K.H.A. Choo

Evolution of α-satellite DNA on human acrocentric chromosomes

Abstract In situ hybridization of five new and one previously described α-satellite sequences isolated from chromosome 21 libraries gave the following chromosomal distribution patterns: (a) two sequences (pTRA-1 and -4) hybridizing to chromosomes 13, 14, 15, 21, and 22 (also 19 and 20); (b) one sequence (pTRA-7) hybridizing to chromosome 14; and (c) three sequences (pTRA-2, -11 and -15) hybridizing to chromosomes 13, 14, and 21, with significant but weaker signals on 15 and 22. These results suggested the sharing of alphoid domains between different acrocentric chromosomes and the coexistence of multiple domains on each chromosome. Analysis of somatic cell hybrids carrying a single human acrocentric chromosome using pTRA-2 demonstrated a higher-order repeating structure common to chromosomes 13, 14, and 21, but not to 15 and 22, providing direct evidence for sequence homogenization in this domain among the former three chromosomes. We present a model of evolution and genetic exchange of α sequences on the acrocentric chromosomes which can satisfactorily explain these and previous observations of (a) two different alphoid subfamilies, one common to chromosomes 13 and 21 and the other common to chromosomes 14 and 22, (b) a different alphoid subfamily on chromosome 22, and (c) nonrandom participation of chromosomes 13 and 14, and 14 and 21 in Robertsonian translocations.

Conservation of centromere protein in vertebrates.

The chicken genome comprises 78 chromosomes which include several macrochromosomes and many microchromosomes. Very little information is currently available concerning chicken centromere structure and function and it is unclear if the two types of chromosomes share a common centromere mechanism or whether this mechanism resembles those in other species. Immunofluorescence studies using antibodies to mammalian constitutive centromere proteins CENP-A, CENP-B, and CENP-C and the passenger proteins CENP-E, and CENP-F revealed the presence of each of these proteins at the centromeres of both macro- and microchromsomes. CENP-A, CENP-B, and CENP-E levels showed variability between metaphase centromeres while CENP-C and CENP-F levels were relatively constant. These results suggest a common centromere mechanism for both types of chromosomes as well as indicating a high degree of conservation of individual proteins between widely divergent vertebrate classes and an overall conservation of centromere function throughout vertebrate evolution.

Esperanto for histones: CENP-A, not CenH3, is the centromeric histone H3 variant.

The first centromeric protein identified in any species was CENP-A, a divergent member of the histone H3 family that was recognised by autoantibodies from patients with scleroderma-spectrum disease. It has recently been suggested to rename this protein CenH3. Here, we argue that the original name should be maintained both because it is the basis of a long established nomenclature for centromere proteins and because it avoids confusion due to the presence of canonical histone H3 at centromeres.

Components of the human spindle checkpoint control mechanism localize specifically to the active centromere on dicentric chromosomes

Abstract. The spindle checkpoint control mechanism functions to ensure faithful chromosome segregation by delaying cell division until all chromosomes are correctly oriented on the mitotic spindle. Initially identified in budding yeast, several mammalian spindle checkpoint-associated proteins have recently been identified and partially characterized. These proteins associate with all active human centromeres, including neocentromeres, in the early stages of mitosis prior to the commencement of anaphase. We have examined the status of proteins associated with the checkpoint protein complex (BUB1, BUBR1, BUB3, MAD2), the anaphase-promoting complex (Tsg24, p55CDC), and other proteins associated with mitotic checkpoint control (ERK1, 3F3/2 epitope, hZW10), on a human dicentric chromosome. Each of these proteins was found to specifically associate with only the active centromere, suggesting that only active centromeres participate in the spindle checkpoint. This finding complements previous studies on multicentric chromosomes demonstrating specific association of structural and motor-related centromere proteins with active centromeres, and suggests that centromere inactivation is accompanied by loss of all functionally important centromere proteins.

Partially functional Cenpa-GFP fusion protein causes increased chromosome missegregation and apoptosis during mouse embryogenesis

CENP-A is an essential histone H3-like protein that localizes to the centromeric region of eukaryotic chromosomes. Heterozygous and homozygous Cenpa–GFP fusion-protein mouse mutants, generated through targeted insertion of the green fluorescent protein (GFP) gene into the mouse Cenpa gene locus, show specific localized fluorescence at all the centromeres. Heterozygous mice are healthy and fertile. Cenpa–GFP homozygotes (Cenpag/g) undergo many cell divisions, giving rise to up to one million cells that show relatively accurate differentiation into distinct mouse embryonic tissues until day 10.5 when significant levels of chromosome missegregation, aneuploidy and apoptosis result in death. Cenpag/g embryos assemble functional kinetochores that bind toa host of centromere-specific structural and mitotic spindle checkpoint proteins (Cenpc, BubR1, Mad2 and Zw10). Examination of the nucleosomal phasing ofcentromeric minor and pericentromeric major satellite sequences indicates that the formation of Cenpag/g homotypic nucleosomes is not accompanied by any overt alteration to the overall size of the monomeric nucleosomal structure or the spacing of these structures. This study provides the first example of an essential centromeric protein gene variant in which subtle perturbation at the centromeric nucleosomal/chromatin level manifests in a significantly delayed lethality when compared with Cenpa null mice.

Neocentromere formation in a stable ring 1p32-p36.1 chromosome

Neocentromeres are functional centromeres formed in chromosome regions outside the normal centromere domains and are found in an increasing number of mitotically stable human marker chromosomes in both neoplastic and non-neoplastic cells. We describe here the formation of a neocentromere in a previously undescribed chromosomal region at 1p32→p36.1 in an oligospermic patient. Cytogenetic GTL banding analysis and the absence of detectable fluorescence in situ hybridisation (FISH) signals using telomeric probes indicate the marker to be a ring chromosome. The chromosome is negative for CBG banding and is devoid of detectable centromeric α satellite and its associated centromere protein CENP-B, suggesting activation of a neocentromere within the 1p32-36.1 region. Functional activity of the neocentromere is shown by the retention of the ring chromosome in 97% of the patients lymphocytes and 100% of his cultured fibroblasts, as well as by the presence of key centromere binding proteins CENP-E, CENP-F, and INCENP. These results indicate that in addition to CENP-A, CENP-C, and CENP-E described in earlier studies, neocentromere activity can further be defined by CENP-F and INCENP binding. Our evidence suggests that neocentromere formation constitutes a viable mechanism for the mitotic stabilisation of acentric ring chromosomes.

Non C-banding variants in some normal families might be homogeneously staining regions.

SummaryThree families are reported showing transmission of a previously described variant, which is not associated with any clinical abnormality. The variant involves additional material at the band 9p12, which shows homogeneous staining of intermediate density with GTL- and RBG-banding, and negative staining with CBG-banding. The region stains positively with Feulgen stain. In situ hybridization with total genomic human DNA, cloned alpha satellite, satellite III, and ribosomal DNA all show no hybridization to the 9p12 variant. Two members of one of the families show the largest 9p12 variant yet reported; two other carriers in this family have inherited a variant of decreased size. It is suggested that the 9p12 variants are homogeneously staining regions. Using the ISCN three-letter convention, this variant could be described as hsr(9)(p12).

Centromeric alpha satellite DNA amplification and translocation in an unusually large chromosome 14p+ variant

SummaryWe report cytogenetic and molecular studies on a family that carries, in the father, an unusually large chromosome 14p+ variant [WSi-var(14)(p+)] and, in one of his children, a translocation [DSi-der(14)] involving the variant chromosome. Increase in the size of WSi-var(14)(p+) was estimated to be approximately 35% that of a normal chromosome 14. Presence of extra chromosomal material in this variant chromosome was demonstrated by G-banding using trypsin and staining with Leishman, G-banding using bromodeoxyuridine (BrdU) and Giemsa, and R-banding using BrdU and Giemsa. This material was positive using C-banding with BaOH and staining with Giemsa and negative in DAPI/distamycin staining, suggesting that it contained repetitive DNA but probably not of the types found in the heterochromatic regions of chromosomes 1, 9, 15, 16, and Y. Staining of the nucleolus organiser region (NOR) with AgNO3 indicated the retention of the NOR in WSi-var(14)(p+) but not in DSi-der(14). In situ hybridisation of metaphase cells with an alpha satellite DNA probe specific for human acrocentric chromosomes demonstrated a significantly increased amount of centromeric alpha sequences in WSi-var(14)(p+). Most or all of the extra alpha sequences were retained in DSi-der(14), indicating translocation near the very distal end of the enlarged region. The extra alpha satellite DNA material may have originated through amplification of some centromeric segments. The possible role of the amplified DNA in chromosomal translocations is discussed.

Molecular distinction between true centric fission and pericentric duplication-fission

Centromere (centric) fission, also known as transverse or lateral centric misdivision, has been defined as the splitting of one functional centromere of a metacentric or submetacentric chromosome to produce two derivative centric chromosomes. It has been observed in a range of organisms and has been ascribed an important role in karyotype evolution; however, the underlying mechanisms remain unknown. We have investigated four cases of apparent centric fission in humans. Two cases show a missing chromosome 22 or 18 that is replaced by two centric ring products, a third case shows two chromosome-10-derived telocentric chromosomes, whereas a fourth case involves the formation of two chromosome-18-derived isochromosomes. In all four cases, results of gross cytogenetic and fluorescence in situ hybridisation analyses were consistent with a simple centric fission event. However, detailed molecular analyses provided evidence in support of centromere duplication as a predisposing mechanism for the observed chromosomal breakage in two of the cases. Results for the third case are consistent with direct centric fission not involving centromere pre-duplication as the likely mechanism. Insufficient material has precluded the further study of the fourth case. The data provide the first molecular evidence for centromere pre-duplication as a possible mechanism to explain the classically assumed simple “centric fission” events in clinical cytogenetics, karyotype evolution and speciation.

Rapid cloning of mouse DNA as yeast artificial chromosomes by transformation-associated recombination (TAR)

Several yeast artificial chromosome (YAC) libraries have been constructed for physical mapping and characterization of the mouse genome (Larin et al. 1991; Chartier et al. 1992; Haldi et al. 1996). These complex libraries were constructed by in vitro ligation of telomere-containing vectors to mouse chromosome fragments generated by endonuclease digestion. Recently a novel approach for in vivo construction of YACs was developed. Transformation-associated recombination (TAR) in yeast has been utilized for the rapid cloning of human DNA as linear and circular YACs (Larionov et al. 1996a, 1996b). In linear TAR cloning (Larionov et al. 1996a), yeast spheroplasts are presented with gently isolated human DNA and one or two, nonreplicating vectors that contain selectable markers, a yeast telomere, and an Alu or LINE sequence(s) to serve as a ‘‘hook’’. Homologous recombination can occur between the co-transformed human DNA and repeats on the vectors to create linear YACs. Propagation of the YACs can occur because one of the vectors contains a yeast centromere and cloned human DNA typically contains sequences that can function as yeast autonomous replicating sequences (ARS). A YAC can also be established when one vector is used because a telomere can be formed at the end of cloned human DNA, possibly by (CA)n microsatellite repeats. This approach has been used to isolate human YACs ranging in size from 70 kb to >600 kb (Larionov et al. 1996a). Circular TAR cloning (Larionov et al. 1996b) differs from linear TAR cloning in that a single linear vector is used that contains a centromere and hooks at each end to generate circular YACs. Circular TAR cloning procedure has produced YACs containing human inserts up to 500 kb (Larionov et al. 1996b). Among the many utilities provided by TAR cloning is the opportunity to selectively isolate large random fragments of human DNA from rodent/human monochromosomal and radiation hybrid cell lines (Larionov et al. 1996a, 1996b). We have adapted TAR cloning for the isolation of mouse DNA as YACs. A series of linear and circular TAR vectors containing mouse repetitive elements B1 and B2 were constructed (Fig. 1). These elements were chosen because of their relative abundance in the mouse genome (80,000 and 180,000 copies respectively; Bennett et al. 1984). For linear TAR cloning the vectors contain a mouse repeat element at one end and a telomere at the other, whereas for circular TAR cloning the vectors contain mouse repeat elements at each end (Fig. 1). All vectors contain the yeast HIS3 gene as a selectable marker (except pLM1, which contains the LYS2 gene), the yeast centromere CEN6 (except pWJ522, which is acentric). Yeast spheroplasts prepared from the strain VL6-48 (the HIS3 gene is deleted) or from the strain YPH857-D1 (the entire LYS2 coding sequence and promoter region are deleted) were cotransformed with TAR vectors plus various genomic DNAs. As shown in Table 1, transformation of yeast spheroplasts by a mixture of mouse DNA plus a linear TAR cloning vector containing a B1 or B2 repeat yielded a high level of transformants, comparable to that previously reported for the cloning of human DNA with Alu-containing vectors (Larionov et al. 1996a, 1996b). Only a few transformants were obtained when mouse DNA was not included. 200 His transformants obtained with the linear vector pVCB2 and mouse genomic DNA (Table 1) were characterized by PFGE separation of chromosome size DNAs following probing with radiolabeled mouse cot-1 DNA (BRL) that was preannealed with unlabeled B2 DNA. Linear YACs (data not shown) ranging in size from 100 to >600 kb were detected in 198 of the 200 transformants examined, indicating that nearly all contained mouse DNA. Similar observations were made with transformants obtained with the linear vector pVC-B1 and mouse DNA. We also analyzed His transformants obtained with the circular TAR cloning vectors, pVC-B1-B2 and pVC-B2-B2. Large circular DNA molecules were detected in the loading wells [as expected with intact large circular molecules (Larionov et al. 1996b)] for 99 among 100 His pVC-B1-B2 and all 40 His pVC-B2-B2 transformants examined. The sizes of these circular YACs [after linearization with ionizing radiation (Larionov et al. 1996b)] ranged between 70 and 300 kb (data not shown). One hundred His transformants obtained with linear and circular TAR cloning B2 vectors were also analyzed by PCR with the primer ‘B1-21’ (Herman et al. 1991). Different fingerprints of multiple inter-B1 products were observed for all transformants (examples are shown in Fig. 2), demonstrating that different regions of the mouse genome had been cloned. The TAR cloning with B1or B2-targeting vectors was specific, since transformation was greatly reduced when chicken DNA was used instead of mouse DNA (Table 1). Transformation results also demonstrate the specificity of the B2 element for cloning of mouse DNA compared with human DNA (Table 1). In contrast, transformation of the B1-targeting vector along with human or hamster DNA occurred with the same efficiency as with mouse DNA (Table 1). In other experiments we have confirmed that mouse DNA can be selectively cloned by the circular TAR vectors pVC-B1-B2, pVC-B2-B2, and pVC-B2-B2-Neo when present in excess of heterologous DNA. On the basis of blot-hybridization analysis of His transformants obtained with pVC-B1-B2 from a mixture of mouse and chicken DNAs, 78% (60/76) contained mouse DNA. None of these transformants contained chicken DNA. From blot-hybridization analysis (with a B1 probe) of His transformants obtained from a mixture of mouse and human DNAs Correspondence to: N. Kouprina Mammalian Genome 9, 157–159 (1998).