CHROMOSOME MICRODISSECTION TECHNIQUES FOR CHROMOSOME EVOLUTION STUDY IN PRIMATES.

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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