Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): Application to sequencing 6·5 kb genome segment of hantavirus strain B-1

Isegawa, Yuji; Sheng, Jun; Sokawa, Yoshihiro; Yamanishi, Koichi; Nakagomi, Osamu; Ueda, Shigeharu

doi:10.1016/0890-8508(92)90043-w

Cited by 122 publications

(68 citation statements)

References 14 publications

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“…Adaptor PCR, a method for amplifying sequences adjacent to any single known site (Isegawa et al, 1992), was used to define the point of cag region insertion more closely. Here, genomic DNAs from two cag ¹ strains (WV99 and Tx30A) were digested with HindIII, and the pools of DNA fragments were ligated with adaptors and amplifed with primers 1 and 2 plus the appropriate adaptor-specific primers (Fig.…”

Section: Dna Sequence Of Cagii and Adjacent Dnasmentioning

confidence: 99%

Analyses of the cag pathogenicity island of Helicobacter pylori

Akopyants¹,

Clifton²,

Kersulyte³

et al. 1998

Molecular Microbiology

674

331

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SummaryMost strains of Helicobacter pylori from patients with peptic ulcer disease or intestinal-type gastric cancer carry cagA, a gene that encodes an immunodominant protein of unknown function, whereas many of the strains from asymptomatically infected persons lack this gene. Recent studies showed that the cagA gene lies near the right end of a Ϸ37 kb DNA segment (a pathogenicity island, or PAI) that is unique to cagA þ strains and that the cag PAI was split in half by a transposable element insertion in the reference strain NCTC11638. In complementary experiments reported here, we also found the same cag PAI, and sequenced a 39 kb cosmid clone containing the left 'cagII' half of this PAI. Encoded in cagII were four proteins each with homology to four components of multiprotein complexes of Bordetella pertussis ('Ptl'), Agrobacterium tumefaciens ('Vir'), and conjugative plasmids ('Tra') that help deliver pertussis toxin and T (tumour inducing) and plasmid DNA, respectively, to target eukaryotic or prokaryotic cells, and also homologues of eukaryotic proteins that are involved in cytoskeletal structure. To the left of cagII in this cosmid were genes for homologues of HslU (heat-shock protein) and Era (essential GTPase); to the right of cagII were homologues of genes for a type I restriction endonuclease and ion transport functions. Deletion of the cag PAI had no effect on synthesis of the vacuolating cytotoxin, but this deletion and several cag insertion mutations blocked induction of synthesis of proinflammatory cytokine IL-8 in gastric epithelial cells. Comparisons among H. pylori strains indicated that cag PAI gene content and arrangement are rather well conserved. We also identified two genome rearrangements with end-points in the cag PAI. One, in reference strain NCTC11638, involved IS605, a recently described transposable element (as also found by others). Another rearrangement, in 3 of 10 strains tested (including type strain NCTC11637), separated the normally adjacent cagA and picA genes and did not involve IS605. Our results are discussed in terms of how cagencoded proteins might help trigger the damaging inflammatory responses in the gastric epithelium and possible contributions of DNA rearrangements to genome evolution.

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Section: Dna Sequence Of Cagii and Adjacent Dnasmentioning

confidence: 99%

Analyses of the cag pathogenicity island of Helicobacter pylori

Akopyants¹,

Clifton²,

Kersulyte³

et al. 1998

Molecular Microbiology

674

331

View full text Add to dashboard Cite

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“…YAC and PAC end-DNA fragments were cloned on the basis of the cassette-ligation-mediated PCR method (11). For isolation of PAC end-fragment clones, we used oligonucleotide primers AKI-2 (5Ј-GGCCGTCGACATTTAGGTGAC-3Ј, for the first PCR of the SP6 promoter side), AKI-4 (5Ј-CCGTCGACATTTAGGTGACACT-3Ј, for the second PCR of the SP6 promoter side), MICHI-1 (5Ј-AAGGAGCTGACT-GGGTTGA-3Ј, for the first PCR of the T7 promoter side), and MICHI-4 (5Ј-CGGTCGAGCTTGACATTGTAGG-3Ј, for the second PCR of the T7 promoter side), as specific primers for the PAC vector.…”

Section: Isolation Of Yeast Artificial Chromosome (Yac) and Pac End-dnamentioning

confidence: 99%

Hd6 , a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

Takahashi

Shomura

Sasaki

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

439

252

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Hd6 is a quantitative trait locus involved in rice photoperiod sensitivity. It was detected in backcross progeny derived from a cross between the japonica variety Nipponbare and the indica variety Kasalath. To isolate a gene at Hd6, we used a large segregating population for the high-resolution and fine-scale mapping of Hd6 and constructed genomic clone contigs around the Hd6 region. Linkage analysis with P1-derived artificial chromosome clone-derived DNA markers delimited Hd6 to a 26.4-kb genomic region. We identified a gene encoding the ␣ subunit of protein kinase CK2 (CK2␣) in this region. The Nipponbare allele of CK2␣ contains a premature stop codon, and the resulting truncated product is undoubtedly nonfunctional. Genetic complementation analysis revealed that the Kasalath allele of CK2␣ increases daysto-heading. Map-based cloning with advanced backcross progeny enabled us to identify a gene underlying a quantitative trait locus even though it exhibited a relatively small effect on the phenotype.

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“…Gene walking for obtaining the whole gene encoding the 16-kDa protein was performed as follows. XbaI-HindIII-digested genomic fragments were ligated to oligonucleotide cassettes containing a restriction site (XbaI or HindIII) and a 35-bp consensus sequence as a primer (Takara, Shiga, Japan) (16). PCR amplification using LA PCR in vitro cloning kit (Takara) with a cassette primer and specific primers designed from the partial DNA sequence of the 16-kDa protein were performed to obtain upstream and downstream DNA sequences.…”

Section: Materials-[␣-mentioning

confidence: 99%

A Magnetosome-specific GTPase from the Magnetic BacteriumMagnetospirillum magneticum AMB-1

Okamura

Takeyama

Matsunaga

2001

Journal of Biological Chemistry

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Magnetic bacteria produce intracellular vesicles that envelope single domain magnetite crystals. Although many proteins are present in this intracellular vesicle membrane, five are specific to this membrane. A 16-kDa protein, designated Mms16, is the most abundant of the magnetosome-specific proteins, and to establish its function we cloned and sequenced its gene from Magnetospirillum magneticum AMB-1. This was achieved by determination of the N-terminal amino acid sequence of the protein following two dimensional polyacrylamide gel electrophoresis, and sequencing of the gene was performed by gene walking using anchored polymerase chain reaction. Mms16 contains a putative ATP/GTP binding motif (P-loop). Recombinant Mms16 with a hemagglutinin tag, was expressed in Escherichia coli and purified. Recombinant Mms16 protein could bind GTP and showed GTPase activity. GTP was the preferred substrate for Mms16-catalyzed nucleotide triphosphate hydrolysis. These results suggest that a novel protein specifically localized on the magnetic particle membrane, Mms16, is a GTPase. Mms16 protein showed similar characteristics to small GTPases involved in the formation of intracellular vesicles. Furthermore, addition of the GTPase inhibitor AlF 4 ؊ also inhibited magnetic particle synthesis, suggesting that GTPase is required for magnetic particles synthesis.Magnetic bacteria synthesize intracellular magnetite particles that are aligned in chains of around 10 particles per cell. They are covered with a thin lipid membrane and have an average diameter of 50 -100 nm. The morphology of these bacterial magnetic particles (BMPs) 1 is species-dependent, and it is therefore reasonable to hypothesize that species-specific biological factors located in the BMP membrane mediate magnetite crystallization (1). However, the mechanism of BMP synthesis, including vesicle formation, still remains unclear. A complete understanding of biomineralization and of membrane vesicle formation at the molecular level will also have important implications for studying biomineralization in general and vesicle formation in prokaryotes in particular.Several processes are involved in BMP synthesis, and one of the most important is vesicle formation. Numerous studies have been devoted to the investigation of eukaryotic intracellular vesicle formation, and hence the molecular machinery is well understood (for reviews, see Refs. 2 and 3). In contrast, there are few molecular studies of vesicle formation or of the events leading to invagination of the cytoplasmic membrane in prokaryotes. We have hypothesized that the BMP membrane is derived from the cytoplasmic membrane and formed through the invagination of the cytoplasmic membrane by a process similar that which occurs in eukaryotes (4).A second process in BMP synthesis is magnetite crystallization, and this has been studied in more detail. It appears that ferric iron is reduced on the cell surface, taken into the cytoplasm, transferred into vesicles (magnetosomes), and finally oxidized to produce magnetite (5...

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Selective amplification of cDNA sequence from total RNA by cassette-ligation mediated polymerase chain reaction (PCR): Application to sequencing 6·5 kb genome segment of hantavirus strain B-1

Cited by 122 publications

References 14 publications

Analyses of the cag pathogenicity island of Helicobacter pylori

Analyses of the cag pathogenicity island of Helicobacter pylori

Hd6 , a rice quantitative trait locus involved in photoperiod sensitivity, encodes the α subunit of protein kinase CK2

A Magnetosome-specific GTPase from the Magnetic BacteriumMagnetospirillum magneticum AMB-1

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