Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

To begin to assess the independent structural and functional characteristics of the mitochondrially encoded subunits of mammalian cytochrome c oxidase, we have converted the cloned mitochondrial gene for rat subunit II (coxII) into its universal codon equivalent (ucoxII) by oligonucleotide-directed, site-specific mutagenesis. This involved synthesizing 12 oligodeoxynucleotides to achieve the 13 ATA to ATG and the 5 TGA to TGG changes needed. To express ucoxII in Escherichia coli, we used a number of different expression vectors in which the promoters and ribosome-binding sequences of the messenger RNA were varied. While ucoxII alone was expressed at a low level, a striking increase in the level of expression resulted when the ucoxII gene was fused to other E. coli genes. The COXII peptide was identified by proteolytic digestion, partial sequencing, and reaction with specific antisera. A cro-beta-galactosidase-COXII fusion protein has been purified, characterized, and used to produce polyclonal antibodies to the COXII peptide. The ucoxII gene was also expressed in a cell-free translation system and in Xenopus oocytes, yielding a nondenatured, membrane-associated peptide with the same apparent molecular weight as authentic subunit II. In oocytes and in a reticulocyte lysate in vitro system supplemented with microsomal membranes, the protein is glycosylated and coisolates with the washed membrane fraction. In both cases, the COXII peptide is soluble under mild conditions in a nonionic detergent and is precipitable by antibodies to subunit II. The production of subunit II in the in vitro translation system is stimulated as strongly by addition of soybean phospholipid vesicles as by microsomal membranes, providing further evidence of membrane insertion and stabilization.(ABSTRACT TRUNCATED AT 250 WORDS)
 ...
PMID:Conversion of a mitochondrial gene for mammalian cytochrome c oxidase subunit II into its universal codon equivalent and expression in vivo and in vitro. 184 93

The fdhF gene encoding the 80-kDa selenopolypeptide subunit of formate dehydrogenase H from Escherichia coli contains an in-frame TGA codon at amino acid position 140, which encodes selenocysteine. We have analyzed how this UGA "sense codon" is discriminated from a UGA codon signaling polypeptide chain termination. Deletions were introduced from the 3' side into the fdhF gene and the truncated 5' segments were fused in-frame to the lacZ reporter gene. Efficient read-through of the UGA codon, as measured by beta-galactosidase activity and incorporation of selenium, was dependent on the presence of at least 40 bases of fdhF mRNA downstream of the UGA codon. There was excellent correlation between the results of the deletion studies and the existence of a putative stem-loop structure lying immediately downstream of the UGA in that deletions extending into the helix drastically reduced UGA translation. Similar secondary structures can be formed in the mRNAs coding for other selenoproteins. Selenocysteine insertion cartridges were synthesized that contained this hairpin structure and variable portions of the fdhF gene upstream of the UGA codon and inserted into the lacZ gene. Expression studies showed that upstream sequences were not required for selenocysteine insertion but that they may be involved in modulating the efficiency of read-through. Translation of the UGA codon was found to occur with high fidelity since it was refractory to ribosomal mutations affecting proofreading and to suppression by the sup-9 gene product.
 ...
PMID:Features of the formate dehydrogenase mRNA necessary for decoding of the UGA codon as selenocysteine. 214 Nov 70

A method is described for genetic detection of mismatch repair products following transformation of Saccharomyces cerevisiae. The method is based on the detection of beta-galactosidase activity in clonal derivatives of cells transformed with heteroduplex plasmid DNA. Heteroduplex plasmid substrates were constructed by insertion of an oligonucleotide heteroduplex into the coding sequence of the Escherichia coli lacZ gene. The plasmid and oligonucleotides were designed so that one strand of the construct would code for a functional beta-galactosidase and the other strand would contain an in-frame nonsense codon. The frequencies of transformed clones containing only Lac+ cells, only Lac- cells, or a mixture of the two Lac phenotypes provided information on the efficiency of the repair reaction. With this method, plasmids carrying single-base substitution mismatches, a single-base frameshift mismatch (T/delta), or a 3-base-pair substitution mismatch (TGA/GAA) were tested. A/C, G/T, G/A, G/G, and T/delta mismatches were repaired with significantly greater efficiencies than C/C, A/A, T/T, and TGA/GAA. T/C was repaired with an intermediate efficiency. The frequencies of products obtained with G/G, G/A, and T/delta mismatches suggested modest inequality of repair in the two possible directions. Strains carrying the repair-deficient pms1-1 mutation were also tested. The efficiencies of repair of A/C, G/T, G/G, and A/A mismatches were reduced in pms1-1 cells compared with wild-type cells. In addition, a change in repair inequality was detected when transformation of the two strains with an A/C mismatch was compared.
 ...
PMID:Specificity of mismatch repair following transformation of Saccharomyces cerevisiae with heteroduplex plasmid DNA. 249 74

pBR322 contains the amp gene encoding beta-lactamase. When Escherichia coli carrying this plasmid is exposed to heat shock, beta-lactamase synthesis is repressed transiently at the translational level. To identify the DNA element responsible for this translational repression, DNA segments containing the translation start region of the amp gene were excised from pAT153 and fused in frame with the lacZ reading frame in the open reading frame vector pORF1. These constructs were introduced into E. coli, and the effect of heat shock of the cells on the synthesis of beta-galactosidase starting from the amp start codon was examined. As is the case for pBR322-encoded synthesis of beta-lactamase, the synthesis of beta-galactosidase encoded by the fused genes also ceased transiently upon heat shock. It is concluded that the heat shock-induced repression of the amp gene occurs at the initiation step of translation. As far as the present study is concerned, the minimum DNA segment responsible for the repression is AT TGA AAA AGG AAG AGT ATG AG, which includes the Shine-Dalgarno sequence (AAGGA) and the initiation codon (ATG).
 ...
PMID:The translation start signal region of TEM beta-lactamase mRNA is responsible for heat shock-induced repression of amp gene expression in Escherichia coli. 250 25

A cDNA library was prepared in lambda gt11 bacteriophage from poly(A)+ RNA isolated from primary cultures of endothelial cells from human umbilical vein. Approximately 2.5 million independent recombinants were screened and 2 of those were found to synthesize a fusion protein with beta-galactosidase that reacted with rabbit antibody against human von Willebrand factor. Comparison of the amino acid sequence translated from the cDNA insert of the two clones with the amino acid sequence determined by Edman degradation of the protein established that both phage isolates code for von Willebrand factor. The first clone (lambda HvWF1) contained an insert of 404 nucleotides that corresponded to amino acid residues 1-110 in the mature protein circulating in blood, in addition to a portion (24 amino acids) of a prepro leader sequence. The second cDNA clone (lambda HvWF3) contained an insert of 4.9 kilobases that coded for the carboxyl-terminal 1525 amino acids of von Willebrand factor, a stop codon of TGA, 134 nucleotides of 3' noncoding sequence, and a poly(A) tail of 150 nucleotides. The two clones together code for greater than 80% of the molecule circulating in blood. The same carboxyl-terminal lysine residue was identified in the mature protein as well as in the cDNA, indicating that all of the proteolytic processing that occurs during the biosynthesis and assembly of von Willebrand factor is associated with the amino-terminal portion of the precursor protein. The amino acid sequence of von Willebrand factor indicates the presence of two different internal gene duplications and one triplication. These repetitive amino acid sequences account for about one-half of the amino acids present in the mature protein. The tetrapeptide sequence of Arg-Gly-Asp-Ser, which mediates the cell attachment and platelet binding activity of fibronectin, was also identified in the carboxyl-terminal portion of von Willebrand factor.
 ...
PMID:Cloning and characterization of two cDNAs coding for human von Willebrand factor. 286 88

An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.
 ...
PMID:Efficiencies of translation in three reading frames of unusual non-ORF sequences isolated from phage display. 1069 76

Expression of Proteus mirabilis urease is governed by UreR, an AraC-like positive transcriptional activator. A poly(A) tract nucleotide sequence, consisting of A(6)TA(2)CA(2)TGGTA(5)GA(6)TGA(5), is located 16 bp upstream of the sigma(70)-like ureR promoter P2. Since poly(A) tracts of DNA serve as binding sites for the gene repressor histone-like nucleoid structuring protein (H-NS), we measured beta-galactosidase activity of wild-type Escherichia coli MC4100 (H-NS(+)) and its isogenic derivative ATM121 (hns::Tn10) (H-NS(-)) harboring a ureR-lacZ operon fusion plasmid (pLC9801). beta-Galactosidase activity in the H-NS(-) host strain was constitutive and sevenfold greater (P < 0.0001) than that in the H-NS(+) host. A recombinant plasmid containing cloned P. mirabilis hns was able to complement and restore repression of the ureR promoter in the H-NS(-) host when provided in trans. Deletion of the poly(A) tract nucleotide sequence from pLC9801 resulted in an increase in beta-galactosidase activity in the H-NS(+) host to nearly the same levels as that observed for wild-type pLC9801 harbored by the H-NS(-) host. Urease activity in strains harboring the recombinant plasmid pMID1010 (encoding the entire urease gene cluster of P. mirabilis) was equivalent in both the H-NS(-) background and the H-NS(+) background in the presence of urea but was eightfold greater (P = 0.0001) in the H-NS(-) background in the absence of urea. We conclude that H-NS represses ureR expression in the absence of urea induction.
 ...
PMID:H-NS is a repressor of the Proteus mirabilis urease transcriptional activator gene ureR. 1076 73

The metanephric kidney is a mesodermal organ that develops as a result of reciprocal interactions between the ureteric bud and the blastema. The generation of embryonic stem (ES) cell-derived progenitors offers potential for regenerative therapies but is often limited by development of tumor formation. Because brachyury (T) denotes mesoderm specification, a mouse ES cell line with green fluorescence protein (GFP) knocked into the functional T locus as well as lacZ in the ROSA26 locus (LacZ/T/GFP) was used in cell selection and lineage tracing. In the absence of leukemia inhibitory factor, mouse ES cells give rise to embryoid bodies that can differentiate into mesoderm. Culture conditions were optimized (4 d, 10 ng/ml Activin-A) to generate maximal numbers of renal progenitor populations identified by expression of the specific combination of renal markers cadherin-11, WT-1, Pax-2, and Wnt-4. LacZ/T/GFP+ cells were further enriched by FACS selection. Five days after injection of LacZ/T/GFP+ cells into embryonic kidney explants in organ culture, beta-galactosidase immunohistochemistry showed incorporation into blastemal cells of the nephrogenic zone. After a single injection into developing live newborn mouse kidneys, co-localization studies showed that the LacZ/T/GFP+ cells were stably integrated into proximal tubules with normal morphology and normal polarization of alkaline phosphatase and aquaporin-1 for 7 mo, without teratoma formation. It is concluded that defined differentiation of ES cells into embryoid bodies with Activin-A and selection for T expression provides a means to isolate and purify renal proximal tubular progenitor cells with the potential for safe use in regenerative therapies.
 ...
PMID:Mouse embryonic stem cell-derived embryoid bodies generate progenitors that integrate long term into renal proximal tubules in vivo. 2199 95