Henning K A, Novotny E A, Compton S T, Guan X Y, Liu P P, Ashlock M A
Genetics and Molecular Biology Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Proc Natl Acad Sci U S A. 1999 Jan 19;96(2):592-7. doi: 10.1073/pnas.96.2.592.
A human artificial chromosome (HAC) vector was constructed from a 1-Mb yeast artificial chromosome (YAC) that was selected based on its size from among several YACs identified by screening a randomly chosen subset of the Centre d'Etude du Polymorphisme Humain (CEPH) (Paris) YAC library with a degenerate alpha satellite probe. This YAC, which also included non-alpha satellite DNA, was modified to contain human telomeric DNA and a putative origin of replication from the human beta-globin locus. The resultant HAC vector was introduced into human cells by lipid-mediated DNA transfection, and HACs were identified that bound the active kinetochore protein CENP-E and were mitotically stable in the absence of selection for at least 100 generations. Microdissected HACs used as fluorescence in situ hybridization probes localized to the HAC itself and not to the arms of any endogenous human chromosomes, suggesting that the HAC was not formed by telomere fragmentation. Our ability to manipulate the HAC vector by recombinant genetic methods should allow us to further define the elements necessary for mammalian chromosome function.
人类人工染色体（HAC）载体由一个1兆碱基对（Mb）的酵母人工染色体（YAC）构建而成。该YAC是从多个YAC中根据其大小挑选出来的，这些YAC是通过用简并α卫星探针筛选人类多态性研究中心（CEPH，位于巴黎）YAC文库的一个随机选择子集而鉴定出来的。这个YAC还包含非α卫星DNA，经过修饰后含有人类端粒DNA和来自人类β-珠蛋白基因座的一个假定复制起点。通过脂质介导的DNA转染将所得的HAC载体导入人类细胞，并鉴定出与活性动粒蛋白CENP-E结合且在无选择条件下至少100代有丝分裂稳定的HAC。用作荧光原位杂交探针的显微切割HAC定位于HAC本身，而非任何内源性人类染色体的臂，这表明HAC不是由端粒断裂形成的。我们通过重组遗传方法操纵HAC载体的能力应使我们能够进一步确定哺乳动物染色体功能所需的元件。
