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At least 919 records · Page 51Linked to original sources
PubMed1990

Immunological characterization of the Prochlorothrix hollandica and Prochloron sp. chlorophyll a/b antenna proteins.

Polyclonal antibodies were prepared against the major antenna chlorophyll (Chl) a/b-binding protein from the prokaryote Prochlorothrix hollandica (Burger-Wiersma et al. (1986) Nature (Lond.) 320, 262-264). Immunoblotting experiments on Triton X-114 phase-partitioned P. hollandica thylakoids revealed that the antibody recognizes intrinsic membrane polypeptides of 33 and 30 kDa, and immunocytochemistry of P. hollandica thin sections showed that the antibody preferentially decorates the thylakoid. The antibody was immunopurified against a LacZ fusion protein produced in Escherichia coli by an immunopositive phage clone retrieved from a lambda ZAP expression library. This purified antibody crossreacted to both the 33 and 30 kDa polypeptides, indicating that these proteins are either structurally related products of different genes, or modified forms of the same gene product. Whereas immunological crossreactivity of Prochlorothrix antibody to the major LHC-II Chl a/b antenna of maize could not be detected, the immunopurified antibody reacted strongly to the major 34 kDa Chl a/b antenna protein from the prokaryote Prochloron sp. (Lewin (1975) Phycologia 14, 153-160). These data confirm the structural similarity of the prochlorophyte photosynthetic antenna systems.

Antibodies↗
PubMed1996

Transcription of the Campylobacter jejuni cell division gene ftsA.

As part of a study of genes whose transcription is maintained in stationary phase, cloned segments of DNA were selected from a Lambda ZAP II library of Campylobacter jejuni NCTC 11,168. One such clone was found to encode a homologue of the Escherichia coli cell division gene ftsA. Examination of mRNA by transcription mapping revealed that the Campylobacter gene has one major and three minor transcription start sites. There were several significant differences in the structure and organisation of the C. jejuni ftsA promoter compared to that of E. coli.

Amino Acid Sequence↗
PubMed1992

Cloning and high-level expression of chitinase-encoding gene of Streptomyces plicatus.

A chitinase (Cht)-encoding gene from Streptomyces plicatus was previously cloned and expressed in Escherichia coli [Robbins et al., J. Biol. Chem., 263 (1988) 442-447]. We have sequenced this gene, compared its sequence with other genes encoding Cht and have explored its expression and regulation when reintroduced into Streptomyces lividans on multicopy plasmids. We have also cloned two other Streptomyces Cht-encoding genes and a beta-hexosaminidase-encoding gene in E. coli by expression in the lambda ZAP-Bluescript vector. The hexosaminidase and one of the Chts were expressed directly from the genomic library in E. coli at a high level as chimeric fusions with the beta-galactosidase alpha-complementing peptide encoded by the vector. Direct cloning and high-level expression of such chimeric proteins, which overcomes the difficulties associated with expressing Streptomyces genes in E. coli, should generally be possible wherever large numbers of transformants can be conveniently screened.

Amino Acid Sequence↗
PubMed1992

Cloning and sequence determination of a cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase.

A partial cDNA encoding Aspergillus nidulans calmodulin-dependent multifunctional protein kinase (ACMPK) was isolated from a lambda ZAP expression library by immunoselection using monospecific polyclonal antibodies to the enzyme. The sequence of both strands of the cDNA (CMKa) was determined. The deduced amino acid (aa) sequence contained all eleven consensus domains found in serine/threonine protein kinases [Hanks et al., Science 241 (1988) 42-52], as well as a putative calmodulin-binding domain. The cDNA contained an intron, lacked an in-frame start codon, and was not polyadenylated. A full-length copy of CMKa was subsequently isolated from a lambda gt10 library of A. nidulans cDNA using a restriction fragment of the first clone as a probe. It contained an in-frame start codon, an open reading frame (ORF) of 1242 bp and was polyadenylated. The ORF encoded a protein of 414 aa residues with an M(r) of 46,895 and an isoelectric point pI = 6.4. These values are in good agreement with that observed for the native enzyme [Bartelt et al., Proc. Natl. Acad. Sci. USA 85 (1988) 3279-3283]. When aligned to optimize homology, 29% of the predicted aa sequence of ACMPK is identical to that of the alpha-subunit of rat brain calmodulin-dependent protein kinase II. ACMPK shares 40 and 44% identity in aa sequence with YCMK1 and YCMK2, respectively, two Ca2+/calmodulin-dependent protein kinases recently cloned from Saccharomyces cerevisiae [Pausch et al., EMBO J. 10 (1991) 1511-1522]. Results of Southern analysis of restriction digests of genomic DNA indicate that ACMPK is encoded by a single-copy gene.

Amino Acid Sequence↗
PubMed1993

An interferon-induced Mx protein: cDNA sequence and high-level expression in the endometrium of pregnant sheep.

We have cloned a 2.4-kb cDNA containing the complete coding sequence of ovine Mx from a lambda ZAP library constructed using RNA from the endometrium of a normal sheep on day 16 of pregnancy. Ovine Mx shows 80% similarity to human MxA. Human and mouse Mx are type-1 interferon (IFN)-induced genes that have previously been shown to confer resistance to influenza virus. The ovine Mx cDNA contains an open reading frame of 1962 nucleotides (nt) coding for a 653-amino-acid (aa) protein. The deduced translated sequence has consensus GTP-binding sites and similarity to the human MxA sequence (RKFLKERLARL) that has been shown to be essential for resistance against vesicular stomatitis virus (VSV). The presence of Mx mRNA was investigated by Northern blot analysis in the endometrium of non-pregnant sheep and between days 8 and 127 of pregnancy. Mx expression was detected at high levels between day 13 and day 20 of pregnancy. Furthermore, eightfold higher levels of Mx mRNA were detected in the pregnant versus the non-pregnant uterine horn in unilaterally pregnant sheep. Mx mRNA can be induced in sheep endometrium by ovine trophoblast interferon (IFN-tau).

Amino Acid Sequence↗
PubMed1993

Polycos vectors: a system for packaging filamentous phage and phagemid vectors using lambda phage packaging extracts.

A new class of hybrid vectors, 'polycos' vectors, incorporate a phage lambda cos site and filamentous phage origin to allow high-efficiency cloning via in vitro lambda packaging extracts. The head-filling mechanism of cos site recognition by lambda packaging proteins permits concatemers of several of these small cos-containing vectors to be packaged per phage particle. Excision of vector monomers after infection is accomplished via a lambda ZAP-like M13 excision process. This system has the advantage of adapting high-efficiency lambda packaging extracts to M13 and phagemid cloning vectors.

Bacteriophage M13↗
PubMed1995

Screening and sequence determination of a cDNA encoding the human brain 4-aminobutyrate aminotransferase.

A human brain cDNA library constructed in the lambda ZAP II vector was screened using a fragment of pig brain cDNA encoding 4-aminobutyrate aminotransferase (pGaba-t). A cDNA that encodes the human brain Gaba-t (hGaba-t) has been isolated from the library and sequenced. Using the GenBank and EMBL databases, comparison of the predicted amino-acid sequence of hGaba-t with the pig enzyme revealed 95.4% homology.

4-Aminobutyrate Transaminase↗
PubMed1994

Cloning and characterization of the human gene encoding aspartyl beta-hydroxylase.

Sequence information for aspartyl beta-hydroxylase (AspH), which specifically hydroxylates one Asp or Asn residue in certain epidermal growth factor (EGF)-like domains of a number of proteins, is so far only described for bovine species. We have isolated a 4.3-kb cDNA encoding the human AspH (hAspH) by immunoscreening of a human osteosarcoma (MG63) cDNA library in lambda ZAP with an antiserum raised against membrane fractions of these cells. Northern blot analyses revealed two transcripts with lengths of 2.6 and 4.3 kb. The deduced amino acid (aa) sequence of this cDNA encodes a protein of 757 aa (85 kDa). Comparison with the deduced bovine AspH (bAspH) aa sequence showed striking differences in the N-terminal portion of this protein. In vitro transcription and translation in the presence of canine pancreas microsomes yielded a 56-kDa protein. Western blot analyses of membrane fractions from MG63 cells with AspH-specific antibodies revealed a protein of the same M(r). These results suggest a posttranslational cleavage of the catalytic C terminus in the lumen of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1994

The thiol-specific antioxidant protein from human brain: gene cloning and analysis of conserved cysteine regions.

The complete cDNA encoding human thiol-specific antioxidant protein (PRP) was isolated from a human brain cDNA library in the lambda Zap expression vector. An open reading frame (ORF) was identified and found to encode a polypeptide of 197 aa with a M(r) of 21,729. The cDNA contained 98 bp of 5'-untranslated sequence (UTR) and 259 bp of 3'-UTR containing a poly(A) signal, AATAAA. Expression of the human PRP cDNA in Escherichia coli yielded a functionally active protein. The observed local sequence homologies between human PRP and other homologous proteins whose functions have not yet been defined give important insight into elucidating the biochemical function of a new protein family which has highly conserved regions containing cysteine.

Amino Acid Sequence↗
PubMed1995

Yeast vectors for the controlled expression of heterologous proteins in different genetic backgrounds.

An expression system for Saccharomyces cerevisiae (Sc) has been developed which, depending on the chosen vector, allows the constitutive expression of proteins at different levels over a range of three orders of magnitude and in different genetic backgrounds. The expression system is comprised of cassettes composed of a weak CYC1 promoter, the ADH promoter or the stronger TEF and GPD promoters, flanked by a cloning array and the CYC1 terminator. The multiple cloning array based on pBIISK (Stratagene) provides six to nine unique restriction sites, which facilitates the cloning of genes and allows for the directed cloning of cDNAs by the widely used ZAP system (Stratagene). Expression cassettes were placed into both the centromeric and 2 mu plasmids of the pRS series [Sikorski and Hieter, Genetics 122 (1989) 19-27; Christianson et al., Gene 110 (1992) 119-122] containing HIS3, TRP1, LEU2 or URA3 markers. The 32 expression vectors created by this strategy provide a powerful tool for the convenient cloning and the controlled expression of genes or cDNAs in nearly every genetic background of the currently used Sc strains.

Cloning, Molecular↗
PubMed1995

Lens epithelial cell mRNA, II. Expression of a mRNA encoding a lipid-binding protein in rat lens epithelial cells.

A lens epithelial (LE) cell cDNA clone, designated pLELBP, was isolated by subtraction-hybridization methods between a 4-week-old rat LE cell and rat lens fiber cell lambda ZAP cDNA libraries. The cDNA contained 683 bp, an ATG at bp 42, and an open reading frame (ORF) encoding a protein of 135 amino acids (aa), and a poly(A) signal at bp 641 (GenBank/EMBL accession No. U13253). Northern blot analysis showed that the lens mRNA was at a concentration that exceeded 100-fold that found in non-ocular tissues examined, except in skin it was found at levels equal to about 1/15 of the lens levels. In the retina, it was also found at about 1/15 of that present in the lens. By in situ hybridization analysis, the sense RNA was localized to the LE cells and to the glial cells of the retina, and negligibly to the lens fiber cells. Search of GenBank, EMBL and SwissProt data bases revealed that the LE cell mRNA and its aa encoded protein showed extensive sequence homology to members of the cytosolic lipid-binding protein (LBP) family. It matched to 91% in aa sequence homology to the protein encoded by ORF of mal1 cDNA, isolated from mouse skin squamous cell carcinoma [Krieg et al., J. Biol. Chem. 268 (1993) 17362-17369], and 99% in aa sequence homology to the protein encoded by ORF of rat skin LBP mRNA [Watanabe et al., Biochem. Biophys. Res. Commun. 200 (1994) 253-259]. Occurrence of high levels of mRNA for a specific LBP in the LE cells relative to other non-ocular tissues is consistent with a hypothesis that the encoded protein may be involved in several lens epithelial cell-specific mechanisms, possibly including differentiation, protective and nutritional processes.

Animals↗
PubMed1995

PCR-mediated cloning and sequencing of the DmOST50 gene, a WBP1/AvOST50/OST48 homologue, from Drosophila melanogaster.

Oligodeoxyribonucleotides were used in a PCR reaction to amplify the conserved region of the DmOST50 cDNA encoding an oligosaccharyltransferase subunit from Drosophila melanogaster (Dm). The amplified fragment was cloned and sequenced, and was then used as a homologous probe to isolate a DmOST50 cDNA from a lambda ZAP library. The deduced amino acid (aa) sequence of DmOst50p shows 27.1% identity with the corresponding sequence of the yeast Wbp1p, 62.4% identity with the avian AvOst50p and 62.7% with the canine Ost48p sequences. 17% of all aa residues were found to be identical among all species tested, indicating a high degree of conservation during evolution.

Amino Acid Sequence↗
PubMed1996

Cloning of a cDNA encoding a new ribosome-inactivating protein from Beta vulgaris vulgaris (mangold).

By means of a lambda ZAP II cDNA library constructed from seedings of Beta vulgaris vulgaris and immunoscreening, a cDNA clone containing a partial sequence of a new ribosome-inactivating protein (RIP) was obtained. As confirmed by Western blot analysis, this clone produced a RIP upon induction with IPTG. We called it betavulgin (Bvg). The recombinant protein (re-Bvg) was somewhat smaller than plant-derived RIP (28 versus 30 and 32 kDa), but showed the specific N-glycosidase activity on tobacco ribosomes, confirming its RIP character. The cDNA was sequenced and the missing 5'-end was established by RACE using bvg-specific primers. The entire cDNA was 1080 nucleotides in length and encoded a protein of 272 amino acids with a sequence identity of 26-40% with other RIP.

Amino Acid Sequence↗
PubMed1990

The use of transgenic mice for short-term, in vivo mutagenicity testing.

In order to develop a short-term, in vivo assay to study the mutagenic effects of chemical exposure, transgenic mice were generated using a lambda shuttle vector containing a lacZ target gene. Following exposure to mutagens, this target can be rescued efficiently from genomic DNA prepared from tissues of the treated mice using restriction minus, in vitro lambda phage packaging extract and restriction minus Escherichia coli plating cultures. Mutations in the target gene appear as colorless plaques on a background of blue plaques when plated on indicator agar. Spontaneous background levels were approximately 1 x 10(-5) in each of three mouse lineages analyzed. Exposure of lambda transgenic mice to N-ethyl-N-nitrosourea resulted in as much as a 14-fold induction in detected mutations over background levels. The assay is currently being modified to incorporate lacI as the target for ease of mutation detection as well as in vivo excision properties of the Lambda ZAP vector, facilitating sequence analysis of mutant plaques.

Animals↗
PubMed1990

Subtraction hybridization cDNA libraries from colon carcinoma and hepatic cancer.

cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 X enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available lambda ZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because lambda libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.

Bacteriophage lambda↗
PubMed1992

Development of cell surface protein associated gene probe specific for Listeria monocytogenes and detection of the bacteria in food by PCR.

A genomic library of L. monocytogenes was constructed using lambda Zap II-Eco RI and screened with a monoclonal antibody which is specific for a Listeria cell surface protein. Three positive clones each contained a 6.5 kb insert which in E. coli could express the same Listeria protein. The 6.5 kb insert was further digested with Hin dIII and the smaller fragments were subcloned into a plasmid vector (pBluescript) and screened with 32P-labelled genomic DNA from L. monocytogenes or L. innocua. Three clones which were positive with L. monocytogenes and negative with L. innocua were screened and each contained a 2.1 kb insert. The 2.1 kb insert was partly sequenced and some candidate oligomer probes from the sequences were selected and compared with sequences in a Genbank computer search. One such oligomer probe (T7-list) was confirmed to be specific for L. monocytogenes. The probe hybridized with all 28 strains of L. monocytogenes tested, but not with any of six other Listeria species nor 11 other bacteria tested. Using this probe-primer, a PCR method was developed which could detect as few as 2 cfu of L. monocytogenes in pure cultures, and as few as 4-10 cfu of L. monocytogenes when inoculated into foods.

Antibodies, Monoclonal↗
PubMed1994

Role of tyrosine kinases in lymphocyte activation.

Interaction of T- and B-cell antigen receptors with cytoplasmic non-receptor tyrosine protein kinases is critical to the activation of lymphocytes by antigen. Both the src-family tyrosine protein kinases Lck, Fyn, Lyn and Blk and the syk-family tyrosine protein kinases Syk and ZAP-70 play a role in lymphocyte activation. The antigen receptors are coupled to this cluster of kinases by the cytoplasmic tails of the gamma, delta, epsilon, zeta, and eta subunits of the T-cell receptor, and the Ig-alpha and Ig-beta subunits of the B-cell receptor. Each of these proteins contains one or more 'tyrosine based activation motifs', with the amino acid sequence D/EX7D/EXXYXXL/IX7YXXL/I. This motif appears to allow binding of one or more src-like kinases, via their unique amino termini, before the onset of lymphocyte activation. Invariant tyrosines in the motif become phosphorylated following the triggering of lymphocyte activation, and this modification induces the binding of the src- and syk-family tyrosine protein kinases, and potentially other signalling molecules, through SH2 domains to the antigen receptors.

Amino Acid Sequence↗
PubMed1995

Protein tyrosine kinases in the initiation of antigen receptor signaling.

Intracellular responses to antigen receptor engagement involve the activation of protein tyrosine kinases and the tyrosine phosphorylation of cellular proteins, including components of the antigen receptor. Phosphorylation of two tyrosine residues within an 18 amino acid segment of the cytoplasmic domain of antigen receptor subunits, and the subsequent association of either the Syk or Zap protein tyrosine kinase, has recently been shown to be required for successful antigen receptor signal propagation. The recent finding that distinct primary human immunodeficiencies result from mutations in genes encoding two non-transmembrane protein tyrosine kinases underscores the importance of this class of enzyme in antigen receptor signal transduction.

Humans↗