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)

CEDIA assays represent a state of the art technique utilizing two genetically engineered, enzymatically inactive fragments of beta-galactosidase as the basis for a homogeneous enzyme immunoassay. The smaller, amino-terminal polypeptide, designated the enzyme donor (ED), can recombine spontaneously with the large residual fragment, called the enzyme acceptor (EA), to form active beta-galactosidase, in a process called complementation. ED have been designed in such a way that a ligand, such as a hormone or drug, can be chemically attached to a specific amino acid residue without affecting the enzyme complementation. However, the binding of a ligand-specific antibody to the ED-ligand conjugate will inhibit complementation. If a sample containing ligand is added to the reaction mixture, the ligand will compete with the ED-ligand conjugate for the limited number of antibody binding sites. Thus, the ligand concentration in the sample will modulate enzymatic activity by influencing the amount of free ED-ligand conjugate available for complementation. The basic technology of CEDIA assays has a number of inherent advantages, the most important of these being a linear calibration curve with high precision over the whole assay range, lack of endogeneous enzyme activity and minimal serum interference, chemically defined conjugates and flexibility in assay design. These provide significant advantages in comparison to other homogeneous immunoassay techniques. As a result, CEDIA assays have been successfully developed for high concentration drugs such as theophylline, phenobarbital and phenytoin as well as for very low concentration analytes such as digoxin, B12 and folate. In a modified assay format, even the determination of binding proteins has been accomplished, an example being thyroxine binding proteins in the CEDIA T-uptake assay. More recently, the methodology has been extended to the measurement of high molecular weight analytes like ferritin.
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PMID:CEDIA in vitro diagnostics with a novel homogeneous immunoassay technique. Current status and future prospects. 161 62

Nontuberculous mycobacteria, particularly Mycobacterium avium, have been isolated from a significant percentage of patients with AIDS. Early detection of M. avium infection is difficult, and treatment regimens are often ineffective. Much needs to be learned about antigens and factors responsible for immunity to and pathogenesis of the disease. Specific antigens and diagnostic procedures for infection need to be developed. To address some of these problems, we have generated 25 different monoclonal antibodies against a serovar 4 strain of M. avium isolated from a patient with AIDS. Protease sensitivity studies have demonstrated that each of these antibodies recognizes a protein-associated epitope. Immunoblot analyses suggest that seven of these monoclonal antibodies react specifically with M. avium and M. intracellular epitopes. Immunoreactive bacteriophages were identified from an M. avium lambda gt11 expression library with two of these monoclonal antibodies (3808 C3 and 3954 B12). Lambda lysogens, generated from the immunoreactive bacteriophages, overproduced beta-galactosidase fusion proteins which were reactive with the two monoclonal antibodies in immunoblot assays. The purified fusion proteins were shown to elicit skin test reactions in sensitized guinea pigs.
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PMID:Immunological characterization of recombinant antigens isolated from a Mycobacterium avium lambda gt11 expression library by using monoclonal antibody probes. 171 96

Synthesis of the Escherichia coli outer membrane protein BtuB, which mediates the binding and transport of vitamin B12, is repressed when cells are grown in the presence of vitamin B12. Expression of btuB-lacZ fusions was also found to be repressed, and selection for constitutive production of beta-galactosidase in the presence of vitamin B12 yielded mutations at btuR. The btuR locus, at 27.9 min on the chromosome map, was isolated on a 952-base-pair EcoRV fragment, and its nucleotide sequence was determined. The BtuR protein was identified in maxicells as a 22,000-dalton polypeptide, as predicted from the nucleotide sequence. Strains mutant at btuR had negligible pools of adenosylcobalamin but did convert vitamin B12 into other derivatives. Although btuB expression in a btuR strain could not be repressed by cyano- or methylcobalamin, it was repressed by adenosylcobalamin. Growth on ethanolamine as the sole nitrogen source requires adenosylcobalamin. btuR mutants grew on ethanolamine but were affected in the length of the lag period before initiation of growth, which suggested that an alternative route for adenosylcobalamin synthesis might exist. No mutations were found that conferred constitutive btuB expression in the presence of adenosylcobalamin. Other genes near btuR may also be involved in cobalamin metabolism, as suggested from the complementation behavior of strains generated by excision of the Tn10 element in btuR. These results indicated that the btuR product is involved in the metabolism of adenosylcobalamin and that this cofactor, or some derivative, controls btuB expression.
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PMID:Altered cobalamin metabolism in Escherichia coli btuR mutants affects btuB gene regulation. 264 87

The transport of vitamin B12 in Escherichia coli requires a specific vitamin B12 receptor protein in the outer membrane and the tonB gene product. In addition, the btuC gene, located at min 38 on the genetic map, has been found to influence vitamin B12 uptake or utilization. The btuC function is required for the growth response to vitamin B12 when the outer membrane transport process (btuB or tonB function) is defective. However, even in a wild-type strain, btuC is required for proper transport of vitamin B12. Additional mutations in the vicinity of btuC were isolated as lac fusions that produced a phenotype similar to that of a btuC mutant. The btuC region was cloned by selection for complementation of a btuC mutation. Complementation testing with plasmids carrying various deletions or transposon Tn1000 insertions demonstrated that the new mutations defined a separate, independently expressed locus, termed btuD. The coding regions for both genes were identified on a 3.4-kilobase HindIII-HincII fragment and were 800 to 1,000 base pairs in length. They were separated by a 600- to 800-base-pair region. The gene order in this portion of the chromosome map was found to be pps-zdh-3::Tn10-btuD-btuC-pheS. Expression of beta-galactosidase in the btuD-lac fusion-bearing strains, whether proficient or defective in vitamin B12 transport, was not regulated by the presence of vitamin B12 in the growth medium.
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PMID:Transport of vitamin B12 in Escherichia coli: cloning of the btuCD region. 298 92

The btuB gene of Escherichia coli codes for a protein (BtuB) located in the outer membrane. BtuB is the receptor for vitamin B12 (cyanocobalamin). We have cloned the btuB gene into pUC8 using transposon Tn5 as the marker to first isolate several parts of the relevant DNA fragment from the specialized transducing phage lambda darg13. After reconstitution of the gene, Tn5 was removed by selecting for spontaneous excision. The partial nucleotide sequence and transcriptional start of the btuB gene were determined. The BtuB+ plasmid allowed a large amplification of the synthesis of BtuB, resulting in a 65-fold increased level of vitamin B12 binding. The level of vitamin B12 binding was reduced by a factor of 22 when cells were grown in the presence of high concentrations of vitamin B12. The regulation of the gene was studied in more detail by the use of a protein fusion between the extreme amino-terminus of BtuB and beta-galactosidase of E. coli. The kinetics of repression and derepression were consistent with the presence in the cells of a large amount of a regulatory molecule exhibiting an apparent Km for vitamin B12 of 3 microM.
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PMID:Regulation of expression of the gene for vitamin B12 receptor cloned on a multicopy plasmid in Escherichia coli. 302 10

Synthesis of the btuB-encoded outer membrane receptor for vitamin B12 and the metE-encoded homocysteine methyltransferase is repressed by growth of Escherichia coli in the presence of vitamin B12. The regulation by vitamin B12 of the production of beta-galactosidase in strains carrying btuB-lac or metE-lac operon fusions indicated that repression of both genes operates at the transcriptional level. Selection for expression of these fusions under repressive conditions allowed isolation of second-site mutations in which repressibility by vitamin B12 had been lost. Mutations in metH and metF prevented vitamin B12-dependent regulation of metE, but not that of btuB. Mutations in btuB and other genes involved in uptake of the vitamin eliminated or reduced repression. Mutations in the newly identified gene, btuR, controlled the repressibility of btuB, but had no effect on metE regulation. The btuR gene resides at 27.9 min on the genetic map in the gene order cysB-topA-btuR-trp; it acts in a trans-dominant manner and appears to encode a repressor of btuB transcription.
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PMID:Separate regulatory systems for the repression of metE and btuB by vitamin B12 in Escherichia coli. 310 27

Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (lambda Flac), in which beta-galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring beta-galactosidase levels in E. coli strains lysogenized with the wild-type lambda Flac phage and mutant lambda Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B12 control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B12 is dependent on a functional metJ gene product.
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PMID:Salmonella typhimurium LT2 metF operator mutations. 314 73

We used a metE-lacZ fusion phage (lambda Elac) to select for mutants with operator-constitutive mutations in the Salmonella typhimurium metE control region. All of the mutations identified were found to lie within a region containing tandemly-repeating 8-bp palindromes with the consensus sequence 5'-AGACGTCT-3', previously proposed to be the binding region for the metJ-encoded repressor. Lysogens carrying mutant lambda Elac phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. Although repression of metE expression by vitamin B12 is not disrupted in metJ+ lysogens, vitamin B12 repression is disrupted in lysogens lacking an active MetJ repressor. These results suggest that there is an interaction between the metJ-encoded repressor and the vitamin B12 repression system mediated by the metH gene product.
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PMID:Salmonella typhimurium metE operator-constitutive mutations. 314 4

Colicins A, E1, E2, E3, E4, E5, E6, and E7 exhibited reduced activity against BtuB mutants of Escherichia coli K-12 and also against wild-type cells in the presence of vitamin B12. Plasmids encoding representatives of these colicins were specifically immune to high levels of the homologous colicin. Col(+) cells grown in media containing mitomycin C accumulated large amounts of colicin polypeptide. ColE2(+), ColE3(+), ColE4(+), ColE5(+), and ColE6(+) cultures also synthesized large amounts of second, lower-molecular-weight protein under these conditions. Colicins E2 through E7, but not A or E1, reacted with antiserum raised against purified colicin E3. Colicins E2 and E7 induced synthesis of beta-galactosidase encoded by lacZ under the control of the colicin Ib gene promotor on a derivative of Col plasmid ColIb.P9. This promotor is usually active only when the cells are treated with agents which damage DNA or block replication. Plasmids encoding various mutant forms of colicin E3 (M. Mock and M. Schwartz, J. Bacteriol. 142:384-390, 1980) recombined with ColE2, ColE4, ColE5, or ColE6 plasmids at a frequency of 10(-4) per cell to produce a colicin active against ColE2(+), E4(+), E5(+), or E6(+) cells. ColE5 and ColE6 plasmids recombined with ColE3 plasmids bearing mutations affecting colicin E3 receptor recognition, envelope penetration, and catalytic activities. ColE2 and ColE4 plasmids recombined only with ColE3 plasmids bearing mutations affecting receptor recognition and envelope penetration. Recombinants between mutant ColE3 plasmids and ColA, ColE1, or ColE7 plasmids were not detected. We propose the designation BtuB group for the colicins described here, and we divide the group into two classes comprising colicins A and E1, which act on the cytoplasmic membrane, and the related colicins E2 through E7, which have known or putative nuclease activities.
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PMID:The BtuB group col plasmids and homology between the colicins they encode. 628 Dec 33

Fusions of the lac genes to the promoters of four structural genes in the methionine biosynthetic pathway, metA, metB, metE, and metF, were obtained by the use of the Mu d(Ap lac) bacteriophage. The levels of beta-galactosidase in these strains could be derepressed by growth under methionine-limiting conditions. Furthermore, growth in the presence of vitamin B12 repressed the synthesis of beta-galactosidase in strains containing a fusion of lacZ to the metE promoter, phi(metE'-lacZ+). Mutations affecting the regulation of met-lac fusions were generated by the insertion of Tn5. Tn5 insertions were obtained at the known regulatory loci metJ and metK. Interestingly, a significant amount of methionine adenosyltransferase activity remained in the metK mutant despite the fact that the mutation was generated by an insertion. Several Tn5-induced regulatory mutations were isolated by screening for high-level beta-galactosidase expression in a phi(metE'-lacZ+) strain in the presence of vitamin B12. Tn5 insertions mapping at the btuB (B12 uptake), metH (B12 dependent tetrahydropteroylglutamate methyltransferase), and metF (5,10-methylenetetrahydrofolate reductase) loci were obtained. The isolation of the metH mutant was consistent with previous suggestions that the metH gene product is required for the repression of metE by vitamin B12. The metF::Tn5 insertion was of particular interest since it suggested that a functional metf gene product was also needed for repression of metE by vitamin B12.
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PMID:Mutations affecting regulation of methionine biosynthetic genes isolated by use of met-lac fusions. 628 5


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