RESUMO
BACKGROUND AND OBJECTIVES: This study provides a unique method for identifying individuals carrying the Rh haplotype cDe, and supports a model for the evolution of Rh haplotypes in which cDe is the progenitor. MATERIALS AND METHODS: DNA from 212 unrelated donors of known Rh serological phenotype was PCR amplified. The resulting products were analysed by denaturing polyacrylamide gel electrophoresis, denaturing gradient gel electrophoresis and DNA sequencing. RESULTS: Two adjacent microsatellite repeat elements of the form (AC)n (GCAC)n were found within the human Rh blood group genes. These display copy number variation which was non-randomly distributed with respect to Rh serological phenotype, and was restricted to alleles of RHCE expressing the c antigen. CONCLUSION: The predominantly Black African allele cDe displayed a unique set of microsatellite alleles, providing a method of identifying individuals carrying this haplotype.
Assuntos
Glicoproteínas/sangue , Glicoproteínas/genética , Repetições de Microssatélites/imunologia , Proteínas Recombinantes de Fusão , África/etnologia , Alelos , Inglaterra/epidemiologia , Europa (Continente)/etnologia , Saúde da Família , Haplótipos , Humanos , Íntrons , Japão/etnologia , Proteínas de Fusão Oncogênica/sangue , Polimorfismo Genético , Sistema do Grupo Sanguíneo Rh-Hr , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
The human chromosome 3 locus coding for hepatocyte growth factor-like protein/macrophage stimulating protein (HGFL/MSP) is homologous to two sets of amplified loci on human chromosome 1 at 1p36. One copy of one of the amplified loci (D1F15S1A) has been further characterized by restriction enzyme and DNA sequence analysis. A total of 8331 bp of continuous sequence was determined for this locus. The first 6878 bp of sequence is 96.1% identical to the HGFL/MSP gene, while there is no homology between the two genes following nucleotide 6878. Based on the presence of a 5 bp deletion in putative exon 2 and several downstream stop codons it is very likely that this gene is a pseudogene. Screening of a human liver cDNA library with a chromosome 1-specific probe indicates that at least several other members of the chromosome 1 loci are transcribed.
Assuntos
Cromossomos Humanos Par 1/genética , Cromossomos Humanos Par 3/genética , Substâncias de Crescimento/genética , Fator de Crescimento de Hepatócito , Proteínas Proto-Oncogênicas , Clonagem Molecular , Sondas de DNA , Humanos , Fígado/metabolismo , Pseudogenes/genética , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência do Ácido NucleicoRESUMO
Almost exactly 50 years ago, R. A. Fisher and R. Race proposed a model for the evolution of the RH (rhesus) genes in which the less common haplotypes were derived from the commoner ones by recombination, and in which the gene order was D-C-E. No direct-evidence bearing on this model was available then, and has not been until now. Here we present evidence for non-reciprocal intergenic exchange (gene conversion) occurring once in human history to generate the common RHCE allele, Ce. We have also used new polymorphisms to construct haplotypes which suggest that intragenic recombination played a major role in the generation of the less common haplotypes, but only if RHD lies 3' of RHCE, i.e. the order is C-E-D. We provide both genetic and physical evidence supporting this arrangement.
Assuntos
Evolução Molecular , Sistema do Grupo Sanguíneo Rh-Hr/genética , Sequência de Bases , Cromossomos Artificiais de Levedura , DNA Complementar , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Haplótipos , Humanos , Íntrons , Dados de Sequência Molecular , Polimorfismo Genético , Sequências Repetitivas de Ácido NucleicoRESUMO
We have studied the arrangement of Rh (rhesus) genes in donors who are completely null for the products of one of them, RHCE. We show that five of six homozygous individuals with the so-called Rh D-- phenotype, who express no red-cell antigens of the C/c and E/e series, have rearranged RHCE genes in which internal sequences have been replaced by the corresponding sequences from RHD. Moreover, although there is heterogeneity at the 3' end, the 5' boundary of this chimerism is within the same small interval around exon 2. This interval is characterized by an exceptionally high degree of sequence homology between RHCE and RHD, a high density of dispersed repetitive elements, and the presence of an alternating purine-pyrimidine copolymer tract. We suggest that these features may explain the mechanistic basis for the origin of the rearrangement.
Assuntos
Doadores de Sangue , Rearranjo Gênico , Sistema do Grupo Sanguíneo Rh-Hr/genética , Alelos , Humanos , Fenótipo , Análise de Sequência de DNARESUMO
Denaturing gradient gel electrophoresis (DGGE) was carried out on PCR products amplified from exons 2 and 5 of RHD and RHCE. Exon 2 of RHD and exon 2 of the C allele of RHCE have an identical sequence, which differs from that of the c allele of RHCE. One band representing D and/or C, and another representing c, could be distinguished by DGGE of exon 2 amplifications of genomic DNA from individuals with the appropriate Rh phenotype. C and c could only be distinguished in D-negative samples. Exon 5 of RHD and exon 5 of the E and e alleles of RHCE all have different nucleotide sequences. Bands representing D, E and e could be distinguished following DGGE of the products of exon 5 amplification of genomic DNA from individuals with red cells of the appropriate Rh phenotype. In samples from individuals with VS+ red cells (V+ or V-) there was a shift of the band representing e. Sequencing demonstrated that VS is associated with a RHCE e sequence with a single base change predicting a Leu245 --> Val substitution in the Rh polypeptide. This substitution may be responsible for the VS and e5 antigens.
Assuntos
DNA/genética , Eletroforese/métodos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Sequência de Bases , Análise Mutacional de DNA , Éxons , Humanos , Dados de Sequência Molecular , Mutação , Ácidos Nucleicos Heteroduplexes , Fenótipo , Reação em Cadeia da Polimerase , Sistema do Grupo Sanguíneo Rh-Hr/imunologiaRESUMO
Initial Rh phenotyping of a man with hemolytic anemia, his wife, and son appeared to exclude paternity. No exclusion was found in other blood groups or in the human leukocyte antigen (HLA) system; excluding Rh, the paternity index was 98.58 percent. Samples from these three family members, and two other family members, were tested with additional Rh antisera. The results indicated that the propositus has an Rhmod phenotype with expression of c, weak e, and very weak D, E, and G antigens. To support this hypothesis, DNA analysis of the RHD and RHCE genes was performed on the five family members. Polymerase chain reaction (PCR) products from exons 2 and 5 were analyzed by denaturing gradient gel electrophoresis (DGGE). The DNA results corroborated the serologic findings and refuted the exclusion of paternity.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 3/genética , Animais , Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 3/ultraestrutura , Expressão Gênica , Doenças Genéticas Inatas/genética , Ligação Genética , Marcadores Genéticos , Humanos , Camundongos , Especificidade da EspécieRESUMO
We describe a PCR-based method of performing RHD and C/c typing in a single reaction. The method is based on an earlier observation of a polymorphism in intron 2 of both genes which, in addition to detecting the RHD deletion responsible for most known D-negative phenotypes, is also associated with C/c serological type. Using this assay, we typed 105 unrelated individuals from at least four different population groups and compared the results to those obtained using conventional serological testing of red cells. An absolute correlation, with no exceptions, was seen. We also showed that the method has potential in the antenatal determination of RH type, as it was possible to type fetal trophoblasts recovered from the endocervical canal at 9 weeks pregnancy.
Assuntos
Tipagem e Reações Cruzadas Sanguíneas/métodos , DNA/química , Reação em Cadeia da Polimerase/métodos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Southern Blotting , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Eletroforese em Gel de Ágar , Feminino , Deleção de Genes , Humanos , Íntrons , Hibridização de Ácido Nucleico , Fenótipo , Polimorfismo de Fragmento de Restrição , GravidezAssuntos
Diagnóstico Pré-Natal/métodos , Sistema do Grupo Sanguíneo Rh-Hr/genética , Trofoblastos/citologia , Sequência de Bases , Tipagem e Reações Cruzadas Sanguíneas , Colo do Útero/citologia , Vilosidades Coriônicas , DNA/análise , Feminino , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , GravidezRESUMO
We report the identification and characterization of a clone for the DNA binding protein SON, which we have isolated from a human keratinocyte cDNA library. Using this clone we have found that the SON gene is expressed in different cell types and that homologous sequences can be detected in vertebrate and insect genomic DNA. Using the polymerase chain reaction (PCR) to amplify SON sequences from a panel of somatic cell hybrids we have assigned the gene encoding human SON to chromosome 21. By use of hybrids containing regions of chromosome 21 the localization has been refined to 21q 22.1-q22.2.
Assuntos
Cromossomos Humanos Par 21 , Proteínas de Ligação a DNA/genética , Genes/genética , Queratinócitos/química , Sequência de Aminoácidos , Animais , Linhagem Celular , Mapeamento Cromossômico , Clonagem Molecular , Sequência Conservada , DNA Complementar , Expressão Gênica , Humanos , Células Híbridas , Antígenos de Histocompatibilidade Menor , Precursores de Proteínas/genética , RNA Mensageiro/análise , Proteínas Recombinantes de Fusão/análise , Proteínas Recombinantes de Fusão/genética , Análise de Sequência de DNA , Especificidade da Espécie , beta-Galactosidase/genéticaRESUMO
We have investigated the arrangement of genes in the rare Rh (Rhesus) partial null condition D--. Southern blot and PCR studies under conditions which distinguish the highly homologous RH D and RH C/E genes show that in an Icelandic family with the D-- haplotype at least 85% of the RH C/E gene is deleted. This finding is in contrast to one other published case of this phenotype, where intact RH D and C/E genes were found, and also to the full amorph Rhnull phenotype, where an intact RH C/E gene was found, accompanied by the deletion of the RH D gene typical of Rh D-negative individuals.
Assuntos
Deleção de Genes , Sistema do Grupo Sanguíneo Rh-Hr/genética , Sequência de Bases , Éxons , Heterozigoto , Homozigoto , Humanos , Islândia , Íntrons , Dados de Sequência Molecular , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de RestriçãoRESUMO
We have studied the RH genes in donors with the RhD-negative haplotype dCes. In contrast to the usual arrangement of genes in RhD-negative individuals, where the lack of antigen expression is due to deletion of the entire RHD gene, we find that the dCes haplotype includes an RHD gene with an internal deletion. Moreover, there appear to be no 5' sequences characteristic of RHC suggesting that the RhC antigen may be encoded by a truncated RHD or a recombinant RHD/CE gene in these dCes/dce genomes.
Assuntos
Sistema do Grupo Sanguíneo Rh-Hr/genética , Deleção de Sequência , Sequência de Bases , População Negra/genética , Tipagem e Reações Cruzadas Sanguíneas , Análise Mutacional de DNA , Haplótipos/genética , Humanos , Íntrons , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Alu element-primed PCR was performed on genomic clones containing human RH blood group genes. When used as a probe, the Alu PCR product detected a restriction fragment-length polymorphism which is in complete linkage disequilibrium with the Rh C/c serological polymorphism, irrespective of the Rh D or E serological type it is coupled with. This provides the opportunity to type individuals for their RH C gene directly at the DNA level. RFLP analysis of two individuals with the amorph Rh null phenotype revealed that in one case this phenotype occurred on an RH C background, whereas in the other it was on an RH c background. Taken together these results indicate that the Rh C/c polymorphism has arisen only once, but that the amorph Rh null phenotype, although exceedingly rare, is the result of at least two independent mutations.
Assuntos
Sistema do Grupo Sanguíneo Rh-Hr/genética , Alelos , Análise Mutacional de DNA , Feminino , Humanos , Desequilíbrio de Ligação , Masculino , Linhagem , Fenótipo , Reação em Cadeia da Polimerase , Polimorfismo de Fragmento de Restrição , Deleção de SequênciaRESUMO
The chromosomal region 3p21 is thought to be the site of a lung tumor suppressor gene. We recently cloned a gene from this region that has greatly reduced expression in almost all lung tumor cell lines examined, in spite of being widely expressed in a variety of other tumor and nontumor cell types. We report here the sequence of this gene and show that it has significant homology to the genes encoding the ubiquitin-activating enzymes of three species, including humans. This suggests it is a second, autosomal member of this gene family in humans and may play a role in the ubiquitin conjugation pathway, which is of central importance in all eukaryotes.
Assuntos
Cromossomos Humanos Par 3 , Genes Supressores de Tumor , Ligases/genética , Neoplasias Pulmonares/genética , Sequência de Aminoácidos , Sequência de Bases , DNA/química , Humanos , Dados de Sequência Molecular , Homologia de Sequência do Ácido Nucleico , Enzimas Ativadoras de Ubiquitina , Ubiquitina-Proteína LigasesRESUMO
We have developed a general PCR-based method to quantify the amount of a specific mRNA present in a given cell line or tissue. We applied this quantitative PCR to analyse the expression of D8, a human gene which we recently identified in the chromosomal region 3p21, the common deletion region of lung cancer. Our PCR-aided assay shows that in most lung-cancer-derived cell lines the amount of D8 transcripts is only 2% or less of that in normal lung tissue. The virtual absence of expression may imply some role of the gene in the development of lung cancer.
Assuntos
Cromossomos Humanos Par 3 , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Reação em Cadeia da Polimerase/métodos , Sequência de Bases , Humanos , Dados de Sequência Molecular , RNA Mensageiro/análise , Células Tumorais CultivadasRESUMO
A combination of cytogenetic and molecular studies has implicated the p21 region of human chromosome 3 as the probable site of a gene the loss of which contributes to the development of small cell lung cancer. We report here the isolation of a gene from this region which is expressed in normal lung tissue and in cell lines derived from a number of different types of tumor, but the expression of which in small cell lung cancer cell lines is undetectable by RNA blot analysis. Although the more sensitive polymerase chain reaction did detect transcripts, a novel quantitative polymerase chain reaction assay showed that their concentration in small cell lung cancer cell lines is less than 3% of that in normal lung.
Assuntos
Carcinoma de Células Pequenas/genética , Cromossomos Humanos Par 3 , Genes Supressores de Tumor , Neoplasias Pulmonares/genética , Mapeamento Cromossômico , DNA de Neoplasias/análise , Humanos , Hibridização de Ácido Nucleico , Reação em Cadeia da Polimerase , RNA Mensageiro/análise , RNA Neoplásico/análiseRESUMO
Using a panel of somatic cell hybrids, we have localised the 30-kDal Rhesus blood group-antigen-related protein to human chromosome 1 in the region p36.13----p34. This confirms the localisation of this protein described previously using cytogenetic and linkage analyses.