Primary Constriction Research Articles

True vs. false inv(Y)(p11q11.2): a familial instance concurrent with trisomy 21

A boy with Down syndrome due to a free trisomy 21 also had a metacentric Y chromosome with an arm euchromatic and the other heterochromatic inherited from his phenotypically normal father. This chromosome was mitotically stable and hybridized with the DYZ3 probe precisely at its primary constriction; in addition, a subtelomeric Xp/Yp probe gave the expected signal near the end of the euchromatic arm. So, the proband’s karyotype was 47,X,inv(Y)(p11q11.2),+21. Given the high frequency of both chromosome anomalies, we regard its concurrence as a mere coincidence. This observation, along with previous reports, allows us to classify the apparent pericentric inversions of the Y chromosome into two types: “true” inversions characterized by an alphoid single centromere and mitotic stability, and “false” inversions in which a nonalphoid centromere has taken over the usual alphoid centromere; indeed, these chromosomes are dicentric and mitotically unstable. Finally, the inv(Y) polymorphism in man compares with that documented in other mammal species, in which the rearranged Y chromosome neither impairs the fertility nor has other phenotypical consequences.

Mitotic and meiotic chromosomes in Somatochlora metallica (Cordulidae, Odonata). The absence of localized centromeres and inverted meiosis.

Spermatogonial metaphase chromosomes were examined in two dragonfly species, Somatochlora metallica (Cordulidae) and Aeshna grandis (Aeshnidae), and the behaviour of male meiotic chromosomes was studied in S. metallica. Both in S. metallica and A. grandis the male mitotic metaphase chromosomes from cells treated with colchicine consisted of two equidistantly aligned chromatids, showing no primary constriction. In meiosis the chromosomes of S. metallica males showed telokinetic activity during the first meiotic division, and kinetic activity was restricted in the middle parts of chromosomes during the second division. The kinetic behaviour of the chromosomes both in mitosis and meiosis showed that they were holocentric. One chiasma arises interstitially in each bivalent in S. metallica male meiosis. The chiasmata retain their interstitial position at metaphase I and do not terminalize. At metaphase I bivalents co-orient with homologous telomere regions towards the opposite poles. Thus genuine dyads segregate at the first anaphase. Meiosis in these male dragonflies is thus pre-reductional or conventional, not post-reductional or inverted, as has been previously proposed.

Extensive cytogenetic analysis of a stable dicentric isochromosome 21, idic(21), formed by fusion of the terminal long arms

The dicentric isochromosome 21 described in this paper was formed by fusion of the terminal parts of the long arms of two chromosomes 21. No interstitial telomeric AGGGTT repeats could be detected at the fusion point, but G-banding, comparative genomic hybridization, and fluorescence in situ hybridization with painting probes for 21qter revealed no loss of other terminal DNA sequences at the fusion point. Thus, only the telomeric repeats seem to have been lost prior to, or as a consequence of, isochromosome formation. Both short arms of the isochromosome were intact with complete NORs, and staining for α-satellite DNA showed that the DNA content of the two centromeres was the same. Antibody staining for the centromeric proteins CENP-C and CENP-E and for topoisomerase IIα and IIβ demonstrated that these proteins were localized predominantly or exclusively at the centromere in the primary constriction. A novel functional in situ assay for topoisomerase activity in vivo similarly demonstrated enzyme activity exclusively at the primary constriction centromere.

A well-differentiated liposarcoma with a new type of chromosome 12-derived markers

Well-differentiated liposarcomas (WDLPS) are cytogenetically characterized by the presence of supernumerary ring or giant rod marker chromosomes. These supernumerary chromosomes are composed of amplified sequences from chromosome 12 (12q14∼15) in association with amplified segments from various other chromosomes, and contain alterations of the alpha satellite sequences. We report a case of WDLPS of the lipoma-like and sclerosing subtype that contains a novel type of supernumerary marker chromosome. Instead of rings or giant rods, these cells had three apparently identical copies of a subtelocentric supernumerary marker with a size and shape similar to C-group chromosomes. Fluorescence in situ hybridization analysis revealed that the markers were composed of amplified material from 12q14∼15, including the genes MDM2 and CDK4. Similar to the rings and giant rods observed in other WDLPS cases, these unusual markers had no alpha satellite repeats at the primary constriction site, but centromeric activity could be demonstrated by using anti-centromere protein C antibodies. These findings show that the supernumerary markers of WDLPS may be variable in size and shape, but consistently share the same genomic structure, specifically 12q amplified sequences together with centromere alterations, and underline the importance of molecular methods in the diagnosis of adipose tissue tumors.

The centromere structure in Robertsonian wheat-rye translocation chromosomes indicates that centric breakage-fusion can occur at different positions within the primary constriction.

Univalent chromosomes at meiotic metaphase I have a tendency to misdivide at the centromeres. Fusion of the misdivision products may produce Robertsonian translocations. The fine structure of the centromeres in Robertsonian wheat-rye translocation chromosomes was analyzed by fluorescence in situ hybridization (FISH) using two centromere-specific DNA clones: pRCS1, derived from rice, and pAWRC1, derived from rye. Clone pRCS1 hybridizes to the centromeres of all grasses including wheat and rye, whereas clone pAWRC1 is rye specific and hybridizes only to the centromeres of rye. Four of the six wheat-rye translocations derived from a single centric misdivision event (1st generation translocations) had hybrid centromeres, with approximately half of the centromere derived from rye and half from wheat. In the two other 1st generation translocations, the entire centromere was derived from rye. Among eight reconstructed wheat and rye chromosomes that originated from two consecutive centric misdivision-fusion events (2nd generation translocations), T1BS.1BL (derived from T1BS.1RL and T1RS.1BL) and one of three T2BS.2BL (derived from T2RS.2BL and T2BS.2RL) had hybrid centromeres. T1RS.1RL (derived from T1BS.1RL and T1RS.1BL), two of three T2BS.2BL, and all three T2RS.2RL (derived from T2RS.2BL and T2BS.2RL) had rye centromeres. All three 3rd generation translocations had hybrid centromeres with approximately half of the centromere derived from rye. There were no indications that the composite structure of the centromere in these chromosomes affected their behavior in mitosis or meiosis. These observations support the notion of a compound structure of the centromere in higher organisms, and indicate that during the centric breakage-fusion event, centromere breakage may occur in different positions along the segment of the chromosome that interacts with the spindle fibers. Normal behavior of the 1st, 2nd, and 3rd generation centric translocations in mitosis and meiosis indicates that, at least in wheat and rye, centromeres are not chromosome specific.

Using Electron Tomography to Determine How Kinetochores Bind and Alter the Conformations of Microtubule Plus Ends

Chromosome alignment during mitosis and meiosis is mediated through the interaction between kinetochores and spindle microtubules (Mts). Kinetochores are fibrous mat-like structures, located at the primary constriction of chromosomes, that bind spindle Mts, generate poleward chromosome motion, and delay progression through mitosis until chromosome alignment is complete. Mts are polar polymers that function as tracks for the movement of a variety of cellular organelles and vesicles. Chromosome alignment requires a unique form of Mt-based motion because kinetochores capture and bind the plus ends of a bundle of Mts, rather than moving along the lateral surface of a single Mt . As a result, the direction of chromosome motion must be coordinated with the growth and shrinkage of Mts. Furthermore, the kinetochore maintains continuous attachment to growing and shrinking Mts and simultaneously permits the addition and dissociation of tubulin subunits within its confines

Centromeres

The centromere is the chromosomal site which associates with spindle microtubules and is responsible for equal division of the chromosomes to both daughter cells. Centromeres can often be recognised cytologically as a distinctive narrow region on metaphase chromosomes known as the primary constriction. The terms ‘centromere’ and ‘kinetochore’ are often used synonymously but it is best to consider the centromere as the DNA and chromatin on which the kinetochore is assembled and the kinetochore as the protein machine, including centromeric chromatin, which mediates attachment to the spindle. Centromeres are found near the centre of the chromosome on metacentric chromosomes, but they can also be submetacentric, between the middle and an end; acrocentric, close to an end; and telocentric, at an end. Some organisms such as Caenorhabditis elegans are holocentric and assemble centromeric chromatin and kinetochores along the entire chromosome. Delving further you discover that centromeres are eccentric! The centromere region is also a site of strong cohesion; in higher eukaryotes it is the last point of contact between sister-chromatids at metaphase prior to their separation at anaphase. Cohesion between sister centromeres is intimately associated with kinetochore function as it counteracts forces of microtubule–kinetochore attachment and possibly aids orientation and attachment of sister kinetochores to opposite poles. Centromeres are frequently associated with heterochromatin. Genes are silenced if placed in these regions by accident or design. It is not known why these regions are transcriptionally silent and whether silencing itself is important for centromere function. In fission yeast, at least, it appears that the silent state is a by-product of full kinetochore assembly. Centromeres range in size and complexity from a relatively minute 125 bp in budding yeast, through more complex structures of 40–120 kb in fission yeast, to enormous arrays of relatively simple repeats extending for 250 kb in flies and 5000 kb in humans. Regardless of size, they all assemble kinetochores that segregate chromosomes with awe-inspiring fidelity. The number of microtubules per metaphase kinetochore varies from one in budding yeast, 2–4 in fission yeast and from 4 to over 40 in mammals. DNA sequence is not conserved between centromeres of different organisms. However, all active centromeres have a histone H3-like protein, such as CENP-A, which must contribute to the assembly of a specialised centromere specific chromatin structure. Yes and no. Yes for yeast. But it's more complicated for mammalian centromeres. In humans vast arrays of so-called alphoid satellite repeats are found at centromeres, and mammalian artificial chromosomes have been created that contain alphoid DNA. So, you think, alphoid DNA is the determinant of centromeric activity! But in some dicentric chromosomes, both centromeres contain alphoid DNA, but only one is active, as assayed by the presence of important kinetochore components such as CENP-A and CENP-C. And in certain rare cases centromere activity has been gained by regions which have never before acted as centromeres. These neocentromeres contain no alphoid DNA but have been propagated through up to three human generations. These strange goings-on indicate that centromeres are epigenetically regulated. Presumably there is a meta-stable mark that normally ensures that centromeres and kinetochores do assemble at the same place division after division. Epigenetic phenomena also contribute to centromere function in fission yeast and Drosophila, and probably other organisms. Where can I find out more?

Mosaic inv dup(8p) marker chromosome with stable neocentromere suggests neocentromerization is a post-zygotic event.

Marker chromosomes containing active human neocentromeres have been described in individuals where the chromosomes are non-mosaic, suggesting that they are mitotically stable, but also in individuals where there is mosaicism, raising the possibility of neocentromere instability. We report two independently ascertained individuals who are mosaic for a supernumerary marker chromosome, shown by reverse chromosome painting to have an 8p origin, resulting in mosaicism for tetrasomy 8p23.1-->pter in the patient. The markers have a primary constriction but show no detectable centromeric alpha-satellite DNA. The marker in Patient 1 demonstrated no centromere protein CENP-B binding, but associated with nine different functionally critical centromere proteins. Investigation of peripheral blood lymphocytes from this patient on five separate occasions over a 13-year period showed 23-46% mosaicism for the marker chromosome with no decrease in incidence. In vitro investigation of primary and secondary sub-clones of a lymphoblast cell line derived from the patient demonstrated 100% stability of the marker chromosome indicating that neocentromere instability is unlikely to be responsible for the mosaicism in the patient. This and other available data support a general model of neocentromerization as a post-zygotic event, irrespective of whether the supernumerary chromosome fragment has arisen during meiosis or post-fertilization at mitosis.

Karyotype evolution and cytotaxonomy in Brazilian species of Rhynchospora Vahl (Cyperaceae)

Karyotypes were analysed in 113 populations of 34 species of Rhynchospora (Cyperaceae). They showed typical holocentric chromosomes lacking primary constrictions. Nucleolar constrictions were observed in the terminal regions of a few chromosomes (except in R. nanuzae, in which they were interstitial). The chromosome numbers varied between 2n =4 and 2 n=50. The primary base number seems to be x=5; x=6 and x=9 are secondary. Dysploidy occurred at low frequency, whereas polyploidy seemed to be the predominant event in the karyotype evolution of the genus. Karyotypic differences were found in R. consanguinea var. speciosa and R. consanguinea var.rigida and the closely related R. emaciata, R. riparia and R. tenuis from sectionTenues . On the other hand, karyotypic similarities occurred among sections Valderugosae, Marisculae,Stenophyllae and Cernuae.

Karyotype evolution and cytotaxonomy in Brazilian species of Rhynchospora Vahl (Cyperaceae)

Abstract Karyotypes were analysed in 113 populations of 34 species of Rhynchospora (Cyperaceae). They showed typical holocentric chromosomes lacking primary constrictions. Nucleolar constrictions were observed in the terminal regions of a few chromosomes (except in R. nanuzae , in which they were interstitial). The chromosome numbers varied between 2 n =4 and 2 n =50. The primary base number seems to be x=5; x=6 and x=9 are secondary. Dysploidy occurred at low frequency, whereas polyploidy seemed to be the predominant event in the karyotype evolution of the genus. Karyotypic differences were found in R. consanguinea var. speciosa and R. consanguinea var. rigida and the closely related R. emaciata , R. riparia and R. tenuis from section Tenues . On the other hand, karyotypic similarities occurred among sections Valderugosae , Marisculae , Stenophyllae and Cernuae .

A New Chromosome Model

We present a new model of the three-dimensional structure of chromosomes. With DNA and protein staining it could be shown by high-resolution scanning electron microscopy that metaphase chromosomes are mainly composed of DNA packed in “chromomeres” (coiled solenoides) and a dynamic matrix formed of parallel protein fibers. In the centromeric region, the chromomeres are less densly packed, giving insight into the matrix fibers. We postulate that chromosome condensation is achieved by the binding of solenoids to matrix fibers which have contact sites to one another and move antiparallel to each other. As condensation progresses, loops of solenoids accumulate to form additional chromomeres, causing chromosomes to become successively shorter and thicker as more chromomeres are formed. For sterical reasons, a tension vertical to the axial direction forces the chromatids apart. The model can simply explain the enormous variety of chromosome morphology in plant and animal systems by varying only a few cytological parameters. Primary and secondary constrictions and deletions are defined as regions devoid of chromomeres. Even in the highly condensed metaphase, all genes would be easily accessible.

A karyological study of the spirurid nematodeMastophorus muris(Nematoda: Spirocercidae)

The karyotype of Mastophorus muris (Gmelin, 1790) comprises four pairs of small autosomal chromosomes and two larger sex X chromosomes in females or one X chromosome in males (2n = 8 + XX/XO). All pairs of chromosomes possess rather uniform morphology without distinct primary or secondary constrictions. No heterochromatin bands were found by C-banding analysis. The absolute chromosome length ranges from 4.02 to 2.24 microns. The mean total length of the haploid complement is 14.34 microns. The course of gametogenesis represents a typical pattern common in the order Spirurida. The recently available karyotypes of spirurid nematodes have been reviewed.

Kinetochores.

What are they? Kinetochores are protein machines that assemble on centromeric DNA. They attach chromosomes to spindle microtubules, helping them to segregate to daughter cells during cell division with incredible precision. If you ever wondered how your 100 trillion cells end up with 46 — and not 45 or 47 — chromosomes, then kinetochores are (part of) the answer. Not to be confused with…centrosomes. But it’s OK to confuse them with centromeres. The centromere is the chromosomal domain where the kinetochore is assembled. Genetically speaking, the centromere is the locus necessary to segregate chromosomes. In many species, the centromere is also defined cytologically as the primary constriction visible on condensed chromosomes (the intersection point of the classical X-shaped chromosome) — this is also the point where one observes connection to spindle microtubules. Probably the most commonsensical definition describes the kinetochore as the protein machine that assembles on centromeric DNA during mitosis/meiosis. What about the DNA sequences on which kinetochores assemble? They’re hugely variable. In some species (for example, budding yeast) they are small (125 base pairs) and specific, much like a transcriptional promoter. In other species they are large chunks of DNA often containing imperfect repeats; phrases such as ‘higher order chromatin structure’ and ‘epigenetic character’ often dominate discussions of their nature. It’s not really known why centromeric DNA varies so much between species, nor how it directs assembly of a kinetochore, but the evolutionary conservation of some kinetochore components suggests some underlying similarities in kinetochore structure. What do they look like? In budding yeastFigure 1, a 125-base-pair DNA element forms a large nucleoprotein complex that, in turn, connects to a single microtubule. The actual molecules mediating this connection remain elusive. Electron microscopy of the cells of several species reveals a multilayered plate structure at the primary constriction of mitotic chromosomes. This textbook ‘trilaminar plate’ view of the kinetochore has been recently challenged by new EM methods, although the concept of a proteinaceous plate remains prevalent. An interesting twist is provided by species that have holocentric chromosomes (such as Caenorhabditis elegans), where the kinetochore does not assemble at a specific point but all along the length of the chromosome. How do they work? Elegant studies in vertebrate and insect cells have provided insights into the mechanics of kinetochore attachment to, and movement on, spindle microtubules but the molecular mechanisms remain largely uncharacterized. The fact that microtubule motor and depolymerizing proteins localize to kinetochores suggests they have a role at the kinetochore–microtubule interface. More progress has been made on how the kinetochore ensures high-fidelity segregation, with the identification of a conserved checkpoint pathway that prevents cells from entering anaphase until all kinetochores are attached to microtubules. The involvement of microtubule motor proteins in the checkpoint pathway points to links between the mechanochemical and attachment-monitoring functions of the kinetochore. All in all, there’s a lot going on in that tiny little space. Do they have commercial potential? Yes. Mutations in the checkpoint pathway monitoring kinetochore–microtubule interactions may underly chromosomal instabilities associated with some forms of cancer. Anti-cancer drugs that target tubulin, such as taxol, may actually work by perturbing the dynamic kinetochore–microtubule interaction. Compounds specifically targeting the kinetochore–microtubule interface could therefore provide new cancer therapy opportunities. Do say… “There’s so much about this tiny, incredibly complex machine that remains to be discovered.” Don’t say… “There are many bleached bones on the shores of the kinetochore field.” Where can I find out more? Clarke L: Centromeres: proteins, protein complexes, and repeated domains at centromeres of simple eukaryotes.Curr Opin Genet Dev 1998, 8:212-218. Karpen GH, Allshire RC: The case for epigenetic effects on centromere identity and function.Trends Genet 1997, 13:489-496. Maney T, Ginkel LM, Hunter AW, Wordeman L: The kinetochore of higher eucaryotes: a molecular view.Int Rev Cytol 2000, 194:67-131. McEwen BF, Hsieh CE, Mattheyses AL, Rieder CL: A new look at kinetochore structure in vertebrate somatic cells using high-pressure freezing and freeze substitution.Chromosoma 1988, 107:366-375. Rieder CL, Salmon ED: The vertebrate cell kinetochore and its roles during mitosis.Trends Cell Biol 1998, 8:310-318.

The pachytene chromosomes of maize as revealed by fluorescence in situ hybridization with repetitive DNA sequences

A repetitive DNA sequence, ZmCR2.6c, was isolated from maize based on centromeric sequence CCS1 of the wild grass Brachypodium sylvaticum. ZmCR2.6c is 309 bp in length and shares 65% homology to bases 421–721 of the sorghum centromeric sequence pSau3A9. Fluorescence in situ hybridization (FISH) localized ZmCR2.6c to the primary constrictions of pachytene bivalents and to the stretched regions of MI/AI chromosomes, indicating that ZmCR2.6c is an important part of the centromere. Based on measurements of chromosome lengths and the positions of FISH signals of several cells, a pachytene karyotype was constructed for maize inbred line KYS. The karyotype agrees well with those derived from traditional analyses. Four classes of tandemly repeated sequences were mapped to the karyotype by FISH. Repeats 180 bp long are present in cytologically detectable knobs on 5L, 6S, 6L, 7L, and 9S, as well as at the termini and in the interstitial regions of many chromosomes not reported previously. A most interesting finding is the presence of 180-bp repeats in the NOR-secondary constriction. TR-1 elements co-exist with 180-bp repeats in the knob on 6S and form alone a small cluster in 4L. 26S and 5S rRNA genes are located in the NOR and at 2L.88, respectively. The combination of chromosome length, centromere position, and distribution of the tandem repeats allows all chromosomes to be identified unambiguously. The results presented form an important basis for using FISH for physical mapping and for investigating genome organization in maize.

Correlation between centromere and chromosome length in human male pronuclear chromosomes: ultrastructural analysis

Ultrastructural and morphometric analyses of centromeric regions by scanning and transmission electron microscopy have been performed in chromosomes from male pronuclei obtained by heterologous fertilisation of hamster oocytes with human spermatozoa. In 1308 of 1323 chromosomes analysed, the primary constriction showed a defined biconcave constriction of variable length (0.56-1.34 microns) and constant width (0.64-0.7 micron). A positive correlation was observed between centromeric length and chromosome length. In some chromosomes, the primary constriction appears as decondensed regions of variable length (1.6-2.51 microns) composed of chromatin fibres with a minimum diameter of 30 nm.

Direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA families

Several repetitive DNA fragments were generated from PCR amplifications of caribou DNA using primer sequences derived from the white-tailed deer satellite II DNA clone OvDII. Two fragments, designated Rt-0.5 and Rt-0.7, were sequenced and found to have 96% sequence similarity. These caribou clones also had 85% sequence similarity with OvDII. Multiple-colored fluorescence in situ hybridization (FISH) studies with satellite I and satellite II DNA probes to caribou metaphase chromosomes and extended chromatin fibers provided direct visualization of the genomic organization of these two satellite DNA families, with the following findings: (1) Cervid satellite I DNA is confined to the centromeric regions of the acrocentric autosomes, whereas satellite II DNA is found at the centromeric regions of all chromosomes except for the Y. (2) For most acrocentric chromosomes, the satellite I signal appeared to be medially located at the primary constriction, in contrast to that of satellite II, which appeared to be oriented toward the lateral sides as two separate fluorescent dots. (3) The satellite II clone Rt-0.7 appeared to be enriched in the centromeric region of the caribou X chromosome, a pair of biarmed autosomes, and a number of other acrocentric autosomes. (4) Fiber-FISH demonstrated that the satellite I and satellite II arrays were juxtaposed. On highly extended chromatin fibers, the total length of the hybridization signals for the two satellite DNA arrays often reached 300–400 μm. The length of a given satellite II array usually reached 200 μm, corresponding to 2 × 10³ kb of DNA in a given centromere.

The use of multivariate analysis of karyotypesto determine relationships between species ofOpuntia(Cactaceae)

Abstract Somatic chromosomes were studied from roots from seven species and two varieties of the genus Opuntia collected from different localities in Eastern India. 2n = 22 chromosomes were observed in four, 2n = 44 in two and 2n = 55 chromosomes in one population. The total chromosome length is maximum in O. Ficus-indica (2n = 55) and minimum in O. microdasys var. lutea (2n = 22). The karyotypes show a similarity in basic number x = 11, except for the horticultural variety, O. monocantha variegata with 2n = 34. The chromosome sizes range from very small (0.97 mm) to medium (3.46 mm); the primary constriction varies from nearly submedian to median and the numbers of chromosomes with secondary constrictions are from four to six. Numerical analysis of data indicates a general similarity of the karyotypes, with O. Ficus-indica occupying a separate position.

Complex Chromosome 9, 20, and 22 Rearrangements in Acute Lymphoblastic Leukemia with Duplication of BCR and ABL Sequences

Cytogenetic analysis was performed on bone marrow cells from a 28-year-old woman who was diagnosed with acute lymphoblastic leukemia (ALL). Her karyotype was: 46,XX,t(9;22)(q34;q11) [6]/47,XX, +8,t(9;22)(q34;q11) [4]/47,XX, +8,t(9;22)(q34;q11),del(20)(q11) [2]/46,XX,t(9;22)(q34;q11),del(20)(q11) [7]/45,XX,der(9)t(9;22)(q34;q11), −20, −22, +mar1 [8]/45,XX,der(9)t(9;22)(q34;q11), −20, −22, +mar2 [3]. Both marker chromosomes are dicentric and have the same size and banding pattern but different primary constrictions. Fluorescence in situ hybridization (FISH) demonstrated that both markers were derived from chromosomes 9, 20, and 22. FISH with the bcr/abl probe showed fusion of the BCR gene with the ABL gene; however, this fusion signal was present in duplicate on both marker chromosomes. To our knowledge, duplication of the BCR/ABL fusion signal on a single chromosome arm has not been reported before, except for the extensive amplification of BCR/ABL fusion signals in the leukemic cell line K-562. These data demonstrate that the marker chromosomes are the result of complex genomic rearrangements. At the molecular level, the BCR/ABL fusion gene encodes the p190 fusion protein. Similar findings have never been observed in any case of ALL.

Variable stability of chromosomes containing amplified alpha-satellite sequences in human mesenchymal tumours.

Alpha-satellite sequences are found in the centromeric region of all human chromosomes and have been implicated in centromeric function. We describe the structure and behaviour of chromosomes containing amplified human alphoid DNA from chromosome 12, in an osteosarcoma cell line (OSA) and an atypical lipomatous tumour (ALT). In OSA, the amplified material was detected in one large marker chromosome, whereas in ALT amplified sequences were observed in chromosomes of variable number and appearance. The marker in OSA was mitotically stable, but those in ALT exhibited a high degree of mitotic instability, forming bridges at anaphase and chromatin strings between interphase nuclei. The amplified alpha-satellite arrays reacted positively with human anti-centromeric antiserum and anti-centromere protein B antibodies in both tumours. Centromere protein C, previously shown to be present only in functional kinetochores, was invariably detected at the constriction of the marker in OSA, while one-fifth of markers in ALT appeared to exhibit additional centromere protein C-positive regions outside the primary constriction, indicating that the observed chromosomal instability in ALT might, at least in part, be a consequence of the occasional formation of more than one functional kinetochore. In OSA the alphoid DNA was coamplified with unique sequences from central 12q and the amplified material was C-band negative but in ALT amplified material from central 12q as well as sequences from proximal 12p were detected, resulting in C-band-positive areas. A propensity for additional kinetochore formation might thus be associated with the coamplification of alphoid DNA and pericentromeric sequences from chromosome 12.

Transmission of a Fully Functional Human Neocentromere through Three Generations

An unusual Y chromosome with a primary constriction inside the long-arm heterochromatin was found in the amniocytes of a 38-year-old woman. The same Y chromosome was found in her husband and brother-in-law, thus proving that it was already present in the father. FISH with alphoid DNA showed hybridization signals at the usual position of the Y centromere but not at the primary constriction. Centromere proteins (CENP)-A, CENP-C, and CENP-E could not be detected at the site of the canonic centromere but were present at the new constriction, whereas CENP-B was not detected on this Y chromosome. Experiments with 82 Y-specific loci distributed throughout the chromosome confirmed that no gross deletion or rearrangement had taken place, and that the Y chromosome belonged to a haplogroup whose members have a mean alphoid array of 770 kb (range 430-1,600 kb), whereas that of this case was approximately 250 kb. Thus, this Y chromosome appeared to be deleted for part of the alphoid DNA. It seems likely that this deletion was responsible for the silencing of the normal centromere and that the activation of the neocentromere prevented the loss of this chromosome. Alternatively, neocentromere activation could have occurred first and stimulated inactivation of the normal centromere by partial deletion. Whatever the mechanism, the presence of this chromosome in three generations demonstrates that it functions sufficiently well in mitosis for male sex determination and fertility and that neocentromeres can be transmitted normally at meiosis.