Functional expression of the gene cu, encoding the phytotoxic hydrophobin cerato-ulmin, enables Ophiostoma quercus, a nonpathogen on elm, to cause symptoms of Dutch elm disease

We studied the involvement of the phytotoxic hydrophobin cerato-ulmin (CU) in pathogenesis and virulence of Dutch elm disease (DED) by expressing its encoding gene (cu) in Ophiostoma quercus, a nonpathogenic species on elm closely related to the DED pathogens O. ulmi and O. novo-ulmi. The production of the toxin was quantitatively determined in culture filtrates and in mycelial extracts of the transformants. Production of CU in vitro was associated with the ability to cause typical DED symptoms, consisting of foliar yellow and wilting and vascular tissue discoloration on a moderately resistant elm genotype. The presence of CU was monitored by enzyme-linked immunosorbent assay in symptomatic leaves of plants inoculated with O. quercus transformants expressing CU and found to be associated with wilt symptoms. In general, the virulence of the cu-expressing transformants, as measured in terms of vascular discoloration and percentage of defoliation, was lower than that of the mildly pathogenic isolate E2 of O. ulmi. However, one transformant (C39) displayed a virulence level intermediate between that of E2 and 182, a highly virulent isolate of O. novo-ulmi. Our results indicate that CU production influences virulence in nonaggressive strains of Ophiostoma fungi.     

Selective acquisition of novel mating type and vegetative incompatibility genes via interspecies gene transfer in the globally invading eukaryote Ophiostoma novo-ulmi

The Dutch elm disease fungus Ophiostoma novo-ulmi, which has destroyed billions of elm trees worldwide, originally invaded Europe as a series of clonal populations with a single mating type (MAT-2) and a single vegetative incompatibility (vic) type. The populations then rapidly became diverse with the appearance of the MAT-1 type and many vegetative incompatibility types. Here, we have investigated the mechanism using isolates from sites in Portugal at which the rapid evolution of O. novo-ulmi populations from clonality to heterogeneity was well established. We show by genetic mapping of vic and MAT loci with AFLP markers and by sequence analysis of MAT loci that this diversification was due to selective acquisition by O. novo-ulmi of the MAT-1 and vic loci from another species, Ophiostoma ulmi. A global survey showed that interspecies transfer of the MAT-1 locus occurred on many occasions as O. novo-ulmi spread across the world. We discuss the possibility that fixation of the MAT-1 and vic loci occurred in response to spread of deleterious viruses in the originally clonal populations. The process demonstrates the potential of interspecies gene transfer for facilitating rapid adaptation of invasive organisms to a new environment.     

Use of insertional mutagenesis to tag putative parasitic fitness genes in the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi

We used insertional mutagenesis to produce genetically tagged mutants of the Dutch elm disease fungus Ophiostoma novo-ulmi subsp. novo-ulmi. We first optimized transformation of O. novo-ulmi protoplasts by the restriction enzyme mediated integration method. A concentration of 80 U of HindIII with 108 fungal protoplasts and 5 mug of plasmid DNA was the most efficient for generating a high number of O. novo-ulmi mutants carrying a single insertion in their genome. Mycelium- and yeast-like growth kinetics of 24 O. novo-ulmi mutants were evaluated in vitro. Flanking sequences were successfully recovered in 8% of the transformants analyzed. Some mutant phenotypes appeared to result from gene disruption events, whereas others likely involved modifications of noncoding regions. Several nuclear loci that control vegetative growth and could potentially impact parasitic fitness were successfully tagged.     

Evolutionary Relationships of the Dutch Elm Disease Fungus Ophiostoma novo-ulmi to Other Ophiostoma Species Investigated by Restriction Fragment Length Polymorphism Analysis of the rDNA Region

Restriction fragment length polymorphisms (RFLPs) in the ribosomal RNA gene (rDNA) region were used to assess relationships between the Dutch elm disease fungi Ophiostoma novo-ulmi and Ophiostoma ulmi , the recently described Himalayan Dutch elm disease pathogen, Ophiostoma himal-ulmi , the morphologically similar sapstain fungi, Ophiostoma piceae and Ophiostoma quercus , and several Ophiostoma species from hardwood trees, including Ophiostoma stenoceras and Ophiostoma proliferum . A distance matrix and cluster analysis indicated that the rDNA region of O. himal-ulmi is more closely related to those of O. novo-ulmi and O. ulmi than to those of O. piceae and O. quercus and is more distantly related to O. stenoceras and the other Ophiostoma species, which formed a separate clade. The rDNA region of O. quercus was found to be at least as closely related to that of O. novo-ulmi and O. ulmi as it is to that of O. piceae . The implications of these results for the evolution of the Dutch elm disease fungi are discussed.     

Sequence analysis of the chitin synthase A gene of the Dutch elm pathogen Ophiostoma novo-ulmi indicates a close association with the human pathogen Sporothrix schenckii.

Degenerate oligonucleotide primers were designed according to conserved regions of the chitin synthase gene family and used to amplify a 621 basepair (bp) fragment from genomic DNA of Ophiostoma novo-ulmi, the causal agent of Dutch elm disease. The amplification product was used as a hybridization probe to screen a library of genomic DNA sequences and to retrieve a full-length chitin synthase gene (chsA). The putative coding region of the gene was 2619 bp long, lacked introns, and encoded a polypeptide of 873 amino acids. Based on the similarity of the predicted amino acid sequence to the full-length chsC gene of Aspergillus nidulans and chsA gene of Ampelomyces quisqualis, the O. novo-ulmi chsA was classified as a Class I chitin synthase. The phylogenies constructed, according to a subregion of all available chitin synthases, showed that O. novo-ulmi consistently clustered most closely with the human pathogen Sporothrix schenckii, recently classified as a member of the mitosporic Ophiostomataceae. Disruption of the chsA gene locus had no obvious effects on the growth or morphology of the fungus.     

Molecular characterization of Irish E. coli O157:H7 isolates of human, bovine, ovine and porcine origin

Aims:  To determine the degree of relatedness between isolates of Escherichia coli O157:H7 of human, bovine, ovine and porcine origin.
Methods and Results:  Escherichia coli O157:H7 isolates were compared using (i) PFGE Xba I patterns, (ii) PCR profiles of virulence genes and (iii) the DNA sequences of genes reported to play a role in pathogenicity. The 77 E. coli O157:H7 isolates demonstrated 49 different PFGE patterns of which, eight were common to multiple isolates, and the remaining 41 were distinct. Isolates of different origin did not correlate, except for one cluster consisting of two human and two beef isolates. The majority of animal isolates had the same PCR profiles of virulence genes as those isolated from clinical patients. Single nucleotide polymorphisms (SNPs) were identified in the sequence of a 255-bp region of the vtx2 subunit A gene.
Conclusions:  Six SNPs were detected in the vtx2 A gene, defining four different haplotypes. One nonsynonymous substitution encoded for an amino acid change from glutamic to aspartic acid.
Significance and Impact of the Study:  Results indicate that although E. coli O157:H7 isolates of differing origin were distinct by PFGE, the DNA sequences of the main virulence genes associated with human clinical illness were conserved.     

Clonally diverse rfb gene clusters are involved in expression of a family of related D-galactan O antigens in Klebsiella species.

Klebsiella species express a family of structurally related lipopolysaccharide O antigens which share a common backbone known as D-galactan I. Serotype specificity results from modification of D-galactan I by addition of domains of altered structure or by substitution with O-acetyl and/or alpha-D-Galp side groups with various linkages and stoichiometries. In the prototype, Klebsiella serotype O1, the his-linked rfb gene cluster is required for synthesis of D-galactan I, but genes conferring serotype specificity are unlinked. The D-galactan I part of the O polysaccharide is O acetylated in Klebsiella serotype O8. By cloning the rfb region from Klebsiella serotype O8 and analyzing the O polysaccharide synthesized in Escherichia coli K-12 hosts, we show that, like rfbO1, the rfbO8 region directs formation of unmodified D-galactan I. The rfbAB genes encode an ATP-binding cassette transporter required for export of polymeric D-galactan I across the plasma membrane prior to completion of the lipopolysaccharide molecule by ligation of the O polysaccharide to lipid A-core. Complementation experiments show that the rfbAB gene products in serotypes O1 and O8 are functionally equivalent and interchangeable. Hybridization experiments and physical mapping of the rfb regions in related Klebsiella serotypes suggest the existence of shared rfb genes with a common organization. However, despite the functional equivalence of these rfb gene clusters, at least three distinct clonal groups were detected in different Klebsiella species and subspecies, on the basis of Southern hybridization experiments carried out under high-stringency conditions. The clonal groups cannot be predicted by features of the O-antigen structure. To examine the relationships in more detail, the complete nucleotide sequence of the serotype O8 rfb cluster was determined and compared with that of the serotype O1 prototype. The nucleotide sequences for the six rfb genes showed variations in moles percent G+C values and in the values for nucleotide sequence identity, which ranged from 66.9 to 79.7%. The predicted polypeptides ranged from 64.3% identity (78.4% total similarity) to 94.3% identity (98.0% similarity). The results presented here are not consistent with dissemination of the Klebsiella D-galactan I rfb genes through recent lateral transfer events.     

Stress-induced mobility of OPHIO1 and OPHIO2, DNA transposons of the Dutch elm disease fungi.

The mobility of transposable elements (TEs) can contribute to genome plasticity, under- or over-expression of genes and ectopic recombination. The data collected in this study provide evidence of stress-induced mobility of OPHIO1 and OPHIO2 transposons, recently detected in Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease (DED). The analyses of OPHIO UTRs and TIRs indicated the presence of two potential binding site motifs and a heat shock protein (hsp) promoter which could be involved in the mobility of OPHIO1 following a heat shock stress. The exact position of the hsp promoter was determined by 5' RACE PCR. After confirmation of the expression by RT-PCR of both OPHIO1 and OPHIO2 transposases in the absence of stress factors, we tested two experimental procedures to induce mobility of OPHIO TEs: (1) an exogenous (cloned) copy of OPHIO1 was introduced into the O. novo-ulmi subsp. americana strain W2 (OPHIO1 free strain) to give mutant strain W2:OPHIO1. After exposure of W2:OPHIO1 to a 55 degrees C heat shock treatment, some of the survivors showed signs of incomplete transposition (excision without reinsertion) of OPHIO1. (2) The O. novo-ulmi subsp. novo-ulmi strain AST27, introgressed from O. ulmi and carrying a distinct endogenous copy of OPHIO2 (OPHIO2-int.), was subjected to a series of abiotic stress treatments. Although a promoter sequence could not be identified, both exposures to UV light and to a 4 degrees C cold treatment caused perfect excision of OPHIO2-int. In contrast to OPHIO1, heat shock stress did not induce OPHIO2-int. mobility. Taken together, these results allow us to hypothesize a potential interspecific invasion of OPHIO transposons due to their mobility in Ophiostoma spp.     

Stress-induced mobility of OPHIO1 and OPHIO2, DNA transposons of the Dutch elm disease fungi

The mobility of transposable elements (TEs) can contribute to genome plasticity, under- or over-expression of genes and ectopic recombination. The data collected in this study provide evidence of stress-induced mobility of OPHIO1 and OPHIO2 transposons, recently detected in Ophiostoma ulmi and O. novo-ulmi, the causal agents of Dutch elm disease (DED). The analyses of OPHIO UTRs and TIRs indicated the presence of two potential binding site motifs and a heat shock protein (hsp) promoter which could be involved in the mobility of OPHIO1 following a heat shock stress. The exact position of the hsp promoter was determined by 5' RACE PCR. After confirmation of the expression by RT-PCR of both OPHIO1 and OPHIO2 transposases in the absence of stress factors, we tested two experimental procedures to induce mobility of OPHIO TEs: (1) an exogenous (cloned) copy of OPHIO1 was introduced into the O. novo-ulmi subsp. americana strain W2 (OPHIO1 free strain) to give mutant strain W2:OPHIO1. After exposure of W2:OPHIO1 to a 55 degrees C heat shock treatment, some of the survivors showed signs of incomplete transposition (excision without reinsertion) of OPHIO1. (2) The O. novo-ulmi subsp. novo-ulmi strain AST27, introgressed from O. ulmi and carrying a distinct endogenous copy of OPHIO2 (OPHIO2-int.), was subjected to a series of abiotic stress treatments. Although a promoter sequence could not be identified, both exposures to UV light and to a 4 degrees C cold treatment caused perfect excision of OPHIO2-int. In contrast to OPHIO1, heat shock stress did not induce OPHIO2-int. mobility. Taken together, these results allow us to hypothesize a potential interspecific invasion of OPHIO transposons due to their mobility in Ophiostoma spp.     

Lateral gene transfer of O1 serogroup encoding genes of Vibrio cholerae

In Gram-negative bacteria, the O-antigen-encoding genes may be transferred between lineages, although mechanisms are not fully understood. To assess possible lateral gene transfer (LGT), 21 Argentinean Vibrio cholerae O-group 1 (O1) isolates were examined using multilocus sequence typing (MLST) to determine the genetic relatedness of housekeeping genes and genes from the O1 gene cluster. MSLT analysis revealed that 4.4% of the nucleotides in the seven housekeeping loci were variable, with six distinct genetic lineages identified among O1 isolates. In contrast, MLST analysis of the eight loci from the O1 serogroup region revealed that 0.24% of the 4943 nucleotides were variable. A putative breakpoint was identified in the JUMPstart sequence. Nine conserved nucleotides differed by a single nucleotide from a DNA uptake signal sequence (USS) also found in Pastuerellaceae . Our data indicate that genes in the O1 biogenesis region are closely related even in distinct genetic lineages, indicative of LGT, with a putative DNA USS identified at the defined boundary for the DNA exchange.     

Fine-resolution analysis of products of intrachromosomal homeologous recombination in mammalian cells.

Mouse Ltk- cell lines that contained a herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene with a 16-bp insertion mutation linked to either a defective HSV-2 tk gene or a hybrid tk sequence comprised of HSV-1 and HSV-2 tk sequences were constructed. HSV-1 and HSV-2 tk genes have 81% nucleotide identity and hence are homeologous. Correction of the insertion mutant HSV-1 tk gene via recombination with the hybrid tk sequence required an exchange between homeologous tk sequences, although recombination could initiate within a region of significant sequence identity. Seven cell lines containing linked HSV-1 and HSV-1-HSV-2 hybrid tk sequences gave rise to tk+ segregants at an average rate of 10(-8) events per cell division. DNA sequencing revealed that each recombinant from these lines displayed an apparent gene conversion which involved an accurate transfer of an uninterrupted block of information between homeologous tk sequences. Conversion tract lengths ranged from 35 to >330 bp. In contrast, cell lines containing linked HSV-1 and HSV-2 tk sequences with no significant stretches of sequence identity had an overall rate of homeologous recombination of <10(-9). One such cell line produced homeologous recombinants at a rate of 10(-8). Strikingly, all homeologous recombinants from this latter cell line were due to crossovers between the HSV-1 and HSV-2 tk genes. Our results, which provide the first detailed analysis of homeologous recombination within a mammalian genome, suggest that rearrangements in mammalian genomes are regulated by the degree of sequence divergence located at the site of recombination initiation.     

Analysis of the enterohemorrhagic Escherichia coli O157 DNA region containing lambdoid phage gene p and Shiga-like toxin structural genes.

In this study, we determined the nucleotide sequence of the p gene contained within a 5-kb EcoRI restriction fragment cloned from Shiga-like toxin II (SLT-II)-converting phage 933W of Escherichia coli O157:H7 strain EDL933. The p gene was 702 bp long and had 95.3% sequence similarity to the p gene of phage lambda. Multiple hybridization patterns were obtained when genomic DNA fragments were hybridized with both p and slt-I, slt-II, or slt-IIc sequences. All O157 isolates also possessed an analog of lambda gene p which was not linked with either slt-I or slt-II. Restriction fragment length polymorphism comparisons of clinical O157 isolates and derivates undergoing genotype turnover during infection were made, and loss of large DNA fragments that hybridized with slt-II and p sequences was observed. To further analyze the DNA region containing the p and slt genes, we amplified fragments by using a PCR with one primer complementary to p and the other complementary to either the slt-I or the slt-II gene. PCR analysis with enterohemorrhagic E. coli O157 and non-O157 strains yielded PCR products that varied in size between 5.1 and 7.8 kb. These results suggest that even within O157 isolates, the genomes of SLT-converting phages differ. The methods described here may assist in further investigation of SLT-encoding phages and their role in the epidemiology of infection with enterohemorrhagic E. coli.     

The murine Slp gene. Additional evidence that sex-limited protein has no biologic function

Sex-limited protein (Slp) is a mouse serum protein of unknown function that has approximately 95% amino acid sequence identity with murine complement component C4 but is inactive in the complement pathway. The gene for Slp lies in the S region of the murine H-2 complex adjacent to the gene Cyp21 that encodes the Cytochrome P-450 enzyme steroid 21-hydroxylase. We report the sequence of a 26,307 bp long segment of the mouse genome that includes both the Slp and Cyp21 genes. The sequence reported was assembled from the sequences of three overlapping lambda phage genomic clones from mouse strain B10.WR, which carries four tandem pairs of Slp and Cyp21 genes. We also report the sequence of a fourth lambda clone, 12,539 bp in length, carrying parts of a distinct pair of Slp and Cyp21 genes from B10.WR mice. The Slp gene at 14.3 kb in length is about 1 kb shorter than the C4 gene; this difference is due primarily to absences of a simple repetitive sequence and a middle repetitive MT element in the corresponding introns 14 and 15, respectively. The gene sequence reveals an intron/exon organization identical to that of the murine C4 gene, and also that the 9 nucleotide deletion in exon 18, which appears to be directly responsible for the absence of complement activity, is unrelated to differences in intron sequences. Detailed comparisons of C4 and Slp gene sequences indicate that nucleotide substitutions in the Slp gene are occurring at approximately the same rate in both exons and introns. This implies that the murine Slp gene resembles a pseudogene and supports previously reported evidence that the Slp protein has no biologic function.     

Identification, cloning and sequencing of Escherichia coli strain χ1378 (O78:K80) iss gene isolated from poultry colibacillosis in Iran

Aims:  To identify, clone and sequence the iss (increased serum survival) gene from E. coli strain χ1378 isolated from Iranian poultry and to predict its protein product, Iss.
Methods and Results:  The iss gene from E. coli strain χ1378 was amplified and cloned into the pTZ57R/T vector and sequenced. From the DNA sequence, the Iss predictive protein was evaluated using bioinformatics. Iss from strain χ1378 had 100% identity with other E. coli serotypes and isolates from different origins and also 98% identity with E. coli O157:H7 Iss protein. Phylogenetic analysis showed no significant different phylogenic groups among E. coli strains.
Conclusions:  The strong association of predicted Iss protein among different E. coli strains suggests that it could be a good antigen to control and detect avian pathogenic E. coli (APEC).
Significance and Impact of the study:  Because the exact pathogenesis and the role of virulence factors are unknown, the Iss protein could be used as a target for vaccination in the future, but further research is required.     

Evidence for recent intergeneric transfer of a new tetracycline resistance gene, tet(W), isolated from Butyrivibrio fibrisolvens, and the occurrence of tet(O) in ruminal bacteria

We have previously reported high-frequency transfer of tetracycline resistance between strains of the rumen anaerobic bacterium Butyrivibrio fibrisolvens . Donor strains were postulated to carry two Tc^R genes, one of which is transferred on a novel chromosomal element. It is shown here that coding sequences within the non-transmissible gene in B. fibrisolvens 1.230 are identical to those of the Streptococcus pneumoniae tet (O) gene. This provides the first evidence for genetic exchange between facultatively anaerobic bacteria and rumen obligate anaerobes. In contrast, the product of the transmissible Tc^R gene shares only 68% amino acid sequence identity with the TetO and TetM proteins and represents a new class of ribosome protection tetracycline resistance determinant, designated Tet W. The tet (W) coding region shows a higher DNA G + C content (53%) than other B. fibrisolvens genes or other ribosome protection-type tet genes, suggesting recent acquisition from a high G + C content genome. Tet (W) genes with almost identical sequences are also shown to be present in Tc^R strains of B. fibrisolvens from Australian sheep and in Tc^R strains of two other genera of rumen obligate anaerobes, Selenomonas ruminantium and Mitsuokella multiacidus . This provides compelling evidence for recent intergeneric transfer of resistance genes between ruminal bacteria. Tet (W) is not restricted to ruminal bacteria, as it was also present in a porcine strain of M. multiacidus .     

Relationships of the Escherichia coli O157, O111, and O55 O-antigen gene clusters with those of Salmonella enterica and Citrobacter freundii, which express identical O antigens

Escherichia coli O157, Salmonella enterica O30, and Citrobacter freundii F90 have identical O-antigen structures, as do E. coli O55 and S. enterica O50. The O-antigen gene cluster sequences for E. coli O157 and E. coli O55 have been published, and the genes necessary for O-antigen biosynthesis have been identified, although transferase genes for glycosidic linkages are only generic and have not been allocated to specific linkages. We determined sequences for S. enterica O30 and C. freundii F90 O-antigen gene clusters and compared them to the sequence of the previously described E. coli O157 cluster. We also determined the sequence of the S. enterica O50 O-antigen gene cluster and compared it to the sequence of the previously described E. coli O55 cluster. For both the S. enterica O30-C. freundii F90-E. coli O157 group and the S. enterica O50-E. coli O55 group of O antigens, the gene clusters have identical or nearly identical organizations. The two sets of gene clusters had comparable overall levels of similarity in their genes, which were lower than the levels determined for housekeeping genes for these species, which were 55 to 65% for the genes encoding glycosyltransferases and O-antigen processing proteins and 75 to 93% for the nucleotide-sugar pathway genes. Nonetheless, the similarity of the levels of divergence in the five gene clusters required us to consider the possibility that the parent gene cluster for each structure was in the common ancestor of the species and that divergence is faster than expected for the common ancestor hypothesis. We propose that the identical O-antigen gene clusters originated from a common ancestor, and we discuss some possible explanations for the increased rate of divergence that is seen in these genes.     

Epstein-Barr virus types 1 and 2 differ in their EBNA-3A, EBNA-3B, and EBNA-3C genes.

The two Epstein-Barr virus (EBV) types, EBV-1 and EBV-2, are known to differ in their EBNA-2 genes, which are 64 and 53% identical in their nucleotide and predicted amino acid sequences, respectively. Restriction endonuclease maps and serologic analyses detect few other differences between EBV-1 and EBV-2 except in the EBNA-3 gene family. We determined the DNA sequence of the AG876 EBV-2 EBNA-3 coding region and have compared it with known B95-8 EBV-1 EBNA-3 sequences to delineate the extent of divergence between EBV-1 and EBV-2 isolates in their EBNA-3 genes. The B95-8 and AG876 EBV isolates had nucleotide and amino acid identity levels of 90 and 84%, 88 and 80%, and 81 and 72% for the EBNA-3A, -3B, and -3C genes, respectively. In contrast, nucleotide sequence identity in the noncoding DNA adjacent to the B95-8 and AG876 EBNA-3 open reading frames was 96%. We used the polymerase chain reaction to demonstrate that five additional EBV-1 isolates and six additional EBV-2 isolates have the type-specific differences in their EBNA-3 genes predicted from the B95-8 or AG876 sequences. Thus, EBV-1 and EBV-2 are two distinct wild-type EBV strains that have significantly diverged at four genetic loci and have maintained type-characteristic differences at each locus. The delineation of these sequence differences between EBV-1 and EBV-2 is essential to ongoing molecular dissection of the biologic properties of EBV and of the human immune response to EBV infection. The application of these data to the delineation of epitopes recognized in the EBV-immune T-cell response is also discussed.     

RNA-dependent RNA polymerase gene sequence from foot-and-mouth disease virus in Hong Kong

A foot-and-mouth disease virus (FMDV, HKN/2002) was isolated in Hong Kong in 2002. The nucleotide sequence of the 3D(pol) gene encoding the viral RNA-dependent RNA polymerase was determined and compared with that of the same gene from other FMDVs. The 3D(pol) gene was 1410 nucleotides in length encoding a protein of 470 amino acid residues. Sequence comparisons indicated that HKN/2002 belonged to serotype O. An evolutionary tree based on the 3D(pol) sequences of 20 FMDV isolates revealed that the nucleotide sequence of the HKN/2002 3D(pol) gene was most similar to those of isolates found in Taiwan in 1997, suggesting that they share a common ancestor. The amino acid sequence of the HKN/2002 3D(pol) gene was determined and aligned with those of representative isolates from seven other Picornaviridae genera. Eight highly conserved regions were detected, indicating a conserved functional relevance for these motifs. Alignment of 20 FMDV 3D(pol) amino acid sequences revealed a hypermutation region near the N-terminus that may help the virus evade host immune systems.     

Latent a1 VH germline genes in an a2a2 rabbit. Evidence for gene conversion at both the germline and somatic levels

We have previously reported the sequences of putative latent a1 cDNA derived from an alpha 2 alpha 2 rabbit. Significant similarity to nominal a1 cDNA sequences was noted, but none of the latent sequences were completely a1-like. We have now probed a genomic library, produced from the same alpha 2 alpha 2 rabbit, for evidence of germline latent a1 VH genes. Four hundred ninety-four VH+ clones were screened with oligonucleotides specific for a1 diagnostic regions of framework region 1 (FR1) and FR3. Twenty-two percent of the VH+ clones hybridized with an a1FR3 oligonucleotide probe. Two a1 FR1 probes yielded weak signals with 6% to 13% of the VH+ clones. Twenty VH genes from clones positive for one or more of the a1-specific oligonucleotide probes were sequenced, revealing 14 unique germline VH genes. All but one of these genes were 85% to 92% identical to the VH1-a1 nominal gene prototype, with sequence identity extending into the leader intron. Most genes displayed extended regions of similarity to a1 in FR1, FR3, or both and expressed 13 to 17 of the 21 allotype-associated residues, consistent with the nominal a1 sequence. The a1-like sequences were variously interspersed with short non-a1 segments, suggestive of germline gene conversion. Although none of the germline a1-like VH genes we have isolated from the alpha 2 alpha 2 rabbits are identical to the known a1 genes or protein sequences from alpha 1 alpha 1 rabbits and 8 of 14 are pseudogenes, most could make significant contributions to the synthesis of a complete nominal a1 sequence by serving as a pool of sequence donors during somatic gene conversion.     

Multilocus sequence typing of Listeria monocytogenes by use of hypervariable genes reveals clonal and recombination histories of three lineages

In an attempt to develop a method to discriminate among isolates of Listeria monocytogenes, the sequences of all of the annotated genes from the fully sequenced strain L. monocytogenes EGD-e (serotype 1/2a) were compared by BLASTn to a file of the unfinished genomic sequence of L. monocytogenes ATCC 19115 (serotype 4b). Approximately 7% of the matching genes demonstrated 90% or lower identity between the two strains, and the lowest observed identity was 80%. Nine genes (hisJ, cbiE, truB, ribC, comEA, purM, aroE, hisC, and addB) in the 80 to 90% identity group and two genes (gyrB and rnhB) with approximately 97% identity were selected for multilocus sequence analysis in two sets of L. monocytogenes isolates (a 15-strain diversity set and a set of 19 isolates from a single food-processing plant). Based on concatenated sequences, a total of 33 allotypes were differentiated among the 34 isolates tested. Population genetics analyses revealed three lineages of L. monocytogenes that differed in their history of apparent recombination. Lineage I appeared to be completely clonal, whereas representatives of the other lineages demonstrated evidence of horizontal gene transfer and recombination. Although most of the gene sequences for lineage II strains were distinct from those of lineage I, a few strains with the majority of genes characteristic of lineage II had some that were characteristic of lineage I. Genes from lineage III organisms were mostly similar to lineage I genes, with instances of genes appearing to be mosaics with lineage II genes. Even though lineage I and lineage II generally demonstrated very distinct sequences, the sequences for the 11 selected genes demonstrated little discriminatory power within each lineage. In the L. monocytogenes isolate set obtained from one food-processing plant, lineage I and lineage II were found to be almost equally prevalent. While it appears that different lineages of L. monocytogenes can share habitats, they appear to differ in their histories of horizontal gene transfer.