RESUMEN
The Escherichia coli bacteriophage P1 packages host chromosome separately from phage DNA, and transfers it to recipient cells at low frequency in a process called generalized transduction. Phage genomes are packaged from concatemers beginning at a specific site, pac. To increase transduction rate, we have inserted pac into the chromosome at up to five equally spaced positions; at least this many are fully tolerated in the absence of P1 infection. A single chromosomal pac greatly increases transduction of downstream markers without decreasing phage yields; 3.5 × as much total chromosomal DNA is packaged. Additional insertions decrease phage yield by > 90% and also decrease phage DNA synthesis, although less dramatically. Packaging of chromosomal markers near to and downstream of each inserted pac site is, at the same time, increased by greater than 10 fold. Transduction of markers near an inserted pac site can be increased by over 1000-fold, potentially allowing identification of such transductants by screening.
Asunto(s)
Bacteriófago P1/fisiología , Cromosomas Artificiales de Bacteriófagos P1/fisiología , Cromosomas Bacterianos/genética , ADN Bacteriano/genética , Escherichia coli/genética , Cromosomas Artificiales de Bacteriófagos P1/genética , ADN Viral/análisis , ADN Viral/fisiología , Hibridación de Ácido Nucleico , Transducción GenéticaRESUMEN
P1 bacteriophages lysogenize bacteria as independent plasmid-like elements. We describe here a P1-like bacteriophage, RCS47, carrying a blaSHV-2 gene, isolated from a clinical strain of Escherichia coli from phylogroup B1, and we report the prevalence of P1-like prophages in natural E. coli isolates. We found that 70% of the sequence of RCS47, a 115-kb circular molecule, was common to the reference P1 bacteriophage under GenBank accession no. AF234172.1, with the shared sequences being 99% identical. RCS47 had acquired two main foreign DNA fragments: a 9,636-bp fragment mobilized by two IS26 elements containing a blaSHV-2 gene, and an 8,544-bp fragment mobilized by two IS5 elements containing an operon encoding a dimethyl sulfoxide reductase. The reference P1 prophage plasmid replication gene belonged to the IncY incompatibility group, whereas that of RCS47 was from an unknown group. The lytic capacity of RCS47 and blaSHV-2 gene transduction, through the lysogenization of RCS47 in the recipient E. coli strains, were not demonstrated. The prevalence of P1-like prophages in various animal and human E. coli strain collections, as determined by the PCR detection of repL, the lytic replication gene, was 12.6%. No differences in the prevalences of these prophages were found between extended-spectrum ß-lactamase (ESBL)-producing and non-ESBL-producing strains (P = 0.69), but this prevalence was lower in phylogroup B2 than in the other phylogroups (P = 0.008), suggesting epistatic interactions between P1 family phages and the genetic background of E. coli strains. P1-like phages are part of the mobile elements that carry antibiotic resistance. The high prevalence of P1-like prophages suggests their role may be underestimated.
Asunto(s)
Cromosomas Artificiales de Bacteriófagos P1/genética , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , beta-Lactamasas/genética , Secuencia de Bases , Infecciones por Escherichia coli/tratamiento farmacológico , Infecciones por Escherichia coli/microbiología , Humanos , Datos de Secuencia Molecular , Análisis de Secuencia de ADNRESUMEN
Vertebrate wnt8a links anteroposterior and dorsoventral axis patterning, but the regulation of wnt8a expression and its relationship to mesoderm induction and maintenance pathways is unclear. To address this, we have generated zebrafish transgenic for a modified genomic PAC clone that expresses EGFP from the wnt8a locus. The EGFP reporter transgene is expressed in a pattern nearly identical to wnt8a, including maternal deposition, expression in the ventrolateral mesoderm and in the yolk syncytial layer. Loss of function studies show that wnt8a expression is under biphasic control by Nodal and No Tail/Brachyury, whereby early phase expression is Nodal-dependent but late phase expression is Ntl/Bra dependent. EGFP fluorescence persists in cells that transcribe the reporter, thus comprising a tracer for ventrolaterally derived mesodermal lineages. We use this property to show that wnt8a expression marks Nodal-independent tail mesoderm formation and that Ntl/Bra predominantly regulates wnt8a in paraxial mesoderm progenitors.
Asunto(s)
Cromosomas Artificiales de Bacteriófagos P1/genética , Proteínas del Citoesqueleto/genética , Mesodermo/embriología , Proteínas Wnt/genética , Proteínas de Pez Cebra/genética , Pez Cebra/embriología , Animales , Animales Modificados Genéticamente , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Proteínas del Citoesqueleto/metabolismo , Embrión no Mamífero , Regulación del Desarrollo de la Expresión Génica/fisiología , Redes Reguladoras de Genes/fisiología , Genes Reporteros/fisiología , Mesodermo/metabolismo , Modelos Biológicos , Células Madre/metabolismo , Células Madre/fisiología , Cola (estructura animal)/embriología , Cola (estructura animal)/metabolismo , Vertebrados/embriología , Vertebrados/genética , Vertebrados/metabolismo , Proteínas Wnt/metabolismo , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Heading date is one of the most important quantitative traits responsible for the domestication of rice. We compared a 155-kb reference segment of the Oryza sativa ssp. japonica cv. Nipponbare genome surrounding Hd1, a major heading date gene in rice, with orthologous regions from nine diploid Oryza species that diverged over a relatively short time frame (â¼16 My) to study sequence evolution around a domestication locus. The orthologous Hd1 region from Sorghum bicolor was included to compare and contrast the evolution in a more distant relative of rice. Consistent with other observations at the adh1/adh2, monoculm1, and sh2/a1 loci in grass species, we found high gene colinearity in the Hd1 region amidst size differences that were lineage specific and long terminal repeat retrotransposon driven. Unexpectedly, the Hd1 gene was deleted in O. glaberrima, whereas the O. rufipogon and O. punctata copies had degenerative mutations, suggesting that other heading date loci might compensate for the loss or nonfunctionality of Hd1 in these species. Compared with the japonica Hd1 region, the orthologous region in sorghum exhibited micro-rearrangements including gene translocations, seven additional genes, and a gene triplication and truncation event predating the divergence from Oryza.
Asunto(s)
Diploidia , Genes de Plantas/genética , Oryza/genética , Homología de Secuencia de Ácido Nucleico , Sorghum/genética , Sintenía/genética , Secuencia de Bases , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Bacteriófagos P1/genética , Secuencia de Consenso/genética , Secuencia Conservada/genética , Elementos Transponibles de ADN/genética , Bases de Datos de Ácidos Nucleicos , Sitios Genéticos/genética , Especiación Genética , Datos de Secuencia Molecular , Filogenia , Especificidad de la Especie , Secuencias Repetidas Terminales/genética , Factores de TiempoRESUMEN
Mutations in the presenilin 1 (PS1) gene are the most commonly recognized cause of familial Alzheimer's disease (FAD). Besides senile plaques, neurofibrillary tangles, and neuronal loss, Alzheimer's disease (AD) is also accompanied by vascular pathology. Here we describe an age-related vascular pathology in two lines of PS1 FAD-mutant transgenic mice that mimics many features of the vascular pathology seen in AD. The pathology was especially prominent in the microvasculature whose vessels became thinned and irregular with the appearance of many abnormally looped vessels as well as string vessels. Stereologic assessments revealed a reduction of the microvasculature in the hippocampus that was accompanied by hippocampal atrophy. The vascular changes were not congophilic. Yet, despite the lack of congophilia, penetrating vessels at the cortical surface were often abnormal morphologically and microhemorrhages sometimes occurred. Altered immunostaining of blood vessels with basement membrane-associated antigens was an early feature of the microangiopathy and was associated with thickening of the vascular basal laminae and endothelial cell alterations that were visible ultrastructurally. Interestingly, although the FAD-mutant transgene was expressed in neurons in both lines of mice, there was no detectable expression in vascular endothelial cells or glial cells. These studies thus have implications for the role of neuronal to vascular signaling in the pathogenesis of the vascular pathology associated with AD.
Asunto(s)
Envejecimiento/patología , Enfermedad de Alzheimer/genética , Vasos Sanguíneos/patología , Mutación/genética , Presenilina-1/metabolismo , Envejecimiento/metabolismo , Animales , Atrofia , Membrana Basal/metabolismo , Vasos Sanguíneos/anomalías , Vasos Sanguíneos/metabolismo , Vasos Sanguíneos/ultraestructura , Encéfalo/irrigación sanguínea , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Cromosomas Artificiales de Bacteriófagos P1/genética , Dendritas/metabolismo , Dendritas/patología , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Microvasos/anomalías , Microvasos/metabolismo , Microvasos/patología , Microvasos/ultraestructura , Proteínas Mutantes/metabolismo , Transgenes/genéticaRESUMEN
BACKGROUND: The Wolf-Hirschhorn syndrome (WHS) is usually caused by terminal deletions of the short arm of chromosome 4 and is phenotypically defined by growth and mental retardation, seizures, and specific craniofacial manifestations. Large variation is observed in phenotypic expression of these features. In order to compare the phenotype with the genotype, we localised the breakpoints of the 4 pter aberrations using a chromosome 4 specific tiling BAC/PAC array. METHODS: In total, DNA from 21 patients was analysed, of which 8 had a cytogenetic visible and 13 a submicroscopic deletion. RESULTS AND CONCLUSION: In addition to classical terminal deletions sized between 1.9 and 30 Mb, we observed the smallest terminal deletion (1.4 Mb) ever reported in a patient with mild WHS stigmata. In addition, we identified and mapped interstitial deletions in four patients. This study positions the genes causing microcephaly, intrauterine and postnatal growth retardation between 0.3 and 1.4 Mb and further refines the regions causing congenital heart disease, cleft lip and/or palate, oligodontia, and hypospadias.
Asunto(s)
Cromosomas Humanos Par 4/genética , Síndrome de Wolf-Hirschhorn/genética , Niño , Rotura Cromosómica , Deleción Cromosómica , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Bacteriófagos P1/genética , Femenino , Genotipo , Humanos , Hibridación Fluorescente in Situ , Masculino , Hibridación de Ácido Nucleico , FenotipoRESUMEN
Deletions in the short arm of chromosome 17 (17p) involving the tumor suppressor TP53 occur in up to 20% of diffuse large B-cell lymphomas (DLBCLs). Although inactivation of both alleles of a tumor suppressor gene is usually required for tumor development, the overlap between TP53 deletions and mutations is poorly understood in DLBCLs, suggesting the possible existence of additional tumor suppressor genes in 17p. Using a bacterial artificial chromosome (BAC) and Phage 1 artificial chromosome (PAC) contig, we here define a minimally deleted region in DLBCLs encompassing approximately 0.8 MB telomeric to the TP53 locus. This genomic region harbors the tumor suppressor Hypermethylated in Cancer 1 (HIC1). Methylation-specific PCR demonstrated hypermethylation of HIC1 exon 1a in a substantial subset of DLBCLs, which is accompanied by simultaneous HIC1 deletion of the second allele in 90% of cases. In contrast, HIC1 inactivation by hypermethylation was rarely encountered in DLBCLs without concomitant loss of the second allele. DLBCL patients with complete inactivation of both HIC1 and TP53 may be characterized by an even inferior clinical course than patients with inactivation of TP53 alone, suggesting a functional cooperation between these two proteins. These findings strongly imply HIC1 as a novel tumor suppressor in a subset of DLBCLs.
Asunto(s)
Deleción Cromosómica , Mapeo Cromosómico , Cromosomas Humanos Par 17/genética , Factores de Transcripción de Tipo Kruppel/genética , Linfoma de Células B Grandes Difuso/genética , Telómero/genética , Proteína p53 Supresora de Tumor/genética , Alelos , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Bacteriófagos P1/genética , Cromosomas Humanos Par 17/metabolismo , Metilación de ADN , ADN de Neoplasias/genética , ADN de Neoplasias/metabolismo , Humanos , Factores de Transcripción de Tipo Kruppel/metabolismo , Linfoma de Células B Grandes Difuso/metabolismo , Sitios de Carácter Cuantitativo/genéticaRESUMEN
The early and frequent occurrence of deletions at 3p21.3 in lung cancer has led to the consideration of this chromosomal region as a lung cancer (LUCA) critical region with tumor suppressor activity. We covered this 19 genes-containing region with overlapping P1 artificial chromosomes (PACs), in which genes are likely accompanied by their own promoters or other regulatory sequences. With these PACs we transfected cells from a small cell lung cancer (SCLC) cell line which readily caused tumors in nude mice. Per PAC we selected two cell clones with a low number of PAC copies integrated at a single genomic site. The selected clones were s.c. injected into nude mice to investigate whether the integrated genes suppressed the tumor-inducing capacity of the original SCLC cell line. We could demonstrate PAC-specific gene expression in the transfected cells. All of the PAC integration sites were different. It appeared that introduction of a PAC or even an empty PAC vector causes some chromosomal instability, which in principle may either promote or inhibit cell growth. However, both cell clones with integration of the same PAC from the centromeric part of the LUCA region in different genomic sites were the sole pair of clones that caused smaller tumors than did the original SCLC cell line. This suggests that rather than the induced chromosomal instability, the DNA sequence of that PAC, which in addition to two protein-encoding genes contains at least one potential miRNA gene, is responsible for the tumor suppressor activity.
Asunto(s)
Carcinoma de Células Pequeñas/genética , Cromosomas Humanos Par 3/genética , Genes Supresores de Tumor , Neoplasias Pulmonares/genética , Animales , Carcinoma de Células Pequeñas/patología , Línea Celular Tumoral , Inestabilidad Cromosómica , Cromosomas Artificiales de Bacteriófagos P1/genética , Femenino , Expresión Génica , Humanos , Neoplasias Pulmonares/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Trasplante de Neoplasias , Transfección , Trasplante HeterólogoRESUMEN
In 2-8% of patients with mental retardation, small copy number changes in the subtelomeric region are thought to be the underlying cause. As detection of these genomic rearrangements is labour intensive using FISH, we constructed and validated a high-density BAC/PAC array covering the first 5 Mb of all subtelomeric regions and applied it in our routine screening of patients with idiopathic mental retardation for submicroscopic telomeric rearrangements. The present study shows the efficiency of this comprehensive subtelomere array in detecting terminal deletions and duplications but also small interstitial subtelomeric rearrangements, starting from small amounts of DNA. With our array, the size of the affected segments, at least those smaller than 5 Mb, can be determined simultaneously in the same experiment. In the first 100 patient samples analysed in our diagnostic practice by the use of this comprehensive telomere array, we found three patients with deletions in 3p, 10q and 15q, respectively, four patients with duplications in 9p, 12p, 21q and Xp, respectively, and one patient with a del 6q/dup 16q. The patients with del 3p and 10q and dup 12p had interstitial rearrangements that would have been missed with techniques using one probe per subtelomeric region chosen close to the telomere.
Asunto(s)
Discapacidad Intelectual/diagnóstico , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Telómero/genética , Adulto , Niño , Preescolar , Aberraciones Cromosómicas , Cromosomas Artificiales de Bacteriófagos P1/genética , Cromosomas Bacterianos/genética , ADN/análisis , Femenino , Dosificación de Gen , Humanos , Masculino , Hibridación de Ácido Nucleico/métodosRESUMEN
Clones from one BAC and one PAC library carrying centromeric alphoid DNA were characterized and found to be stable but to differ according to the enzyme used to make the library. Five different clones with homogeneous alphoid DNA, derived from chromosomes 13/21, 14/22, 17 and 18, were all shown to form minichromosomes de novo after transfection into the human cell line HT1080 in greater than 29% of the cell lines analysed. Similarly sized alphoid arrays (110-160 kb) from chromosomes 17, 13/21 and 14/22 all formed minichromosomes in about 50% of the cell lines analysed while a smaller array (50 kb) of 14/22 alphoid was less efficient (29% of cell lines) and a larger array (200 kb) from chromosome 18 was more efficient (2/2 cell lines). Thus the larger arrays of alphoid DNA gave higher percentages of cell lines with minichromosomes. However, smaller arrays may be preferable for gene expression as there appeared to be more EGFP expression from these minichromosomes.
Asunto(s)
Cromosomas/genética , Cromosomas/metabolismo , ADN Satélite/genética , Centrómero/metabolismo , Cromosomas/química , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Bacteriófagos P1/genética , Clonación Molecular , Humanos , Análisis de Secuencia de ADNRESUMEN
During this study, we analysed the pericentric inversion that distinguishes human chromosome 12 (HSA12) from the homologous chimpanzee chromosome (PTR10). Two large chimpanzee-specific duplications of 86 and 23 kb were observed in the breakpoint regions, which most probably occurred associated with the inversion. The inversion break in PTR10p caused the disruption of the SLCO1B3 gene in exon 11. However, the 86-kb duplication includes the functional SLCO1B3 locus, which is thus retained in the chimpanzee, although inverted to PTR10q. The second duplication spans 23 kb and does not contain expressed sequences. Eleven genes map to a region of about 1 Mb around the breakpoints. Six of these eleven genes are not among the differentially expressed genes as determined previously by comparing the human and chimpanzee transcriptome of fibroblast cell lines, blood leukocytes, liver and brain samples. These findings imply that the inversion did not cause major expression differences of these genes. Comparative FISH analysis with BACs spanning the inversion breakpoints in PTR on metaphase chromosomes of gorilla (GGO) confirmed that the pericentric inversion of the chromosome 12 homologs in GGO and PTR have distinct breakpoints and that humans retain the ancestral arrangement. These findings coincide with the trend observed in hominoid karyotype evolution that humans have a karyotype close to an ancestral one, while African great apes present with more derived chromosome arrangements.
Asunto(s)
Centrómero/genética , Rotura Cromosómica/genética , Inversión Cromosómica/genética , Cromosomas Humanos Par 12/genética , Pan troglodytes/genética , Homología de Secuencia de Ácido Nucleico , Animales , Línea Celular , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Bacteriófagos P1/genética , Cromosomas de los Mamíferos/genética , Evolución Molecular , Duplicación de Gen , Reordenamiento Génico/genética , Genes/genética , Gorilla gorilla/genética , Humanos , Hibridación Fluorescente in Situ/métodos , Datos de Secuencia MolecularRESUMEN
Comparative FISH mapping of PAC clones covering almost 3 Mb of the human AZFa region in Yq11.21 to metaphases of human and great apes unravels breakpoints that were involved in species-specific Y chromosome evolution. An astonishing clustering of evolutionary breakpoints was detected in the very proximal region on the long arm of the human Y chromosome in Yq11.21. These breakpoints were involved in deletions, one specific for the human and another for the orang-utan Y chromosome, in a duplicative translocation/transposition specific for bonobo and chimpanzee Y chromosomes and in a pericentric inversion specific for the gorilla Y chromosome. In addition, our comparative results allow the deduction of a model for the human Y chromosome evolution.
Asunto(s)
Rotura Cromosómica/genética , Cromosomas Humanos Y/genética , Evolución Molecular , Primates/genética , Cromosoma Y/genética , Animales , Mapeo Cromosómico/métodos , Cromosomas Artificiales de Bacteriófagos P1/genética , Cromosomas Humanos X/genética , Cromosomas de los Mamíferos/genética , Gorilla gorilla/genética , Humanos , Hibridación Fluorescente in Situ/métodos , Linfocitos/química , Linfocitos/citología , Linfocitos/metabolismo , Macaca nemestrina/genética , Masculino , Metafase/genética , Pan troglodytes/genética , Pongo pygmaeus/genética , Cromosoma X/genéticaRESUMEN
Clones of a PAC contig encompassing the human AZFa region in Yq11.21 were comparatively FISH mapped to great ape Y chromosomes. While the orthologous AZFa locus in the chimpanzee, the bonobo and the gorilla maps to the long arm of their Y chromosomes in Yq12.1-->q12.2, Yq13.1-->q13.2 and Yq11.2, respectively, it is found on the short arm of the orang-utan subspecies of Borneo and Sumatra, in Yp12.3 and Yp13.2, respectively. Regarding the order of PAC clones and genes within the AZFa region, no differences could be detected between apes and man, indicating a strong evolutionary stability of this non-recombining region.
Asunto(s)
Evolución Molecular , Primates/genética , Proteínas de Plasma Seminal/genética , Animales , Línea Celular , Cromosomas Artificiales de Bacteriófagos P1/genética , Cromosomas Humanos X/genética , Cromosomas Humanos Y/genética , Cromosomas de los Mamíferos/genética , Mapeo Contig/métodos , Sitios Genéticos , Gorilla gorilla/genética , Humanos , Hibridación Fluorescente in Situ/métodos , Linfocitos/química , Linfocitos/citología , Linfocitos/metabolismo , Macaca nemestrina/genética , Masculino , Pan troglodytes/genética , Pongo pygmaeus/genética , Cromosoma X/genética , Cromosoma Y/genéticaRESUMEN
Transfer of P1-derived artificial chromosome (PAC) deoxyribonucleic acid (DNA) into keratinocytes is an extremely important technique that enables functional studies of keratinocyte-specific genes to be performed and genomic gene therapy for inherited and acquired diseases to be attempted. Ex vivo gene therapy approaches are possible using well-established conditions for keratinocyte culture and grafting, whilst the skin is the most accessible organ for administering in vivo therapy. PAC vectors lack relevant reporter genes to distinguish transfected mammalian cells from the non-transfected background, or to select clones in which the PAC construct has stably integrated into the genome. In this chapter, protocols to retrofit a reporter gene cassette will be described, together with techniques for transfecting large PAC constructs into keratinocytes without breakage. Protocols to select for stable integrants and to assess the integration event(s) within the keratinocyte genome will also be provided.
Asunto(s)
Cromosomas Artificiales de Bacteriófagos P1/genética , Vectores Genéticos , Queratinocitos/metabolismo , Recombinación Genética , Southern Blotting , Células Cultivadas , Clonación Molecular , HumanosRESUMEN
Human artificial chromosomes (HACs) were generated by transfer of telomerized PAC constructs containing alpha satellite DNA of various human chromosomes. To monitor which cells took up constructs and subsequently formed stable clones under blasticidin S (BS) selection, a CMV/EGFP expression cassette was inserted into a HAC construct based on chromosome 5 alpha satellite DNA (142 kb). Lipofection into HT1080 cells resulted in a small proportion of cells exhibiting bright green fluorescence on day 1. Areas containing such early green cells were marked, and plates monitored over 2 weeks. In only one out of 41 marked areas, a viable clone developed. In the remaining 40 areas, the green cells ceased division at 1-8 cells. In contrast, outside the marked areas, 16 stable clones formed which did not exhibit green fluorescence during the first cell divisions, but all cells of each became green around day 4-6. Fluorescence in situ hybridization (FISH) analysis of isolated clonal lines demonstrated low copy HAC formation without integration. We conclude that transient expression of an EGFP marker on HAC DNA is not a suitable means for the identification of the proportion of transfected cells which are capable of forming viable clones. One explanation could be that the high copy number required to consistently detect transient EGFP expression (Schindelhauer and Laner, 2002) impairs viability and clone formation.
Asunto(s)
Cromosomas Artificiales Humanos/genética , Citomegalovirus/genética , Proteínas Fluorescentes Verdes/genética , Línea Celular , Cromosomas Artificiales de Bacteriófagos P1/genética , Proteínas Fluorescentes Verdes/biosíntesis , Humanos , Telómero/genética , Transfección/métodosRESUMEN
BACKGROUND: Microarray-based comparative genomic hybridisation (array CGH) is a technique by which variation in relative copy numbers between two genomes can be analysed by competitive hybridisation to DNA microarrays. This technology has most commonly been used to detect chromosomal amplifications and deletions in cancer. Dedicated tools are needed to analyse the results of such experiments, which include appropriate visualisation, and to take into consideration the physical relation in the genome between the probes on the array. RESULTS: M-CGH is a MATLAB toolbox with a graphical user interface designed specifically for the analysis of array CGH experiments, with multiple approaches to ratio normalization. Specifically, the distributions of three classes of DNA copy numbers (gains, normal and losses) can be estimated using a maximum likelihood method. Amplicon boundaries are computed by either the fuzzy K-nearest neighbour method or a wavelet approach. The program also allows linking each genomic clone with the corresponding genomic information in the Ensembl database http://www.ensembl.org. CONCLUSIONS: M-CGH, which encompasses the basic tools needed for analysing array CGH experiments, is freely available for academics http://www.uio.no/~junbaiw/mcgh, and does not require any other MATLAB toolbox.
Asunto(s)
Hibridación de Ácido Nucleico/genética , Análisis de Secuencia por Matrices de Oligonucleótidos/estadística & datos numéricos , Cromosomas Artificiales Bacterianos/genética , Cromosomas Artificiales de Bacteriófagos P1/genética , Cromosomas Humanos Par 1/genética , Gráficos por Computador , ADN de Neoplasias/genética , Lógica Difusa , Genes Relacionados con las Neoplasias/genética , Genes Supresores de Tumor , Genoma Humano , Humanos , Hibridación de Ácido Nucleico/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oncogenes/genética , Interfaz Usuario-ComputadorRESUMEN
Deletion of chromosome arm 1p is one of the most frequent genetic alterations in neuroblastoma. However, using conventional comparative genomic hybridization, we have observed amplifications on 1p in 2 neuroblastoma tumors at bands 1p34.2 and 1p36.3, respectively. Using a medium-resolution genomic array containing 178 PACs/BACs from 1p and then 2 high-resolution arrays containing contigs of overlapping PACs/BACs from the amplified regions, we could precisely map and delineate both amplicons. The 1p34.2 amplicon appeared as a homogeneous amplification unit, whereas the 1p36.3 amplicon had a more complex structure, with 2 noncontiguous, highly amplified regions and several moderate amplification units. In this case, fluorescence in situ hybridization analysis confirmed the amplification of several clones and indicated that the 2 highest amplification units corresponded to 2 populations of double minute chromosomes, one of which also contained the MYCN locus. This is the first report of 1p amplifications in primary neuroblastomas.