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)

In this report a method for transfection and selection of mammalian cells in serum-free medium is described. Chinese hamster ovary (CHO) cells were grown in serum-free medium in plastic dishes coated with one of the following attachment factors: poly-D-lysine, Cell-Tak (polyphenolic proteins extracted from the marine mussel Mytilus edulis), fibronectin or laminin. Cells grown to 80% confluence were transfected with an expression vector encoding the hygromycin resistance gene as a selectable marker and beta-galactosidase as the reporter gene. Transfectants were selected using hygromycin at a concentration of 500 micrograms/ml. Both fibronectin and laminin supported colony formation following selection in serum-free medium. However, poly-D-lysine and Cell-Tak did not. This method can, thus, be used to select for clones producing a recombinant product in cells that are growing in serum-free medium from the onset to provide a better system from which to purify proteins.
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PMID:Serum-free transfection and selection in Chinese hamster ovary (CHO) cells. 826 82

Late transcription of bacteriophage Mu initiates at four promoters, P(lys), PI, PP and Pmom, and requires the Mu C protein and the host RNA polymerase. Promoter-containing DNA fragments extending approximately 200 bp upstream and downstream of the 5' starts of the lys, I and P transcripts were cloned into a multicopy lacZ-expression plasmid. Promoter activity, assayed by beta-galactosidase expression, was determined under two different conditions: (1) with C provided from a compatible plasmid in the absence of other Mu factors and (2) with C provided from an induced Mu prophage. beta-galactosidase activities were greatest for P(lys), intermediate for PI, and lowest for PP. Similar analysis of plasmids containing nested sets of deletions removing 5' or 3' sequences of P(lys) demonstrated that a 68-bp region was sufficient for full activity. Point mutations were generated within the 68-bp region by mutagenic oligonucleotide-directed PCR (Mod-PCR). Properties of the lys promoter mutants indicated that, in addition to the -10 region, a 19-bp region from -52 to -34 containing the C footprint is required for C-dependent promoter activity.
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PMID:Mutational analysis of a C-dependent late promoter of bacteriophage Mu. 829 68

The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue. Distinct versions of the N-end rule operate in bacteria, fungi, and mammals. We report the cloning and analysis of aat, the Escherichia coli gene that encodes leucyl, phenylalanyl-tRNA-protein transferase (L/F-transferase), a component of the bacterial N-end rule pathway. L/F-transferase is required for the degradation of N-end rule substrates bearing an N-terminal arginine or lysine. The aat gene maps to the 19-min region of the E. coli chromosome and encodes a 234-residue protein whose sequence lacks significant similarities to sequences in data bases. In vitro, L/F-transferase catalyzes the posttranslational conjugation of leucine or phenylalanine to the N termini of proteins that bear an N-terminal arginine or lysine. However, the isolation and sequence analysis of a beta-galactosidase variant engineered to expose an N-terminal arginine in vivo revealed the conjugation of leucine but not of phenylalanine to the N terminus of the beta-galactosidase variant. Thus, the specificity of L/F-transferase in vivo may be greater than that in vitro. The aat gene is located approximately 1 kb from clpA, which encodes a subunit of ATP-dependent protease Clp. Although both aat and clpA are required for the degradation of certain N-end rule substrates, their nearly adjacent genes are convergently transcribed. The aat gene lies downstream of an open reading frame that encodes a homolog of the mammalian multidrug resistance P glycoproteins.
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PMID:The N-end rule in Escherichia coli: cloning and analysis of the leucyl, phenylalanyl-tRNA-protein transferase gene aat. 833 Oct 68

We have combined a receptor-mediated DNA delivery system with the endosomal lysis ability of adenovirus and shown that DNA can be delivered into primary hepatocytes, resulting in a high level of gene expression. When asialoorosomucoid conjugated with poly(L-lysine) was used to deliver the Escherichia coli beta-galactosidase gene into primary hepatocytes through binding with the hepatic asialoglycoprotein receptor, only a low level of beta-galactosidase was detectable, with less than 0.1% of the hepatocytes being transfected. This level of activity can be greatly enhanced by the cointernalization of the DNA.protein complex with a replication-defective adenovirus, resulting in 100% of the hepatocytes staining blue with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. Quantitative analysis of beta-galactosidase expression also showed a 1000-fold enhancement of activity. To test the applicability of this DNA delivery system for the correction of phenylketonuria, a metabolic disorder that causes severe mental retardation in children, we have delivered the human phenylalanine hydroxylase (PAH) gene to hepatocytes derived from a PAH-deficient mouse strain and demonstrated complete reconstitution of enzymatic activity. This method shows great promise for efficient gene delivery to the liver for correction of hepatic disorders.
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PMID:Hepatic gene therapy: adenovirus enhancement of receptor-mediated gene delivery and expression in primary hepatocytes. 838 12

The 3', 5' cyclic adenosine monophosphate (cAMP) binding pocket of the cAMP receptor protein (CRP) of Escherichia coli was mutagenized to substitute leucine, glutamine, or aspartate for glutamate 72; and lysine, histidine, leucine, isoleucine, or glutamine for arginine 82. Substitutions were made in wild-type CRP and in a CRP*, or cAMP-independent, form of the protein to assess the effects of the amino acid substitutions on CRP structure. Cells containing the binding pocket residue-substituted forms of CRP were characterized through beta-galactosidase activity and by measurement of cAMP binding activity. This study confirms a role for both glutamate 72 and arginine 82 in cAMP binding and activation of CRP. Glutamine or leucine substitution of glutamate 72 produced forms of CRP having low affinity for the cAMP and unresponsive to the nucleotide. Aspartate substituted for glutamate 72 produced a low affinity cAMP-responsive form of CRP. CRP has a stringent requirement for the positioning of the position 72 glutamate carboxyl group within the cyclic nucleotide binding pocket. Results of this study also indicate that there are differences in the binding requirements of cAMP and cGMP, a competitive inhibitor of cAMP binding to CRP.
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PMID:Mutagenesis of the cyclic AMP receptor protein of Escherichia coli: targeting positions 72 and 82 of the cyclic nucleotide binding pocket. 838 97

The aerobactin gene cluster in pColV-K30 consists of five genes (iucABCD iutA); four of these (iucABCD) are involved in aerobactin biosynthesis, whereas the fifth one (iutA) encodes the ferriaerobactin outer membrane receptor. iucD encodes lysine:N6-hydroxylase, which catalyzes the first step in aerobactin biosynthesis. Regardless of the method used for cell rupture, we have consistently found that IucD remains membrane bound, and repeated efforts to achieve a purified and active soluble form of the enzyme have been unsuccessful. To circumvent this problem, we have constructed recombinant IucD proteins with modified amino termini by creating three in-frame gene fusions of IucD to the amino-terminal amino acids of the cytoplasmic enzyme beta-galactosidase. Two of these constructs resulted in the addition to the iucD coding region of a hydrophilic leader sequence of 13 and 30 amino acids. The other construct involved the deletion of the first 47 amino acids of the IucD amino terminus and the addition of 19 amino acids of the amino terminus of beta-galactosidase. Cells expressing any of the three recombinant IucD forms were found to produce soluble N6-hydroxylysine. One of these proteins, IucD439, was purified to homogeneity from the soluble fraction of the cell lysates, and it was capable of participating in the biosynthesis of aerobactin, as determined in vitro by a cell-free system and in vivo by a cross-feeding bioassay. A medium ionic strength of 0.25 (250 mM NaCl) or higher was required to maintain the protein in a catalytically functional, tetrameric state.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Construction and biochemical characterization of recombinant cytoplasmic forms of the IucD protein (lysine:N6-hydroxylase) encoded by the pColV-K30 aerobactin gene cluster. 842 34

The last enzymatic step for L-cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) which synthesizes L-cysteine from O-acetylserine and "sulfide." We have isolated and characterized a full-length cDNA (1432 bp) from a lambda gt11 library of spinach leaf encoding the complete precursor of the chloroplast isoform. The 1149-nucleotide open reading frame coding for O-acetylserine(thiol)lyase was in the direction opposite that of the lambda gt11 beta-galactosidase gene. The derived amino acid sequence indicates that the protein precursor consists of 383 amino acid residues including a N-terminal presequence peptide of 52 residues. The amino acid sequence of mature spinach chloroplast O-acetylserine(thiol)lyase shows 40 and 57% homology with its bacterial counterparts. Sequence comparison with several pyridoxal 5'-phosphate-containing proteins reveals the presence of a lysine residue assumed to be involved in cofactor binding. A synthetic cDNA was constructed, coding for the entire 331-amino-acid mature O-acetylserine(thiol)lyase and for an initiating methionine. A high level of expression of the active mature chloroplast isoform was achieved in an Escherichia coli strain carrying the T7 RNA polymerase system (F. W. Studier, A. H. Rosenberg, J. J. Dunn, and J. W. Dubendorff, 1990, in Methods in Enzymology, D. V. Goeddel, Ed., Vol. 185, pp. 60-89, Academic Press, San Diego, CA). Addition of pyridoxine to the bacterial growth medium enhanced the enzyme activity due to the recombinant protein. The extent of production is 25-fold higher than in chloroplast from spinach leaves and the recombinant protein presents the relative molecular mass and immunological properties of the natural enzyme from spinach leaf chloroplast. This work, together with our previous biochemical studies, are in accordance with a prokaryotic type enzyme for L-cysteine biosynthesis in higher plant chloroplasts. Southern blot analysis indicated that O-acetylserine(thiol)lyase is encoded by multiple genes in the spinach leaf genomic DNA.
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PMID:O-acetylserine(thiol)lyase from spinach (Spinacia oleracea L.) leaf: cDNA cloning, characterization, and overexpression in Escherichia coli of the chloroplast isoform. 842 55

Screening of a cDNA library (prepared in lambda gt11) of the blood stages of Plasmodium chabaudi chabaudi (AS) with immune serum has revealed an antigen the elicits a strong antibody response in infected mice. The clone (clone 6) expressing that antigen contains a 0.7 kb insert and produces a beta-galactosidase fusion protein of about 150 kDa. In Western blot analysis performed on parasite extracts, monoclonal antibodies and polyclonal sera prepared against the fusion protein revealed that the fusion protein contains part of a malarial protein of 93 kDa. Northern hybridization with clone 6 insert as probe detected a plasmodial RNA of about 3.2 kb, which could well code for a protein of this size. The insert hybridized to a single EcoRI fragment and a single HindIII fragment in genomic Southern blotting, suggesting that the gene is present in one copy in the P. chabaudi genome. The DNA sequence of clone 6 insert predicts a hydrophilic, acidic polypeptide consisting of seven repeats of 23-34 amino acids rich in lysine (24%) and aspartic acid (17.5%).
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PMID:Cloning and sequencing of a cDNA fragment from Plasmodium chabaudi chabaudi that contains repetitive sequences coding for a potentially lysine-rich aspartic acid-rich protein. 847 31

Correct folding of newly synthesized proteins is proposed to be assisted by molecular chaperones and folding catalysts. To identify cellular factors involved in the initial stages of this process we searched for proteins associated with nascent polypeptide chains. In an Escherichia coli transcription/translation system synthesizing beta-galactosidase we identified a 58-kDa protein which associated with translating ribosomes but dissociated from these ribosomes upon release of nascent beta-galactosidase. N-terminal sequencing identified it as trigger factor, previously implicated in protein secretion. Direct evidence for association of trigger factor with nascent polypeptide chains was obtained by crosslinking. In a wheat germ translation system complemented with E. coli lysates, epsilon-4-(3-trifluoromethyldiazirino)benzoic acid-lysine residues were incorporated into nascent secretory preprolactin and a nonsecretory preprolactin mutant. Trigger factor crosslinked to both types of nascent chains, provided they were ribosome bound. Trigger factor contains key residues of the substrate-binding pocket of FK506-binding protein-type peptidyl-prolyl-cis/trans-isomerases and has prolyl isomerase activity in vitro. We propose that trigger factor is a folding catalyst acting cotranslationally.
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PMID:Escherichia coli trigger factor is a prolyl isomerase that associates with nascent polypeptide chains. 863 85

Although it is clear that gene transfection is a potentially valuable approach in the study of cardiac cell function and differentiation, classic transfection methods are limited by their poor efficiencies in cardiac cells. Recent studies show that recombinant replication-defective human adenovirus can transfect primary cardiac cultures with near 100% efficiency. Since such recombinants are time consuming to prepare, the goal of this study was to develop a plasmid/viral transfection system that would capitalize on the advantages of adenovirus. We have found that a "component system" formed by preincubation of Ad5dl312 adenovirus, poly-L-lysine, and an expression plasmid (lacZ reporter gene under control of the human cytomegalovirus (HCMV) major immediate early promoter) can transfect cultured cardiac cells. Optimal conditions were determined by quantifying beta-galactosidase expression. Histochemical analysis of cultures revealed that the component system transfected 70% of the cells under these conditions. LacZ-positive myocytes could be identified in intact myocytes with the fluorescent substrate C12-fluorescein di-beta-galactopyranoside. Functional studies with such cells indicated that contractile behavior was maintained in transfected cardiocytes. Furthermore, the component system was used to transfect a DNA vector expressing a physiologically relevant protein, protein kinase C delta. In summary, this powerful and simple approach can promote the expression of heterologous genes that can be studied at the biochemical and cellular level in cardiac cells.
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PMID:Novel adenovirus component system that transfects cultured cardiac cells with high efficiency. 863 47


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