Abstract
This thesis describes the characterization of several cervid centromeric satellite DNA families and their use in elucidating the karyotypic evolution within the Asian muntjac species. Cervid satellite I DNA has been well characterized previously in a number of deer species. Cervid satellite II and III DNA are relatively novel and have not been study in detail. The first part of thesis research involves the direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA, satellites I and II. Two cervid satellite II DNA clones of the Canadian woodland caribou (Rangifer tarandus caribou) were generated by PCR using primer sequences derived from the white tailed deer satellite II clone OvDII (Qureshi and Blake, 1995). These two clones were designated as Rt-0.5 and Rt-0.7, respectively, and found to share 96% sequence similarity between each other. The caribou satellite II clones are 63% GC-rich, and comprises some 3.9% of the caribou genome. Dual-color fluorescence in situ hybridisation (FISH) studies were performed with caribou satellite I DNA (Rt-Pst3) (Lee et al., 1994) and caribou satellite II DNA (Rt-0.7) probes to caribou metaphase chromosomes and extended chromatin fibers. Direct visualization of the genomic organization of these two satellite DNA families revealed the following: (a) Sat. I and sat.II co-localized at the centromeres of acrocentric chromosomes, whereas the centromeres of bi-armed chromosomes revealed only sat. II signals. The centromere of the Y chromosome appeared to be devoid of either satellite DNA repeat. (b) Rt-0.5 and Rt-0.7 repeats could represent specific subsets of caribou satellite II DNA that have a differential chromosomal distribution in addition to higher-order organization. (c) FISH studies on highly extended chromatin fibers demonstrated that satellite I and satellite II arrays were juxtaposed the length of a given satellite II array usually reached 200 mm, corresponding to 2 x 103 kb of DNA at a given centromere. Results of this study have been published (Li et al., 2000a). In the second part of this thesis, a cervid satellite II DNA clone was generated from PCR amplification of Indian muntjac genomic DNA using primer sequences derived from OvDII. The Indian Muntjac satellite II clone (Mmv-0.7) was characterized by a tandem repetition of 0.7-kb monomers, 62.1% GC-rich, and comprised approximately 2.1% of the Indian muntjac genome. Dual colored FISH studies were performed with the Indian muntjac satellite I DNA (C5 clone) (Lin et al., 1991) and this satellite II DNA (Mmv-0.7 clone) as probes to Indian muntjac metaphase chromosomes. The results obtained enabled us to more precisely define the chromosome breakage and fusion sites that are likely associated with the formation of the present-day Indian muntjac karyotype (2n=6/7). Furthermore, the study showed a total of 27 distinct interstitial hybridization sites, in addition to pericentromeric signals. This is remarkably close to the theoretical maximum number of 29 interstitial sites expected from chromosome fusions involving a deer species with 70 acrocentric chromosomes. This new finding further hints at the possibility that the Indian muntjac karyotype may have evolved directly from a ancestral deer species with a 2n=70 karyotype rather than from an intermediate Chinese muntjac-like species with a 2n=46 karyotype (Lin et al., 1991). Results from this part of study have also now been published (Li et al., 2000b). The third part of this thesis research deals with genomic organization of several cervid satellite DNA families in the Chinese water deer (Hydropotes inermis), the muntjac species (Muntiacus muntjak vaginalis and Muntiacus reevesi) and Columbian black tailed deer (Odocoileus hemionus hemionus). The water deer appeared to have two types of centromeric heterochromatin which are resolvable by FISH analysis using cervid satellite I, II and III DNAs (water deer satellite III clone was derived from PCR products using primer sequences of the roe deer satellite III DNA (Buntjer et al., 1998) probes to metaphase chromosomes or resting nuclear preparations. The large cluster of hybridization signal in the centromeric and pericentromeric region produced by satellite I and satellite III DNA probes appeared as a group of small fluorescent spots. This unique hybridization signal pattern was also observed in the resting nuclei. These findings suggested that the satellite I and III DNA chromatin is more diffuse with chromatin fiber extended out over a large area. Whereas, the hybridization signal with satellite II DNA appeared as pairs of distinct fluorescent spots located at the primary constrictions. These pairs of satellite DNA II signals also co-localized with the immunofluorescent signals produced by the human CREST anti-sera. These observations suggested that satellite II chromatin is more condensely packaged. Such a manner of chromatin packing may be a prerequisite for CENPs binding. During the PCR cloning experiments of cervid satellite II DNAs, another satellite DNA organized as 1-kb monomer repeat was also obtained. This 1-kb satellite DNAs has little significant homology to satellites I, II and III and is thought to potentially be a new cervid satellite DNA family which exists in the pericentric regions of the majority of the Munticus chromosomes and in the centromeric regions of certain chromosomes of the mule deer as well. Part of the results from the above study have been presented in the 50th Annual Meeting of the American Society of Human Genetics, Philadelphia, Oct. 3-7, 2000 （American Journal of Human Genetics 67:A155, 2000）