RESUMEN
We have created a kappa transgene in which a polymerase (pol) III promoter replaces the pol II promoter. Two independent transgenic lines show somatic hypermutation of the transgene in B cells from hyperimmunized mice. Both lines transcribe transgenes from the pol III promoter in the liver. However, in spleen and spleen B cell-derived hybridomas, they also transcribe mRNA from pol II promoters located within the 3' kappa enhancer of the preceding transgene copy in a tandem transgene array. The findings demonstrate that in an array of multiple transgenes the expression (and somatic hypermutation) of an individual transgene copy must be considered in the context of the other copies. We also show that sequences around the 3' kappa enhancer in endogenous genes are transcribed. The possible role of these promoters in endogenous kappa gene expression is discussed. An unrelated finding in this study was a novel RNA splice in one hybridoma.
Asunto(s)
Elementos de Facilitación Genéticos , Cadenas kappa de Inmunoglobulina/genética , Regiones Promotoras Genéticas , ARN Polimerasa II/genética , Animales , Secuencia de Bases , ADN/genética , Cartilla de ADN/genética , Expresión Génica , Genes de Inmunoglobulinas , Hibridomas , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación , ARN Polimerasa III/genética , Empalme del ARNRESUMEN
Gene-targeted mice were generated with a loxP-neomycin resistance gene (neo(r)) cassette inserted upstream of the Jlambda1 region and replacement of the glycine 154 codon in the Clambda1 gene with a serine codon. This insertion dramatically increases Vlambda1-Jlambda1 recombination. Jlambda1 germline transcription levels in pre-B cells and thymus cells are also greatly increased, apparently due to the strong housekeeping phosphoglycerine kinase (PGK) promoter driving the neo gene. In contrast, deletion of the neo gene causes a significant decrease in VJlambda1 recombination to levels below those in normal mice. This reduction is due to the loxP site left on the chromosome which reduces the Jlambda1 germline transcription in cis. Thus, the correlation between germline transcription and variable (V), diversity (D), and joining (J) recombination is not just an all or none phenomenon. Rather, the transcription efficiency is directly associated with the recombination efficiency. Furthermore, Jlambda1 and Vlambda1 germline transcription itself is not sufficient to lead to VJ recombination in T cells or early pre-B cells. The findings may suggest that in vivo: (a) locus and cell type-specific transactivators direct the immunoglobulin or T cell receptor loci, respectively, to a "recombination factory" in the nucleus, and (b) transcription complexes deliver V(D)J recombinase to the recombination signal sequences.
Asunto(s)
Reordenamiento Génico de Cadena Ligera de Linfocito B , Región de Unión de la Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Cadenas lambda de Inmunoglobulina/genética , Alelos , Animales , Secuencia de Bases , Mapeo Cromosómico , Marcación de Gen , Genes de Inmunoglobulinas , Células Germinativas , Recuento de Linfocitos , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Mutagénesis Insercional , Neomicina , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Regiones Promotoras GenéticasRESUMEN
Somatic hypermutation (SHM) of immunoglobulin (Ig) genes is a highly specific mechanism restricted to B lymphocytes during only a few cell generations. Data presented here suggest that transcription of the target genes is required, but not sufficient for SHM. Presumably, cis-acting elements, such as those present in the Ig enhancers, are required to target a mutator factor (MuF) to Ig and human BCL-6 genes. It is postulated that the MuF travels with the transcribing RNA polymerase and is deposited on the target gene when the polymerase pauses. Point mutations, and rare deletions and insertions, are created by the combined actions of MuF and certain DNA polymerases. A subset of the mutations is corrected during SHM by DNA mismatch repair.
Asunto(s)
Linfocitos B/fisiología , Proteínas de Unión al ADN/genética , Inmunoglobulinas/genética , Mutación , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Animales , Reparación del ADN/genética , Humanos , Ratones , Modelos Genéticos , Proteínas Proto-Oncogénicas c-bcl-6 , Transcripción GenéticaRESUMEN
Large-scale genomic sequencing projects have provided DNA sequence information for many genes, but the biological functions for most of them will only be known through functional studies. Bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) are large genomic clones stably maintained in bacteria and are very important in functional studies through transfection because of their large size and stability. Because most BAC or PAC vectors do not have a mammalian selection marker, transfecting mammalian cells with genes cloned in BACs or PACs requires the insertion into the BAC/PAC of a mammalian selectable marker. However, currently available procedures are not satisfactory in efficiency and fidelity. We describe a very simple and efficient procedure that allows one to retrofit dozens of BACs in a day with no detectable deletions or unwanted recombination. We use a BAC/PAC retrofitting vector that, on transformation into competent BAC or PAC strains, will catalyze the specific insertion of itself into BAC/PAC vectors through in vivo cre/loxP site-specific recombination.
Asunto(s)
Bacteriófago P1/genética , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales/genética , Transfección/métodos , Proteínas Virales , Animales , Fusión Artificial Génica , Línea Celular , Marcadores Genéticos/genética , Vectores Genéticos/síntesis química , Humanos , Integrasas/genética , Ratones , Recombinación Genética , Reproducibilidad de los Resultados , Células Madre/fisiología , Transformación GenéticaAsunto(s)
Diversidad de Anticuerpos/genética , Citidina Desaminasa/metabolismo , Genes de Inmunoglobulinas/genética , Animales , Citidina Desaminasa/genética , Activación Enzimática , Humanos , Cambio de Clase de Inmunoglobulina/genética , Síndromes de Inmunodeficiencia/genética , Modelos Genéticos , Mutación/genética , Edición de ARN , Recombinación Genética/genéticaAsunto(s)
Cromosomas/genética , Metilación de ADN , Animales , Southern Blotting , Mapeo Cromosómico , Cruzamientos Genéticos , ADN/genética , ADN/metabolismo , Femenino , Ligamiento Genético , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones Transgénicos , Repeticiones de MicrosatéliteRESUMEN
Immunoglobulin (IG:) genes are hypermutated in mature B cells after interaction with antigen and T cells in a germinal center reaction. We and others have recently shown that the human BCL6 gene is also hypermutated in human peripheral blood memory B cells and tonsils. A preliminary analysis of other non-Ig genes (c-MYC:, S14 and AFP) suggested that they were not mutated in memory B cells. We have now performed an in-depth analysis of three non-Ig genes that are expressed in germinal center B cells in two human donors in whom BCL6 is highly mutated. It was found that the TATA binding protein (TBP), c-MYC: and survivin genes are not hypermutated. This lack of targeting by the Ig hypermutation mechanism must be due to the lack of regulatory DNA elements, since the primary sequences of the three tested genes have at least as high intrinsic mutability indices as the BCL6 gene.
Asunto(s)
Linfocitos B/fisiología , Proteínas de Unión al ADN/genética , Genes de Inmunoglobulinas , Genes myc , Memoria Inmunológica , Proteínas Asociadas a Microtúbulos , Mutación , Proteínas/genética , Proteínas Proto-Oncogénicas/genética , Factores de Transcripción/genética , Secuencia de Bases , Humanos , Proteínas Inhibidoras de la Apoptosis , Datos de Secuencia Molecular , Proteínas de Neoplasias , Proteínas Proto-Oncogénicas c-bcl-6 , Survivin , TATA Box , Proteína de Unión a TATA-BoxRESUMEN
Mice carrying a gamma2b transgene have been shown previously to be deficient in B cell development. In particular, a developmental block exists at the pre-B cell stage. The few B cells that develop all express endogenous micro heavy chains. The phenotype suggests that gamma2b exerts a strong feedback inhibition on endogenous Ig gene rearrangement, but, unlike micro, cannot support further B cell development. In this study we have created hybrid transgenes between gamma2b and micro. Transgenic mice with a C(H)1 domain of micro, or both a C(H)1 and transmembrane/cytoplasmic domain of micro replacing the respective domains of a gamma2b transgene, have the same B cell defect as gamma2b transgenic mice. Interestingly, the severity of the defect is correlated with the level of expression of the transgene, suggesting that the degree of feedback inhibition of Ig gene rearrangement depends on the level and timing of Ig production. Crossing the gamma2b/micro transgenes into a Bcl-x(L) transgenic line allows immature gamma2b B cells to survive, but not to develop to maturity. Therefore, the missing function of micro is not simply an anti-apoptotic effect.
Asunto(s)
Linfocitos B/citología , Cadenas Pesadas de Inmunoglobulina/fisiología , Cadenas gamma de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/fisiología , Animales , Linfocitos B/inmunología , Southern Blotting , Células de la Médula Ósea/inmunología , Diferenciación Celular , Citometría de Flujo , Cadenas Pesadas de Inmunoglobulina/genética , Cadenas mu de Inmunoglobulina/genética , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Proteínas Proto-Oncogénicas c-bcl-2/genética , ARN/análisis , Bazo/citología , Factores de Tiempo , Proteína bcl-XRESUMEN
During B and T lymphocyte development, immunoglobulin and T cell receptor genes are assembled from the germline V, (D) and J gene segments (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). These DNA rearrangements, responsible for immune system diversity, are mediated by a site specific recombination machinery via recognition signal sequences (RSSs) composed of conserved heptamers and nonamers separated by spacers of 12 or 23 nucleotides (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). Recombination occurs only between a RSS with a 12mer spacer and a RSS with a 23mer spacer (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Adv. Immunol. 56, 27-150). RAG1 and RAG2 proteins cleave precisely at the RSS-coding sequence border leading to flush signal ends and coding ends with a hairpin structure (Eastman, M., Leu, T., Schatz, D., 1996. Initiation of V(D)J recombination in vitro obeying the 12/23 rule. Nature 380, 85-88; Roth, D.B., Menetski, J.P., Nakajima, P.B., Bosma, M.J., Gellert, M., 1992. V(D)J recombination: broken DNA molecules with covalently sealed (hairpin) coding ends in scid mouse thymocytes. Cell 983-991: Roth, D.B., Zhu, C., Gellert. M., 1993. Characterization of broken DNA molecules associated with V(D)J recombination. Proc. Natl. Acad. Sci. USA 90, 10,788-10,792; van Gent, D., McBlane, J.. Sadofsky, M., Hesse, J., Gellert, M., 1995. Initiation of V(D)J recombination in a cell-free system. Cell 81, 925-934). Signal ends join, forming a signal joint. The hairpin coding ends are opened by a yet unknown endonuclease, and are further processed to form the coding joint (Lewis, S.M., 1994. The mechanism of V(D)J joining: lessons from molecular, immunological and comparative analyses. Ad. Immunol. 56, 27-150.) The murine scid mutation has been shown to affect coding joints, but much less signal joint formation. In this study we demonstrate that the murine scid mutation inhibits correct signal joint formation when both coding ends contain homopolymeric sequences. We suggest that this finding may be due to the function of the SCID protein as an assembly component in V(D)J recombination.
Asunto(s)
Proteínas de Unión al ADN , Reordenamiento Génico de Linfocito B , Inmunodeficiencia Combinada Grave/genética , Inmunodeficiencia Combinada Grave/inmunología , Animales , Linfocitos B/enzimología , Linfocitos B/inmunología , Secuencia de Bases , Línea Celular , ADN/genética , Cartilla de ADN/genética , Proteína Quinasa Activada por ADN , Fibroblastos/enzimología , Fibroblastos/inmunología , Genes de Inmunoglobulinas , Células Madre Hematopoyéticas/enzimología , Células Madre Hematopoyéticas/inmunología , Ratones , Ratones SCID , Plásmidos/genética , Reacción en Cadena de la Polimerasa , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/inmunología , Recombinación Genética , Inmunodeficiencia Combinada Grave/enzimología , TransfecciónRESUMEN
Somatic hypermutation of Ig genes is probably dependent on transcription of the target gene via a mutator factor associated with the RNA polymerase (Storb, U., E.L. Klotz, J. Hackett, Jr., K. Kage, G. Bozek, and T.E. Martin. 1998. J. Exp. Med. 188:689-698). It is also probable that some form of DNA repair is involved in the mutation process. It was shown that the nucleotide excision repair proteins were not required, nor were mismatch repair (MMR) proteins. However, certain changes in mutation patterns and frequency of point mutations were observed in Msh2 (MutS homologue) and Pms2 (MutL homologue) MMR-deficient mice (for review see Kim, N., and U. Storb. 1998. J. Exp. Med. 187:1729-1733). These data were obtained from endogenous immunoglobulin (Ig) genes and were presumably influenced by selection of B cells whose Ig genes had undergone certain mutations. In this study, we have analyzed somatic hypermutation in two MutL types of MMR deficiencies, Pms2 and Mlh1. The mutation target was a nonselectable Ig-kappa gene with an artificial insert in the V region. We found that both Pms2- and Mlh1-deficient mice can somatically hypermutate the Ig test gene at approximately twofold reduced frequencies. Furthermore, highly mutated sequences are almost absent. Together with the finding of genome instability in the germinal center B cells, these observations support the conclusion, previously reached for Msh2 mice, that MMR-deficient B cells undergoing somatic hypermutation have a short life span. Pms2- and Mlh-1-deficient mice also resemble Msh2-deficient mice with respect to preferential targeting of G and C nucleotides. Thus, it appears that the different MMR proteins do not have unique functions with respect to somatic hypermutation. Several intrinsic characteristics of somatic hypermutation remain unaltered in the MMR-deficient mice: a preference for targeting A over T, a strand bias, mutational hot spots, and hypermutability of the artificial insert are all seen in the unselectable Ig gene. This implies that the MMR proteins are not required for and most likely are not involved in the primary step of introducing the mutations. Instead, they are recruited to repair certain somatic point mutations, presumably soon after these are created.
Asunto(s)
Disparidad de Par Base , Reparación del ADN , Proteínas Adaptadoras Transductoras de Señales , Animales , Linfocitos B/metabolismo , Secuencia de Bases , Proteínas Portadoras , Codón de Terminación , Femenino , Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Masculino , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Homólogo 1 de la Proteína MutL , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteínas Nucleares , Mapeo Restrictivo , TransgenesRESUMEN
Although an inverse correlation between CpG methylation and V(D)J recombination has been demonstrated for both artificial substrates and endogenous genes, it is not known whether all hypomethylated targets are competent to rearrange or if other factors are required. We have created several artificial V(D)J recombination substrate transgenes whose methylation can be controlled by breeding into different genetic backgrounds. A transgene which contains the immunoglobulin heavy chain intronic enhancer rearranges efficiently in B lymphocytes when the transgene loci are unmethylated. When the same loci become methylated, upon breeding into a different mouse strain, no rearrangement can be detected. A similar transgene, but lacking the enhancer, also shows no evidence of V(D)J recombination when it is methylated. Even when this enhancerless transgene is hypomethylated, however, no V(D)J recombination can be detected in B lymphocytes. Thus, hypomethylation is required to permit V(D)J recombination but not all hypomethylated targets are capable of recombination. The results may indicate that the immunoglobulin enhancer is required for the assembly of factors involved in V(D)J recombination.
Asunto(s)
Metilación de ADN , Genes de Inmunoglobulinas , Recombinación Genética , Animales , Secuencia de Bases , Islas de CpG , Cartilla de ADN/genética , Elementos de Facilitación Genéticos , Reordenamiento Génico de Linfocito B , Reordenamiento Génico de Linfocito T , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos DBA , Ratones TransgénicosRESUMEN
This review describes studies on somatic hypermutation of immunoglobulin genes that were started in the mid-80s in collaboration with Ralph Brinster. Almost all of the experiments were carried out using Ig transgenes as targets for the somatic mutation mechanism. Ig transgenes can be very good targets of somatic mutation, despite many different transgene integration sites. Thus, the required cis-acting elements must be present within the approximately 10 kb of the transgene. Only the Ig variable region and its proximate flanks are mutated, not the constant region in unmanipulated sequences. Several Ig gene enhancers are permissive for somatic mutation and they do not have to be associated with the Ig promoter they normally interact with. However, the mutation process does seem to be specific for Ig genes. No mutations were found in several housekeeping genes isolated from cells that had very high levels of somatic hypermutation of their Ig genes. This suggests that the Ig enhancers provide the lg gene specificity. An exception is the Bcl-6 gene, encoding a transcription factor, which was found to be mutated in normal human memory B cells. When the transcriptional promoter that is located upstream of the variable region is duplicated upstream of the constant region, this region is mutated as well. This suggests a transcription coupled model in which a mutator factor associates with the RNA polymerase at the initiation of transcription, travels with the polymerase during elongation, and causes mutations during polymerase pausing. Our recent data with an artificial substrate for somatic mutation suggest that the mutations are increased by increased stability of the secondary structures in the nascent RNA, and the specific nucleotides that are mutated are due to preferences of a mutator factor.
Asunto(s)
Genes de Inmunoglobulinas , Transgenes , Animales , Humanos , Ratones , Ratones Transgénicos , MutagénesisRESUMEN
The Ig lambda light chain gene enhancer has two unique essential motifs, lambdaA and lambdaB. The transcription factors that bind the AB motif have been identified as Pu.1 and Pu.1-interacting partner (Pip). We report here that the lambdaA site includes a binding site for the myocyte-specific enhancer factor 2 (Mef2) family of transcription factors. Mef2 proteins were first described in muscle cells and, in vertebrates, include four known members designated A to D. Using a AA electrophoretic-mobility shift assay (EMSA), in conjunction with a high affinity Mef2 binding site and anti-Mef2 Abs, we show that members of the Mef2 family are present in nuclear extracts of lambda-producing B cells and bind the AA site. Functional assays using the chloramphenicol acetyltransferase (CAT) reporter construct containing three copies of the lambdaA motif demonstrate that the AA sequence can function as an enhancer in conjunction with the thymidine kinase (TK) promoter and is regulated by Mef2 proteins. Extrapolating from other systems where transcriptional regulation by Mef2 has been studied, other transcription factors may be involved along with Mef2 in transcriptional regulation at the lambdaA site.
Asunto(s)
Linfocitos B/metabolismo , Proteínas de Unión al ADN/fisiología , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Genes de Inmunoglobulinas , Cadenas lambda de Inmunoglobulina/genética , Proteínas Musculares/fisiología , Factores de Transcripción/fisiología , Células 3T3 , Animales , Secuencia de Bases , Sitios de Unión , Diferenciación Celular , Cloranfenicol O-Acetiltransferasa/biosíntesis , Cloranfenicol O-Acetiltransferasa/genética , Proteínas de Unión al ADN/metabolismo , Genes Reporteros , Factores de Transcripción MEF2 , Ratones , Datos de Secuencia Molecular , Familia de Multigenes , Mieloma Múltiple/patología , Proteínas Musculares/metabolismo , Factores Reguladores Miogénicos , Unión Proteica , Proteínas Recombinantes de Fusión/biosíntesis , Factores de Transcripción/metabolismo , Activación Transcripcional , Células Tumorales CultivadasRESUMEN
Silencing of chromosomal domains has been described in diverse systems such as position effect variegation in insects, silencing near yeast telomeres, and mammalian X chromosome inactivation. In mammals, silencing is associated with methylation at CpG dinucleotides, but little is known about how methylation patterns are established or altered during development. We previously described a strain-specific modifier locus, Ssm1, that controls the methylation of a complex transgene. In this study we address the questions of the nature of Ssm1's targets and whether its effect extends into adjacent sequences. By examining the inheritance of methylation patterns in a series of mice harboring deletion derivatives of the original transgene, we have identified a discrete segment, derived from the gpt gene of Escherichia coli, that is a major determinant for Ssm1-mediated methylation. Methylation analysis of sequences adjacent to a transgenic target indicates that the influence of this modifier extends into the surrounding chromosome in a strain-dependent fashion. Implications for the mechanism of Ssm1 action are discussed.
Asunto(s)
Proteínas Fúngicas/genética , Proteínas Ribosómicas/genética , Proteínas de Saccharomyces cerevisiae , Animales , Mapeo Cromosómico , Metilación de ADN , Femenino , Eliminación de Gen , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Linaje , TransgenesRESUMEN
Immunoglobulin (Ig) genes expressed in mature B lymphocytes can undergo somatic hypermutation upon cell interaction with antigen and T cells. The mutation mechanism had previously been shown to depend upon transcription initiation, suggesting that a mutator factor was loaded on an RNA polymerase initiating at the promoter and causing mutations during elongation (Peters, A., and U. Storb. 1996. Immunity. 4:57-65). To further elucidate this process we have created an artificial substrate consisting of alternating EcoRV and PvuII restriction enzyme sites (EPS) located within the variable (V) region of an Ig transgene. This substrate can easily be assayed for the presence of mutations in DNA from transgenic lymphocytes by amplifying the EPS insert and determining by restriction enzyme digestion whether any of the restriction sites have been altered. Surprisingly, the EPS insert was mutated many times more frequently than the flanking Ig sequences. In addition there were striking differences in mutability of the different nucleotides within the restriction sites. The data favor a model of somatic hypermutation where the fine specificity of the mutations is determined by nucleotide sequence preferences of a mutator factor, and where the general site of mutagenesis is determined by the pausing of the RNA polymerase due to secondary structures within the nascent RNA.
Asunto(s)
Región Variable de Inmunoglobulina/genética , Cadenas kappa de Inmunoglobulina/genética , Mutagénesis Insercional , ARN , Animales , Secuencia de Bases , Sitios de Unión , ADN Complementario , Desoxirribonucleasas de Localización Especificada Tipo II , Región de Unión de la Inmunoglobulina/genética , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN/química , ARN/metabolismo , TransgenesRESUMEN
The renal countercurrent bundles of elasmobranch fish were studied by light and electron microscopy. The kidneys of the lesser spotted dogfish, Scyliorhinus caniculus Blainville, and the little skate, Raja erinacea Mitchill, were investigated. Three-dimensional reconstruction with computer assistance revealed the spatial association of the renal tubular segments and their relationships to each other, as well as to the microvasculature. Regular association between structures was assessed by quantification of contact points on histological sections. The bundles contain a hairpin loop of neck segment and the beginning of the proximal tubule, PIa. The limbs of this loop closely adhere to each other, and a second loop (the early distal tubule) coils around the first loop at the tip of the bundle. The collecting tubule runs between the two loops, and merges with the collecting duct inside the end portion of the bundle. A single lymph capillary-like vessel originates from a few blind-ended rami at the tip of the bundle and runs in close contact with the collecting tubule along the entire bundle. This central vessel merges via several side branches with the venous sinusoid capillaries of the peritubular blood circulation. Thereby the central vessel provides a channel for convective flow of NaCl-rich fluid unidirectionally to the venous portal system of the mesial tissue zone of the kidney. By the close spatial arrangement of the collecting tubule and the central vessel countercurrent exchange of urea from the collecting tubule urine to the fluid in the central vessel is feasible. Thus, the spatial organisation of renal tubular segments and the central vessel is considered to represent the morphological correlate to urea retention by the kidney of Elasmobranchii.
Asunto(s)
Cazón/anatomía & histología , Túbulos Renales Colectores/irrigación sanguínea , Rajidae/anatomía & histología , Animales , Femenino , Procesamiento de Imagen Asistido por Computador , Túbulos Renales Colectores/fisiología , Túbulos Renales Colectores/ultraestructura , Microcirculación/ultraestructura , Microscopía Electrónica , Nefronas/ultraestructura , Urea/metabolismoRESUMEN
We have characterized a novel substrate for somatic hypermutation, confirming that non-Ig sequences can be targeted for mutation and demonstrating that this substrate allows for the rapid assay for mutations. An artificial sequence containing alternating EcoRV and PvuII sites (EPS) was inserted into the Vkappa167 transgene, which is known to be a target for mutation. To assay for somatic hypermutation, the EPS is amplified using flanking transgene primers, and the PCR product is subsequently digested with either EcoRV or PvuII. A mutation is seen as the appearance of a larger fragment, indicating a base change in a restriction enzyme site. The original transgene, Vkappa167/EPS, showed evidence of a low level of mutation in both splenic hybridomas and Peyer's patch-derived or immunized splenic B220+ cells with high peanut agglutinin levels. Two derivative lines of Vkappa167/EPS were made, Vkappa167/POX and Vkappa167/PEPS. While none of the Vkappa167/POX transgenic lines demonstrated mutation, the Vkappa167/PEPS transgene was highly mutated in B220+ splenic B cells with high peanut agglutinin levels at a frequency similar to that of endogenous Ig genes. An analysis of splenic RNA from the unimmunized transgenic mice indicated that the levels of stable message in splenic B cells could not be correlated with the mutation seen in GC B cells. The mutable Vkappa167/PEPS transgenic line is a unique tool to study somatic hypermutation in vivo.