Mapping And Gene Content Of The Mouse T-complex

小鼠17号染色体上的t复合体一直是发育生物学家关注的焦点。由于许多胚胎致死突变位于该区域，因此推断发育中重要的相应基因位于该区域，因此它是高优先级序列分析的主要目标。我们的合作者的发现，该地区包含大量的其他未知的基因，包括许多选择性表达的外胎盘锥，既强调了该地区的重要性，并提供探针，以筛选底物的远程序列分析。到目前为止，我们已经在BAC中回收了11.6 Mb DNA，仅占估计的30 Mb区域的1/3以上。这包括从Brachyury至Sod 2的5 Mb区域和覆盖Fgd 2至Tbp的1.4 Mb重叠群、MGD CM区域10.0-12.0之间的2.5 Mb区域以及覆盖tw 5和Tir 1关键区域的1.7 Mb重叠群的连续延伸。剩余部分存在于正在被主动延伸的种子重叠群中。总的来说，当前地图被格式化为具有592个STS（即，5.0 STS/100 kb）和400个BAC。目前，已经从38个BAC克隆中产生了总共7.5 Mb的序列，准确率大于99.9%。该数据库现在包含5.8 Mb序列，其中1.75 Mb已被完全注释，其余序列表示为HTGS阶段1。剩余的1.7 Mb正在注释以供提交。测序区域的GC含量从40%到高达50%不等，具有相应的基因浓度范围。我们用于序列分析的主要工具是与Genbank EST数据库、外显子预测程序GRAIL、重复序列发现程序CENSOR的比较，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、Qk II、Qk-1的多种同种型、Plg、Slca 22 a1、Slca 22 a2、Slca 22 a3、Igf 2 r、Thbs 2、Dll 1、Psmb 1、Tbp、Pdcd 2、RabIIb和MyoIf、Zfp 54和Zfp 51。我们还指定以前已知的基因没有精确定位的t-复合体在这个重叠群中的定义的地方。这些基因包括帕金森相关基因2（Parkin 2）、Brp 44、溶血磷脂酸酰基转移酶、Mekk 4、Zfp 118、溶基质素、Clc-7、泛素结合酶和Pdpk 1基因以及锌金属内肽酶基因。已发表的来自一个BAC的序列已经鉴定了11个基因，其中5个是已知的。它们是Rsp 29、Als、Nubp 3、Jsap 1和Ndk 3。剩下的6个预测基因是新的。目前预测的其他新基因包括与人脑表达的KIAA 0183同源的基因、两种金属蛋白酶、两种含有Krab-Zinc finger的基因、两种具有Zinc finger基序的基因、一种具有PHD finger结构域的基因以及一种类似于小鼠精子发生所需的核蛋白MORC的基因。具有来自胚胎致死基因座的候选基因的BAC被用于构建转基因以研究它们通过互补作用的作用。

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.