Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P01034 (cystatin C)
 3,397 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In order to study the conversion of UV lesions into frameshift and base substitution mutations, M13mp2 phage DNA was altered by the addition of extra pyrimidines, or by construction of a nonsense codon preceded by a run of pyrimidines within the beta-galactosidase complementing region. The normal sequence 5' GTC GTT TTA CAA 3' was changed to GTC GTT T TTA CAA (MIDT) or GTC GTT C TTA CAA (MIDC) to study frameshifts and to GTC GTT CTT TAA (OCHRE) to study reversion of the ochre (TAA) codon. Escherichia coli pol I Kf and T7 DNA polymerase mutant enzymes devoid of 3'-->5' exonuclease activity produced UV-induced revertants at higher frequency than did their exonuclease proficient counterparts. Removal of cyclobutane dimers with photolyase before in vitro synthesis did not greatly affect mutant frequency although such treatment led to significantly increased DNA synthesis by the wild-type T7 DNA polymerase on UV-irradiated substrate. Reversions of the in frame ochre sequence GTT CTT TAA produced by the delta 28 T7 DNA polymerase were mainly by base substitution in the TAA codon. About half of the E. coli Kf exo- enzyme ochre revertants had a TTA deletion. Five mutant T7 DNA polymerases with varying exonuclease activity gave revertant frequencies that correlated better with published values of enzyme velocity than with exonuclease activity or with measured bypass synthesis. Our data indicate that loss of proofreading activity increases the frequency of UV-induced frameshifts, but lack of such activity is not sufficient for their production. We suggest that frameshifts occur more frequently when nucleotide addition opposite the lesion is slow. The same lesion can give rise to a different spectrum of mutations depending on the polymerase.
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PMID:Production of UV-induced frameshift mutations in vitro by DNA polymerases deficient in 3'-->5' exonuclease activity. 802 6

We describe a gene system allowing the facile production of multiply substituted reverse transcriptases (RTs), the enzymatic characterization of these purified RTs, and the study of these mutations in the defined genetic background of the macrophagetropic, non-laboratory-adapted human immunodeficiency virus type 1 (HIV-1) AD8 strain. Thirteen unique silent restriction sites were introduced in the pol gene encoding HIV-1 RT, allowing easy introduction of mutations. To simplify genetic manipulation and generate p66/p51 heterodimers in Escherichia coli, a gene construct of the viral protease alone was optimized for expression from a separate vector carrying a p15A origin of replication. Active-site titration experiments using pre-steady-state kinetics showed that our system yields a higher proportion of active enzyme than that obtained by alternate methods. To facilitate phenotype/genotype correlations, the modified RT gene was designed to be easily reintroduced into a recombinant proviral AD8 HIV-1 DNA. Infectious viruses made from this vector were undistinguishable from wild-type AD8 HIV-1, an isolate able to infect peripheral blood mononuclear cells and macrophages. Thus, the pol gene can tolerate many silent mutations in the polymerase domain without affecting the functionality of the HIV-1 genome. The system was validated biochemically and virologically using the V75T substitution associated with stavudine resistance.
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PMID:An integrated system to study multiply substituted human immunodeficiency virus type 1 reverse transcriptase. 1131 28

Transcription is an important control point in the transposable element mobilization process. To better understand the regulation of the plant SINE (Short Interspersed Elements) S1, its promoter sequence was studied using an in vitro pol III transcription system derived from tobacco cells. We show that the internal S1 promoter can be functional although upstream external sequences were found to enhance this basal level of transcription. For one putative 'master' locus (na7), three CAA triplets (in positions -12, -7 and -2) and two overlapping TATA motifs (in positions -54 to -43) were important to stimulate transcription. For this locus, two transcription initiation regions were characterized, one centered on position + 1 (first nucleotide of the S1 element) and one centered on position - 19 independently of the internal motifs. The CAA triplets only influence transcription in + 1 and work in association with the internal motifs. We show that methylation can inhibit transcription at the na7 locus. We also observe that S1 RNA is cleaved in a smaller Poly (A) minus product by a process analogous to the maturation of mammalian SINEs.
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PMID:Analysis of the SINE S1 Pol III promoter from Brassica; impact of methylation and influence of external sequences. 1143 18

DNA structural perturbations that are induced by site specifically and stereospecifically defined benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (BPDE) adducts are directly correlated with mutagenesis, leading to cellular transformation. Although previous investigations had established that replication of DNAs containing N(6) -BPDE dA adducts at the second position in the N-ras codon 61(CAA) (61(2) ) resulted exclusively in A to G transitions, NMR analyses not only established the structural basis for this transition mutation but also predicted that if the adduct were positioned at the third position in the same codon, an expanded spectra of mutations was possible. To test this prediction, replication of DNAs containing C10 S-BPDE and C10 R-BPDE lesions linked through the N(6) position of adenine in the sequence context N-ras codon 61, position 3 (C10 S-BPDE and C10 R-BPDE at 61(3) ) was carried out in Escherichia coli, and these data revealed a wide mutation spectrum. In addition to A to G transitions produced by replication of both lesions, replication of the C10 S-BPDE and C10 R-BPDE adducts also yielded A to C and A to T transversions, respectively. Analyses of single nucleotide incorporation using Sequenase 2.0 and exonuclease-deficient E. coli Klenow fragment and pol II not only revealed high fidelity synthesis but also demonstrated the same hierarchy of preference opposite a particular lesion, independent of the sequence context. Primer extension assays with the two lesions at N-ras 61(3) resulted in truncated products, with the C10 S-BPDE adducts being more blocking than C10 R-BPDE lesions, and termination of synthesis was more pronounced at position 61(3) than at 61(2) for each of the lesions.
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PMID:Sequence context modulation of polycyclic aromatic hydrocarbon-induced mutagenesis. 2391 16