We have developed a methodology for identification and fine mapping of chromosome-specific transcripts. Combining digestion of DNA with different restriction enzymes, ligation to “bubble” linkers, ...and PCR amplification fromAluand “bubble” primers, we have synthesized human chromosome 1-specific sequences from DNA of a somatic cell hybrid, A9Neo1. After hybridization to human fetal brain cDNA, we could efficiently capture chromosome 1-specific cDNAs. The cDNAs were sequenced and used as probes in hybridizations to high-density filters containing the arrayed CEPH Mega-YAC library and to the arrayed cDNA library from infant brain made by B. Soares, which has been extensively sequenced. By this approach we have been able to select chromosome 1-specific cDNAs, to map them to chromosome 1 YAC contigs, and to identify and map corresponding longer cDNAs and ESTs.
Thirtytwo probes for CpG islands of the distal long arm of the human X chromosome have been Identified. From a genomic library of ONA of the hamster-human cell hybrid X3000.1 digested with the rare ...cutter restriction enzyme Eagl, 53 different human clones have been isolated and characterized by methylation and sequence analysis. The characteristic pattern of DNA methylation of CpG islands at the 5′ end of genes of the X chromosome has been used to distinguish between Eagl sites In CpG islands versus isolated Eagl sites. The sequence analysis has confirmed and completed the characterization showing that sequences at the 5′ end of known genes were among the clones defined CpG islands and that the non-CpG islands clones were mostly repetitive sequences with a non-methylated or variably methylated Eagl site. Thus, since clones corresponding to repetitive sequences can be easily identified by sequencing, such libraries are a very good source of CpG islands. The methylation analysis of 28 different new probes allows to state that demethylatlon of CpG islands of the active X and methylation of those on the inactive X chromosome are the general rule. Moreover, the finding, in all instances, of methylation differences between male and female DNA is in very strong support of the notion that most genes of the distal long arm of the X chromosome are subject to X inactivation.
The transcriptional organization of the region of the mouse X chromosome between the G6pd and the Fln1 genes was studied in detail, and it was compared with the syntenic region of the human ...chromosome. A cosmid contig of 250 kb was constructed by screening mouse cosmid libraries with probes for human genes and with whole cosmids. Overlapping cosmids were aligned by comparing
EcoRI and rare-cutter restriction enzyme digestions. The gene order and the orientation of transcription were determined by hybridization with fragments from the 5′ and 3′ moieties of each cDNA. Our work demonstrates that all of the new genes identified in human are present in the mouse. The size of the region, 250 kb, is also very similar, as are gene order and gene organization: the transcriptional organization in "domains" described in human is found to be identical in the mouse. The major difference detected is the much lower content in rare-cutter restriction sites, which is related to the lower G+C and CpG content of mouse DNA. The very high conservation that we have described suggests that a potent selective pressure has contributed to such conservation of gene organization.
Two genes encode actin-binding protein 280 isoforms. ABP-280 or filamin (FLN1) is present in the cytoskeleton of many cell types, whereas expression of FLN2 is limited to skeletal muscle and heart. ...FLN1 maps to human chromosome Xq28, and, by physical mapping in YAC clones, we have mapped the homologous murine locus (Fln1) to mouse chromosome X, in a region of syntenic homology with human chromosome X. We mapped FLN2 to human chromosome 7q32-q35 by analysis of somatic cell hybrids containing portions of chromosome 7, and, by using a mapping panel from an interspecific murine cross, we mapped the corresponding murine locus (Fln2) to murine chromosome 6 in a region homologous to human chromosome 7.
We have isolated and characterized 55 EagI-containing genomic DNA clones from the distal long arm of the human X chromosome. The presence of additional sites for rare-cutter restriction enzymes and ...the demethylation of the corresponding genomic DNA demonstrate that at least 30 clones correspond to CpG islands of the Xq24-Xqter region. All clones were regionally mapped with a hybrid panel. The majority are in Xq28 and Xq24 (18 and 14 clones, respectively), 15 are in the Xq26-Xq27 interval, and none is in Xq25. This analysis demonstrates a nonuniform distribution of CpG islands that may reflect the distribution of coding regions in this part of the genome.
The severe hemoglobinopathies, including beta-thalassemia major and sickle cell anemia, are candidate diseases for a genetic treatment based on the transfer of a regulated globin gene in autologous ...hematopoietic stem cells. Two years ago, May et al reported that an optimized beta-globin transcription unit containing multiple proximal and distal regulatory elements harbored by a recombinant lentiviral vector could efficiently integrate into murine hematopoietic stem cells and express therapeutic levels of the human beta-globin gene. Here, we review the advantages afforded by lentivirus-mediated globin gene transfer and recent studies based on this strategy.
β-thalassemia is an inherited disorder due to mutations found in the β-globin gene, leading to anemia and requiring sporadic or chronic blood transfusions for survival. Without proper chelation, ...β-thalassemia results in iron overload. Ineffective erythropoiesis can lead to iron overload even in untransfused patients who are affected by β-thalassemia intermedia. Better understanding of the molecular biologic aspects of this disorder has led to improvements in population screening and prenatal diagnosis, which, in turn, have led to dramatic reductions in the number of children born with β-thalassemia major in the Mediterranean littoral. However, as a consequence of decreases in neonatal and childhood mortality in other geographical areas, β-thalassemia has become a worldwide clinical problem. A number of unsolved pathophysiological issues remain, such as ineffective erythropoieis, abnormal iron absorption, oxidative stress, splenomegaly and thrombosis. In the last few years, novel studies have the potential to introduce new therapeutic approaches that might reduce these problems and limit the need for blood transfusion.
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