common release mechanism. Alkaline protease had separate mechanisms for regulation and release, since this protease was found in culture supernatants of all but one of the mutants, and none of the strains had cell-bound enzyme.

Bacterial Proteins↗

Genetic mapping of katG, a locus that affects synthesis of the bifunctional catalase-peroxidase hydroperoxidase I in Escherichia coli.

A locus unlinked to either katE or katF that affected catalase levels in Escherichia coli was identified and localized between metB and ppc at 89.2 min on the genome. The locus was named katG. Mutations in katG which prevented the formation of both isoenzyme forms of the bifunctional catalase-peroxidase HPI were created both by nitrosoguanidine and by transposon Tn10 insertions. All katG+ recombinants and transductants contained both HPI isoenzymes. Despite the common feature of little or no catalase activity in four of the catalase-deficient strains, subtle differences in the phenotypes of each strain resulted from the different katG mutations. All three mutants caused by nitrosoguanidine produced a protein with little or no catalase activity but with the same subunit molecular weight and with similar antigenic properties to HPI, implying the presence of missense mutations rather than nonsense mutations in each strain. Indeed one mutant produced an HPI-like protein that retained peroxidase activity, whereas the HPI-like protein in a second mutant exhibited no catalase or peroxidase activity. The third mutant responded to ascorbate induction with the synthesis of near normal catalase levels, suggesting a regulatory defect. The Tn10 insertion mutant produced no catalase and no protein that was antigenically similar to HPI.

Catalase↗

Genetic mapping and characterization of Pseudomonas aeruginosa mutants that hyperproduce exoproteins.

We isolated two mutants of Pseudomonas aeruginosa PAO with defective iron uptake. In contrast to the wild-type strain, the mutants produced extracellular protease activity in media containing high concentrations of salts or iron and hyperproduced elastase, staphylolytic enzyme, and exotoxin A in ordinary media (Xch mutants). The mutations were located in the 55' region of the chromosome, between the markers met-9011 and pyrD.

Bacterial Proteins↗