Genome-wide sequence divergence of satellite DNA could underlie meiotic failure in male hybrids of bighead catfish and North African catfish (Clarias, Clariidae).

Hybrid sterility, a hallmark of postzygotic isolation, arises from parental genome divergence disrupting meiosis. While chromosomal incompatibility is often implicated, the underlying mechanisms remain unclear. This study investigated meiotic behavior and genome-wide divergence in bighead catfish (C. macrocephalus), North African catfish (C. gariepinus), and their sterile male hybrids (important in aquaculture). Repetitive DNA analysis using bioinformatics and cytogenetics revealed significant divergence in satellite DNA (satDNA) families between parental species. Notably, one hybrid exhibited successful meiosis and spermatozoa production, suggesting potential variation in sterility expression. Our findings suggest that genome-wide satDNA divergence, rather than chromosome number differences, likely contributes to meiotic failure and male sterility in these catfish hybrids.

Spaghetti connections: synaptonemal complexes as a tool to explore chromosome structure, evolution, and meiotic behavior in fish.

Backgound The synaptonemal complex (SC) is a protein axis formed along chromosomes during meiotic prophase to ensure proper pairing and crossing over. SC analysis has been widely used to study the chromosomes of mammals, and less frequently of birds, reptiles, and fish. It is a promising method to investigate the evolution of fish genomes and chromosomes as a part of complex approach. Summary Compared with conventional metaphase chromosomes, pachytene chromosomes are less condensed and exhibit pairing between homologous chromosomes. These features of SCs facilitate the study of the small chromosomes that are typical in fish. Moreover, it allows the study of heteromorphisms in sex chromosomes and supernumerary chromosomes. In addition, it enables the investigation of the pairing between orthologous chromosomes in hybrids, which is crucial for uncovering the causes of hybrid sterility and asexual reproduction, such as gynogenesis or hybridogenesis. However, the application of SC analysis to fish chromosomes is limited by the associated complications. First, in most fish, meiosis does not occur during every season and life stage. Second, different SC preparation methods are optimal for different fish species. Third, commercial antibodies targeting meiotic proteins have been primarily developed against mammalian antigens, and not all of them are suitable for fish chromosomes. Key messages In the present review, we provide an overview of the methods for preparing fish SCs and highlight important studies using SC analysis in fish. This study will be valuable for planning and designing research that applies SC analysis to fish cytogenetics and genomics.

Hi-C sequencing unravels dynamic three-dimensional chromatin interactions in muntjac lineage: insights from chromosome fusions in Fea's muntjac genome.

Eukaryotes have varying numbers and structures of characteristic chromosomes across lineages or species. The evolutionary trajectory of species may have been affected by spontaneous genome rearrangements. Chromosome fusion drastically alters karyotypes. However, the mechanisms and consequences of chromosome fusions, particularly in muntjac species, are poorly understood. Recent research-based advancements in three-dimensional (3D) genomics, particularly high-throughput chromatin conformation capture (Hi-C) sequencing, have allowed for the identification of chromosome fusions and provided mechanistic insights into three muntjac species: Muntiacus muntjak, M. reevesi, and M. crinifrons. This study aimed to uncover potential genome rearrangement patterns in the threatened species Fea's muntjac (Muntiacus feae), which have not been previously examined for such characteristics. Deep Hi-C sequencing (31.42 × coverage) was performed to reveal the 3D chromatin architecture of the Fea's muntjac genome. Patterns of repeated chromosome fusions that were potentially mediated by high-abundance transposable elements were identified. Comparative Hi-C maps demonstrated linkage homology between the sex chromosomes in Fea's muntjac and autosomes in M. reevesi, indicating that fusions may have played a crucial role in the evolution of the sex chromosomes of the lineage. The species-level dynamics of topologically associated domains (TADs) suggest that TAD organization could be altered by differential chromosome interactions owing to repeated chromosome fusions. However, research on the effect of TADs on muntjac genome evolution is insufficient. This study generated Hi-C data for the Fea's muntjac, providing a genomic resource for future investigations of the evolutionary patterns of chromatin conformation at the chromosomal level.

MicrosatNavigator: exploring nonrandom distribution and lineage-specificity of microsatellite repeat motifs on vertebrate sex chromosomes across 186 whole genomes.

Microsatellites are short tandem DNA repeats, ubiquitous in genomes. They are believed to be under selection pressure, considering their high distribution and abundance beyond chance or random accumulation. However, limited analysis of microsatellites in single taxonomic groups makes it challenging to understand their evolutionary significance across taxonomic boundaries. Despite abundant genomic information, microsatellites have been studied in limited contexts and within a few species, warranting an unbiased examination of their genome-wide distribution in distinct versus closely related-clades. Large-scale comparisons have revealed relevant trends, especially in vertebrates. Here, "MicrosatNavigator", a new tool that allows quick and reliable investigation of perfect microsatellites in DNA sequences, was developed. This tool can identify microsatellites across the entire genome sequences. Using this tool, microsatellite repeat motifs were identified in the genome sequences of 186 vertebrates. A significant positive correlation was noted between the abundance, density, length, and GC bias of microsatellites and specific lineages. The (AC)n motif is the most prevalent in vertebrate genomes, showing distinct patterns in closely related species. Longer microsatellites were observed on sex chromosomes in birds and mammals but not on autosomes. Microsatellites on sex chromosomes of non-fish vertebrates have the lowest GC content, whereas high-GC microsatellites (≥ 50 M% GC) are preferred in bony and cartilaginous fishes. Thus, similar selective forces and mutational processes may constrain GC-rich microsatellites to different clades. These findings should facilitate investigations into the roles of microsatellites in sex chromosome differentiation and provide candidate microsatellites for functional analysis across the vertebrate evolutionary spectrum.

Small but Mighty: Genetic Diversity of the Thai Ridgeback Dog Population.

Originating in Thailand, the Thai Ridgeback dog is known for its unique fur ridge that grows in the opposite direction along its back. Selective breeding and a limited populations in Thailand have led to significant close inbreeding among related individuals. The current Thai Ridgeback population is assumed to have experienced a loss of genetic diversity and bottleneck events. Furthermore, studies on the genetic diversity and structure of Thai Ridgeback dogs are limited. Therefore, the aim of this study was to assess the genetic diversity in Thai Ridgeback dogs. Microsatellite genotyping and mitochondrial DNA D-loop sequences were used to assess genetic diversity in 105 Thai Ridgeback dogs from various farms throughout Thailand. Significant genetic diversity and minimal inbreeding were observed in the current Thai Ridgeback population. Signs of bottlenecks were not observed because the exchange of genetic material among Thai Ridgeback owners effectively preserved the genetic diversity. Moreover, the genetic parameters in this study supported owner-to-owner exchanges animals for mating programs. To sustain the genetic diversity of Thai Ridgeback dogs, the use of genetic parameters to manage genetic closeness while preserving breed characteristics is essential. These data are crucial for ensuring demographic stability, which is pivotal for long-term conservation and effective population management.

Importance of Thai macaque bioresources for biological research and human health.

During the past century, macaque bioresources have provided remarkable scientific and biomedical discoveries related to the understanding of human physiology, neuroanatomy, reproduction, development, cognition, and pathology. Considerable progress has been made, and an urgent need has arisen to develop infrastructure and viable settings to meet the current global demand in research models during the so-called new normal after COVID-19 era. This review highlights the critical need for macaque bioresources and proposes the establishment of a designated primate research center to integrate research in primate laboratories for the rescue and rehabilitation of wild macaques. Key areas where macaque models have been and continue to be essential for advancing fundamental knowledge in biomedical and biological research are outlined. Detailed genetic studies on macaque bioresources of Thai origin can further facilitate the rapid pace of vaccine discovery.

Genome-wide SNP analysis of Siamese cobra (Naja kaouthia) reveals the molecular basis of transitions between Z and W sex chromosomes and supports the presence of an ancestral super-sex chromosome in amniotes.

Elucidation of the process of sex chromosome differentiation is necessary to understand the dynamics of evolutionary mechanisms in organisms. The W sex chromosome of the Siamese cobra (Naja kaouthia) contains a large number of repeats and shares amniote sex chromosomal linkages. Diversity Arrays Technology provides an effective approach to identify sex-specific loci that are epoch-making, to understand the dynamics of molecular transitions between the Z and W sex chromosomes in a snake lineage. From a total of 543 sex-specific loci, 90 showed partial homology with sex chromosomes of several amniotes and 89 loci were homologous to transposable elements. Two loci were confirmed as W-specific nucleotides after PCR amplification. These loci might result from a sex chromosome differentiation process and involve putative sex-determination regions in the Siamese cobra. Sex-specific loci shared linkage homologies among amniote sex chromosomes, supporting an ancestral super-sex chromosome.

Do sex chromosomes of snakes, monitor lizards, and iguanian lizards result from multiple fission of an "ancestral amniote super-sex chromosome"?

Sex chromosomes in some amniotes share linkage homologies with distantly related taxa in regions orthologous to squamate reptile chromosome 2 (SR2) and the snake W sex chromosome. Thus, the SR2 and W chromosomes may formerly have been part of a larger ancestral amniote super-sex chromosome. Comparison of various sex chromosomal linkage homologies in Toxicofera with those in other amniotes offers an excellent model to assess key cytological differences, to understand the mechanisms of amniote sex chromosome evolution in each lineage and the existence of an ancestral amniote super-sex chromosome. Chromosome maps of four species of Toxicofera were constructed using bacterial artificial chromosomes (BACs) derived from chicken and zebra finch libraries containing amniote sex chromosomal linkages. Different macrochromosome linkage homologies were highly conserved among Toxicofera, and at least two BACs (CH261-125F1 and CH261-40D6) showed partial homology with sex chromosomes of amniotes associated with SR2, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial linkage homologies. The present data also suggest a possible multiple fission mechanism of an ancestral super-sex chromosome, which resulted in further development of various sex chromosomal linkages of Toxicofera based on particular properties that favored the role of sex chromosomes.

Characterization of centromeric satellite DNAs (MALREP) in the Asian swamp eel (Monopterus albus) suggests the possible origin of repeats from transposable elements.

Centromeric satellite DNA (cen-satDNA) sequences of the Asian swamp eel (Monopterus albus) were characterized. Three GC-rich cen-satDNA sequences were detected as a 233 bp MALREP-A and a 293 bp MALREP-B localized to all chromosomes, and a 293 bp MALREP-C distributed on eight chromosome pairs. Sequence lengths of MALREP-B and MALREP-C were 60 bp larger than that of MALREP-A, showing partial homology with core sequences (233 bp). Size differences between MALREP-A and MALREP-B/C suggest the possible occurrence of two satDNA families. The presence of an additional 60 bp in MALREP-B/C resulted from an ancient dimer of 233 bp monomers and subsequent mutation and homogenization between the two monomers. All MALREPs showed partial homology with transposable elements (TEs), suggesting that the MALREPs originated from the TEs. The MALREPs might have been acquired in the Asian swamp eel, thereby promoting fixation in the species.

Chromosome map of the Siamese cobra: did partial synteny of sex chromosomes in the amniote represent "a hypothetical ancestral super-sex chromosome" or random distribution?

BACKGROUND: Unlike the chromosome constitution of most snakes (2n=36), the cobra karyotype shows a diploid chromosome number of 38 with a highly heterochromatic W chromosome and a large morphologically different chromosome 2. To investigate the process of sex chromosome differentiation and evolution between cobras, most snakes, and other amniotes, we constructed a chromosome map of the Siamese cobra (Naja kaouthia) with 43 bacterial artificial chromosomes (BACs) derived from the chicken and zebra finch libraries using the fluorescence in situ hybridization (FISH) technique, and compared it with those of the chicken, the zebra finch, and other amniotes. RESULTS: We produced a detailed chromosome map of the Siamese cobra genome, focusing on chromosome 2 and sex chromosomes. Synteny of the Siamese cobra chromosome 2 (NKA2) and NKAZ were highly conserved among snakes and other squamate reptiles, except for intrachromosomal rearrangements occurring in NKA2. Interestingly, twelve BACs that had partial homology with sex chromosomes of several amniotes were mapped on the heterochromatic NKAW as hybridization signals such as repeat sequences. Sequence analysis showed that most of these BACs contained high proportions of transposable elements. In addition, hybridization signals of telomeric repeat (TTAGGG)n and six microsatellite repeat motifs ((AAGG)8, (AGAT)8, (AAAC)8, (ACAG)8, (AATC)8, and (AAAAT)6) were observed on NKAW, and most of these were also found on other amniote sex chromosomes. CONCLUSIONS: The frequent amplification of repeats might involve heterochromatinization and promote sex chromosome differentiation in the Siamese cobra W sex chromosome. Repeat sequences are also shared among amniote sex chromosomes, which supports the hypothesis of an ancestral super-sex chromosome with overlaps of partial syntenies. Alternatively, amplification of microsatellite repeat motifs could have occurred independently in each lineage, representing convergent sex chromosomal differentiation among amniote sex chromosomes.

Lack of satellite DNA species-specific homogenization and relationship to chromosomal rearrangements in monitor lizards (Varanidae, Squamata).

BACKGROUND: Satellite DNAs (stDNAs) are highly repeated sequences that constitute large portions of any genome. The evolutionary dynamics of stDNA (e.g. copy number, nucleotide sequence, location) can, therefore, provide an insight into genome organization and evolution. We investigated the evolutionary origin of VSAREP stDNA in 17 monitor lizards (seven Asian, five Australian, and five African) at molecular and cytogenetic level. RESULTS: Results revealed that VSAREP is conserved in the genome of Asian and Australian varanids, but not in African varanids, suggesting that these sequences are either differentiated or lost in the African varanids. Phylogenetic and arrangement network analyses revealed the existence of at least four VSAREP subfamilies. The similarity of each sequence unit within the same VSAREP subfamily from different species was higher than those of other VSAREP subfamilies belonging to the same species. Additionally, all VSAREP subfamilies isolated from the three Australian species (Varanus rosenbergi, V. gouldii, and V. acanthurus) were co-localized near the centromeric or pericentromeric regions of the macrochromosomes, except for chromosomes 3 and 4 in each Australian varanid. However, their chromosomal arrangements were different among species. CONCLUSIONS: The VSAREP stDNA family lack homogenized species-specific nucleotide positions in varanid lineage. Most VSAREP sequences were shared among varanids within the four VSAREP subfamilies. This suggests that nucleotide substitutions in each varanid species accumulated more slowly than homogenization rates in each VSAREP subfamily, resulting in non-species-specific evolution of stDNA profiles. Moreover, changes in location of VSAREP stDNA in each Australian varanid suggests a correlation with chromosomal rearrangements, leading to karyotypic differences among these species.

Origin of Amniote Sex Chromosomes: An Ancestral Super-Sex Chromosome, or Common Requirements?

The diversity of sex chromosomes among amniotes is the product of independent evolution of different systems in different lineages, defined by novel sex-determining genes. Convergent evolution is very common, suggesting that some genes are particularly adept at taking on a sex-determining role. Comparative gene mapping, and more recently whole genome sequencing, have now turned up other surprising relationships; different regions of the amniote genome that have become sex determining in some taxa seem to share synteny, or share sequence, in others. Is this, after all, evidence that these regions were once linked in a super-sex chromosome that underwent multiple fission in different ways in different amniote lineages? Or does it signify that special properties of sex chromosomes (paucity of active genes, low recombination, epigenetic regulation to achieve dosage compensation) predispose particular chromosomes to a sex-determining role?

Evolutionary Dynamics of the Gametologous CTNNB1 Gene on the Z and W Chromosomes of Snakes.

Snakes exhibit genotypic sex determination with female heterogamety (ZZ males and ZW females), and the state of sex chromosome differentiation also varies among lineages. To investigate the evolutionary history of homologous genes located in the nonrecombining region of differentiated sex chromosomes in snakes, partial sequences of the gametologous CTNNB1 gene were analyzed for 12 species belonging to henophid (Cylindrophiidae, Xenopeltidae, and Pythonidae) and caenophid snakes (Viperidae, Elapidae, and Colubridae). Nonsynonymous/synonymous substitution ratios (Ka/Ks) in coding sequences were low (Ka/Ks < 1) between CTNNB1Z and CTNNB1W, suggesting that these 2 genes may have similar functional properties. However, frequencies of intron sequence substitutions and insertion­deletions were higher in CTNNB1Z than CTNNB1W, suggesting that Z-linked sequences evolved faster than W-linked sequences. Molecular phylogeny based on both intron and exon sequences showed the presence of 2 major clades: 1) Z-linked sequences of Caenophidia and 2) W-linked sequences of Caenophidia clustered with Z-linked sequences of Henophidia, which suggests that the sequence divergence between CTNNB1Z and CTNNB1W in Caenophidia may have occurred by the cessation of recombination after the split from Henophidia.

Molecular cloning and characterization of satellite DNA sequences from constitutive heterochromatin of the habu snake (Protobothrops flavoviridis, Viperidae) and the Burmese python (Python bivittatus, Pythonidae).

Highly repetitive DNA sequences of the centromeric heterochromatin provide valuable molecular cytogenetic markers for the investigation of genomic compartmentalization in the macrochromosomes and microchromosomes of sauropsids. Here, the relationship between centromeric heterochromatin and karyotype evolution was examined using cloned repetitive DNA sequences from two snake species, the habu snake (Protobothrops flavoviridis, Crotalinae, Viperidae) and Burmese python (Python bivittatus, Pythonidae). Three satellite DNA (stDNA) families were isolated from the heterochromatin of these snakes: 168-bp PFL-MspI from P. flavoviridis and 196-bp PBI-DdeI and 174-bp PBI-MspI from P. bivittatus. The PFL-MspI and PBI-DdeI sequences were localized to the centromeric regions of most chromosomes in the respective species, suggesting that the two sequences were the major components of the centromeric heterochromatin in these organisms. The PBI-MspI sequence was localized to the pericentromeric region of four chromosome pairs. The PFL-MspI and the PBI-DdeI sequences were conserved only in the genome of closely related species, Gloydius blomhoffii (Crotalinae) and Python molurus, respectively, although their locations on the chromosomes were slightly different. In contrast, the PBI-MspI sequence was also in the genomes of P. molurus and Boa constrictor (Boidae), and additionally localized to the centromeric regions of eight chromosome pairs in B. constrictor, suggesting that this sequence originated in the genome of a common ancestor of Pythonidae and Boidae, approximately 86 million years ago. The three stDNA sequences showed no genomic compartmentalization between the macrochromosomes and microchromosomes, suggesting that homogenization of the centromeric and/or pericentromeric stDNA sequences occurred in the macrochromosomes and microchromosomes of these snakes.

Hypervariability of Nucleolus Organizer Regions in Bengal Slow Lorises, Nycticebus bengalensis (Primates, Lorisidae).

Slow lorises are a cryptic species complex, and thus genetic markers are needed to identify distinct evolutionary lineages or species. We examined the nucleolus organizer regions (NORs) of Bengal slow lorises (Nycticebus bengalensis) using FISH with 18S rDNA (rDNA-FISH) and silver nitrate staining (Ag-NOR stain). Ten individuals of the putatively single species N. bengalensis showed higher variability in localization than 3 other congeners, though their overall karyotypes were similar. The rDNA-FISH analysis detected a total of 18 loci, in contrast to previous studies of other slow loris species that revealed far fewer (6-10) loci. Eight of the 18 loci detected in the present analysis were found to be semi-stable localizations at 4 different chromosomes, while 10 were found to be unstable localizations at 5 other chromosomes. The semi-stable locations showed occasional presence/absence of variations for rDNA-FISH, and unstable locations were polymorphic among individuals, contributing to the higher variability of NORs in this taxon. We hypothesize that the larger numbers of rDNA loci found in N. bengalensis were introduced by genomic dispersion through ectopic recombination in association with terminal regions including rDNA. Such differences are potentially very powerful chromosomal markers to be used in species identification and conservation.

CENP-B box, a nucleotide motif involved in centromere formation, occurs in a New World monkey.

Centromere protein B (CENP-B) is one of the major proteins involved in centromere formation, binding to centromeric repetitive DNA by recognizing a 17 bp motif called the CENP-B box. Hominids (humans and great apes) carry large numbers of CENP-B boxes in alpha satellite DNA (AS, the major centromeric repetitive DNA of simian primates). Only negative results have been reported regarding the presence of the CENP-B box in other primate taxa. Consequently, it is widely believed that the CENP-B box is confined, within primates, to the hominids. We report here that the common marmoset, a New World monkey, contains an abundance of CENP-B boxes in its AS. First, in a long contig sequence we constructed and analysed, we identified the motif in 17 of the 38 alpha satellite repeat units. We then sequenced terminal regions of additional clones and found the motif in many of them. Immunostaining of marmoset cells demonstrated that CENP-B binds to DNA in the centromeric regions of chromosomes. Therefore, functional CENP-B boxes are not confined to hominids. Our results indicate that the efficiency of identification of the CENP-B box may depend largely on the sequencing methods used, and that the CENP-B box in centromeric repetitive DNA may be more common than researchers previously thought.

Molecular cloning and characterization of Siamese crocodile (Crocodylus siamensis) copper, zinc superoxide dismutase (CSI-Cu,Zn-SOD) gene.

Superoxide dismutase (SOD, EC 1.15.1.1) is an antioxidant enzyme found in all living cells. It regulates oxidative stress by breaking down superoxide radicals to oxygen and hydrogen peroxide. A gene coding for Cu,Zn-SOD was cloned and characterized from Siamese crocodile (Crocodylus siamensis; CSI). The full-length expressed sequence tag (EST) of this Cu,Zn-SOD gene (designated as CSI-Cu,Zn-SOD) contained 462bp encoding a protein of 154 amino acids without signal peptides, indicated as intracellular CSI-Cu,Zn-SOD. This agreed with the results from the phylogenetic tree, which indicated that CSI-Cu,Zn-SOD belonged to the intracellular Cu,Zn-SOD. Chromosomal location determined that the CSI-Cu,Zn-SOD was localized to the proximal region of the Siamese crocodile chromosome 1p. Several highly conserved motifs, two conserved signature sequences (GFHVHEFGDNT and GNAGGRLACGVI), and conserved amino acid residues for binding copper and zinc (His(47), His(49), His(64), His(72), His(81), Asp(84), and His(120)) were also identified in CSI-Cu,Zn-SOD. Real-time PCR analysis showed that CSI-Cu,Zn-SOD mRNA was expressed in all the tissues examined (liver, pancreas, lung, kidney, heart, and whole blood), which suggests a constitutively expressed gene in these tissues. Expression of the gene in Escherichia coli cells followed by purification yielded a recombinant CSI-Cu,Zn-SOD, with Km and Vmax values of 6.075mM xanthine and 1.4×10(-3)mmolmin(-1)mg(-1), respectively. This Vmax value was 40 times lower than native Cu,Zn-SOD (56×10(-3)mmolmin(-1)mg(-1)), extracted from crocodile erythrocytes. This suggests that cofactors, protein folding properties, or post-translational modifications were lost during the protein purification process, leading to a reduction in the rate of enzyme activity in bacterial expression of CSI-Cu,Zn-SOD.

Identification of the linkage group of the Z sex chromosomes of the sand lizard (Lacerta agilis, Lacertidae) and elucidation of karyotype evolution in lacertid lizards.

The sand lizard (Lacerta agilis, Lacertidae) has a chromosome number of 2n = 38, with 17 pairs of acrocentric chromosomes, one pair of microchromosomes, a large acrocentric Z chromosome, and a micro-W chromosome. To investigate the process of karyotype evolution in L. agilis, we performed chromosome banding and fluorescent in situ hybridization for gene mapping and constructed a cytogenetic map with 86 functional genes. Chromosome banding revealed that the Z chromosome is the fifth largest chromosome. The cytogenetic map revealed homology of the L. agilis Z chromosome with chicken chromosomes 6 and 9. Comparison of the L. agilis cytogenetic map with those of four Toxicofera species with many microchromosomes (Elaphe quadrivirgata, Varanus salvator macromaculatus, Leiolepis reevesii rubritaeniata, and Anolis carolinensis) showed highly conserved linkage homology of L. agilis chromosomes (LAG) 1, 2, 3, 4, 5(Z), 7, 8, 9, and 10 with macrochromosomes and/or macrochromosome segments of the four Toxicofera species. Most of the genes located on the microchromosomes of Toxicofera were localized to LAG6, small acrocentric chromosomes (LAG11-18), and a microchromosome (LAG19) in L. agilis. These results suggest that the L. agilis karyotype resulted from frequent fusions of microchromosomes, which occurred in the ancestral karyotype of Toxicofera and led to the disappearance of microchromosomes and the appearance of many small macrochromosomes.

No Interstitial Telomeres on Autosomes but Remarkable Amplification of Telomeric Repeats on the W Sex Chromosome in the Sand Lizard (Lacerta agilis).

Telomeres are repeat (TTAGGG) n sequences that form terminal ends of chromosomes and have several functions, such as protecting the coding DNA from erosion at mitosis. Due to chromosomal rearrangements through evolutionary history (e.g., inversions and fusions), telomeric sequences are also found between the centromere and the terminal ends (i.e., at interstitial telomeric sites, ITSs). ITS telomere sequences have been implicated in heritable disease caused by genomic instability of ITS polymorphic variants, both with respect to copy number and sequence. In the sand lizard (Lacerta agilis), we have shown that telomere length is predictive of lifetime fitness in females but not males. To assess whether this sex specific fitness effect could be traced to ITSs differences, we mapped (TTAGGG) n sequences using fluorescence in situ hybridization in fibroblast cells cultured from 4 specimens of known sex. No ITSs could be found on autosomes in either sex. However, females have heterogametic sex chromosomes in sand lizards (ZW, 2n = 38) and the female W chromosome showed degeneration and remarkable (TTAGGG) n amplification, which was absent in the Z chromosomes. This work warrants further research on sex chromosome content, in particular of the degenerate W chromosome, and links to female fitness in sand lizards.

Reduction in the structural instability of cloned eukaryotic tandem-repeat DNA by low-temperature culturing of host bacteria.

Summary For accurate analyses of eukaryotic tandem-repeat DNA, it is often required to clone a genomic DNA fragment into a bacterial plasmid. It is, however, a serious problem that tandem-repeat DNA is frequently subjected to structural changes during maintenance or amplification in the host bacteria. Here, we show an example of a clear difference in the instability of tandem-repeat DNA between different culturing temperatures. A fragment of monkey centromeric DNA carried by pUC19 was considerably degraded by culturing bacteria at 37 °C, but the damage was reduced at 25 °C. Thus, culturing temperature is a significant factor for avoiding degradation, in addition to the genotype of the host bacteria.