RESUMEN
Migraine is a prevalent neurovascular disease with a significant genetic component. Linkage studies have so far identified migraine susceptibility loci on chromosomes 1, 4, 6, 11, 14, 19 and X. We performed a genome-wide scan of 92 Australian pedigrees phenotyped for migraine with and without aura and for a more heritable form of "severe" migraine. Multipoint non-parametric linkage analysis revealed suggestive linkage on chromosome 18p11 for the severe migraine phenotype (LOD*=2.32, P=0.0006) and chromosome 3q (LOD*=2.28, P=0.0006). Excess allele sharing was also observed at multiple different chromosomal regions, some of which overlap with, or are directly adjacent to, previously implicated migraine susceptibility regions. We have provided evidence for two loci involved in severe migraine susceptibility and conclude that dissection of the "migraine" phenotype may be helpful for identifying susceptibility genes that influence the more heritable clinical (symptom) profiles in affected pedigrees. Also, we concluded that the genetic aetiology of the common (International Headache Society) forms of the disease is probably comprised of a number of low to moderate effect susceptibility genes, perhaps acting synergistically, and this effect is not easily detected by traditional single-locus linkage analyses of large samples of affected pedigrees.
Asunto(s)
Cromosomas Humanos Par 18 , Perfilación de la Expresión Génica , Genómica , Migraña sin Aura/genética , Ligamiento Genético , Predisposición Genética a la Enfermedad , Humanos , Linaje , FenotipoRESUMEN
The cytochrome p450 enzyme, CYP2D6, metabolises approximately 20% of marketed drugs. CYP2D6 multiple variants are associated with altered enzyme activities. Genotyping 1018 Caucasians for CYP2D6 polymorphisms (G1846A, delT1707, delA2549 and A2935C), known to result in the recessive CYP2D6 poor drug metaboliser (PM) phenotype, identified 41 individuals with predicted PM phenotype. These 41 individuals were classified as 'cases'. Single nucleotide polymorphisms (SNPs) mapping within an 880 kb region flanking CYP2D6, were identified to evaluate potential association between genetic variation and the CYP2D6 PM phenotype. The 41 PM cases and 977 controls were genotyped and analysed for 27 SNPs. Associations were observed across a 390 kb region between 14 SNPs and the PM phenotype (P values from 6.20 x 10(-4) to 4.54 x 10(-35)). Haplotype analysis revealed more significant levels of association (P = 3.54 x 10(-56)). Strong (D' > 0.7) linkage disequilibrium (LD) between SNPs was observed across the same 390 kb region associated with the CYP2D6 phenotype. The observed phenotype:genotype association reached genome-wide levels of significance, and supports the strategy for potential application of LD mapping and whole genome association scans to pharmacogenetic studies.
Asunto(s)
Mapeo Cromosómico/métodos , Citocromo P-450 CYP2D6/genética , Desequilibrio de Ligamiento/genética , Preparaciones Farmacéuticas/metabolismo , Cromosomas/genética , Frecuencia de los Genes , Marcadores Genéticos , Genotipo , Haplotipos , Humanos , Fenotipo , Polimorfismo Genético/genéticaRESUMEN
We have identified a migraine locus on chromosome 19p13.3/2 using linkage and association analysis. We isolated 48 single-nucleotide polymorphisms within the locus, of which we genotyped 24 in a Caucasian population comprising 827 unrelated cases and 765 controls. Five single-nucleotide polymorphisms within the insulin receptor gene showed significant association with migraine. This association was independently replicated in a case-control population collected separately. We used experiments with insulin receptor RNA and protein to investigate functionality for the migraine-associated single-nucleotide polymorphisms. We suggest possible functions for the insulin receptor in migraine pathogenesis.
Asunto(s)
Alelos , Trastornos Migrañosos/genética , Polimorfismo de Nucleótido Simple , Receptor de Insulina/genética , Secuencia de Bases , Estudios de Casos y Controles , Mapeo Cromosómico , Cromosomas Artificiales Bacterianos , Cromosomas Humanos Par 19 , Cartilla de ADN , Femenino , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Desequilibrio de Ligamiento , Masculino , Unión Proteica , Receptor de Insulina/metabolismo , Reproducibilidad de los Resultados , Población Blanca/genéticaRESUMEN
This unit describes several polymerase chain reaction (PCR)-based methods to obtain DNA fragments from clones with large inserts without prior knowledge of the insert DNA sequence. The protocols can be categorized into three groups: (1) methods to generate DNA fragments at random representing the entire length of the cloned insert, (2) methods to generate DNA fragments representing the extremities of an insert, and (3) methods to generate complex probes suitable for fluorescence in situ hybridization. Support protocols describe direct cloning of these PCR products and the isolation of total yeast DNA from yeast artificial chromosome (YAC) clones.
Asunto(s)
Técnicas de Sonda Molecular , Reacción en Cadena de la Polimerasa/métodos , Elementos Alu , Cromosomas Artificiales de Bacteriófagos P1/genética , Cromosomas Artificiales de Levadura/genética , Clonación Molecular , Sondas de ADN/genética , Sondas de ADN/aislamiento & purificación , Vectores Genéticos , Genética Médica , Humanos , Hibridación Fluorescente in SituRESUMEN
The numerous successes using positional cloning to identify genes mutated in monogenic disorders has galvanised geneticists to start using similar techniques to tackle common complex diseases such as asthma, osteoarthritis, depression and early onset heart disease. The technology is currently at an intermediate stage in which linkage in family studies is being supplemented with locus-specific association studies in populations, enabling accurate localisation of the disease causing or susceptibility gene. These studies are often labour and time intensive unless focus is placed on biological candidate genes. In general, most candidate gene studies for common diseases have been unrewarding. However, single nucleotide polymorphisin (SNP) mapping has accelerated complex disease gene localisation, providing a tool to narrow the linkage region by the detection of multiple SNPs associated with the disease in a relatively small linkage disequilibriuln (LD) region. Identification of susceptibility genes will enable a better understanding of the mechanisms of the disease processes and will facilitate the discovery of new and more efficacious medicines. Whole genome SNP maps will also allow abbreviated SNP profiles to be developed for pharmacogenetic applications, enabling physicians to tailor therapeutic regimens (i.e., identify patients likely to receive therapeutic benefit and not suffer adverse reactions).
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Polimorfismo de Nucleótido Simple/genética , ADN/genética , Marcadores Genéticos , Genoma Humano , HumanosRESUMEN
A loxP-transposon retrofitting strategy for generating large nested deletions from one end of the insert DNA in bacterial artificial chromosomes and P1 artificial chromosomes was described recently [Chatterjee, P. K. & Coren, J. S. (1997) Nucleic Acids Res. 25, 2205-2212]. In this report, we combine this procedure with direct sequencing of nested-deletion templates by using primers located in the transposon end to illustrate its value for position-specific single-nucleotide polymorphism (SNP) discovery from chosen regions of large insert clones. A simple ampicillin sensitivity screen was developed to facilitate identification and recovery of deletion clones free of transduced transposon plasmid. This directed approach requires minimal DNA sequencing, and no in vitro subclone library generation; positionally oriented SNPs are a consequence of the method. The procedure is used to discover new SNPs as well as physically map those identified from random subcloned libraries or sequence databases. The deletion templates, positioned SNPs, and markers are also used to orient large insert clones into a contig. The deletion clone can serve as a ready resource for future functional genomic studies because each carries a mammalian cell-specific antibiotic resistance gene from the transposon. Furthermore, the technique should be especially applicable to the analysis of genomes for which a full genome sequence or radiation hybrid cell lines are unavailable.
Asunto(s)
Polimorfismo de Nucleótido Simple , Eliminación de Secuencia , Secuencia de Bases , Cromosomas Bacterianos , Cartilla de ADN , Elementos Transponibles de ADN , Plásmidos , Recombinación GenéticaRESUMEN
We have constructed YAC, PAC, and cosmid contigs in the ataxia-telangiectasia gene region and used the assembled clones to isolate expressed sequences by exon trapping and hybridization selection. In the interval between D11S1819 and D11S2029, exons and cDNAs for potentially 13 different genes were identified. Three of these genes, F37, K28, and 6.82, are large novel genes expressed in a variety of different tissues. K28 shows sequence homology to the Rab GTP binding protein family and gene 6.82 homology to the rabbit vasopressin activated calcium mobilizing receptor, while gene F37 has no homology to any known sequence in the database. Three further clones, exon 6.41 and cDNAs K22 and E74, from the interval between D11S1819 and D11S2029, appear to be expressed endogenous retrovirus sequences. The fourth large novel genes, E14, together with two further possible novel genes, E13 and E3, was identified from exons and cDNAs in the more telomeric 300-kb interval between markers D11S2029 and D11S2179. These are in addition to the genes for mitochondrial acetoacetyl-CoA-acetyltransferase (ACAT) and the ATM gene in the same region. Genes E3, E13, and E14 do not show homology to any known genes. K28, 6.82, ACAT, and ATM all appear to have the same transcriptional orientation toward the telomere.
Asunto(s)
Ataxia Telangiectasia/genética , Mapeo Cromosómico/métodos , Cromosomas Humanos Par 11/genética , Proteínas Serina-Treonina Quinasas , Proteínas/genética , Transcripción Genética/genética , Acetil-CoA C-Acetiltransferasa/genética , Secuencia de Aminoácidos , Proteínas de la Ataxia Telangiectasia Mutada , Proteínas de Ciclo Celular , ADN Complementario/genética , Proteínas de Unión al ADN , Exones/genética , Proteínas de Unión al GTP/genética , Humanos , Datos de Secuencia Molecular , Especificidad de Órganos , ARN Mensajero/análisis , ARN Mensajero/genética , Retroviridae/genética , Homología de Secuencia de Aminoácido , Proteínas Supresoras de TumorRESUMEN
The inherited metabolic disorder trimethylaminuria (fish-odor syndrome) is associated with defective hepatic N-oxidation of dietary-derived trimethylamine catalyzed by flavin-containing monooxygenase (FMO). As FMO3 encodes the major form of FMO expressed in adult human liver, it represents the best candidate gene for the disorder. The structural organization of FMO3 was determined by sequencing the products of exon-to-exon and vectorette PCR, the latter through the use of vectorette libraries constructed directly from genomic DNA. The gene contains one noncoding and eight coding exons. Knowledge of the exon/intron organization of the human FMO3 gene enabled each of the coding exons of the gene, together with their associated flanking intron sequences, to be amplified from genomic DNA and will thus facilitate the identification of mutations in FMO3 in families affected with fish-odor syndrome.
Asunto(s)
Errores Innatos del Metabolismo/genética , Metilaminas/metabolismo , Oxigenasas/genética , Exones , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Intrones , Datos de Secuencia Molecular , NADP/metabolismo , Odorantes , Oxigenasas/metabolismo , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADNRESUMEN
Due to its unique biology of partial sex linkage and high recombination rates, the pseudoautosomal region (PAR1) on both X and Y chromosomes has attracted considerable interest. In addition, an extremely high level of YAC instability has been observed in this region. We have derived 82 YAC clones from six different YAC libraries mapping to this 2.6-Mb region. Of these a subset of 22 YACs was analyzed in detail. YAC contigs were assembled using 67 pseudoautosomal probes, of which 64 were unambiguously ordered. All markers are well distributed over the entire region, including the middle part of the region, which has previously been found difficult to contig. Two gaps of less than 50 kb within the genomic locus of CSF2RA and around XE7 remain, which could not be covered with YACs, cosmids, or phages. This YAC contig anchored on the physical map of PAR1 represents one of the best characterized large regions of the human genome with a map completion greater than 90% at 100-kb resolution and has permitted the accurate localization of all known genes within this region.
Asunto(s)
Cromosomas Artificiales de Levadura , Cromosoma X , Cromosoma Y , Quimera , Deleción Cromosómica , Mapeo Cromosómico , Clonación Molecular , Femenino , Biblioteca de Genes , Marcadores Genéticos , Humanos , Hibridación Fluorescente in Situ , MasculinoRESUMEN
Although IL-12 has been reported to synergize with c-kit ligand (KL) in promoting hematopoietic stem cell proliferation in vitro, administration of recombinant mouse IL-12 (rIL-12) to normal mice caused a dose- and time-dependent anemia, leukopenia, and thrombocytopenia in vivo. Decreased numbers of bone marrow cells were recovered from the tibiae of IL-12-treated mice, and histologic examination of the marrow revealed a loss of mature neutrophils and red blood cell precursors. However, simultaneously with the suppression of hematopoiesis in the bone marrow, the IL-12-treated mice developed splenomegaly, which was largely caused by a marked enhancement of splenic extramedullary hematopoiesis of the erythroid, myeloid, and megakaryocytic lineages. These histologic observations were confirmed by colony-forming cell assays in which administration of IL-12 was shown to cause a time-dependent decrease in bone marrow CFU-GM, CFU-E, and BFU-E hematopoietic colony-forming cells while causing an increase in splenic CFU-GM and BFU-E colony-forming cells. All these effects were reversible upon cessation of IL-12 treatment. The observation that in IL-12-treated mice hematopoiesis was suppressed in the marrow but enhanced in the spleen suggests that myelosuppression was not caused by a direct effect of IL-12 on hematopoietic progenitors. It seems likely that myelosuppression was caused instead by an IL-12-induced alteration in the local environment of the marrow.
Asunto(s)
Médula Ósea/efectos de los fármacos , Hematopoyesis Extramedular/efectos de los fármacos , Hematopoyesis/efectos de los fármacos , Interleucina-12/farmacología , Bazo/efectos de los fármacos , Anemia/inducido químicamente , Animales , Relación Dosis-Respuesta a Droga , Leucopenia/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Proteínas Recombinantes/farmacología , Valores de Referencia , Trombocitopenia/inducido químicamenteRESUMEN
BACKGROUND: Interleukin-12 is a novel heterodimeric cytokine that stimulates the proliferation of activated T and NK cells and induces lymphokine-activated killer cell activity in vitro. To investigate the biological effects of recombinant human IL-12 (rHuIL-12) in vivo, two exploratory studies were conducted in squirrel monkeys (Sciureus saimiri), which have been shown to be pharmacologically responsive to rHuIL-12 in vitro. EXPERIMENTAL DESIGN: In the first study, 18 monkeys (3/sex/group) were given daily subcutaneous injections of 0 (vehicle control), 10, or 50 micrograms/kg/day rHuIL-12 for 14 days. In the second study, 18 monkeys were given 0, 0.1, or 1 micrograms/kg/day rHuIL-12 for 14 days The animals were monitored for clinical signs, hematology and clinical chemistry changes, and sacrificed on day 15 to evaluate gross and histopathologic changes. One monkey in the high dose group was sacrificed moribund on day 14. RESULTS: Monkeys given rHuIL-12 had dose-related hematologic changes characterized by mild to moderate anemia and leukocytosis. Serum chemistry changes included hypoproteinemia, hypoalbuminemia, hypophosphatemia, and hypocalcemia. Gross pathologic findings included generalized lymph node enlargement and splenomegaly with pulmonary edema and peritoneal effusions in two high dose monkeys. Dose-related histopathologic findings included thymic cortical atrophy, splenic lymphoid hyperplasia with histiocytic hyperplasia and extramedullary hematopoiesis of red pulp, Kupffer cell hypertrophy and hyperplasia, trilineage bone marrow hyperplasia, and reactive hyperplasia of lymph nodes. Animals in the 10 and 50 micrograms/kg/day dose groups developed high titers of anti-rHuIL-12 antibodies by day 15. CONCLUSIONS: These studies indicate that rHuIL-12 is bioactive over a wide dose range and induces prominent hyperplasia of hematopoietic and lymphohistiocytic tissues in squirrel monkeys. Moreover, positive immunomodulatory activity (enhanced lymphocyte lytic activity) was detected at a dose of rHuIL-12 that is 500-fold less than the dose causing severe toxicity.
Asunto(s)
Interleucina-12/farmacología , Animales , Células Sanguíneas/efectos de los fármacos , Análisis Químico de la Sangre , Pruebas Inmunológicas de Citotoxicidad , Femenino , Humanos , Técnicas para Inmunoenzimas , Interleucina-12/sangre , Interleucina-12/toxicidad , Hígado/efectos de los fármacos , Hígado/patología , Linfocinas/sangre , Masculino , Proteínas Recombinantes/sangre , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/toxicidad , Saimiri , Bazo/efectos de los fármacos , Bazo/patologíaRESUMEN
Treacher Collins syndrome (TCOF1) is an autosomal dominant disorder of craniofacial development the features of which include conductive hearing loss and cleft palate. Previous studies have localized the TCOF1 locus between D5S519 (proximal) and SPARC (distal), a region of 22 centirays as estimated by radiation hybrid mapping. In the current investigation we have created a contig across the TCOF1 critical region, using YAC clones. Isolation of a novel short tandem repeat polymorphism corresponding to the end of one of the YACs has allowed us to reduce the size of the critical region to approximately 840 kb, which has been covered with three nonchimeric YACs. Restriction mapping has revealed that the region contains a high density of clustered rare-cutter restriction sites, suggesting that it may contain a number of different genes. The results of the present investigation have further allowed us to confirm that the RPS14 locus lies proximal to the critical region and can thereby be excluded from a role in the pathogenesis of TCOF1, while ANX6 lies within the TCOF1 critical region and remains a potential candidate for the mutated gene.
Asunto(s)
Anexina A6/genética , Cromosomas Artificiales de Levadura , Cromosomas Humanos Par 5 , Disostosis Mandibulofacial/genética , Secuencia de Bases , Paseo de Cromosoma , Clonación Molecular/métodos , Cartilla de ADN , Biblioteca de Genes , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos Nucleicos , Mapeo Restrictivo , Lugares Marcados de SecuenciaRESUMEN
We have constructed a long-range physical map for 12 markers, including genes for GRIA4, IL1BC, and ACAT, across 9 Mb of chromosome 11q22-q23 in the region of the major locus for ataxia-telangiectasia (A-T). The markers fall into proximal and distal groups with respect to the centromere. We have linked the proximal and distal groups by hybridization to a 2.7-Mb NotI fragment and a 4.6-Mb MluI fragment. The following locus order was obtained: centromere-CJ52.75-J12.1C2-Y11B11R-IL1BC-+ ++hbcDNA-GRIA4-CJ52.3-Y11B29L-ACAT- CJ52.193-J12.8-Y11B06R-telomere. We show that hbcDNA/GRIA4 and CJ52.3 are very closely linked to each end, respectively, of the 2.7-Mb NotI fragment, thereby fixing the position of the complete contig. Our results indicate that the gene for A-T is flanked by the markers GRIA4 and J12.8, which are no more than 3 Mb apart, on a 4.6-Mb MluI fragment. The physical map allows rapid positioning of markers, and this will facilitate the construction of a YAC contig across the region.
Asunto(s)
Ataxia Telangiectasia/genética , Cromosomas Humanos Par 11 , Secuencia de Bases , Línea Celular , Centrómero , Mapeo Cromosómico , ADN/genética , Cartilla de ADN , Ligamiento Genético , Marcadores Genéticos , Humanos , Escala de Lod , Linfocitos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Recombinación Genética , Mapeo RestrictivoAsunto(s)
Cromosomas Humanos Par 11 , Genes Fúngicos , Leucemia/genética , Translocación Genética , Levaduras/genética , Enfermedad Aguda , Adulto , Anciano , Niño , Mapeo Cromosómico , Femenino , Biblioteca de Genes , Humanos , Hibridación in Situ , Lactante , Masculino , Reacción en Cadena de la Polimerasa , Tenipósido/efectos adversosRESUMEN
A cluster of metalloproteinase genes, stromelysin, fibroblast collagenase, and stromelysin 2 together with the anonymous DNA marker D11S385, was mapped using pulsed-field gel electrophoresis to a 135-kb region of chromosome 11q22-q23. The physical proximity of these markers was subsequently confirmed using two YAC clones, and their relative order was established as stromelysin 2-collagenase-stromelysin-D11S385. The pattern of marker representation in a panel of radiation-reduced chromosome 11 hybrids suggests that the metalloproteinase gene/D11S385 cluster is orientated with STMY2 closest to the centromere.
Asunto(s)
Cromosomas Humanos Par 11 , Metaloendopeptidasas/genética , Familia de Multigenes , Animales , Secuencia de Bases , Colagenasas/genética , Cricetinae , ADN/genética , Sondas de ADN , Marcadores Genéticos , Glicoproteínas/genética , Humanos , Células Híbridas , Metaloproteinasa 10 de la Matriz , Metaloproteinasa 3 de la Matriz , Datos de Secuencia MolecularRESUMEN
We have constructed the physical map of the 0.8 megabase DNA fragment which contains the 3' 64 variable region (V) gene segments of the human immunoglobulin heavy chain (H) locus. The organization of the VH locus showed several features that indicate dynamic reshuffling of this locus. The sequenced 64 VH segments include 31 pseudogenes, of which 24 are highly conserved except for a few point mutations. Comparison of the 64 germline VH sequences shows that each VH family has conserved sequences, suggesting that there might be some genetic or selection mechanisms involved in maintenance of each family. The total number of the human VH segments was estimated to be about 120, including at least 7 orphons.
Asunto(s)
Cadenas Pesadas de Inmunoglobulina/genética , Región Variable de Inmunoglobulina/genética , Secuencia de Aminoácidos , Secuencia de Bases , Southern Blotting , Línea Celular , Cromosomas Humanos Par 14 , ADN , Expresión Génica , Humanos , Datos de Secuencia Molecular , Mapeo RestrictivoRESUMEN
The yeast artificial chromosome (YAC-13HH4), which spans a 440-kb region of DNA just distal to the CD3 locus on chromosome 11 at band q23, has been used to characterize a range of chromosomal translocations in acute leukemias from both adults and infants. In situ hybridization was performed on metaphase cells from bone marrow of 17 leukemias and two cell lines with a variety of chromosome 11q23 abnormalities. It was established that in infant leukemias the translocations t(11;19), t(4;11), and t(5;11) had occurred in the region defined by YAC 13HH4. Additionally, the translocations t(4;11), t(6;11), t(9;11), t(X;11), and t(10;11) in other leukemias were found to disrupt the same region of chromosome 11q23, although an exception was found in one t(6;11) translocation for which the breakpoint was distal to the YAC. One patient had a t(9;11) translocation in a therapy-related leukemia, suggesting that this class of etoposide-related malignancy has similar breakpoints to those occurring in de novo leukemias. An example of a lymphoma-derived translocation t(4;11) was shown to involve a deletion of the region defined by YAC 13HH4. A leukemia with a deletion on chromosome 11 (q23-q25) was also studied and it was shown that the YAC sequence was unaffected. It was concluded that, with a few exceptions, the translocations at 11q23 in a wide range of acute infant and adult leukemias occur in a common region and may result from a common underlying mechanism.
Asunto(s)
Cromosomas Humanos Par 11 , Leucemia/genética , Translocación Genética , Enfermedad Aguda , Adulto , Cromosomas Fúngicos , Femenino , Humanos , Hibridación in Situ , Lactante , Cariotipificación , Masculino , Saccharomyces cerevisiae/genética , Células Tumorales Cultivadas , Cromosoma XRESUMEN
Three overlapping yeast artificial chromosomes (YACs) spanning a 780 kb region of DNA around the CD3 locus on chromosome 11 have been isolated and characterised. The individual cloned regions have been mapped by in situ hybridisation to chromosome band 11q23, and a restriction enzyme map of this region has been constructed. The positions of these clones in relation to a series of leukaemia-associated chromosomal translocations has also been determined. It was concluded that, although two clones lay entirely proximal to the breakpoints examined, the third clone (13HH4) encompassed the breakpoints for the translocations t(4;11), t(6;11), and t(9;11). The t(9;11) was observed in an acute myeloid leukaemia in a patient previously treated for an unrelated malignancy. It would thus appear that the breakpoints at chromosome band 11q23 occurring in therapy-related leukaemias are in the same region as those found in adult and childhood acute leukaemias and may result from a common underlying mechanism.
Asunto(s)
Cromosomas Humanos Par 11 , Leucemia/genética , Translocación Genética/genética , Antígenos de Diferenciación de Linfocitos T/genética , Secuencia de Bases , Southern Blotting , Cromosomas Humanos Par 4 , Cromosomas Humanos Par 6 , Cromosomas Humanos Par 9 , Clonación Molecular , Humanos , Hibridación Fluorescente in Situ , Datos de Secuencia Molecular , Sondas de Oligonucleótidos/genéticaRESUMEN
Vectors have been constructed for the introduction of the neomycin resistance gene (neo) into the left arm, right arm or human insert DNA of yeast artificial chromosomes (YACs) by homologous recombination. These vectors contain a yeast selectable marker Lys-2, i.e. the alpha-aminoadipidate reductase gene, and a mammalian selection marker, neo, which confers G418 resistance. The vectors can be used to modify YACs in the most commonly used yeast strain for YAC library construction, AB1380. Specific targeting can be carried out by transfection of restriction endonuclease treated linear plasmids, with highly specific recombinogenic ends, into the YAC containing yeast cells. Analysis of targeted YACs confirmed that all three vectors can target correctly in yeast. Introduction of one of the targeted YACs into V79 (Chinese hamster fibroblast) cells showed complete and intact transfer of the YAC.