CELL-SURFACE GLYCOCONJUGATES IN HEMATOLOGICAL DISORDERS

HEMPAS（遗传性成红细胞异常性多核性， 酸化血清溶解试验阳性）是一种人类遗传性疾病 特征在于聚乳糖胺聚糖蛋白质的糖基化缺陷 在红细胞中。 先前获得的数据表明， 每名患者的三种酶之一，N-乙酰葡糖胺转移酶II (GnT II）、半乳糖基转移酶（GT）或α-甘露糖苷酶II（α-MII）。 在接下来的五年里，每一个HEMPAS的基因缺陷将被确定。 建立HEMPAS的分子遗传学。 首先，HEMPAS变体G.K.的GT基因将决定他的GT是否是 有缺陷 为了实现这一点，将来自变异体的GT的核苷酸序列 G.K.将通过采用聚合酶链式反应（PCR）来扩增。 的 G.K.的GT序列将与正常的GT序列进行比较， 先前通过cDNA克隆确定。 如果GT的突变是 鉴定了具有GT cDNA的表达载体，所述GT cDNA具有与 在G.K.将构建变量。 突变GT的贩运 将检查用载体转染的COS-1细胞中是否 如在G. K.细胞 第二，在HEMPAS G.C.中发现α-MII基因突变。将分析 进一步. G.C.细胞表达低水平的α-MII poly（A）+mRNA。 调控区如启动子区和转录区 将研究α-MII基因起始位点， 将确定导致低α-MII mRNA产生的基因。 为了 将低α-MII活性与HEMPAS的形态异常相关联 红系细胞，体外培养的成红细胞的形态， 将研究苦马豆素（α-MII抑制剂）的存在。 第三个项目将是确定HEMPAS的大多数成员是否 患者GnT II有缺陷。 人胎盘cDNA文库的构建 筛选并分离GnT II的cDNA，以确定 正常GnT II。 然后，HEMPAS患者的DNA和mRNA将通过 分别为Southern和北方分析。 GnT II cDNA序列在 将通过基于mRNA的PCR确定HEMPAS患者。 确定HEMPAS的主要缺陷将提供一个分子 为今后该病的诊断和基因治疗提供依据。

HEMPAS (Hereditary dyserythroblastic multinuclearity associated with positive acidified serum lysis test) is a human genetic disease characterized by defective glycosylation of polylactosaminoglycan proteins in erythroid cells. Previously obtained data are implicating a defect in each patient of one of three enzymes, N-acetylglucosaminyltransferase II (GnT II), galactosyltransferase (GT) or alpha-mannosidase II (alpha-MII). Over the next five years, the gene defect in each HEMPAS will be determined to establish molecular genetics of HEMPAS. First, HEMPAS variant G.K.'s GT gene will be determined whether his GT is defective. To accomplish this, nucleotide sequence of the GT from variant G.K. will be amplified by employing a polymerase chain reaction (PCR). The G.K.'s GT sequence will be compared to the normal GT sequence which has been determined previously by cDNA cloning. If a mutation of GT is identified, an expression vector having GT cDNA with the same mutation as in the G.K. variant will be constructed. The trafficking of the mutated GT in COS-1 cells transfected by the vector will be examined as to whether mutated GT is secreted from the cells as observed in G.K. cells. Second, gene mutation of alpha-MII found in HEMPAS G.C. will be analyzed further. G.C. cells express a low level of alpha-MII poly(A)+mRNA. Regulatory regions such as the promoter region and the transcription initiation site of alpha-MII gene will be investigated and genetic mutation leading to the low alpha-MII mRNA production will be determined. In order to correlate low alpha-MII activity and morphological abnormality of HEMPAS erythroid cells, the morphology of erythroblasts cultured in vitro in the presence of swainsonine (alpha-MII inhibitor) will be investigated. The third project will be to determine whether a majority of HEMPAS patients are defective in GnT II. A human placenta cDNA library will be screened and cDNA for GnT II will be isolated to determine the sequence of normal GnT II. Then the HEMPAS patients' DNA and mRNA will be examined by Southern and Northern analysis, respectively. The GnT II cDNA sequence in HEMPAS patients will be determined through mRNA-based PCR. Determination of the primary defect of HEMPAS will provide a molecular basis for future diagnosis and genetic therapy of this disease.