Meiosis and chromosome painting of sex chromosome systems in Ceboidea

The identity of the chromosomes involved in the multiple sex system of Alouatta caraya (Aca) and the possible distribution of this system among other Ceboidea were investigated by chromosome painting of mitotic cells from five species and by analysis of meiosis at pachytene in two species. The identity of the autosome #7 (X2) involved in the multiple system of Aca and its breakage points were demonstrated by both meiosis and chromosome painting. These features are identical to those described by Consigliere et al. [1996] in Alouatta seniculus sara (Assa) and Alouatta seniculus arctoidea (Asar). This multiple system was absent in the other four Ceboidea species studied here. However, data from the literature strongly suggest the presence of this multiple in other members of this genus. The presence of this multiple system among several species and subspecies that show high levels of chromosome rearrangements may suggest a special selective value of this multiple. The meiotic features of the sex systems of Aca and Cebus apella paraguayanus (Cap) are strikingly different at pachytene, as the latter system is similar to the sex pair of man and other primates. The relatively large genetic distances between species presently showing this multiple system suggest that its origin is not recent. Other members of the same genus should be investigated at meiosis and by chromosome painting in order to know the extent and distribution of this complex sex-chromosome system.     

High-resolution chromosome banding studies in Cebus apella,Cebus albifrons,and Lagothrix lagothricha: Comparison with the human karyotype

Karyotypes of three species of South American primates (Cebus apella, Cebus albifrons, and Lagothrix lagothricha) were studied using high-resolution banding techniques, and were compared to the human karyotype. The number of homologies was very high for the three species. Some of the breakpoints implicated in chromosome rearrangements corresponded with human fragile sites. The fragile sites in human chromosomes often correspond with the localization of latent centromeres in the platyrrhines or with large heterochromatic regions that may have been lost or newly added during evolution.     

Effect of CO2 on short-term human lymphocyte culture in vitro

Summary There was no significant difference in the mitotic indices of the cultures maintained at different CO₂ concentrations, i.e. 0%, 5% and 10%. However, considerable variation was recorded among different individuals. Supported by National Cancer Institute Contract No. 1 CP 43251.     

Frequencies of chromosome aberrations in a control population determined by G banding

Control data on chromosome aberration frequencies determined by G banding from several studies undertaken in this laboratory have been re-analysed in relation to age and smoking. The combined study group comprised a total of 162 men (90 non-smokers and 72 smokers) with ages ranging from 20 to 72 years. For the group as a whole, significant increases were observed in translocations and all symmetrical exchanges with increasing age and also when smokers were compared with non-smokers.     

Visualization of early chromosome condensation: a hierarchical folding, axial glue model of chromosome structure

Current models of mitotic chromosome structure are based largely on the examination of maximally condensed metaphase chromosomes. Here, we test these models by correlating the distribution of two scaffold components with the appearance of prophase chromosome folding intermediates. We confirm an axial distribution of topoisomerase IIalpha and the condensin subunit, structural maintenance of chromosomes 2 (SMC2), in unextracted metaphase chromosomes, with SMC2 localizing to a 150-200-nm-diameter central core. In contrast to predictions of radial loop/scaffold models, this axial distribution does not appear until late prophase, after formation of uniformly condensed middle prophase chromosomes. Instead, SMC2 associates throughout early and middle prophase chromatids, frequently forming foci over the chromosome exterior. Early prophase condensation occurs through folding of large-scale chromatin fibers into condensed masses. These resolve into linear, 200-300-nm-diameter middle prophase chromatids that double in diameter by late prophase. We propose a unified model of chromosome structure in which hierarchical levels of chromatin folding are stabilized late in mitosis by an axial "glue."     

Reduction of chromosome number in Eleocharis subarticulata (Cyperaceae) by multiple translocations

Eleocharis subarticulata is recorded as the third species of Cyperaceae with a reduced chromosome number ( n  = 3), following reports on Rhynchospora tenuis ( n  = 2) and Fimbristylis umbellaris ( n  = 3). For Eleocharis, the numbers recorded to date vary from 2 n  = 10 to 2 n  = c. 196, with x  = 5 as the possible basic number. The karyotype of E. subarticulata was studied using conventional staining (mitosis and meiosis), C-CMA₃/DAPI banding, and FISH with 45S rDNA and telomere probes. The chromosomes showed no primary constrictions, as expected in the holocentric chromosomes of Cyperaceae. The meiotic behaviour was abnormal, with a single multivalent ring of six chromosomes at metaphase I, resulting from multiple translocations. At anaphase I six chromatids migrated to each pole, evidencing the inverted meiosis, and these groups were also visible at metaphase II. The C-CMA₃/DAPI banding technique showed only four terminal GC-rich blocks. FISH with 45S rDNA probes revealed four terminal signals, probably associated with GC-rich blocks. The telomeric probe located terminal signals in all the chromosomes, besides a hybridization site in the middle of the large pair. The occurrence of ectopic telomeric sites has not been described previously for plants with holokinetic karyotypes and with reduced chromosome numbers. These data reinforce the hypothesis of the reduction in chromosome number by multiple translocations.  © 2005 The Linnean Society of London, Botanical Journal of the Linnean Society , 2005, 149 , 457–464.     

Chromosome evolution in the cercopithecidae and its relationship to human fragile sites and neoplasia

Seven species of the family Cercopithecidae have been studied using highresolution banding techniques. Comparative studies allowed us to identify the main chromosomal reorganizations in this group, as well as to establish the phylogenetic relationships between species. Some of the regions involved in evolutionary rearrangements correspond to human fragile sites and/or chromosomal rearrangements related to neoplasia.     

A novel combined 15q11.2 duplication and a bisatellited supernumerary marker derived from chromosome 22: Molecular characterization of the marker

Supernumerary marker chromosomes (SMC) are heterogeneous group of chromosomes which are reported in variable phenotypes. Approximately 70% originate from acrocentric chromosomes. Here we report a couple with recurrent miscarriages and a SMC originating from an acrocentric chromosome. The cytogenetic analysis of the husband revealed a karyotype of 47,XY+marker whereas the wife had a normal karyotype. Analysis of SMC with C-banding showed the presence of a big centromere in the center and silver staining showed prominent satellites on both sides of the marker. Apparently, microarray analysis revealed a 2.1 Mb duplication of 15q11.2 region but molecular cytogenetic analysis by fluorescence in situ hybridization (FISH) with whole chromosome paint (WCP) 15 showed that the SMC is not of chromosome 15 origin. Subsequently, FISH with centromere 22 identified the SMC to originate from chromosome 22 which was also confirmed by WCP 22. Additional dual FISH with centromere 22 and Acro-p-arm probes confirmed the centromere 22 and satellites on the SMC. Further fine mapping of the marker with Bacterial Artificial Chromosome (BAC) clones; two on chromosome 22 and four on chromosome 15 determined the marker to possess only centromere 22 sequences and that the duplication 15 exists directly on chromosome 15. In our study, we had identified and characterized a SMC showing inversion duplication 22(p11.1) combined with a direct tandem duplication of 15q11.2. The possible genotype–phenotype in relation with the two rearrangements is discussed.     

Genomic characterization of the human heterotrimeric G protein alpha, beta, and gamma subunit genes.

Heterotrimeric guanine nucleotide binding proteins (G proteins) transduce extracellular signals received by transmembrane receptors to effector proteins. Each subunit of the G protein complex is encoded by a member of one of three corresponding gene families. Currently, 16 different members of the alpha subunit family, 5 different members of the beta subunit family, and 11 different members of the gamma subunit family have been described in mammals. Here we have identified and characterized Bacterial Artificial Chromosomes (BACs) containing the human homologs of each of the alpha, beta, and gamma subunit genes as well as a G alpha11 pseudogene and a previously undiscovered G gamma5-like gene. The gene structure and chromosome location of each gene was determined, as were the orientations of paired genes. These results provide greater insight into the evolution and functional diversity of the mammalian G protein subunit genes.     

Prematurely condensed chromosome fragments in human lymphocytes induced by high doses of high-linear-energy-transfer irradiation

This study provides a useful biodosimetry protocol for radiation accidents that involve high doses of heavy particle radiation. Human peripheral blood lymphocytes (PBLs) were irradiated in vitro with high doses (5–50 Gy) of charged heavy-ion particles (carbon ions, at an effective linear-energy-transfer (LET) of 34.6 keV/μm), and were then stimulated to obtain dividing cells. PBLs were treated with 100 nM calyculin A to force chromosomes to condense prematurely, and chromosome spreads were obtained and stained with Giemsa. The G2 prematurely condensed chromosome (G2-PCC) index and the number of G2-PCC including fragments (G2-PCC-Fs) per cell for each radiation dose point were scored. Dose-effect relationships were obtained by plotting the G2-PCC indices or G2-PCC-Fs numbers against radiation doses. The G2-PCC index was greater than 5% up to doses of 15 Gy; even after a 30 Gy radiation dose, the index was 1 to 2%. At doses higher than 30 Gy, however, the G2-PCC indices were close to zero. The number of G2-PCC-Fs increased steeply for radiation doses up to 30 Gy at a rate of 1.07 Gy⁻¹. At doses higher than 30 Gy, the numbers of G2-PCC-Fs could not be accurately indexed because of the limited numbers of cells for analysis. Therefore, the number of G2-PCC-Fs could be used to estimate radiation doses up to 30 Gy. In addition, a G2-PCC index close to zero could be used as an indicator for radiation doses greater than 40 Gy.     

Karyotype of Mesembryanthemum crystallinum (Aizoaceae) studied by chromosome banding,FISH with rDNA probes and immunofluorescence detection of DNA methylation

The karyotype of the common ice plant Mesembryanthemum crystallinum L. (Aizoaceae) was studied using Chromomycin A₃ (CMA)/4′,6-diamidino-2-phenylindole (DAPI) staining, fluorescence in situ hybridization with 5S and 18S–5.8S–25S rDNA probes, DAPI/C-banding and immunodetection of 5-methylcytosine. A single bright CMA-band was revealed on the satellite chromosome, whose location was coincided with a position of a site of 18S–5.8S–25S rRNA genes. A site of 5S rRNA genes was observed on one of the other chromosomes. Relatively large DAPI/C-bands were mainly localized in the pericentromeric regions of the chromosomes. DAPI/C-banding patterns allowed us to identify all the chromosomes in the karyotype of M. crystallinum. The methylation of euchromatic chromosome regions was weaker as compared with heterochromatic DAPI/C-bands, which were hypermethylated. The obtained results may provide opportunities for investigating, at the chromosomal level, the genomic changes occurring in M. crystallinum either under salinization or under the action of other stress factors.     

Clustering of multiple specific genes and gene-rich R-bands around SC-35 domains: evidence for local euchromatic neighborhoods

Typically, eukaryotic nuclei contain 10-30 prominent domains (referred to here as SC-35 domains) that are concentrated in mRNA metabolic factors. Here, we show that multiple specific genes cluster around a common SC-35 domain, which contains multiple mRNAs. Nonsyntenic genes are capable of associating with a common domain, but domain "choice" appears random, even for two coordinately expressed genes. Active genes widely separated on different chromosome arms associate with the same domain frequently, assorting randomly into the 3-4 subregions of the chromosome periphery that contact a domain. Most importantly, visualization of six individual chromosome bands showed that large genomic segments ( approximately 5 Mb) have striking differences in organization relative to domains. Certain bands showed extensive contact, often aligning with or encircling an SC-35 domain, whereas others did not. All three gene-rich reverse bands showed this more than the gene-poor Giemsa dark bands, and morphometric analyses demonstrated statistically significant differences. Similarly, late-replicating DNA generally avoids SC-35 domains. These findings suggest a functional rationale for gene clustering in chromosomal bands, which relates to nuclear clustering of genes with SC-35 domains. Rather than random reservoirs of splicing factors, or factors accumulated on an individual highly active gene, we propose a model of SC-35 domains as functional centers for a multitude of clustered genes, forming local euchromatic "neighborhoods."     

Nuclear and territorial topography of chromosome telomeres in human lymphocytes

Nuclear and territorial positioning of p- and q-telomeres and centromeres of chromosomes 3, 8, 9, 13, and 19 were studied by repeated fluorescence in situ hybridization, high-resolution cytometry, and three-dimensional image analysis in human blood lymphocytes before and after stimulation. Telomeres were found on the opposite side of the territories as compared with the centromeres for all chromosome territories investigated. Mutual distances between telomeres of submetacentric chromosomes were very short, usually shorter than centromere-to-telomere distances, which means that the chromosome territory is nonrandomly folded. Telomeres are, on average, much nearer to the center of the cell nucleus than centromeres; q-telomeres were found, on average, more centrally localized as compared with p-telomeres. Consequently, we directly showed that chromosome territories in the cell nucleus are (1) polar and (2) partially oriented in cell nuclei. The distributions of genetic elements relative to chromosome territories (territorial distributions) can be either narrower or broader than their nuclear distributions, which reflects the degree of adhesion of an element to the territory or to the nucleus. We found no tethering of heterologous telomeres of chromosomes 8, 9, and 19. In contrast, both pairs of homologous telomeres of chromosome 19 (but not in other chromosomes) are tethered (associated) very frequently.     

Sexual differentiation in the developing mouse brain: contributions of sex chromosome genes

Neural sexual differentiation begins during embryogenesis and continues after birth for a variable amount of time depending on the species and brain region. Because gonadal hormones were the first factors identified in neural sexual differentiation, their role in this process has eclipsed investigation of other factors. Here, we use a mouse with a spontaneous translocation that produces four different unique sets of sex chromosomes. Each genotype has one normal X‐chromosome and a unique second sex chromosome creating the following genotypes: XY^*x, XX, XY^*, XX^Y*. This Y^* mouse line is used by several laboratories to study two human aneuploid conditions: Turner and Klinefelter syndromes. As sex chromosome number affects behavior and brain morphology, we surveyed brain gene expression at embryonic days 11.5 and 18.5 to isolate X‐chromosome dose effects in the developing brain as possible mechanistic changes underlying the phenotypes. We compared gene expression differences between gonadal males and females as well as individuals with one vs. two X‐chromosomes. We present data showing, in addition to genes reported to escape X‐inactivation, a number of autosomal genes are differentially expressed between the sexes and in mice with different numbers of X‐chromosomes. Based on our results, we can now identify the genes present in the region around the chromosomal break point that produces the Y^* model. Our results also indicate an interaction between gonadal development and sex chromosome number that could further elucidate the role of sex chromosome genes and hormones in the sexual differentiation of behavior.     

Cytogenetic applications of high resolution secondary ion imaging microanalysis: detection and mapping of tracer isotopes in human chromosomes.

Analytical imaging by secondary ion mass spectrometry (SIMS) using a state-of-the-art scanning ion microprobe enables the detection and mapping of tracer isotopes in human metaphase chromosomes. The stimulated mitosis of cells cultured in media containing labelled nucleosides, typically 14C-labelled thymidine or adenosine, and BrDU, yields chromosomes that have incorporated the labelled molecule in their constituent DNA. The label is subsequently detected and localized by SIMS imaging. The relative label signal intensities of sister chromatids can be quantified. The occurrence of sister chromatid exchanges (SCE) can be detected. The distribution of specific nucleosides can be directly mapped. This is non-uniform along the chromatids, giving rise to characteristic banding patterns (SIMS bands) that seem to correspond to the well known G- or Q-bands resulting from conventional staining methods. The study of a number of cytogenetic problems is expected to benefit from the use of this new method of approach, similar in principle, but potentially more sensitive and capable of higher spatial resolution than autoradiography.     

Meiotic sex chromosome inactivation in the marsupial Monodelphis domestica

In eutherian mammals, the X and Y chromosomes undergo meiotic sex chromosome inactivation (MSCI) during spermatogenesis in males. However, following fertilization, both the paternally (Xp) and maternally (Xm) inherited X chromosomes are active in the inner cell mass of the female blastocyst, and then random inactivation of one X chromosome occurs in each cell, leading to a mosaic pattern of X-chromosome activity in adult female tissues. In contrast, marsupial females show a nonrandom pattern of X chromosome activity, with repression of the Xp in all somatic tissues. Here, we show that MSCI also occurs during spermatogenesis in marsupials in a manner similar to, but more stable than that in eutherians. These findings support the suggestion that MSCI may have provided the basis for an early dosage compensation mechanism in mammals based solely on gametogenic events, and that random X-chromosome inactivation during embryogenesis may have evolved subsequently in eutherian mammals.     

The evolutionary origin of a novel karyotype in Timarcha (Coleoptera, Chrysomelidae) and general trends of chromosome evolution in the genus

In this work, we have analysed the karyotypes of six species of Timarcha for the first time and updated the cytological information for two additional taxa, for one of them confirming previous results ( Timarcha erosa vermiculata ), but not for the other ( T. scabripennis ). We describe the remarkable karyotype of T. aurichalcea , the lowest chromosome number in the genus (2 n  = 18), distinctive as well for the presence of an unusual chiasmatic sexual bivalent hitherto unreported for Timarcha . This study increases the number of species studied cytologically in this genus to forty. Additional cytogenetic analyses are performed on several species, including Ag-NOR staining and fluorescent in situ hybridization (FISH) studies with ribosomal DNA probes. Karyotype evolution is analysed by tracing different karyotype coding strategies on a published independent phylogenetic hypothesis for Timarcha based on the study of three genetic markers. The implementation of a likelihood model of character change optimized onto the phylogeny is tentatively used to detect possible drifts in chromosome changes. These analyses show that karyotype is conservative in the evolution of the genus and that there is an apparent trend to reducing chromosome number. Cytological and phylogenetic data are used to explain the evolutionary origin of the karyotype of T. aurichalcea by two centric fusions involving one pair of acrocentric autosomes and the sexual chromosomes.     

Sex-dependent selection differentially shapes genetic variation on and off the guppy Y chromosome

Because selection is often sex-dependent, alleles can have positive effects on fitness in one sex and negative effects in the other, resulting in intralocus sexual conflict. Evolutionary theory predicts that intralocus sexual conflict can drive the evolution of sex limitation, sex-linkage, and sex chromosome differentiation. However, evidence that sex-dependent selection results in sex-linkage is limited. Here, we formally partition the contribution of Y-linked and non-Y-linked quantitative genetic variation in coloration, tail, and body size of male guppies (Poecilia reticulata)-traits previously implicated as sexually antagonistic. We show that these traits are strongly genetically correlated, both on and off the Y chromosome, but that these correlations differ in sign and magnitude between both parts of the genome. As predicted, variation in attractiveness was found to be associated with the Y-linked, rather than with the non-Y-linked component of genetic variation in male ornamentation. These findings show how the evolution of Y-linkage may be able to resolve sexual conflict. More generally, they provide unique insight into how sex-specific selection has the potential to differentially shape the genetic architecture of fitness traits across different parts of the genome.     

Chromosomal rearrangements in cattle and pigs revealed by chromosome microdissection and chromosome painting

A pericentric inversion of chromosome 4 in a boar, as well as a case of (2q-;5p+) translocation mosaicism in a bull were analysed by chromosome painting using probes generated by conventional microdissection. For the porcine inversion, probes specific for p arms and q arms were produced and hybridised simultaneously on metaphases of a heterozygote carrier. In the case of the bovine translocation, two whole chromosome probes (chromosome 5, and derived chromosome 5) were elaborated and hybridised independently on chromosomal preparations of the bull who was a carrier of the mosaic translocation. The impossibility of differentiating chromosomes 2 and der(2) from other chromosomes of the metaphases did not allow the production of painting probes for these chromosomes. For all experiments, the quality of painting was comparable to that usually observed with probes obtained from flow-sorted chromosomes. The results obtained allowed confirmation of the interpretations proposed with G-banding karyotype analyses. In the bovine case, however, the reciprocity of the translocation could not be proven. The results presented in this paper show the usefulness of the microdissection technique for characterising chromosomal rearrangements in species for which commercial probes are not available. They also confirmed that the main limiting factor of the technique is the quality of the chromosomal preparations, which does not allow the identification of target chromosomes or chromosome fragments in all cases.