RESUMEN
A fundamental problem in the identification and isolation of tumor suppressor and other growth-inhibiting genes is the loss of power of genetic complementation at the subchromosomal level. A direct genetic strategy was developed to isolate subchromosomal transferable fragments (STFs) from any chromosome, each containing a selectable marker within the human DNA, that could be transferred to any mammalian cell. As a test of the method, several overlapping STFs from 11p15 were shown to cause in vitro growth arrest of rhabdomyosarcoma cells. This activity mapped between the beta-globin and insulin genes.
Asunto(s)
Cromosomas Humanos Par 11 , ADN/genética , Genes Supresores de Tumor , Técnicas Genéticas , Rabdomiosarcoma/patología , Animales , Secuencia de Bases , Células CHO , División Celular , Línea Celular , Cricetinae , Marcadores Genéticos , Globinas/genética , Humanos , Insulina/genética , Ratones , Datos de Secuencia Molecular , Células Tumorales CultivadasRESUMEN
We have isolated cDNAs for three members of a family of seven-pass transmembrane cadherins in mouse (Celsr1, 2 and 3). These three genes represent vertebrate homologues of flamingo/starry night, recently identified as an essential component of the Drosophila planar cell polarity pathway and for the correct formation of dendritic fields within the Drosophila peripheral nervous system. In this study, we show that each member of the mouse Celsr family exhibit distinct patterns of expression within a range of different tissues within the developing embryo. Celsr1 and Celsr2 expression is observed during gastrulation and within the developing nervous system. Celsr3 transcripts, however, are found only at sites of active neurogenesis.
Asunto(s)
Cadherinas/genética , Proteínas Fetales , Expresión Génica , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Animales , Drosophila , Proteínas de Drosophila , Desarrollo Embrionario y Fetal , Perfilación de la Expresión Génica , Humanos , RatonesRESUMEN
Mcelsr1 encodes a protein of 3034 amino acids predicted to contain seven membrane spanning domains having homology to a group of peptide hormone binding G-protein coupled receptors. Its extracellular domain comprises epidermal growth factor-like repeats, laminin A G-domains and cadherin repeats. Homologous genes have been identified in C. elegans and D. melanogaster suggesting that the Celsr gene family is ancient. mCelsr1 mRNA expression precedes gastrulation, is subsequently restricted primarily to ectodermal derivatives and is tightly regulated in the developing central nervous system (CNS). We observe segmentally-restricted gene expression in the developing hindbrain and in the spinal cord dynamic dorso-ventrally restricted 'stripes' of expression.
Asunto(s)
Cadherinas/genética , Desarrollo Embrionario y Fetal/genética , Proteínas Fetales/genética , Regulación del Desarrollo de la Expresión Génica , Receptores de Superficie Celular/genética , Receptores Acoplados a Proteínas G , Secuencia de Aminoácidos , Animales , Blastocisto/metabolismo , Cadherinas/biosíntesis , Cadherinas/química , Cadherinas/fisiología , Caenorhabditis elegans/genética , Sistema Nervioso Central/embriología , Sistema Nervioso Central/metabolismo , ADN Complementario/genética , Drosophila melanogaster/genética , Evolución Molecular , Proteínas Fetales/biosíntesis , Proteínas Fetales/química , Proteínas Fetales/fisiología , Gástrula/metabolismo , Ratones , Datos de Secuencia Molecular , Morfogénesis/genética , Proteínas del Tejido Nervioso/biosíntesis , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/fisiología , Conformación Proteica , Estructura Terciaria de Proteína , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Receptores de Superficie Celular/biosíntesis , Receptores de Superficie Celular/química , Receptores de Superficie Celular/fisiología , Erizos de Mar/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Especificidad de la EspecieRESUMEN
We have constructed a transcription map covering a 2 Mb region beginning approximately 1 Mb distal to HLA-F. Cosmids isolated from a chromsome 6 library were positioned by YAC hybridisation, STS and fingerprint analysis. Using direct cDNA selection, exon trapping, and direct genomic sequence analysis, we identified 42 potential exonic fragments in this region. Six fragments corresponded to previously characterised genes, four previously broadly mapped to this region. Five fragments were similar to known genes, eight fragments matched ESTs and 10 of the remaining 23 novel fragments, gave a positive signal on northern analysis. All cDNA fragments were mapped to the YAC and cosmid contig covering the region and with respect to other known genes and STS in this area. The distribution of the cDNA fragments indicated their organisation in three clusters around CpG islands.
Asunto(s)
Antígenos HLA/genética , Antígenos de Histocompatibilidad Clase I/genética , Transcripción Genética , Secuencia de Bases , Clonación Molecular , Mapeo Contig , Cósmidos , Cartilla de ADN , HumanosRESUMEN
We describe the construction of a cosmid, LoristB, that contains SP6 and T7 phage-encoded RNA polymerase promoter sequences that are oriented towards and immediately adjacent to HindIII and BamHI cloning sites. We describe techniques for rapidly generating RNA probes from these promoters that must be complementary to the extreme left or right ends of the cloned DNA and can be used for library screening. Probe preparation requires neither prior knowledge of restriction sites nor fragment isolation. We also make extensive use of cos mapping restriction-mapping protocols that we have devised for our cosmid vectors for generation and alignment of steps in a cosmid walk.
Asunto(s)
Mapeo Cromosómico , Clonación Molecular , Cósmidos , ADN Recombinante/metabolismo , Escherichia coli/genética , Vectores Genéticos , Fagos T/genética , Secuencia de Bases , ARN Polimerasas Dirigidas por ADN/genética , Genes , Genes Virales , Plásmidos , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
Transcription terminators have been included in a phage-lambda-replicon-based cosmid vector, Lorist2, to insulate vector genes against transcriptional interference from cloned insert DNA. DNA yields of recombinant clones containing Escherichia coli genomic DNA inserts are more even for Lorist2 than with its progenitor LoristB. However, the terminators provide only a partial reduction in the over-representation of r X DNA-containing clones generally observed in cosmid libraries of Caenorhabditis elegans DNA, suggesting that causes other than transcriptional readthrough into the vector contribute to this problem.
Asunto(s)
Clonación Molecular/métodos , Cósmidos , Replicación del ADN , Genes Reguladores , Vectores Genéticos , Regiones Terminadoras Genéticas , Animales , Caenorhabditis/genética , División Celular , Escherichia coli/genética , Regiones Promotoras GenéticasRESUMEN
We describe a new phage-lambda-replicon-based cosmid vector suitable for both chromosome walking and P-element-mediated transformation in Drosophila. Its unique BamHI cloning site is flanked by the promoters for the SP6 and T7-encoded RNA polymerases, permitting the synthesis of probes complementary to the ends of the cloned inserts for library screening. The selectable marker is tet for bacterial cell transformation and neo for Drosophila transformation expressed under the control of the Drosophila hsp70 promoter.
Asunto(s)
Cósmidos , Drosophila/genética , Vectores Genéticos , Regiones Promotoras Genéticas , Transfección , Animales , Bacteriófago lambda/genética , Clonación Molecular , Proteínas de Choque Térmico/genética , ReplicónRESUMEN
The gene responsible for hereditary haemochromatosis (HH) has recently been identified. One mutation in this gene, termed HFE, has been found in all Australian HH patients. We previously identified a predominant HH ancestral haplotype covering 4.5Mb at 6p21.3, and showed that patients with two copies of this haplotype express a more severe form of the disorder. One key question to now be resolved is why haplotype related variation in phenotypic expression of HH is present if all patients tested have the same HFE mutation. A cosmid resource covering the 4.5Mb HH ancestral haplo type region was obtained. These cosmids provide the material for the completion of a transcript map of this region, and will assist the identification of candidate modifiers of HFE expression.
Asunto(s)
Cromosomas Humanos Par 6/genética , Antígenos HLA/genética , Hemocromatosis/genética , Antígenos de Histocompatibilidad Clase I/genética , Proteínas de la Membrana , Cromosomas Artificiales de Levadura , Cósmidos , Femenino , Proteína de la Hemocromatosis , Humanos , MasculinoAsunto(s)
Mapeo Cromosómico/métodos , Clonación Molecular/métodos , Animales , Cósmidos , ADN , Biblioteca de GenesRESUMEN
We report that a number of related zinc finger protein genes are closely linked on mouse chromosome 17. At least four of these genes are transcribed in the 8.5-day postcoitum embryo and are deleted in the t complex early acting embryonic lethal mutation tw18. We have evidence that additional finger protein genes are located in this region. These findings demonstrate that related finger protein genes can be clustered in the murine genome and identify genes that may be considered as candidates for the tw18 mutation.
Asunto(s)
Genes Letales , Dedos de Zinc , Secuencia de Bases , Deleción Cromosómica , Mapeo Cromosómico , Clonación Molecular , ADN/genética , Sondas de ADN , Genes , Datos de Secuencia Molecular , Oligonucleótidos/química , Polimorfismo de Longitud del Fragmento de Restricción , Homología de Secuencia de Ácido NucleicoRESUMEN
We describe the construction and use of a cosmid vector, loric, which is derived from the phage lambda origin of replication and appears to be more stable than ColE1-derived cosmids. Loric recombinants can be efficiently packaged in vivo to yield 100-300 micrograms of DNA per liter that is linear and has single-stranded cos ends. We call such molecules "phosmids." Phosmid restriction maps can be rapidly generated by labeling either the left or right cos site by annealing on a 32P-labeled oligonucleotide complementary to either cos-L or cos-R. Partial restriction enzyme digestion, agarose gel electrophoresis, and autoradiography are used to size restriction fragments of increasing length, all of which terminate at the labeled cos site. The procedures have been tested by isolating and mapping a region of the H-2 locus of mouse chromosome 17.
Asunto(s)
Mapeo Cromosómico , Enzimas de Restricción del ADN , Vectores Genéticos , Plásmidos , Bacteriófago lambda/genética , Secuencia de Bases , Replicación del ADN , Peso MolecularRESUMEN
A large body of evidence that links alterations of chromosome 11p13 to tumor formation and various developmental disorders has been accumulated. To address the underlying genetic events it would be helpful to have a comprehensive gene map of the region, and this is most readily achieved by generating the complete genomic sequence. Building upon previous mapping and YAC contig analysis we have established a 3-Mb sequence-ready PAC contig. It was constructed by chromosome walking and independently verified by fingerprint analysis of individual clones. The contig starts from the catalase gene on the centromeric side and reaches beyond the PAX6 gene at the 11p13/p14.1 boundary. Additional smaller contigs on either side were identified, but still have to be linked up. The 3-Mb contig spans the central region of deletions encompassing 16 chromosomal breakpoints in patients with WAGR syndrome (Wilms tumor, aniridia, genitourinary malformation, mental retardation), and its construction is an important step in facilitating functional analysis of these genes.
Asunto(s)
Mapeo Cromosómico/métodos , Cromosomas Humanos Par 11 , Clonación Molecular/métodos , Mapeo Contig , Tumor de Wilms/genética , Aberraciones Cromosómicas , Dermatoglifia del ADN , Biblioteca de Genes , Humanos , Análisis de Secuencia de ADNRESUMEN
The region p13 of the short arm of human chromosome 11 has been studied intensely during the search for genes involved in the etiology of the Wilms' tumor, aniridia, genitourinary abnormalities, mental retardation (WAGR) syndrome, and related conditions. The gene map for this region is far from being complete, however, strengthening the need for additional gene identification efforts. We describe the extension of an existing contig map with P1-derived artificial chromosomes (PACs) to cover 7.5 Mb of 11p13-14.1. The extended sequence-ready contig was established by end probe walking and fingerprinting and consists of 201 PAC clones. Utilizing bins defined by overlapping PACs, we generated a detailed gene map containing 20 genes as well as 22 anonymous ESTs which have been identified by searching the RH databases. RH maps and our established gene map show global correlation, but the limits of resolution of the current RH panels are evident at this scale. Initial expression studies on the novel genes have been performed by Northern blot analyses. To extend these expression profiles, corresponding mouse cDNA clones were identified by database search and employed for Northern blot analyses and RNA in situ hybridizations to mouse embryo sections. Genomic sequencing of clones along a minimal tiling path through the contig is currently under way and will facilitate these expression studies by in silico gene identification approaches.
Asunto(s)
Cromosomas Humanos Par 11 , Expresión Génica , Animales , Bacteriófago P1/genética , Northern Blotting/métodos , Mapeo Cromosómico , Paseo de Cromosoma/métodos , Cromosomas Artificiales de Levadura/genética , Mapeo Contig/métodos , Dermatoglifia del ADN/métodos , Sondas de ADN/genética , ADN Complementario/genética , ADN Viral/genética , Bases de Datos Factuales , Embrión de Mamíferos , Etiquetas de Secuencia Expresada , Humanos , Hibridación in Situ/métodos , Ratones , Mapeo Físico de Cromosoma , ARN/análisisRESUMEN
We describe the construction and characterization of miniplasmid vectors that can be used to isolate and express normal and mutant alleles of the human beta-globin gene. These vectors, designated pi SV beta plasmids, contain a bacterial origin of replication and selectable marker, a 5'-flanking beta-globin DNA fragment that can be used for recombination screening (Seed, 1983), and simian virus 40 (SV40) sequences that allow accurate and efficient expression of the beta-globin gene transfected into mammalian cells. We show that pi SV beta plasmids can be used to select cloned beta-globin genes from a bacteriophage lambda library of genomic DNA, and that plasmids containing the beta-globin gene linked to the SV40 enhancer sequence can be excised from the phage, circularized and recovered by transformation of Escherichia coli. Analysis of the beta-globin transcripts produced by the recovered pi SV beta recombinant plasmids after transfection into COS cells and replication to high copy number, indicates that the beta-globin gene is accurately transcribed, but a substantial fraction of the transcripts are the result of readthrough from sites within the vector. In contrast, when these plasmids are transferred into HeLa cells beta-globin RNA is accurately initiated and little readthrough transcription is observed. These results indicate that HeLa cells are more suitable than COS cells for studying mutant beta-globin genes, even though the copy number of the pi SV beta plasmids is much higher in COS cells.
Asunto(s)
Vectores Genéticos , Globinas/genética , Plásmidos , Alelos , Animales , Células Cultivadas , Genes , Humanos , Mutación , Recombinación Genética , Virus 40 de los Simios/genética , Transcripción GenéticaRESUMEN
Type II collagen is the major extracellular matrix component of cartilage and correct expression of the alpha 1(II) collagen gene is important for vertebrate skeletal development. In order to provide the basis for studying the control of type II collagen gene expression in embryogenesis and in mouse models of human connective tissue disease, the complete mouse Col2-a1 gene has been isolated in a single cosmid clone, cosMco1.2, and partially characterized. The gene is approximately 30 kb and is highly conserved in exon/intron structure and nucleotide and amino acid sequence (greater than 80% homology) when compared with the human, rat, bovine and chicken equivalents. A high degree of conservation was also found in the 5' flanking region of the rat, human and mouse alpha 1(II) collagen genes, including the presence of several G + C and C + T rich, direct repeat motifs. The sites of transcription start, termination codon and polyadenylation have also been identified. Unlike chicken, bovine and human, where polyA attachment is at a single site, for the mouse Col2a-1 gene two polyadenylation sites are utilized. Col2a-1 has also been localized by interspecies backcross analysis to the central portion of mouse Chromosome (Chr) 15, approximately 8 centiMorgans (cM) proximal of Int-1 and 18 cM distal of Myc. Col2a-1 is therefore included in a linkage group which is conserved on human Chr 12q.
Asunto(s)
Colágeno/genética , Ratones/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , Cruzamientos Genéticos , Datos de Secuencia Molecular , Poli A/metabolismo , Homología de Secuencia de Ácido Nucleico , Transcripción Genética , Vertebrados/genéticaRESUMEN
We have previously reported the identification of a novel transforming gene, hst, in DNA samples taken from human stomach cancers and a noncancerous portion of stomach. Five clones, containing the genomic hst gene, were isolated from a human cosmid library constructed from leukocyte DNA from a patient with acute leukemia. All clones possessed transforming activity when transfected to NIH3T3 cells. From one clone, an 8.7 kb BamHI fragment was subcloned into pBR322, and this subclone was active in transforming NIH3T3 cells. This is the first isolation of transformation-competent genomic hst clones directly from a human genomic library, that is, without prior passage through NIH3T3 cells.
Asunto(s)
Transformación Celular Neoplásica , ADN Recombinante , ADN/genética , Leucemia/genética , Enfermedad Aguda , Línea Celular , Enzimas de Restricción del ADN , Desoxirribonucleasa BamHI , Humanos , Leucocitos/análisis , Hibridación de Ácido Nucleico , TransfecciónRESUMEN
Proteins that share conserved "zinc finger" motifs represent a class of DNA-binding proteins that have been shown to play a fundamental role in regulating gene expression and to be involved in a number of human hereditary and malignant disease states. We have isolated, characterized, and mapped zinc finger-encoding genes specific to human chromosome 11q to investigate their possible association in the molecular pathogenesis of several disease loci mapped to this chromosome. An arrayed chromosome 11q cosmid library was screened using a degenerate oligonucleotide corresponding to the H/C link consensus sequence of the Drosophila Kruppel zinc finger gene, resulting in the isolation of six putative zinc finger genes. Three of the genes (ZNF123, ZNF125, and ZNF126) were analyzed and shown to contain tandemly repeated zinc finger motifs of the C2-H2 class. All three novel genes were found to be expressed in normal adult human tissues, although the tissue-specific pattern of expression differs markedly. Isolated zinc finger genes were regionally mapped on chromosome 11 using fluorescence in situ suppression hybridization and demonstrated clustering of the genes at 11q13.3-11q13.4 and 11q23.1-11q23.2. Analysis of in situ hybridization to interphase nuclei demonstrated a maximum distance of 1 Mb separating distinct finger genes. This analysis defines two linked multigene families of zinc finger genes to chromosome bands associated with a high frequency of specific translocations associated with malignancies.
Asunto(s)
Cromosomas Humanos Par 11 , Familia de Multigenes , Dedos de Zinc/genética , Secuencia de Aminoácidos , Secuencia de Bases , Northern Blotting , Mapeo Cromosómico , ADN , Expresión Génica , Humanos , Hibridación Fluorescente in Situ , Cariotipificación , Datos de Secuencia Molecular , Homología de Secuencia de AminoácidoRESUMEN
We have used cosmid "fingerprinting" to construct an overlapping DNA clone "map" of the human DNA in a mouse/human hybrid cell line, E65-9, that contains about 4 x 10(6) bp, including the H-Ras gene, as its human component. We have additionally used 32P-labeled RNA probes to establish linkage of particular sets of clones, and the final map comprises about 300,000 bp and is contained in three nonoverlapping segments. The reasons for failure to close the gaps by direct probing are discussed. We have developed techniques to search for rare cutting restriction enzyme cleavage sites in large numbers of cloned DNAs and have positioned sites for EagI and BssHII on our clone map. The methods we used are capable of considerable scale-up and are currently being applied to the short arm of human chromosome 11.