Molecular and cytological characterization of mammalian centromes, kinetochores and chromosomal arms in metaphase
Creators: name: Wong, Alexander Kin-Choi
Publisher: name: University of Calgary
Year: 1990
Subjects: subject: Molecular genetics
subject: Genetics - Research

description: Bibliography: p. 197-213.
description: An enriched fraction of mouse centromere/kinetochore sequences was isolated by an in situ chromosome digestion protocol. Cloning of these sequences has helped to identify a 120 bp genomic consensus sequence for the minor satellite. With the use of high resolution non-isotopic fluorescent in situ hybridization analysis the minor satellite was localized specifically to the outer surface of the centromere of all chromosomes of the M. musculus karyotype with the possible exception of the Y chromosome. This pattern of distribution also coincides with the sites of anti-kinetochore staining, thus suggesting a close association with the kinetochore domain. The mapping of sequences flanking the minor satellite has provided some information on the sequence makeup of the kinetochore structure. Further investigation of the cytological distribution of the major and mmor satellite has revealed that the chromosomal location of these two mouse satellite classes is not conserved through evolution in the genus Mus. The minor satellite was absent in M. caroli and present throughout the entire centromere in M. spretus while the major satellite was not limited to centromere in these two species, but instead distributed throughout the chromosomal arm regions. There is an apparent correlation between the differential distribution of the major and minor satellite and phylogeny. A 19 bp motif of the mouse minor satellite is homologous to a segment of the human alphoid sequence containing the CENP-B box. Indirect immunofluorescent detection with anti-human CENP-B and Southern analysis of the 19 bp distribution in several mammalian genomes have established a positive correlation of their presence only in recently evolved primate and Mus species. This observation is in agreement with the report that the CENP-B protein interacts with the CENP-B box in human. Comparative analysis of the satellite sequences suggests that the 19 bp motif arose from a progenitor sequence in recent evolution. A class of CT-rich tracts has been shown to be under-represented if not excluded from the centromeres of mouse and other vertebrate chromosomes with the exception of chicken. They are found throughout the chromosomal arm regions and in some karyotypes they are clustered, producing a banding pattern. The 56 bp mixed homopyrimidine sequence used in the in situ hybridization analyses has the ability to form a triplex structure as detected by S 1 nuclease mapping. Besides the association of unusual DNA conformation with homopyrimidine tracts, a novel single-stranded CT DNA binding activity has been identified from the 500 mM KCl nuclear extracts of mouse, human, muntjac, African green monkey and chimpanzee by southwestern blotting and gel retardation assays. A set of proteins with apparent molecular weights of 54000, 55500 and 57000 was found to be highly conserved and likely modified by a post-translational process. The remarkable conservation of this protein class in various mammalian species and the non-random distribution of its target sequence, CT tracts, indicates that they play an as yet defined role in the nucleus.