CHROMOSOME 3G ABNORMALITIES AND MEGAKARYOCYTOPOIESIS

调节巨核细胞分化的生物因素和 体内的血小板生成还没有被确定。我们建议使用 一些创新的分子生物学技术来分离和 描述其基因产物涉及的一系列候选基因 在巨核细胞生成中。与重排相关的人类白血病 带3g21(通常带有3g26)具有典型的巨核细胞生成异常 和血小板增多，表明该区域的基因(S0可能调节 巨核细胞生成。我们将精确地对3g21带进行显微解剖，采用 我们开发的高分辨率系统，并放大显微解剖 用含有6个简并基因的“通用”引物进行染色体DNA的聚合酶链式反应 本垒打。扩增的产物将被放射性同位素标记并使用 为了探索，1)正常人骨髓cDNA库，2)cDNA库 来源于携带t(3；3)的白血病成髓细胞(G21；26.2) 易位，两个细胞来源预测表达的基因(S) 利息位于3G21。隔离克隆将映射到中期 利用荧光原位杂交技术对染色体进行鉴定 插入片段大小、限制性内切酶长度分析和DNA测序。一个 将使用显微切割条带分离额外的一组cDNA克隆 3g26.2 DNA为探针。新克隆的初级结构将是 通过计算机同源检索和分析来阐明 与已知基因的结构/功能关系，特别是 造血生长因子和质膜受体。这 方法论为分离小说提供了一种快速而直接的方法 表达特定染色体区域的基因。美国人的基因产品 在骨髓和特定白血病细胞中表达的分离基因可能 在巨核细胞和BE的病理状态中起重要作用 在治疗药物引起的血小板减少症方面很有用。

The biological factors which regulate megakaryocyte differentiation and platelet production in vivo have not been identified. We propose to use a number of innovative molecular biological techniques to isolate and characterize a series of candidate genes whose gene products are involved in megakaryocytopoiesis. Human leukemias associated with rearrangements of band 3g21 (often with 3g26) have characteristic dysmegakaryocytopoiesis and thrombocytosis, indicating that gene(s0 in this region may regulate megakaryocytopoiesis. We shall precisely microdissect band 3g21, employing a high resolution system we have developed, and amplify microdissected chromosomal DNA by PCR using "universal" primers containing six degenerate bases. The amplified product will be radioisotopically labelled and used to probe, 1) a normal human bone marrow cDNA library, and 2) a cDNA library derived from leukemic myeloblasts carrying the t (3;3) (g21;26.2) translocation, two cell sources predicted to express the gene(s) of interest located at 3g21. Isolated clones will be mapped to metaphase chromosomes using fluorescent in situ hybridization an characterized by insert size, restriction length analysis and by DNA sequencing. An additional set of cDNA clones will be isolated using microdissected band 3g26.2 DNA as probe. The primary structure of novel clones will be subjected to computer homology search and analysis to elucidate structure/function relationships with known genes, particularly hematopoietic growth factors and plasma membrane receptors. This methodology provides a rapid and direct approach for isolating novel expressed genes from specific chromosome regions. The gene products of the isolated genes expressed in bone marrow and specific leukemic cells may have an important role in pathological conditions of megakaryocytes and be useful in the treatment of drug-induced thrombocytopenia.