AN ATTEMPT TO DEVELOP DIRECT CENOME MAPPING METHODS IN SCHISTOSOMA JAPONICUH

在本项目中，我们开发了以下三个项目：(1)染色体显微解剖和鱼类：染色体制备在24x60mm ^2的盖玻璃上，用乙醇（99.5%）固定细胞。RPMI 1640含20% FCS、3mug/ml LPS和2mug/ml conA。在倒置显微镜下，用直径为2微米的微玻璃刀解剖染色体。将微解剖的染色体片段收集到0.5ml的试管中，并滴注收集液。得到的PCR产物用fish技术进行了定位。(2) PCR步骤：将切片转移到0.5ml微管中，微管中含有1ul收集液（2.5mm EDTA，1mg/ml蛋白酶K，41%聚乙二醇6000）。脱蛋白后，使用通用引物A （5-gga aac agc, gac ctg aat tcn nnn nna tgt gg-3）进行第一次PCR扩增。第二次PCR扩增使用引物B （5-gga aac agc即gac ctg aat tc-3）进行。最后用引物B和生物素进行PCR标记。标记的产品被鱼类定位以检查其质量。(3)染色体显微解剖技术的应用：a)染色体显微解剖：对粳稻的1号染色体（短臂和长臂）、2号染色体（SJ2）和ZW染色体进行解剖，并尝试用FISH方法进行定位。只有SJ2染色体与所有染色体的每个端粒区杂交。对马来西亚血吸虫的中间寄主Robertsiella sp.和日本血吸虫的中间寄主Oncomelania hupensis的第1和第2染色体进行了解剖和染色。在两种寄主的全染色体上都检测到许多强信号。B) PRINS：根据绘制区域，观察到三种类型。(1)所有染色体的每一端：S.japonicum S.sinensium(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Less

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