CELL-SURFACE GLYCOCONJUGATES IN HEMATOLOGICAL DISORDERS

遗传性红细胞增多性多核症 酸化血清裂解试验阳性)是一种人类遗传病 以多聚乳糖胺聚糖蛋白糖基化缺陷为特征的 在红系细胞中。之前获得的数据表明 每名患者三种酶中的一种，N-乙酰氨基葡萄糖转移酶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，将确定每个HEMPA中的基因缺陷 目的：建立HEMPAS的分子遗传学。 首先，HEMPAS变异的G.K.‘S的GT基因将被确定他的GT是否是 有缺陷的。要做到这一点，GT的核苷酸序列来自变体 G.K.将通过聚合酶链式反应(PCR)扩增。这个 G.K.‘S GT序列将与具有 先前通过克隆cdna来确定。如果GT的突变是 经鉴定，具有与GT相同突变的GT基因的表达载体 在G.K.变种中将被构造。突变的GT的贩运 在COS-1细胞中，将检测由该载体转导的 正如在G.K.细胞中观察到的那样，突变的GT是从细胞中分泌出来的。 其次，对在HEMPAS G.C.中发现的α-MII基因突变进行分析。 再远一点。GC细胞表达低水平的α-MII Poly(A)+mRNA。 调控区域，如启动子区域和转录 将调查α-MII基因的起始点和基因突变 导致α-MII mRNA产量低的原因将被确定。按顺序 低α-MII活性与HEMPAS形态异常的关系 体外培养的红系细胞的形态特征 将调查是否存在苦马豆素(α-MII抑制剂)。 第三个项目将确定是否大多数HEMPAS 患者存在GnT II缺陷。人胎盘cDNA库将被 GnT II的筛选和cDNA的分离，以确定其序列 GnT II正常。然后对HEMPAS患者的DNA和mRNA进行检测 南方和北方分别进行了分析。GnT II的cDNAs序列 HEMPAS患者将通过基于mRNA的聚合酶链式反应进行检测。 对HEMPAS原发缺陷的确定将提供一个分子 为今后该病的诊断和基因治疗奠定了基础。

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.