Gene/Protein Disease Symptom Drug Enzyme Compound
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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)

The recent advances in the field of immunology and molecular biology allow to consider not distant the identification of protective antigens to be used in new prophilactic and diagnostic tools. In our laboratory we have analysed a M. tuberculosis expression library by murine monoclonal antibodies, human sera and human T lymphocytes. We have identified a 35 Kd protein present only on the M. tuberculosis complex (M. tuberculosis, M. bovis, M. africanum). This 35 Kd protein is also detected, by immunohistological analysis, inside the alveolar macrophage of pulmonary tuberculosis patients. Both recombinant beta-galactosidase and MS2 polymerase-fused proteins have been produced and used to perform western blotting and T cell proliferation assay. As the cloned fragment contains an internal EcoRI restriction site, it was possible to identify two fragments of 1.7 and 2.2 kb respectively. Southern blot analysis showed that both the fragments hibridized with the genomic DNA from M. tuberculosis complex but not with the DNA from other mycobacterial isolates from 12 pulmonary tuberculous patients hibridized with the 1.7 kb fragment. The possible use of this insert for the molecular diagnosis of tuberculosis is under investigation.
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PMID:Identification of a specific Mycobacterium tuberculosis protein able to activate T lymphocytes. 172 Feb 94

The nucleotide sequence of the neuC gene of the Escherichia coli K1 capsule gene cluster encodes a protein with a predicted molecular weight of 44,210 containing 391 amino acids. A chimeric protein with beta-galactosidase fused to the carboxy terminus of the neuC gene product (P7) was constructed and purified. Its amino-terminal sequence confirmed the prediction from the nucleotide sequence that the neuC gene overlaps the distal end of the neuA gene by a single base pair. Both the neuA and neuC genes are coexpressed under the control of a single upstream T7 or tac promoter, suggesting that neuA and neuC are part of an operon.
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PMID:Sequence and expression of the Escherichia coli K1 neuC gene product. 172 18

To examine the role of primary amino acid sequence in the localization of proteins within the nucleus, we studied the nucleolar protein NO38 of amphibian oocytes. We synthesized NO38 transcripts in vitro, injected them into the oocyte cytoplasm, and followed the distribution of the translation products. The injected RNA contained a short sequence encoding an epitope derived from the human c-myc protein. We used an mAb against this epitope to detect translation products from injected RNAs by Western blots and by immunofluoresent staining of cytological preparations. When full-length transcripts of NO38 were injected into oocytes, the translation products accumulated efficiently in the germinal vesicle, and a major fraction was localized in the multiple nucleoli. To identify protein domains involved in this nucleolus-specific accumulation, we prepared a series of carboxy-terminal deletions of the cDNA. Oocytes injected with RNA encoding truncated forms of NO38 were examined for altered patterns of protein accumulation. We defined a domain of about 24 amino acids near the carboxy terminus that was essential for nucleolar localization of NO38. This domain is separated by more than 70 amino acids from two putative nuclear localization signals near the middle of the molecule. Hybrid constructs were made which encoded part of Escherichia coli beta-galactosidase or pyruvate kinase fused to a long segment of NO38 containing the essential domain. Injection of RNA from these constructs showed that the essential domain was not sufficient to target the hybrid proteins to the nucleolus. We suggest that nucleolar accumulation of NO38 requires more than a single linear domain.
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PMID:Localization of the nucleolar protein NO38 in amphibian oocytes. 173 Jul 39

The closely related Drosophila serendipity (sry) beta and delta zinc finger proteins display consensus in vitro DNA recognition sequences differing by 4 of 13 nucleotide positions and bind in vivo to distinct sets of sites on polytene chromosomes. We compared the pattern of in vivo chromosomal binding of deleted forms of the sry delta protein fused to beta-galactosidase and expressed in Drosophila transgenic lines. Results show that the carboxy-terminal DNA-binding finger domain is required and sufficient for binding at specific chromosomal sites but that this binding does not nearly reproduce the wild-type pattern. An NH2-terminal domain of the sry delta protein is essential to its specificity of in vivo interaction with chromatin. In vitro and in vivo experiments using reciprocal finger swap between the sry beta and delta proteins suggest that the in vivo specificity is dependent on selective protein-protein contacts at defined chromosomal sites, in addition to DNA specific recognition.
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PMID:Zinc fingers and other domains cooperate in binding of Drosophila sry beta and delta proteins at specific chromosomal sites. 173 41

The guaBA operon determines production of the two enzymes required to convert hypoxanthine to guanine at the nucleotide level during guanine nucleotide biosynthesis. Two DnaA boxes, binding sites for the DNA replication-initiating DnaA protein, are present in the gua operon, one at the gua promoter (guaP) and the other within the guaB coding sequence. Regulation of the guaBA operon by DnaA protein was studied using strains carrying chromosomal gua-lacZ fusions. In these strains beta-galactosidase acts as a reporter enzyme for transcription initiated at guaP. When the intracellular levels of DnaA were increased (by induction of a multicopy plasmid carrying the dnaA gene fused to the tac promoter) transcription from the gua promoter was repressed. Reducing the intracellular level of DnaA, either by sequestration with an oriC plasmid or by placing a temperature-sensitive dnaA mutant at the restrictive temperature, resulted in increased transcription from guaP. Thus the transcriptional activity of the gua operon is coupled, through the DnaA protein, to the DNA replication cycle. Repression of guaP by DnaA was dependent on the presence of both boxes in the gua-lacZ fusion; constructs containing only the box at guaP were unaffected by DnaA.
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PMID:Regulation of the gua operon of Escherichia coli by the DnaA protein. 173 96

We have constructed a series of mutations in the membrane and linker domains of Syrian hamster 3-hydroxy-3-methylglutaryl-(HMG) CoA reductase in order to determine the regions critical for the regulated degradation of the enzyme. In transfected Chinese hamster ovary cells, we have expressed a fusion protein, HMGal, which consists of the membrane and linker domains of the Syrian hamster HMG-CoA reductase fused to beta-galactosidase. Using this fusion protein, we have determined that a deletion of 64 amino acids from the central region of the membrane domain causes the protein to be degraded extremely rapidly. In addition, deletion of PEST sequences has little effect on degradation, but deletion of the linker domain makes the protein's degradation insensitive to sterols and mevalonate. In addition to deletion mutations, we have systematically replaced each hydrophobic, putative membrane spanning region of the membrane domain with the first transmembrane sequence from bacteriorhodopsin. Replacement of span 4 has no effect on degradation. Replacements of spans 5 or 6 result in a protein which has a normal basal rate of degradation, but this rate of degradation is not accelerated by mevalonate, low density lipoprotein, or 25-hydroxycholesterol. Replacement of span 3 results in a protein whose degradation is similarly not accelerated by sterols or mevalonate, but since this protein might be mislocalized, these results are inconclusive. Replacement of span 7 yields a short-lived protein which is degraded more rapidly in response to mevalonate but not in response to exogenous sterols. Replacement of span 8 extends both the basal and mevalonate-accelerated half-life about 5-fold. This work begins to define the critical regions for regulated degradation within the membrane domain of HMG-CoA reductase.
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PMID:The role of the membrane domain in the regulated degradation of 3-hydroxy-3-methylglutaryl coenzyme A reductase. 174 Apr 63

The plasmid pSK1002 carrying the fused gene umuC'-'lacZ could increase the number of revertants induced by methyl methanesulfonate (MMS) and 4-nitroquinoline-1-oxide (4NQO) in S. typhimurium LT2 TA 1535(his-). Maximum revertants were 4.35 and 3.96 times that of the controls without the plasmid. The values induced by N-methyl-N'-nitrosoquanidine (MN-NG) were about the same with or without plasmid. However, the plasmid pKM101-mediated mutagenesis-enhancing effect was much greater than that through the mediation of pSK1002 as induced by the three mutagens mentioned above. Moreover, the plasmid pSK1002 could induce umu-mediated SOS response in the presence, of either mutagen stated above and mitomycin C(MMC). Maximum levels of beta-galactosidase (beta-gal) activity induced by four mutagens respectively were 4.56, 7.14, 4.94, and 3.42 times that of the controls, and a dose-response relationship was evident. The sensitivity of SOS response was superior to mutagenesis-enhancing effect. It showed that pSK1002 (umuC'-'lacZ) had a dual biological effect, namely, mutator effect and the effect of inducing SOS response. Besides, this study has proved SOS mutagenesis of 2,5-dichloronitrobenzol (2,5-DCNB). Because of the dual indicator nature of pSK1002, it is probable that pSK1002 would be further developed and applied in studying the relation between SOS response and mutagenesis, and monitoring environmental SOS mutagens.
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PMID:[Plasmid pSK1002-mediated mutator effect and SOS response in Salmonella typhimurium and its use for detection of mutagens]. 177 74

Secretion of haemolysin (HlyA) is secA independent, but depends upon two accessory membrane proteins, HlyB and HlyD, encoded by the hly determinant. A fourth (cytoplasmic) protein, HlyC, is required to activate HlyA post-translationally, but has no role in export. Deletion studies have previously shown that the HlyA molecule contains a targeting signal close to the C-terminus which specifically directs its secretion to the medium. This targeting signal has been variously located within the terminal 27, 53, 60 or 113 amino acids. In this paper, we have sought to confirm the presence of a C-terminal targeting signal and to analyse the specificity of the Hly transport system through fusion of C-terminal fragments of HlyA to heterologous polypeptides. A C-terminal fragment (23 kDa) of HlyA, when fused at the C-terminus, efficiently promoted the secretion of the eukaryotic protein prochymosin (PCM) to the medium via HlyB and HlyD. This result is in contrast to previous findings that prochymosin, preceded by the alkaline phosphatase signal sequence, cannot be translocated across the Escherichia coli inner membrane. The HlyA targeting domain was also used to secrete to the medium varying portions of chloramphenicol acetyltransferase (CAT) and 98 per cent of the beta-galactosidase (LacZ) molecule (both E. coli cytoplasmic proteins). In the case of the PCM and CAT fusions the efficiency of secretion was reduced as the proportion of the PCM and CAT molecule increased. This result is consistent with inhibition of secretion through the irreversible folding of the larger passenger protein fragments, or the occlusion of the HlyA targeting signal by upstream sequences. Analysis of the nature of the C-terminal domain promoting secretion of prochymosin, demonstrated that shortening the signal domain from 218 to 113 amino acids significantly reduced the efficiency of secretion. This result may also reflect the importance of maintaining an independently folded signal motif well separated from a passenger domain.
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PMID:Analysis of the haemolysin transport process through the secretion from Escherichia coli of PCM, CAT or beta-galactosidase fused to the Hly C-terminal signal domain. 179 66

Strategies for the expression of precursors of eukaryotic secreted proteins as part of fused proteins in Escherichia coli have been explored. A fusion protein with beta-galactosidase at the N-terminal end and honeybee prepromelittin at the C-terminal end (beta-gal-pM) was expressed in low amounts as a cleaved polypeptide, from which the promelittin portion had been removed. Inclusion in the induction culture of 10 mM MgCl2 or 8.3% (v/v) ethanol, inhibitors of signal peptidase, gave rise to the full-length beta-gal-pM fusion protein. The results suggest that a soluble recombinant fusion protein with a signal peptide in an internal location 660 residues from the N-terminus is recognized by the E. coli translocation apparatus in the inner membrane and by leader peptidase. High-level production (about 45% of total cellular proteins) of prepromelittin was achieved when it was part of a fusion protein at the C-terminus of a truncated insoluble polypeptide from bacteriophage gene 10. This fusion protein separated into inclusion bodies in an aggregated form. In contrast, attempts to express prepromelittin by itself or at the N-terminal end of a fusion with mouse dihydrofolate reductase (pM-DHFR) proved unsuccessful.
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PMID:Expression of honeybee prepromelittin as a fusion protein in Escherichia coli. 182 10

The sequence of Escherichia coli UvrA protein suggests that it may fold into two functional domains each possessing DNA binding and ATPase activities. We have taken two approaches to physically isolate polypeptides corresponding to the two putative domains. First, a 180 base pair DNA segment encoding multiple collagenase recognition sequences was inserted into UvrA's putative interdomain hinge region. This UvrA derivative was purified and digested with collagenase, and the resulting 70-kDa N-terminal and 35-kDa C-terminal fragments were purified. Both fragments possessed nonspecific DNA binding activity, but only the N-terminal domain retained its nucleotide binding capacity as evidence by measurements of ATP hydrolysis and by ATP photo-cross-linking. Together, the two fragments failed to substitute for UvrA in reconstituting (A)BC excinuclease and, therefore, were presumed to be unable to load UvrB onto damaged DNA. Second, the DNA segments encoding the two domains were fused to the beta-galactosidase gene. The UvrA N-terminal domain-beta-galactosidase fusion protein was overproduced and purified. This fusion protein had ATPase activity, thus confirming that the amino-terminal domain does possess an intrinsic ATPase activity independent of any interaction with the carboxy terminus. Our results show that UvrA has two functional domains and that the specificity for binding to damaged DNA is provided by the proper three-dimensional orientation of one zinc finger motif relative to the other and is not an intrinsic property of an individual zinc finger domain.
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PMID:Isolation and characterization of functional domains of UvrA. 182 51


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