Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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 gene coding for a Trichosanthes trypsin inhibitor analog (Ala-6-TTI) in which methionine at position 6 was replaced by alanine was synthesized chemically. The synthetic gene was cloned into plasmid pWR590-1 and expressed in Escherichia coli as a fusion protein composed of beta-galactosidase fragment of 590 amino acid residues and (Ala-6)-TTI, with methionine as a connecting residue. After cyanogen bromide cleavage and reduction of the fusion protein, followed by refolding with trypsin-Sepharose 4B as a matrix and affinity chromatography on the immobilized enzyme, the fully active (Ala-6)-TTI was obtained. The trypsin inhibitory activity and amino acid composition of the recombinant (Ala-6)-TTI were consistent with those of the natural one. The (Ala-6)-TTI gene was also cloned into the secretion expression vector, pVT102U/alpha, in Saccharomyces cerevisiae. In order to make the reading frame of the gene compatible with the vector, a nucleotide was inserted into the (Ala-6)-TTI gene via site-directed mutagenesis. The secreted (Ala-6)-TTI was purified and found to be correctly processed at the junction between the alpha-factor leader peptide and (Ala-6)-TTI downstream. Of the two expression systems, the latter is more advantageous in the high yield (greater than 2 mg/liter), easy purification and needlessness of disulfide refolding.
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PMID:Chemical synthesis, molecular cloning, and expression of the gene coding for the Trichosanthes trypsin inhibitor--a squash family inhibitor. 142 10

The beta-galactosidase activities arising from Tn5lac insertions in several genes required for antibiotic TA production were measured under different growth conditions. In all of the non-TA-producing mutants, the beta-galactosidase specific activity was higher when the cells were grown in nutrient-limited 0.5CTS medium (0.5% Casitone plus alanine, serine, and glucose) than in rich 2CT medium (2% Casitone). One of the mutants, 420, had low beta-galactosidase specific activity in both media. The other seven mutants containing inserts in genes essential for TA production had specific activities of 139 to 367 U/mg of protein in 0.5CTS medium and 11 to 48 U/mg of protein in 2CT medium. The beta-galactosidase specific activities of two strains, 1030 and 420, increased during exponential growth in 0.5CTS medium. The beta-galactosidase specific activities of both strains increased greatly when the cells were grown in the presence of magnesium phosphate, which traps ammonium ions. The Tn5lac insertions in 1030 and 420 were used to screen for mutants with increased levels of transcription. An N-methyl-N'-nitro-N-nitrosoguanidine-induced mutation in 1030 that mapped 17 kb from the omega 1010 insert increased the specific activity of beta-galactosidase 21 times in 2CT medium. The regulatory mutation appears to release the repression caused by 2CT medium. A UV-induced mutation in 420 increased the beta-galactosidase specific activity 1.4 to 2.4 times. Medium conditions that affect the transcription of TA genes are discussed in terms of enhanced antibiotic TA production.
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PMID:Use of Tn5lac to study expression of genes required for production of the antibiotic TA. 144 12

The yellow (y) gene of Drosophila melanogaster is required for the pigmentation of larval and adult cuticle structures. The deduced y protein sequence includes two putative N-linked glycosylation sites and a putative signal peptide, suggesting that it might be a secreted molecule. Consistent with the characteristics of a secreted protein, our in vitro translation studies using RNA synthesised from the y cDNA demonstrate that the nascent y polypeptide is a preprotein that cotranslationally translocates into the endoplasmic reticulum (ER) membrane and becomes glycosylated. The N-terminal peptide is cleaved from the preprotein between the two alanine residues at positions 21 and 22, to release the final product into the lumen of the ER. Antibodies raised against the y polypeptide detect the protein starting at 13 h post-fertilization in epidermal cells and in the cuticle structures secreted by them that later become pigmented; in addition, yellow protein is detected in the cuticle structures associated with Keilin's organs. The embryonic beta-galactosidase staining pattern of a transgene, bearing a construct in which expression of the lacZ gene is driven by the y promoter, is also described and is similar to that of the y protein. Our results indicate that the y gene product is an apically secreted protein which becomes an immobilised structural component of the pigmented cuticle.
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PMID:Apical secretion and association of the Drosophila yellow gene product with developing larval cuticle structures during embryogenesis. 146 12

Adenovirus DNA polymerase (AdPol) contains three clusters of basic amino acids within the N-terminal 48 amino acids: RARR, which begins at amino acid 8, RRRVR, which begins at amino acid 25, and RARRRR, which begins at amino acid 41. These clusters are designated BS I, BS II, and BS III, respectively. (The amino acid codes are: R, arginine; A, alanine; V, valine.) Mutational analysis of these noncontiguous clusters showed that AdPol contains a novel organization of bipartite nuclear localization signals (NLS) that interact differentially to serve in the nuclear targeting of AdPol or of chimeric proteins in which AdPol is linked to Escherichia coli beta-galactosidase (beta-gal). The region containing BS I and BS II functioned interdependently as an NLS for the nuclear targeting of AdPol, for which BS III was dispensible. However, the region containing BS II and BS III constituted a second and more efficient bipartite NLS for the nuclear targeting of the AdPol-E. coli beta-gal fusion protein. Moreover, deletion or limited insertion of amino acids in the spacer region between BS II and BS III did not affect their nuclear targeting function for these fusion proteins. Chou-Fasman predictive analysis of protein secondary structure in the vicinity of the bipartite NLS sequences supports a model in which protein conformation in the spacer region may play an important role in bringing these clusters of basic amino acids into close proximity, allowing them to function as nuclear targeting signals for this class of nuclear proteins.
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PMID:Three basic regions in adenovirus DNA polymerase interact differentially depending on the protein context to function as bipartite nuclear localization signals. 177 81

The fibronectin-related region of the 72 kDa type IV procollagenase has been expressed in E. coli as a beta-galactosidase fusion product. The fragment containing the three type II units of the protein was found to have affinity for denatured collagen, suggesting that these domains may be responsible for the collagen-affinity of type IV collagenase. We have also shown that segment Ala-Ala-His-Glu of type IV collagenase (residues 372-375), which is similar to a fibronectin-segment previously implicated in collagen-binding, is not essential for binding activity.
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PMID:Evidence for the involvement of type II domains in collagen binding by 72 kDa type IV procollagenase. 185 Nov 8

Partial sequences of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli, containing the catalytic domain, were cloned in pUC plasmids and over-expressed in E. coli TG2. A high expression of a homogeneous protein was only detectable for E2p mutants consisting of the catalytic domain and the alanine-proline-rich sequence between a putative binding region for the peripheral components and the catalytic domain (apa-4). Most of the catalytic domain from A. vinelandii without the apa-4 sequence was degraded intracellularly, probably due to incorrect folding. Fusion proteins of six amino acids from beta-galactosidase, the apa-4 region and the catalytic domains of A. vinelandii or E. coli E2p could be highly purified. Both catalytic domains were assembled in 24-subunit structures with a molecular mass of approximately 670 kDa. The expression of catalytic domain from A. vinelandii E2p is more than twice as high as found for wild-type E2p. This can be explained by intracellular degradation of over-expressed wild-type E2p, whereas the catalytic domains are stable against proteolysis in vivo and in vitro. The interaction of the peripheral components pyruvate dehydrogenase (E1p) and dihydrolipoamide dehydrogenase (E3) with the catalytic domains was studied, using gel filtration on Superose-6 and sedimentation velocity experiments. No binding of either E1p or E3 to the catalytic domain of either organism was detectable. Crystals of the catalytic domain of A. vinelandii E2p could be grown to a maximum size of 0.6 x 0.6 x 0.4 mm. They diffract up to a resolution of 0.28 nm.
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PMID:The catalytic domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii and Escherichia coli. Expression, purification, properties and preliminary X-ray analysis. 193 51

The gerA operon of Bacillus subtilis 168 comprises three genes concerned with the triggering of spore germination by L-alanine and its analogues. The expression of this operon has been characterized using chromosomal lacZ fusions to the gerA promoter. The gerA promoter is switched on 2.5-3 hours after the initiation of sporulation, in parallel with glucose dehydrogenase. A high proportion of the gerA-driven beta-galactosidase detected in sporulating cells is found in the mature spore; the gerA promoter is therefore active in the forespore compartment of the sporulating cell. The gerA promoter is not expressed in spoO, spoII or spoIIIA, B, E and G mutant backgrounds, but is expressed in spoIIIC and D and in spoIV and V mutants. The in vivo transcriptional startpoint of the operon has been mapped by primer extension experiments; sequences upstream from this startpoint show significant homology with recognition sequences for RNA polymerase containing sigma G (E sigma G). The gerA operon was transcribed in vitro by E sigma G with a startpoint identical to that used in vivo, and expression of the gerA operon was rapidly induced in vegetative cells by induction of sigma G synthesis. These data indicate that the gerA operon is an additional member of the sigma G regulon, which includes a number of genes expressed in parallel only in the forespore compartment of sporulating B. subtilis cells.
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PMID:The regulation of transcription of the gerA spore germination operon of Bacillus subtilis. 211 Sep 96

Several fusion proteins of our previously chemically synthesized gene encoding the interleukin-2-receptor alpha subunit (IL-2R alpha or Tac protein) were constructed. They were designed in order to be cleavable by cyanogen bromide. Thus, the original internal methionines of the IL-2R alpha were replaced by either alanine, valine, leucine or isoleucine, based on secondary structure predictions. Additionally, aspartate at position 6 was substituted for glutamate in order to stabilize the acid-labile Asp-Pro bond. Direct C-terminal fusion of total beta-galactosidase and portions thereof did not result in substantial amounts of the expected construct. Ternary fusions consisting of beta-galactosidase domains N- and C-terminally fused to the mutant synthetic methionine-free interleukin-2 receptor alpha subunit (synIL-2R alpha) yielded inclusion bodies amounting to 4-7% of the total protein. This first overexpression of a type I membrane receptor can be rationalized by the known beta-galactosidase structure models. The fusion protein can be cleaved with cyanogen bromide, isolated and the resulting synIL-2R alpha detected by Western blot analysis.
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PMID:Overexpression in Escherichia coli of a methionine-free designed interleukin-2 receptor (Tac protein) based on a chemically cleavable fusion protein. 212 81

The SSN6 protein functions as a negative regulator of a variety of genes in Saccharomyces cerevisiae and is required for normal growth, mating, and sporulation. It is a member of a family defined by a repeated amino acid sequence, the TPR (tetratricopeptide repeat) motif. Here, we have used specific antibody to identify and characterize the SSN6 protein. Both SSN6 and a bifunctional SSN6-beta-galactosidase fusion protein were localized in the nucleus by immunofluorescence staining. The N-terminal one-third of the protein containing the TPR units was identified as the region that is important for SSN6 function. Analysis of four nonsense alleles, isolated as intragenic suppressors of an ssn6::URA3 insertion, revealed that polypeptides truncated after TPR unit 7 provide SSN6 function. Deletion analysis suggested that TPR units are required but that 4 of the 10 TPR units are sufficient. In addition, deletion studies indicated that three very long, homogeneous tracts of polyglutamine and poly(glutamine-alanine) are dispensable. Previous genetic evidence suggested the SSN6 protein as a possible target of the SNF1 protein kinase. Here, we show that the C terminus of SSN6 is phosphorylated in vivo and that the SNF1 kinase is not responsible for most of the phosphorylation. Finally, SSN6 has a modest effect on the maintenance of minichromosomes.
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PMID:The N-terminal TPR region is the functional domain of SSN6, a nuclear phosphoprotein of Saccharomyces cerevisiae. 220 1

We have examined transcriptional start sites responsible for expression of the transposase and transposition inhibitor proteins encoded by IS50R, and determined the likely translational start site of transposase. Amino-terminal analysis of a transposase-beta-galactosidase fusion protein gave the sequence Met-Ile-Thr-Ser-Ala, which corresponds to the predicted amino acid sequence starting at position 93 of IS50. S1 nuclease mapping of IS50 RNA produced in vivo indicated that three transcripts, T1, T2 and T3, start near this position. Only T1 starts upstream from the transposase amino terminus. T2 corresponds to an in-vitro transcript described previously. Analysis of the transcripts and proteins produced from deletion derivatives of an IS50-lacZ construct suggested that the three transcripts initiate at independent but overlapping promoters clustered near the end of IS50. This analysis confirmed that only T1 can encode transposase, and that T2 is largely responsible for expression of the inhibitor protein. The coding capacity of T3 was not determined. Finally, transcripts that originate outside of IS50 are prevented from expressing transposase because of a secondary structure that is present in these transcripts only.
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PMID:Transcriptional and translational initiation sites of IS50. Control of transposase and inhibitor expression. 243 19


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