Yan Christopher M, Dobie Kenneth W, Le Hiep D, Konev Alexander Y, Karpen Gary H
Molecular and Cell Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, California 92037, USA.
Genetics. 2002 May;161(1):217-29. doi: 10.1093/genetics/161.1.217.
Approximately one-third of the human and Drosophila melanogaster genomes are heterochromatic, yet we know very little about the structure and function of this enigmatic component of eukaryotic genomes. To facilitate molecular and cytological analysis of heterochromatin we introduced a yellow(+) (y(+))-marked P element into centric heterochromatin by screening for variegated phenotypes, that is, mosaic gene inactivation. We recovered >110 P insertions with variegated yellow expression from approximately 3500 total mobilization events. FISH analysis of 71 of these insertions showed that 69 (97%) were in the centric heterochromatin, rather than telomeres or euchromatin. High-resolution banding analysis showed a wide but nonuniform distribution of insertions within centric heterochromatin; variegated insertions were predominantly recovered near regions of satellite DNA. We successfully used inverse PCR to clone and sequence the flanking DNA for approximately 63% of the insertions. BLAST analysis of the flanks demonstrated that either most of the variegated insertions could not be placed on the genomic scaffold, and thus may be inserted within novel DNA sequence, or that the flanking DNA hit multiple sites on the scaffold, due to insertions within different transposons. Taken together these data suggest that screening for yellow variegation is a very efficient method for recovering centric insertions and that a large-scale screen for variegated yellow P insertions will provide important tools for detailed analysis of centric heterochromatin structure and function.
人类和黑腹果蝇基因组中约三分之一是异染色质，但我们对真核生物基因组这一神秘组成部分的结构和功能却知之甚少。为便于对异染色质进行分子和细胞学分析，我们通过筛选斑驳表型（即镶嵌基因失活），将一个带有黄色（+）（y（+））标记的P因子导入着丝粒异染色质中。在约3500次总的转座事件中，我们获得了>110个黄色表达斑驳的P插入片段。对其中71个插入片段进行荧光原位杂交（FISH）分析表明，69个（97%）位于着丝粒异染色质中，而非端粒或常染色质。高分辨率带型分析显示，着丝粒异染色质内的插入片段分布广泛但不均匀；斑驳插入片段主要在卫星DNA区域附近获得。我们成功地使用反向PCR克隆并测序了约63%插入片段的侧翼DNA。对侧翼序列的BLAST分析表明，要么大多数斑驳插入片段无法定位到基因组支架上，因此可能插入到新的DNA序列中，要么侧翼DNA在支架上命中多个位点，这是由于插入到不同转座子内所致。综合这些数据表明，筛选黄色斑驳是一种非常有效的回收着丝粒插入片段的方法，并且大规模筛选黄色斑驳P插入片段将为详细分析着丝粒异染色质的结构和功能提供重要工具。
