Fusion of two apparently intact human X chromosomes

Summary Cytological studies have been presented from a 15-year-old girl with short stature and failure of puberty. Buccal mucosa preparations revealed X-chromatin mass approximately double in size of that of a normal female. Leukocyte metaphases suggested a two cell line composition of the patient. One population of cells conformed with 45,X chromosome distribution. The chromosome complement of her other cell line had a modal number of 46. In this cell line a C chromosome was replaced by an exceptionally large submetacentric chromosome. This abnormal element exhibited late DNA replicating pattern. G-banding study revealed that the abnormal chromosome was produced as a result of fusion involving telomeric ends of long arms of 2 intact X chromosomes. This translocation X was bearing 2 C-banded areas; one around the centromere and the other at the distal end of the long arm. The distal C-band area did not show any evidence for centromeric function. It appears that a centromere becomes latent in the presence of another centromere in a translocation bearing 2 total chromosomes. Such a change of state in the additional centromere is vital for the stability of the translocation chromosome.     

Cytogenetic evidence for the absence of an inactivated X chromosome in a human female (XX) breast carcinoma cell line

Summary Barr body staining procedures were applied directly to the chamber slide cultures of female amniotic cells, WI38 fibroblasts, normal female kidney cells, and a human breast carcinoma cell line, Elco. A high frequency of Barr bodies was found in all the normal female control cells; however, no Barr bodies were observed in the Elco cells. By trypsin G-banding analysis, two normal X chromosomes were identified in all Elco cells. The late DNA replication pattern of the cell line was then studied with the terminal BrdU pulse method. Both X chromosomes in the Elco cell line were found to be euchromatic with a characteristic R-banding pattern; no late-replicating X chromosome was observed. The absence of both a Barr body and a late-replicating heterocyclic X chromosome provides strong cytogenetic evidence that an inactivated X chromosome is absent in the human breast carcinoma cells bearing two X chromosomes.     

Bloom's syndrome. III. Analysis of the chromosome aberration characteristic of this disorder

The following hypothesis is put forward: X chromatin in man condenses around a center which is situated on Xq at a short distance from the centromere. The hypothesis is based on, and explains, two classes of observations. (1) Abnormal X chromosomes that have the assumed center in duplicate form bipartite Barr bodies in part of the cells. The frequency of bipartite bodies and the distance between the two parts seem to be determined by the distance between the postulated centers. (2) A large number of variously abnormal X chromosomes have been described. Almost all of them possess the postulated center and it seems possible that the very few apparent exceptions represent misidentifications of chromosome Xq — as isochromosome i(Xp). According to the hypothesis, chromosomes lacking the center would form no Barr body and therefore presumably would not be inactivated, thus leaving the cell severely unbalanced. Furthermore, absence of the center might interfere with the viability of the chromosome itself.     

Immunocytochemical evidence for methylation of the inactive X chromosome in human fetal oogonia

The state of DNA methylation of the X chromosomes of human interphase oogonia from a 46,XX and a 46,XX/47,XXX fetus at 17 weeks of gestation was tested immunocytochemically with an antibody to 5-methylcytosine (5MeC). Of 1637 oogonial nuclei from the 46,XX fetal ovary, 313 (19.1%) contained Barr bodies, of which 93.6% were positive for 5MeC. Of 1780 oogonia from the 46,XX/47,XXX fetus 327 (18.4%) contained Barr bodies; 175 oogonia had one Barr body and 152 had two. Of the single Barr bodies 145 (82.8%) had positive 5MeC reaction product. Of the 152 oogonia from the XXX line, 97 (63.8%) had positive 5MeC on both Barr bodies, 35 (23%) had one positive and one negative, and 20 (13.1%) had no product on either Barr body. This immunocytochemical evidence supports the hypothesis that the DNA of the inactive X-chromosome of the human 17-week gestation oogonium is methylated.     

Cytogenetics of the South American Akodont rodents (Cricetidae) X. Akodon mollis: a species with XY females and B chromosomes

The morphology, G- and C-banding pattern of the Akodon mollis chromosome complement is analysed. Over a total of 14 males and 10 females studied, 8 males and 7 females had a modal chromosome number of 22, while 6 males and 3 females showed a modal number of 23 chromosomes. In the animals with 23 chromosomes the odd element was considered a B chromosome on the basis of: (a) its small size, (b) the lack of an homologous chromosome and the subsequent formation of univalents at diakinesis and metaphase I from testes, (c) the weak or null genetic action as evidenced by the lack of any obvious variation in the phenotype of carriers.Four females exhibited a sex-pair dimorphism indistinguishable from that observed in males. The G-banding analysis showed homology between the pattern found in the Y chromosome and that detected in the short arm of the X. The study of C-band distribution showed that several autosome pairs and the X chromosomes had small masses of centromeric heterochromatin. On the other hand, the Y and B chromosomes were C-band negative. The Y-like chromosome in females with dimorphism of the sex pair was also C-band negative. Accordingly these females were considered to be XY and not Xx (the x being an extensively deleted X chromosome).This work was supported by grants from UNESCO, OEA, CONICET and CIC. Requests for reprints should be addressed to N.O. Bianchi.     

黄鳝体内寄生胃瘤线虫的染色体核型及G-带分析

采用常规空气干燥法制片,对寄生于黄鳝(Monopterus albus)体腔内的胃瘤线虫(Eustrongylidesignotus)染色体核型进行分析。结果表明:胃瘤线虫体细胞有12条染色体,为二倍体,核型公式为2n=12=10 m+2 sm。由5对常染色体和1对性染色体组成,性别决定模式为XX-XY,其中X、Y和1～4号染色体都为中着丝粒染色体,5号为亚中着丝粒染色体。每对染色体都有特定的G-带带型。     

A possible active segment on the inactive human X chromosome

An idic(Xp-) in which the two X chromosomes are attached short arm to short arm, and which thus has two b regions (the Q-dark segment next to the centromere on Xp) between the inactivation centers, assumed to be situated on the Q-dark region next to the centromere on Xq, showed 63.8% bipartite Barr bodies as compared with 22.2% formed by idic(Xq-). In addition, the mean distance of the two parts of the Barr bodies in the fibroblasts of a patient with idic(Xp-) is significantly greater than in the cases with one or no b region. Contrary to the other patients with abnormal X chromosomes, the buccal cells of a woman idic(Xp-) showed a number of bipartite Barr bodies. — To explain these observations we have put forward the hypothesis that the b region on the Xp always remains active and thus, when the rest of the chromosome forms a Barr body, this segment is extended, allowing the two parts of the X chromatin to get farther apart and at the same time increasing the percentage of bipartite bodies.     

Late replicating X chromosomes in human triploidy.

The status of X-chromosome replication was studied in twenty-seven 69,XXY and nine 69,XXX human triploids in which the parental origin of the additional haploid set was known from the study of chromosome heteromorphisms. Among the 69,XXY triploids, fourteen had no late replicating X, two had one late replicating X in all cells examined, and eleven had two populations of cells, one with late replicating X chromosome, and one without any. Among the 69,XXX triploids, four had a single late replicating X, and five had two populations of cells, one with one late replicating X, and one with two late replicating X chromosomes. There was no correlation between the parental origin of the triploidy and the type of X-chromosome inactivation. However the number of late replicating X chromosomes was significantly lower in cultures grown from fetal tissue when compared with those grown from extra-embryonic tissue. In cultures derived from extra-embryonic tissue there was a significant correlation between the gestational age of the sample and the proportion of late replicating X chromosomes. The older the specimen, the greater the number of late replicating X chromosomes.     

Structure and Barr body formation of an Xp + chromosome with two inactivation centers.

A patients with seizures, Von Willebrand disease, and symptoms of Turner syndrome was a chromosomal mosaic. In blood culture (1974), 56% of the cells were 45, X 33% 46, XXp+ and 11% 47,XXp + Xp +; in the skin, no cells with 47 chromosomes were found. Presumably the Xp + chromosome arose through a break in the Q-banded dark region next to the centromere on Xp to which an Xq had been attached. The abnormal X was late-labeling and formed a larger than normal Barr body. Of the chromatin-positive fibroblasts, 18.2% showed bipartite Barr bodies, which agrees with the hypothesis that the X inactivation center lies on the proximal part of the Xq. On the basis of the structure and behavior of the bipartite bodies in the present patient, as compared to those formed by other chromosomes with two presumed inactivation centers, we propose that the dark region next to the centromere of Xp remains active in the inactive X. In cells with 45,X and 46,XY, this region has the same relative size, whereas it is significantly shorter in the active X of three females, including the present patient, with one abnormal X. We propose that this region on the active X reveals different states of activity, as reflected in its length, depending on how many other X chromosomes are in the cell.     

Changes in chromosome positioning may contribute to the development of diseases related to X-chromosome aneuploidy

The radial positions of the centromeric regions of chromosomes 1 and X were determined in normal male fibroblasts (XY) and in fibroblasts from a patient with a rare case of XXXXY polysomy. The centromeric regions and presumably the whole territories of active X chromosomes were demonstrated to occupy similar, although not identical, positions in XY and XXXXY cells. The centromeres of inactive X chromosomes (Barr bodies) were located closer to the nuclear periphery as compared with the centromeres of active X chromosomes. In addition, it was established that the nuclear radial position of gene-rich chromosome 1 was changed in XXXXY cells as compared to normal XY cells. The data are discussed in the context of the hypothesis postulating that changes in nuclear positioning of chromosomal territories induced by the presence of extra copies of individual chromosomes may contribute to the development of diseases related to different polysomies.     

Genetic recombination and adaptation to fluctuating environments: selection for geotaxis in Drosophila melanogaster

Heritable variation in fitness is the fuel of adaptive evolution, and sex can generate new adaptive combinations of alleles. If the generation of beneficial combinations drives the evolution of recombination, then the level of recombination should result in changes in the response to selection. Three types of lines of Drosophila melanogaster varying in their level of genetic recombination were selected over 38 generations for geotaxis. The within-chromosome recombination level of these lines was controlled for 60% of the genome: chromosome X and chromosome II. The full recombination lines had normal, unmanipulated levels of recombination on these two chromosomes. Conversely, nonrecombination lines had recombination effectively eliminated within the X and second chromosomes. Finally, partial recombination lines had the effective rate of within-chromosome recombination lowered to 10% of natural levels for these two chromosomes. The rate of response to selection was measured for continuous negative geotaxis and for a fluctuating environment (alternating selection for negative and positive geotaxis). All selected Drosophila lines responded to selection and approximately 36% of the response to selection was because of the X and second chromosomes. However, recombination did not accelerate adaptation during either directional or fluctuating selection for geotaxis.     

Fusion of Tumour Cells with Host Cells

THE A9 cell is an 8-azaguanine-resistant derivative of the L cell line¹. It lacks the enzyme inosinic acid pyrophosphorylase and is thus unable to grow in media such as HAT² in which endogenous synthesis of nucleic acid is blocked by aminopterin. The A9 line has little ability to grow progressively in vivo. Inocula of 5 × 10⁴ to 2 × 10⁶ cells produced progressive tumours in only 12% of X-irradiated newborn syngeneic C3H mice³. One of these tumours was explanted as a cell suspension into Eagle's minimal essential medium containing 15% foetal calf serum and then subcultivated in this medium with 5% foetal calf serum. At each passage, cells were inoculated into X-irradiated newborn syngeneic C3H or semi-allogeneic C3H×X F₁ mice (X designates a number of different allogeneic parents). Between 80 and 90% of the inoculated animals developed progressive tumours. The cell line was therefore designated A9HT (high take incidence). The karyotype of the A9HT line was found to be similar to that of the A9 line, but with a slightly reduced total chromosome number. The modal chromosome number of A9HT was about 53, compared with about 57 for A9 (see ref. 4). A9 and A9HT both had between 20 and 30 bi-armed chromosomes and a number of marker chromosomes in common. A detailed comparison of the karyotypes of the two lines examined by the quinacrine fluorescence technique has been made⁵. The A9HT line, like its A9 parent, lacks inosinic acid pyrophos-phorylase and is unable to grow in HAT medium.     

Origin of mono- and binucleate cells with heterochromatic chromosomes in the malpighian tubules of the european elm scale,Gossyparia spuria (Coccoidea: Homoptera)

Males of the European elm scale, Gossyparia spuria (Erioccoccidae) have two Malphigian tubules, each made up of mononucleate and binucleate cells. Both types of cells may contain heterochromatic (H) chromosomes which form an H body. The cells with H bodies (H cells) usually appeared singly anywhere along the tubule. However, when two or more H cells were present they tended to be closer to each other than would be expected by chance. The possible origin of this tendency is discussed. Following squashing, the nuclei of the binucleate cells were much larger than those of most other somatic cells, suggesting that they were highly endopolyploid. However, the H bodies of the cells of the tubules were of about the same size as those of the other cells. These observations suggested that the H chromosomes of the binucleate cells did not replicate while the euchromatic chromosomes of these cells replicated several times. The great majority of the nuclei of the H cells contained a single H body per nucleus. An analysis of the number of H bodies in binucleate cells indicated that when two H bodies were present in the same nucleus they usually did not fuse. Thus, they were believed also not to fuse in the mononucleate cells. Since almost all the mononucleate H cells had only a single H body (rather than 2) it was concluded that they did not originate from binucleate cells by nuclear fusion.     

A restriction-fragment-length difference detected by the anonymous probe DXS199 exhibits non-Mendelian inheritance.

Anonymous DNA probes were isolated from an X chromosome-enriched flow-sorted library. One of these probes, DXS199, identified a restriction-fragment difference that failed to show Mendelian segregation. All normal females were found to have two AvaII fragments of 6.5 kb and 6.0 kb, whereas all normal males had only the 6.5-kb fragment. DNA from a 49,XXXXY male was found to have both 6.0- and 6.5-kb AvaII fragments, in the same 3:1 ratio as seen in the inactive:active number of X chromosomes. This variant, which reflects a structural difference between active and inactive X chromosomes, is likely to be due to a methylation site on the active X chromosome.     

Heterochromatin patterns in triatomines of the genus Panstrongylus

Spermatogenesis was analysed by C-banding in two species of triatomines, Panstrongylus megistus and P. herreri. Both species revealed interstitial and terminal bands in the autosomes, which is a common pattern in Heteroptera. The terminal bands corroborated the hypothesis that in holocentric chromosomes the heterochromatin is preferentially located at the telomere. The sex chromosomes in P. herreri were totally heterochromatic in spermatogenesis, and in P. megistus the X chromosomes alternated between positive and negative banding.     

The origin of the genetical diversity of Microtus mandarinus chromosomes

Here we describe our comparative studies on two types of X chromosomes, namely X(M) and X(SM,) of the mandarin vole (Microtus mandarinus). By chromosome G- and C-banding analysis, we have found that two different types of X chromosomes exist in mandarin voles. The two types of X chromosomes present two different G- and C-banding patterns: the X(M) chromosome is a longer metacentric X chromosome which is C-band negative; and the X(SM) is a shorter submetacentric X chromosome which has one C-band at the centromere and another one at the middle part of the short arm. The X(SM) has 6 G-bands including one on the kinetochore, one in the middle of the short arm, and four on the long arm. The X(M) has 7 G-bands including one on the kinetochore, two on the short arm, and four on the long arm. We have further found that female voles can be grouped into three types based on the composition of the X chromosome but the male voles have only one type. The three female groups are: (1) female voles (X(M)X(SM)), in which the two X chromosomes are different, the longer one is metacentric and the shorter is submetacentric; (2) female vole (X(SM)X(SM)), in which the two X chromosomes are both submetacentric; (3) female vole (X(M)O), in which there is only one X chromosome that is metacentric. Surprisingly, we have never found female voles with X(M)X(M), females with X(SM)O or males with X(M)Y. We hypothesize that the X(SM) chromosome is derived from the X(M) through its breakage and re-joining. The paper also discusses the formation of X(M)O females.     

Cytogenetic analysis of the neotropical spider Nephilengys cruentata (Araneomorphae, Tetragnathidae): standard staining nors, C-bands and base-specific fluorochromes.

The aim of this work is to characterize Nephilengys cruentata in relation to the diploid number, chromosome morphology, type of sex determination chromosome system, chromosomes bearing the Nucleolar Organizer Regions (NORs), C-banding pattern, and AT or GC repetitive sequences. The chromosome preparations were submitted to standard staining (Giemsa), NOR silver impregnation, C-banding technique, and base-specific fluorochrome staining. The analysis of the cells showed 2n = 24 and 2n = 26 chromosomes in the embryos, and 2n = 26 in the ovarian cells, being all the chromosomes acrocentric. The long arm of the pairs 1, 2 and 3 showed an extensive negative heteropycnotic area when the mitotic metaphases were stained with Giemsa. The sexual chromosomes did not show differential characteristics that allowed to distinguish them from the other chromosomes of the complement. Considering the diploid numbers found in N. cruentata and the prevalence of X1X2 sex determination chromosome system in Tetragnathidae, N. cruentata seems to possess 2n = 24 = 22 + X1X2 in the males, and 2n = 26 = 22 + X1X1X2X2 in the females. The pairs 1, 2 and 3 showed NORs which are coincident with the negative heteropycnotic patterns. Using the C-banding technique, the pericentromeric region of the chromosomes revealed small quantity or even absence of constitutive heterochromatin, differing of the C-banding pattern described in other species of spiders. In N. cruentata the fluorochromes DAPI/DA, DAPI/MM and CMA3/DA revealed that the constitutive heterochromatin is rich in AT bases and the NORs possess repetitive sequences of GC bases.     

Three-dimensional analysis of the arrangement of compact chromatin in the nucleus of G0 rat lymphocytes

The arrangement of compact chromatin of G₀ lymphocytes was studied in three-dimensional reconstructions of the ensemble of the chromatin and of individual compact chromatin bodies. Rat spleen was serially cut and sections were contrasted with procedures preferential for DNA. Electron microscopy images were digitized, processed, and displayed using a commercial soft-ware package, complemented by a system for three-dimensional reconstruction and analysis developed by us on an IBM-compatible microcomputer provided with an image acquisition board. The reconstructions showed a continuous layer of compact chromatin in contact with the nuclear envelope that prevents the automatic recognition of individual chromatin clumps. The ensemble of the arrangement of compact chromatin was found to be very similar in different lymphocytes. After morphological filtering procedures, the initial mass was divided into individual bodies of compact chromatin, which were tagged. Most of these bodies contact the nuclear envelope. The number of bodies as well as the number of contacts with the envelope are similar and correspond to a haploid number of chromosomes. The largest body is always the one containing nucleolus-associated chromatin. When the cell has two nucleoli, the nucleolus-associated chromatin bodies contact the envelope in diametrically opposed areas. This feature was also described in rat liver cells. It is concluded that: (a) the individualized compact chromatin bodies do not correspond to an entire chromosome or to a pair of chromosomes; (b) the arrangement of compact chromatin is not identical in each G₀ lymphocyte, but there are patterns that are repeated with limited changes; and (c) there are common features that appear in different cell types of individuals of the same species.     

Studies on the activity of the X chromosomes in female teratocarcinoma cells in culture

Embryonal carcinoma cells derived from murine teratocarcinomas are able to differentiate into the same variety of tissue types as early embryonic cells. Because embryonal carcinoma cells resemble those of the embryo at a stage before X chromosome inactivation has occurred in females embyronal carcinoma cells containing two X chromosomes were examined to determine whether both are genetically active. The specific activities of X-linked enzymes were measured in embryonal carcinoma cells containing either one or two X chromosomes. The activities in both cell types were similar, suggesting that only one X chromosome was active in the female cells. Further support for this conclusion came from experiments in which azaguanine-resistant mutants were recovered with similar frequencies from embryonal carcinoma cell lines containing one and two X chromosomes. Late replication of an X chromosome DNA was detected in one embryonal carcinoma cell line with two X chromosomes but not in another. This suggests that cells of these two lines were arrested at different developmental stages, and that late DNA replication may not be a necessary adjunct of X inactivation. Evidence is presented which suggests that X chromosome reactivation does not occur during differentiation of the cells in vitro.