Molecular Genetics of a New Tumor Suppressor Gene in Drosophila

我们提出了一种新发现的果蝇肿瘤抑制基因(WARTS)的分子遗传学分析。该基因是通过有丝分裂-重组克隆的肿瘤表型来鉴定的，这些克隆是WTS基因小染色体缺陷的纯合。假想盘细胞中WTS基因的缺失导致细胞过度增殖，可能通过干扰定向细胞分裂的控制而导致克隆变圆，并允许角质层在细胞之间和顶端表面分泌。从细胞遗传学图谱得知，WTS基因存在于已被克隆的DNA中。我们将使用一种新的方法分离WTS和其他未知抑癌基因的新突变，以揭示有丝分裂重组克隆中隐性突变的纯合子表型。我们将通过在DNA水平上定位缺失断裂点和WTS突变来确定克隆的DNA中与WTS对应的转录单位。我们将分离与WTS相对应的cDNA，并测定其核苷酸序列，以预测该基因产物的氨基酸序列和可能的功能。针对细菌中产生的融合基因产物将产生抗体，这些抗体将用于在光镜和电子显微镜水平上定位基因产物。我们将在未分化的成像盘中标记WTS克隆，以进一步检测突变对克隆生长以及细胞骨架和细胞表面蛋白分布的影响。在这个简单的果蝇模型系统中发现和鉴定新的抑癌基因可能会导致在人类中发现新的抑癌基因。

We propose a molecular genetic analysis of a newly discovered tumor-suppressor gene (warts,abb.wts) in Drosophila. The gene is identified by the tumorous phenotype of mitotic-recombination clones that are homozygous for small chromosome deficiencies of the wts gene. Absence of the wts gene from imaginal disc cells causes excess cell proliferation, causes clone rounding presumably by interfering with control of oriented cell divisions, and allows cuticle to be secreted between cells as well as over their apical surfaces. The wts gene is known, from cytogenetic mapping, to be present in DNA that has already been cloned. We will isolate new mutations in wts and other unidentified tumor suppressor genes using a new method for revealing the homozygous phenotype of recessive mutations in mitotic recombination clones. We will identify the transcription unit corresponding to wts in the cloned DNA by mapping deficiency breakpoints and wts mutations at the DNA level. We will isolate cDNAs corresponding to wts and determine their nucleotide sequence in order to predict the amino acid sequence and possible function of the gene product. Antibodies will be raised against a fusion gene product made in bacteria, and the antibodies will be used to localize the gene product at both light and electron microscopic levels. We will mark wts clones in the undifferentiated imaginal disc in order to further examine the effects of the mutation on clone growth and on the distribution of cytoskeletal and cell-surface proteins. %%% The identification and characterization of novel tumor-suppressor genes in this simple model system of the fruit fly may lead to the identification of new tumor-suppressor genes in man.