CHROMOSOME MICRODISSECTION TECHNIQUES FOR CHROMOSOME EVOLUTION STUDY IN PRIMATES.

（1）染色体显微切割和FISH技术：染色体制备采用盖玻片（24 × 60 mm ^2），细胞经乙醇（99.5%）固定。用包含20%FCS、3 μ g/ml LPS和2 μ g/ml Con A的RPMI 1640培养细胞。在倒置显微镜上用直径为2 μ m的微型玻璃刀切割制备物上的染色体。将显微切割的染色体片段收集到0.5ml试管中。通过PCR处理获得的产物（2）通过FISH技术定位。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）显示出对Y染色体短臂的特异性。Ohters与许多染色体杂交。MffY-2对分析日本猴Y和X染色体配对方式非常有用。(2)PCR程序：将显微切割的片段置于1穆尔l 0.5ml/ml的收集液滴（25 mMEDTA，1 mg/ml蛋白酶K，41%聚乙二醇6000）中， 关于我们 微管。脱蛋白后，使用通用引物A（5 '-GGAAA C AG CTATGACCTGAATTCNNNNNNATGTGG-3'）进行第一次PCR，然后使用引物B（5 '-GGAAACAGCTATGACCTGAATTC-3'）进行第二次PCR，最后使用引物B和生物素进行PCR标记。通过FISH对贴有标签的产品进行本地化，以检查产品的质量。我们的程序变得能够从日本猴Y染色体的一个解剖片段扩增DNA。(3)染色体显微切割技术的应用：应用显微切割-FISH技术分析了人（肺癌、卵巢癌）和大鼠（卵巢癌）癌细胞染色体畸变的机制，该技术对检测癌细胞中HSRs和双微体的来源非常有用。(4)染色体进化的新理论：Imai et al（1986）提出了染色体进化的新理论，“最小相互作用”理论。这一次，该理论被全面概括，并进行了更详细的汇编。这一理论对灵长类动物的染色体进化也是非常有用的。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