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)

It was demonstrated previously that a deoxyribophosphodiesterase (dRpase) activity is associated with the DNA repair enzyme exonuclease I, and that this activity is stimulated by the addition of the E. coli single-stranded DNA-binding protein (Ssb). This activity catalyzes the release of deoxyribose-phosphate groups at apurinic/apyrimidinic (AP) sites in the DNA that have been cleared by the action of an AP endonuclease. We have now used the yeast two-hybrid system to demonstrate that a protein-protein interaction occurs between exonuclease I and Ssb. When the E. coli ssb gene was fused in frame to the DNA-activating domain of the GAL4 transcriptional activator and the exonuclease I gene was fused in frame to the DNA-binding domain, a functional GAL4 transcriptional activator was produced as determined by growth of yeast on selective medium and the measurement of beta-galactosidase activity. We have also demonstrated that Ssb can stimulate the dRpase activity of exonuclease I using double-stranded bacteriophage M13 DNA containing several strand interruptions at incised AP sites. These results suggest that Ssb may be required for efficient base-excision repair in bacteria.
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PMID:Protein-protein interactions between the Escherichia coli single-stranded DNA-binding protein and exonuclease I. 861 28

The yeast two-hybrid system was used to isolate a clone from a 17-day-old mouse embryo cDNA library that codes for a novel 812-aa long protein fragment, glucocorticoid receptor-interacting protein 1 (GRIP1), that can interact with the hormone binding domain (HBD) of the glucocorticoid receptor. In the yeast two-hybrid system and in vitro, GRIP1 interacted with the HBDs of the glucocorticoid, estrogen, and androgen receptors in a hormone-regulated manner. When fused to the DNA binding domain of a heterologous protein, the GRIP1 fragment activated a reporter gene containing a suitable enhancer site in yeast cells and in mammalian cells, indicating that GRIP1 contains a transcriptional activation domain. Overexpression of the GRIP1 fragment in mammalian cells interfered with hormone-regulated expression of mouse mammary tumor virus-chloramphenicol acetyltransferase gene and constitutive expression of cytomegalovirus-beta-galactosidase reporter gene, but not constitutive expression from a tRNA gene promoter. This selective squelching activity suggests that GRIM can interact with an essential component of the RNA polymerase II transcription machinery. Finally, while a steroid receptor HBD fused with a GAL4 DNA binding domain did not, by itself, activate transcription of a reporter gene in yeast, coexpression of this fusion protein with GRIP1 strongly activated the reporter gene. Thus, in yeast, GRIP1 can serve as a coactivator, potentiating the transactivation functions in steroid receptor HBDs, possibly by acting as a bridge between HBDs of the receptors and the basal transcription machinery.
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PMID:GRIP1, a novel mouse protein that serves as a transcriptional coactivator in yeast for the hormone binding domains of steroid receptors. 864 9

Subunit interactions among the F1-ATPase subunits were studied by the yeast two-hybrid system. Various pairwise combinations of genes encoding alpha, beta, gamma, delta and epsilon subunits of Escherichia coli H+-ATPase fused to the DNA-binding or activation domain of the yeast GAL4 gene were introduced into yeast and expression of a reporter gene encoding beta-galactosidase was detected. Combinations of the alpha and beta subunit genes, and of the epsilon and gamma subunit genes showed high levels of reporter gene expression, while those of alpha and delta, beta and delta, gamma and delta, and delta and epsilon demonstrated weak but significant reporter gene expression. However, combinations of alpha and gamma, beta and gamma, alpha and epsilon, and beta and epsilon did not induce reporter expression. None of the fused genes alone induced reporter gene expression. These results suggested that specific and strong interactions between the alpha and beta, gamma and epsilon, and weak interactions between the alpha and delta, beta and delta, and gamma and delta subunits occurred in yeast cells in the two-hybrid system. Effects of previously identified mutant beta subunits with Leu-40 to Pro. Glu-41 to Lys or Pro-332 to Gln substitutions which caused defects in molecular assembly of F1-ATPase were analyzed with regard to alpha-beta interactions. No interaction of the alpha and beta subunits was observed in this system using the beta subunit with mutation of Pro-332 to Gln. However, for the other two mutations, alpha-beta interactions were observed. This system may be useful for isolating mutants which have defects in interaction of F1-ATPase subunits.
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PMID:Interactions of the F1-ATPase subunits from Escherichia coli detected by the yeast two-hybrid system. 864 96

HSV-1 B capsids are composed of seven major proteins, designated VP5, VP19C, 21, 22a, VP23, VP24, and VP26. VP indicates that the capsid protein is also a component of the infectious virion. Capsid proteins 21, 22a, and VP24 are specified by a single open reading frame (UL26) that encodes 635 amino acids. An objective of the work in our laboratory is to identify and map interactions among and between capsid proteins. In the present studies we employed the yeast GAL4 two-hybrid system developed by Fields and his colleagues (Nature 240, 245-246 (1989)) for this purpose. DNA corresponding to the capsid open reading frames was derived as a PCR product and fused to sequences of the GAL4 activation and DNA binding domains. Using this system each of the capsid proteins has been tested for interactions with all of the other capsid proteins. Three interactions have been identified: a relatively strong self-interaction between 22a molecules (residues 307-635 of UL26), bimolecular interactions between 22a and VP5, and another between VP19C and VP23. The interactions were detected by the expression of beta-galactosidase enzyme activity, and yielded 289, 86, and 63 units of enzyme activity, respectively. For the 22a self-interaction, elimination of residues 611-635 resulted in an approximately twofold decrease in enzyme activity. The C-terminal 25 amino acids of 22a were also essential for the bimolecular interaction between 22a and VP5.
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PMID:Molecular interactions between the HSV-1 capsid proteins as measured by the yeast two-hybrid system. 866 4

Arsenical resistance (ars) operons produce resistance to trivalent and pentavalent salts of the metalloids arsenic and antimony in cells of Escherichia coli. The first gene in the operon, arsR, was previously shown to encode a homodimeric trans-acting metalloregulatory repressor protein. Dimerization of ArsR was investigated using the yeast two-hybrid system in which the ArsR protein was fused to the Saccharomyces cerevisiae GAL4 DNA-binding domain and GAL4 activation domain to produce chimeric proteins. Transcriptional activation of lacZ reporter indicated that dimerization of the ArsR is stable in yeast. The results indicated that residues 1-8 and 90-117 are not required for ArsR dimerization. The genes for a series of truncated ArsR proteins containing six histidine tags were constructed and the proteins purified. The mass of each recombinant protein, as determined by size exclusion chromatography, was consistent with the results from two-hybrid analysis. The results of beta-galactosidase assays in vivo and gel mobility shift assays in vitro showed that dimers retained the ability to bind to the ars promoter and to respond to inducer, whereas monomeric ArsRs did neither. These results suggest that a core sequence of about 80 residues has all of the information necessary for dimerization, repression, and metal recognition.
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PMID:Dimerization is essential for DNA binding and repression by the ArsR metalloregulatory protein of Escherichia coli. 918 67

To investigate the biological function of hepatitis C virus (HCV)-NS5A, the NS5A was fused at its N-terminus with the DNA binding domain (DBD) of yeast transcriptional activator GAL4 (GAL4-DBD). The GAL4-DBD alone had no transcriptional activation function. However, a mutant of the GAL4-DBD/NS5A fusion protein, in which 129 amino acid residues were deleted from the N-terminus of NS5A, exhibited strong transcriptional activation in yeast cells, bearing the Escherichia coli lacZ reporter gene encoding the beta-galactosidase under the transcriptional control of GAL4 promoter and TATA box. Further mutational analysis of NS5A revealed that the region between the amino acid residues 130 to 352 were critical for optimal level of transactivation. This region includes two acidic domains and one proline-rich region which have been shown to be involved in the function of several transcriptional activators.
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PMID:The amino terminal deletion mutants of hepatitis C virus nonstructural protein NS5A function as transcriptional activators in yeast. 924 Apr 41

Interaction between a peptide hormone and extracellular domains of its receptor is a crucial step for initiation of hormone action. We have developed a modification of the yeast two-hybrid system to study this interaction and have used it to characterize the interaction of insulin-like growth factor 1 (IGF-1) with its receptor by using GAL4 transcriptional regulation with a beta-galactosidase assay as readout. In this system, IGF-1 and proIGF-1 bound to the cysteine-rich domain, extracellular domain, or entire IGF-1 proreceptor. This interaction was specific. Thus, proinsulin showed no significant interaction with the IGF-1 receptor, while a chimeric proinsulin containing the C-peptide of IGF-1 had an intermediate interaction, consistent with its affinity for the IGF-1 receptor. Over 2000 IGF-1 mutants were generated by PCR and screened for interaction with the color assay. About 40% showed a strong interaction, 20% showed an intermediate interaction, and 40% give little or no signal. Of 50 mutants that were sequenced, several (Leu-5 --> His, Glu-9 --> Val, Arg-37 --> Gly, and Met-59 --> Leu) appeared to enhance receptor association, others resulted in weaker receptor interaction (Tyr-31 --> Phe and Ile-43 --> Phe), and two gave no detectable signal (Leu-14 --> Arg and Glu-46 --> Ala). Using PCR-based mutagenesis with proinsulin, we also identified a gain of function mutant (proinsulin Leu-17 --> Pro) that allowed for a strong IGF-1-receptor interaction. These data demonstrate that the specificity of the interaction between a hormone and its receptor can be characterized with high efficiency in the two-hybrid system and that novel hormone analogues may be found by this method.
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PMID:Analysis of a peptide hormone-receptor interaction in the yeast two-hybrid system. 937

We report the analysis of a number of lines of Drosophila melanogaster containing insertions of the yeast gal4 gene. By crossing a UAS-lacZ fusion gene as a reporter into these lines, we analysed the expression patterns of beta-galactosidase during oogenesis. Since there is no expression of GAL4 in the germ-line in these experiments, this is an ideal system for the analysis of expression patterns in sub-sets of follicle cells. These lines provide ideal markers for sets of follicle cells, e.g. anterior or posterior polar cells for studying genetic interactions in oogenesis; however, they can also be used in the same way as conventional enhancer traps to clone nearby genes with similar expression patterns. The advantages of this dual gal4/UAS system over conventional enhancer trapping includes the possibility of GAL4-directed misexpression and antisense expression studies to establish the function of the genes we identified during follicle cell determination and differentiation. These studies could lead to the isolation of homologous genes crucial in mammalian oogenesis. Understanding how the somatic cells and germ cells interact to promote growth and maturation of the mammalian follicle and oocyte could well be crucial for improving the fertility of eggs used for in-vitro fertilization programmes, and could provide methods for assessing the quality of eggs.
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PMID:Analysis of P[gal4] insertion lines of Drosophila melanogaster as a route to identifying genes important in the follicle cells during oogenesis. 939 63

In order to evaluate the efficiency of the tetracycline-regulated gene expression system in Drosophila, we have generated transgenic lines expressing a tetracycline-controlled transactivator protein (tTA), with specific expression patterns during embryonic and larval development. These lines were used to direct expression of a tTA-responsive promoter fused to the coding region of either the beta-galactosidase or the homeotic protein Antennapedia (ANTP), under various conditions of tetracycline treatment. We found that expression of beta-galactosidase can be efficiently inhibited in embryos and larvae with tetracycline provided in the food, and that a simple removal of the larvae from tetracycline exposure results in the induction of the enzyme in a time- and concentration-dependent manner. Similar treatments can be used to prevent the lethality associated with the ectopic expression of ANTP in embryos and, subsequently, to control the timing of expression of the homeoprotein ANTP specifically in the antennal imaginal disc. Our results show that the expression of a gene placed under the control of a tetracycline-responsive promoter can be tightly controlled, both spatially by the regulatory sequences driving the expression of tTA and temporally by tetracycline. This provides the basis of a versatile binary system for controlling gene expression in Drosophila, with an additional level of regulation as compared to the general method using the yeast transcription factor GAL4.
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PMID:Spatial and temporal targeting of gene expression in Drosophila by means of a tetracycline-dependent transactivator system. 958 19

In the filamentous fungus Neurospora crassa during conditions of sulfur limitation, CYS3, a major positive-acting regulatory protein, turns on the expression of an entire set of genes which encode permeases and enzymes involved in the acquisition of sulfur from environmental sources. CYS3 functions as a homodimeric protein and possesses a b-Zip domain that confers sequence-specific DNA binding. Expression of various hybrid GAL4-CYS3 fusion proteins in yeast was used to detect regions involved in gene activation. An amino-terminal serine/threonine-rich domain of CYS3 alone strongly activated expression of beta-galactosidase, the yeast reporter. Moreover, mutant CYS3 proteins with amino-acid substitutions in this region that showed increased expression in Neurospora also displayed an enhanced activation potential in yeast. The cys-3 gene of the exotic N. crassa Mauriceville strain and of N. intermedia were cloned and demonstrated to be functional for gene activation and for sulfur-mediated regulation by complementation of a loss-of-function cys-3 mutation. The amino-terminal serine/threonine-rich region is highly conserved in these two CYS3 proteins, in agreement with the possibility that it serves as the activation domain. Surprisingly, an extended promoter region of the cys-3 gene in the Mauriceville strain and in N. intermedia was very well conserved with that of the standard N. crassa gene, including the presence of three CYS3-binding sites possibly involved in autogenous control. Results are presented which indicate that synthesis of the CYS3 regulatory protein is highly regulated and can be detected in the nucleus of cells subjected to sulfur de-repression, but is not found in the nucleus or the cytoplasm of S-repressed cells. The amino-acid substitutions of the CYS3 protein present in a temperature-sensitive cys-3 mutant and in a second-site revertant of a cys-3 null mutation are presented and are shown to affect their DNA-binding activities.
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PMID:Functional analysis of different regions of the positive-acting CYS3 regulatory protein of Neurospora crassa. 964 2


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