A high-molecular-mass (120- to 128-kDa) Helicobacter pylori antigen has been associated with peptic ulcer disease. We created a bank of 40,000 random chromosomal fragments of H. pylori 84-183 by using lambda ZapII. Screening of this bank in Escherichia coli XL1-Blue with absorbed serum from an H. pylori-infected person permitted the isolation and purification of a clone with a 3.5-kb insert. Subcloning of this insert (pMC3) permitted the expression of a recombinant H. pylori protein that had a mass of approximately 96 kDa and that was recognized by the human serum. Sera that were obtained from H. pylori-infected persons and that recognized the native 120- to 128-kDa H. pylori antigen recognized the recombinant 96-kDa pMC3 protein to a significantly greater extent than did sera that did not recognize the native H. pylori antigen. All 19 H. pylori isolates producing the 120- to 128-kDa antigen hybridized with pMC3; none of 13 nonproducers did so (P < 0.001). Because all 15 isolates producing the vacuolating cytotoxin hybridized with pMC3, we called the gene cagA (cytotoxin-associated gene). Sequence analysis of pMC3 identified an open reading frame of 859 amino acids, without a termination codon. Parallel screening of a lambda gt11 library with human serum revealed positive plaques with identical 0.6-kb inserts and sequences matching the sequence of the downstream region of pMC3. To clone the full-length gene, we used the 0.6-kb fragment as a probe and isolated a clone with a 2.7-kb insert from the lambda ZapII genomic library. Nucleotide sequencing of this insert (pYB 2) revealed a 785-bp sequence that overlapped the downstream region of pMC3. Translation of the complete nucleotide sequence of cagA revealed an open reading frame of 1,181 amino acids yielding a protein of 131,517 daltons. There was no significant homology with any previously reported protein sequence. These findings indicate the cloning and characterization of a high-molecular-mass H. pylori antigen potentially associated with virulence and with cytotoxin production.

https://iai.asm.org/content/iai/61/5/1799.full.pdf

Cloning and expression of a high-molecular-mass major antigen of Helicobacter pylori: evidence of linkage to cytotoxin production.

During progression of Moloney murine leukemia virus (Mo-MuLV)-induced rat T cell lymphomas, growth selection results in the expansion of cell clones carrying increasing numbers of integrated proviruses. These new provirus insertions reproducibly contribute to enhanced growth, allowing the emergence of cell clones from the initially heterogeneous population of tumor cells. The Mo-MuLV-induced rat T cell lymphoma lines 2780d and 5675d, which are dependent on interleukin-2 (IL-2) for growth in culture (IL-2d), were placed in IL-2-free medium to select for IL-2-independent (IL-2i) mutants. Southern blot analysis of genomic DNA from these mutants, which was hybridized to a Mo-MuLV long terminal repeat probe, revealed that all mutants carried new provirus insertions (from one to four new proviruses per cell line). A locus of integration identified through cloning of the single new provirus detected in one of the IL-2i mutants, 2780i.5, was found to be the target of provirus insertion in 1 additional IL-2i cell line of 24 tested. A full-length cDNA of a gene (growth factor independence-1 [Gfi-1]) activated by promoter insertion in the 2780i.5 cells was cloned and shown to encode a novel zinc finger protein. Gfi-1 is expressed at low levels in IL-2d cell lines cultured in IL-2-containing medium and at high levels in most IL-2i cell lines, including the two harboring a provirus at this locus. Gfi-1 expression in adult animals is restricted to the thymus, spleen, and testis. In mitogen-stimulated splenocytes, Gfi-1 expression begins to rise at 12 h after stimulation and reaches very high levels after 50 h, suggesting that it may be functionally involved in events occurring after the interaction of IL-2 with its receptor, perhaps during the transition from the G1 to the S phase of the cell cycle. In agreement with this, Gfi-1 does not induce the expression of IL-2. Expression of Gfi-1 in 2780d cells following transfer of a Gfi-1/LXSN retrovirus construct contributes to the emergence of the IL-2i phenotype.

Progression of interleukin-2 (IL-2)-dependent rat T cell lymphoma lines to IL-2-independent growth following activation of a gene (Gfi-1) encoding a novel zinc finger protein.

We isolated andsequenced aclone forCandida albicans enolase fromaC.albicans cDNAlibrary byusing molecular genetic techniques. The1.4-kbp cDNAencoded onelongopenreading frameof440aminoacids whichwas87and75%similar topredicted enolases ofSaccharomyces cerevisiae andenolases fromother organisms, respectively. ThecDNAincluded theentire coding region andpredicted aprotein ofmolecular weight 47,178. Thecodonusage washighly biased andsimilar tothatfoundforthehighly expressed EF-la proteins ofC.albicans. Northern (RNA)blot analysis showed that theenolase cDNAhybridized toanabundant C.albicans mRNA of1.5kbpresent inbothyeast andhyphal growth forms. Thepolypeptide product ofthe cloned cDNA,whichwaspurified asarecombinant protein fused toglutathione S-transferase, hadenolase enzymatic activity andinhibited radioimmunoprecipitation ofasingle C.albicans protein ofmolecular weight 47,000. Analysis ofthepredicted C.albicans enolase showed strong conservation inregions ofa helices, 13 sheets, andaturns, asdetermined bycomparison withthecrystal structure ofapo-enolase A ofS.cerevisiae. Thelackofcysteine residues andatwo-amino-acid insertion inthemaindomaindifferentiated C.albicans enolase fromS.cerevisiae enolase. Immunofluorescence ofwhole C.albicans cells byusing amouseantiserum generated against thepurified fusion protein showed that enolase isnotlocated onthesurface ofC.albicans. Recombinant C.albicans enolase will beuseful inunderstanding thepathogenesis andhostimmuneresponse indisseminated candidiasis, since enolase isanimmunodominant antigen whichcirculates during disseminated infections.

Molecular Cloning ofcDNA andAnalysis ofProtein Secondary Structure ofCandida albicans Enolase, an Abundant, Immunodominant Glycolytic Enzyme

The complete sequence of a beta-mannanase gene from an anaerobic extreme thermophile was determined, and it shows that the expressed protein consists of two catalytic domains and two binding domains separated by spacer regions rich in proline and threonine residues. The amino-terminal catalytic domain has beta-mannanase activity, and the carboxy-terminal domain acts as an endoglucanase. Neither domain shows homology with any other cellulase or hemicellulase sequence at the nucleic acid or protein level.

https://aem.asm.org/content/aem/58/12/3864.full.pdf

The beta-mannanase from "Caldocellum saccharolyticum" is part of a multidomain enzyme.

The Escherichia coli helD (encoding helicase IV) and uvrD (encoding helicase II) genes have been deleted, independently and in combination, from the chromosome and replaced with genes encoding antibiotic resistance. Each deletion was verified by Southern blots, and the location of each deletion was confirmed by P1-mediated transduction. Cell strains containing the single and double deletions were viable, indicating that helicases II and IV are not essential for viability. Cell strains lacking helicase IV (delta helD) exhibited no increase in sensitivity to UV irradiation but were slightly more resistant to methyl methanesulfonate (MMS) than the isogenic wild-type cell strain. As expected, cell strains containing the helicase II deletion (delta uvrD) were sensitive to both UV irradiation and MMS. The introduction of the helicase IV deletion into a delta uvrD background had essentially no effect on the UV and MMS sensitivity of the cell strains analyzed. The double deletions, however, conferred a Rec- mutant phenotype for conjugational and transductional recombination in both recBC sbcB(C) and recBC sbcA backgrounds. The Rec- mutant phenotype was more profound in the recBC sbcB(C) background than in the recBC sbcA background. The recombination-deficient phenotype indicates the direct involvement of helicase II and/or helicase IV in the RecF pathway [recBC sbcB(C) background] and RecE pathway (recBC sbcA background) of recombination. The modest decrease in the recombination frequency observed in single-deletion mutants in the recBC sbcB(C) background suggests that either helicase is sufficient. In addition, helicase IV has been overexpressed in a tightly regulated system. The data suggest that even modest overexpression of helicase IV is lethal to the cell.

Double helicase II (uvrD)-helicase IV (helD) deletion mutants are defective in the recombination pathways of Escherichia coli.

Alambda insertion typecDNAcloning vector, LambdaZAP,hasbeenconstructed. InE.coli a phagemid, pBluescnpt SK(-), contained within thevector, canbeexcised byf1orM13helper phage. The excision process eliminates theneedtosubclone DNAinserts fromthelambda phageinto aplasmid by restrction digestion andligation. This ispossible because LambdaZAPincorporates thesignals forboth initiation andtermination ofDNAsynthesis fromthefIbacteriophage origin ofreplication (1). Sixof21 restriction sites intheexcised pBluescript SKpolyflinker, contained within theNH2-portion ofthelacZ gene, areunique inlambda ZAP.Coding sequences inserted into these restriction sites, inthe appropriate reading frame, canbeexpressed fromthelacZpromoter asfusion proteins. Thefeatures of this vector significantly increase therate atwhich clones canbeisolated andanalyzed. Thelambda ZAPvector wastested bythepreparation ofachicken liver cDNAlibrary andthe isolation ofactin clones byscreening with oligonucleotide probes. Putative actin clones wereexcised fromthelambda vector andidentified byDNAsequencing. Theability oflambda ZAPtoserve asa vector fortheconstnuction ofcDNAexpression libraries wasdetermined bydetecting fusion proteins from clones containing glucocerbrosidase cDNA's using rabbit IgGanti-glucocerbrosidase antibodies.

https://biosci-batzerlab.biology.lsu.edu/Hum_Mol_Gen_Lectures/Short_et_al_1988_-_Lambda_zap.pdf

D. Huse

Papers

XZAP:abacteriophage Xexpression vector withinvivo excision properties