Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.30.2 (endonuclease)
 18,621 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The GAL4 locus encodes a positive regulator of the inducible galactose and melibiose genes of yeast. Using the yeast plasmid vector YEp13, we have cloned GAL4 by complementation of a gal4 mutation. Restriction endonuclease mapping of subclone DNA has delimited the region sufficient for complementation to a 3.2-kilobase segment of DNA. The GAL4 mRNA is 2.8 kilobases long, sufficient to encode a protein as large as 105,000 daltons. The concentration of the GAL4 transcript is about 0.1 per cell and is almost identical in galactose-induced and noninduced cells. This result is consistent with a previously proposed model in which the activity of the GAL4 protein and not the transcription of the GAL4 gene is modulated by galactose induction.
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PMID:Isolation and preliminary characterization of the GAL4 gene, a positive regulator of transcription in yeast. 629 56

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

We examine the generality of transcription factor-mediated chromatin remodeling by monitoring changes in chromatin structure in a yeast (Saccharomyces cerevisiae) episome outside of the context of a natural promoter. The episome has a well defined chromatin structure and a binding site for the transcription factor GAL4 but lacks a nearby functional TATA element or transcription start site, so that changes in chromatin structure are unlikely to be caused by transcription. To separate changes caused by binding and by activation domains, we use both GAL4 and a chimeric, hormone-dependent activator consisting of the GAL4 DNA-binding domain, an estrogen receptor (ER) hormone-binding domain, and a VP16 activation domain (Louvion, J.-F., Havaux-Copf, B. and Picard, D. (1993) Gene (Amst.) 131, 129-134). Both GAL4 and GAL4.ER.VP16 show very little perturbation of chromatin structure in their nonactivating configurations. Substantial additional perturbation occurs upon activation. This additional perturbation is marked by changes in micrococcal nuclease cleavage patterns, restriction endonuclease accessibility, and DNA topology and is not seen with the nonactivating derivative GAL4.ER. Remodeling by GAL4.ER.VP16 is detectable within 15 min following hormone addition and is complete within 45 min, suggesting that replication is not required. We conclude that activation domains can exert a major influence on chromatin remodeling by increasing binding affinity and/or by recruitment of other chromatin remodeling activities and that this remodeling can occur outside the context of a bona fide promoter.
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PMID:Chromatin remodeling by transcriptional activation domains in a yeast episome. 911 Oct 67

By using a yeast two-hybrid system, a yeast two-hybrid bait vector was constructed and identified for screening of the HPV18 E6-interacting proteins, and its effects on the growth of yeast cells and the activation of reporter genes were investigated. Total mRNA extracted from Hela cells was reversely transcribed into cDNA. Fragment of HPV18 E6 cDNA was amplified using RT-PCR and directly ligated to the pGBKT7 vector. The recombinant plasmid was confirmed by restriction endonuclease analysis and DNA sequencing. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector were transformed into the yeast cell AH109, respectively. After they were cultured respectively in YPDA liquid medium and nutrition-deficient culture medium, their toxicity and transcriptional activation were tested by both the phenotype assay and the color assay. The bait plasmid HPV18 E6 was successfully obtained. After being cultured in YPDA liquid medium for 16h, the A (600 nm) values of two yeast fluids were 0.98+/-0.03 and 0.99+/-0.02, respectively. The recombinant pGBKT7-HPV18 E6 plasmid and empty pGBKT7 vector could grow to white colonies on SD/-Trp/X-alpha-gal plates, while no colony could survive on SD/-His/-Trp/X-alpha-gal, SD/-Ade/-Trp/X-alpha-gal plates, indicating that the bait plasmid pGBKT7-HPV18 E6 was constructed successfully and expressed correctly, and could not activate the transcription of reporter gene alone. The yeast two-hybrid GAL4 system 3 can be utilized to find HPV18 E6 interacting proteins.
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PMID:Construction and identification of a yeast two-hybrid bait vector and its effect on the growth of yeast cells and the self-activating function of reporter genes for screening of HPV18 E6-interacting protein. 2015 48

DNA repair declines with age and correlates with longevity in many animal species. In this study, we investigated the effects of GAL4-induced overexpression of genes implicated in DNA repair on lifespan and resistance to stress factors in Drosophila melanogaster. Stress factors included hyperthermia, oxidative stress, and starvation. Overexpression was either constitutive or conditional and either ubiquitous or tissue-specific (nervous system). Overexpressed genes included those involved in recognition of DNA damage (homologs of HUS1, CHK2), nucleotide and base excision repair (homologs of XPF, XPC and AP-endonuclease-1), and repair of double-stranded DNA breaks (homologs of BRCA2, XRCC3, KU80 and WRNexo). The overexpression of different DNA repair genes led to both positive and negative effects on lifespan and stress resistance. Effects were dependent on GAL4 driver, stage of induction, sex, and role of the gene in the DNA repair process. While the constitutive/neuron-specific and conditional/ubiquitous overexpression of DNA repair genes negatively impacted lifespan and stress resistance, the constitutive/ubiquitous and conditional/neuron-specific overexpression of Hus1, mnk, mei-9, mus210, and WRNexo had beneficial effects. This study demonstrates for the first time the effects of overexpression of these DNA repair genes on both lifespan and stress resistance in D. melanogaster.
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PMID:Lifespan and Stress Resistance in Drosophila with Overexpressed DNA Repair Genes. 2647 11

The CRISPR/Cas9 system has revolutionized genomic editing. The Cas9 endonuclease targets DNA via an experimentally determined guide RNA (gRNA). This results in a double-strand break at the target site . We generated transgenic Drosophila melanogaster in which the CRISPR/Cas9 system was used to target a GAL4 transgene in vivo To our surprise, progeny whose genomes did not contain CRISPR/Cas9 components were still capable of mutating GAL4 sequences. We demonstrate this effect was caused by maternal deposition of Cas9 and gRNAs into the embryo, leading to extensive GAL4 mutations in both somatic and germline tissues. This serves as a cautionary observation on the effects of maternal contributions when conducting experiments using genomically encoded CRISPR/Cas9 components. These results also highlight a mode of artificial inheritance in which maternal contributions of DNA editing components lead to transmissible mutant defects even in animals whose genomes lack the editing components. We suggest calling this a dominant maternal effect to reflect it is caused by the gain of maternally contributed products. Models of CRISPR-mediated gene drive will need to incorporate dominant maternal effects in order to accurately predict the efficiency and dynamics of gene drive in a population.
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PMID:Non-Mendelian Dominant Maternal Effects Caused by CRISPR/Cas9 Transgenic Components in Drosophila melanogaster. 2763 86