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 constructed a synthetic oligonucleotide corresponding to the previously proposed consensus binding site for the Fur protein, a central iron-regulatory protein of Escherichia coli. When this oligonucleotide was introduced at the start of transcription of an operon fusion between the ompF promoter and the lacZ structural gene, beta-galactosidase activity became iron regulated. This consensus sequence is sufficient to function as an operator site for the binding of Fur protein in vivo.
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PMID:Confirmation of the Fur operator site by insertion of a synthetic oligonucleotide into an operon fusion plasmid. 282 7

Clones carrying the iron uptake region of the Vibrio anguillarum plasmid pJM1 were subjected to insertion mutagenesis, using transposon Tn3::HoHo1 which carries a promoterless lacZ gene and can thus generate lacZ transcriptional fusions if inserted downstream from an indigenous promoter. Four classes of insertion mutants were obtained based on the level of expression of components of the iron uptake system, and six genetic units were defined according to the phenotype of the mutants. Five of the six genetic units were crucial for biosynthesis of the siderophore anguibactin. Insertions in the remaining genetic unit led to an iron uptake-deficient phenotype and showed either reduced levels of the outer membrane protein OM2 as well as anguibactin activity or a complet shutoff of both OM2 and anguibactin biosyntheses. Analysis of beta-galactosidase production by cells carrying the lacZ fusion derivatives identified iron-regulated and constitutive transcriptional units as well as their orientation in the genetic units. Molecular cloning of pJM1 plasmid DNA noncontiguous to the iron uptake region also identified genetic determinants for a trans-acting factor required for full expression of anguibactin activity. Evidence obtained from bioassays, spectrophotometric measurements, and the lacZ fusion mutants suggested that the trans-acting factor is a novel activator of siderophore biosynthesis at the transcriptional level.
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PMID:Genetic analysis of the iron uptake region of the Vibrio anguillarum plasmid pJM1: molecular cloning of genetic determinants encoding a novel trans activator of siderophore biosynthesis. 283 88

Two clones containing inserts in pBR322 that together include the entire 1074-base open reading frame coding for the 358 amino acids of rat liver stearyl-CoA desaturase have been used to construct expression vectors for residues 3-358 and 27-358 fused to the first 6 residues of beta-galactosidase and several amino acids of the multiple cloning site of pUC8. Growth of transformed Escherichia coli under conditions for suppression of the lac promoter, followed by subsequent induction of these cultures results in the synthesis of higher levels of desaturase proteins than those found in induced rat liver. The proteins are almost completely associated with the membrane fraction of cell homogenates. Posttranslational iron insertion into the apoproteins, either in vitro with membrane preparations or by iron addition during induction, results in the formation of active holoenzyme which can be reconstituted with NADH cytochrome b5 reductase and cytochrome b5 to form an active stearyl-CoA desaturase system. The deletion of the first 26 amino-terminal amino acid residues does not affect either enzyme activity or membrane binding. Therefore, the unusual sequence of 11 residues containing 10 amino acids with hydroxyl groups plays no apparent significant role in either protein insertion into membranes or iron chelation. Since the protein product for residues 3-358 is processed even further to delete the initial 33 amino-terminal residues, the limiting polypeptide primary structure required for an active membrane-bound catalyst is even smaller than this initial deletion mutation indicates.
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PMID:Bacterial synthesis of active rat stearyl-CoA desaturase lacking the 26-residue amino-terminal amino acid sequence. 289 38

A lambda gt11 expression library containing cDNA inserts prepared from porcine endometrial mRNA was immunologically screened by using an antiserum developed against porcine uteroferrin (Uf), a glycoprotein that has been strongly implicated in transplacental iron transport in the pregnant pig. Antibody reactive clones (lambda 4a3, 13.1, and 2.2) were isolated after screening 1.5 x 10(5) recombinant phages. Clones 4a3 and 13.1 expressed Uf antigenic determinants in beta-galactosidase fusion proteins and specifically selected antibody which reacted with Uf in immunoblots prepared from uterine cytosolic extracts. In addition, all three cDNA clones collectively contained DNA sequences that encoded an 85-amino acid peptide which corresponded to a region within the carboxyterminal portion of the Uf protein. Northern blot hybridization of these cDNAs to RNAs extracted from whole uterine tissue of pregnant pigs revealed a single uterine poly(A)+ RNA of approximately 1.7 kilobases in length, which was not found in liver and mammary tissue RNAs. The concentration of the Uf mRNA changed in a temporal fashion during pregnancy in a manner that was distinct from that of the progesterone receptor mRNAs. Highest levels of Uf mRNA were found at mid and late pregnancy (days 45-110) and were about 50-fold greater than at day 30 of pregnancy. By contrast, RIA analysis of the uterine tissue extracts showed that maximum amounts of Uf were present at day 60 and then declined sharply. Thus the pattern of Uf mRNA present in the uterus did not parallel the amount of Uf polypeptide that could be recovered from the tissue. The tissue specific and temporal regulation of Uf gene expression emphasizes that the protein plays an important role in uterine activity and/or fetal development.
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PMID:Molecular cloning and temporal expression during pregnancy of the messenger ribonucleic acid encoding uteroferrin, a progesterone-induced uterine secretory protein. 296 54

The Escherichia coli entF gene, which encodes the serine-activating enzyme involved in enterobactin synthesis, has been localized to a 4.7-kilobase-pair DNA fragment inserted in the vector pBR328. This recombinant molecule, pITS32, restored the ability of an entF mutant to grow on low-iron medium and to produce enterobactin. Examination of its translation products by minicell and electrophoretic analyses revealed a protein of approximately 160,000 daltons, which we identified as the EntF protein. A small DNA segment from pITS32 containing the translational start site for entF allowed the low constitutive expression of beta-galactosidase when cloned (pITS301) upstream of the lacZ structural gene in the vector pMC1403. In contrast, a clone (pITS312) containing the identical entF-lacZ fusion and a larger region upstream of entF including the entire fes gene and extending into the fepA gene (whose transcription is in the opposite direction relative to entF) expressed beta-galactosidase in high yet inducible amounts in response to fluctuations in the metabolic iron concentration. Transposon insertion mutations in the fes gene but not an insertion near the 5' region of fepA in pITS312 reduced this high inducible expression to the low constitutive level seen for pITS301. These observations are most readily explained by the presence of a regulatory region located upstream of fes which mediates the iron-regulated expression of a transcript that includes the fes and entF genes.
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PMID:Molecular characterization of the Escherichia coli enterobactin cistron entF and coupled expression of entF and the fes gene. 304 Jun 79

The fusion-generating phage lambda plac Mu1 was used to produce fusions of lacZ to fhuA, the gene encoding the ferrichrome-iron receptor (FhuA protein) in the outer membrane of Escherichia coli K-12. Fusions to the fhuA gene in a delta (lac) strain were selected by their resistance to bacteriophage phi 80 vir. Ten independent (fhuA'-'lacZ) fusions were all Lac+ and were resistant to the lethal agents which require the FhuA protein as receptor, i.e., phi 80 vir, T5, T1, UC-1, and colicin M; none could utilize ferrichrome as the sole iron source. Specialized transducing phages were obtained by illegitimate excision from the chromosome of each of the fusion-bearing strains, and EcoRI fragments which encoded the fusions were subcloned into the high-copy plasmid pMLB524. Physical mapping of the fusion-containing plasmids confirmed the presence of three restriction sites which were also located on the chromosomal DNA of sequences near the fhuA gene. The direction of transcription of the fhuA gene was deduced from the direction of transcription of the (fhuA'-'lacZ) gene fusion. Identification of the chimeric proteins was made by both radiolabeling cells and immunoprecipitating the LacZ-containing proteins with antibody to beta-galactosidase and by preparing whole cell extracts from Lac+ cells containing the cloned gene fusions. Two sizes of (FhuA'-'LacZ) proteins were detected, 121 kDa and 124 kDa. The DNA sequences at the unique fusion joints were determined. The sequence information allowed us to identify three distinct fusion joints which were grouped as follows, type I fusions, 5'-ACT GCT CAG CCA A-3'; type IIa fusions, 5'-GCG GTT GAA CCG A-3'; and type IIb fusions: 5'-ACC GCT GCA CCT G-3'. To orient these fhuA fusion joints, the complete nucleotide sequence of the fhuA gene was determined from a 2,902-base-pair fragment of DNA. A single open reading frame was found which translated into a 747-amino acid polypeptide. The signal sequence of 33 amino acids was followed by a mature protein with a molecular weight of 78,992. Alignment of the amino acid sequence of the FhuA protein with the amino acid sequences presented for two other tonB-dependent receptor proteins in the outer membrane of E. coli showed an area of local homology at the amino terminus of all three proteins.
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PMID:Protein fusions of beta-galactosidase to the ferrichrome-iron receptor of Escherichia coli K-12. 307 47

Iron is known to depress Shiga toxin production by Shigella dysenteriae 1, and temperature has been shown to regulate several genes required for Shigella invasiveness. In this study, the influence of iron and temperature on regulation of a highly related toxin, Shiga-like toxin I (SLT-I) of enterohemorrhagic Escherichia coli, was examined in strains lysogenic for the toxin-converting coliphage 933J and in strains carrying the cloned slt-I genes on a high-copy-number plasmid vector. For comparison, S. dysenteriae 1 was included in these studies. As expected, iron suppressed Shiga toxin synthesis, and reduced growth temperature was also found to decrease Shiga toxin production. Iron also suppressed SLT-I synthesis in E. coli lysogenized with phage 933J but did not demonstrably repress toxin synthesis in E. coli strains carrying the cloned slt-I genes. Temperature had no effect on SLT-I synthesis. Mini-Mu lac operon fusions were then isolated in the cloned slt-I genes and used to test for regulation of beta-galactosidase by iron. Iron did not decrease beta-galactosidase production in strains that harbored these operon fusion plasmids. Taken together, these results indicate that iron but not temperature represses SLT-I synthesis when the slt-I genes are phage associated but this suppression is not easily demonstrated when the slt-I genes are cloned on a high-copy-number plasmid.
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PMID:Effects of iron and temperature on Shiga-like toxin I production by Escherichia coli. 312 8

Protein and operon fusions between the manganese superoxide dismutase (MnSOD) gene, sodA, and genes of the lactose operon were constructed in an attempt to explore the effects of various factors on MnSOD expression and the level at which they operate. In sodA-lacZ protein fusions, induction of beta-galactosidase perfectly mimicked MnSOD induction (i.e., beta-galactosidase was not expressed in anaerobiosis and was induced by oxygen, redox-cycling compounds in aerobiosis, and iron chelators in anaerobiosis). In tac-sodA operon fusions, MnSOD induction was monitored only by the lactose operon inducer isopropyl-beta-D-thiogalactopyranoside. Various plasmids carrying part or all of the sodA regulatory and structural region inhibited aerobic beta-galactosidase induction in sodA-lacZ fusions. This included plasmids carrying only the transcription start and upstream region and also plasmids which did not contain this region and in which MnSOD was under foreign transcriptional control. The role of metal ions was also investigated. Addition of Mn(II) enhanced MnSOD activity but did not affect induction. The anaerobic expression of MnSOD from the oxygen-insensitive tac promoter was enhanced threefold by iron-chelating agents, implying a posttranscriptional or most likely a posttranslational modulation of enzyme activity via metal ions. To accommodate all these data, multiregulation of MnSOD is proposed.
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PMID:Transcriptional and posttranscriptional regulation of manganese superoxide dismutase biosynthesis in Escherichia coli, studied with operon and protein fusions. 313 2

A selection procedure using Mn2+ is described. A high percentage of the Mn2+ resistant mutants had constitutive iron transport systems. By P1 transduction, and complementation with the cloned fur gene it could be shown that nearly all the mutants constitutive in the expression of the operon fusion fiu::lambda placMu were only defective in fur. High concentrations of manganese inhibited the derepression of an iron-regulated lac operon fusion. In another iron-regulated lac operon fusion that was inducible by iron, manganese also induced the production of beta-galactosidase. Most of the fur mutants isolated (80%) were not able to grow on succinate, fumarate or acetate. After transformation with a fur+ plasmid all 39 mutants tested were able to grow on succinate. In fur mutants the presence of succinate in the growth medium reduced succinate uptake rates by 50%-70%. Succinate dehydrogenase activity was reduced to 10% of that of the parent strain.
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PMID:Selection procedure for deregulated iron transport mutants (fur) in Escherichia coli K 12: fur not only affects iron metabolism. 332 34

The expression of the diphtheria tox228 gene encoding the nontoxic, serologically related CRM228 mutant diphtheria toxin has been analyzed in Corynebacterium diphtheriae and Escherichia coli. The diphtheria toxin promoter has been used to direct the expression of beta-galactosidase in E.coli, and the efficiency of promotion has been compared to that obtained with the lac promoter. Expression in C.diphtheriae is known to be dependent on the absence of iron, and we present for the first time direct evidence that this regulation occurs at the level of transcription. The 5' end of toxin mRNA maps at the same position in C.diphtheriae and E.coli, suggesting identical sequences to be recognized by C.diphtheriae and E.coli RNA polymerase. The diphtheria toxin promoter carries at position -34 a TTGATT sequence closely related to the E.coli -35 consensus sequence and in the -14 to -8 region a set of overlapping sequences with complete or partial homology to the E.coli -10 consensus sequence.
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PMID:Diphtheria toxin promoter function in Corynebacterium diphtheriae and Escherichia coli. 392 42


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