Great Abundance of Satellite DNA in Proceratophrys (Anura, Odontophrynidae) Revealed by Genome Sequencing.

Most eukaryotic genomes contain substantial portions of repetitive DNA sequences. These are located primarily in highly compacted heterochromatin and, in many cases, are one of the most abundant components of the sex chromosomes. In this sense, the anuran Proceratophrys boiei represents an interesting model for analyses on repetitive sequences by means of cytogenetic techniques, since it has a karyotype with large blocks of heterochromatin and a ZZ/ZW sex chromosome system. The present study describes, for the first time, families of satellite DNA (satDNA) in the frog P. boiei. Its genome size was estimated at 1.6 Gb, of which 41% correspond to repetitive sequences, including satDNAs, rDNAs, transposable elements, and other elements characterized as non-repetitive. The satDNAs were mapped by FISH in the centromeric and pericentromeric regions of all chromosomes, suggesting a possible involvement of these sequences in centromere function. SatDNAs are also present in the W sex chromosome, occupying the entire heterochromatic area, indicating a probable contribution of this class of repetitive DNA to the differentiation of the sex chromosomes in this species. This study is a valuable contribution to the existing knowledge on repetitive sequences in amphibians. We show the presence of repetitive DNAs, especially satDNAs, in the genome of P. boiei that might be of relevance in genome organization and regulation, setting the stage for a deeper functional genome analysis of Proceratophrys.

Chromosome size-correlated and chromosome size-uncorrelated homogenization of centromeric repetitive sequences in New World quails.

Many families of centromeric repetitive DNA sequences isolated from Struthioniformes, Galliformes, Falconiformes, and Passeriformes are localized primarily to microchromosomes. However, it is unclear whether chromosome size-correlated homogenization is a common characteristic of centromeric repetitive sequences in Aves. New World and Old World quails have the typical avian karyotype comprising chromosomes of two distinct sizes, and C-positive heterochromatin is distributed in centromeric regions of most autosomes and the whole W chromosome. We isolated six types of centromeric repetitive sequences from three New World quail species (Colinus virginianus, CVI; Callipepla californica, CCA; and Callipepla squamata, CSQ; Odontophoridae) and one Old World quail species (Alectoris chukar, ACH; Phasianidae), and characterized the sequences by nucleotide sequencing, chromosome in situ hybridization, and filter hybridization. The 385-bp CVI-MspI, 591-bp CCA-BamHI, 582-bp CSQ-BamHI, and 366-bp ACH-Sau3AI fragments exhibited tandem arrays of the monomer unit, and the 224-bp CVI-HaeIII and 135-bp CCA-HaeIII fragments were composed of minisatellite-like and microsatellite-like repeats, respectively. ACH-Sau3AI was a homolog of the chicken nuclear membrane repeat sequence, whose homologs are common in Phasianidae. CVI-MspI, CCA-BamHI, and CSQ-BamHI showed high homology and were specific to the Odontophoridae. CVI-MspI was localized to microchromosomes, whereas CVI-HaeIII, CCA-BamHI, and CSQ-BamHI were mapped to almost all chromosomes. CCA-HaeIII was localized to five pairs of macrochromosomes and most microchromosomes. ACH-Sau3AI was distributed in three pairs of macrochromosomes and all microchromosomes. Centromeric repetitive sequences may be homogenized in chromosome size-correlated and -uncorrelated manners in New World quails, although there may be a mechanism that causes homogenization of centromeric repetitive sequences primarily between microchromosomes, which is commonly observed in phasianid birds.

Epigenetic, genetic and maternal effects enable stable centromere inheritance.

Centromeres are defined epigenetically by the histone H3 variant CENP-A. The propagation cycle by which pre-existing CENP-A nucleosomes serve as templates for nascent assembly predicts the epigenetic memory of weakened centromeres. Using a mouse model with reduced levels of CENP-A nucleosomes, we find that an embryonic plastic phase precedes epigenetic memory through development. During this phase, nascent CENP-A nucleosome assembly depends on the maternal Cenpa genotype rather than the pre-existing template. Weakened centromeres are thus limited to a single generation, and parental epigenetic differences are eliminated by equal assembly on maternal and paternal centromeres. These differences persist, however, when the underlying DNA of parental centromeres differs in repeat abundance, as assembly during the plastic phase also depends on sufficient repetitive centromere DNA. With contributions of centromere DNA and the Cenpa maternal effect, we propose that centromere inheritance naturally minimizes fitness costs associated with weakened centromeres or epigenetic differences between parents.

A paralog of the MtN3/saliva family recessively confers race-specific resistance to Xanthomonas oryzae in rice.

Approximately one third of the identified 34 rice major disease resistance (R) genes conferring race-specific resistance to different strains of Xanthomonas oryzae pv. oryzae (Xoo), which causes rice bacterial blight disease, are recessive genes. However, only two of the recessive resistance genes have been characterized thus far. Here we report the characterization of another recessive resistance gene, xa25, for Xoo resistance. The xa25, localized in the centromeric region of chromosome 12, mediates race-specific resistance to Xoo strain PXO339 at both seedling and adult stages by inhibiting Xoo growth. It encodes a protein of the MtN3/saliva family, which is prevalent in eukaryotes, including mammals. Transformation of the dominant Xa25 into a resistant rice line carrying the recessive xa25 abolished its resistance to PXO339. The encoding proteins of recessive xa25 and its dominant allele Xa25 have eight amino acid differences. The expression of dominant Xa25 but not recessive xa25 was rapidly induced by PXO339 but not other Xoo strain infections. The nature of xa25-encoding protein and its expression pattern in comparison with its susceptible allele in rice-Xoo interaction indicate that the mechanism of xa25-mediated resistance appears to be different from that conferred by most of the characterized R proteins.

Neocentromere marker chromosome of distal 3q mimicking dup(3q) syndrome phenotype.

Supernumerary marker chromosomes (SMCs) lacking alpha-satellite sequences and possessing a newly derived functional centromere are referred to as neocentromere marker chromosomes (NMCs). Although the delineation of the chromosome content of these NMCs would be helpful for genetic counseling, such fine mapping has been difficult because of the limited sizes of the involved segments. We report on a female patient with mosaic NMC involving 3q26.3-3qter, the content of which was determined using an array CGH analysis. Our results support the validity of an array CGH-based approach to investigating the origins of SMCs. Further FISH analyses revealed that the NMC is characterized by an asymmetric inv-dup structure separated by a single-copy region. The present case had many manifestations of dup(3q) syndrome, the critical interval of which is considered to be 3q26.3-q27. Common features included mental and growth retardation, hirsutism, synophrys, a broad nasal root, anteverted nares, downturned corners of the mouth, and malformed ears. The observation gives further credence to the concept that the critical region responsible for the dup(3q) phenotype to 3q26.3-q27.

Centromeres, CENP-B and Tigger too.

The highly conserved centromere-associated protein CENP-B is a common feature of mammalian centromeres. Binding sites for CENP-B, so-called 'CENP-B boxes', are present in the otherwise unrelated centromeric satellite DNAs of humans, Mus musculus, Mus caroli, ferrets, giant pandas, tree shrews and gerbils, suggesting a role for CENP-B in centromere function. However, CENP-B and its binding sites are not detected at the centromeres of mammalian Y chromosomes and few, if any, binding sites seem present on African green monkey chromosomes. There is extensive sequence similarity between CENP-B and transposase proteins encoded by the pogo superfamily of transposable elements, which includes the human Tigger elements. Intriguingly, Tigger 2 has an almost perfect match to the CENP-B-binding site within its terminal inverted repeat. Comparison of the amino acid sequence of CENP-B with related proteins raises the possibility that CENP-B might share the ability to cause single-stranded DNA breaks. Such nicks could promote recombination, as has been suggested for the Charcot-Marie-Tooth disease duplication where a recombination hotspot exists close to a mariner-like element. We suggest that by promoting nicks adjacent to CENP-B boxes, CENP-B might facilitate the evolution and maintenance of satellite sequence arrays, rather than have a direct role in centromere function.

Comparative genomic hybridisation using a proximal 17p BAC/PAC array detects rearrangements responsible for four genomic disorders.

BACKGROUND: Proximal chromosome 17p is a region rich in low copy repeats (LCRs) and prone to chromosomal rearrangements. Four genomic disorders map within the interval 17p11-p12: Charcot-Marie-Tooth disease type 1A, hereditary neuropathy with liability to pressure palsies, Smith-Magenis syndrome, and dup(17)(p11.2p11.2) syndrome. While 80-90% or more of the rearrangements resulting in each disorder are recurrent, several non-recurrent deletions or duplications of varying sizes within proximal 17p also have been characterised using fluorescence in situ hybridisation (FISH). METHODS: A BAC/PAC array based comparative genomic hybridisation (array-CGH) method was tested for its ability to detect these genomic dosage differences and map breakpoints in 25 patients with recurrent and non-recurrent rearrangements. RESULTS: Array-CGH detected the dosage imbalances resulting from either deletion or duplication in all the samples examined. The array-CGH approach, in combination with a dependent statistical inference method, mapped 45/46 (97.8%) of the analysed breakpoints to within one overlapping BAC/PAC clone, compared with determinations done independently by FISH. Several clones within the array that contained large LCRs did not have an adverse effect on the interpretation of the array-CGH data. CONCLUSIONS: Array-CGH is an accurate and sensitive method for detecting genomic dosage differences and identifying rearrangement breakpoints, even in LCR-rich regions of the genome.

Quantitative trait loci on chromosomes 10 and 11 influencing mandible size of SMXA RI mouse strains.

Predicting the mandible size before the termination of growth of the maxillofacial bones is essential in pedodontics as well as for the predictions needed for genetic analysis. Here, Quantitative Trait Locus (QTL) analysis was used to detect the chromosomal regions responsible for the mandible length between the menton and gonion in an SMXA recombinant inbred strain of mice. Around the region 60 cM from the centromere in chromosome 10, the logarithm of the odds score showed a higher than suggestive level. Around the regions 13 cM and 16 cM in chromosome 11, two significant QTLs were detected. Analysis of genotypes from loci corresponding to those QTLs revealed a large mandible when the region between the markers Hba and D11Mit163 and D10Mit70 and D10Mit136 indicated the genotype from the A/J and SM/J alleles, respectively. These results suggest that the major gene(s) responsible for mandible length are located in these regions.

The molecular phylogeny of the Sparidae (Pisces, Perciformes) based on two satellite DNA families.

In this study, the phylogenetic relationships and which the taxonomic status of the species belonging to the Sparidae family (Pisces: Perciformes) are analysed and revised. This study includes species of this family that are distributed by the North-eastern Atlantic and Mediterranean coasts, is based on the analysis of two satellite DNA families. While one satellite DNA, the centromeric EcoRI family, extends to all the species analysed, the other, the subtelomeric DraI family, is restricted to only six of the 16 species studied. Based on phylogenetic use of these two markers, we conclude that the Sparidae family is composed by two major lineages: one comprising the species of the genera Sparus, Diplodus, Lithognathus, Boops, Sarpa and Spondyliosoma, and one species of Pagellus (P. bogaraveo); and the other lineage is comprised of the species of Pagrus and Dentex, and one species of Pagellus (P. erythrinus). This classification is consistent across the two markers used and clearly contradicts previous morphological phylogenies based mainly on dentition. In addition, the current status and the phylogenetic position of some of the species analysed (i.e. species of Pagrus, Dentex and Pagellus) are not supported by our analyses. Finally, we discuss the value of the morphological characters used until now for the classification of this group of fish.

A girl with duplication 17p10-p12 associated with a dicentric chromosome.

We report a 7 1/2-year-old girl with an approximately 9.5 Mb duplication of proximal 17p. Her clinical features include moderately severe developmental delay, absence of speech, talipes, congenital dislocation of the hips, premature adrenarche, dysmorphic facial features, deep palmar creases, and signs and symptoms of peripheral neuropathy consistent with Charcot-Marie-Tooth disease type 1A (CMT1A). Chromosome analysis revealed a partially duplicated 17p with two centromeres on the derivative chromosome. Fluorescence in situ hybridization (FISH) analysis demonstrated the tandemly duplicated segment spans 17p10-p12, including the entire Smith-Magenis syndrome (SMS) critical region and a portion of the CMT1A critical region. One breakpoint mapped within the centromere and the second breakpoint mapped within the CMT1A critical region, distal to the PMP22 gene. Microsatellite polymorphism studies showed that the duplicated chromosome is of maternal origin. We compare the clinical features of our patient to those of individuals with partial trisomy of proximal 17p to further delineate the genotype-phenotype correlation associated with segmental duplication of this chromosomal region.

Construction of an 800-kb contig in the near-centromeric region of the rice blast resistance gene Pi-ta2 using a highly representative rice BAC library.

We constructed a rice Bacterial Artificial Chromosome (BAC) library from green leaf protoplasts of the cultivar Shimokita harboring the rice blast resistance gene Pi-ta. The average insert size of 155 kb and the library size of seven genome equivalents make it one of the most comprehensive BAC libraries available, and larger than many plant YAC libraries. The library clones were plated on seven high density membranes of microplate size, enabling efficient colony identification in colony hybridization experiments. Seven percent of clones carried chloroplast DNA. By probing with markers close to the blast resistance genes Pi-ta2(closely linked to Pi-ta) and Pi-b, respectively located in the centromeric region of chromosome 12 and near the telomeric end of chromosome 2, on average 2.2 +/- 1.3 and 8.0 +/- 2.6 BAC clones/marker were isolated. Differences in chromosomal structures may contribute to this wide variation in yield. A contig of about 800 kb, consisting of 19 clones, was constructed in the Pi-ta2 region. This region had a high frequency of repetitive sequences. To circumvent this difficulty, we devised a "two-step walking" method. The contig spanned a 300 kb region between markers located at 0 cM and 0.3 cM from Pi-ta. The ratio of physical to genetic distances (> 1,000 kb/cM) was more than three times larger than the average of rice (300 kb/cM). The low recombination rate and high frequency of repetitive sequences may also be related to the near centromeric character of this region. Fluorescent in situ hybridization (FISH) with a BAC clone from the Pi-b region yielded very clear signals on the long arm of chromosome 2, while a clone from the Pi-ta2 region showed various cross-hybridizing signals near the centromeric regions of all chromosomes.

A 1-Mb critical region in six patients with 9q34.3 terminal deletion syndrome.

Patients with 9q34.3 terminal deletion usually show a clinically recognizable phenotype characterized by specific facial features (microcephaly, flat face, arched eyebrows, hypertelorism, short nose, anteverted nostrils, carp mouth and protruding tongue) in combination with severe mental retardation, hypotonia, and other anomalies. We analyzed six unrelated patients with a various 9q34.3 terminal deletion. While having different-sized 9q34.3 deletions, all of these patients shared several distinctive anomalies. These anomalies are likely to arise from a commonly deleted region at distal 9q34.3. Fluorescence in situ hybridization (FISH) analysis using a dozen BAC clones mapped at the 9q34.13-q34.3 region defined the shortest region of deletion overlap (SRO) as a 1-Mb segment proximal to 9qter containing eight known genes. Possible candidate genes delineating specific phenotypes of the 9q34.3 terminal deletion syndrome are discussed.

Mapping of the breakpoints on the short arm of chromosome 17 in neoplasms with an i(17q).

Isochromosomes are monocentric or dicentric chromosomes with homologous arms that are attached in a reverse configuration as mirror images. With an incidence of 3-4%, the i(17q) represents the most frequent isochromosome in human cancer. It is found in a variety of tumors, particularly in blast crisis of chronic myeloid leukemia (CML-BC), acute myeloid leukemia (AML), non-Hodgkin's lymphoma (NHL), and medulloblastoma (MB), and indicates a poor prognosis. To determine the breakpoints on the molecular genetic level, we analyzed 18 neoplasms (six CML, four AML, one NHL, and seven MB) with an i(17q) and two MB with a pure del(17p) applying fluorescence in situ hybridization (FISH) with yeast artificial chromosome (YAC) clones, P1-artificial chromosome (PAC) clones, and cosmids from a well-characterized contig covering more than 6 Mb of genomic DNA. We identified four different breakpoint cluster regions. One is located close to or within the centromere of chromosome 17 and a second in the Charcot-Marie-Tooth (CMT1A) region at 17(p11.2). A third breakpoint was found telomeric to the CMT1A region. The fourth, most common breakpoint was detected in MB, AML, and in CML-BC specimens and was bordered by two adjacent cosmid clones (clones D14149 and M0140) within the Smith-Magenis syndrome (SMS) region. These results indicate that the low copy number repeat gene clusters which are present in the CMT and SMS regions may be one of the factors for the increased instability that may trigger the formation of an i(17q).

Evidence for a Turner syndrome locus or loci at Xp11.2-p22.1.

Turner syndrome is the complex human phenotype associated with complete or partial monosomy X. Principle features of Turner syndrome include short stature, ovarian failure, and a variety of other anatomic and physiological abnormalities, such as webbed neck, lymphedema, cardiovascular and renal anomalies, hypertension, and autoimmune thyroid disease. We studied 28 apparently nonmosaic subjects with partial deletions of Xp, in order to map loci responsible for various components of the Turner syndrome phenotype. Subjects were carefully evaluated for the presence or absence of Turner syndrome features, and their deletions were mapped by FISH with a panel of Xp markers. Using a statistical method to examine genotype/phenotype correlations, we mapped one or more Turner syndrome traits to a critical region in Xp11.2-p22.1. These traits included short stature, ovarian failure, high-arched palate, and autoimmune thyroid disease. The results are useful for genetic counseling of individuals with partial monosomy X. Study of additional subjects should refine the localization of Turner syndrome loci and provide a rational basis for exploration of candidate genes.