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)

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

A 3.3-kilobase DNA complementary to human microtubule-associated protein 2 (MAP2) was sequenced by the dideoxy method. The 3' end terminates at an internal EcoRI site before the polyA tail. Due to the arrangement of the cDNA insert in the lambda gt11 vector, the MAP2 fragment is not fused to beta-galactosidase when expressed. The Chou Fasman algorithm for the initial 58 amino acids from the first in-frame methionine predicts an alpha helix. Beyond this point, a series of turns is predicted until amino acid 160. The frequent presence of basic residues in proximity to serines or threonines is consistent with multiple phosphorylation sites. The minimum specificity determinant for Ca2+/calmodulin-dependent kinase is repeated 13 times. The sequence of a region containing a MAP2 epitope that is shared with the Alzheimer neurofibrillary tangle was determined by DNase treatment of the cDNA and antibody selecting the small resultant clones in a lambda gt11 sublibrary. Likewise, a MAP2 epitope that is not shared with the neurofibrillary tangle also has been located. Both epitopes are in the projection portion of the molecule. A bovine MAP2 cyanogen bromide fragment, which contains the epitope shared with the neurofibrillary tangle, is partially insoluble under aqueous conditions, probably due to the aggregation of oppositely charged residues. Thus, rapid cleavage of MAP2 to small peptides is probably necessary in vivo to prevent the aggregation of larger cleavage fragments.
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PMID:Partial sequence of MAP2 in the region of a shared epitope with Alzheimer neurofibrillary tangles. 245 76

The chondroitin sulfate proteoglycans of brain contain several core proteins bearing HNK-1 antibody epitopes. Endo-beta-galactosidase treatment resulted in the almost complete disappearance of HNK-1 staining of proteoglycan immunoblots, indicating that a significant portion of the 3-sulfated sugar residues recognized by this antibody are present on poly(N-acetyllactosaminyl) oligosaccharides. However, after treatment with chondroitinase ABC followed by endo-beta-galactosidase, several proteoglycan species showed HNK-1 reactivity, presumably due to the presence of this epitope on other oligosaccharides which are both resistant to endo-beta-galactosidase and inaccessible to the antibody in the native proteoglycan. Immunostaining of the endo-beta-galactosidase degradation products after separation by thin-layer chromatography demonstrated that HNK-1 reactivity was confined to a minor population of large oligosaccharides. Only a relatively small portion of the native chondroitin sulfate proteoglycans of brain enter a 6-12% SDS-polyacrylamide gel. However, after treatment of the proteoglycans with chondroitinase ABC (or chondroitinase and endo-beta-galactosidase) in the presence of protease inhibitors, seven bands with molecular sizes ranging from 80 to 200 kDa appear in Coomassie Blue stained gels, and two additional bands with molecular sizes of 67 and 350-400 kDa are apparent in fluorographs of sodium [35S]sulfate labeled proteoglycans. Most of these components probably represent individual proteoglycan species rather than different degrees of nonchondroitin sulfate/keratan sulfate glycosylation of a single protein core, since [35S]methionine-labeled proteins of comparable molecular size were synthesized by an in vitro translation system. These findings suggest that chondroitin sulfate proteoglycans which differ in molecular size and composition may be specific to particular cell types in brain.
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PMID:Presence of the HNK-1 epitope on poly(N-acetyllactosaminyl) oligosaccharides and identification of multiple core proteins in the chondroitin sulfate proteoglycans of brain. 247 68

We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (lambda Clac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying lambda Clac mutant phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.
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PMID:Salmonella typhimurium metC operator-constitutive mutations. 250 6

The beta-galactosidase-based assay for lysine developed by Tuffnell & Payne was used to measure the bioavailabilities of cyst(e)ine, methionine, threonine and tryptophan in pronase digests of 17 foods. The digests were assayed by estimating the beta-galactosidase synthesis responses of five Escherichia coli mutants, each requiring one of the respective amino acids for protein synthesis. Deletion mutants were used whenever possible in order to ensure strain stability. Single digests of each food were assayed with 3 or 4 separate cultures of each mutant and the results were compared with those from the corresponding chemical assay. Omitting the anomalously low values for one food, the rank correlation coefficients of the bio- and chemo-assay values were 0.61 (cysteine), 0.91 (lysine), 0.95 (methionine), 0.64 (threonine) and 0.85 (tryptophan). Mean (+/- S.D.) relative amino acid bioavailabilities (casein = 100%) for the 17 foods were: cysteine, 53 +/- 23; lysine, 90 +/- 10; methionine, 95 +/- 18; threonine, 89 +/- 13; and tryptophan, 89 +/- 25. The cysteine mutant appeared to give unusually low bioavailability values except for the milk products. These amino acid mutants afford a rapid method for assaying the bioavailabilities of at least four of the five amino acids studied.
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PMID:The use of Escherichia coli mutants to measure the bioavailability of essential amino acids in foods. 265 34

A synthetic gene encoding Cerebratulus lacteus neurotoxin B-IV has been designed, cloned, and expressed in Escherichia coli. Although expression of the toxin alone appears to be incompatible with host viability, large amounts could be synthesized as a fusion protein with either E. coli beta-galactosidase or the gene 9 protein of bacteriophage T7, the latter system being the more efficient. The fusion protein has been purified, and, after Factor Xa-catalyzed hydrolysis at a customized linker site, we have obtained the equivalent of 12 mg of pure toxin B-IV per liter of bacterial culture. The recombinant protein is identical with B-IV isolated from Cerebratulus with respect to high performance liquid chromatography mobility and secondary structure, and its amino acid composition differs only by the presence of an amino-terminal methionine residue and replacement of Hyp10 by Pro. Quantal bioassay indicates that the cloned protein is comparable to the natural toxin in specific toxicity. The small differences observed in comparing the activities of the two proteins are most likely due to the presence of the methionine extension at the amino terminus of the recombinant, although lack of hydroxylation of Pro10 may also contribute.
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PMID:Cloning and expression of a synthetic gene for Cerebratulus lacteus neurotoxin B-IV. 267 Sep 34

In a significant fraction of the Escherichia coli cytosolic proteins, the N-terminal methionine residue incorporated during the translation initiation step is excised. The N-terminal methionine excision is catalyzed by methionyl-aminopeptidase (MAP). Previous studies have suggested that the action of this enzyme could depend mainly on the nature of the second amino acid residue in the polypeptide chain. In this study, to achieve a systematic analysis of the specificity of MAP action, each of the 20 amino acids was introduced at the penultimate position of methionyl-tRNA synthetase of E. coli and the extent of in vivo methionine excision was measured. To facilitate variant protein purification and N-terminal sequence determination, an expression shuttle vector based on protein fusion with beta-galactosidase was used. From our results, methionine excision catalyzed by MAP is shown to obey the following rule: the catalytic efficiency of MAP, and therefore the extent of cleavage, decreases in parallel with the increasing of the maximal side-chain length of the amino acid in the penultimate position. This molecular model accounts for the rate of N-terminal methionine excision in E. coli, as deduced from the analysis of 100 protein N-terminal sequences.
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PMID:Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. 268 40

The objective of this study was to identify and characterize the gene encoding a protein of approximately 12 kDa that is secreted from cells infected with the vaccinia virus. The absence of this protein from the medium of cells infected with a spontaneous deletion mutant (6/2) suggested that the open reading frame (ORF) was located within a 12,800-base pair segment near the left end of the genome (G. Kotwal and B. Moss, Nature (London) 335, 176-178, 1988). Antibody to the 12-kDa protein immunoprecipitated an appropriate size in vitro translation product of mRNA that hybridized to a DNA segment containing an ORF (N1L) that could encode a 13.8-kDa polypeptide. The similarity in the sizes of the in vitro translation product and the secreted protein was consistent with the absence of processing. Transcriptional analysis revealed major and minor early RNA start sites preceding the N1L ORF as well as a late RNA start site with an atypical TAAAAT sequence. The N1L gene was interrupted by replacing a segment of the ORF with the Escherichia coli beta-galactosidase gene. When two-dimensional polyacrylamide gel electrophoretic patterns of [35S]methionine-labeled proteins secreted from cells infected with parental and recombinant viruses were compared, a spot missing from the latter corresponded in molecular weigh and isoelectric point with that predicted from the N1L ORF. The latter analysis revealed the presence of other secreted proteins of similar molecular weight but different isoelectric points that also appear to map within the left end of the vaccinia genome. The recombinant virus was attenuated as judged by the increased intracranial LD50 for mice but nevertheless induced antibody and cytotoxic responses after intradermal and intraperitoneal injections. Relative to the parental virus, the recombinant was also more attenuated for immunodeficient nude mice, based on their survival time after infection.
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PMID:Mapping and insertional mutagenesis of a vaccinia virus gene encoding a 13,800-Da secreted protein. 276 67

Escherichia coli and Salmonella typhimurium preferentially utilize sugar substrates of the phosphoenol-pyruvate:glycose phosphotransferase system (PTS) when the growth medium also contains other sugars. This phenomenon, diauxic growth, is regulated by the crr gene, which encodes the PTS protein IIIGlc (Saffen, D.W., Presper, K.A., Doering, T.L., and Roseman, S. (1987) J. Biol. Chem. 16241-16253). We have proposed that non-PTS permeases are regulated by their interaction with IIIGlc, and in vitro studies from other laboratories have provided support for this model, but the in vivo effects of excess IIIGlc are not known. In the present studies, transformed cells that overproduced IIIGlc 2- and 10-fold, respectively, were constructed from a pts+ strain of E. coli and plasmids containing the crr gene. In the 2-fold overproducer, fermentation of, and growth on the non-PTS carbohydrates glycerol, lactose, maltose, and melibiose was generally more sensitive to the glucose analogue methyl-alpha-D-glucopyranoside than in a control strain containing normal levels of IIIGlc. In addition, inhibition of lactose permease activity by methyl-alpha-glucoside (inducer exclusion) was more effective in the 2-fold overproducer than in the control strain, particularly when the permease activity was high. The 10-fold IIIGlc overproducing strain had a requirement for the amino acids methionine, isoleucine, leucine, and valine that may or may not be related to the increased concentration of IIIGlc. Fermentation of non-PTS carbohydrates was also poor in the latter strain. Finally, lactose permease activity was 50% of that in control cells containing the same levels of beta-galactosidase, and the lactose permease activity in the IIIGlc overproducer was reduced to an extremely low level in the presence of methyl alpha-glucoside. Thus there is an inverse relationship between the cellular concentration of IIIGlc and the ability to metabolize non-PTS substrates. The results are consistent with the model where inducer exclusion is affected by a direct interaction between IIIGlc and a non-PTS transport system.
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PMID:Sugar transport by the bacterial phosphotransferase system. In vivo regulation of lactose transport in Escherichia coli by IIIGlc, a protein of the phosphoenolpyruvate:glycose phosphotransferase system. 282 84

Forty-nine amino acid substitutions were made at four positions in the Escherichia coli enzyme beta-galactosidase; three of the four targeted amino acids are thought to be part of the active site. Many of the substitutions were made by converting the appropriate codon in lacZ to an amber codon, and using one of 12 suppressor strains to introduce the replacement amino acid. Glu-461 and Tyr-503 were replaced, independently, with 13 amino acids. All 26 of the strains containing mutant enzymes are Lac-. Enzyme activity is reduced to less than 10% of wild type by substitutions at Glu-461 and to less than 1% of wild type by substitutions at Tyr-503. Many of the mutant enzymes have less than 0.1% wild-type activity. His-464 and Met-3 were replaced with 11 and 12 amino acids, respectively. Strains containing any one of these mutant proteins are Lac+. The results support previous evidence that Glu-461 and Tyr-503 are essential for catalysis, and suggest that His-464 is not part of the active site. Site-directed mutagenesis was facilitated by construction of an f1 bacteriophage containing the complete lacZ gene on a single EcoRI fragment.
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PMID:Effects of amino acid substitutions at the active site in Escherichia coli beta-galactosidase. 290 3


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