Showing papers on "XhoI published in 2002"

A Whole-Genome Shotgun Optical Map of Yersinia pestis Strain KIM

Abstract: Yersinia pestis is the causative agent of the bubonic, septicemic, and pneumonic plagues (also known as black death) and has been responsible for recurrent devastating pandemics throughout history. To further understand this virulent bacterium and to accelerate an ongoing sequencing project, two whole-genome restriction maps (XhoI and PvuII) of Y. pestis strain KIM were constructed using shotgun optical mapping. This approach constructs ordered restriction maps from randomly sheared individual DNA molecules directly extracted from cells. The two maps served different purposes; the XhoI map facilitated sequence assembly by providing a scaffold for high-resolution alignment, while the PvuII map verified genome sequence assembly. Our results show that such maps facilitated the closure of sequence gaps and, most importantly, provided a purely independent means for sequence validation. Given the recent advancements to the optical mapping system, increased resolution and throughput are enabling such maps to guide sequence assembly at a very early stage of a microbial sequencing project.

Molecular and cytological characterization of SspI-family repetitive sequence on the chicken W chromosome.

Abstract: A genomic clone, pWS44, isolated from the chicken W chromosome-specific genomic library contained a partial (226-bp) sequence of a novel SspI-family repetitive sequence. A genomic clone, pWPRS09, containing a 508-bp SspI fragment (a repeating unit of the family) was subsequently obtained and sequenced. This 0.5-kb unit is tandemly repeated about 11 300 times. FISH to mitotic and lampbrush W chromosomes indicates that the SspI-family is located on the chromomere 6 between heterochromatic and distal non-heterochromatic regions on the short arm. The SspI-family sequence was proved to be a good positional marker in FISH mapping of active genes in the non-heterochromatic region on the lampbrush W chromosome. The presence of SspI-family repetitive sequence is limited to the genus Gallus (chickens and jungle fowls). The 0.5-kb repeating unit contains a 120-bp stretch of polypurine/polypyrimidine sequence (GGAGA repeats), shows no DNA curvature, and rapid electrophoretic mobility in 4% polyacrylamide gel at 4°C. The SspI-family forms a relatively diffused chromatin structure in nuclei. These features are distinctly different from those of XhoI- and EcoRI-family sequences on the W chromosome. The total amount of non-repetitive DNA in the chicken W chromosome is estimated to be about 10 Mb.

Molecular Organization of Intrinsic Restriction and Modification Genes BsuM of Bacillus subtilis Marburg

Abstract: Transcriptional analysis and disruption of five open reading frames (ORFs), ydiO, ydiP, ydiR, ydiS, and ydjA, in the prophage 3 region of the chromosome of Bacillus subtilis Marburg revealed that they are component genes of the intrinsic BsuM restriction and modification system of this organism. The classical mutant strain RM125, which lacks the restriction and modification system of B. subtilis Marburg, lacks the prophage 3 region carrying these five ORFs. These ORFs constitute two operons, the ydiO-ydiP operon and the ydiR-ydiS-ydjA operon, both of which are expressed during the logarithmic phase of growth. The predicted gene products YdiO and YdiP are the orthologues of cytosine DNA methyltransferases. The predicted YdiS product is an orthologue of restriction nucleases, while the predicted YdiR and YdjA products have no apparent paralogues and orthologues whose functions are known. Disruption of the ydiR-ydiS-ydjA operon resulted in enhanced transformation by plasmid DNA carrying multiple BsuM target sequences. Disruption of ydiO or ydiP function requires disruption of at least one of the following genes on the chromosome: ydiR, ydiS, and ydjA. The degrees of methylation of the BsuM target sequences on chromosomal DNAs were estimated indirectly by determining the susceptibility to digestion with XhoI (an isoschizomer of BsuM) of DNAs extracted from the disruptant strains. Six XhoI (BsuM) sites were examined. XhoI digested at the XhoI sites in the DNAs from disruptants with disruptions in both operons, while XhoI did not digest at the XhoI sites in the DNAs from the wild-type strain or from the disruptants with disruptions in the ydiR-ydiS-ydjA operon. Therefore, the ydiO-ydiP operon and the ydiR-ydiS-ydjA operon are considered operons that are responsible for BsuM modification and BsuM restriction, respectively.

Partially inverted tandem repeat isolated from pericentric region of chicken chromosome 8.

Abstract: The majority of chicken repetitive sequence is nuclear-membrane-associated sequence (CNM), which resides in a large number of microchromosomes (chromosomes 11–39) and is absent from macrochromosomes 1–5, ZW, and some of the intermediate chromosomes 6–10. Two repetitive families, EcoRI/XhoI, are confined to the female-specific W chromosome. The core repeat units of the three families are 21 bp, containing (A)3–5 and (T)3–5 clusters separated by 5–7-bp sequences. In this article, we describe the isolation and initial characterization of a novel repeat family that is related to CNM/EcoRI/XhoI families. The novel family, designated as PIR, consists of multiple types of partially inverted repeat units of about 1.2, 1.4 and 1.6 kb. The PIR sequence is restricted to chicken chromosome 8, and accounts for about 3.8 mb, or 2500 copies of the 1.4-kb units, of the chicken genome. The evolution of PIR and related sequences is discussed.

High resolution genotyping of Campylobacter jejuni strains by macrorestriction analysis with XhoI and polymerase chain reaction targeting enterobacterial repetitive intergenic consensus sequences: can we predict the zoonotic potential of strains?

Abstract: Campylobacter jejuni isolates of human, canine, feline, bovine and poultry origin were investigated for their genomic diversity using O-antigen typing (n = 271), SmaI (n = 158) and XhoI (n = 158) macrorestriction analysis and ERIC-PCR (n = 107). The O-antigens 0: 1/44, 0: 2, 0:4 complex, 0: 37, 0:40 were identified and 53.7% of the human and 56.1% of the animal strains were typable with the available antisera. Two ERIC-PCR pattern groups were generated representing human and animal strains as well as those exclusively of animal origin. XhoI macrorestriction analysis also distinguished 'human' and 'non-human' strain clusters, but by Smal restriction mainly serotype-associated clusters were found. In conclusion, genomic differences may occur between 'human' and 'non-human' strains and this may reflect their potential to overcome the barrier from animals to humans

Rapid sex identification of chicken by fluorescence in situ hybridization using a W chromosome-specific DNA probe

Abstract: It has been known that the sex of chicken cells can be most accurately identified by fluorescence in situ hybridization (FISH). However, the presently available FISH has not been widely used for sex identification, because the procedures for cell preparation and FISH itself are complicated and time-consuming. The present study was undertaken to test a rapid FISH procedure for sexing chicken. A FISH probe was simultaneously synthesized and labeled with digoxigenin by polymerase chain reaction (PCR) targeting a 416 bp segment of the 717 bp XhoI family fragment which is repeated over 10 thousand times exclusively in the W chromosome. Sexing by FISH was performed on cytological preparations of early embryos, adult lymphocytes and feather pulps of newly hatched chicks. The DNA probe hybridized to all types of uncultured interphase as well as metaphase female but not male cells that had been examined. Moreover, consistent with the known site of the XhoI family, the hybridization signal was localized to the pericentromeric region of the W chromosome. We, therefore, conclude that the present PCR-based FISH can be used as a rapid and reliable sex identification procedure for chicken. (Asian-Aust. J. Anim. Sci. 2002. Vol 15, No. 11 : 1531-1535)

Isolasi, Karakterisasi, dan Kloning Gen Penyandi α-Amilase Bakteri Halofil Moderat asal Bledug Kuwu

Abstract: A moderately halophilic bacteria, BK-05, was isolated from Bledug Kuwu, a saline terrestrial area at Central Java. Based on partial sequence of 16S-rRNA gene, the isolate was closely related to Halobacillus litoralis. This isolate showed amylolytic activity when grown on saline media [15% (w/v) NaCl] suplemented with starch. A pair of primer was designed based on the sequence of amyH gene from Halomonas meridiana and Pseudoalteromonas haloplanktis. PCR amplification using these primers showed three DNA bands with each size approximately 1.50, 1.00, and 0.75 kb. Partial DNA sequencing analysis based on its deduced protein sequence revealed that the 1.50 kb band was closely related to the sequence of metalloprotease from Bacillus subtilis (approximately 54.3% identity in 184 amino acid overlap). Southern hybridization analysis showed that the 1.50 kb fragment was located within a 4.0 kb fragment of BamHI, 4.8 kb of EcoRI, 4.3 kb of HindIII, and 4.0 kb of XhoI digestion of BK-05 genomic DNA, respectively.

Cloning and expression of nucleoprotein gene of transmissible gastroenteritis virus

Abstract: The viral subgenome mRNA of porcine transmissible gastroenteristic virus (TGEV) was amplified by RT-PCR using primer pairs Li01 and Li02 which designed according to the reported reference. A DNA fragment was amplified which contains completely ORF gene and the size is about 1174bp and then the DNA fragement was cloned site specially into pProEXHTb expression vector between the NspV and XhoI cleavage site. The recombinant plasmid(PHN) was verified by restriction endonuclease analysis and nucleotide sequencing. Then it was transformed into E. coli strain TG1 for N gene expression. The expression product was identified by SDS-PAGE and Western-blot test, a fusion protein about 47KD as we expected was found. The results shows that the vitro expressed protein of N gene via recombinant plasmid vector in the E. coli maintains antigenicity of TGEV.

Cloning and Expression of Nucleoprotein Gene of Transmissible Gastroenteritis Virus

Abstract: The viral subgenome mRNA of porcine transmissible gastroenteristis virus(TGEV)was amplified by RT PCR using primer pairs Li01 and Li02 which designed according to the reported reference.A DNA fragment was amplified which contains completely ORF gene and the size is about 1174bp and then the DNA fragement was cloned site specially into pProEXHTb expression vector between the NspV and XhoI cleavage site.The recombinant plasmid(PHN)was verified by restriction endonuclease analysis and nucleotide sequencing.Then it was transformed into E.coli strain TG1 for N gene expression.The expression product was identified by SDS PAGE and Western blot test,a fusion protein about 47KD as we expected was found.The results shows that the vitro expressed protein of N gene via recombinant plasmid vector in the E.coli maintains antigenicity of TGEV.