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 oxygen-dependent promoter of the Vitreoscilla hemoglobin (VHb) gene has been shown to be functional in E. coli. Earlier studies established that the promoter is maximally induced under microaerobic conditions and that its activity is also influenced by the cAMP-CAP complex. We demonstrate here that the promoter can be used for regulated, high-level expression of recombinant proteins in two-stage fed-batch fermentations. The promoter is maximally induced at dissolved oxygen levels lower than 5% air saturation. Despite the influence of catabolite repression, glucose and glycerol-containing media give comparable product levels under carbon-limited conditions such as those encountered in typical fed-batch fermentations. The possibility of a third level of control of promoter activity is also indicated. This mode of induction can be repressed by addition of a complex nitrogen source such as yeast extract to the medium. The observed promoter activity can be modulated at least 30-fold over the course of high-cell density fermentations producing either cloned beta-galactosidase or cloned chloramphenicol acetyltransferase (CAT). Densitometer scanning of SDS-polyacrylamide gels revealed that beta-galactosidase was expressed to a level of approximately 10% of total cellular protein.
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PMID:Expression of recombinant proteins in Escherichia coli using an oxygen-responsive promoter. 136 36

The Escherichia coli lac promoter mutation Pr115, an A X T to T X A transversion at +1 (the transcription initiation site of the lac wild-type and lac UV5 promoters), creates a new "-10 region"-like sequence starting at +1. We show that this mutation activates a new RNA polymerase binding site (P115) that overlaps with, and is shifted 12 base-pairs downstream from, the wild-type RNA polymerase binding site (P1). Nuclease S1 mapping studies and RNA polymerase protection experiments in vitro indicate that, in the absence of CAP-cAMP, this new site is used preferentially over the P1 site. In vivo, beta-galactosidase assays of the Pr115 mutation in combination with mutations of the P1 "-35 region" demonstrate that the P1 -35 region sequences are not involved in the interaction between RNA polymerase and P115 in the absence of CAP-cAMP; therefore P115 is an independent binding site. The presence of CAP-cAMP in vivo stimulates polymerase binding and initiation at P1, which serves to block polymerase from binding at P115.
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PMID:Lactose promoter mutation Pr115 activates an overlapping promoter within the lactose control region. 241 53

We have isolated a new class of mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that alter the transcription initiation properties of RNA polymerase holoenzyme. The rpoD(Lac) mutations increase expression of the lac operon in the absence of CAP-cAMP, allowing a strain lacking adenyl cyclase to grow on lactose. Four of the six alleles isolated have three- to fivefold increases in the amount of lac mRNA and beta-galactosidase per cell. We show that these four mutations increase transcription initiation from the same promoter used by wild-type RNA polymerase. The mutations were mapped and sequenced. One mutation occurs in the codon for amino acid 389 of the sigma 70 polypeptide. The remaining five mutations are clustered, affecting residues 570, 571 and 575. These five mutations are within or near a proposed helix-turn-helix motif in the C terminus of sigma 70.
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PMID:Mutations in rpoD, the gene encoding the sigma 70 subunit of Escherichia coli RNA polymerase, that increase expression of the lac operon in the absence of CAP-cAMP. 284 53

Uninduced malPQ transcription, as followed by measuring beta-galactosidase expression in a strain carrying a malP-lacZ hybrid gene and grown in the absence of maltose, requires the presence of CAP. However this requirement is lost when the expression of malT, positive regulator gene of the maltose regulon, is rendered independent of CAP by a mutation in the malT promoter. This result suggests that the effect of CAP on uninduced malPQ expression is mediated through a modulation of MalT protein synthesis. The effect of CAP on the induced expression of malPQ is presumably mediated, in addition, through a modulation of the synthesis of the maltose transport system and, hence, of the entry of the inducer. Therefore the effect of CAP on malPQ expression seems to be merely indirect, and this is surprising since a CAP binding site is present at the malPQ promoter.
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PMID:Indirect effects of the 3'-5' cyclic adenosine monophosphate binding protein (CAP) on the transcription of the malPQ operon in Escherichia coli. 298 27

The Escherichia coli lac promoter has been shown to contain an RNA polymerase binding site (P2) that overlaps with, and is shifted 22 base-pairs upstream from the normal lac promoter (P1). In this paper, we provide RNA polymerase protection data obtained in vitro that show that, in the absence of CAP-cAMP, in vitro P2 is the preferred polymerase binding site on the P+ template. In the presence of CAP-cAMP, polymerase binding to P2 is reduced and more polymerase is bound at P1. Two lac P1 "-35 region" mutations, L157 and 4, which increase the homology between this region and the consensus "-10 region" sequence, are both shown to have an increased affinity for polymerase binding at P2. CAP-cAMP is also able to decrease the amount of polymerase bound to P2 and to increase the amount bound to P1 on these mutant promoter fragments. P2 does not initiate transcription efficiently in vivo. Nuclease S1 mapping experiments detect only a low level of transcription from one of the P2 "up" mutations, but no beta-galactosidase synthesis is directed by this mutant. Mutations such as L157 and 4, which alter the P2-10 region, also alter lac P sensitivity to CAP-cAMP in vivo, suggesting that the P2 sequence plays a role in CAP-cAMP regulation of lac P. Possible roles for P2 in vivo are discussed.
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PMID:Properties of lac P2 in vivo and in vitro. An overlapping RNA polymerase binding site within the lactose promoter. 299 53

Kline et al. (1980) have reported that indole-3-acetic acid (IAA) and four other indole derivatives are able to substitute for cAMP in activating expression of the ara regulon of E. coli. We have examined this phenomenon in detail, utilizing fusions between the structural gene for beta-galactosidase and the promoters for the araBAD, araE, and araFG operons. We confirm that IAA potently stimulates transcription from the araBAD promoter. The effect is highly specific to araBAD, as IAA has no, or only slight, effects on the araE and araFG operons. However, contrary to the results of Kline et al., we find that the action of IAA does not require CAP. Thus, IAA fully stimulates the transcription of araBAD in a strain which bears a complete deletion of the crp gene.
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PMID:The catabolite gene activator protein (CAP) is not required for indole-3-acetic acid to activate transcription of the araBAD operon of Escherichia coli K-12. 299 82

The polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus was analysed with respect to which sequences are required upstream of the mRNA transcription initiation (CAP) site for efficient promoter activity. Insertions (8, 95 and 785 nucleotides) were made in this region at an EcoR V site between the CAAT- and TATA-like boxes. When these mutations were introduced into the virus they did not affect the activity of the polyhedrin promoter as judged by expression of the beta-galactosidase (lacZ) gene inserted in lieu of the polyhedrin coding sequences. Deletions were made in the promoter which progressively removed sequences upstream from the CAP site. Removal of the TATA motif did not affect lacZ gene expression. A sequence 69 nucleotides upstream to the normal position of the polyhedrin ATG translation initiation codon was sufficient for maximum promoter activity but this was reduced by 90% when only 56 nucleotides upstream remained. The normal CAP site was utilized by each deletion mutant. Promoter activity was undetectable when the CAP site was deleted. The results are discussed in relation to other eukaryotic promoters.
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PMID:Analysis of the polyhedrin gene promoter of the Autographa californica nuclear polyhedrosis virus. 332 Sep 64

S1 nuclease was used to generate a series of deletions which extend into the CAP-cAMP binding site from upstream of the Escherichia coli lactose operon promoter (lacP). Deletion and insertion mutations were also created which changed the spacing region between the CAP-cAMP binding site and the lacP -35 region. The promoter activities of these mutations were compared by measuring the levels of beta-galactosidase gene expression in vivo. The results show that sequence information prior to 74 base pairs (-74) upstream from the transcription start site (designated as +1) is not necessary for the full activation of the lac promoter by the CAP-cAMP complex. However, the deletion which extends to the -71 position retains only one third of the promoter activity in the presence of the CAP-cAMP complex. Removal of one symmetrical element from the two fold symmetry in the CAP-cAMP binding site abolished the CAP-cAMP stimulation of the lac promoter. Spacer mutations which increase by one base pair or decrease by two base pairs the length of the spacing region between the CAP-cAMP binding site and the lacP -35 region drastically reduced the CAP-cAMP stimulation of the lac promoter. This suggests that the distance between the lac promoter transcription start site and CAP-cAMP binding site is crucial for the function of the lac promoter, despite the fact that this distance varies in other E. coli promoters positively regulated by CAP-cAMP. A deletion which extends to the -59 position results in a two fold enhanced expression of lac in the absence of CAP-cAMP. This is consistent with the existance of a competitive RNA polymerase binding site in this region which would normally act to inhibit RNA polymerase binding.
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PMID:Deletion analysis of the CAP-cAMP binding site of the Escherichia coli lactose promoter. 608 87

The synthesis of exotoxin A (ETA) by Pseudomonas aeruginosa is a complex, regulated event. Several ETA putative regulatory mutants of P. aeruginosa PA103 have previously been characterized (S. E. H. West, S. A. Kaye, A. N. Hamood, and B. H. Iglewski, Infect. Immun. 62:897-903, 1994). In addition to ETA production, these mutants, PA103-15, PA103-16, and PA103-19, were also deficient in the production of protease and in regA P1 promoter activity. RegA is a positive regulator of ETA transcription. We cloned a gene, designated vfr for virulence factor regulator, that restored ETA and protease production to parental levels in these mutants. In addition, transcription from the regA P1 promoter was restored. In Escherichia coli, when vfr was overexpressed from a phage T7 promoter, a protein with an apparent molecular mass of 28.5 kDa was produced. Analysis of the deduced amino acid sequence of vfr revealed that the expected protein is 67% identical and 91% similar over a 202-amino-acid overlap to the E. coli cyclic AMP receptor protein (CAP or Crp). The cloned vfr gene complemented the beta-galactosidase- and tryptophanase-deficient phenotypes of E. coli RZ1331, a crp deletion mutant. However, the E. coli crp gene under the control of the tac promoter did not complement the ETA-deficient or protease-deficient phenotype of PA103-15 or PA103-16. The ability of vfr to restore both ETA and protease production to these mutants suggests that vfr is a global regulator of virulence factor expression in P. aeruginosa.
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PMID:The vfr gene product, required for Pseudomonas aeruginosa exotoxin A and protease production, belongs to the cyclic AMP receptor protein family. 800 77

Current clinical gene therapy protocols for the treatment of human immunodeficiency virus type 1 (HIV-1) infection often involve the ex vivo transduction and expansion of CD4+ T cells derived from HIV-positive patients at a late stage in their disease (CD4 count <400). These protocols involve the transduction of T cells by murine leukemia virus (MLV)-based vectors encoding antiviral constructs such as the rev m10 dominant negative mutant or a ribozyme directed against the CAP site of HIV-1 RNA. We examined the efficiency and stability of transduction of CD4+ T cells derived from HIV-infected patients at different stages in the progression of their disease, from seroconversion to AIDS. CD4+ T cells from HIV-positive patients and uninfected donors were transduced with MLV-based vectors encoding beta-galactosidase and an intracellular antibody directed against gp120 (sFv 105) or Tat. (sFvtat1-Ckappa). The expression of marker genes and the effects of the antiviral constructs were monitored in vitro in unselected transduced CD4+ T cells. Efficiency and stability of transduction varied during the course of HIV infection; CD4+ T cells derived from asymptomatic patients were transducible at higher efficiencies and stabilities than CD4+ T cells from patients with acquired immunodeficiency syndrome (AIDS). Expression of the anti-tat intracellular antibody was more effective at stably inhibiting HIV-1 replication in transduced cells from HIV-infected individuals than was sFv 105. The results of this study have important implications for the development of a clinically relevant gene therapy for the treatment of HIV-1 infection.
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PMID:Inhibition of human immunodeficiency virus replication and growth advantage of CD4+ T cells from HIV-infected individuals that express intracellular antibodies against HIV-1 gp120 or Tat. 952 10


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