RESUMO
Here we describe a new procedure (cloning of polymorphisms, COP) for enrichment of single nucleotide polymorphisms (SNPs) that represent restriction fragment length polymorphisms (RFLPs). COP would be applicable to the isolation of SNPs from particular regions of the genome, e.g. CpG islands, chromosomal bands, YACs or PAC contigs. A combination of digestion with restriction enzymes, treatment with uracil-DNA glycosylase and mung bean nuclease, PCR amplification and purification with streptavidin magnetic beads was used to isolate polymorphic sequences from the genomes of two human samples. After only two cycles of enrichment, 80% of the isolated clones were found to contain RFLPs. A simple method for the PCR detection of these polymorphisms was also developed.
Assuntos
Clonagem Molecular/métodos , DNA Glicosilases , Polimorfismo de Fragmento de Restrição , Polimorfismo de Nucleotídeo Único , DNA/genética , DNA/isolamento & purificação , Eletroforese em Gel de Ágar , Genoma Humano , Humanos , N-Glicosil Hidrolases/metabolismo , Reação em Cadeia da Polimerase , Endonucleases Específicas para DNA e RNA de Cadeia Simples/metabolismo , Uracila-DNA GlicosidaseRESUMO
Not I linking clones contain sequences flanking Not I recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of Not I clones in genome research, high density grids with 50 000 Not I linking clones originating from six representative Not I linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of Not I sites in the human genome. A total of 3437 sequences flanking Not I sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity < or =90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of Not I sites with the first exon and suggest that the so-called 3' ESTs can actually be generated from 5'-ends of genes that contain Not I sites in their first exon.
Assuntos
Desoxirribonucleases de Sítio Específico do Tipo II , Genoma Humano , Sequência de Bases , Clonagem Molecular , Ilhas de CpG , Primers do DNA/genética , Bases de Dados Factuais , Etiquetas de Sequências Expressas , Biblioteca Gênica , Humanos , Dados de Sequência Molecular , RNA Mensageiro/genética , Mapeamento por RestriçãoRESUMO
Twenty-three unique NotI-linking clones, mainly isolated from the NRL1 library, were mapped and ordered by fluorescence in situ hybridization to human chromosome 3. All these clones were partially sequenced around the NotI sites and thus represent sequence-tagged sites. The EMBL nucleotide database was then searched with sequences from the NotI-linking clones using the FASTA program. This search revealed that the NRL-090 clone (at 3q24) contains the gene encoding human guanosine 5'-monophosphate synthetase (GMPS-PEN). To our knowledge, this is the first localization of this gene. Clone NL1-320 (at 3p21.3) contains a gene encoding arginine tRNA (97.3% identity in 73 bp), while clones NRL-063, NRL-097 and NRL-143 contain expressed sequences with unknown functions. Other clones displayed 60-85% similarities to cDNAs, CpG islands and other genes.
Assuntos
Carbono-Nitrogênio Ligases , Cromossomos Humanos Par 3/genética , Desoxirribonucleases de Sítio Específico do Tipo II/genética , Ligases/genética , Sequência de Bases , Mapeamento Cromossômico , Clonagem Molecular , Sequência Conservada , Ilhas de CpG , Expressão Gênica , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência Molecular , Mapeamento por RestriçãoRESUMO
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
Normal tissue contamination of tumors may eclipse the detection of loss of heterozygosity (LOH) by microsatellite analysis and may also hamper isolation of tumor suppressor genes. To test the potential impact of this problem, we prepared artificial mixtures of mouse-human microcell hybrid lines that carried different alleles of the same chromosome 3 marker. After performing an allele titration assay, we found a consistent difference between the LOH of a high molecular weight (H) allele and the LOH of a low molecular weight (L) allele of the same CA repeat marker. It follows that normal tissue admixtures will be less of a problem when LOH affects a H allele than with a L allele. Random screening of 100 papers published between 1994 and 1999 revealed that the loss of a L allele was recorded at about half the frequency (52%) of loss of a H allele. To avoid this bias, we have developed rules for the evaluation of LOH data. We suggest that the loss of a L allele should be given more weight than the loss of a H allele in LOH studies using microsatellite markers.
Assuntos
Biomarcadores Tumorais , Perda de Heterozigosidade , Repetições de Microssatélites/genética , Alelos , Animais , Células Cultivadas , Cromossomos Humanos Par 3 , Estudos de Avaliação como Assunto , Humanos , Células Híbridas , Camundongos , Controle de Qualidade , Reprodutibilidade dos TestesRESUMO
By applying the 'recognition mask' strategy to 300 mammalian sequences containing NotI sites we demonstrated that 5' ends of genes are highly enriched in NotI sites. A NotI linking clone NL2-252 (D3S1678) containing transferrin receptor (TFRC) gene was used as an initial point for chromosomal jumping. One of the jumping clones, J21-045 traverses 210 kbp and links NL2-252 to NL26 (D3S1632), a NotI linking clone containing highly polymorphic sequences. The TFRC gene was mapped to 3q29, close to the telomeric marker D3S2344, by linkage analysis, a panel of hybrid cell lines, GeneBridge 4 panel and FISH. Clone NLM-007 (D3S4302) was found to contain ras-homologous gene RAB7. By FISH and a panel of hybrid cell lines this gene was mapped to 3q21. This region is of particular interest due to frequent rearrangements in different types of leukemia. Clone L2-081 (D3S4283) containing new member of ubiquitin-specific proteases (HAUSP gene) was localized in 3p21 inspiring further investigation of involvement of this gene in development of lung and renal carcinomas.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 3 , Endopeptidases/genética , Proteínas de Ligação ao GTP/genética , Biblioteca Gênica , Genoma Humano , Leucemia/genética , Neoplasias/genética , Receptores da Transferrina/genética , Proteínas rab de Ligação ao GTP , Clonagem Molecular , Rearranjo Gênico , Humanos , Dados de Sequência Molecular , Ubiquitina Tiolesterase , Peptidase 7 Específica de Ubiquitina , proteínas de unión al GTP Rab7RESUMO
For construction of a NotI restriction map of the human genome, the isolation and mapping of unique NotI linking clones represent important and critical steps. Recently we have shown that an Alu-PCR approach can be used for isolation of NotI linking clones from defined regions of the chromosomes. This represents a useful method for isolating and analyzing a small number of clones, but it would be laborious to use it for mapping many NotI linking clones simultaneously. Here we suggest another modification of Alu-PCR for rapid concurrent mapping of many NotI linking clones. The results clearly demonstrate the utility of this approach. Seventy-one random NotI linking clones were analyzed. Among them, 65 clones (91.5%) were correctly selected and mapped using this approach. With differential hybridization and Alu-PCR, a significant portion of all human NotI linking clones (> 30%) can be rapidly mapped to particular chromosomes or to defined regions of these chromosomes.
Assuntos
Southern Blotting , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Genoma Humano , Reação em Cadeia da Polimerase/métodos , Sequências Repetitivas de Ácido Nucleico , Mapeamento por Restrição , Animais , Sequência de Bases , Cromossomos Humanos Par 3 , Primers do DNA , Ligação Genética , Humanos , Células Híbridas , Camundongos , Dados de Sequência MolecularRESUMO
NotI linking clones represent valuable tools for both physical and genetic mapping. Using procedures that we have previously described, several chromosome 3-specific NotI linking libraries have been constructed. Here, we describe the construction of six independent NotI linking libraries specific for the total human genome. These libraries were made using three different vectors and two combinations of restriction enzymes. Altogether, these six libraries contain more than 1 million recombinant phages. Considering that the human genome contains about 3000-5000 NotI sites, it is likely that all clonable NotI sites are present in these libraries. Two of the six libraries were transferred into plasmid form. At the same time, a chromosome 3-specific EcoRI-NotI library (NRL1) was constructed. This library considerably increases the representation of cloned NotI sites in combination with previously constructed libraries that were made using BamHI-NotI digestion. All libraries are available on request.
Assuntos
Cromossomos Humanos Par 3 , Biblioteca Gênica , Ligação Genética , Genoma Humano , Southern Blotting , Clonagem Molecular , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , HumanosRESUMO
We have recently proposed a strategy for construction of long-range physical maps based on random sequencing of NotI linking and jumping clones. Here, we present results of sequence comparison between 168 NotI linking (100 of them were sequenced from both sides) and 81 chromosome 3-specific jumping clones. We were able to identify 14 NotI jumping clones (17%), each joined with two NotI linking clones. The average size of chromosomal jumps was about 650 kb. The assembled 42 NotI genomic fragments correspond to 12-15% of chromosome 3. These results demonstrate the value of random sequencing of NotI linking and jumping clones for genome mapping. This mapping proposal can be used for connecting physical and genetic maps of the human genome and will be a valuable supplement to YAC and cosmid library based mapping projects.
Assuntos
Mapeamento Cromossômico , Passeio de Cromossomo , Genoma Humano , Animais , Sequência de Bases , Linhagem Celular , Clonagem Molecular , Primers do DNA , Desoxirribonucleases de Sítio Específico do Tipo II , Biblioteca Gênica , Ligação Genética , Humanos , Células Híbridas , Camundongos , Dados de Sequência Molecular , Projetos Piloto , Mapeamento por Restrição , Homologia de Sequência do Ácido NucleicoRESUMO
Development of the methods permitting cloning of identical sequences between two sources of DNA can be very useful for many purposes, including isolation of disease genes. Here we describe a new method called CIS (cloning of identical sequences). A combination of digestion with MvnI, treatment with mung bean nuclease, UDG (uracil-DNA glycosylase) and PCR with 5'-methyl-dCTP and dUTP was used to isolate identical sequences between two micro-cell hybrid lines (MCH). In a control experiment, mouse MCH903.1 and MCH939.2 containing human chromosome 3 from different individuals, were compared using the CIS procedure. Only background fluorescence in-situ hybridization (FISH) was achieved. In another experiment, mouse MCH903.1, containing complete human chromosome 3, and rat MCH429.11, containing a part of human 3q from the same chromosome were compared. The experiment showed that the original MCH429.11 and the DNA purified using the CIS procedure had identical FISH patterns to human metaphase chromosomes, thus demonstrating the efficiency of CIS.
Assuntos
Clonagem Molecular/métodos , Animais , DNA/química , DNA/isolamento & purificação , Humanos , Células Híbridas , Camundongos , Ratos , Homologia de Sequência do Ácido NucleicoRESUMO
In the mammalian genome CpG islands are associated with functional genes and cloning of these islands could be an alternative approach for cloning functional genes. Recently we have developed a new approach for cloning CpG islands and constructing NotI linking libraries. We have initiated the construction of a NotI restriction map for chromosome 3, especially focusing on the rearrangements in the 3p14-p21 region, which are associated with different malignancies. CpG islands from this region are useful for isolation of candidate tumor suppressor genes that map to this region and for isolating NotI-linking clones from 3p14-p21 for mapping purposes. Here we suggest a modification of Alu-PCR as an approach to isolating NotI sites (e.g., CpG islands) from defined regions of the chromosome. Instead of using whole chromosomal DNA for Alu-PCR, we have used representative NotI-linking libraries from hybrid cell lines containing either whole or deleted human chromosome 3 (MCH903.1 and MCH924.4, respectively). This decreases the complexity of the Alu-PCR products 10-100 times compared to the whole human genome. Using this modification, we can isolate NotI-linking clones, which are natural markers on the chromosome, rather than random genomic fragments. Among eight clones selected by this method, seven were from the region deleted in MCH924.4. The results clearly demonstrate the feasibility of Alu-PCR for isolating CpG islands from defined regions of the genome.
Assuntos
Carcinoma de Células Renais/genética , Deleção Cromossômica , Cromossomos Humanos Par 3 , Neoplasias Renais/genética , Sequências Repetitivas de Ácido Nucleico , Animais , Bacteriófago lambda/genética , Sequência de Bases , Clonagem Molecular , DNA , Desoxirribonucleases de Sítio Específico do Tipo II/metabolismo , Biblioteca Gênica , Ligação Genética , Humanos , Células Híbridas , Camundongos , Dados de Sequência Molecular , Plasmídeos , Reação em Cadeia da PolimeraseRESUMO
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.