A new molecular method for the rapid subtyping of bovine herpesvirus 1 field isolates

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.

Gene Stacking for Fungal Resistance in Plant Transformation Vector

ABSTRACT: Fungal diseases like early blight, late blight, fusarium wilt cause 30-40 per cent loss in fruit production. Form past decade many transgenic plants had been developed using genes encoding chitinases and glucanases with the objective of imparting fungal disease resistance. Since the genes encoding chitinase and glucanase act synergistically. The study was performed to construct plant transformation vector pRAGS carrying both ech42 and bgn under single T-DNA region. Initially, HindIII site at 5' end of earlier cloned bgn (T. harzianum) was removed using primers during reamplification of the gene. The amplicons were cloned into pTZ57R/T containing T overhangs at Eco321 site and transferred to E. coli DH5a and further to plant transformation vector pBI121 which was named as pRA121. In order to clone another gene (ech42) into pRA121, expression cassette from iHP vector was transferred to pRA121 and named as pRAG121. Further in order to gain XhoI site for cloning ech42 gene into pRAG121, ech42 (pSUM1) was cloned into pYES2/CT, named as pSAG1, ech42 from pSAG1 cloned with KpnI and XhoI in pRAG121 and named as pRAGS121. The vector constructed in the present study can be used to transform important crop plants to have enhanced resistance to fungal diseases.

Putative Polyketide Synthase and Laccase Genes for Biosynthesis of Aurofusarin in Gibberella zeae

ABSTRACT Mycelia of Gibberella zeae (anamorph, Fusarium graminearum), an important pathogen of cereal crops, are yellow to tan with white to carmine red margins. We isolated genes encoding the following two proteins that are required for aurofusarin biosynthesis from G. zeae: a type I polyketide synthase (PKS) and a putative laccase. Screening of insertional mutants of G. zeae, which were generated by using a restriction enzyme-mediated integration procedure, resulted in the isolation of mutant S4B3076, which is a pigment mutant. In a sexual cross of the mutant with a strain with normal pigmentation, the pigment mutation was linked to the inserted vector. The vector insertion site in S4B3076 was a HindIII site 38 bp upstream from an open reading frame (ORF) on contig 1.116 in the F. graminearum genome database. The ORF, designated Gip1 (for Gibberella zeae pigment mutation 1), encodes a putative laccase. A 30-kb region surrounding the insertion site and Gip1 contains 10 additional ORFs, including a putative ORF identified as PKS12 whose product exhibits about 40% amino acid identity to the products of type I fungal PKS genes, which are involved in pigment biosynthesis. Targeted gene deletion and complementation analyses confirmed that both Gip1 and PKS12 are required for aurofusarin production in G. zeae. This information is the first information concerning the biosynthesis of these pigments by G. zeae and could help in studies of their toxicity in domesticated animals.

Construction and characterization of a bacterial artificial chromosome (BAC) library of hexaploid wheat (Triticum aestivum L.) and validation of genome coverage using locus-specific primers

A BAC library of hexaploid wheat was constructed using the spring wheat cultivar Triticum aestivum L. 'Glenlea'. Fresh shoot tissue from 7- to 10-day-old seedlings was used to obtain HMW DNA. The library was constructed using the HindIII site of pIndigoBAC-5 and the BamHI site of pIndigoBAC-5 and pECBAC1. A total of 12 ligations were used to construct the entire library, which contains over 650 000 clones. Ninety-six percent of the clones had inserts. The insert size ranged from 5 to 189 kb with an average of 79 kb. The entire library was gridded onto 24 high-density filters using a 5 × 5 array. A subset of these membranes was hybridized with two intergenic chloroplast probes and the percentage of clones containing chloroplast DNA (cpDNA) was calculated to be 2.2%. The genome coverage was estimated to be 3.1 × haploid genome equivalents, giving a 95.3% probability of identifying a clone corresponding to any wheat DNA sequence. BAC pools were constructed and screened using markers targeting the Glu-B1 locus (1BL), the hardness loci (5AS, 5BS, 5DS), the leaf rust resistance locus Lr1 (5DL), and the major fusarium head blight QTL locus located on 3BS. These markers were either locus-specific amplicons or microsatellites. A total of 49 BAC clones were identified for 14 markers giving an average of 3.5 clones/marker, thereby corroborating the estimated 3.1× genome coverage. An example using the gene encoding the HMW glutenin Bx7 is illustrated.Key words: BAC library, BAC pools, hexaploid wheat, locus-specific primers, HMW glutenin.

Sheltering of gamma-globin expression from position effects requires both an upstream locus control region and a regulatory element 3' to the A gamma-globin gene.

Integration position-independent expression of human globin transgenes in transgenic mice requires the presence of regulatory elements from the beta-globin locus control region (LCR) in the transgene construct. However, several recent studies have suggested that, while clearly necessary, such elements are not by themselves sufficient to realize this effect. In the case of the human fetal gamma-globin genes, previous results have indicated that additional regulatory information required for sheltering of gamma-globin transgene expression from position effects may reside downstream from the A gamma gene. To investigate this possibility, we established 17 lines of transgenic mice carrying constructs comprising a micro-LCR (microLCR) element, an A gamma-globin gene fragment, and a variable length of 3' sequence information beyond the A gamma 3' HindIII site. gamma-Globin expression during development was studied in 170 individual F2 progeny from these lines. We find that gamma-globin expression becomes sheltered from position effects when the normally position-sensitive microLCR-A gamma construct is extended by 600 bp beyond the 3' HindIII site to include a previously identified regulatory sequence (the A gamma-globin enhancer), the functional significance of which in vivo had heretofore been unclear. The results suggest that the mechanism whereby an upstream LCR achieves sheltering of globin gene expression from position effects involves cooperation with a gene-proximal regulatory element distinct from the promoter region.

B-cell non-Hodgkin's lymphoma cell line (Karpas 1106) with complex translocation involving 18q21.3 but lacking BCL2 rearrangement and expression

A B-cell non-Hodgkin's lymphoma (B-NHL) cell line (Karpas 1106) with an unusual three-way translocation involving 18q21.3 has been derived from a patient with mediastinal lymphoblastic B-NHL. Although conventional cytogenetics showed a derivative 18q-identical to that seen in cases with t(14;18)(q32.3;q21.3), no translocations of either chromosome 14 could be detected. Instead fluorescent in situ hybridization analysis using a chromosome-18 paint showed that the segment 18q21.3–18qter had become sandwiched on a derivative chromosome X between segments Xqter-c- Xq28 and 13q12-qter, with the centrometric site of 18q21.3 subband juxtaposed to the X sequences. Pulsed-field DNA blots failed to detect rearrangement of the BCL2 gene. Conventional DNA blots using a variety of restriction digests and both 52 and 32 BCL2 and FVT 1 probes also failed to detect rearrangement in Karpas 1106. A rearranged fragment seen only in HindIII digests with 52 BLC2 probes may represent a local microalteration, which is either a mutation or small deletion involving the HindIII site as seen in other cases of B-NHL. Neither BCL2 RNA nor BCL2 protein expression were detected. These and other data suggest that genes at 18q21.3, other than BCL2 and FVT1, may be targets for translocation in certain subgroups of B-NHL.

B-cell non-Hodgkin's lymphoma cell line (Karpas 1106) with complex translocation involving 18q21.3 but lacking BCL2 rearrangement and expression

A B-cell non-Hodgkin's lymphoma (B-NHL) cell line (Karpas 1106) with an unusual three-way translocation involving 18q21.3 has been derived from a patient with mediastinal lymphoblastic B-NHL. Although conventional cytogenetics showed a derivative 18q-identical to that seen in cases with t(14;18)(q32.3;q21.3), no translocations of either chromosome 14 could be detected. Instead fluorescent in situ hybridization analysis using a chromosome-18 paint showed that the segment 18q21.3–18qter had become sandwiched on a derivative chromosome X between segments Xqter-c- Xq28 and 13q12-qter, with the centrometric site of 18q21.3 subband juxtaposed to the X sequences. Pulsed-field DNA blots failed to detect rearrangement of the BCL2 gene. Conventional DNA blots using a variety of restriction digests and both 52 and 32 BCL2 and FVT 1 probes also failed to detect rearrangement in Karpas 1106. A rearranged fragment seen only in HindIII digests with 52 BLC2 probes may represent a local microalteration, which is either a mutation or small deletion involving the HindIII site as seen in other cases of B-NHL. Neither BCL2 RNA nor BCL2 protein expression were detected. These and other data suggest that genes at 18q21.3, other than BCL2 and FVT1, may be targets for translocation in certain subgroups of B-NHL.

Ribosomal DNA studies in poplars: Populus deltoides, P. nigra, P. trichocarpa, P. maximowiczii, and P. alba

The nuclear rDNA physical maps of Populus nigra, P. deltoides, P. trichocarpa, P. maximowiczii, and P. alba have been built up for the restriction enzymes EcoRI, EcoRV, BamHI, PstI, and SacI. There is no HindIII site. A large variability appeared between the species in the intergenic spacer mainly for EcoRI and PstI. For several clones of each species two to three major unit types coexist in the genome, while several minor units as well as length variant units or new unit types have been found. The variability between the species is due to different major unit types, while the variability between clones in one species is due to the minor unit types. Every species carries several rDNA unit types either different in size (variable length unit) or variable in restriction sites (variable unit types). Four thousands copies of rDNA units were found in P. nigra, P. deltoides, and P. maximowiczii. The clones belonging to the same species carry the same major unit types but are different in their minor variable length units or minor unit types. The hybrid clones carry the sum of the major unit types of the two parental species. These facts suggested the existence of three rDNA loci. The combination of one enzyme EcoRI and one probe (flax entire rDNA unit) allowed to easily recognize each of the five species. Conversely, we detected the presence of rDNA fragments characteristic to P. deltoides in some clones belonging to P. nigra. We conclude that these clones are likely to have a hybridization in their stands but without observable phenotypic consequences. This technique will be used to verify the purity of each P. nigra candidate clone before exploiting it in a breeding or propagation program.Key words: Populus, nuclear rDNA, intergenic spacer variation, introgression.

Identification of recombination events resulting in three hybrid genes encoding human MiV, MiV(J.L.), and Sta glycophorins

MiV, MiV(J.L.), and Sta glycophorins specify the respective variant phenotypes of the human MNSs blood group system. We report that unequal but homologous crossing-over between alpha and delta glycophorin genes results in three hybrid genes encoding MiV, MiV(J.L.), and Sta glycophorins. Restriction mapping and allele-specific oligonucleotide hybridization grossly defined the third intron as the probable crossing- over site and showed that MiV and MiV(J.L.) genes are arranged in the same 5′ alpha-delta 3′ frame whereas Sta gene is in a reciprocal 5′delta-alpha 3′ configuration. Genomic sequences spanning the extracellular domain exons 2 to 4 were amplified from each variant gene by polymerase chain reaction and determined by direct DNA sequencing. Comparison of nucleotide sequences encompassing the third intron showed that the three hybrid genes differed in location of crossing-over sites. The alpha-delta breakpoints in MiV and MiV(J.L.) genes were localized to the 3′ end of the HindIII site downstream from exon 3 and to the 5′ end immediately upstream from exon 4, respectively, whereas the delta-alpha breakpoint in Sta gene resided in between. An AAAGT sequence oriented in either forward or reverse direction was identified within the crossing-over region of each hybrid gene whose surrounding sequences bear a strong local strand asymmetry. The single nucleotide substitution in exon 4 of MiV and MiV(J.L.) genes (ACG [Thr] to ATG [Met]) demonstrated that the two genes differed in the delta glycophorin alleles that must have participated in the recombination.

Identification of recombination events resulting in three hybrid genes encoding human MiV, MiV(J.L.), and Sta glycophorins

MiV, MiV(J.L.), and Sta glycophorins specify the respective variant phenotypes of the human MNSs blood group system. We report that unequal but homologous crossing-over between alpha and delta glycophorin genes results in three hybrid genes encoding MiV, MiV(J.L.), and Sta glycophorins. Restriction mapping and allele-specific oligonucleotide hybridization grossly defined the third intron as the probable crossing- over site and showed that MiV and MiV(J.L.) genes are arranged in the same 5′ alpha-delta 3′ frame whereas Sta gene is in a reciprocal 5′delta-alpha 3′ configuration. Genomic sequences spanning the extracellular domain exons 2 to 4 were amplified from each variant gene by polymerase chain reaction and determined by direct DNA sequencing. Comparison of nucleotide sequences encompassing the third intron showed that the three hybrid genes differed in location of crossing-over sites. The alpha-delta breakpoints in MiV and MiV(J.L.) genes were localized to the 3′ end of the HindIII site downstream from exon 3 and to the 5′ end immediately upstream from exon 4, respectively, whereas the delta-alpha breakpoint in Sta gene resided in between. An AAAGT sequence oriented in either forward or reverse direction was identified within the crossing-over region of each hybrid gene whose surrounding sequences bear a strong local strand asymmetry. The single nucleotide substitution in exon 4 of MiV and MiV(J.L.) genes (ACG [Thr] to ATG [Met]) demonstrated that the two genes differed in the delta glycophorin alleles that must have participated in the recombination.