AN ATTEMPT TO DEVELOP DIRECT CENOME MAPPING METHODS IN SCHISTOSOMA JAPONICUH

（1）染色体显微切割和FISH：染色体制备采用乙醇（99.5%）固定的细胞在盖玻片（24 × 60 mm ^2）上进行。用含20%FCS、3 μ g/ml LPS和2 μ g/ml conA的RPMI 1640培养细胞。显微切割的染色体片段收集于0.5ml试管中。PCR产物经FISH技术定位。(2)PCR程序：将显微切割的碎片转移到含有1 μ l收集液滴（2.5mm EDTA，1 mg/ml蛋白酶K，41%聚乙二醇6，000）的0.5ml微管中。脱蛋白后，使用通用引物A（5-gga aac tagatagac ctg aat tcn nnn nna tgt gg-3）进行第一次PCR扩增。用引物B（5-gga aac达特gac ctg aat tc-3）进行第二次PCR扩增。PCR标记为最终结果 ...更多信息 使用引物B和生物素进行。贴上标签的产品被鱼定位，以检查它们的质量。(3)染色体显微切割技术的应用：a）染色体显微切割：对日本血吸虫第1号染色体（包括长臂和短臂）、第2号染色体（SJ 2）和ZW染色体进行了显微切割，并尝试用FISH方法进行定位。只有SJ 2染色体与所有染色体的每个端粒区域杂交。本文对马来血吸虫中间宿主Robertsiella sp.的第1和Y染色体以及日本血吸虫中间宿主钉螺的第1和第2染色体进行了解剖和描绘。在两种寄主的整条染色体上都检测到许多强信号。B）PRINS：基于着色区域观察到三种类型。(1)所有染色体的每一端：S.japonicum S. sinensis（2）所有染色体的每一端，W染色体的着丝粒和异染色质：S.mansoni（3）所有染色体的每一端和W染色体的异染色质减

In this project, we developed three items as following : (1) Chromosome microdissection and fish : chromosome preparation was made on a cover glass (24x60 mm^2) using cells fixed by ethanol (99.5%). The cells were cultured with RPMI 1640 included 20% FCS,3mug/ml LPS,and 2mug/ml conA.Chromosomes on the preparations were dissected by using a micro glass knife with 2mum diameter on an inverted microscope. The microdissected chromosome fragments were collected into 0.5ml tube with collection drop. PCR products obtained were localized by the fish techniques. (2) PCR procedure : microdissected fragments were transferred into 0.5ml microtube containing 1ul collection drop(2.5mm EDTA,1mg/ml proteinase K,41% polyethylene glycol 6,000). After deproteinization, the 1st PCR amplification was carried out using universal primer A (5-gga aac agc tat gac ctg aat tcn nnn nna tgt gg-3). The second PCR anplification was performed using primer B (5-gga aac agc tat gac ctg aat tc-3). PCR Iabeling was final … More ly done using the primer B and biotin. The labeled products were localized by fish to check their qualities. (3) Application of the chromosome microdissection techniclue : a) Chromosome microdissection : in S.japonicum, the 1st chromosome (both of short and long arms) , the 2nd chromosome (SJ2) and the ZW chromosome were dissected and tried to be localized by the FISH method. Only the SJ2 chromosome was hybridized with every telomer region of all of the chromosomes. For Robertsiella sp. (the intermediate host of S.malaynsis), the 1st and Y chromosomes, and, the 1st and 2nd chromosomes for Oncomelania hupensis (the intermediate host of S.japonicum) were tried to dissect and paint. Many strong signals were detected on the whole chromosomes in both host species. B) PRINS : three types were observed on the basis of painted regions. (1) every end of all the chromosomes : S.japonicum S.sinensium (2) every end of all the chromosomes, centromere and heterochromotin of the W chromosome : S.mansoni (3) every end of all the chromosomes and heterochromotin of the W chromosome Less