Differential suppressor effects of the ssb-1 and ssb-113 alleles on uvrD mutator of Escherichia coli in DNA repair and mutagenesis

We have constructed double mutants carrying either ssb-1 or ssb-113 alleles, which encode temperature-sensitive single strand DNA binding proteins (SSB), and the uvrD::Tn5 allele causing deficiency in DNA helicase II, and have examined sensitivity to ultraviolet light (UV), recombination and spontaneous as well as UV-induced mutagenesis. We have found in a recA+ background that (i) none of the ssb uvrD double mutants was more sensitive to UV than either single mutant; (ii) the ssb-1 allele partially suppressed the strong UV sensitivity of uvrD::Tn5 mutants; (iii) in the recA730 background with constitutive SOS expression, the ssb-1 and ssb-113 alleles suppressed the strong UV-sensitivity caused by the uvrD::Tn5 mutation; (iv) in ssb-113 mutants, the level of recombination was reduced only 10-fold but 100-fold in ssb-1 mutants, showing that there was no correlation between the DNA repair deficiency and the recombination deficiency; (v) the hyper-recombination phenotype of the uvrD::Tn5 mutant was suppressed by the addition of either the ssb-1 or the ssb-113 allele; (vi) no addition of the spontaneous mutator effects promoted by the uvrD::Tn5 and the ssb-113 alleles was observed. These results suggest a possible functional interaction between SSB and Helicase II in DNA repair and mutagenesis.     

Cloning-Free PCR-Based Allele Replacement Methods

Efficient homologous recombination permits the directed introduction of specific mutations into the yeast genome. Here we describe a cloning-free, PCR-based allele replacement method that simplifies allele transfer between yeast strains. The desired allele from one strain is amplified by PCR, along with a selectable/counterselectable marker. After transformation, the resident allele in the target strain is replaced by creating a duplication of the new allele. Selection for direct repeat recombinants results in a single copy of the new allele in the target strain. Specifically, the desired allele is amplified by PCR with a pair of adaptamers, which are chimeric oligonucleotides that are used to amplify the allele and differentially tag its 5′ and 3′ ends. These tags allow the directed fusion to two different, but overlapping, regions of an appropriately tagged selectable/counterselectable marker after a second round of PCR amplification. Following cotransformation of the two fusion fragments into yeast, homologous recombination efficiently generates a duplication of the amplified allele flanking the intact selectable marker in the genome. After counterselection, only the desired allele is retained as a result of direct repeat recombination. A simple modification of this method allows the creation of de novo mutations in the genome.     

Identification of novel virulence determinants in Mycobacterium paratuberculosis by screening a library of insertional mutants

Johne's disease, caused by Mycobacterium paratuberculosis infection, is a worldwide problem for the dairy industry and has a possible involvement in Crohn's disease in humans. To identify virulence determinants of this economically important pathogen, a library of 5,060 transposon mutants was constructed using Tn5367 insertion mutagenesis, followed by large-scale sequencing to identify disrupted genes. In this report, 1,150 mutants were analyzed and 970 unique insertion sites were identified. Sequence analysis of the disrupted genes indicated that the insertion of Tn5367 was more prevalent in genomic regions with G+C content (50.5 to 60.5%) lower than the average G+C content (69.3%) of the rest of the genome. Phenotypic screening of the library identified disruptions of genes involved in iron, tryptophan, or mycolic acid metabolic pathways that displayed unique growth characteristics. Bioinformatic analysis of disrupted genes identified a list of potential virulence determinants for further testing with animals. Mouse infection studies showed a significant decrease in tissue colonization by mutants with a disruption in the gcpE, pstA, kdpC, papA2, impA, umaA1, or fabG2_2 gene. Attenuation phenotypes were tissue specific (e.g., for the umaA1 mutant) as well as time specific (e.g., for the impA mutant), suggesting that those genes may be involved in different virulence mechanisms. The identified potential virulence determinants represent novel functional classes that could be necessary for mycobacterial survival during infection and could provide suitable targets for vaccine and drug development against Johne's and Crohn's diseases.     

The ompA gene in Chlamydia trachomatis differs in phylogeny and rate of evolution from other regions of the genome

Strains of Chlamydia trachomatis are classified into serovars based on nucleotide sequence differences in ompA, the gene that encodes the major outer membrane protein. Phylogenetic characterization of strains based on ompA, however, results in serovar groupings that are inconsistent with the distinguishing features of C. trachomatis pathobiology, e.g., tissue tropisms and disease presentation. We have compared nucleotide sequences at multiple sites distributed around the chlamydial genome from 18 strains representing 16 serovars; sampled regions included genes encoding housekeeping enzymes (totaling 2,073 bp), intergenic noncoding segments (1,612 bp), and a gene encoding a second outer membrane protein (porB; 1,023 bp), with the ompA sequence (1,194 bp) used for reference. These comparative analyses revealed substantial variation in nucleotide substitution patterns among the sampled regions, with average pairwise sequence differences ranging from 0.15% for the housekeeping genes to 12.1% for ompA. Phylogenetic characterization of the sampled genomic sequences yielded a strongly supported tree that divides the strains into groupings consistent with C. trachomatis biology and which has a topology quite distinct from the ompA tree. This phylogenetic incongruity can be accounted for by recombination of the ompA gene between different genomic backgrounds. We found, however, no evidence of recombination within or between any of the sampled regions around the C. trachomatis genome apart from ompA. Parallel analysis of published sequence data on four members of the pmp gene family are consistent with the phylogenetic analyses reported here.     

Allelic recombination and de novo deletions in sperm in the human beta-globin gene region

Meiotic recombination is of fundamental importance in creating haplotype diversity in the human genome and has the potential to cause genomic rearrangements by ectopic recombination between repeat sequences and through other changes triggered by recombination-initiating events. However, the relationship between allelic recombination and genome instability in the human germline remains unclear. We have therefore analysed recombination and DNA instability in the delta-, beta-globin gene region and its associated recombination hotspot. Sperm typing has for the first time accurately defined the hotspot and shown it to be the most active autosomal crossover hotspot yet described, although unusually inactive in non-exchange gene conversion. The hotspot just extends into a homology block shared by the delta- and beta-globin genes, within which ectopic exchanges can generate Hb Lepore deletions. We developed a physical selection method for recovering and validating extremely rare de novo deletions in human DNA and used it to characterize the dynamics of these Hb Lepore deletions in sperm as well as other deletions not arising from ectopic exchanges between homologous DNA sequences. Surprisingly, both classes of deletion showed breakpoints that avoided the beta-globin hotspot, establishing that it possesses remarkable fidelity and does not play a significant role in triggering these DNA rearrangements. This study also provides the first direct analysis of de novo deletion in the human germline and points to a possible deletion-controlling element in the beta-globin gene separate from the crossover hotspot.     

Identification of essential and non-essential genes of the guinea pig cytomegalovirus (GPCMV) genome via transposome mutagenesis of an infectious BAC clone

We report application of a transposition methodology that allows the easy characterization and mutation of genes encoded on an infectious bacterial artificial chromosome (BAC) clone. We characterized mutants generated by transposome (Tn) mutagenesis of a BAC clone of guinea pig cytomegalovirus (GPCMV). A pool of Tn mutant GPCMV BACs were screened initially by restriction profile analysis to verify they were full-length, and subsequently GPCMV BAC DNA from individual mutants was transfected onto guinea pig lung fibroblast cells in order to generate virus. Tn GPCMV BAC mutants were classed as either essential or non-essential gene insertions, depending upon their ability to regenerate viable, replication-competent virus. Representative mutants were more fully characterized. Analysis by sequencing the Tn insertion site on the mutated BACs, and by regeneration of virus using transfection of guinea pig fibroblasts (GPL), demonstrated that a recombinant with a Tn insertion in the UL35 homolog gene (GP35) was a non-essential gene for viral replication in tissue culture. A mutant with an insertion in the UL46 homolog (GP46) was nonviable, a phenotype which could be rescued by homologous recombination of BAC DNA with wild-type UL46 sequences, suggesting an essential role of this putative capsid gene in virus replication.     

Identification of a virulence-associated determinant, dihydrolipoamide dehydrogenase (lpd), in Mycoplasma gallisepticum through in vivo screening of transposon mutants

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.     

Linkage Analysis of Affected Sib Pairs Allowing for Parent-of-Origin Effects

Parent‐of‐origin effects, also known as genomic imprinting, exist for many mammalian genes. For imprinted genes the expression of an allele depends upon the sex of the transmitting parent. Here we have developed a method based on alleles that are shared identical by descent by affected sib pairs, that allows for parent‐of‐origin effects. Our method allows for sex‐specific recombination rates, an important consideration in studying imprinted genes. We have also derived a tetrahedron for the true identical‐by‐descent frequencies accounting for parent‐of‐origin effects. Using this tetrahedron, we propose a robust generalized minmax test for linkage and discuss its properties in the presence of genomic imprinting. We have also performed power comparisons of various allele sharing tests and provide regions of the tetrahedron in which the different tests are optimal. We also provide useful strategies to determine the optimal tests to use while performing a genome scan.     

Staphylococcus aureus virulence factors identified by using a high-throughput Caenorhabditis elegans-killing model

Staphylococcus aureus is an important human pathogen that is also able to kill the model nematode Caenorhabditis elegans. We constructed a 2,950-member Tn917 transposon insertion library in S. aureus strain NCTC 8325. Twenty-one of these insertions exhibited attenuated C. elegans killing, and of these, 12 contained insertions in different genes or chromosomal locations. Ten of these 12 insertions showed attenuated killing phenotypes when transduced into two different S. aureus strains, and 5 of the 10 mutants correspond to genes that have not been previously identified in signature-tagged mutagenesis studies. These latter five mutants were tested in a murine renal abscess model, and one mutant harboring an insertion in nagD exhibited attenuated virulence. Interestingly, Tn917 was shown to have a very strong bias for insertions near the terminus of DNA replication.     

Transposon Tn5 mutagenesis of Brucella abortus.

We demonstrate that transposon-mediated mutagenesis can be used to construct mutations in the pathogen Brucella abortus. We have used both a plasmid and a bacteriophage to introduce either Tn5 or Tn5 lac into the Brucella chromosome. B. abortus is naturally sensitive to kanamycin. We have selected 22 independent kanamycin-resistant colonies in strain US-19 and 19 colonies in strain 2308. We have demonstrated by Southern hybridization that Tn5 was inserted into the chromosome.     

Does recombination shape the distribution and evolution of tandemly arrayed genes (TAGs) in the Arabidopsis thaliana genome?

Tandemly arrayed genes (TAGs) are an important genomic component. However, most previous studies have focused on individual TAG families, and a broader characterization of their genomic distribution is not yet available. In this study, we examined the distribution of TAGs in the Arabidopsis thaliana genome and examined TAG density with relation to recombination rates. Recombination rates along A. thaliana chromosomes were estimated by comparing a genetic map with the genome sequence. Average recombination rates in A. thaliana are high, and rates vary more than threefold among chromosomal regions. Comparisons between TAG density and recombination indicate a positive correlation on chromosomes 1, 2, and 3. Moreover, there is a consistent centromeric effect. Relative to single-copy genes, TAGs are proportionally less frequent in centromeres than on chromosomal arms. We also examined several factors that have been proposed to affect the sequence evolution of TAG members. Sequence divergence is related to the number of members in the TAG, but genomic location has no obvious effect on TAG sequence divergence, nor does the presence of unrelated genes within a TAG. Overall, the distribution of TAGs in the genome is not consistent with theoretical models predicting the accumulation of repeats in regions of low recombination but may be consistent with stabilizing selection models of TAG evolution.     

A Gateway recombination herpesvirus cloning system with negative selection that produces vectorless progeny

Crossover recombination based on the lambda phage integration/excision functions enables insertion of a gene of interest into a specific locus by a simple one-step in vitro recombination reaction. Recently, a highly efficient recombination system for targeted mutagenesis, which utilizes lambda phage crossover recombination cloning, has been described for a human herpesvirus 2 bacterial artificial chromosome (BAC). The disadvantages of the system are that it allows only neutral selection (loss of green fluorescent protein) of desired recombinants and that it regenerates herpesvirus progeny containing the BAC sequence inserted in the herpesvirus genome. In this study, the existing channel catfish herpesvirus (CCV) infectious clone (in the form of overlapping fragments) was modified to allow introduction of foreign genes by modified lambda phage crossover recombination cloning. This novel system enables negative and neutral selection and regenerates vectorless herpesvirus progeny. Construction of two CCV mutants expressing lacZ, one from the native CCV ORF5 promoter and the other from the immediate-early cytomegalovirus promoter, demonstrated the efficiency and reliability of this system. This novel cloning system enables rapid incorporation, direct delivery and high-level expression of foreign genes by a herpesvirus. This system has broad utility and could be used to facilitate development of recombinant viruses, viral vectors and better vaccines.     

A streamlined process to phenotypically profile heterologous cDNAs in parallel using yeast cell-based assays.

To meet the demands of developing lead drugs for the profusion of human genes being sequenced as part of the human genome project, we developed a high-throughput assay construction method in yeast. A set of optimized techniques allows us to rapidly transfer large numbers of heterologous cDNAs from nonyeast plasmids into yeast expression vectors. These high- or low-copy yeast expression plasmids are then converted quickly into integration-competent vectors for phenotypic profiling of the heterologous gene products. The process was validated first by testing proteins of diverse function, such as p38, poly(ADP-ribose) polymerase-1, and PI 3-kinase, by making active-site mutations and using existing small molecule inhibitors of these proteins. For less well-characterized genes, a novel random mutagenesis scheme was developed that allows a combination selection/screen for mutations that retain full-length expression and yet reverse a growth phenotype in yeast. A broad range of proteins in different functional classes has been profiled, with an average yield for growth interference phenotypes of approximately 30%. The ease of manipulation of the yeast genome affords us the opportunity to approach drug discovery and exploratory biology on a genomic scale and shortens assay development time significantly.     

Rapid and efficient construction of recombinant bovine herpesvirus 1 genomes

Bovine herpesvirus 1 (BoHV-1) is an important pathogen of cattle. Recombinant bovine herpesvirus 1 viruses (rBoHV) have been studied extensively as potential vaccines for BoHV-1 associated diseases. A method is described which advances protocols used currently for constructing rBoHV by producing recombinant viruses free of parent virus. The method, restriction endonuclease mediated recombination (REMR), utilises a unique NsiI site in the BoHV-1 genome. Following NsiI digestion the two genomic fragments are prevented from recombining by dephosphorylation. However, when the genomic fragments are co-transfected into a susceptible cell-line with a third DNA fragment (DNA bridge), which encodes DNA homologous to the digested viral termini, the three DNA molecules are able to undergo homologous recombination and produce infectious BoHV-1. During the recombination process foreign DNA within the DNA bridge is incorporated into the BoHV-1 genome, producing rBoHV. In the absence of the DNA bridge virus reconstitution does not occur thus eliminating contamination by the nonrecombinant parent virus. As REMR used an NsiI site occurring naturally in the BoHV-1 genome it can be used for the insertion of foreign DNA into the genome without any prior modifications. REMR could also be applied to any herpesvirus for which the genome sequence is known.     

Identification and cloning of genes from Porphyromonas gingivalis after mutagenesis with a modified Tn4400 transposon from Bacteroides fragilis

Porphyromonas gingivalis is a gram-negative, black-pigmented, oral anaerobe strongly associated with adult periodontitis. Previous transposon mutagenesis studies with this organism were based on the Bacteroides transposon Tn4351. Characterization of Tn4351-disrupted genes by cloning has not been an efficient way to analyze large numbers of mutants and is further complicated by the high rate of cointegration of the suicide delivery vector containing Tn4351. In this study, we mutagenized P. gingivalis with a modified version of the Bacteroides fragilis transposon Tn4400. Plasmid pYT646B carrying the transposon was mobilized from Escherichia coli to P. gingivalis ATCC 33277 by conjugation. Both normal and inverse transposition frequencies were similar (3 x 10(-8)). However, the inverse transposon (Tn4400') contains a pBR322 replicon and a beta-lactamase gene; thus, the cloning of disrupted genomic DNAs from inverse transposition mutants was easily accomplished after ligation of genomic fragments and transformation into E. coli. Thousands of transconjugants could be obtained in a single mating experiment, and inverse transposition was random as demonstrated by Southern hybridization. By this procedure the disrupted genes from P. gingivalis pleiotropic mutants were quickly cloned, sequenced, and identified.     

Functional analysis of the yeast genome: use of two-dimensional gel electrophoresis to detect genes in randomly cloned DNA sequences

Summary Genes are overexpressed when present in yeast cells on multicopy plasmids. Taking advantage of the protein amplification which results from this overexpression, a method has been developed for large scale detection of yeast genes on randomly cloned DNA sequences. It is based on the analysis, by two-dimensional gel electrophoresis, of the proteins from yeast cells transformed with a yeast genomic DNA library constructed in a multicopy vector. We demonstrate here the applicability of this method for exploring the yeast genome. In addition, we report results which suggest that this method may also be useful for detecting regulatory genes.     

Identification of new genes involved in the virulence of Listeria monocytogenes by signature-tagged transposon mutagenesis

Listeria monocytogenes is a gram-positive, facultative intracellular pathogen that can cause severe food-born infections in humans and animals. We have adapted signature-tagged transposon mutagenesis to L. monocytogenes to identify new genes involved in virulence in the murine model of infection. We used transposon Tn1545 carried on the integrative vector pAT113. Forty-eight tagged transposons were constructed and used to generate banks of L. monocytogenes mutants. Pools of 48 mutants were assembled, taking one mutant from each bank, injected into mice, and screened for those affected in their multiplication in the brains of infected animals. From 2,000 mutants tested, 18 were attenuated in vivo. The insertions harbored by these mutants led to the identification of 10 distinct loci, 7 of which corresponded to previously unknown genes. The properties of four loci involving putative cell wall components were further studied in vitro and in vivo. The data suggested that these components are involved in bacterial invasion and multiplication in the brain.     

Multiple LTR-retrotransposon families in the asexual yeast Candida albicans

We have begun a characterization of the long terminal repeat (LTR) retrotransposons in the asexual yeast Candida albicans. A database of assembled C. albicans genomic sequence at Stanford University, which represents 14.9 Mb of the 16-Mb haploid genome, was screened and >350 distinct retrotransposon insertions were identified. The majority of these insertions represent previously unrecognized retrotransposons. The various elements were classified into 34 distinct families, each family being similar, in terms of the range of sequences that it represents, to a typical Ty element family of the related yeast Saccharomyces cerevisiae. These C. albicans retrotransposon families are generally of low copy number and vary widely in coding capacity. For only three families, was a full-length and apparently intact retrotransposon identified. For many families, only solo LTRs and LTR fragments remain. Several families of highly degenerate elements appear to be still capable of transposition, presumably via trans-activation. The overall structure of the retrotransposon population in C. albicans differs considerably from that of S. cerevisiae. In that species, retrotransposon insertions can be assigned to just five families. Most of these families still retain functional examples, and they generally appear at higher copy numbers than the C. albicans families. The possibility that these differences between the two species are attributable to the nonstandard genetic code of C. albicans or the asexual nature of its genome is discussed. A region rich in retrotransposon fragments, that lies adjacent to many of the CARE-2/Rel-2 sub-telomeric repeats, and which appears to have arisen through multiple rounds of duplication and recombination, is also described.     

Vaccinia virus DNA ligase is nonessential for virus replication: Recovery of plasmids from virus-infected cells

The essentiality of the vaccinia virus DNA ligase gene, SalF 15R, for virus growth was tested by insertional mutagenesis. A plasmid containing E. coli gpt inserted within a large deletion in the DNA ligase gene was transfected into vaccinia virus-infected cells and recombinant viruses selected by three cycles of plaque purification in the presence of mycophenolic acid (MPA). Surprisingly, in some isolates, which replicated in a manner indistinguishable from wild type (WT) virus, the WT gene was replaced by the gpt allele, demonstrating that the DNA ligase gene is nonessential for growth in cultured cells. In other isolates the entire plasmid was integrated into the virus genome by a single crossover event and a functional copy of the DNA ligase was retained. Southern blot analyses of the latter, drug-resistant viruses indicated extra DNA fragments, of sizes inconsistent with predicted viral structures, which represent the plasmid products of homologous recombination. Hirt extracts from cells infected with such multiply plaque purified virus isolates yielded plasmids that produced ampicillin-resistant colonies after transformation of E. coli. These plasmids were of two structures, representing either the original plasmid used for transfection, or a plasmid containing the WT ligase gene rescued by recombination with the virus genome. Similarly, insertional mutagenesis of the vaccinia virus thymidine kinase (TK) gene with gpt yielded plasmids containing mutant or wild type TK alleles when recombinant viruses were selected in MPA. Such plasmids were not isolated when TK minus viruses were selected in 5-bromodeoxyuridine (BUdR).