K. H. A. Choo

Contrasting roles of condensin I and condensin II in mitotic chromosome formation

In vertebrates, two condensin complexes exist, condensin I and condensin II, which have differing but unresolved roles in organizing mitotic chromosomes. To dissect accurately the role of each complex in mitosis, we have made and studied the first vertebrate conditional knockouts of the genes encoding condensin I subunit CAP-H and condensin II subunit CAP-D3 in chicken DT40 cells. Live-cell imaging reveals highly distinct segregation defects. CAP-D3 (condensin II) knockout results in masses of chromatin-containing anaphase bridges. CAP-H (condensin I)-knockout anaphases have a more subtle defect, with chromatids showing fine chromatin fibres that are associated with failure of cytokinesis and cell death. Super-resolution microscopy reveals that condensin-I-depleted mitotic chromosomes are wider and shorter, with a diffuse chromosome scaffold, whereas condensin-II-depleted chromosomes retain a more defined scaffold, with chromosomes more stretched and seemingly lacking in axial rigidity. We conclude that condensin II is required primarily to provide rigidity by establishing an initial chromosome axis around which condensin I can arrange loops of chromatin.

A satellite III sequence shared by human chromosomes 13,14, and 21 that is contiguous with α satellite DNA

We report the isolation of a clone (pTR9) from a human chromosome 21 lambda phage library, which was found to contain two distinct components: (1) a previously unreported subfamily of human satellite III (pTR9-s3; 1,485 bp) and (2) an alpha satellite sequence (pTR9-alpha; 250 bp) containing 1.5 copies of a 171-bp alphoid unit that shows 88.4% homology to a previously reported alpha satellite consensus sequence. The two components are separated by two direct repeats of 9 bp. Use of the polymerase chain reaction (PCR) to amplify across the junction between pTR9-s3 and pTR9-alpha established that these two sequences are contiguous in total human genomic DNA and in DNA derived from somatic cell hybrids carrying human chromosomes 13, 14, or 21. A related, but considerably more diverged, sequence was also detected on chromosome 15. Southern analysis of somatic cell hybrids at high stringency revealed a common structure of the pTR9-s3 sequence on chromosomes 13, 14, and 21 but not on 15 or 22. This sequence should be useful for the study of the structural organisation of the centromere of these chromosomes and the mechanism of their involvement in Robertsonian translocations.

Construction of neocentromere-based human minichromosomes for gene delivery and centromere studies

Human neocentromeres are fully functional centromeres that arise naturally in non-centromeric regions devoid of α-satellite DNA. We have successfully produced a series of minichromosomes by telomere-associated truncation of a marker chromosome mardel(10) containing a neocentromere. The resulting minichromosomes are either linear or circular in nature, and range in size from approximately 650 kb to 2 Mb. These minichromosomes exhibit full centromeric activity, bind to essential centromere proteins, and are mitotically stable over many generations. They provide a useful system for dissecting the functional domains of complex eukaryotic centromeres and as vectors for therapeutic gene delivery.

Mouse mitotic spindle checkpoint Bub3 gene maps to the distal region of Chromosome 7 by interspecific backcross analysis

Human mitotic spindle checkpoint BUB3 gene encodes a 37-kDa protein (Taylor et al., 1998) and is a member of the BUB (budding uninhibited by benzimidazole)-family of genes, first identified in Saccharomyes cerevisiae (Hyot et al., 1991). BUB3 has been shown to interact with BUB1 in mammalian cells and like BUB1, the protein localises to the kinetochore before chromosome alignment takes place on the mitotic spindle during cell division (Roberts et al., 1994; Basu et al., 1998; Taylor et al., 1998; Martinez-Exposito et al., 1999). Mutations of the BUB1 gene have been identified in colon cancer cell lines that display an altered mitotic checkpoint status as well as chromosome instability phenotype (Cahill et al., 1998). BUB3 has been mapped to human chromosome 10q24 (Seeley et al., 1999) and 10q24→q26 (Cahill et al., 1999). Chromosome deletions in the 10q24 region appear in cancers from a number of tissues suggesting that BUB3 may function as a tumour suppressor gene (Seeley et al., 1999). Here we report the use of a mouse genomic fragment to map the chromosome position of the mouse Bub3 gene. Materials and methods

ZNF397, a new class of interphase to early prophase-specific, SCAN-zinc-finger, mammalian centromere protein

The centromere is a complex structure required for equal segregation of newly synthesised sister chromatids at mitosis. One of the significant objectives in centromere research is to determine the complete repertoire of protein components that constitute the kinetochore. Here, we identify a novel centromere protein using a centromere-positive autoimmune serum from a patient with watermelon stomach disease. Western blot and screening of a lambda phage expression library revealed a 60-kDa protein, ZNF397. This protein belongs to the classical Cys2His2 group of the zinc-finger protein superfamily and contains two conserved domains: a leucine-rich SCAN domain and nine Cys2His2 zinc fingers. Bioinformatic analysis shows that ZNF397 is conserved in placental mammals. Stable GFP:ZNF397-expressing human cells show co-localisation of ZNF397 with the constitutive centromere protein CENP-A during interphase and early prophase. Deletion and domain-swap constructs indicate that the SCAN domain is necessary but not sufficient for centromere localisation. Gene-knockout studies in mice using the mouse orthologue (Zfp397) reveal that ZNF397 is a non-essential protein. These properties define ZNF397 as a member of a new class of interphase to early prophase-specific and SCAN domain-containing mammalian centromere protein. The possible role of this protein in transcription at the centromere is discussed.

Assignment1 of the Centromere Protein H (Cenph) gene to mouse chromosome band 13D1 by in situ hybridization and interspecific backcross analyses

The centromere plays an important role in the accurate and coordinated segregation of chromosomes during mitosis and meiosis. In all higher eukaryotes investigated, the normal centromere consists of highly repetitive DNA complexes with proteins forming the kinetochore (reviewed in Choo, 1997). The cDNA of a new member of the constitutive centromere proteins, Cenph, was recently cloned from the mouse erythroleukemia cell line SKT6 (Sugata et al., 1999). Its constitutive association with the kinetochore throughout the whole cell cycle and the predicted presence of a coiled-coil region suggests its importance in kinetochore organization and possible interactions with other kinetochore components. Here, we report the mapping of the mouse Cenph gene by fluorescence in situ hybridization (FISH) and establishing flanking markers using interspecific backcross analysis.