Simplified preparation of unidirectional deletion clones

Hoheisel, Jörg D.; Pohl, Fritz M.

doi:10.1093/nar/14.8.3605

Cited by 60 publications

(33 citation statements)

References 2 publications

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“…lovirus (CMV) early promoter/enhancer as described (31 Several 5' deletions of pACCAT12 were made by digesting the plasmid with Sac 1 (5' flanking polylinker site), Sac I and Xho I, or Sac I and Sma I followed by exonuclease III/S1 nuclease digestion (33). This yielded the constructs pACCATA&SacI, pACCATAXhoI, and pACCATe3O, which was further treated with EcoRI, BAL-31, and, finally, HindIII to release the desired promoter fragments.…”

Section: Methodsmentioning

confidence: 99%

Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1.

Gashler

Bonthron

Madden

et al. 1992

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

208

112

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Section: Methodsmentioning

confidence: 99%

Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1.

Gashler

Bonthron

Madden

et al. 1992

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

208

112

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“…Purified pUC plasmid DNA was sequenced (12) with M13 universal primers. For randomdeletion subcloning (13), purified DNA inserts with two 5' protruding ends (EcoRI) were digested with exonuclease III, made blunt-ended with S1 nuclease, and cloned into pUC19 that was linearized with HincII.…”

Section: Methodsmentioning

confidence: 99%

Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes.

Baehr

Zhang

Joseph

et al. 1988

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

292

247

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Chlamydia trachomatis is an obligate prokaryotic intracellular pathogen of humans that infects mucosal epithelial cells. Exposed domains of its major outer membrane protein (MOMP) are both serotyping and protective antigenic determinants. To identify these domains, we have cloned and epitope-mapped the genes of serovars A, C (C serogroup) and L2, B (B serogroup) with a panel of monoclonal antibodies (mAbs). Predominantly conserved regions of the genes of both serogroups are interspersed with four short variable domains (I-IV). Recombinant phage clones expressing specific MOMP antigenic determinants revealed that protective serotypespecific mAbs recognized epitopes in variable domains I and fl. Protective subspecies and serogroup-specific mAbs recognized overlapping determinants in variable domain IV near the C terminus. A nonprotective species-specific mAb mapped to an invariant peptide of nine residues contained within variable domain IV. In the intact chlamydial organism of serovar B, variable domains HI and IV were susceptible to proteolytic digestion, whereas both N and C termini were protected. These results suggest an arrangement of MOMP in the outer membrane in which three of the four variable domains are exposed to the outside and in which both N and C termini are presumably oriented toward the periplasmic space. This molecular analysis of MOMP antigenic determinants and their surface topology on intact chlamydiae will be useful toward the development of a recombinant subunit or synthetic chlamydial vaccine.

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“…pcHN22 and pcHNF31 were treated with exonuclease I11 and mung bean nuclease to obtain unidirectional-deletion subclones as described [23]. After single-stranded DNAs were isolated with the aid of helper phages (M13K07), both strands of all regions were sequenced by the dideoxynucleotide chaintermination method [24] using the SequenaseIM DNA-sequence kit (United States Biochemical Corp., Cleveland, OH).…”

Section: N a Sequencingmentioning

confidence: 99%

Primary structure of human placental 5′‐nucleotidase and identification of the glycolipid anchor in the mature form

Misumi

Ogata

Ohkubo

et al. 1990

European Journal of Biochemistry

144

103

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A cDNA was cloned coding for human placental 5'-nucleotidase. The 3547-bp cDNA contains an open reading frame that encodes a 574-residue polypeptide with a calculated size of 63 375 Da. The NH2-terminal 26 residues comprise a signal peptide, which is followed by the NH,-terminal sequence of the purified protein. Four potential N-linked glycosylation sites are found in the molecule, accounting for a larger mass of the mature form (71 kDa). The predicted structure contains a hydrophobic amino acid sequence at the COOH terminus, a possible signal for the post-translational modification by glycophospholipid. To confirm this possibility, we tried to isolate and characterize the membrane-anchoring domain of 5'-nucleotidase. BrCN-cleaved fragments of the protein were extracted with hexane and subjected to HPLC, resulting in purification of a single component of 2.3 kDa. Chemical analyses revealed that the purified fragment contains the tetradecapeptide Lys-Val-Ile-Tyr-Pro-Ala-Val-Glu-GlyArg-Ile-Lys-Phe-Ser, ethanolamine, glucosamine, mannose, inositol, palmitic acid, and stearic acid. The peptide sequence determined is identified at positions 510 -523 in the primary structure deduced from the cDNA sequence, which predicts a further extension to position 548, containing the hydrophobic amino acid sequence. Thus, it is concluded that the mature 5'-nucleotidase lacks the predicted COOH-terminal peptide extension (524 -548), which has been replaced by the glycophospholipid functioning as the membrane anchor of 5'-nucleotidase. The NH2-terminal hydrophobic domain of these enzymes functions as both a translocation signal and membrane anchor (uncleavable signal peptide). On the other hand, recent studies with cloning and sequencing of cDNAs have demonstrated that alkaline phosphatase, another representative ectoenzyme, has a hydrophobic domain at the COOH terminus which could participate in membrane localization [7,8]. However, in purified alkaline phosphatase, the COOH-terminal hydrophobic domain is replaced by a glycosyl-phosphatidylinositol (glycosyl-PtdIns) moiety [9, 101. It is now suggested that the COOH-terminal hydrophobic domain is post-translationally cleaved and replaced by glycosyl-PtdIns for membrane anchoring in many proteins expressed on the cell surface [ l l , 121.

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Simplified preparation of unidirectional deletion clones

Cited by 60 publications

References 2 publications

Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1.

Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1.

Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes.

Primary structure of human placental 5′‐nucleotidase and identification of the glycolipid anchor in the mature form

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