CHROMOSOME MICRODISSECTION TECHNIQUES FOR CHROMOSOME EVOLUTION STUDY IN PRIMATES.

(1)染色体显微切割和FISH：用乙醇(99.5%)固定的细胞，在盖玻片(24×60 mm^2)上制备染色体。用含20%胎牛血清、3µg/mlLPS和2µg/mlConA的RPMI1640培养细胞，倒置显微镜下用直径为2µm的微型玻璃刀解剖染色体。将显微切割的染色体片段用收集液滴入0.5ml试管中。用FISH技术对扩增产物(2)进行定位。此时，在从日本猴Y染色体获得的14个产物中，只有一个(MffY-2)显示了对Y染色体短臂的特异性。杂交种与多条染色体杂交。MffY-2对分析日本猴Y和X的配对方式非常有用。(2)聚合酶链式反应程序：将显微切割的片段放入1滴(25 mM乙二胺四乙酸乙二酯，1 mg/ml蛋白酶K，41%聚乙二醇6000)0.5mlm…中更多的微管。去蛋白后，用通用引物A(5‘-GGAAA C AG CTATGACCTGAATTCNNNNNAGTGG-3’)进行第一次聚合酶链式反应，然后用引物B(5‘-GGAAACAGCTATGACCTGAATTC-3’)进行第二次聚合酶链式反应，最后用引物B和生物素进行聚合酶链式反应标记。对标有标签的产品进行鱼类定位，以检查产品质量。我们的程序能够从日本猴子的Y染色体的一个分离片段中扩增出DNA。(3)染色体显微切割技术的应用：利用显微切割-FISH技术分析了人类(肺癌、卵巢)和大鼠(卵巢)癌症中染色体异常的机制。该技术对于检测HSR的起源和这些癌细胞中的双分钟非常有用。(4)染色体进化的新理论：Imai等人(1986)提出了一种新的染色体进化理论--“最小相互作用”理论。这一次，该理论得到了全面的概括和更详细的汇编。这一理论对灵长类染色体的进化也一定非常有用。较少

In this project, we developed four items as following :(1) Chromosome microdissection and FISH : Chromosome preparation was made on a cover glass (24 X 60 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 Con A.Chromosomes on the preparation were dissected using by a micro glass knife with 2mum diameter on an inverted microscope. The microdissected chromosome fragments were collected into 0.5 ml tube with collection drop. Products obtained through PCR treatment (2) were localized by FISH technique. At this time, of 14 products obtained from Japanese monkey Y chromosome only one (MffY-2) showed specificity to the short arm of Y chromosome. Ohters hybridized with many chromosomes. MffY-2 was very useful to analyze a pairing manner between Y and X of Japanese monkey.(2) PCR procedure : Microdissected fragments were put in 1 mul collection drop (25 mM EDTA,lmg/ml proteinase K,41% Polyethylene glycol 6000) of 0.5 ml m … More icrotube. After deproteinization, 1st PCR was performed using universal primer A (5'-GGAAA C AG CTATGACCTGAATTCNNNNNNATGTGG-3'), afterward 2nd PCR,using primer B (5'-GGAAACAGCTATGACCTGAATTC-3'), and finally PCR labeling, using primer B and biotin. The labeled products were localized by FISH to check the quality of the products. Our procedure became to be able to amplify DNA from one dissected fragment of Y chromosome of Japanese monkey.(3) Application of chromosome microdissection technique : Mechanisms of chromosome aberrations in human (lung, ovary) and rat (ovary) cancers were analyzed using microdissection-FISH.This technique was very useful to detect the derivation of HSRs and double minutes in those cancer cells.(4) New theory of chromosome evolution : Imai et al (1986) has proposed a new theory of chromosome evolution, "Minimum Interaction" theory. This time the theory was overall generalized and compiled in more detail. This theory must be very available also for primate chromosome evolution. Less