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)

mRNA maturation in Trypanosoma brucei depends upon trans splicing, and variations in trans-splicing efficiency could be an important step in controlling the levels of individual mRNAs. RNA splicing requires specific sequence elements, including conserved 5' splice sites, branch points, pyrimidine-rich regions [poly(Y) tracts], 3' splice sites (3'SS), and sometimes enhancer elements. To analyze sequence requirements for efficient trans splicing in the poly(Y) tract and around the 3'SS, we constructed a luciferase-beta-galactosidase double-reporter system. By testing approximately 90 sequences, we demonstrated that the optimum poly(Y) tract length is approximately 25 nucleotides. Interspersing a purely uridine-containing poly(Y) tract with cytidine resulted in increased trans-splicing efficiency, whereas purines led to a large decrease. The position of the poly(Y) tract relative to the 3'SS is important, and an AC dinucleotide at positions -3 and -4 can lead to a 20-fold decrease in trans splicing. However, efficient trans splicing can be restored by inserting a second AG dinucleotide downstream, which does not function as a splice site but may aid in recruitment of the splicing machinery. These findings should assist in the development of improved algorithms for computationally identifying a 3'SS and help to discriminate noncoding open reading frames from true genes in current efforts to annotate the T. brucei genome.
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PMID:Systematic study of sequence motifs for RNA trans splicing in Trypanosoma brucei. 1622 7

Reversible competitive inhibitors of the three enzymes beta-galactosidase, trypsin, and serum cholinesterase have been covalently attached to nonionic ethoxylated surfactants. The binding of the resulting affinity-derivatized surfactants to the respective enzymes has been quantified by measuring Michaelis-Menten inhibition constants with kinetic assays. The surfactant-inhibitor of serum cholinesterase, octaethylene glycol monohexadecy ether pyridinium (C(16)E(8)-PYR), was adsorbed in aqueous solution to an octadecyl-bonded reverse-phase silica packing in a 2 x 0.2 cm stainless steel test column. The ability of the test column to function as a high-performance affinity chromatography (HPAC) column was determined by applying a mixture of bovine serum albumin and cholinesterase (4:1 w/w). Virtually all of the cholinesterase bound and was eluted by applying a gradient in ionic strength. The applied cholinfesterase was recovered with a yield of over 90% and an 11-fold purification. An aliquot of raw horse serum was then purified in the same fashion with a yield of 84% and a 280-fold purification. The surfactant-inhibitor was easily removed from the column with an alcohol wash for sterilization, cleaning, or application of a different affinity ligand. Moreover, the ligand density on the column can be easily manipulated by adsorbing mixtures of derivatized and underivatized surfactants. Leakage of ligands from the support seems to be minimal since the cholinesterase affinity column was operated efficiently after being exposed to 24,000 column volumes of buffer. The application of this technique to high-capacity, high-throughput reversible affinity purifications is limited only by the ability to identify suitable ligands.
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PMID:Water-soluble nonionic surfactants for affinity bioseparations. 1858 59

Saccharomyces cerevisiae autoselection strains with mutations in the ura3, fur1, and urid-k genes have been obtained through a sequential isolation procedure. This autoselection system is an extension of one described by Loison et al. The mutations effectively block both the pyrimidine biosynthetic and salvage pathways and in combination are lethal to the host. Therefore, a plasmidencoded URA3 gene is essential for cell viability regardless of the growth conditions, and complex (traditionally nonselective) media can be employed without the risk of plasmid loss. The effects of medium enrichment on growth and cloned gene product synthesis were examined in batch culture for two autoselection strains. The plasmid gene product beta-galactosidase was under the control of the yeast GAL1 promoter, and two methods of induction were employed; one strain was induced via temperature shift while the other was induced by galactose addition. Three nutrient media were investigated: a lean selective medium (SD), a richer semidefined medium (SDC), and a rich complex medium (YPD). The results demonstrated the improvements in cloned gene productivity possible when the growth medium is enriched, with up to 10-fold increases in beta-galactosidase productivity observed. Plasmid instability and mutation reversion were not problems for the autoselection strains, even in uracil-containing medium. Short-term plasmid stabilities were approximately 90% in all three media tested. During continuous culture of the autoselection temperature-sensitive strain, long-term plasmid stability was excellent and beta-galactosidase expression remained high after more than 25 residence times under inducing conditions. In contrast, both beta-galactosidase specific activity and plasmid stability decreased linearly with time for an analogous nonautoselection strain. The introduced fur1 and uridk mutations were very stable; after more than 50 generations of growth in complex medium, stability values of 99-100% were measured.
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PMID:Enhancement of cloned gene product synthesis via autoselection in recombinant Saccharomyces cerevisiae. 1860 24

Transcription-coupled nucleotide excision repair (TC-NER) removes certain kinds of lesions from the transcribed strand of expressed genes. The signal for TC-NER is thought to be RNA polymerase stalled at a lesion in the DNA template. In Escherichia coli, the stalled polymerase is dissociated from the lesion by the transcription repair coupling factor (Mfd protein), which also recruits excision repair proteins to the site resulting in efficient removal of the lesion. TC-NER has been documented in cells from a variety of organisms ranging from bacteria to humans. In each case, the RNA polymerase involved has been a multimeric protein complex. To ascertain whether a gene transcribed by the monomeric RNA polymerase of bacteriophage T7 could be repaired by TC-NER, we constructed strains of E. coli in which the chromosomal lacZ gene is controlled by a T7 promoter. In the absence of T7 RNA polymerase, little or no beta-galactosidase is produced, indicating that the E. coli RNA polymerase does not transcribe lacZ efficiently, if at all, in these strains. By introducing a plasmid (pAR1219) carrying the T7 gene 1 under control of the E. coli lac UV5 promoter into these strains, we obtained derivatives in which the level of T7 RNA polymerase could be regulated. In cultures containing upregulated levels of the polymerase, beta-galactosidase was actively produced indicating that the T7 RNA polymerase transcribes the lacZ gene efficiently. Under these conditions, we observed that UV-induced cyclobutane pyrimidine dimers were removed more rapidly from the transcribed strand of lacZ than from the nontranscribed strand, supporting the conclusion that TC-NER occurred in this gene. This response was absent in an mfd-1 mutant, indicating that the underlying mechanism may be similar to that for the bacterial RNA polymerase.
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PMID:Transcription-coupled nucleotide excision repair of a gene transcribed by bacteriophage T7 RNA polymerase in Escherichia coli. 2063 14


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