EcoRI-digested Genomic DNA Research Articles

Single Nucleotide Polymorphism-Based Diagnostic System for Crop-Associated Sclerotinia Species

A molecular diagnostic system using single nucleotide polymorphisms (SNPs) was developed to identify four Sclerotinia species: S. sclerotiorum (Lib.) de Bary, S. minor Jagger, S. trifoliorum Erikss., and the undescribed species Sclerotinia species 1. DNAs of samples are hybridized with each of five 15-bp oligonucleotide probes containing an SNP site midsequence unique to each species. For additional verification, hybridizations were performed using diagnostic single nucleotide substitutions at a 17-bp sequence of the calmodulin locus. The accuracy of these procedures was compared to that of a restriction fragment length polymorphism (RFLP) method based on Southern hybridizations of EcoRI-digested genomic DNA probed with the ribosomal DNA-containing plasmid probe pMF2, previously shown to differentiate S. sclerotiorum, S. minor, and S. trifoliorum. The efficiency of the SNP-based assay as a diagnostic test was evaluated in a blind screening of 48 Sclerotinia isolates from agricultural and wild hosts. One isolate of Botrytis cinerea was used as a negative control. The SNP-based assay accurately identified 96% of Sclerotinia isolates and could be performed faster than RFLP profiling using pMF2. This method shows promise for accurate, high-throughput species identification.

High rate of restriction fragment length polymorphisms between two population of the nematode Pristionchus pacificus (Diplogastridae)

Pristionchus pacificus (Diplogastridae, Nematoda) has recently been described as a ‘satelite’ organism for a functinal comparative approach, becuase genetic, molecular and cell-biological tools can be used in way similar to the genetic model organism Caenorhabditis elegans. This tudy describes the analysis of two previously isolated strains of P. pacificus for the occurrence of restriction fragment length polymorphisms (RFLPs). In all, 14 of 17 randomly chosen cDNA clones give polymorphisms after hybridization to EcoRI digested genomic DNA of the populations from California and Washington. This polymorphism is much higher than polymorphism found among different strains in C. elegans. Therefore this study compares most of the nucleotide sequence of the Ppa-let-60/ras gene between the two strains. No base-pair substitutions were found between these two sequences within the coding regions. However, within the untranslated region, four base-pair substitutions in introns and the deletion of three base-pairs in the 5′ sequence and in intron 4 have been observed. Since the two strains interbreed, RFLPs can be used as molecular markers for future chromosomal walking and gene cloning.

Genetic Diversity of Trichomonas vaginalis Clinical Isolates Determined by EcoRI Restriction Fragment Length Polymorphism of Heat-Shock Protein 70 Genes

Restriction fragment length polymorphism (RFLP) analysis using a multilocus heat-inducible cytoplasmic heat-shock protein 70 (Hsp70) hybridization probe with EcoRI-digested genomic DNA was used in molecular typing of 129 Trichomonas vaginalis isolates. Results indicate that Trichomonas organisms exhibit considerable polymorphism in their Hsp70 RFLP patterns. Analysis of seven American Type Culture Collection reference strains and 122 clinical isolates, including 84 isolates from Jackson, Mississippi, 18 isolates from Atlanta, Georgia, and 20 isolates from throughout the United States, showed 105 distinct Hsp70 RFLP pattern subtypes for Trichomonas. Phylogenetic analysis of the Hsp70 RFLP data showed that the T. vaginalis isolates were organized into two clonal lineages. These results illustrate the substantial genomic diversity present in T. vaginalis and indicate that a large number of genetically distinct Trichomonas isolates may be responsible for human trichomoniasis in the United States.

New families of site-specific repetitive DNA sequences that comprise constitutive heterochromatin of the Syrian hamster (Mesocricetus auratus, Cricetinae, Rodentia)

We molecularly cloned new families of site-specific repetitive DNA sequences from BglII- and EcoRI-digested genomic DNA of the Syrian hamster (Mesocricetus auratus, Cricetrinae, Rodentia) and characterized them by chromosome in situ hybridization and filter hybridization. They were classified into six different types of repetitive DNA sequence families according to chromosomal distribution and genome organization. The hybridization patterns of the sequences were consistent with the distribution of C-positive bands and/or Hoechst-stained heterochromatin. The centromeric major satellite DNA and sex chromosome-specific and telomeric region-specific repetitive sequences were conserved in the same genus (Mesocricetus) but divergent in different genera. The chromosome-2-specific sequence was conserved in two genera, Mesocricetus and Cricetulus, and a low copy number of repetitive sequences on the heterochromatic chromosome arms were conserved in the subfamily Cricetinae but not in the subfamily Calomyscinae. By contrast, the other type of repetitive sequences on the heterochromatic chromosome arms, which had sequence similarities to a LINE sequence of rodents, was conserved through the three subfamilies, Cricetinae, Calomyscinae and Murinae. The nucleotide divergence of the repetitive sequences of heterochromatin was well correlated with the phylogenetic relationships of the Cricetinae species, and each sequence has been independently amplified and diverged in the same genome.

Inhibition of Nucleotide Excision Repair by High Mobility Group Protein HMGA1

The mammalian non-histone "high mobility group" A (HMGA) proteins are the primary nuclear proteins that bind to the minor groove of AT-rich DNA. They may, therefore, influence the formation and/or repair of DNA lesions that occur in AT-rich DNA, such as cyclobutane pyrimidine dimers (CPDs) induced by UV radiation. Employing both stably transfected lines of human MCF7 cells containing tetracycline-regulated HMGA1 transgenes and primary Hs578T tumor cells, which naturally overexpress HMGA1 proteins, we have shown that cells overexpressing HMGA1a protein exhibit increased UV sensitivity. Moreover, we demonstrated that knockdown of intracellular HMGA1 concentrations via two independent methods abrogated this sensitivity. Most significantly, we observed that HMGA1a overexpression inhibited global genomic nucleotide excision repair of UV-induced CPD lesions in MCF-7 cells. Consistent with these findings in intact cells, DNA repair experiments employing Xenopus oocyte nuclear extracts and lesion-containing DNA substrates demonstrated that binding of HMGA1a markedly inhibits removal of CPDs in vitro. Furthermore, UV "photo-foot-printing" demonstrated that CPD formation within a long run of Ts (T(18)-tract) in a DNA substrate changes significantly when HMGA1 is bound prior to UV irradiation. Together, these results suggest that HMGA1 directly influences both the formation and repair of UV-induced DNA lesions in intact cells. These findings have important implications for the role that HMGA protein overexpression might play in the accumulation of mutations and genomic instabilities associated with many types of human cancers.

Targeted Disruption of MAIL, a Nuclear IκB Protein, Leads to Severe Atopic Dermatitis-like Disease

MAIL (molecule-possessing ankyrin repeats induced by lipopolysaccharide) is a nuclear IkappaB protein that is also termed interleukin-1-inducible nuclear ankyrin repeat protein or inhibitor of nuclear factor kappaB (IkappaB) zeta. In this study, we generated Mail-/- mice to investigate the roles of MAIL in whole organisms. Mail-/- mice grew normally until 4-8 weeks after birth, when they began to develop lesions in the skin of the periocular region, face, and neck. MAIL mRNA and protein were constitutively expressed in the skin of wild type controls, especially in the keratinocytes. Serum IgE was higher in Mail-/- mice than in normal. Histopathological analysis indicated that the Mail-/- skin lesions appeared to be atopic dermatitis (AD) eczema with inflammatory cell infiltration. In addition, markedly elevated expression of some chemokines such as thymus and activation-regulated chemokine was detected in the Mail-/- skin lesions, similar to that observed in the skin of patients with AD. In Mail-/- mice, MAIL-deficient keratinocytes might be activated to produce chemokines and induce intraepidermal filtration of inflammatory cells, resulting in the onset of the AD-like disease. These findings suggest that MAIL is an essential molecule for homeostatic regulation of skin immunity. The Mail-/- mouse is a valuable new animal model for research on AD.

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes).

We isolated a new family of satellite DNA sequences from HaeIII- and EcoRI-digested genomic DNA of the Blakiston's fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.

Development of a Specific Polymerase Chain Reaction-Based Assay for the Identification of Fusarium oxysporum f. sp. ciceris and Its Pathogenic Races 0, 1A, 5, and 6

ABSTRACT Specific primers and polymerase chain reaction (PCR) assays that identify Fusarium oxysporum f. sp. ciceris and each of the F. oxysporum f. sp. ciceris pathogenic races 0, 1A, 5, and 6 were developed. F. oxysporum f. sp. ciceris- and race-specific random amplified polymorphic DNA (RAPD) markers identified in a previous study were cloned and sequenced, and sequence characterized amplified region (SCAR) primers for specific PCR were developed. Each cloned RAPD marker was characterized by Southern hybridization analysis of Eco RI-digested genomic DNA of a subset of F. oxysporum f. sp. ciceris and nonpathogenic F. oxysporum isolates. All except two cloned RAPD markers consisted of DNA sequences that were found highly repetitive in the genome of all F. oxysporum f. sp. ciceris races. F. oxysporum f. sp. ciceris isolates representing eight reported races from a wide geographic range, nonpathogenic F. oxysporum isolates, isolates of F. oxysporum f. spp. lycopersici, melonis, niveum, phaseoli, and pisi, and isolates of 47 different Fusarium spp. were tested using the SCAR markers developed. The specific primer pairs amplified a single 1,503-bp product from all F. oxysporum f. sp. ciceris isolates; and single 900- and 1,000-bp products were selectively amplified from race 0 and race 6 isolates, respectively. The specificity of these amplifications was confirmed by hybridization analysis of the PCR products. A race 5-specific identification assay was developed using a touchdown-PCR procedure. A joint use of race 0- and race 6-specific SCAR primers in a single-PCR reaction together with a PCR assay using the race 6-specific primer pair correctly identified race 1A isolates for which no RAPD marker had been found previously. All the PCR assays described herein detected up to 0.1 ng of fungal genomic DNA. The specific SCAR primers and PCR assays developed in this study clearly identify and differentiate isolates of F. oxysporum f. sp. ciceris and of each of its pathogenic races 0, 1A, 5, and 6.

Molecular cloning and characterization of novel centromeric repetitive DNA sequences in the blue-breasted quail (Coturnix chinensis, Galliformes)

A new family of centromeric highly repetitive DNA sequences was isolated from EcoRI-digested genomic DNA of the blue-breasted quail (Coturnix chinensis, Galliformes), and characterized by filter hybridization and chromosome in situ hybridization. The repeated elements were divided into two types by nucleotide length and chromosomal distribution; the 578-bp element predominantly localized to microchromosomes and the 1,524-bp element localized to chromosomes 1 and 2. The 578-bp element represented tandem arrays and did not hybridize to genomic DNAs of other Galliformes species, chicken (Gallus gallus), Japanese quail (Coturnix japonica) and guinea fowl (Numida meleagris). On the other hand, the 1,524-bp element was not organized in tandem arrays, and did hybridize to the genomic DNAs of three other Galliformes species, suggesting that the 1,524-bp element is highly conserved in the Galliformes. The 578-bp element was composed of basic 20-bp internal repeats, and the consensus nucleotide sequence of the internal repeats had homologies to the 41–42 bp CNM repeat and the XhoI family repeat of chicken. Our data suggest that the microchromosome-specific highly repetitive sequences of the blue-breasted quail and chicken were derived from a common ancestral sequence, and that they are one of the major and essential components of chromosomal heterochromatin in Galliformes species.

Development of interspecific somatic hybrid cell lines in cultivated jute and their early characterization using jute chloroplast RFLP marker

AbstractA strong sexual incompatibility barrier that exists between the two cultivated jute species, Corchorus capsularis and Corchorus olitorius, limits the scope for improvement through genetic introgression. Protoplast fusion was carried out to generate interspecific hybrid cell lines. Cotyledonary cell protoplasts of C. capsularis and anthocyaninpigmented hypocotyl protoplasts of C. olitorius were used in the fusion experiments, which appeared to be visually useful in the early selection of the fused products. A chloroplast DNA (cpDNA) marker was developed in jute, which showed species‐specific hybridization patterns with EcoRI‐digested total genomic DNA of C. capsularis and C. olitorius. This cpDNA marker was used in the characterization of the somatic hybrid cell lines at their early stages of growth. Evidence for the presence of both types of cpDNA in the hybrid cell lines was obtained when the total genomic DNA of 4‐ to 7‐month‐old hybrid cell lines was challenged with the chloroplast DNA marker through Southern analysis. It was shown that the early segregation of the parental chloroplasts did not occur in jute, although this is common in other plant species. The hybrid nature of the fused cell lines could also be identified through peroxidase isozyme analysis. Isozyme banding patterns were complex and varied among the hybrid cell lines.

Identification and characterization of a putative light-harvesting chlorophyll a/b-binding protein gene encoded at a fertility restorer locus for the Ogura CMS in Brassica napus L.

The nuclear-encoded fertility restorer locus for Ogura radish cytoplasmic male sterility (CMS) was previously transferred from radish into Brassica napus through intergeneric crosses, with the putative restorer gene(s) being expressed in leaves and flower buds. To identify transcripts corresponding to this restorer locus, we performed a PCR-selected cDNA subtraction using leaf mRNA from the restorer line and its recurrent parent. The resulting library was differentially screened using the subtracted cDNAs for the library itself and the cDNAs from the reverse subtraction (recurrent parent subtracted against the restorer line). Forty-eight cDNA clones were isolated based on their specific hybridization with cDNAs for the library construction. Sequence analyses revealed that these 48 clones correspond to only three different genes: two light-harvesting chlorophyll a/b-binding protein (Cab) genes and one chloroplast-encoded gene (a chloroplast 50S ribosomal protein gene). Of the two putative Cab genes, one showed polymorphism on the Southern blot with the EcoRI-digested genomic DNA from the restorer line and its recurrent parent. Segregation analyses confirmed that this putative Cab gene comes from radish DNA and co-segregates with the restorer locus. A sequence comparison of this putative Cab gene with its homeolog in Brassica napus revealed that (1) their coding regions share a 94% similarity, (2) they differ in the size of the intron, and (3) they are identical in the deduced amino acid sequences, except that the one encoded at the restorer locus contained an additional alanine at position 34.

Molecular typing of Trichomonas vaginalis isolates by HSP70 restriction fragment length polymorphism.

Subtyping isolates of Trichomonas vaginalis is an essential tool for understanding the epidemiology of this common sexually-transmitted disease. Restriction fragment length polymorphism (RFLP) analysis employing a probe from the heat-inducible cytoplasmic HSP70 gene family hybridized with EcoR I-digested genomic DNA was used in the molecular typing of Trichomonas isolates. Analysis of five American Type Culture Collection (ATCC) reference strains and 31 Jackson, Mississippi, isolates from six male and 21 female patients, revealed 10 distinct RFLP pattern subtypes of Trichomonas. The subtypes were temporally stable and cosmopolitan. The RFLP profiles seen in Maryland, Ohio, Massachusetts, and New York ATCC strains were identical to those of some Mississippi isolates, even though the samples were isolated 10-35 years apart. There was no correlation between metronidazole resistance and RFLP subtype with resistant isolates from eight patients distributed among six different subtypes.

Early-replicating DNA from mosquito cells is associated with a distinct EcoRI fragment

In an effort to define an origin of bi-directional DNA replication (OBR) in mosquito genomic DNA, we applied methods that take advantage of characteristic features of single-stranded DNA to methotrexate-resistant Aedes albopictus cells. The Mtx-5011-256 cells contained approximately 1000 copies of a 200 kb amplicon containing the dihydrofolate reductase locus, which likely contained one or more replication origins. When Mtx-5011-256 cells were synchronized by treatment with hydroxyurea, released into the S phase of the cell cycle, and labeled in vivo with tritiated DNA precursors, a 1.9 kb EcoRI fragment was preferentially labeled in EcoRI-digested genomic DNA. Similarly, we detected a 1.9 kb EcoRI fragment in DNA from wild type cells after cell cycle synchronization and in vivo labeling. In a complementary method, unlabeled single-stranded DNA was isolated from Mtx-5011-256 cells, labeled in vitro, and hybridized to EcoRI-digested genomic DNA from mosquito cells. The labeled probe hybridized preferentially to a 1.9 kb fragment. Finally, a 1.9 kb EcoRI fragment was detected when nascent DNA was recovered from unsynchronized cells, made double-stranded by in vitro labeling, and digested with EcoRI. Taken together, these results suggest that in Aedes albopictus mosquito cells, many replication origins used at different times during S are flanked by EcoRI sites that define a 1.9 kb fragment, which has become more abundant in Mtx-5011-256 cells because it occurs in the dhfr amplicon. Tentative mapping of this origin to amplicon DNA remains ambiguous, further suggesting that a repeated sequence element occurs at or near the origin of replication.

Homologous recombination and non-homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells.

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ). Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54(-/-), KU70(-/-) and RAD54(-/-)/KU70(-/-), generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing gamma-radiation-induced DSBs during G1-early S phase while recombinational repair is preferentially used in late S-G2 phase. RAD54(-/-)/KU70(-/-) cells were profoundly more sensitive to gamma-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54(-/-) and RAD54(-/-)/KU70(-/-) cells, with RAD54(-/-)/KU70(-/-) cells exhibiting significantly higher levels of chromosomal aberrations than RAD54(-/-) cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.

Reiterated repeat region variability in the ciliary adhesin gene of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is a highly prevalent pathogen which colonizes the ciliated epithelial lining of the porcine respiratory tract. Expression libraries constructed from genomic DNA of the non-pathogenic strain M. hyopneumoniae J were screened with porcine hyperimmune antiserum against M. hyopneumoniae. One clone expressed a 28 kDa protein which was also reactive with monospecific antiserum raised against a putative M. hyopneumoniae-specific 94 kDa antigen derived from strain J. Trypsin digestion of whole M. hyopneumoniae cells showed the 94 kDa antigen to be surface-accessible. DNA sequence analysis of the gene encoding the 94 kDa antigen revealed greater than 90% homology to two adhesin genes, encoding P97 and Mhp1, cloned from pathogenic strain 232 and strain P5722 of M. hyopneumoniae, respectively. Two regions of repetitive DNA sequence were identified in the gene encoding the 94 kDa antigen. The first encoded the deduced amino acid sequence A(T)-K-P-E(V)-A(T) arranged as nine tandem repeats (RR1). The second region of repetitive DNA sequence encoded the deduced amino acid sequence G-A(E,S)-P-N(S)-Q-G-K-K-A-E arranged as five tandem repeats (RR2). Comparison of the three M. hyopneumoniae adhesin genes revealed that the genes encoding P97 and Mhp1, and the strain J gene encoding the 94 kDa antigen contained 15, 12 and 9 tandem repeats, respectively, in RR1, and 4, 5 and 5 tandem repeats, respectively, in RR2. Southern hybridization analysis of EcoRI-digested genomic DNA probed with an 820 bp fragment spanning RR1 and RR2 identified a strongly hybridizing fragment ranging in size from 2.15 to 2.30 kb among seven geographically diverse strains of M. hyopneumoniae but failed to hybridize with DNA from four strains of Mycoplasma hyorhinis or Mycoplasma flocculare strain Ms42. PCR primers flanking the DNA sequence encoding RR1 and RR2 were used to amplify DNA from the seven strains of M. hyopneumoniae and DNA sequence analysis of the amplification products showed that the number of tandem amino acid repeats in RR1 varied considerably between strains. RR1 from M. hyopneumoniae strains YZ, Beaufort, Sue, OMZ407 and C1735/2 comprised 11, 15, 12, 15 and 8 tandem copies, respectively, of the 5-aa repeat whilst RR2 comprised 4, 3, 4, 3 and 4 tandem copies, respectively, of the 10-aa repeat. Two putative integrin binding sites (L-E-T and R-X-X-X-D) were identified in the 94 kDa ciliary adhesin. Variability in the number of amino acid repeats in RR1 amongst strains of M. hyopneumoniae may influence ciliary binding.

Cloning and characterization of a novel macrolide efflux gene, mreA, from Streptococcus agalactiae.

A strain of Streptococcus agalactiae displayed resistance to 14-, 15-, and 16-membered macrolides. In PCR assays, total genomic DNA from this strain contained neither erm nor mef genes. EcoRI-digested genomic DNA from this strain was cloned into lambda Zap II to construct a library of S. agalactiae genomic DNA. A clone, pAES63, expressing resistance to erythromycin, azithromycin, and spiramycin in Escherichia coli was recovered. Deletion derivatives of pAES63 which defined a functional region on this clone that encoded resistance to 14- and 15-membered, but not 16-membered, macrolides were produced. Studies that determined the levels of incorporation of radiolabelled erythromycin into E. coli were consistent with the presence of a macrolide efflux determinant. This putative efflux determinant was distinct from the recently described Mef pump in Streptococcus pyogenes and Streptococcus pneumoniae and from the multicomponent MsrA pump in Staphylococcus aureus and coagulase-negative staphylococci. Its gene has been designated mreA (for macrolide resistance efflux).

DNA fragments from regions involved in surface antigen expression specifically identify Listeria monocytogenes serovar 4 and a subset thereof: cluster IIB (serotypes 4b, 4d, and 4e).

Listeria monocytogenes serotype 4b has frequently been implicated in sporadic as well as epidemic listeriosis. On the basis of pulsed-field fingerprinting, serotype 4b strains, along with strains of serotypes 4d and 4e, constitute one genomic cluster (IIB). We have identified two genomic regions essential for the expression of surface antigens which previously were shown to be specific to cluster IIB strains. A DNA probe of 1.1 kb derived from one of the regions (probe 1) hybridized only with strains of serotypes 4b, 4d, and 4e in Southern blots and dot blots. A different DNA probe of 0.3 kb (probe 2), derived from the other region, hybridized with all serovar 4 strains (serotypes 4b, 4a, 4c, 4d, and 4e). All other L. monocytogenes serotypes were negative with probe 1 or 2. Use of probe 1 in Southern blots of EcoRI-digested genomic DNA revealed a restriction fragment length polymorphism in serotype 4b strains, with the hybridizing EcoRI fragments being 4.5 kb (strains of the epidemic clone) and either 4.5 or 5.0 kb (all other serotype 4b strains). Although the probes hybridized with a special group of Listeria innocua strains which also expressed the surface antigens, the latter could be readily distinguished by the size of the hybridizing EcoRI fragment with probe 1 (ca. 2.2 kb). These data suggest that the combined use of these probes with L. monocytogenes can readily and specifically identify cluster IIB strains as well as the entire serovar 4 complex.

Identification of simian varicella virus homologues of varicella zoster virus genes.

Clinical, pathologic, immunologic and virologic features of simian varicella virus (SVV) infection in primates resemble human varicella-zoster virus (VZV) infection. Further, the SVV and VZV genomes are similar in size and structure, show striking homology in their configuration and DNA sequences, and encode antigenically related polypeptides. Although the entire VZV genome is present during latency in human ganglia, transcription is limited. VZV genes 21, 29, 62 and 63 are transcribed during latency, while genes 4, 10, 40, 51 and 61 are not transcribed. The entire VZV genome has been sequenced, but the SVV genome has not. Thus, to analyze SVV genes transcribed during latency, we have begun to identify SVV homologues of the above VZV genes. We used nick-translated [32p]-labeled-VZV open reading frame (ORF)-specific probes to screen Southern blots containing EcoRI-digested SVV genomic DNA and recombinant clones of SVV EcoRI and BamHI DNA fragments spanning approximately 97% of the virus genome. We showed that SVV homologues of VZV ORFs 4, 10, 29, 40, 51 and 61 mapped to SVV DNA fragments EcoRI I, A, N, BamHI E, EcoRI D and E, respectively. We also confirmed earlier reports that SVV homologues of VZV genes 21 and 63 mapped to SVV EcoRI DNA fragments H and C, respectively. Viral genes on the SVV and VZV genomes seem to be collinear.

Organization and chromosomal localization of a B1-like containing repeat of Microtus subarvalis

A repetitive DNA sequence, MS2, was isolated from EcoRI-digested genomic DNA of the vole, Microtus subarvalis. The fragment was cloned and sequenced. Sequence analysis of this 1194-bp fragment revealed a 156-bp region demonstrating a 55% homology with the mouse B1 repeat. The remaining MS2 sequence shows no significant homology with other known GenBank sequences. The results of in situ hybridization of MS2 on vole metaphase chromosomes indicate the fragment is confined to heterochromatin blocks of the sex chromosomes in all but one species (M. arvalis). Distribution of MS2 sequences provides evidence for heterogeneity of the giant heterochromatin blocks of the XY Chromosomes (Chrs) in voles, for the unique cluster-like localization of MS2 within these blocks.

Cloning of genes required for hypersensitivity and pathogenicity inPseudomonas syringae pv.aptata

A genomic library of Pseudomonas syringae pv. aptata strain NCPPB 2664, which causes bacterial blight of sugar beet, lettuce and other plants, was constructed in the cosmid vector pCPP31. The 13.4 kb EcoRI fragment of the cosmid pHIR11, containing the hrp (hypersensitive response and pathogenicity) gene cluster of the closely related bacterium Pseudomonas syringae pv. syringae strain 61, was used as a probe to identify a homologous hrp gene cluster in P. syringae pv. aptata. Thirty of 2500 cosmid clones, screened by colony hybridization, gave a strong hybridization signal with the probe, but none of these conferred to the non-pathogenic bacterium, Pseudomonas fluorescens, the ability to elicit the hypersensitive response (HR) in tobacco. Southern blot analysis of EcoRI-digested genomic DNA of P. syringae pv. aptata showed hybridizing bands of 12 kb and 4.4 kb. Only a 12 kb fragment hybridized in digests of the cosmids. Cosmid clone pCPP1069 was mutagenized with Tn10-minitet and marker-exchanged into the genome of P. syringae pv. aptata. Three resulting prototrophic mutant strains failed to elicit the HR in tobacco and to cause disease in lettuce. The DNA flanking the Tn10-minitet insertions from mutated derivatives of pCPP1069 hybridized with the 10.6 kb Bg/II fragment of pHIR11. These results indicate that P. syringae pv. aptata harbours hrp genes that are similar to, but arranged differently from, homologous hrp genes of P. syringae pv. syringae.