RESUMO
In vivo genetic footprinting was developed in the yeast Saccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting for Escherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10 transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage lambda transposon delivery system.
Assuntos
Pegada de DNA , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/genética , Genes Bacterianos , Bacteriófago lambda/genética , Bacteriófago lambda/fisiologia , Sequência de Bases , Meios de Cultura , Elementos de DNA Transponíveis , Escherichia coli/metabolismo , Genes Essenciais/genética , Dados de Sequência Molecular , Mutagênese Insercional , Plasmídeos/genética , Transposases/genética , Transposases/metabolismoRESUMO
Uterine leiomyomata are the most common tumors in women and can cause abnormal uterine bleeding, pelvic pain, and infertility. Approximately 200,000 hysterectomies are performed annually in the U.S. to relieve patients of the medical sequelae of these benign neoplasms. Our efforts have focused on cloning the t(12;14)(q14-q15;q23-q24) breakpoint in uterine leiomyoma to further our understanding of the biology of these tumors. Thirty-nine YACs and six cosmids mapping to 12q14-q15 have been mapped by fluorescence in situ hybridization to tumor metaphase chromosomes containing a t(12;14). One YAC spanned the translocation breakpoint and was mapped to tumor metaphases from a pulmonary chondroid hamartoma containing a t(12;14)(q14-q15;q23-q24) and a lipoma containing a t(12;15)(q15;q24); this YAC also spanned the breakpoint in these two tumors, suggesting that the same gene on chromosome 12 may be involved in the pathobiology of these distinct benign neoplasms.
Assuntos
Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 12 , Hamartoma/genética , Leiomioma/genética , Lipoma/genética , Pneumopatias/genética , Translocação Genética , Neoplasias Uterinas/genética , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Humanos Par 12/ultraestrutura , Cromossomos Humanos Par 14/ultraestrutura , Cromossomos Humanos Par 15/ultraestrutura , Feminino , Humanos , Hibridização in Situ Fluorescente , Dados de Sequência MolecularRESUMO
We have mapped five genes encoding protein tyrosine kinases (PTKs) to the pericentromeric region of human chromosome 4. PTK4 and TYRO4, which encode nonreceptor intracellular PTKs, are located at 4p12 and 4q13, respectively. The other three genes, PDGFRA, KIT, and KDR, encode type III transmembrane receptor PTKs for known ligands. We have developed a contig of 29 yeast artificial chromosomes (YACs) spanning approximately 2 Mb of DNA at 4q12 that includes PDGFRA, KIT, and KDR, and we have used this YAC contig to map 12 different sequence-tagged sites in this region. PDGFRA, KIT, and KDR thus constitute a cluster of genes at 4q12 encoding closely related type III receptor PTKs. Mutations of the human KIT gene result in piebaldism, an autosomal dominant disorder of melanocyte development.
Assuntos
Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 4 , Genes , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Receptores de Fator Estimulador de Colônias/genética , Receptores do Fator de Crescimento Derivado de Plaquetas/genética , Sequência de Bases , Passeio de Cromossomo , Humanos , Dados de Sequência Molecular , Família Multigênica , Reação em Cadeia da Polimerase , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-kitRESUMO
One hundred eighty-three YACs carrying human chromosome 10 sequences were isolated from multigenome equivalent libraries by PCR-based screening for the presence of 80 different chromosome 10-specific STSs. Ninety-four of the isolated YACs, representing 52 genes and DNA segments, were mapped to regions of chromosome 10 by fluorescence in situ hybridization. The results localized 26 DNA segments to cytogenetic bands for the first time. About 37% (35/94) of the YACs hybridized to more than one chromosomal location: 31 to other chromosomes in addition to chromosome 10 and 4 to 2 distinct locations on chromosome 10. These results are consistent with the number of chimeric YACs expected from these libraries but may also reflect the presence of 2 or more YACs within a single clone or the presence of low copy repeated elements within the genome. This STS anchor screening effort resulted in the identification of 69 contigs, with 7 contigs consisting of 2 anchors each and 1 contig consisting of 5 anchors. All linked STSs were multiply linked by at least 2 independent YACs. These anchored YACs span the entire chromosome and appear to cover 15% of chromosome 10.
Assuntos
Mapeamento Cromossômico , Cromossomos Artificiais de Levedura/genética , Cromossomos Humanos Par 10 , Biblioteca Gênica , Ligação Genética , Marcadores Genéticos , Projeto Genoma Humano , Humanos , Hibridização in Situ Fluorescente , Sitios de Sequências RotuladasRESUMO
A total of 124 new chromosome 10-specific sequence-tagged sites (STSs) were derived from two sources: (1) DNA sequences obtained from anonymous clones in new libraries enriched for human chromosome 10 inserts, and (2) published sequences of genes and other loci already known to map to chromosome 10. Libraries were constructed from a somatic cell hybrid carrying human chromosomes 10 and Y. A cosmid library was made from total DNA of the hybrid and probed with labeled total human DNA to identify clones with human DNA inserts. Two hundred seventeen cosmids were mapped to regions of human chromosome 10 by fluorescence in situ hybridization. Twenty-five cosmids represent probes that have been placed on the genetic map previously. One hundred ninety-two cosmids represent new probes that have not been mapped previously. Cosmids carrying inserts with CA repeats were identified by hybridization with a labeled poly(dC-dA)-poly(dG-dT) probe and subcloned to yield microsatellite STS markers. Two small insert plasmid libraries were made, the first by subcloning inserts from a chromosome 10-enriched lambda phage library (LL10NS01) and the second by cloning Alu element-mediated PCR products amplified from hybrid DNA. STSs were generated from the DNA sequences of clone inserts. Chromosome 10-specific STSs were distinguished from Y chromosome STSs by one or both of the following criteria: (1) successful PCR amplification from a template consisting of DNA from another chromosome 10-containing cell line, NA10926B, or (2) FISH localization to chromosome 10 of the source cosmid or of YACs isolated by PCR screening with the STS. These libraries were the source of 90 new chromosome 10-specific STSs, 42 of which contain CA repeats.
Assuntos
Cromossomos Humanos Par 10 , Sitios de Sequências Rotuladas , Sequência de Bases , Mapeamento Cromossômico , Cromossomos Artificiais de Levedura , Clonagem Molecular , Cosmídeos , Citogenética , Primers do DNA/genética , DNA Satélite/genética , Biblioteca Gênica , Marcadores Genéticos , Humanos , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos/genética , Sequências Repetitivas de Ácido NucleicoRESUMO
An overlapping set of 21 yeast artificial chromosomes (YACs) spanning the RET proto-oncogene [Takahashi et al., Oncogene 3 (1988) 571-578] and D10S102 markers on human chromosome 10 was isolated in a series of hybridization-based chromosomal walks in a YAC library. Genetic linkage analyses implicate this chromosomal region as the location of the gene (MEN2A) responsible for multiple endocrine neoplasia type 2A. Four YACs carrying a RET sequence-tagged site (STS) and two YACs carrying a D10S102 STS were used to initiate chromosome walks. These were based on hybridization of Alu element-mediated polymerase chain reaction (Alu-PCR) products from YACs to dot blots of Alu-PCR products from complex pools of YAC clones. The hybridization anchor content of YACs identified in the walks was confirmed by probing blots of Alu-PCR products from individual YACs and by comparing Alu-PCR fingerprints of each YAC. Ten hybridization-based Alu-PCR anchors and three STS anchors were ordered within eleven intervals created by the 21 overlapping YACs. The order of anchors requiring the fewest gaps in the YACs is consistent with the walking results and establishes the STS anchor order as D10S102-D10S94-RET. The overlapping set of YACs represents about 1.55 Mb of the human genome according to restriction mapping of four representative YACs in the contig. These results demonstrate the power of Alu-PCR hybridization for chromosomal walking and provide a rich source of overlapping YACs which can be used to identify candidate MEN2A genes.
Assuntos
Cromossomos Artificiais de Levedura , Cromossomos Humanos Par 10 , Neoplasia Endócrina Múltipla/genética , Sequências Repetitivas de Ácido Nucleico , Sequência de Bases , Passeio de Cromossomo , Primers do DNA , Ligação Genética , Marcadores Genéticos , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proto-Oncogene Mas , Mapeamento por RestriçãoRESUMO
An experimental yeast artificial chromosome (YAC) library consisting of one genome equivalent of human DNA was prepared in a selectable high-copy-number (hcn) YAC vector. Screening for unique loci was accomplished by PCR of successively smaller DNA pools and by hybridization to high-density microcolony blots. Inserts averaged 200 kb in size, but several YACs with inserts averaging about 650 kb were obtained when polyamines were added prior to yeast transformation. YACs were identified for 17 out of 29 sequence-tagged sites (STS) screened by a PCR-based approach. All YACs in the size range of 100-600 kb that were examined could be obtained at significantly elevated copy numbers following growth of the clones in methotrexate/sulfanilamide/thymidine-supplemented medium. The hcn YACs could also be selected during growth in microtiter dishes, and the resulting clones were used to prepare high-density microcolony DNA blots for hybridization with radiolabeled PCR products. DNA pools for the PCR-based screening of this experimental library are available to investigators interested in applications of hcn YACs.
Assuntos
Cromossomos Fúngicos , Biblioteca Gênica , Vetores Genéticos , Genoma Humano , Sequência de Bases , Clonagem Molecular/métodos , DNA de Cadeia Simples , Eletroforese em Gel de Campo Pulsado , Humanos , Dados de Sequência Molecular , Reação em Cadeia da PolimeraseRESUMO
Human cells contain two topoisomerase II isozymes named topo II alpha and topo II beta. The complementary DNAs for both enzymes have been cloned. The topo II alpha and topo II beta complementary DNAs hybridized to unique sequences of human, rodent, and chicken DNAs in Southern blots. The human topo II alpha gene has previously been mapped to chromosome 17. We confirmed the chromosomal location of topo II alpha and mapped the topo II beta gene to chromosome 3. In addition, topo II beta exhibits genetic polymorphism as has been reported for topoisomerases I and II alpha.
Assuntos
Mapeamento Cromossômico , Cromossomos Humanos Par 17/enzimologia , Cromossomos Humanos Par 3/enzimologia , DNA Topoisomerases Tipo II/genética , Isoenzimas/genética , Southern Blotting , HumanosRESUMO
The genes for two new P-type ATPases, PMR1 and PMR2, have been identified in yeast. A comparison of the deduced sequences of the PMR proteins with other known ion pumps showed that both proteins are very similar to Ca2+ ATPases. PMR1 is identical to SSC1, a gene previously identified by its effect on secretion of some foreign proteins from yeast. Proteins secreted from pmr1 mutants lack the outer chain glycosylation that normally results from passage through the Golgi. Loss of PMR1 function suppresses the lethality of ypt1-1, a mutation that blocks the secretion pathway. These data suggest that PMR1 functions as a Ca2+ pump affecting transit through the secretory pathway.
