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)

A previously constructed trp-S-lacZ fusion encoding a hybrid protein with beta-galactosidase activity was subcloned from a multicopy plasmid onto a lambda vector. Single-copy lysogens of lambda trpS-lacZ were used to determine whether trpS was regulated in a manner similar to that of other aminoacyl-tRNA synthetases. trpS regulation was found to resemble that of the majority of synthetases, in that expression of the lysogen-encoded hybrid beta-galactosidase varied with growth rate; beta-galactosidase activity increased 2.5-fold as the generation time decreased from 150 to 37 min. This regulatory response was confirmed by DNA/RNA hybridization experiments, which also suggested that this form of metabolic regulation occurred at the transcriptional level. No alteration in the level of hybrid beta-galactosidase was observed, however, when cells were starved for tryptophan.
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PMID:Regulation of tryptophanyl-tRNA synthetase formation. 704

We constructed a trpR-lacZ gene fusion that specifies a hybrid protein that has full beta-galactosidase activity. The gene fusion was associated with the unaltered trpR transcription and translation control region; thus, hybrid beta-galactosidase production was an indicator of expression of the trp aporepressor (trpR) operon. To facilitate in vivo expression studies, a DNA segment containing the trpR-lacZ gene fusion and the trpR controlling region was transferred to bacteriophage lambda and subsequently inserted into the bacterial chromosome. Analyses of hybrid beta-galactosidase production showed that the trpR operon is regulated autogenously but that the rate of synthesis of aporepressor varies only 4- to 5-fold in response to changes in the intracellular concentration of tryptophan. Under comparable conditions, the trp operon is regulated by trp repressor approximately 70-fold. Therefore, the operators of the trp operon and the trpR operon must have very different affinities for trp repressor in vivo. The promoter controlling trpR expression was found to be moderately active. Nevertheless, there are only about 50-300 molecules of trp aporepressor per cell. The low aporepressor level appears to be due to inefficient translation of trpR mRNA.
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PMID:Trp aporepressor production is controlled by autogenous regulation and inefficient translation. 704 1

The requirements for efficient translation termination are incompletely understood. Since the local context surrounding stop codons can influence the efficiency of translation termination, premature termination codons introduced by random mutation may not always terminate at the optimal efficiencies expected of naturally occurring stop codons. To investigate whether this could result in physiologically significant levels of read through, we examined the suppression of premature translation termination mutations within a sequence motif of the yeast Ste6 protein (Ste6p) that is highly conserved among members of the ATP-binding cassette (ABC) transporter family. The human cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in individuals with the disease cystic fibrosis, is also a member of this protein family. The mutations examined in Ste6p were chosen because a premature termination codon at the corresponding residue of CFTR has previously been reported to cause less severe pulmonary involvement than some missense mutations, suggesting that low level suppression of this stop codon could be occurring. Our results indicate that these premature stop codons in Ste6p can be suppressed at frequencies as high as 10%. Characterization of this phenomenon using a beta-galactosidase read through assay system showed that a limited sequence context surrounding this site contained information that was sufficient to cause suppression of translation termination. Amino acid sequence analysis of the full-length translation products produced by read through of an amber codon demonstrated that termination suppression was mediated by near-cognate tRNA mispairing that resulted in the insertion of tyrosine, lysine, or tryptophan.
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PMID:Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family. 751 33

GPD1 (encoding glyceraldehyde-3-phosphate dehydrogenase) is a constitutively expressed gene in Cochliobolus heterostrophus that produces a single transcript. The steady state level of GPD1 mRNA is 14-fold greater than that of the constitutively-expressed TRP1 gene (encoding a tryptophan biosynthesis enzyme) indicating that GPD1 has a stronger promoter and/or a more stable mRNA. A set of lacZ translational fusion vectors was constructed to compare the gene expression signals of GPD1, TRP1 and PRO1 (a C. heterostrophus genomic fragment selected for promoter activity) in C. heterostrophus as single copies at the same site in the chromosome. Under conditions that repressed endogenous beta-galactosidase expression, beta-galactosidase activity in transformants was constitutive and required the GPD1, TRP1 or PRO1 expression signals. In-frame GPD1::lacZ activities were 6-fold greater than in-frame TRP1::lacZ and PRO1::lacZ activities, indicating that GPD1 has more efficient expression signals.
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PMID:Relative strengths of promoters from Cochliobolus heterostrophus. 792 7

Two early and potential rate-limiting steps in the biosynthesis of isoquinoline alkaloids, such as morphine and codeine, in opium poppy (Papaver somniferum) involve decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-dopa) to yield tyramine and dopamine, respectively. A DNA fragment was amplified by polymerase chain reaction (PCR) using degenerate primers designed to two highly conserved domains found in other aromatic amino acid decarboxylases. A poppy seedling cDNA library was screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dopa decarboxylase (TYDC/DODC) was isolated. Two other independent cDNAs (cTYDC2 and cTYDC3) encoding TYDC/DODC were isolated by heterologous screening with a plant tryptophan decarboxylase (TDC) cDNA as probe. A poppy genomic library was screened with cTYDC1 and two intronless genomic clones (gTYDC1 and gTYDC4) were isolated. The deduced amino acid sequences of all poppy clones share extensive identity with other reported pyridoxal phosphate-dependent decarboxylases from both plants and animals. Based on sequence homology, members of the gene family were divided into two subsets (cTYDC1 and gTYDC4; cTYDC2 and cTYDC3) of proteins with predicted M(r) = 56,983 and 59,323, respectively. Within each subset the clones exhibit greater than 90% identity, whereas clones between subsets share less than 75% identity. Expression of gTYDC1 and cTYDC2 as beta-galactosidase fusion proteins in Escherichia coli resulted in catalytically active enzymes immunodetectable with TDC-specific polyclonal antibodies. Each enzyme showed marginally higher substrate specificity for L-dopa over L-tyrosine, but did not accept L-tryptophan and L-phenylalanine as substrates. Genomic DNA blot-hybridization analysis revealed 6 to 8 genes homologous to cTYDC1 and 4 to 6 genes homologous to cTYDC2 in the tetraploid poppy genome. A premature translation stop codon was found in the gTYDC4 clone suggesting that it may not encode a functional protein. RNA blot-hybridization with probes specific to the gTYDC1- or cTYDC2-like subsets showed that members of the TYDC gene family are differentially expressed in various plant tissues.
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PMID:Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy. 792 1

Tryptophan biosynthesis in Escherichia coli is regulated by the product of the trpR gene, the tryptophan (Trp) repressor. Trp aporepressor binds the corepressor, L-tryptophan, to form a holorepressor complex, which binds trp operator DNA tightly, and inhibits transcription of the tryptophan biosynthetic operon. The conservation of trp operator sequences among enteric Gram-negative bacteria suggests that trpR genes from other bacterial species can be cloned by complementation in E. coli. To clone trpR homologues, a deletion of the E. coli trpR gene, delta trpR504, was made on a plasmid by site-directed mutagenesis, then crossed onto the E. coli genome. Plasmid clones of the trpR genes of Enterobacter aerogenes and Enterobacter cloacae were isolated by complementation of the delta trpR504 allele, scored as the ability to repress beta-galactosidase synthesis from a prophage-borne trpE-lacZ gene fusion. The predicted amino acid sequences of four enteric TrpR proteins show differences, clustered on the backside of the folded repressor, opposite the DNA-binding helix-turn-helix substructures. These differences are predicted to have little effect on the interactions of the aporepressor with tryptophan, holorepressor with operator DNA, or tandemly bound holorepressor dimers with one another. Although there is some variation observed at the dimer interface, interactions predicted to stabilize the interface are conserved. The phylogenetic relationships revealed by the TrpR amino acid sequence alignment agree with the results of others.
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PMID:The tryptophan repressor sequence is highly conserved among the Enterobacteriaceae. 820 6

By expressing a mutant trpR gene in an Escherichia coli strain that is trpR and has beta-galactosidase activity fused to the trp promoter/operator, thus putting the beta-galactosidase activity under the control of the Trp repressor, we can determine quantitatively the relative repression activity of such mutant(s). We used this technique to analyse the biological consequences of substituting certain amino acid residues in only one of the two corepressor binding pockets. By combining two compatible plasmids in this strain, one expressing the mutant T44M and the other expressing only one substitution at a time at position 85, we analysed the repression activity of the resulting interactions in vivo. This approach allowed us to engineer active dimer repressors made of two inactive or partially active monomers. Amino acid substitutions at position 85 with a positive or with an indole ring (W) appeared to complement T44M, which amino acids with a negative charge did not. Only L substitution at position 85 appeared to restore activity among the hydrophobic amino acids tested. Similar to the wild-type repressor activity, the successful mutant-mutant interactions were L-tryptophan dependent. In vivo regulation by three known L-tryptophan analogues demonstrated the same trend of regulation among the wild-type repressors and the active mutant-mutant combinations.
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PMID:In vivo interaction between mutated tryptophan repressors of Escherichia coli. 860 40

Indolicidin is a cationic antimicrobial peptide isolated from bovine neutrophils. It consists of only 13 amino acids, has the highest tryptophan content of any known protein, and is amidated at the carboxyl terminus in nature. By circular dichroism spectroscopy a weak poly-L-proline II extended helix structure was observed that became substantially more pronounced upon interaction with liposomes. Indolicidin bound purified surface lipopolysaccharide with high affinity and permeabilized the outer membrane of Escherichia coli to the small hydrophobic molecule 1-N-phenylnapthylamine (Mr 200), results consistent with indolicidin crossing the outer membrane via the self-promoted uptake pathway. The methyl esterification of indolicidin's carboxyl terminus increased its activity for Gram-negative and Gram-positive bacteria. In Gram-negative bacteria this was associated with an increased binding to lipopolysaccharide and increased permeabilization of the outer membrane. The cytoplasmic membrane was the site of action of indolicidin as assayed in E. coli by the unmasking of cytoplasmic beta-galactosidase due to membrane permeabilization. The mechanism for this activity was shown to be the ability of the peptide to cause an increase in the transmembrane current of planar lipid bilayers. This current increase was activated by transmembrane potentials in excess of -70 to -80 mV. Consistent with this, there was a substantial decrease in indolicidin-mediated bacterial killing and permeabilization of the cytoplasmic membrane of E. coli that had been pretreated with the uncoupler carbonyl cyanide-m-chlorophenyl hydrazone. In planar bilayers, indolicidin induced the formation of discrete channels, which ranged in conductance from 0.05-0.15 nS. Thus despite the small size and unique composition of indolicidin, it was capable of killing Gram-negative bacteria by crossing the outer membrane and causing disruption of the cytoplasmic membrane by channel formation.
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PMID:Mode of action of the antimicrobial peptide indolicidin. 870 13

A new method for obtaining HIV-I protease was suggested. Fusion proteins composed of the N-terminal fragment of human gamma-interferon and HIV-I protease connected with (Asp)4Lys (protein I) or Asp-Pro (protein II) linkers were expressed in Escherichia coli cells. The fusion proteins were produced as insoluble inclusion bodies in the 20% yield of total cell protein. Protein I was cleaved by enterokinase. The solubility of protein I was increased by treating with Na-sulfite/Na-tetrathionate under denaturing conditions. Optimal conditions for efficient acidic hydrolysis of protein II at Asp-Pro bond were found. The hydrolysis products were separated by reversed-phase FPLC. The amount of tryptophan and cysteine residues in the enzyme obtained was estimated. The activity of HIV-I protease was determined using the chromogenic peptide. AlaArgVal NleNphGluAlaNleNH2 and a high-mol-wt substrate consisting of beta-galactosidase and a fragment of gag proteins, including p17-p24 processing site.
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PMID:HIV-I protease. Cloning, expression, and purification. 910 Mar 48

I. Benhar and H. Engelberg-Kulka reported that a 55 nucleotide translational bypass occurs in decoding a fusion of the Escherichia coli tryptophan repressor, trpR, and lacZ genes. The start of the bypass occurred in the trpR gene and coding resumed in the lacZ gene. It was considered that bypassing likely occurred in expression of trpR itself to produce an additional 10 kDa product which may be biologically important. We report here that bypass is undetectable in the same and related trpR'-lacZ' fusions. The beta-galactosidase activity derived from the fusions is accounted for by unusual internal initiation and +1 frameshifting, both of which occur in the lacZ part of the fusion. The 10 kDa product reportedly encoded by the trpR gene was not detectable to a level of 1% of the full-length 12 kDa tryptophan repressor product, at least when expressed from a T7 promoter.
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PMID:Reported translational bypass in a trpR'-lacZ' fusion is accounted for by unusual initiation and +1 frameshifting. 928 21


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