Montanaro V, Casamassimi A, D'Urso M, Yoon J Y, Freije W, Schlessinger D, Muenke M, Nussbaum R L, Saccone S, Maugeri S
International Institute of Genetics and Biophysics, Naples, Italy.
Am J Hum Genet. 1991 Feb;48(2):183-94.
From the collection described by Abidi et al., 102 yeast artificial chromosomes (YACs) with human DNA inserts more than 300 kb in length were assigned to chromosomal band positions on early metaphase chromosomes by in situ hybridization using the biotin-avidin method. All the YACs hybridized within the Xq24-Xqter region, supporting the origin of the vast majority of the YACs from single human X-chromosomal sites. With assignments precise to +/- 0.5 bands, YACs were distributed among cytogenetic bands to roughly equal extents. Thus, there is no gross bias in the cloning of DNA from different bands into large YACs. To test band assignments further, hybridizations were carried out blind, and band positions were then compared with (1) probe localizations in cases in which a reported location was present in one of the YACs; (2) cross-hybridization of a labeled YAC with others in the collection; and (3) hybridization to a panel of DNAs from a series of hybrid cells containing Xq DNA truncated at various regions. Of 31 cases in which YACs contained a probe with a previously reported location, 28 in situ assignments were in agreement, and 14 other assignments, including one of the three discordant with probe localization, were confirmed by YAC cross-hybridization studies. Results with a group of nine YACs were further confirmed with a panel of somatic cell hybrid DNAs from that region. Five YACs hybridized both to Xq25 and to a second site (four in Xq27 and one in Xq28), suggestive of some duplication of DNA of the hybrid cell and perhaps in normal X chromosomes. The in situ assignments are thus sufficient to place YACs easily and systematically within bins of about 7-10 Mb and to detect some possible anomalies. Furthermore, on the basis of expectations for random cloning of DNA in YACs, the assigned YACs probably cover more than 50% of the total Xq24-Xq28 region. This provides one way to initiate the assembly of YAC contigs over extended chromosomal regions.
从阿比迪等人描述的文库中，使用生物素 - 抗生物素蛋白方法，通过原位杂交将102个插入了长度超过300 kb人类DNA的酵母人工染色体（YAC）定位到早中期染色体的染色体带位置上。所有YAC都在Xq24 - Xqter区域内杂交，这支持了绝大多数YAC来源于单个人类X染色体位点。YAC精确地定位到±0.5个带，在细胞遗传学带之间大致均匀分布。因此，将不同带的DNA克隆到大型YAC中没有明显偏差。为了进一步检验带的定位，进行了盲法杂交，然后将带位置与以下情况进行比较：（1）在YAC之一中存在报道位置的情况下探针的定位；（2）标记的YAC与文库中其他YAC的交叉杂交；（3）与一系列含有在不同区域截断的Xq DNA的杂交细胞的DNA文库杂交。在31个YAC含有先前报道位置探针的案例中，28个原位定位一致，另外14个定位，包括与探针定位不一致的三个中的一个，通过YAC交叉杂交研究得到了证实。来自该区域的一组九个YAC的结果通过该区域的一组体细胞杂交DNA进一步得到证实。五个YAC既与Xq25杂交，也与第二个位点杂交（四个在Xq27，一个在Xq28），这表明杂交细胞以及可能正常X染色体中的DNA存在一些重复。因此，原位定位足以轻松且系统地将YAC定位在约7 - 10 Mb的区间内，并检测到一些可能的异常情况。此外，基于YAC中DNA随机克隆的预期，已定位的YAC可能覆盖了Xq24 - Xq28区域总数的50％以上。这为在扩展的染色体区域上启动YAC重叠群的组装提供了一种方法。
