RESUMEN
We tested the hypothesis that brown-headed cowbirds (Molothrus ater) harbor Sarcocystis neurona, the agent of equine protozoal myeloencephalitis (EPM), and act as intermediate hosts for this parasite. In summer 1999, wild caught brown-headed cowbirds were collected and necropsied to determine infection rate with Sarcocystis spp. by macroscopic inspection. Seven of 381 (1.8%) birds had grossly visible sarcocysts in leg muscles with none in breast muscles. Histopathology revealed two classes of sarcocysts in leg muscles, thin-walled and thick-walled suggesting two species. Electron microscopy showed that thick-walled cysts had characteristics of S. falcatula and thin-walled cysts had characteristics of S. neurona. Thereafter, several experiments were conducted to confirm that cowbirds had viable S. neurona that could be transmitted to an intermediate host and cause disease. Specific-pathogen-free opossums fed cowbird leg muscle that was enriched for muscle either with or without visible sarcocysts all shed high numbers of sporocysts by 4 weeks after infection, while the control opossum fed cowbird breast muscle was negative. These sporocysts were apparently of two size classes, 11.4+/-0.7 microm by 7.6+/-0.4 microm (n=25) and 12.6+/-0.6 microm by 8.0+/-0 microm (n=25). When these sporocysts were excysted and introduced into equine dermal cell tissue culture, schizogony occurred, most merozoites survived and replicated long term and merozoites sampled from the cultures with long-term growth were indistinguishable from known S. neurona isolates. A cowbird Sarcocystis isolate, Michigan Cowbird 1 (MICB1), derived from thin-walled sarcocysts from cowbirds that was passaged in SPF opossums and tissue culture went on to produce neurological disease in IFNgamma knockout mice indistinguishable from that of the positive control inoculated with S. neurona. This, together with the knowledge that S. falcatula does not cause lesions in IFNgamma knockout mice, showed that cowbird leg muscles had a Sarcocystis that fulfills the first aim of Koch's postulates to produce disease similar to S. neurona. Two molecular assays provided further support that both S. neurona and S. falcatula were present in cowbird leg muscles. In a blinded study, PCR-RFLP of RAPD-derived DNA designed to discriminate between S. neurona and S. falcatula showed that fresh sporocysts from the opossum feeding trial had both Sarcocystis species. Visible, thick-walled sarcocysts from cowbird leg muscle were positive for S. falcatula but not S. neurona; thin-walled sarcocysts typed as S. neurona. In 1999, DNA was extracted from leg muscles of 100 wild caught cowbirds and subjected to a PCR targeting an S. neurona specific sequence of the small subunit ribosomal RNA (SSU rRNA) gene. In control spiking experiments, this assay detected DNA from 10 S. neurona merozoites in 0.5g of muscle. In the 1999 experiment, 23 of 79 (29.1%) individual cowbird leg muscle samples were positive by this S. neurona-specific PCR. Finally, in June of 2000, 265 cowbird leg muscle samples were tested by histopathology for the presence of thick- and thin-walled sarcocysts. Seven percent (18/265) had only thick-walled sarcocysts, 0.8% (2/265) had only thin-walled sarcocysts and 1.9% (5/265) had both. The other half of these leg muscles when tested by PCR-RFLP of RAPD-derived DNA and SSU rRNA PCR showed a good correlation with histopathological results and the two molecular typing methods concurred; 9.8% (26/265) of cowbirds had sarcocysts in muscle, 7.9% (21/265) had S. falcatula sarcocysts, 1.1% (3/265) had S. neurona sarcocysts, and 0.8% (2/265) had both. These results show that some cowbirds have S. neurona as well as S. falcatula in their leg muscles and can act as intermediate hosts for both parasites.
Asunto(s)
Enfermedades de las Aves/parasitología , Sarcocystis/aislamiento & purificación , Sarcocistosis/veterinaria , Pájaros Cantores/parasitología , Animales , Caballos , Interacciones Huésped-Parásitos , Interferón gamma/genética , Interferón gamma/metabolismo , Ratones , Ratones Noqueados , Músculo Esquelético/parasitología , Zarigüeyas/parasitología , Filogenia , Reacción en Cadena de la Polimerasa/veterinaria , Polimorfismo de Longitud del Fragmento de Restricción , Sarcocystis/genética , Sarcocistosis/parasitología , Sensibilidad y Especificidad , Piel/citología , Piel/parasitología , Organismos Libres de Patógenos EspecíficosRESUMEN
A hallmark of human DNA polymerase iota (poliota) is the asymmetric fidelity of replication at template A and T when the enzyme extends primers annealed to a single-stranded template. Here, we report on the efficiency and accuracy of poliota-dependent replication at a nick, a gap, the very end of a template and from a mispaired primer. Poliota cannot initiate synthesis on a nicked DNA substrate, but fills short gaps efficiently. Surprisingly, poliota's ability to blunt-end a 1 bp recessed terminus is dependent upon the template nucleotide encountered and is highly erroneous. At template G, both C and T are inserted with roughly equal efficiency, whilst at template C, C and A are misinserted 8- and 3-fold more often than the correct base, G. Using substrates containing mispaired primer termini, we show that poliota can extend all 12 mispairs, but with differing efficiencies. Poliota can also extend a tandem mispair, especially when it is located within a short gap. The enzymatic properties of poliota appear consistent with that of a somatic hypermutase and suggest that poliota may be one of the low-fidelity DNA polymerases hypothesized to participate in the hypermutation of immunoglobulin variable genes in vivo.
Asunto(s)
Disparidad de Par Base , Replicación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Animales , Linfocitos B/enzimología , Secuencia de Bases , Cartilla de ADN , Humanos , Cinética , Ratones , Ratones Endogámicos BALB C , Datos de Secuencia Molecular , Bazo/enzimología , Bazo/inmunología , Especificidad por Sustrato , Moldes Genéticos , ADN Polimerasa iotaRESUMEN
The third complementarity-determining region (CDR3) of immunoglobulin variable genes for the heavy chain (VH) has been shown to be shorter in length in hypermutated antibodies than in non-hypermutated antibodies. To determine which components of CDR3 contribute to the shorter length, and if there is an effect of age on the length, we analysed 235 cDNA clones from human peripheral blood of VH6 genes rearranged to immunoglobulin M (IgM) constant genes. There was similar use of diversity (D) and joining (JH) gene segments between clones from young and old donors, and there was similar use of D segments among the mutated and non-mutated heavy chains. However, in the mutated heavy chains, there was increased use of shorter JH4 segments and decreased use of longer JH6 segments compared to the non-mutated proteins. The overall length of CDR3 did not change with age within the mutated and non-mutated categories, but was significantly shorter by three amino acids in the mutated clones compared to the non-mutated clones. Analyses of the individual components that comprise CDR3 indicated that they were all shorter in the mutated clones. Thus, there were more nucleotides deleted from the ends of VH, D, and JH gene segments, and fewer P and N nucleotides added. The results suggest that B cells bearing immunoglobulin receptors with shorter CDR3s have been selected for binding to antigen. A smaller CDR3 may allow room in the antibody binding pocket for antigen to interact with CDRs 1 and 2 as well, so that as the VDJ gene undergoes hypermutation, substitutions in all three CDRs can further contribute to the binding energy.
Asunto(s)
Regiones Determinantes de Complementariedad/genética , Genes de Inmunoglobulinas/inmunología , Mutación , Adulto , Anciano , Envejecimiento/genética , Envejecimiento/inmunología , Diversidad de Anticuerpos/genética , Secuencia de Bases , Codón/genética , ADN Complementario/genética , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Nucleótidos/genética , Relación Estructura-ActividadRESUMEN
Werner syndrome (WS) is a human premature aging syndrome, which is associated with high frequencies of neoplasia and genetic instability. We have examined the occurrence of microsatellite instability, which may result from defective mismatch repair, in lymphoblastoid cell lines derived from nine WS patients. Instability was measured at the D2S123 locus by gel analysis of PCR products. Three WS cell lines had 4-13% altered alleles, compared with 0% in the other six lines. The increased frequency of microsatellite instability could not readily be associated with overt cancer or any other known clinical condition in the three patients. To examine whether the WS defect affected the humoral immune system, we measured the hypermutation of immunoglobulin variable genes in peripheral blood cells from the WS patient who donated the cell line with the highest frequency of microsatellite instability. The frequency and pattern of mutation was similar to that from normal individuals, suggesting that the Werner protein is not involved in generating hypermutation.
Asunto(s)
Reordenamiento Génico de Cadena Pesada de Linfocito B , Región Variable de Inmunoglobulina/genética , Repeticiones de Microsatélite , Mutación , Síndrome de Werner/genética , Secuencia de Bases , Línea Celular , Regiones Determinantes de Complementariedad , ADN Complementario , Humanos , Datos de Secuencia Molecular , Síndrome de Werner/inmunologíaRESUMEN
Chronological aging is associated with an accumulation of DNA mutations that results in cancer formation. The effect of aging on spontaneous mutations in humans is difficult to study because mutations are infrequent in the overall genome and tumors are relatively rare. In contrast, somatic mutations in immunoglobulin variable genes are abundant and can be studied in peripheral blood lymphocytes. To determine if aging alters the frequency and pattern of hypermutation, we sequenced 331 cDNA clones with rearranged V(H)6 genes and compared 452 mutations from young humans to 570 mutations from old humans. There were more mutated clones in the young population compared to the old population. Among the mutated clones, the frequency, location, and types of substitutions were similar between the young and the old groups. However, the ratio of replacement-to-silent mutations was much higher in the complementarity-determining regions of heavy chains from old people, which indicates that their B cells had been selected by antigen. Among individuals, there was variability in the frequency of tandem mutations, which we have observed in mice defective for the PMS2 mismatch repair protein. Microsatellite variability in DNA, which is caused by impaired mismatch repair, was then measured, and there was a strong correlation between the frequency of tandem mutations and microsatellite alterations. The data suggest that individuals vary in their mismatch repair capacity, which can affect the mutational spectra in their antibodies.
Asunto(s)
Envejecimiento/genética , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , MutaciónRESUMEN
To determine whether DNA polymerase eta plays a role in the hypermutation of immunoglobulin variable genes, we examined the frequency and pattern of substitutions in variable VH6 genes from the peripheral blood lymphocytes of three patients with xeroderma pigmentosum variant disease, whose polymerase eta had genetic defects. The frequency of mutation was normal but the types of base changes were different: there was a decrease in mutations at A and T and a concomitant rise in mutations at G and C. We propose that more than one polymerase contributes to hypermutation and that if one is absent, others compensate. The data indicate that polymerase eta is involved in generating errors that occur predominantly at A and T and that another polymerase(s) may preferentially generate errors opposite G and C.
Asunto(s)
ADN Polimerasa Dirigida por ADN/metabolismo , Genes de Inmunoglobulinas , Mutación , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Emparejamiento Base , Secuencia de Bases , ADN/genética , ADN Polimerasa Dirigida por ADN/genética , Humanos , Región Variable de Inmunoglobulina/genética , Ratones , Datos de Secuencia Molecular , Xerodermia Pigmentosa/enzimología , Xerodermia Pigmentosa/genética , Xerodermia Pigmentosa/inmunologíaRESUMEN
Affinity maturation of the humoral immune response is based on the ability of immunoglobulin variable genes to undergo a process of rapid and extensive somatic mutation followed by antigenic selection for antibodies with higher affinity. While the behaviour of this somatic hypermutation phenomenon has been well characterized over the last 20 years, the molecular mechanism responsible for inserting mutations has remained shrouded. To better understand this mechanism, we studied the interplay between hypermutation and other DNA associated activities such as DNA repair. There was no effect on the frequency and pattern of hypermutation in mice deficient for nucleotide excision repair, base excision repair and ataxia-telangiectasia mutated gene repair of double strand breaks. However, variable genes from mice lacking some components of mismatch repair had an increased frequency of tandem mutations and had more mutations of G and C nucleotides. These results suggest that the DNA polymerase(s) involved in the hypermutation pathway produces a unique spectra of mutations, which is then altered by mismatch repair and antigenic selection. We, also describe the differential pattern of expression of some nuclear DNA polymerases in hypermutating versus non-hypermutating B lymphocytes. The rapidly dividing germinal centre B cells expressed DNA polymerases alpha, beta, delta, epsilon and zeta, whereas the resting non-germinal centre cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta and zeta. The lack of expression of polymerase epsilon in the non-germinal centre cells suggests that this enzyme has a critical role in chromosomal replication but does not participate in DNA repair in these cells.
Asunto(s)
Reparación del ADN/genética , Ratones Mutantes , Mutación , Animales , Proteínas de la Ataxia Telangiectasia Mutada , Disparidad de Par Base , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Ratones , Proteínas Serina-Treonina Quinasas/genética , Proteínas Supresoras de TumorRESUMEN
Substantial antibody variability is created when nucleotide substitutions are introduced into immunoglobulin variable genes by a controlled process of hypermutation. Evidence points to a mechanism involving DNA repair events at sites of targeted breaks. In vertebrate cells, there are many recently identified DNA polymerases that inaccurately copy templates. Some of these are candidates for enzymes that introduce base changes during hypermutation. Recent research has focused on possible roles for DNA polymerases zeta (POLZ), eta (POLH), iota (POLI), and mu (POLM) in the process.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Genes de Inmunoglobulinas , Mutación , Animales , Diversidad de Anticuerpos , Reparación del ADN/genética , Reparación del ADN/inmunología , ADN Polimerasa Dirigida por ADN/metabolismo , Humanos , Región Variable de Inmunoglobulina/genética , Modelos Genéticos , Modelos InmunológicosRESUMEN
The REV3 gene encodes the catalytic subunit of DNA polymerase (pol) zeta, which can replicate past certain types of DNA lesions [1]. Saccharomyces cerevisiae rev3 mutants are viable and have lower rates of spontaneous and DNA-damage-induced mutagenesis [2]. Reduction in the level of Rev31, the presumed catalytic subunit of mammalian pol zeta, decreased damage-induced mutagenesis in human cell lines [3]. To study the function of mammalian Rev31, we inactivated the gene in mice. Two exons containing conserved DNA polymerase motifs were replaced by a cassette encoding G418 resistance and beta-galactosidase, under the control of the Rev3l promoter. Surprisingly, disruption of Rev3l caused mid-gestation embryonic lethality, with the frequency of Rev3l(-/-) embryos declining markedly between 9.5 and 12.5 days post coitum (dpc). Rev3l(-/-) embryos were smaller than their heterozygous littermates and showed retarded development. Tissues in many areas were disorganised, with significantly reduced cell density. Rev3l expression, traced by beta-galactosidase staining, was first detected during early somitogenesis and gradually expanded to other tissues of mesodermal origin, including extraembryonic membranes. Embryonic death coincided with the period of more widely distributed Rev3l expression. The data demonstrate an essential function for murine Rev31 and suggest that bypass of specific types of DNAlesions by pol zeta is essential for cell viability during embryonic development in mammals.
Asunto(s)
ADN Polimerasa Dirigida por ADN/genética , Proteínas Fúngicas/genética , Regulación del Desarrollo de la Expresión Génica , Genes Letales , Proteínas de Saccharomyces cerevisiae , Animales , Humanos , RatonesRESUMEN
DNA polymerases may be differentially expressed by cells during periods of quiescence and proliferation. Murine B cells are an ideal population to study because their division time varies widely in vivo, and different subsets can be easily isolated. Consequently, we analyzed RNA from resting cells (B220(+)peanut agglutinin(-)) and activated germinal center cells (B220(+)peanut agglutinin(+)) from spleens by reverse transcriptase-PCR using primers for five nuclear polymerases and their associated subunits. Gel analyses of the amplified products showed that the rapidly-dividing germinal center B cells expressed DNA polymerases alpha, beta, delta, epsilon, and zeta. The resting B cells did not express polymerases alpha or epsilon at detectable levels, although they did express polymerases beta, delta, and zeta. Thus, polymerase epsilon, as well as alpha, appears to have a primary role in chromosomal replication of murine B lymphocytes. Further, the lack of expression of polymerase epsilon in resting cells indicates that this enzyme is not used in any DNA repair pathways by these cells. The expression of polymerase zeta by resting cells suggests that it has another role in DNA repair, perhaps recombination, in addition to its function of bypassing damage during chromosomal replication.
Asunto(s)
Subgrupos de Linfocitos B/metabolismo , ADN Polimerasa II/metabolismo , Reparación del ADN , Replicación del ADN , Activación de Linfocitos/genética , Animales , Separación Celular , Centro Germinal/citología , Inmunización , Ratones , Ratones Endogámicos BALB C , Aglutinina de Mani , Bazo/citologíaRESUMEN
We studied tumorigenesis and p53 immunostaining in a murine transgenic model introducing E1A/E1B under the control of the mouse mammary tumor virus-long terminal repeat (MMTV-LTR) promoter in which adenocarcinoma occurs at the squamocolumnar junction in the foregut, predominantly in males, and at no other site. Mutations of p53 are frequent in human esophageal adenocarcinoma and the E1B gene product interferes with p53-mediated apoptosis, inhibiting tumor suppression at the G(1)/S checkpoint. Transgenic animals were generated utilizing a purified linear 6.7 kb fragment of plasmid DNA containing MMTV-LTR/E1A/E1B and were confirmed by dot blot hybridization of tail DNA to (32)P-labeled E1A/E1B probe and polymerase chain reaction (PCR) amplification of E1A. Transgenic and control animals were observed for morbidity and weight changes. Eleven of 45 animals were transgenic (24% efficiency) with an estimated 5 to 57 copies of the gene per genome. Profound weight loss (>20%) led to sacrifice or death of one of five females (at 12 weeks) and four of six males (at 16 to 17 weeks). Grossly visible tumors (2 to 10 mm) were noted in the forestomach at the visible margin between the proximal (squamous-lined) stomach and the distal glandular stomach. Histologic sections confirmed adenocarcinoma arising in each case at the squamocolumnar junction with glandular formation, pleomorphism, and frequent mitotic figures. Immunostaining was positive for p53 indicating accumulation of mutated or altered p53 protein. E1A/E1B transgenic animals developed macroscopic and microscopic adenocarcinoma at the squamocolumnar junction, which corresponds to adenocarcinoma at the human esophagogastric junction. Disruption of p53 was present in the transgenic model as in the human cancer.
Asunto(s)
Adenocarcinoma/genética , Neoplasias Esofágicas/genética , Genes p53/genética , Neoplasias Gástricas/genética , Adenocarcinoma/patología , Proteínas E1A de Adenovirus/genética , Proteínas E1B de Adenovirus/genética , Animales , Modelos Animales de Enfermedad , Neoplasias Esofágicas/patología , Femenino , Inmunohistoquímica , Masculino , Ratones , Ratones Transgénicos , Mutación , Plásmidos , Reacción en Cadena de la Polimerasa , Regiones Promotoras Genéticas , Neoplasias Gástricas/patologíaRESUMEN
Severe combined immunodeficient (Scid) mice have a mutation in the catalytic subunit of the DNA binding protein kinase that is involved in repair of double-strand breaks in DNA. To determine if the protein also influences repair of single-strand breaks, we examined the ability of Scid cells to repair lesions introduced by ultraviolet light and gamma-ray irradiation. DNA repair was measured both in total genomic DNA and in specific genes from murine Scid and wildtype fibroblast cell lines. The removal of pyrimidine dimers and repair of strand breaks in genes was measured using quantitative Southern blot analyses. After ultraviolet irradiation, there was no significant difference in the repair of photoproducts in bulk DNA between Scid and wildtype cells, as measured by cellular survival and unscheduled DNA synthesis. However, deficient repair was evident in genes, where Scid cells had 25-50% less repair in the c-myc and dihydrofolate reductase genes. After gamma-irradiation, Scid fibroblasts had 20-35% less repair of DNA breaks in immunoglobulin kappa and heavy constant genes than wildtype cells. The data suggest that intact DNA-PK enzyme is needed for the efficient operation of cellular repair of pyrimidine dimers and single-strand breaks in genes, as well as in its established role in rejoining double-strand breaks.
Asunto(s)
Reparación del ADN/genética , Regulación de la Expresión Génica , Ratones SCID/genética , Animales , Línea Celular Transformada , Daño del ADN/efectos de la radiación , Ratones , Piridinas , Rayos UltravioletaRESUMEN
During somatic hypermutation of Ig V genes, mismatched nucleotide substitutions become candidates for removal by the DNA mismatch repair pathway. Previous studies have shown that V genes from mice deficient for the MSH2 and PMS2 mismatch repair proteins have frequencies of mutation that are comparable with those from wild-type (wt) mice; however, the pattern of mutation is altered. Because the absence of MSH2 and PMS2 produced different mutational spectra, we examined the role of another protein involved in mismatch repair, MLH1, on the frequency and pattern of hypermutation. MLH1-deficient mice were immunized with oxazolone Ag, and splenic B cells were analyzed for mutations in their V kappa Ox1 light chain genes. Although the frequency of mutation in MLH1-deficient mice was twofold lower than in wt mice, the pattern of mutation in Mlh1-/- clones was similar to wt clones. These findings suggest that the MLH1 protein has no direct effect on the mutational spectrum.
Asunto(s)
Disparidad de Par Base/inmunología , Reparación del ADN/inmunología , Genes de Inmunoglobulinas , Región Variable de Inmunoglobulina/genética , Mutación/inmunología , Proteínas de Neoplasias/deficiencia , Proteínas Adaptadoras Transductoras de Señales , Animales , Linfocitos B/inmunología , Composición de Base/inmunología , Proteínas Portadoras , Células Clonales/inmunología , Análisis Mutacional de ADN , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Ratones Noqueados , Homólogo 1 de la Proteína MutL , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/fisiología , Proteínas NuclearesRESUMEN
DNA fragmentation during apoptosis is characterized by endonucleolytic cleavage of chromosomal DNA into an oligonucleosomal ladder. To determine if actively transcribed genes are more susceptible to cleavage during apoptosis than non-transcribed genes, the rate of fragmentation of differentially expressed genes was measured in B-lymphocyte hybridoma cells. Five genes were studied based on their transcriptional activity and/or nuclear localization, and mitochondrial DNA was assayed as a negative control for apoptotic fragmentation. Apoptosis was induced in the hybridoma cells by ultraviolet light, and DNA was prepared at multiple time points after ultraviolet irradiation. Degradation into an oligonucleosomal ladder appeared as early as 2 h after treatment, showing that fragmentation is rapidly activated in hybridoma cells. The DNA was then digested with restriction enzymes, separated by gel electrophoresis and hybridized with the gene-specific probes for Southern blot analyses. Loss of gene-specific signals was measured by quantitation of autoradiographs. The results show all of the nuclear genes were degraded at the same rate regardless of their transcriptional status or nuclear localization. The data suggest that once the cell activates its destruction program, nuclear DNA is rapidly degraded in a homogeneous manner.
Asunto(s)
Apoptosis/genética , Fragmentación del ADN , ADN/genética , ADN/metabolismo , Animales , Apoptosis/efectos de la radiación , Linfocitos B/patología , Linfocitos B/fisiología , Hibridomas , Ratones , Rayos UltravioletaRESUMEN
BACKGROUND: Topical application of growth factors to wounds has proven to be suboptimal in achieving epithelial growth and accelerating healing. We propose transfection of fibroblasts with a gene for acidic fibroblast growth factor (aFGF) which will allow continuous, local delivery of the growth factor to wounds, ulcerative lesions, or healing tissues. METHODS: We utilized a pMEXneo vector containing the human aFGF gene with a secretory signal sequence from the hst/KS3 gene to obtain continuous secretion of therapeutic doses of aFGF. NIH 3T3 fibroblasts were transfected using a liposomal transfection reagent and grown in selective media. RESULTS: Dot blot hybridization with labeled complementary DNA probes revealed the presence of plasmid DNA in transfected but not wild type fibroblasts. Intracellular concentrations of aFGF remained low in transfected cells; however, the media contained high levels (32 +/- 7 nM) of aFGF as measured by ELISA. Concentrations of aFGF capable of stimulating cell proliferation were maintained for several weeks. CONCLUSIONS: The aFGF cDNA was transcribed and translated into a functional polypeptide that is secreted from NIH 3T3 cells at physiologically significant concentrations. Stable transfection with a eukaryotic vector which induces secretion of aFGF at levels promoting cell growth holds promise for clinical application in wounds or healing tissue. Transfection could be achieved by topical or endoscopic injection of this type of vector.
Asunto(s)
Factor 1 de Crecimiento de Fibroblastos/genética , Técnicas de Transferencia de Gen , Plásmidos , Cicatrización de Heridas/fisiología , Células 3T3/citología , Células 3T3/metabolismo , Animales , ADN Complementario , Expresión Génica , Immunoblotting , Ratones , Transfección/métodosRESUMEN
Rearranged immunoglobulin variable genes are extensively mutated after stimulation of B lymphocytes by antigen. Mutations are likely generated by an error-prone DNA polymerase, and the mismatch repair pathway may process the mispairs. To examine the role of the MSH2 mismatch repair protein in hypermutation, Msh2-/- mice were immunized with oxazolone, and B cells were analyzed for mutation in their VkappaOx1 light chain genes. The frequency of mutation in the repair-deficient mice was similar to that in Msh2+/+ mice, showing that MSH2-dependent mismatch repair does not cause hypermutation. However, there was a striking bias for mutations to occur at germline G and C nucleotides. The results suggest that the hypermutation pathway frequently mutates G.C pairs, and a MSH2-dependent pathway preferentially corrects mismatches at G and C.
Asunto(s)
Citosina , Reparación del ADN , Proteínas de Unión al ADN , Guanina , Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Mutación , Proteínas Proto-Oncogénicas/fisiología , Animales , Composición de Base , Secuencia de Bases , Eliminación de Gen , Reordenamiento Génico de Linfocito B , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteína 2 Homóloga a MutS , Ácidos Nucleicos Heterodúplex , Oxazolona/inmunología , Proteínas Proto-Oncogénicas/deficiencia , Proteínas Proto-Oncogénicas/genéticaRESUMEN
Mutations are introduced into rearranged Ig variable genes at a frequency of 10(-2) mutations per base pair by an unknown mechanism. Assuming that DNA repair pathways generate or remove mutations, the frequency and pattern of mutation will be different in variable genes from mice defective in repair. Therefore, hypermutation was studied in mice deficient for either the DNA nucleotide excision repair gene Xpa or the mismatch repair gene Pms2. High levels of mutation were found in variable genes from XPA-deficient and PMS2-deficient mice, indicating that neither nucleotide excision repair nor mismatch repair pathways generate hypermutation. However, variable genes from PMS2-deficient mice had significantly more adjacent base substitutions than genes from wild-type or XPA-deficient mice. By using a biochemical assay, we confirmed that tandem mispairs were repaired by wild-type cells but not by Pms2(-/-) human or murine cells. The data indicate that tandem substitutions are produced by the hypermutation mechanism and then processed by a PMS2-dependent pathway.
Asunto(s)
Adenosina Trifosfatasas , Anticuerpos/genética , Enzimas Reparadoras del ADN , Proteínas de Unión al ADN , Genes de Inmunoglobulinas , Mutación , Proteínas/genética , Animales , Anticuerpos/inmunología , Secuencia de Bases , Reparación del ADN , Humanos , Región Variable de Inmunoglobulina/genética , Ratones , Ratones Noqueados/genética , Ratones Noqueados/inmunología , Endonucleasa PMS2 de Reparación del Emparejamiento Incorrecto , Datos de Secuencia MolecularRESUMEN
The immunoglobulin loci are uniquely unstable regions of the genome which undergo as much mutation and selection in a matter of days as a species can undergo in generations of evolution. We have studied the mutational pattern and targeting of this unusual hypermutation process over the past 16 years. The pattern of somatic mutations in rearranged variable (V) genes differs from the pattern of meiotic mutations, indicating that a different mechanism generates hypermutation than generates spontaneous mutation. Hypermutations begin on the 5' end of rearranged V genes downstream of the transcription initiation site and continue through the V exon and into the 3'-flanking region before tapering off. Mutations are located randomly throughout the DNA sequence and exhibit strand bias. The targeting of mutations to the region in and around the rearranged V gene appears to require interactions between the promoter and downstream intronic DNA sequences. The same mechanism that initiates hypermutation around V genes may also produce double-strand breaks that catalyze homologous recombination between rearranged V genes on two chromosomal alleles. With this data we have built a model of hypermutation which predicts that V-region DNA is destabilized at the nuclear matrix during transcription and undergoes strand breaks.