RESUMO
We describe a mammalian artificial mini-chromosome lacking human alphoid DNA and mouse minor and major satellite DNA repeats. This mini-chromosome, initially recovered in a mouse embryonic stem (ES) cell line (CGR8), is 2.6 Mb in size and consists of sequences derived from the human Y chromosome and mouse chromosomes 12 and 15. It is not stable in the CGR8 cells but replicates and segregates with high fidelity after transfer into chicken DT40 cells. Combined analysis by immunocytochemistry/fluorescence in situ hybridisation (FISH) on metaphase spreads detected an active neo-centromere on the mini-chromosome in these cells. Further analysis by immunocytochemistry/FISH on stretched chromatin allowed the localisation of the CENP-C protein to the DNA sequence derived from interval 5 of the human Y chromosome.
Assuntos
Centrômero/fisiologia , Cromossomos/química , Cromossomos/metabolismo , Animais , Southern Blotting , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Camundongos , Modelos Genéticos , Reação em Cadeia da Polimerase , Sequências Repetitivas de Ácido Nucleico , Fatores de Tempo , Cromossomo YRESUMO
The cuckoo chromosome 4S(L) from Aegilops sharonensis is preferentially transmitted when introduced by hybridization into common wheat, Triticum aestivum. Gametocidal (Gc) factors carried in 4S(L) induce chromosome breakage in meiospores not containing them, ensuring their transmission to the progeny. Chromosome breakage and break-fusion-bridge (BFB) cycles can also be observed during early embryo sac development of chromosome 4S(L) addition lines to wheat, often leading to the presence of dicentric chromosomes in the subsequent progeny. However, the process responsible for inducing the primary chromosomal breaks only appears to occur during the initial divisions of the embryo and endosperm. In the presence of chromosome 4S(L), treatment with the hypomethylating agent 5-azacytidine induces chromosome breakage in root tips. This suggests that the process of chromosome fragmentation, induced by the Gc factors during early seed development, is repressed at later stages by DNA methylation.
Assuntos
Azacitidina/farmacologia , Quebra Cromossômica/genética , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Poaceae/genética , Triticum/efeitos dos fármacos , Triticum/genética , Anáfase/efeitos dos fármacos , Metilação de DNA , DNA de Plantas/genética , Impressão Genômica , Hibridização Genética/genética , Raízes de Plantas/citologia , Triticum/citologiaRESUMO
Mammalian genome characterization and biotechnology each require the mobilization of large DNA segments to produce transgenic animals. We recently showed that mouse metaphase II (mII) oocytes could efficiently promote transgenesis (mII transgenesis) when coinjected with sperm and small (<5 kilobases) ubiquitously expressed transgenes (tgs). We have extended this work and now report that mII transgenesis can readily be applied to a range of larger tgs (11.9-170 kilobases), including bacterial and mammalian artificial chromosome (BAC and MAC) constructs. The efficiency of large-construct mII transgenesis was at least as high as that with small constructs; 11-47% of offspring carried the large tgs. More than 95% of these transgenic founders transmitted the tg to offspring. These data demonstrate the ability of mII transgenesis to deliver large tgs efficiently.
