RESUMO
We have partially sequenced more than 1000 NotI linking clones isolated from human chromosome 3-specific libraries. Of these clones, 152 were unique chromosome 3-specific clones. The clones were precisely mapped using a combination of fluorescence in situ hybridization (FISH) and hybridization to somatic cell or radiation hybrids. Two- and three-color FISH was used to order the clones that mapped to the same chromosomal region, and in some cases, chromosome jumping was used to resolve ambiguous mapping. When this NotI restriction map was compared with the yeast artificial chromosome (YAC) based chromosome 3 map, significant differences in several chromosome 3 regions were observed. A search of the EMBL nucleotide database with these sequences revealed homologies (90-100%) to more than 100 different genes or expressed sequence tags (ESTs). Many of these homologies were used to map new genes to chromosome 3. These results suggest that sequencing NotI linking clones, and sequencing CpG islands in general, may complement the EST project and aid in the discovery of all human genes by sequencing random cDNAs. This method may also yield information that cannot be obtained by the EST project alone; namely, the identification of the 5' ends of genes, including potential promoter/enhancer regions and other regulatory sequences
Assuntos
Cromossomos Humanos Par 3/genética , DNA/genética , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Biblioteca Gênica , Animais , Linhagem Celular , Mapeamento Cromossômico , DNA/química , DNA/metabolismo , Bases de Dados Factuais , Etiquetas de Sequências Expressas , Humanos , Células Híbridas , Hibridização in Situ Fluorescente , Camundongos , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
We have cloned earlier a short human genomic fragment which showed strong similarity with the mouse cDNA encoding lung Kruppel-like zinc finger transcription factor (LKLF), predominantly expressed in mouse developing lung, spleen, and vascular system, which might play a key role in programming the quiescent state of single positive T cells and blood vessel wall morphogenesis. Here we report the successful cloning of the human LKLF cDNA, its genomic structure and chromosomal localization at the 19p13.11-p13.13 locus. The full-length human LKLF cDNA has longer 5'-UTR with higher GC content than mouse cDNA and encodes a predicted protein of 355 amino acids which has three zinc fingers at the C-terminus and a proline-rich N-terminal domain. Human and mouse proteins share 87.3% identity and 90.2% amino acid similarity. The human LKLF gene consists of three exons. From the proximal promoter to the end of the second exon, we have found a CpG island with an average 76% GC content and two regions of unusually high GC density.
Assuntos
Cromossomos Humanos Par 19 , Ilhas de CpG , Transativadores/genética , Dedos de Zinco , Sequência de Aminoácidos , Animais , Sequência de Bases , Mapeamento Cromossômico , DNA Complementar , Humanos , Fatores de Transcrição Kruppel-Like , Camundongos , Dados de Sequência Molecular , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Long-range restriction site maps are of central importance for mapping the human genome. The use of clones from linking and jumping libraries for genome mapping offers a promising alternative to the laborious procedures used up until now. In the present review, this research field is analyzed with particular emphasis on the implementation of a shot-gun sequencing strategy for genome mapping and the use of NotI linking clones for analysis of rearrangements in tumors and tumor cell lines.