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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report the molecular cloning and DNA sequence of the gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. The biotin carboxylase gene encodes a protein of 449 residues that is strikingly similar to amino-terminal segments of two biotin-dependent carboxylase proteins, yeast pyruvate carboxylase and the alpha-subunit of rat propionyl-CoA carboxylase. The deduced biotin carboxylase sequence contains a consensus ATP binding site and a cysteine-containing sequence preserved in all sequenced bicarbonate-dependent biotin carboxylases that may play a key catalytic role. The gene encoding the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase is located upstream of the biotin carboxylase gene and the two genes are cotranscribed. As previously reported by others, the BCCP sequence encoded a protein of 16,688 molecular mass. However, this value is much smaller than that (22,500 daltons) obtained by analysis of the protein. Amino-terminal amino acid sequencing of the purified BCCP protein confirmed the deduced amino acid sequence indicating that BCCP is a protein of atypical physical properties. Northern and primer extension analyses demonstrate that BCCP and biotin carboxylase are transcribed as a single mRNA species that contains an unusually long untranslated leader preceding the BCCP gene. We have also determined the mutational alteration in a previously isolated acetyl-CoA carboxylase (fabE) mutant and show the lesion maps within the BCCP gene and results in a BCCP species defective in acceptance of biotin. Translational fusions of the carboxyl-terminal 110 or 84 (but not 76) amino acids of BCCP to beta-galactosidase resulted in biotinated beta-galactosidase molecules and production of one such fusion was shown to result in derepression of the biotin biosynthetic operon.
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PMID:The gene encoding the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase. 137 Apr 69

Tomato cDNA and genomic clones were isolated by using as a probe a cDNA clone that had originally been identified by its ability to direct the synthesis of a biotin-containing polypeptide in Escherichia coli. The nucleotide sequences of the newly isolated cDNAs indicate that they are clones of a single mRNA molecule. However, one of the cDNA clones contains an insertion of a sequence which we identified as an unspliced intron. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed similarity to regions of previously sequenced biotin enzymes, indicating that the isolated cDNAs code for a biotin-containing protein. Portions of the cDNAs were expressed in E. coli as glutathione S-transferase or beta-galactosidase fusion proteins. Each fusion protein was purified and used to immunize rabbits. The resulting antisera recognized a 78-kDa biotin-containing polypeptide in tomato leaf extracts. In addition, both antisera specifically inhibited beta-methylcrotonyl-CoA carboxylase activity in extracts from tomato leaves. These characterizations have identified the isolated tomato cDNAs and genes as coding for the 78-kDa biotin subunit of beta-methylcrotonyl-CoA carboxylase. Comparison of the deduced amino acid sequence of the biotin subunit of beta-methylcrotonyl-CoA carboxylase with other biotin enzymes suggest that this subunit contains the biotin carboxylase and biotin carboxyl-carrier domains.
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PMID:Molecular cloning of cDNAs and genes coding for beta-methylcrotonyl-CoA carboxylase of tomato. 816 72

We describe an in vivo approach for the isolation of proteins interacting with a protein of interest. The protein of interest is "tagged" with a portion of the biotin carboxylase carrier protein (BCCP), encoded on a specially constructed plasmid, so that it becomes biotinylated in vivo. The "query" proteins (e.g., those in a cDNA library) are tagged by fusing them to the 3' end of the lacZ gene on a lambda vector in such a way that the beta-galactosidase activity is not disrupted. These phage are transfected into cells containing the plasmid encoding the BCCP-tagged protein. The infection lyses the cells and exposes the protein complexes. The BCCP-tagged protein and any associated protein(s) are "captured" by using avidin, streptavidin, or anti-biotin antibody-coated filters. The detection of bound protein is accomplished by directly assaying for beta-galactosidase activity on the filters. Positive plaques can be plaque-purified for DNA sequencing. We have tested this approach by using c-Fos and c-Jun as our model system. We show that avidin, streptavidin, or polyclonal anti-biotin (but not a monoclonal anti-biotin) antibody is capable of specifically capturing in vivo biotinylated beta-galactosidase and c-Jun and that this capture is dependent upon the presence of both avidin and the BCCP moiety. Further, complexes containing c-Jun and c-Fos can also be isolated in this manner, and the isolation of this complex is dependent on the presence of c-Fos, c-Jun, avidin, and the BCCP moiety. We discuss the possible uses and limitations of this technique for isolating proteins that interact with a known protein.
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PMID:Screening for in vivo protein-protein interactions. 843 Jan 8

We report the construction of three new vectors which can be used for the 'double-tagging' assay previously reported [Germino et al., Proc. Natl. Acad. Sci. USA 90 (1993) 933-937]. The vectors include two plasmids (pTrc.BCCP and pTrc.EZZ::BCCP) which encode different 'tags' for the capture of a target protein of interest on a filter coated with either avidin or IgG, respectively. The first plasmid (pTrc.BCCP) encodes the C terminus of the biotin carboxylase carrier protein (BCCP) under the control of the Ptac promoter, while the second produces fusions to an IgG-binding domain (EZZ). The gene encoding a protein of interest can be inserted into these plasmids and thereby direct the production of a fusion protein which is biotinylated in vivo and can bind to avidin, or a fusion protein which can bind to IgG. The third is a positive-selection, phase lambda expression vector (lambdaFJG2) which permits the construction of lacZ::cDNA fusion proteins which retain beta-galactosidase activity. The insertion of an active ecoRVR gene between the cloning sites (EcoRI and HindII or NotI) permits the positive selection of inserts. The C-terminal two-thirds of the mouse retinoblastoma-encoding gene (containing the E1A-binding pocket) was cloned into pTrc.BCCP and pTrc.EZZ::BCCP, while the 13S E1a gene was cloned into lambdaFJG2. We show that the interaction between these two proteins can be detected using the 'double-tagging' filter assay, and that this assay has high sensitivity and specificity for detecting this interaction. Finally, we have used these vectors to localize the CDK2-binding domain of the cyclin-dependent kinase inhibitor, p21. These results closely correspond to those obtained using the yeast two-hybrid assay, as well as in vitro binding assays.
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PMID:Vectors for a 'double-tagging' assay for protein-protein interactions: localization of the CDK2-binding domain of human p21. 896 91