AN ATTEMPT TO DEVELOP DIRECT CENOME MAPPING METHODS IN SCHISTOSOMA JAPONICUH

开发日本血吸虫直接CENOME作图方法的尝试

基本信息

批准号：
07557205
负责人：
AGATSUMA Takeshi
金额：
$ 3.71万
依托单位：
OBIHIRO UNIVERSITY OF AGRICULTURE AND VETERINARY MEDICINE
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-07557205/
关键词：
SCHISTOSOMA JAPONICUM GENOME MAPPING CHROMOSOME DISSECTION FISH PRINS PAINTING PROBE C-BANDING PATTERN SNAIL HOST 染色体 DNA ゲノム解析 FISH 染色体顕微切断 PRINS

项目摘要

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

在该项目中，我们开发了三个类似：（1）使用乙醇（99.5％）固定的细胞在盖玻片（24x60 mm^2）上（24x60 mm^2）进行染色体微分解和鱼类：染色体制备。用RPMI 1640培养细胞，其中包括20％FC，3mug/ml LPS和2mug/ml cona。在制剂上使用染色体上的染色体，通过在倒置显微镜上使用2mum直径的微玻璃刀进行解剖。收集下降，将微切除的染色体片段收集到0.5ml管中。获得的PCR产品通过鱼类技术定位。（2）PCR程序：将微解析的片段转移到包含1ul收集液的0.5ml微管中（2.5mm EDTA，1mg/ml蛋白酶K，41％聚乙烯甘油6,000）。 After deproteinization, the 1st PCR amplification was carried out using universal primer A (5-gga aac agc tat gac ctg aat tcn nnnnnnnnn tgt gg-3).使用引物B（5-GGA AAC AGC TAT GAC CTG AAT TC-3）进行第二个PCR灭菌。 PCR i -Incieling是最终的……使用底漆B和生物素进行了更多的工作。标记的产品被鱼类定位，以检查其品质。（3）染色体显微解剖技术的应用：a）染色体显微解剖：在S.japonicum中，第1染色体（短臂和长臂），第二个染色体（SJ2）和ZW染色体被解剖，并试图通过鱼方法定位。仅将SJ2染色体与所有染色体的每个电视区域杂交。对于Robertsiella sp。（s.malaynsis的中间宿主），第一和Y染色体以及hupensis（japonicum的中间宿主）的第一和第二染色体被试图解剖和涂漆。在两个宿主物种的整个染色体上都检测到许多强信号。 B）Prins：根据涂漆区域观察到三种类型。（1）所有染色体的每一端：S。japonicum s. sinensium（2）W染色体的所有染色体，Centromere和Hetrolomotrophin的每一端：S.mansoni：S.mansoni（3）WW染色体的每一末