Detecting the Y chromosome with C2banding and chromosome pa inting technique in the mandarin vole Microtus mandarinus 3

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1 50 (2) : , 2004 A cta Zoologica S inica 3 C2 Y , C2 Y, : C2, X, C, C, Y Y, : 0-2 % 3 % - 5 %,,, Y DNA Y DNA, Y [ 50 (2) : , 2004 ] Y C2 Y Detecting the Y chromosome with C2banding and chromosome pa inting technique in the mandarin vole Microtus mandarinus 3 ZHU Bi2Cai 33, GAO Jian2Guo 333, GAO J un2fang, ZHAN G Yong, DON G Yu2Wei HOU Jin2Hui College of Life Science, Xuzhou Normal University, Xuzhou , Jiangsu, China Abstract The Y chromosome of Microtus m andarinus was detected with the C2banding technique and whole Y chromo2 some specific probe. Two C2bands on X chromosome were distinguished, a distinct one on the kinetochore, and a pale one on the short arm in the metaphase chromosome of the male Microtus m andarinus. We did not find the Y2chromosome which was dyed deeply. Metaphase chromosome and interphase nucleus of marrow cells of Microtus m andarinus were painted with the whole Y chromosome specific probe of mouse, and the metaphase chromosome and interphase nucleus of mouse marrow cells was used as the control. The painting results showed that the signal detection rate is about 0 to 2 % in the metaphase chromosomes and 3 % to 5 % in the interphase nucleus of marrow cell of Microtus m andarinus, and the re2 sults are negative, but the control experiment was positive. From the results, we deduce that there are no homologic se2 quences of whole Y chromosome special DNA sequences of mouse in Y chromosome and whole genome of Microtus m an2 darinus. Perhaps, there are no important genes which determine male sex in the Y chromosome [ Acta Zoologica Sinica 50 (2) : , 2004 ]. Key words Mandarin vole, Microtus m andarinus, Y chromosome, C2banding, Chromosome painting, Whole Y chromosome specific probe of mouse Y,, Y, Y,, Y C (,, 2n = 49-52, 1994) Y XY XX, XY, X0, X , ( ) [ This research was funded by grants from the National Natural Science Foundation of China (No and No ) ]. 33 (Corresponding author). 333 (Contribute to this study equally) ν 2004 Acta Zoologica Sinica E2mail :

2 , X0 (, mol/ L KCl, ), XX X0, min (4) : g 15 min, ( (5) : ( = 3 1), 2004), X 40 min (6) : g 15 min, 1 2, =, 1, ( 7) :, 10 %Giemsa 30 min, (, 1993), ; Y, C Y,, X ( C2 BSG, 1999), Y, (1) : mol/ L HCl 1 h,, (2) : 50 5 % Ba (OH) 2, Y min, (3) : 60 2 Y, SSC 60 min, (4) Y (, : 5 %Giemsa min, 2003) C2 Y Y, (1) : 3 l Y, Y 7 l, 70 5 min, min % 100 % 1 min, , (3) : 10 l 56, 4, 22 mm 22 mm, ; 1, : Y,,, (Whole Y chromosome specific painting probe),, Cambio : Avidin2FITC, Cambio, PNM buffer ( ) SSC 2 min (5) : 100 l min (6) : 4 SSC/ 0105 % Tween 20,, 3, 2 min, 4, SSC 2 min (7) : PI, (1) : (5) : (2) : 70 (70 %, 2 SSC, p H 710) 2 min, 70 % 20 h (4) : 45 (50 %, 2 SSC) 3, 5 min ; 4 SSC/ 0105 % Tween 20 3, 2 min, B1, 24 mm 60 mm,, 40 3 h, 011 % 0101 ml/ g (1) (2) : : Leica DMRA2 ;, (3) :,

3 2 : C2 Y 4, : (1) M,,, ; (2) H, ; (3) T,, (2), : DC 350F,, QFISH : 14 No11 (1),, X 25 (2), 17 (3),, 56 No No11, 2n = 50 ( 1 : a) ; No11 (2), Table 1 The chromosome number and the type of chromo2 2n = 51 ( 1 : b) ; No11 (3) some No11 in individual male M. Sex mandarinus No11 (2n) Type of chromosome No11 Chromosome number (2n) Quantity of specimens (XY) (1) M (M, M) (XY) (2) H (M, T, T) (XY) (3) T ( T, T, T, T) M : H : T : M : metacentric chromosome. H : heterozygosity. T : telocentric chromosome. X, X, 56 : No11 (1), 2n = 52 ( 1 : c) 212 X C2 X, C, C, Y C2 ( 1 : d) 213 Y Y,,, 2 2 Y Table 2 The painting results with whole Y chromosome specif ic probe of the mouse 247 Specimen of chromosome a ( ) Signal distribution (metaphase) ( ) Signal distribution (interphase) ( ) Material No. Annealing time (min) ( ) Painting temperature b ( %) Total Detection rate ( %) Detection Total rate Mouse Microt us mandarinus a : : b : : a : Signal distribution means the number of metaphases or interphases showing 0, 1, 2, 3 signals. b : Detection rate means the ration of the number of metaphases or interphases showing expected signals to the number examined.

4 C2 No11 (M) X a. 2n = 50, No11 b1 2n = 51, No11 c1 2n = 52, d1 C2, 2n = 51, No11 (M) Fig11 The metaphases and C2banding metaphase of inaivsoulal male M. Arrow shows No11 chromosome and X sex chromosome. mandarinus a. 2n = 50, with a pair of metacentric chromosomes. b1 2n = 51, with one metacentric chromosome. c. 2n = 52, without meta2 centric chromosome. d. The C2banding metaphase, 2n = 51, with one metacentric chromosome ,, 100,, 92 1, Y ( 2 : a) 1 92 % ; 100, 94, Y ( 2 : b) 1, 94 % 1, 92 %, (2), 94 % 1 2, , ( 2 : c), 1 % 2 %, % ; 1, 1 2 ( 2 : d), 3 % - 5 %,, 4 % Y 3 % ( 2) (3), 40 (3 ) 42 (4 ), (1) 1 8 min, 3 4 0

5 2 : C2 Y Y a. (2n = 40), Y b. c d : (2n = 52) Fig12 Painting results with whole Y chromosome specif ic probe of mouse a. The metaphase (2n = 40) shows painting signal on whole Y chromosome. b. The interphase shows a painting signal on every interphase nucleus. c and d. Both the metaphase chromosome ( 2n = 52) and the interphase nucleus of Microt us mandarinus show no hybridization signal. Arrows show hybridization signal. 2, 0, 2 %, 3 4 1, Y DNA, Y DNA, 4 1 2, Y, 4 %, Y ( 5 % ( 2),, 2001),,, Y Y ( Human Genome Pro2 gram 3, 1997), Y D NA Y D NA 2,, 94 %, 92 % Y, Y DNA Y Y Y 312 Y, X Y,, Y, Y Y 3 Human Genome Program, Mouse and Human Genetic Similarities. In : US Department of Energy ed, Human Genome Program Re2 port, 32 (www. ornl. gov/ TechResources/ Human2Genome/ graphics/ slides/ ttmouse human. html).

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