Studies on mat1 Imprinting

用单细胞家系分析时，~lt；i&gt；S.pombe&lt；/i&gt；细胞的转换模式是非随机的。在连续两次不对称细胞分裂后，每四个“孙女”细胞中就有一个经历交配型转换。以前，我们证明这种模式是由于&lt；i&gt；mat1&lt；/i&>；印记只以链特定的方式标记一个姐妹染色单体，并且与&lt；i&gt；mat1&lt；/i&gt；处的位点特异性双链DNA断裂有关。我们现在证明，印记是链特定的，碱不稳定的&lt；I&gt；mat1&lt；/i&gt；.&lt；BR&gt；&lt；BR&gt；The DNA修饰DNA断裂是一种人工制品，从印记在DNA纯化过程中创建。我们还提出并检验了&lt；i&gt；mat1&lt；/i&gt；优先由着丝粒-远端起源(S)复制的模型，因此只有在滞后链合成过程中才会出现链特异性印记。通过倒置或引入复制起点来改变复制起点会以预测的方式影响印迹和切换效率。二维凝胶分析证实&lt；i&gt；mat1&lt；/i&gt；优先由着丝粒-远端起源(S)复制。因此，DNA复制机制可能赋予姐妹细胞不同的发育潜力。我们最近的工作发现了&lt；i&gt；sw1&gt；和&lt；i&gt；sw3&lt；/i&gt；基因的生化功能。我们发现SWIF1P和SWIF3P通过在&lt；i&gt；mat1&lt；/i&gt；暂停和终止DNA复制来执行印记。我们的工作表明：(1)Swi1p和Swi3p因子通过在印迹部位暂停复制分叉来发挥作用；以及(2)Swi1p和Swi3p参与复制的近端极终止点(Rt1；rt1；/i&gt；)的终止。用于识别终止因子的遗传筛查确定了一个等位基因，该等位基因将sw1p的暂停/印记和功能终止分开。结果表明，SWIF1P和SWIF3P通过在&lt；i&gt；mat1；/i&gt；暂停复制和在&lt；i&gt；rt1&lt；/i&gt；终止复制，以新颖的方式促进印记。我们还发现，Swi1和Swi3蛋白在体内形成一个复合体，都与染色体上的RTS1和mat1复制暂停位点结合。未来的研究旨在确定印记的机制。我们已经定义了大量影响印记的mat1突变。他们的分子分析应该有助于我们确定印记的机制。

The pattern of switching of <I>S. pombe</i> cells is nonrandom when assayed by single cell pedigrees. After two consecutive asymmetric cell divisions, one in four "granddaughter" cells undergoes a mating-type switch. Previously, we showed that this pattern is due to <I>mat1</i> imprinting that marks only one sister chromatid in a strand-specific manner, and that is related to site-specific, double-stranded DNA break at <I>mat1</i>. We now show that the imprint is a strand-specific, alkali-labile DNA modification at <I>mat1</i>.<BR><BR>The DNA break is an artifact, created from the imprint during DNA purification. We also proposed and tested the model that <I>mat1</i> is preferentially replicated by a centromere-distal origin(s), so that the strand-specific imprint occurs only during lagging-strand synthesis. Altering the origin of replication, by inverting <I>mat1</i> or introducing an origin of replication, affects the imprinting and switching efficiencies in predicted ways. Two-dimensional gel analysis confirmed that <I>mat1</i> is preferentially replicated by a centromere-distal origin(s). Thus, the DNA replication machinery may confer different developmental potential to sister cells.<BR><BR> Our recent work has discovered biochemical functions of <I>swi1</i> and <I>swi3</i> genes. We found that swi1p and swi3p perform imprinting by pausing and termination of DNA replication at <I>mat1</i>. Our work shows (1) that the factors swi1p and swi3p act by pausing the replication fork at the imprinting site; and (2) that swi1p and swi3p are involved in termination at the <I>mat1</i>-proximal polar-terminator of replication (<I>RTS1</i>). A genetic screen to identify termination factors identified an allele that separated pausing/imprinting and termination of functions of swi1p. The results suggest that swi1p and swi3p promote imprinting in novel ways both by pausing replication at <I>mat1</i> and by terminating replication at <I>RTS1</i>. We also showed that Swi1 and Swi3 proteins form a complex in vivo and both bind to the RTS1 and the mat1 replication pause sites on the chromosome. Future studies are designed to define the mechanism of imprinting. We have defined a large number of mat1 mutations that affect imprinting. Their molecular analysis should help us define the mechanism of imprinting.