RESUMO
To construct a mammalian artificial chromosome (MAC), telomere repeats and selectable markers were introduced into a 100 kb yeast artificial chromosome (YAC) containing human centromeric DNA. This YAC, which has a regular repeat structure of alpha-satellite DNA and centromere protein B (CENP-B) boxes, efficiently formed MACs that segregated accurately and bound CENP-B, CENP-C, and CENP-E. The MACs appear to be about 1-5 Mb in size and contain YAC multimers. Structural analyses suggest that the MACs have not acquired host sequences and were formed by a de novo mechanism. The accurate segregation of the MACs suggests they have potential as vectors for introducing genes into mammals.
Assuntos
Autoantígenos , Cromossomos Artificiais de Levedura/genética , Cromossomos/genética , Proteínas de Ligação a DNA , Animais , Linhagem Celular , Linhagem Celular Transformada , Centrômero/genética , Proteína B de Centrômero , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Cromossomos Humanos Par 21/genética , Clonagem Molecular , DNA Satélite/química , DNA Satélite/genética , Vetores Genéticos/genética , Humanos , Hibridização in Situ Fluorescente , Cinetocoros , Reação em Cadeia da Polimerase , Telômero/genética , TransfecçãoRESUMO
Telomeric DNA in the skin cells of 21 human subjects aged between 0 and 92 years was quantified by determining the length of the telomeric smear and the relative amount of TTAGGG repeat sequences. Both telomere length and quantity of telomeric repeat sequences were found to decrease significantly with age. Telomere loss has previously been postulated to be a caused of cell senescence.
Assuntos
Envelhecimento/genética , Sequências Repetitivas de Ácido Nucleico/genética , Telômero/fisiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Southern Blotting , Criança , Pré-Escolar , Cromossomos/fisiologia , Células Epidérmicas , Humanos , Lactente , Recém-Nascido , Pessoa de Meia-IdadeRESUMO
A DNA fragment isolated from a human genomic library, was reported to be present at all human centromeres and present at 16-32 copies per genome. Reintroduction of this DNA into mammalian cells as a concatenated phage clone gave rise to dicentric chromosomes which gave rise to a new, stable, chromosome. Taken together these observations could mean that this DNA is part of a native centromere. We have reexamined the location and copy number of this sequence and find it to be present at 1-2 copies per genome with a single site of in situ hybridisation at 9qter.
Assuntos
Centrômero/fisiologia , Cromossomos Humanos Par 9 , DNA/genética , Southern Blotting , Mapeamento Cromossômico , Clonagem Molecular , DNA/isolamento & purificação , Desoxirribonuclease EcoRI , Biblioteca Genômica , Células HeLa , Humanos , Hibridização In Situ , Linfócitos/citologia , Mapeamento por RestriçãoRESUMO
Telomeres confer stability on chromosomes by protecting them from degradation and recombination and by allowing complete replication of the end. They are genetically important as they define the ends of the linkage map. Telomeres of lower eukaryotes contain short repeats consisting of a G-rich and a C-rich strand, the G-rich strand running 5'-3' towards the telomere and extending at the end. Telomeres of human chromosomes share characteristics with those of lower eukaryotes including sequence similarity as detected by cross-hybridization. Telomeric repeats from many organisms can provide telomere function in yeast. Here we describe a modified yeast artificial chromosome (YAC) vector with only one telomere which we used to clone human telomeres by complementation in yeast. YACs containing human telomeres were identified by hydridization to an oligonucleotide of the trypanosome telomeric repeat. A subcloned human fragment from one such YAC is immediately subtelomeric on at least one human chromosome.
Assuntos
Cromossomos Humanos/ultraestrutura , Clonagem Molecular , Animais , Cromossomos Fúngicos , DNA/sangue , DNA/genética , Enzimas de Restrição do DNA , Genes Fúngicos , Marcadores Genéticos , Vetores Genéticos , Humanos , Masculino , Hibridização de Ácido Nucleico , Sondas de Oligonucleotídeos , Recombinação Genética , Saccharomyces cerevisiae/genética , Homologia de Sequência do Ácido Nucleico , Espermatozoides/análise , Transformação Genética , Trypanosoma/genéticaRESUMO
We have investigated the potential of PAC-based vectors as a route to the incorporation of a gene in a mammalian artificial chromosome (MAC). Previously we demonstrated that a PAC (PAC7c5) containing alpha-satellite DNA generated mitotically stable MACs in human cells. To determine whether a functional HPRT gene could be assembled in a MAC, PAC7c5 was co-transfected with a second PAC containing a 140 kb human HPRT gene into HPRT-deficient HT1080 cells. Lines were isolated containing a MAC hybridizing with both alpha-satellite and HPRT probes. The MACs segregated efficiently, associated with kinetochore proteins and stably expressed HPRT message after 60 days without selection. Complementation of the parental HPRT deficiency was confirmed phenotypically by growth on HAT selection. These results suggest that MACs could be further developed for delivering a range of genomic copies of genes into cells and that stable transgene expression can be achieved.
Assuntos
Cromossomos Artificiais , Expressão Gênica , Técnicas Genéticas , Hipoxantina Fosforribosiltransferase/genética , Northern Blotting , Divisão Celular , Linhagem Celular , DNA Complementar/metabolismo , Humanos , Hibridização in Situ Fluorescente , Repetições de Microssatélites , Microscopia de Fluorescência , Mitose , Fenótipo , Fatores de Tempo , Transfecção , TransgenesRESUMO
In a subset of infertile men, a spectrum of spermatogenic defects ranging from a complete absence of germ cells (sertoli cell only) to oligozoospermia is associated with microdeletions of the DAZ (deleted in azoospermia) gene cluster on human distal Yq. DAZ encodes a testis-specific protein with RNA-binding potential recently derived from a single-copy gene DAZL1 (DAZ-like) on chromosome 3. Y chromosomal DAZ homologues are confined to humans and higher primates. It remains unclear which function unique to higher primate spermatogenesis DAZ may serve, and the functional status of the gene recently has been questioned. To assess the extent of functional conservation we have tested the capacity of a human DAZ gene contained in a 225-kb yeast artificial chromosome to complement the sterile phenotype of the Dazl null mouse (Dazl-/-), which is characterized by severe germ-cell depletion and meiotic failure. Although Dazl-/- mice remained infertile when the DAZ transgene was introduced, histological examination revealed a partial and variable rescue of the mutant phenotype, manifest as a pronounced increase in the germ cell population of the seminiferous tubules and survival to the pachytene stage of meiosis. As well as constituting definitive proof of the spermatogenic role of the DAZ gene product, these findings confirm the high degree of functional conservation between the DAZ and DAZL1 genes, suggesting they may constitute a single target for contraceptive intervention and raising the possibility of therapeutic up-regulation of the DAZL1 gene in infertile men.