Molecular cytogenetics and allotetraploidy in the red vizcacha rat, Tympanoctomys barrerae (Rodentia, Octodontidae)

The theoretical impossibility of polyploidy in mammals was overturned by the discovery of tetraploidy in the red vizcacha rat, Tympanoctomys barrerae (2n = 102). As a consequence of genome duplication, remarkably increased cell dimensions are observed in the spermatozoa and in different somatic cell lines of this species. Locus duplication had been previously demonstrated by in situ PCR and Southern blot analysis of single-copy genes. Here, we corroborate duplication of loci in multiple-copy (major rDNAs) and single-copy (Hoxc8) genes by fluorescence in situ hybridization. We also demonstrate that nucleolar dominance, a large-scale epigenetic silencing phenomenon characteristic of allopolyploids, explains the presence of only one Ag-NOR chromosome pair in T. barrerae. Nucleolar dominance, together with the chromosomal heteromorphism detected in the G-banding pattern and synaptonemal complexes of the species' diploid-like meiosis, consistently indicates allotetraploidy. Allotetraploidization can coherently explain the peculiarities of gene silencing, cell dimensions, and karyotypic features of T. barrerae that remain unexplained by assuming diploidy and a large genome size attained by the dispersion of repetitive sequences.     

Whole-genome duplications in South American desert rodents (Octodontidae)

The discovery of tetraploidy in the red viscacha rat, Tympanoctomys barrerae (4 n  = 102) has emphasized the evolutionary role of genome duplication in mammals. The tetraploid status of this species is corroborated here by in situ PCR and Southern blot analysis of a single-copy gene. The species meiotic configuration strongly suggests a hybrid derivation. To investigate the origin of T. barrerae further, the recently described Pipanacoctomys aureus was studied. This 92-chromosome species also has a duplicated genome size, redundant gene copy number and diploid-like meiotic pairing, consistent with an event of allotetraploidization. Phylogenetic analysis of mitochondrial sequences indicates sister-group relationships between these two tetraploid rodents. The new karyotypic data and the phylogenetic relationships suggest the participation of the ancestral lineages of Octomys mimax in the genesis of P. aureus . The high overall DNA similarity and shared band homology revealed by genomic Southern hybridization as well as matching chromosome numbers between O. mimax and the descendant tetraploid species support the notion of introgressive hybridization between these taxa.  © 2004 The Linnean Society of London, Biological Journal of the Linnean Society , 2004, 82 , 443–451.     

Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss

A colony color assay that measures chromosome stability is described and is used to study several parameters affecting the mitotic maintenance of yeast chromosomes, including ARS function, CEN function, and chromosome size. A cloned ochre-suppressing form of a tRNA gene, SUP11, serves as a marker on natural and in vitro-constructed chromosomes. In diploid strains homozygous for an ochre mutation in ade2, cells carrying no copies of the SUP11 gene are red, those carrying one copy are pink, and those carrying two or more copies are white. Thus, the degree of red sectoring in colonies reflects the frequency of mitotic chromosome loss. The assay also distinguishes between chromosome loss (1:0 segregation) and nondisjunction (2:0 segregation). The most dramatic effect on improving mitotic stability is caused by increasing chromosome size. Circular chromosomes increase in stability through a size range up to approximately 100 kb, but do not continue to be stabilized above this value. However, linear chromosomes continue to increase in mitotic stability throughout the size range tested (up to 137 kb). It is possible that the mitotic stability of linear chromosomes is proportional to chromosome length, up to a plateau value that has not yet been reached in our synthetic constructions.     

Sex chromosomes and origin of males and sex mosaics of the parthenogenetic stick insect Carausius morosus Br.

The chromosome complements of sporadic males and masculinized females of the thelytokous phasmid Carausius morosus Br. could be analysed in spermatogonia and ovarian follicle cells. Masculinized females with ovaries, ovotestes or testes have the female chromosome number, i.e., 61 autosomes and three sex chromosomes. The sex chromosomes, one being longer than the other two, are metacentric. In one masculinized female with testes the three sex chromosomes were different, apparently through a reciprocal translocation. The masculinized females are considered to be intersexes (phenotypic sex determination). The chromosome complement of males differs from that of females by lacking either one of the sex chromosomes or only a segment of one of these chromosomes (genotypic sex determination). The deleted sex chromosomes appear as acrocentrics and may have arisen through a chiasma between a translocated segment in one sex chromosome and its untransposed homologous region in another sex chromosome. One apparently telocentric sex chromosome may have originated from centric fission together with loss of the other arm. The sex chromosomes are positively heteropycntoic in the psermatogonia, also in those of masculinized females. En bloc heterochromatinization of the sex chromosomes, which seems to be under the direct or indirect control of one or more sites on the sex chromosomes themselves, functions in sex determination. The sex determination does not give a decisive answer to the question whether di-, tri-, or tetraploidy is involved.     

Chromosomal gene movements reflect the recent origin and biology of therian sex chromosomes

Mammalian sex chromosomes stem from ancestral autosomes and have substantially differentiated. It was shown that X-linked genes have generated duplicate intronless gene copies (retrogenes) on autosomes due to this differentiation. However, the precise driving forces for this out-of-X gene “movement” and its evolutionary onset are not known. Based on expression analyses of male germ-cell populations, we here substantiate and extend the hypothesis that autosomal retrogenes functionally compensate for the silencing of their X-linked housekeeping parental genes during, but also after, male meiotic sex chromosome inactivation (MSCI). Thus, sexually antagonistic forces have not played a major role for the selective fixation of X-derived gene copies in mammals. Our dating analyses reveal that although retrogenes were produced ever since the common mammalian ancestor, selectively driven retrogene export from the X only started later, on the placental mammal (eutherian) and marsupial (metatherian) lineages, respectively. Together, these observations suggest that chromosome-wide MSCI emerged close to the eutherian–marsupial split approximately 180 million years ago. Given that MSCI probably reflects the spread of the recombination barrier between the X and Y, crucial for their differentiation, our data imply that these chromosomes became more widely differentiated only late in the therian ancestor, well after the divergence of the monotreme lineage. Thus, our study also provides strong independent support for the recent notion that our sex chromosomes emerged, not in the common ancestor of all mammals, but rather in the therian ancestor, and therefore are much younger than previously thought.     

A stably transmitted pair of translocated supernumerary chromosomes in maize.

A pair of stably transmitted supernumerary chromosomes of unknown source has been found in a maize stock carrying a desynaptic mutant. The presence of the supernumerary chromosome appears to be unrelated to the meiotic mutant, but is believed to have been derived from a translocated B chromosome contaminant. The supernumerary chromosomes carry a segment of a A chromosome in this stock where there appear to be two normal copies of each of the 10 A chromosomes. Thus, this A chromosome segment is present in quadruplicate. Surprisingly, a quadrivalent configuration is formed in most microsporocytes, which involves not only synapsis but also chiasma formation in the A chromosome segments involved in the quadrivalent. This represents a strong preferential pairing of supernumeraries with the normal A chromosome segments. Such nonrandom association and crossing over might provide information on the nature of early homologue alignment at meiosis.     

Relationships between vertebrate ZW and XY sex chromosome systems

The peculiar cytology and unique evolution of sex chromosomes raise many fundamental questions. Why and how sex chromosomes evolved has been debated over a century since H.J. Muller suggested that sex chromosome pairs evolved ultimately from a pair of autosomes. This theory was adapted to explain variations in the snake ZW chromosome pair and later the mammal XY. S. Ohno pointed out similarities between the mammal X and the bird/reptile Z chromosomes forty years ago, but his speculation that they had a common evolutionary origin, or at least evolved from similar regions of the genome, has been undermined by comparative gene mapping, and it is accepted that mammal XY and reptile ZW systems evolved independently from a common ancestor. Here we review evidence for the alternative theory, that ZW<-->XY transitions occurred during evolution, citing examples from fish and amphibians, and probably reptiles. We discuss new work from comparative genomics and cytogenetics that leads to a reconsideration of Ohno's idea and advance a new hypothesis that the mammal XY system may have arisen directly from an ancient reptile ZW system.     

A new look at the evolution of avian sex chromosomes

Birds have a ubiquitous, female heterogametic, ZW sex chromosome system. The current model suggests that the Z chromosome and its degraded partner, the W chromosome, evolved from an ancestral pair of autosomes independently from the mammalian XY male heteromorphic sex chromosomes--which are similar in size, but not gene content (Graves, 1995; Fridolfsson et al., 1998). Furthermore the degradation of the W has been proposed to be progressive, with the basal clade of birds (the ratites) possessing virtually homomorphic sex chromosomes and the more recently derived birds (the carinates) possessing highly heteromorphic sex chromosomes (Ohno, 1967; Solari, 1993). Recent findings have suggested an alternative to independent evolution of bird and mammal chromosomes, in which an XY system took over directly from an ancestral ZW system. Here we examine recent research into avian sex chromosomes and offer alternative suggestions as to their evolution.     

Different origins of bird and reptile sex chromosomes inferred from comparative mapping of chicken Z-linked genes

Recent progress of chicken genome projects has revealed that bird ZW and mammalian XY sex chromosomes were derived from different autosomal pairs of the common ancestor; however, the evolutionary relationship between bird and reptilian sex chromosomes is still unclear. The Chinese soft-shelled turtle (Pelodiscus sinensis) exhibits genetic sex determination, but no distinguishable (heteromorphic) sex chromosomes have been identified. In order to investigate this further, we performed molecular cytogenetic analyses of this species, and thereby identified ZZ/ZW-type micro-sex chromosomes. In addition, we cloned reptile homologues of chicken Z-linked genes from three reptilian species, the Chinese soft-shelled turtle and the Japanese four-striped rat snake (Elaphe quadrivirgata), which have heteromorphic sex chromosomes, and the Siam crocodile (Crocodylus siamensis), which exhibits temperature-dependent sex determination and lacks sex chromosomes. We then mapped them to chromosomes of each species using FISH. The linkage of the genes has been highly conserved in all species: the chicken Z chromosome corresponded to the turtle chromosome 6q, snake chromosome 2p and crocodile chromosome 3. The order of the genes was identical among the three species. The absence of homology between the bird Z chromosome and the snake and turtle Z sex chromosomes suggests that the origin of the sex chromosomes and the causative genes of sex determination are different between birds and reptiles.     

A desert octodontid rodent,Tympanoctomys barrerae,uses modified hairs for stripping epidermal tissue from leaves of halophytic plants

Desert rodents that consume halophytic plants must have adaptations for coping with the high salt content of the leaves. A kidney capable of excreting very concentrated urine is one method. Another is removal of the hypersaline epidermis by means of chisel-like incisors prior to ingestion of the leaves. Tympanoctomys barrerae has evolved a unique refinement of the latter adaptation. It possesses two bundles of stiffened hairs on either side of the palate just caudal to the incisors. The bundles vibrate against the lower incisors, removing the epidermis from the leaves. The efficiency of the operation is significantly greater than with the use of incisors alone. Such a device has not been described in any other mammal. The facial muscles associated with the lips, the cheek vibrissae, and the oral cavity are described in T. barrerae and the nonhalophilic octodontid Octomys mimax. M. buccinatorius pars intermaxillaris is the only muscle in direct contact with the bristle bundles. Other anatomical features found in T. barrerae that may be associated with this feeding device are: 1) a much enlarged and mobile lower labial pad operated by Mm. buccinatorius pars orbicularis oris, pars longitudinalis profunda, and mandibularis cranialis profunda; 2) two oral glands not described in other rodents; and 3) a shortened tongue. Although, taken as a whole, this epidermal stripping device is unique to T. barrerae, most of its features have evolved by modification of structures present in the facial region of more generalized rodents.     

The number of alleles in multigene families

The probability distribution and moments of the number of alleles present in a sample of homologous chromosomes are studied. It is assumed that there are multiple copies of the gene on each chromosome. When there are only two copies per chromosome or when there are only two or three chromosomes, it is possible to use analytic methods to tackle the problem. Otherwise, a simulation method is suggested.     

Multiple and independent cessation of recombination between avian sex chromosomes

Birds are characterized by female heterogamety; females carry the Z and W sex chromosomes, while males have two copies of the Z chromosome. We suggest here that full differentiation of the Z and W sex chromosomes of birds did not take place until after the split of major contemporary lineages, in the late Cretaceous. The ATP synthase alpha-subunit gene is now present in one copy each on the nonrecombining part of the W chromosome (ATP5A1W) and on the Z chromosome (ATP5A1Z). This gene seems to have evolved on several independent occasions, in different lineages, from a state of free recombination into two sex-specific and nonrecombining variants. ATP5A1W and ATP5A1Z are thus more similar within orders, relative to what W (or Z) are between orders. Moreover, this cessation of recombination apparently took place at different times in different lineages (estimated at 13, 40, and 65 million years ago in Ciconiiformes, Galliformes, and Anseriformes, respectively). We argue that these observations are the result of recent and traceable steps in the process where sex chromosomes gradually cease to recombine and become differentiated. Our data demonstrate that this process, once initiated, may occur independently in parallel in sister lineages.     

Contrasting levels of nucleotide diversity on the avian Z and W sex chromosomes

Sex chromosomes may provide a context for studying the local effects of mutation rate on molecular evolution, since the two types of sex chromosomes are generally exposed to different mutational environments in male and female germ lines. Importantly, recent studies of some vertebrates have provided evidence for a higher mutation rate among males than among females. Thus, in birds, the Z chromosome, which spends two thirds of its time in the male germ line, is exposed to more mutations than the female-specific W chromosome. We show here that levels of nucleotide diversity are drastically higher on the avian Z chromosome than in paralogous sequences on the W chromosome. In fact, no intraspecific polymorphism whatsoever was seen in about 3.4 kb of CHD1W intron sequence from a total of >150 W chromosome copies of seven different bird species. In contrast, the amount of genetic variability in paralogous sequences on the Z chromosome was significant, with an average pairwise nucleotide diversity (d) of 0.0020 between CHD1Z introns and with 37 segregating sites in a total of 3.8 kb of Z sequence. The contrasting levels of genetic variability on the avian sex chromosomes are thus in a direction predicted from a male-biased mutation rate. However, although a low gene number, as well as some other factors, argues against background selection and/or selective sweeps shaping the genetic variability of the avian W chromosome, we cannot completely exclude selection as a contributor to the low levels of variation on the W chromosome.     

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.     

Gradual and quantum genome size shifts in the hystricognath rodents

We assessed genome size variation by flow cytometry within and among 31 species of nine families of African and South American hystricognath rodents. Interspecific variation was extensive and genome size was relatively high among the South American radiation whereas only moderate variation and smaller estimates of genome size were observed in the African counterparts. The largest genome size, indicating tetraploidy was recorded in the South American octodontid, Tympanoctomys barrerae (16.8 pg DNA). This quantum shift in DNA content represents a novel mechanism of genome evolution in mammals. As expected in polyploid organisms, varying nucleotypic effects were observed in the dimensions of the sperm cells and lymphocytes of T. barrerae. The role of control mechanisms that influence cell dimensions in polyploid organisms is discussed.     

Origin and spread of the SRY gene on the X and Y chromosomes of the rodent Microtus cabrerae: role of L1 elements

In the rodent species Microtus cabrerae, males as well as females present several copies of the SRY gene, a single-copy gene located on the Y chromosome in most mammals. Using different PCR approaches, we have characterized the sequence, structure, and organization of the SRY copies and their flanking regions distributed on the X and Y chromosomes of this species. All copies of SRY analyzed, including those from the Y chromosome, proved to be nonfunctional pseudogenes, as they have internal stop codons. In addition, we demonstrated the association of SRY pseudogenes with different fragments of L1 and LTR retroelements in both sex chromosomes of M. cabrerae. Examining the possible origin of SRY pseudogene and retroposons association, we propose that retroposons could have been involved in the mechanism of SRY gene amplification on the Y chromosome and in the transference of the Y-linked SRY copies to the X-chromosome heterochromatin.     

Novel gene acquisition on carnivore Y chromosomes

Despite its importance in harboring genes critical for spermatogenesis and male-specific functions, the Y chromosome has been largely excluded as a priority in recent mammalian genome sequencing projects. Only the human and chimpanzee Y chromosomes have been well characterized at the sequence level. This is primarily due to the presumed low overall gene content and highly repetitive nature of the Y chromosome and the ensuing difficulties using a shotgun sequence approach for assembly. Here we used direct cDNA selection to isolate and evaluate the extent of novel Y chromosome gene acquisition in the genome of the domestic cat, a species from a different mammalian superorder than human, chimpanzee, and mouse (currently being sequenced). We discovered four novel Y chromosome genes that do not have functional copies in the finished human male-specific region of the Y or on other mammalian Y chromosomes explored thus far. Two genes are derived from putative autosomal progenitors, and the other two have X chromosome homologs from different evolutionary strata. All four genes were shown to be multicopy and expressed predominantly or exclusively in testes, suggesting that their duplication and specialization for testis function were selected for because they enhance spermatogenesis. Two of these genes have testis-expressed, Y-borne copies in the dog genome as well. The absence of the four newly described genes on other characterized mammalian Y chromosomes demonstrates the gene novelty on this chromosome between mammalian orders, suggesting it harbors many lineage-specific genes that may go undetected by traditional comparative genomic approaches. Specific plans to identify the male-specific genes encoded in the Y chromosome of mammals should be a priority.     

Cytogenetic mapping of the W chromosome in the genus Leporinus (Teleostei, Anostomidae) using a highly repetitive DNA sequence

The distribution of the Leporinus elongatus LeSpeI repetitive sequence in other Leporinus species was studied in an attempt to elucidate the evolutionary history of sex chromosomes in this genus using chromosome fluorescence in situ hybridization. The presence of fluorescent signals only in species that have differentiated sex chromosomes suggests that this sequence is related to the differentiation of sex chromosomes in this genus. Thus, these data will contribute to a better understanding of chromosome evolution, especially for sex chromosomes, in the Leporinus genus.     

Differences in recombination frequencies during female and male meioses of the sex chromosomes of the medaka, Oryzias latipes

In the medaka, Oryzias latipes, sex is determined chromosomally. The sex chromosomes differ from those of mammals in that the X and Y chromosomes are highly homologous. Using backcross panels for linkage analysis, we mapped 21 sequence tagged site (STS) markers on the sex chromosomes (linkage group 1). The genetic map of the sex chromosome was established using male and female meioses. The genetic length of the sex chromosome was shorter in male than in female meioses. The region where male recombination is suppressed is the region close to the sex-determining gene y, while female recombination was suppressed in both the telomeric regions. The restriction in recombination does not occur uniformly on the sex chromosome, as the genetic map distances of the markers are not proportional in male and female recombination. Thus, this observation seems to support the hypothesis that the heterogeneous sex chromosomes were derived from suppression of recombination between autosomal chromosomes. In two of the markers, Yc-2 and Casp6, which were expressed sequence-tagged (EST) sites, polymorphisms of both X and Y chromosomes were detected. The alleles of the X and Y chromosomes were also detected in O. curvinotus, a species related to the medaka. These markers could be used for genotyping the sex chromosomes in the medaka and other species, and could be used in other studies on sex chromosomes.     

MreB is important for cell shape but not for chromosome segregation of the filamentous cyanobacterium Anabaena sp. PCC 7120

MreB is a bacterial actin that plays important roles in determination of cell shape and chromosome partitioning in Escherichia coli and Caulobacter crescentus. In this study, the mreB from the filamentous cyanobacterium Anabaena sp. PCC 7120 was inactivated. Although the mreB null mutant showed a drastic change in cell shape, its growth rate, cell division and the filament length were unaltered. Thus, MreB in Anabaena maintains cell shape but is not required for chromosome partitioning. The wild type and the mutant had eight and 10 copies of chromosomes per cell respectively. We demonstrated that DNA content in two daughter cells after cell division in both strains was not always identical. The ratios of DNA content in two daughter cells had a Gaussian distribution with a standard deviation much larger than a value expected if the DNA content in two daughter cells were identical, suggesting that chromosome partitioning is a random process. The multiple copies of chromosomes in cyanobacteria are likely required for chromosome random partitioning in cell division.