Yeast artificial chromosomes for the molecular analysis of the familial polyposis APC gene region.

Abstract

Two yeast artificial chromosomes (YACs) spanning a total distance of 1.1 megabase pairs of DNA around the MCC (for mutated in colorectal carcinoma) and APC (for adenomatous polyposis coli) genes at 5q21 have been isolated and characterized. Starting from the MCC gene, a strategy was undertaken to identify constitutional submicroscopic deletions in familial adenomatous polyposis patients that might considerably narrow down the position of the APC gene. To this end, YACs identified by the MCC gene were screened across a chromosome 5-specific cosmid library to provide a source of DNA probes for genomic sanning. The cosmids isolated from these experiments were used to screen a panel of somatic cell hybrids containing chromosome 5 segregated from patients suspected to carry putative interstitial deletions. This screening approach led to the confirmation of a small heterozygous deletion in a polyposis patient that overlaps one of the two isolated YACs. This YAC has been shown to contain the entire APC gene, in addition to a significant portion of DNA flanking the 5' end of the gene, and should therefore prove a valuable resource for functional studies by transfer to colorectal tumorderived cell lines.Familial adenomatous polyposis (FAP) is an autosomal dominant predisposition to colorectal cancer, affecting about 1 in 5000 to 1 in 10,000 individuals in all populations studied (1).The corresponding gene, designated APC [for adenomatous polyposis coli (2)], was mapped to 5q21-q22 by linkage analysis (3, 4) following a cytogenetic report of a male patient with polyposis and an interstitial deletion on Sq (5). The high incidence of allele loss at 5q21-q22 in carcinomas of sporadic patients (6-9) suggests that APC mutations are common in sporadic colorectal adenocarcinomas.To clone the APC gene, our initial studies used two deletions similar to that originally identified by Herrera et al.(5) to localize probes to a region around the APC gene (10). Subsequently, landmark clones were derived by chromosomal microdissection and microcloning around 5q21-q22 (11,12). Clones that were localized within the two APC-related interstitial deletions were then used to isolate yeast artificial chromosomes (YACs) covering about 4 megabases (ref. 12; G.M.H. and J.R.T.J.W., unpublished results) in order to narrow the search for the APC gene among potential candidates.During this investigation, however, a candidate gene, MCC (for mutated in colorectal carcinoma), was identified by using a randomly isolated cosmid, L5.71, exhibiting a high frequency of allele loss (13) and which also identified a heterozygous 260-kilobase (kb) deletion in one polyposis family (14). After the discovery of MCC, Kinzler et al. (15) and Groden et al. (16) isolated the APC gene itself. In this paper we describe our approach to identifying the APC gene, which has led to the isolation of two overlapping YACs containing the MCC gene, one of which also includes the complete APC gene. MATERIALS AND METHODS YACMethods. The YAC library used in these studies (17)...

Large submicroscopic genomic APC deletions are a common cause of typical familial adenomatous polyposis

“…Cytogenetically detectable interstitial deletions at the chromosomal region 5q22 have been reported in several patients exhibiting FAP and some degree of mental retardation and dysmorphism, [13][14][15][16][17][18] and were the key observation for mapping and cloning of APC. [19][20][21][22][23] Since then, a few submicroscopic deletions have been detected in FAP patients of normal intelligence and without dysmorphic features by different methods, including apparent non-parental segregation of intragenic or flanking polymorphic marker alleles, fluorescent in situ hybridisation (FISH) analysis with clones from chromosomal region 5q22, quantitative PCR, comparative genomic hybridisation, or RNA analysis. 14 24-28 All of these methods examine part of the coding sequence or flanking regions.…”

Use of <I>Apc</I>(Min/+) mouse model in the studies of intestinal tumors

“…目前也有许多靶向敲除的小鼠如 Msh2 敲除小 鼠 [7] 、k-ras 敲除小鼠 [8] 、p53 敲除小鼠等 [9] 。研究者 将几种突变或敲除小鼠的后代杂交繁殖, 形成新品 系的突变-敲除小鼠, 用来研究几种不同蛋白同时发 生失活和/或激活的效应。迄今, 和肠道肿瘤相关的 基因突变小鼠或敲除小鼠大约有 30 多种 [10] 育过程中关键的信号转导途径 [11] , 也对肿瘤的发生 发展起到不同寻常的作用 [12] 。诸多研究支持结直肠 [16] 。大量研究表明多于 75% 的已知突变落于第 15 外显子的突变成簇区(mutation…”

Identification of an alternative 5? untranslated region of the adenomatous polyposis coli gene

“…Mutations within the adenomatous polyposis coli (APC) gene have been demonstrated to represent the molecular basis of FAP Nishisho et al 1991). The APC gene has been cloned in yeast artificial chromosomes (Hampton et al 1992) and the cDNA sequence of the APC transcript has been reported . The initial analysis of APC-specific transcripts revealed that heterogeneity of mRNAs existed because of alternative splicing events, affecting APC exon 9.…”