Mapping And Gene Content Of The Mouse T-complex

小鼠17号染色体上的t复合体长期以来一直是发育生物学家关注的焦点。由于许多胚胎致死突变都定位于该区域，因此推断在发育中起重要作用的相应基因位于该区域，因此它是高优先序列分析的主要目标。我们的合作者发现该区域包含大量未知的基因，包括许多在胎盘外锥体中选择性表达的基因，这都强调了该区域的重要性，并为筛选底物进行远程序列分析提供了探针。到目前为止，我们已经在BACs中恢复了11.6 Mb的DNA，占估计的30Mb区域的三分之一多一点。这包括从Brachyury到Sod2的连续5mb区域，覆盖Fgd2到Tbp的1.4 Mb区域，MGD CM区域10.0-12.0之间的2.5 Mb区域和覆盖tw5和Tir1关键区域的1.7 Mb区域。剩余的部分存在于正在积极扩展的种子群中。总的来说，目前的地图格式为592个STSs（即5.0个STSs/100kb）和400个bac。目前，已从38个BAC克隆中生成了7.5 Mb的序列，准确率超过99.9%。该数据库现在包含5.8 Mb的序列，其中1.75 Mb已被完全注释，其余序列表示为HTGS阶段1。剩下的1.7 Mb正在进行注释以供提交。测序区GC含量从40%到50%不等，具有相应的基因浓度范围。我们进行序列分析的主要工具是与Genbank EST数据库、外显子预测程序GRAIL、重复序列查找程序CENSOR进行比较，以及使用CpG含量作为CpG岛和基因的测定。迄今为止，对17个bac的分析和注释已经确定了该区域中以下已知基因的基因结构：T， T2, QkII, Qk-1, Plg, Slca22a1, Slca22a2, Slca22a3, Igf2r, Thbs2, Dll1, Psmb1, Tbp, Pdcd2， RabIIb和MyoIf， Zfp54和Zfp51。我们还将以前已知的没有精确定位的基因分配到t复合体在这个基因组中确定的位置。这些基因包括帕金森相关基因2 （Parkin2）、Brp44、溶血磷脂酸酰基转移酶、Mekk4、Zfp118、Stromelysin、Clc-7、泛素偶联酶、Pdpk1基因和锌金属内肽酶基因。已发表的一份BAC的序列确定了11个基因，其中5个是已知的。它们是Rsp29、Als、Nubp3、Jsap1和Ndk3。剩下的6个被预测的基因是新的。到目前为止，研究中预测的其他新基因包括与人脑表达KIAA0183同源的基因，两个金属蛋白酶，两个含有蟹锌指的基因，两个带有锌指基序的基因，一个带有PHD指结构域的基因以及一个类似于MORC的基因，MORC是小鼠精子发生所需的核蛋白。具有胚胎致死位点候选基因的BACs被用于构建转基因，以通过互补研究它们的作用。

The mouse t-complex on chromosome 17 has long been a focus of interest for developmental biologists. Because many embryonic lethal mutations are localized in the region, and corresponding genes of importance in development are therefore inferred to reside there, it is a prime target for high priority sequence analysis. The findings of our collaborators that the region contains a large number of otherwise unknown genes, including many expressed selectively in the ectoplacental cone, both underlines the importance of the region and provides probes to screen for substrates for long-range sequence analysis. We have thus far recovered 11.6 Mb DNA in BACs, representing just over 1/3rd of the estimated 30Mb region. This includes contiguous stretch a 5 Mb region from Brachyury to Sod2 and 1.4 Mb contig covering Fgd2 through Tbp, 2.5 Mb region between MGD CM region 10.0-12.0 and 1.7 Mb contig covering tw5 and Tir1 critical regions. The remaining portions exist in seed contigs which are being actively extended. Overall the current map is formatted with 592 STSs (i.e., 5.0 STSs/100kb) and 400 BACs. Currently a total of 7.5 Mb of sequence has been generated with greater than 99.9% accuracy from 38 BAC clones. The database now contains 5.8 Mb sequence of which 1.75 Mb has been fully annotated, with the rest of the sequence represented as HTGS phase 1. The remaining 1.7 Mb is being annotated for submission. The GC content of the sequenced region varies from 40% to a high of 50%, with a corresponding range of gene concentrations. Our main tools for sequence analysis are comparison with the Genbank EST database, the exon-predicting program GRAIL, the repetitive sequence-finding program CENSOR, and the use of CpG content as an assay for CpG islands and genes. Thus far analysis and annotation of 17 BACs has defined the gene structures for the following known genes in the region, T, T2, QkII, multiple isoforms of Qk-1, Plg, Slca22a1, Slca22a2, Slca22a3, Igf2r, Thbs2, Dll1, Psmb1, Tbp, Pdcd2, RabIIb and MyoIf, Zfp54, and Zfp51. We have also assigned previously known genes without precise localization to the t-complex at defined places in this contig. These include Parkinson-related-2 (Parkin2), Brp44, Lysophosphatidic acid acyltransferase, Mekk4, Zfp118, Stromelysin, Clc-7, Ubiquitin conjugating enzyme, and Pdpk1 genes and a gene for Zn metalloendopeptidase. The sequence from one BAC, which has been published, has identified 11 genes of which 5 are known. They are Rsp29, Als, Nubp3, Jsap1, and Ndk3. The remaining 6 predicted genes are new. Other novel genes predicted in the studies thus far include genes homologous to human brain expressed KIAA0183, two metalloproteases, two Krab-Zinc finger containing genes, two genes with Zinc-finger motifs, a gene with a PHD finger domain as well as a gene similar to MORC, a nuclear protein required for mouse spermatogenesis. BACs with candidate genes from the embryonic lethal loci are being used to construct transgenes to investigate their role by complementation.