RESUMO
The chromosomal location of human constant region light chain immunoglobulin (Ig) genes has been determined by analyzing a group of human fibroblast/rodent somatic cell hybrids with nucleic acid probes prepared from cloned human kappa and lambda constant region genes. Human chromosomes in each cell line were identified by isoenzyme analysis. The DNA from hybrid cells was digested with restriction endonucleases, size fractionated by gel electrophoresis, transferred to nitrocellulose or DBM paper, and hybridized with (32)P-labeled nucleic acid probes. The C(kappa) gene was assigned to human chromosome 2 and the C(lambda) genes to chromosome 22, based upon analysis of these hybrid cell lines, and these assignments were confirmed by analysis of subclones. A group of previously unassigned loci can be mapped to chromosome 2 by virtue of their close linkage to C(kappa). The lambda and kappa light chain and heavy chain Ig genes have now been assigned to all three human chromosomes that are involved in translocations with chromosome 8 in human B cell neoplasms. These techniques and probes provide a means to study the detailed arrangement of human Ig genes and their pseudogenes.
Assuntos
Cromossomos Humanos 1-3 , Cromossomos Humanos 21-22 e Y , Cadeias Leves de Imunoglobulina/genética , Cadeias kappa de Imunoglobulina/genética , Cadeias lambda de Imunoglobulina/genética , Animais , Cricetinae , Fibroblastos/citologia , Fibroblastos/imunologia , Genes , Humanos , Células Híbridas/citologia , Células Híbridas/imunologia , Hibridização Genética , Regiões Constantes de Imunoglobulina/genética , CamundongosRESUMO
Labeled unique sequence human X chromosomal DNA has been isolated by hybridization of labeled human DNA with DNA from a human-mouse hybrid cell line, A9/HRBC2-A, which contains a single human chromosome, the X. Homopolymer tails of poly(dA) were added to nick-translated unique sequence human DNA to permit separation of the labeled DNA from unlabeled driver DNA by binding to oligo(dT)-cellulose. Human DNA sequences homologous with mouse DNA were removed from this labeled poly-(dA)-tailed human probe by hybridization with excess unlabeled mouse DNA. The labeled human DNA which did not hybridize with mouse DNA was then hybridized with excess unlabeled A9/HRBC2-A DNA, with which only X chromosomal human DNA will hybridize. After hybridization, the labeled human X chromosomal DNA was separated from the unlabeled A9/HRBC2-A DNA by binding to oligo(dT)-cellulose. The purity of the final X chromosomal DNA preparation was greater than 90%, and the hybridization with mouse DNA was less than 2%. When carried out under standard conditions for DNA reassociation, this procedure is complicated by the formation of hybrids between the poly(dA) tails of the probe and T-rich sequences in mouse and human DNA. However, these imperfectly paired hybrids are less stable than those of unique sequence DNA and can be eliminated by carrying out the hydroxylapatite chromatography at an elevated temperature of 71 degrees C.
