The contrasting role of heterochromatin in the differentiation of sex chromosomes: an overview from Neotropical fishes

During the evolutionary process of the sex chromosomes, a general principle that arises is that cessation or a partial restriction of recombination between the sex chromosome pair is necessary. Data from phylogenetically distinct organisms reveal that this phenomenon is frequently associated with the accumulation of heterochromatin in the sex chromosomes. Fish species emerge as excellent models to study this phenomenon because they have much younger sex chromosomes compared to higher vertebrates and many other organisms making it possible to follow their steps of differentiation. In several Neotropical fish species, the heterochromatinization, accompanied by amplification of tandem repeats, represents an important step in the morphological differentiation of simple sex chromosome systems, especially in the ZZ/ZW sex systems. In contrast, multiple sex chromosome systems have no additional increase of heterochromatin in the chromosomes. Thus, the initial stage of differentiation of the multiple sex chromosome systems seems to be associated with proper chromosomal rearrangements, whereas the simple sex chromosome systems have an accumulation of heterochromatin. In this review, attention has been drawn to this contrasting role of heterochromatin in the differentiation of simple and multiple sex chromosomes of Neotropical fishes, highlighting their surprising evolutionary dynamism.

Saethre-Chotzen phenotype with learning disability and hyper IgE phenotype in a patient due to complex chromosomal rearrangement involving chromosomes 3 and 7

The authors describe on a Brazilian girl with coronal synostosis, facial asymmetry, ptosis, brachydactyly, significant learning difficulties, recurrent scalp infections with marked hair loss, and elevated serum immunoglobulin E. Standard lymphocyte karyotype showed a small additional segment in 7p21[46,XX,add(7)(p21)]. Deletion of the TWIST1 gene, detected by Multiplex Ligation Probe-dependent Amplification (MPLA) and array-CGH, was consistent with phenotype of SaethreChotzen syndrome. Array CGH also showed deletion of four other genes at 7p21.1 (SNX13, PRPS1L1, HD9C9, and FERD3L) and the deletion of six genes (CACNA2D2, C3orf18, HEMK1, CISH, MAPKAPK3, and DOCK3) at 3p21.31. Our case reinforces FERD3L as candidate gene for intellectual disability and suggested that genes located in 3p21.3 can be related to hyper IgE phenotype. (C) 2012 Wiley Periodicals, Inc.

A complex chromosomal rearrangement involving chromosomes 2, 5, and X in autism spectrum disorder

Here, we describe a female patient with autism spectrum disorder and dysmorphic features that harbors a complex genetic alteration, involving a de novo balanced translocation t(2;X)(q11;q24), a 5q11 segmental trisomy and a maternally inherited isodisomy on chromosome 5. All the possibly damaging genetic effects of such alterations are discussed. In light of recent findings on ASD genetic causes, the hypothesis that all these alterations might be acting in orchestration and contributing to the phenotype is also considered. (C) 2012 Wiley Periodicals, Inc.

Three linked enzyme loci in fishes: implications in the evolution of vertebrate chromosomes.

A three-point linkage group comprised of loci coding for adenosine deaminase (ADA), glucose-6-phosphate dehydrogenase (G6PDH), and 6-phospho-gluconate dehydrogenase (6PGD) is described in fish of the genus Xiphophorus (Poeciliidae). The alleles at loci in this group were shown to assort independently from the alleles at three other loci--isocitrate dehydrogenase 1 and 2, and glyceraldehyde-3-phosphate dehydrogenase 1. Alleles at the latter three loci also assort independently from each other. Data were obtained by observing the segregation of electrophoretically variant alleles in reciprocal backcross hybrids derived from crosses between either X. helleri guentheri or X. h. strigatus and X. maculatus. The linkage component of chi2 was significant (less than 0.01) in all crosses, indicating that the linkage group is conserved in all populations of both species of Xiphophorus examined. While data from X. h. guentheri backcrosses indicate the linkage relationship ADA--6%--G6PDH--24%--6PGD, and ADA--29%--6PGD (30% when corrected for double crossovers), data from backcrosses involving strigatus, while supporting the same gene order, yielded significantly different recombination frequencies. The likelihood of the difference being due to an inversion could not be separated from the possibility of a sex effect on recombination in the present data. The linkage of 6PGD and G6PDH has been shown to exist in species of at least three classes of vertebrates, indicating the possibility of evolutionary conservation of this linkage.

The Oxytricha trifallax macronuclear genome: a complex eukaryotic genome with 16,000 tiny chromosomes.

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor "silent" germline micronuclear genome by a process of "unscrambling" and fragmentation. The tiny macronuclear "nanochromosomes" typically encode single, protein-coding genes (a small portion, 10%, encode 2-8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.

Defining the regions of homology between human chromosome 16p12--13 and mouse chromosomes

In this study, the evolutionary relationship between human chromosome 16p12-p13 and mouse chromosomes was investigated by determining the order of marker loci in the region and then identifying the chromosomal locations of the homologous loci in mice. Eighteen genes from human 16 were mapped to fifteen subchromosomal regions by a variety of mapping approaches.^ Thirteen of the genes were mapped in the mouse. Linkage analysis with backcross mice and segregation analysis in a mouse - Chinese Hamster Ovary (CHO) somatic cell hybrid panel informative for different regions of mouse genome were used. The results assigned the thirteen genes to three different mouse chromosomes.^ A group of six genes on mouse 16 was found to be closely linked to Scid. The order of Myh11 and Mrp remains ambiguous since no recombination was detected in backcross analysis. Their relative position in human is also uncertain since they were shown to be very close to each other. For the other mouse loci, an unambiguous gene order could be determined and was found to be identical to that in human. Therefore, they comprise a new conserved linkage group between the two species. The orientation of the group was inverted relative to the centromeres, i.e. the proximal loci in one species become distal in another. The size of the group was estimated to be from 4.4 to 8 Mb and 10 to 32 cM in human. In mouse, it was about 21 cM in the backcross analysis. The two boundaries of the conserved linkage were defined within a 1 Mb range. It is now possible to predict the locations of mouse homologs for some human disease genes based on their locations on human 16p.^ The six human 16p genes that map to MMU7 showed a different gene order in mouse than in human. No recombination was found between Crym and Umod while Crym was distal to D16S79A and proximal to D16S92. The location of Stp and Cdr2 with respect to the above four loci was not determined since they were not mapped in the same set of backcross mice. These genes greatly expanded an existing conserved synteny group between the human 16p12-p13 region and the MMU7. It now consists of eleven loci that span a region of probably more than 10 Mb in human. The gene order derived from this study provided further evidence for chromosomal rearrangements within the conserved synteny. (Abstract shortened by UMI.) ^

PREMATURE CONDENSATION OF SPERMATOGENIC CELL CHROMOSOMES

The technique of premature chromosome condensation (PCC) has been used primarily to study interphase chromosomes of somatic cells. In this study, mitotic cells were fused to cells from the mouse testes to examine the chromosomes of germ cells. The testes contain various types of cells, both germinal and nongerminal. In these initial studies, four types of PCC morphologies were observed. Chromosome morphology of the PCC and labeling experiments demonstrated the mouse cell origin of various PCC. Attempts were next made to determine the cell types producing the PCC. Spermatogonia, diplotene spermatocytes, secondary spermatocytes and round spermatids are proposed to be the origin of the PCC morphologies. Some PCC could be banded by G and C banding techniques and the mouse chromosomes identified.^ Studies were subsequently undertaken to evaluate this technique as a method of evaluating damage to germ cells. Testicular cells from irradiated mice were fused to mitotic cells and the PCC examined. Both round spermatids and secondary spermatocytes exhibited chromosome damage in the form of chromatid breaks. A linear correlation was found between the dose of irradiation and the number of breaks per cell. This technique may develop into a useful method for evaluating the clastogenic effect of agents on the germ cells. ^

THE MICROCELL-MEDIATED TRANSFER OF SINGLE HUMAN CHROMOSOMES INTO RECIPIENT MOUSE CELLS

Microcell-mediated chromosome transfer is a method of gene transfer which allows for the introduction of single or small groups of intact chromosomes into recipient host cells. Microcell transfer was first performed by Fournier and Ruddle using rodent microcells and various recipient cells. Expansion of this technology to include the transfer of normal human genetic material has been hindered because large micronucleate populations from diploid human cells have been unobtainable. This dissertation research describes, however, the methods for production of micronuclei in 40-60% of normal human fibroblasts. Once micronucleate cells were obtained, they were enucleated by centrifugation in the presence of Cytochalasin B; the microcells were then purified and fused to recipient mouse (LMTK('-)) cells using a new fusion protocol employing polyethylene glycol containing phytohemagglutinin. Microcell clones were isolated from the HAT selection system. Alkaline Giemsa staining performed on these hybrids indicated the presence of a single human chromosome in each of seven microcell clones from three separate experiments. That chromosome was further identified by G banding analysis to be human chromosome #17, which codes for thymidine kinase. The time course for production of these hybrids from fusion to karyotypic analysis was 6 weeks. The viability of the transferred human genetic material was assessed by electrophoretic isozyme analysis.^ Subsequent experiments were performed in an attempt to optimize the transfer frequency for the thymidine kinase gene using this system. Results indicated that the frequency could be increased from < 1 x 10('-6) in initial experiments to 2 x 10('-5) in the latest experiment. Analyses were also conducted to determine the number of chromosomes per isolated microcell as well as to investigate the stability of the transferred human chromosome in the mouse genome. ^

CYTOLOGICAL, ULTRASTRUCTURAL, AND BIOCHEMICAL STUDIES OF THE STRUCTURE OF PREMATURELY CONDENSED CHROMOSOMES

The phenomenon of premature chromosome condensation, resulting from fusion between mitotic and interphase cells, includes dissolution of the interphase nuclear framework, thus allowing a direct visualization of interphase chromosomes. Light microscope morphology of prematurely condensed chromosomes (PCC) from synchronized HeLa cells supports the model of an interphase "chromosome condensation cycle". PCC are increasingly attenuated as cells progress through G(,1). A maximum degree of decondensation is observed at active sites of DNA replication during S phase, and a condensed morphology is rapidly resumed following completion of replication of a chromosome segment.^ To permit ultrastructural and biochemical studies of PCC, a procedure was developed to induce premature chromosome condensation at high frequency. This was achieved by polyethylene glycol (PEG)-mediated fusion of a dense monolayer of mitotic and interphase cells induced by centrifugation onto lectin-coated culture dishes. Using this method, PCC induction frequencies of 60-90% are routinely obtained.^ Scanning electron microscope analysis of PCC spreads revealed that the extension of PCC during progression through G(,1) is accompanied by a transition of the basic 30 nm chromatin fiber from tightly packed looping fibers to extended longitudinal fibers. Sites of active DNA replication is S-PCC were indicated to be organized a single longitudinal fibers. Following replication of a chromosome segment, a rapid reorganization from the extended longitudinal fiber to packed looping fibers occurs. The postreplication maturation process appears to include the assembly of a chromosome core consisting of multiple longitudinal fibers.^ The role of histone H1 phosphorylation in PCC formation was investigated by acidurea polyacrylamide gel electrophoresis of total histone extracted from metaphase chromosomes and PCC following high frequency fusion. This investigation failed to demonstrate an extensive phosphorylation of H1 associated with PCC formation. However, significant dephosphorylation of superphosphorylated metaphase chromosome H1 was observed, indicating that interphase H1-phosphatase activity is dominant over metaphase H1 kinase activity. These observations provide evidence against models suggesting a role for H1 superphosphorylation in triggering mitotic condensation of chromosomes. ^