RESUMO
Neuronal nicotinic alpha7 subunits assemble into cell-surface complexes that neither function nor bind alpha-bungarotoxin when expressed in tsA201 cells. Functional alpha-bungarotoxin receptors are expressed if the membrane-spanning and cytoplasmic domains of the alpha7 subunit are replaced by the homologous regions of the serotonin-3 receptor subunit. Bgt-binding surface receptors assembled from chimeric alpha7/serotonin-3 subunits contain subunits in two different conformations as shown by differences in redox state and other features of the subunits. In contrast, alpha7 subunit complexes in the same cell line contain subunits in a single conformation. The appearance of a second alpha7/serotonin-3 subunit conformation coincides with the formation of alpha-bungarotoxin-binding sites and intrasubunit disulfide bonding, apparently within the alpha7 domain of the alpha7/serotonin-3 chimera. In cell lines of neuronal origin that produce functional alpha7 receptors, alpha7 subunits undergo a conformational change similar to alpha7/serotonin-3 subunits. alpha7 subunits, thus, can fold and assemble by two different pathways. Subunits in a single conformation assemble into nonfunctional receptors, or subunits expressed in specialized cells undergo additional processing to produce functional, alpha-bungarotoxin-binding receptors with two alpha7 conformations. Our results suggest that alpha7 subunit diversity can be achieved postranslationally and is required for functional homomeric receptors.
Assuntos
Dissulfetos/química , Receptores Nicotínicos/química , Sequência de Aminoácidos , Animais , Sítios de Ligação , Bungarotoxinas/metabolismo , Linhagem Celular , Galinhas , Dissulfetos/metabolismo , Etilmaleimida/farmacologia , Humanos , Proteínas de Membrana/biossíntese , Proteínas de Membrana/química , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Nicotina/farmacologia , Oxirredução , Técnicas de Patch-Clamp , Conformação Proteica/efeitos dos fármacos , Dobramento de Proteína , Processamento de Proteína Pós-Traducional , Receptores Nicotínicos/biossíntese , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Receptores de Serotonina/biossíntese , Receptores de Serotonina/química , Receptores de Serotonina/genética , Receptores de Serotonina/metabolismo , Receptores 5-HT3 de Serotonina , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Receptor Nicotínico de Acetilcolina alfa7RESUMO
We determined O6-alkylguanine-DNA alkyltransferase (AGT) activity in the peripheral blood lymphocytes (PBLs) of normal controls and patients with Hodgkin's disease or non-Hodgkin's lymphoma and compared these values with those of Epstein-Barr virus (EBV)-transformed cell lines prepared from the same PBL samples. PBLs have an AGT level characteristic of the individual from whom the cells were obtained. The AGT activity of lymphoblastoid cell lines obtained from a control group of PBLs was significantly correlated with the activity of the PBLs from which they were derived (r = 0.742). There was no significant correlation between PBLs and EBV-transformed lines derived from these PBLs in Hodgkin's disease/non-Hodgkin's lymphoma patients (r = 0.407, -0.225, and 0.270 for patients prior to, during, or after therapy, respectively). The lack of significant correlation between lines and PBLs was not due to random fluctuations in AGT activity, because multiple lines prepared from the same PBL sample were found to be highly correlated in AGT activity. In order to account for these results, we suppose that PBLs from a given individual are a heterogeneous population with respect to AGT activity. In normal individuals, the AGT activity of early passages of the multi-clonal EBV-transformed cell lines reflect the AGT activity of the PBLs from which they were derived. Malignancy and/or treatment with chemotherapeutic agents may selectively affect those lymphocytes which are targets for EBV-transformation so that the resultant cell line is no longer representative (with respect to AGT activity) of the total PBL population. Long-term culture of lymphoblastoid cell lines results in changes in AGT activity in some but not all cell lines suggesting that with time in culture, subsets with different AGT activities may be selected. There appears to be no growth advantage of low AGT activity and only rarely have we obtained lines with no measurable AGT activity, even after long periods in culture.
Assuntos
Doença de Hodgkin/enzimologia , Linfócitos/enzimologia , Linfoma não Hodgkin/enzimologia , Metiltransferases/análise , Transformação Celular Viral , Seguimentos , Herpesvirus Humano 4 , Doença de Hodgkin/sangue , Doença de Hodgkin/tratamento farmacológico , Humanos , Linfoma não Hodgkin/sangue , Linfoma não Hodgkin/tratamento farmacológico , O(6)-Metilguanina-DNA MetiltransferaseRESUMO
Chemotherapeutic agents such as procarbazine, which produce methylated bases in DNA, are used to treat many Hodgkin's disease (HD) and non-Hodgkin's lymphoma (NHL) patients. A small proportion of such patients develop secondary malignancy. We examined the possibility that those patients who develop secondary malignancy have low endogenous levels of O6-alkylguanine DNA alkyltransferase (AGT) activity and are therefore more sensitive to the mutagenic and carcinogenic effects of their treatment. We assayed AGT activity in peripheral blood lymphocytes from patients with HD, NHL, acute nonlymphocytic leukemia (ANLL) de novo, and therapy-related ANLL, as well as a group of normal control subjects. Studies in normal controls showed that at least over a short term of 1 week, individuals have characteristic AGT levels, although some individuals sampled repeatedly over several months showed high variation. Mean AGT activities +/- SE for the various groups studied were (fmol/micrograms of DNA): normal control group, 7.05 +/- 0.36; HD and NHL patients (prior to treatment), 4.97 +/- 0.42; HD-NHL patients receiving procarbazine, 3.88 +/- 0.44; ANLL de novo, 7.78 +/- 1.72; and therapy-related ANLL, 4.30 +/- 0.58. AGT activity decreased in the peripheral blood lymphocytes of some individuals taking procarbazine. The mean AGT activity in the procarbazine-treated patients was low, as was the activity for the therapy-related ANLL patients.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/efeitos adversos , Doença de Hodgkin/enzimologia , Leucemia/induzido quimicamente , Linfócitos/enzimologia , Linfoma não Hodgkin/enzimologia , Metiltransferases/sangue , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Bleomicina/administração & dosagem , Bleomicina/efeitos adversos , Dacarbazina/administração & dosagem , Dacarbazina/efeitos adversos , Doxorrubicina/administração & dosagem , Doxorrubicina/efeitos adversos , Doença de Hodgkin/tratamento farmacológico , Humanos , Leucemia/enzimologia , Linfoma não Hodgkin/tratamento farmacológico , Mecloretamina/administração & dosagem , Mecloretamina/efeitos adversos , O(6)-Metilguanina-DNA Metiltransferase , Prednisona/administração & dosagem , Prednisona/efeitos adversos , Procarbazina/administração & dosagem , Procarbazina/efeitos adversos , Valores de Referência , Vimblastina , Vincristina/administração & dosagem , Vincristina/efeitos adversosRESUMO
The RNA associated with poly(A) sequences from Euglena gracilis and Vicia faba has been isolated by binding to millipore filters and characterized by sedimentation velocity centrifugation and electrophoretic mobility. Poly(A)-associated RNA as isolated in solution was highly aggregated. When denatured, it sedimented as a broad peak with a mean value of 16-18 S. This RNA was shown to be covalently linked to poly(A) sequences which are 150-250 nucleotides long. Our size estimates for plant poly(A) and poly(A)-associated RNA are similar to those obtained for animal cells.
Assuntos
Plantas/genética , RNA de Plantas/isolamento & purificação , Animais , Composição de Bases , Euglena gracilis , RNA Mensageiro/química , RNA de Plantas/químicaRESUMO
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.
Assuntos
DNA Viral/efeitos da radiação , DNA Polimerase Dirigida por DNA/metabolismo , Exodesoxirribonucleases/metabolismo , Mutação da Fase de Leitura , Raios Ultravioleta , Bacteriófago M13/genética , Sequência de Bases , DNA/metabolismo , DNA Viral/biossíntese , Exodesoxirribonuclease V , Micrococcus luteus/enzimologia , Dados de Sequência Molecular , Moldes Genéticos , Regiões Terminadoras GenéticasRESUMO
In vitro DNA synthesis on phi X174 or M13 templates with non-instructional lesions such as UV dimers or AP (apurinic/apyrimidinic) sites terminates one base before the site of the lesion when synthesis is catalyzed by T4 DNA polymerase or E. coli polymerase I. E. Coli polymerase I also produces termination bands at the site of AP lesions. Substitution of Mn2+ for Mg2+ and increasing the concentration of dNTP's results in elongation of the newly synthesized strand opposite the site of the lesion and beyond. Purine deoxynucleoside triphosphates are utilized for insertion opposite lesions to a greater extent than are pyrimidine deoxynucleoside triphosphates. Deoxy ATP is used almost exclusively for elongation opposite AP sites with pol I-Klenow fragment in the presence of Mg2+. We suppose that these results illustrate the previously observed greater affinity of polymerases under template-free conditions for purine nucleotides. We also suppose that the results can be used to account for mutagenic base selection on noninstructional DNA templates. If purines are preferentially selected by polymerases, then treatments which inactivate pyrimidines will lead to an excess of transitions whereas inactivation of purines will produce more transversions. Data in the literature support this hypothesis.
Assuntos
Replicação do DNA , DNA Polimerase Dirigida por DNA/metabolismo , Mutação , Bacteriófago phi X 174/genética , Sequência de Bases , Colífagos/genética , DNA Polimerase I/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , Cinética , Magnésio/farmacologia , Manganês/farmacologia , Fagos T/enzimologia , Moldes GenéticosRESUMO
A mismatch repair, proofreading deficient mutant of Escherichia coli lost a C from a C8 run at a rate 10 times higher than the loss of A from an A8 sequence in the same double mutant. This greater frameshift instability of a homopolymeric run of C's may be due to stabilization of a stacked intermediate. Gain of a (CA) unit in a similarly constructed (CA)15 sequence occurred at a rate about 1/3 that previously reported for a (CA)14 construct losing a (CA) repeat unit.
Assuntos
DNA Bacteriano/metabolismo , Escherichia coli/genética , Mutação da Fase de Leitura/genética , Sequência de Bases , DNA Bacteriano/genética , Escherichia coli/metabolismo , Dados de Sequência Molecular , Mutagênese Insercional , Sequências Repetitivas de Ácido Nucleico , Moldes GenéticosRESUMO
A small fraction of those individuals exposed to cytotoxic chemotherapy or radiation for the treatment of a primary malignant disease will develop a second malignancy some time later. Although exposure to the cytotoxic agents is believed to be the causative factor, the reason only certain individuals develop the second malignancy is unknown. Some studies have suggested that these individuals might be predisposed to cancer because of an inherent sensitivity to the alkylating agents used in cancer therapy. We have reported that these individuals with therapy-related acute nonlymphocytic leukemia (t-ANLL) have reduced endogenous levels of the repair protein O6-alkylguanine alkyltransferase (AGT). To further investigate the etiology of this disease, alkylation-induced sister-chromatid exchange (SCE) formation in individuals who developed second malignancies, was compared to other patient groups and normal controls. Peripheral blood lymphocytes from patients with (1) t-ANLL, (2) primary forms of acute nonlymphocytic leukemia (ANLL de novo), (3) patients with primary malignancies at risk of developing secondary disease, and (4) unexposed, healthy controls were treated in vitro with N-methyl-N'-nitro-nitrosoguanidine or mitomycin C. Baseline and mutagen-induced frequencies of SCEs were determined. These studies failed to detect any increased sensitivity in those patients who developed second malignancies as compared to controls or patients with de novo forms of the same disease. Also, no correlation between sensitivity to the alkylating agent N-methyl-N'-nitro-nitrosoguanidine and endogenous levels of the AGT repair protein was found. These results suggest that t-ANLL patients are not sensitive to SCE induction by either MNNG or MMC.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antineoplásicos/efeitos adversos , Leucemia Mieloide Aguda/induzido quimicamente , Linfócitos/efeitos dos fármacos , Troca de Cromátide Irmã/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Doença de Hodgkin/sangue , Doença de Hodgkin/tratamento farmacológico , Doença de Hodgkin/enzimologia , Humanos , Leucemia Mieloide Aguda/sangue , Leucemia Mieloide Aguda/enzimologia , Linfócitos/enzimologia , Linfócitos/patologia , Linfoma não Hodgkin/sangue , Linfoma não Hodgkin/tratamento farmacológico , Linfoma não Hodgkin/enzimologia , Masculino , Metilnitronitrosoguanidina , Metiltransferases/sangue , Mitomicina , Mitomicinas , O(6)-Metilguanina-DNA MetiltransferaseAssuntos
DNA/efeitos dos fármacos , Mutação , 2-Acetilaminofluoreno/farmacologia , Sequência de Bases , DNA/genética , DNA/efeitos da radiação , Dano ao DNA , DNA de Cadeia Simples/efeitos dos fármacos , DNA de Cadeia Simples/efeitos da radiação , DNA Viral/efeitos dos fármacos , DNA Viral/efeitos da radiação , Dados de Sequência MolecularRESUMO
MSRA (EC 1.8.4.6) is a member of the methionine sulfoxide reductase family that can reduce methionine sulfoxide (MetO) in proteins. This repair function has been shown to protect cells against oxidative damage. In this study we have assembled the complete gene structure of msrA and identified the presence of two distinct putative promoters that generate three different transcripts. These transcripts were cloned by 5'RACE and code for three MSRA isoforms with different N-termini. The different forms of MSRA target to distinct intracellular regions. The main MSRA transcript (msrA1) had been previously shown to target the mitochondria. MsrA2 and 3 originate from a second promoter and target the cytosol and nuclei. In the monkey retina msrA message was detected mainly in the macular RPE-choroid region while its activity was measured mainly in the soluble fractions of fractionated neural retina and RPE-choroid. The MSRA protein is found throughout the retina but is especially abundant at the photoreceptor synapses, ganglion and Müller cells. Interestingly, MSRA was not detected in the mitochondria of the photoreceptor inner segments. The RPE in the peripheral retina shows very low levels of expression but the RPE in the macular region is strongly labeled. Targeted silencing of msrA message rendered cultured RPE cells more sensitive to oxidative damage suggesting a role for MSRA in RPE protection against oxidative stress. Collectively these data suggest MSRA may play an important role in protecting macular RPE from oxidative damage.
Assuntos
Macaca mulatta/metabolismo , Estresse Oxidativo/fisiologia , Oxirredutases/genética , Retina/enzimologia , Sequência de Aminoácidos , Animais , Morte Celular , Expressão Gênica , Inativação Gênica , Humanos , Macula Lutea/enzimologia , Metionina Sulfóxido Redutases , Dados de Sequência Molecular , Fases de Leitura Aberta/genética , Oxirredutases/metabolismo , Oxirredutases/fisiologia , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Alinhamento de Sequência , Transcrição GênicaRESUMO
DNA with abasic sites has been prepared by deamination of cytosine followed by treatment of the product with uracil N-glycosylase. Termination in vitro on such templates does not occur until treatment with uracil N-glycosylase. DNA terminated one base before abasic sites created from C's has been used as a template in "second stage" reactions. With enzymes devoid or deficient in 3' greater than 5' exonuclease activity purines, particularly adenine, are preferentially added opposite the putative abasic site. 2-Aminopurine behaves more like adenine than like guanine in these experiments. Polymerase beta preferentially incorporates A opposite abasic sites produced from T, and G opposite abasic sites produced from C. We have eliminated an obvious artefact (e.g. strand switching) which might account for this observation.
Assuntos
Citosina/fisiologia , DNA Glicosilases , Replicação do DNA , Sequência de Bases , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/enzimologia , Escherichia coli/genética , N-Glicosil Hidrolases/metabolismo , Sulfitos , Moldes Genéticos , Uracila-DNA GlicosidaseRESUMO
M13 DNA containing 20-30 apurinic/apyrimidinic (AP) sites per intact circular molecule was prepared by growing phage on an ung- dut- Escherichia coli mutant and treating the DNA with uracil N-glycosylase. AP sites obstruct in vitro DNA synthesis catalyzed by E. coli pol I. The position at which termination of synthesis occurs was determined for four enzymes. T4 DNA polymerase terminates one nucleotide before putative AP sites. DNA pol I, AMV reverse transcriptase, and DNA polymerase alpha terminate synthesis either before or at the site of an AP lesion depending on the particular sequence. We determined the identity of the nucleotide inserted opposite an AP site by synthesizing up to the lesion in a first-stage reaction using T4 DNA polymerase and then determining elongation in a second stage. Purines are inserted opposite AP sites more readily than pyrimidines, and dATP is more efficient than dGTP in promoting such elongation. The DNA-dependent conversion of dNTP to dNMP was determined in mixtures of all four dNTP's by using AP DNA. The production of dAMP from dATP occurs most readily. We conclude that there is an inherent specificity for the incorporation of adenine nucleotides opposite AP sites in this in vitro system. Insofar as the model system reflects in vivo mutational events, our data suggest that depurination should produce transversions and depyrimidination should produce transitions.
Assuntos
Ácido Apurínico/genética , Colífagos/genética , DNA Glicosilases , Replicação do DNA , DNA Viral/genética , DNA Polimerase Dirigida por DNA/metabolismo , Escherichia coli/genética , Polinucleotídeos/genética , Linfoma de Burkitt , Linhagem Celular , DNA Polimerase I/metabolismo , Replicação do DNA/efeitos dos fármacos , Escherichia coli/enzimologia , Humanos , Cinética , Magnésio/farmacologia , Manganês/farmacologia , N-Glicosil Hidrolases/metabolismo , Moldes Genéticos , Uracila-DNA GlicosidaseRESUMO
RNA from wild-type Euglena, aplastidic mutant cells, and purified chloroplasts was chromatographed on oligo(dT)-cellulose at 4 degrees. The poly(A)-containing and poly(A)-lacking fractions from each were then tested in a cell-free protein-synthesizing system from wheat germ for their abilities to specifically stimulate synthesis of large subunit ribulosebisphosphate carboxylase [EC 4.1.1.39; 3-phospho-D-glycerate carboxy-lyase (dimerizing)]. The large subunit polypeptide (59,000 molecular weight) was identified in the in vitro reaction by two-dimensional electrophoresis involving isoelectric focusing and size filtration on polyacrylamide gels. Template activity for the large subunit was detected in wild-type cells but not in aplastidic mutant strains; it was highly enriched in the isolated chloroplasts. This messenger was present only in the poly(A)-lacking RNA fraction, where it constituted the most prominent template species of chloroplast RNA. The large subunit message was freed of considerable non-messenger RNA contamination and localized to the 10-20S fraction by sucrose gradient centrifugation.
Assuntos
Carboxiliases/biossíntese , Cloroplastos/análise , Herança Extracromossômica , RNA Mensageiro/isolamento & purificação , Ribulose-Bifosfato Carboxilase/biossíntese , Euglena gracilisRESUMO
Ribulose 1,5-diphosphate carboxylase (RUDPcase) from autotrophically grown Euglena gracilis was purified to homogeneity as measured by analytical ultracentrifugation, polyacrylamide gel electrophoresis, and immunoprecipitation reactions. The enzyme represented about 9% of total protein and 24% of soluble protein in the autotrophic cell. Light-grown, heterotrophic cells seemed to contain considerably less RUDPcase. Native carboxylase from autotrophic Euglena showed an s(20, w) at low protein concentrations of 17 to 17.5, suggesting a molecular weight of >500,000 daltons. Upon denaturation, the enzyme dissociated into two subunits having different amino acid compositions and molecular weights of 59,000 and 12,000 daltons. Based upon the amino acid mass ratios, a quaternary organization of 7 to 8 large and 8 to 10 small subunits per native enzyme molecule was indicated.The phylogenetic relationship of carboxylase from Euglena and from three higher plants was investigated. In general, the size, subunit formation, and quaternary structure of RUDPcase from the various sources seemed to be similar. A partial immunochemical reaction between anti-RUDPcase serum from Euglena and the enzymes from lettuce, cucumber, and New Zealand spinach suggested that the algal and higher plant carboxylases were related but not identical. This was borne out by amino acid analyses which showed a close correspondence between the large, but not the small, subunits of Euglena and lettuce.
RESUMO
We investigated the relationship between the ability to repair the O6-alkylguanine lesions and sister chromatid exchange (SCE) induction. Six human lymphoblastoid cell lines, with O6-alkylguanine alkyltransferase (AGT) activities ranging from 0 to 13.2 fmol/micrograms DNA, were tested for their sensitivity to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-, methyl methanesulfonate (MMS)- and 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-induced SCEs. L33, a long established lymphoblastoid cell line with no AGT activity, was sensitive to all three alkylating agents. In the other more recently established Epstein-Barr virus transformed cell lines, no correlation between AGT activity (ranging from 2.4 to 13.2 fmol/micrograms DNA) and sensitivity to MMS or MNNG was noted. In fact, of these five cell lines, the cell line with the highest AGT activity, line 852A, was the most sensitive to MNNG-induced SCEs. While cell lines differed in overall alkylation by MNNG, no relationship between overall akylation and sensitivity to MNNG-induced SCE formation was noted. In contrast to the results with the monofunctional alkylating agents, there was a correlation between AGT activity and BCNU-sensitivity to SCE induction. Cell lines with low AGT activities were more sensitive to the bifunctional alkylating agent than cells with higher activities. Therefore, while DNA interstrand cross-links produced by BCNU exposure probably underlie SCE induction by this agent, the lesions and processes that lead to SCE induction after exposure to monofunctional alkylating agents remain unclear.
Assuntos
Alquilantes/farmacologia , Linfócitos/efeitos dos fármacos , Metiltransferases/metabolismo , Troca de Cromátide Irmã , Carmustina/farmacologia , Linhagem Celular Transformada , Humanos , Metanossulfonato de Metila/farmacologia , Metilnitronitrosoguanidina/farmacologia , O(6)-Metilguanina-DNA MetiltransferaseRESUMO
The role of the proofreading exonuclease in maintaining the stability of multiply repeated units in DNA was studied in Escherichia coli. Reversion of plasmids in which the beta-galactosidase alpha complementing sequence was moved +2 out of frame by inserts containing (CA)14, (CA)5, (CA)2 or (TA)6 or +1 by creating a run of 8 C was compared in mutS and mutSdnaQ strains. Proofreading corrects at least half of the frameshift errors for all the plasmids and at least 99% of the errors in the (CA)2 plasmid. The (CA)2 plasmid reverts mostly by +1 frameshifts in the restriction sites flanking the insert. With the (CA)14, (TA)6, (CA)5 and 8C plasmids, reversion is mainly by loss of a repeat unit. The data support the hypothesis that the dnaQgene product recognizes frameshifts close to the DNA growing point. Frameshifts distal to the growing point are mainly corrected by mismatch repair. We speculate that mismatches in mononucleotide repeats are susceptible to proofreading because they can either migrate to a point where they are recognized by the exonuclease or, alternatively, because single nucleotide distortions are more readily detected than dinucleotides.
Assuntos
Reparo do DNA , Repetições de Microssatélites , Bacteriófago M13 , Sequência de Bases , DNA Polimerase III/genética , Escherichia coli/genética , Exonucleases/genética , Mutação da Fase de Leitura , Dosagem de Genes , Dados de Sequência Molecular , Mutagênese , PlasmídeosRESUMO
The bypass of lesions in DNA with insertion of nucleotides opposite damaged bases has been studied as a model for mutagenesis in an in vitro system. Lesions introduced by dimethyl sulfate at adenines and by ultraviolet light at pyrimidine dimers act as termination sites on both double- and single-stranded DNA templates. Base selection opposite noninformational lesions is, in part, a property of the polymerases: different polymerases have different selectivities although all polymerases tested seem to prefer purines. The ability to insert "incorrect" bases is determined in part by the sequence 5' to the lesion on the template strand. The hypothesis that damaged purines tend to result in transversions can be applied to published data on activation of the c-ras oncogene.
Assuntos
Mutação , Sequência de Bases , Reparo do DNA , Replicação do DNA , DNA de Cadeia Simples/genética , Técnicas In Vitro , Cinética , Modelos Biológicos , Moldes GenéticosRESUMO
DNA repair reactions are under cellular control. In bacteria, the reactions removing 0(6)-methylguanine and 3-methyladenine are inducible. It is not clear whether similar inducibility occurs in human lymphoblastoid cells. Nonetheless, the ability to manufacture the 0(6)-methylguanine acceptor protein does seem to be controlled by some chromosomal mechanism which is superimposed on the structural gene. This control system may affect reactions other than the removal of 0(6)-methylguanine. Insofar as this is so, transformed human lymphoblastoid cells have a system reminiscent of that found in bacteria.
Assuntos
Reparo do DNA , Guanina/análogos & derivados , Animais , Linhagem Celular , Guanina/metabolismo , Humanos , Hibridização Genética , MutaçãoRESUMO
Previous studies have shown that alkylation of MS2 RNA by certain derivatives of polycyclic aromatic hydrocarbons renders it noninfectious. Since phage RNA serves as a template for translation and transcription, either of these RNA-directed processes, or both, could be responsible in vivo for the inhibition of phage replication by metabolically activated hydrocarbons. The present study correlates the degree of inhibition of MS2 RNA infectivity, at various levels of alkylation by (+/-)-trans, 7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzol[a]pyrene, with the translation efficiency in vitro of the same alkylated RNA for the synthesis of viral synthetase and of maturation and coat proteins. The results indicate that dihydroxyepoxy-tetrahydrobenzo[a]pyrene modification of MS2 RNA impairs its template capacity for the synthesis of phage-specific proteins; this inhibition is insufficient, however, to account for the loss of RNA infectivity at lower molar ratios of alkylation. For the three viral proteins synthesized in vitro, the translation of RNA synthetase is much more sensitive to MS2 RNA modification than either coat or maturation protein synthesis. Our results also indicate that the loss of viral RNA infectivity follows a single-hit inactivation mechanism, whereas several alkylation events in the viral RNA synthetase cistron may be necessary to block translation of this gene product.