High-resolution organization of mouse centromeric and pericentromeric DNA

We studied the organization of mouse satellite 3 and 4 (MS3 and MS4) in comparison with major (MaSat) and minor (MiSat) DNA sequences, located in the centromeric and pericentromeric regions of mouse telocentric chromosomes by fiber-FISH. The centromeric region consists of a small block of MiSat and MS3 followed by a pericentromeric block of MaSat with MS4. Inside the block of the long-range cluster, MaSat repeats intermingle mostly with MS4, while MiSat intermingle with MS3. The distribution of GC-rich satellite DNA fragments is less strict than that of AT-rich fragments; it is possible to find MS3 fragments in the MaSat array and MS4 fragments in the MiSat array. The methylation pattern does not fully correspond to one of the four families of satellite DNA (satDNA). In each satDNA fragment only part of the DNA is methylated. MS3 and MS4 are heavily methylated being GC-rich. Pericentomeric satellite DNA fragments are more methylated than centromeric ones. Among the four families of satDNA MS4 is the most methylated while MiSat is methylated only to a minimal extent. Estimation of the average fragment length and average distance between fragments shows that the range of the probes used does not cover the whole centromeric region. The existence of unknown sequences in the mouse centromere is likely.     

Localization of satellite DNA and associated proteins in respect to nucleolar precursor bodies in one- and two-cell mouse embryos

Nucleolar precursor bodies (NPB) are discrete entities in zygotic pronuclei and in the nuclei of two-cell mouse embryos. Centromeric (CEN) and pericentromeric (periCEN) chromosome regions are associated with the chromatin layer surrounding NPB. Four types of satellite DNA (satDNA) are currently known in Mus musculus, including mouse minor satellite 4 (MiSat), mouse satellite 3 (MS3) in the CEN region, mouse major satellite (MaSat), and mouse satellite (MS4) in the periCEN region. We determined the localization of these four types of mouse satDNA and associated proteins (RNA-helicase p68, SMC3, Rad21 subunits of the cohesin complex and SYCP3 subunit of the synaptonemal complex) in respect to NPB. Partially flattened nuclei of the one- and two-cell intact embryos and embryos treated with okadaic acid (OA) were used. It was found that different satDNA are localized in different regions at the NPB surface: periCEN MaSat occupied almost the whole NPB surface; CEN MiSat, MS3 and periCEN MS4 were located more peripherally. All four satDNA did not cover the entire NPB area, which indicates the presence of other DNA sequences involved in the association with NPB periphery. Among the proteins probed, RNA-helicase p68 and components of multiprotein cohesin and synaptonemal complexes (SCs) showed the most prominent colocolization with NPB. Our results support the idea that NPB are chromocenter precursors.     

A centromeric tandem repeat family originating from a part of Ty3/gypsy-retroelement in wheat and its relatives

From a wild diploid species that is a relative of wheat, Aegilops speltoides, a 301-bp repeat containing 16 copies of a CAA microsatellite was isolated. Southern blot and fluorescence in situ hybridization revealed that approximately 250 bp of the sequence is tandemly arrayed at the centromere regions of A- and B-genome chromosomes of common wheat and rye chromosomes. Although the DNA sequence of this 250-bp repeat showed no notable homology in the databases, the flanking or intervening sequences between the repeats showed high homologies (>82%) to two separate sequences of the gag gene and its upstream region in cereba, a Ty3/gypsy-like retroelement of Hordeum vulgare. Since the amino acid sequence deduced from the 250 bp with seven CAAs showed some similarity ( approximately 53%) to that of the gag gene, we concluded that the 250-bp repeats had also originated from the cereba-like retroelements in diploid wheat such as Ae. speltoides and had formed tandem arrays, whereas the 300-bp repeats were dispersed as a part of cereba-like retroelements. This suggests that some tandem repeats localized at the centromeric regions of cereals and other plant species originated from parts of retrotransposons.     

Identification of repetitive, genome-specific probes in crucifer oilseed species.

Direct amplification of minisatellite DNA by PCR (DAMD PCR) was used to amplify and subsequently clone several fragments of DNA from crucifer species. The PCR-derived fragments of DNA were generated using known minisatellite core sequences as PCR primers. Southern hybridization of these putative minisatellite DNA fragments revealed that many were genome-specific; they hybridized with high affinity only to the genomic DNA of the species from which they were cloned. The DNA fragments were believed to be dispersed in the genome, based on smear-like hybridization signals on EcoRI-, BamHI-, and HindIII-digested genomic DNA. Genome-specific probes were specifically isolated from Brassica rapa (A genome), Brassica nigra (B genome), and Sinapis alba in addition to several other crucifer species. The sequence of a B. rapa specific probe (pBr17.1.3A) contained a minisatellite region that could be divided into three tandem repeats; each repeat contained between two and five subrepeats and each subrepeat shared a highly conserved core region of 29 bp. This minisatellite sequence also hybridized with high affinity to the A genome species B. napus and B. juncea. This research showed that dispersed, genome-specific probes can be isolated using DAMD PCR and that these probes could be used to detect and quantify alien DNA present in progeny from intergeneric or interspecific crosses.     

Identification of genetic markers for Korean native cattle (Hanwoo) by RAPD analysis

In order to develop the specific genetic marker for Korean native cattle (Hanwoo), randomly amplified polymorphic DNA (RAPD) analysis of 6 different cattle breeds was attempted by using 38 decamer primers. In comparison of RAPD patterns, two distinctive DNA bands specific for Hanwoo were detected. One was 296 bp of DNA fragment found to be specific only for female Hanwoo when primer GTCCACACGG was employed. In individual analysis of this RAPD marker was observed only in female individuals with the possibility of 85.3%. The other was 521 bp of RAPD marker amplified using TCGGCGATAG and AGCCAGCGAA primers, which showed 83.0% of genetic frequency in 85 male and 68 female individuals tested. Nucleotide sequencing of these genetic markers revealed that 296 bp marker has a short microsatellite-like sequence, ACCACCACAC, and a tandem repeat sequence of microsatellite GAAAAATG in the determined sequence. Two distinctive tandem repeats of microsatellite sequences, AAC and GAAGA, were also appeared in 521 bp DNA marker. In BLAST search, any gene having high homology with these markers was not found     

The centromere composition of multiple repetitive sequences on rice chromosome 5

The large-scale primary structure of the centromeric region of rice chromosome 5 was analyzed, the first example in a cereal species. The yeast artificial chromosome (YAC) and bacterial artificial chromosome (BAC) contigs aligned on the centromere of rice chromosome 5 (CEN5) covered a distance of more than 670 kb. Strong suppression of genetic recombination, one of the features of a functional centromere, occurred along the contig region. The most remarkable feature of CEN5 is the composition of the multiple repetitive elements. Oryza-specific RCS2 short tandem repeats were clustered along less than 100 kb at one end of the contig. At least 15 copies of the conserved domain of the 1.9 kb RCE1 centromeric repeats, which are similar to the long terminal repeats (LTRs) of gypsy-type retrotransposon RIRE7, were dispersed mainly in 320 kb stretches next to RCS2 tandem clusters. Many copies of the LTR-like sequences of RIRE3 and RIRE8, another gypsy-type retrotransposon, were also found throughout the contig. On the other hand, the gagpol region was less conserved in the contig. These results indicate that the rice centromere is composed of multiple repetitive sequences with the RCS2 tandem cluster probably being situated as the core of a functional centromere of some hundreds of kilobases to megabases in length.     

Mapping of mouse intracisternal A-particle proviral markers in an interspecific backcross

We present a linkage map of intracisternal A-particle (IAP) proviral loci. The IAP family consists of 2000 endogenous proviral elements that are widely dispersed in the mouse genome. The map was constructed by using an interspecific backcross and markers defined by oligonucleotide probes specific for subclasses of expressed IAP elements. In genomic DNA from C57BL/6J mouse, these probes each detected from 12 to 44 HindIII restriction fragments that represent junctions between proviral and 5-flanking DNA. The fragments have characteristic strain distribution patterns (SDPs) that are particularly polymorphic in the DNAs of C57BL/6J and Mus spretus mice used for the backcross. IAP loci were placed on the map by comparison of their distribution patterns with those of known genetic markers in the backcross. The map includes 51 IAP loci that have not been previously mapped and 23 IAP proviruses that had been previously mapped in recombinant inbred (RI) strains. Comparable map positions were obtained with the IAP markers in the interspecific backcross and the RI strains. The mapped IAP loci were widely dispersed on the X Chromosome (Chr) and all of the autosomes except Chrs 9 and 19, providing useful genetic markers for linkage studies.     

茶树EST-SSRs分布特征及引物开发

为了在茶树中开发EST-SSRs功能性标记,利用生物信息学方法对NCBI网上公开的3288奈茶树（Camellia subebsus）ESTs序列进行EST-SSRs特征分析。剔除冗余序列,得到非冗余序列2083条。在非冗余序列中发现含不同重复基元SSRs的EST序列有385条,共486个EST-SSRs,平均相隔2．10kb出现1个SSR。在2～6bp的重复基元中,二核苷酸重复基元的SSRs出现频率最高（51．97％）,其次是三核苷酸（19．55％）。对所有的重复基元类型进行统计分析发现,所占比例最高的是AG／CT（47．74％）,其次分别是AT／TA（4．73％）和AAG／CTT（4．73％）。利用Prime5软件,设计了206对EST-SSRs引物,随机选用72对引物进行SSR扩增,发现31对引物可以扩增出条带,其中29对引物具有多态性,多态性比率为93．5％。这些EST-SSRs将有助于茶树基因组学方面的研究。     

A complete mitochondrial genome sequence of Asian black bear Sichuan subspecies (Ursus thibetanus mupinensis)

We obtained the complete mitochondrial genome of U.thibetanus mupinensis by DNA sequencing based on the PCR fragments of 18 primers we designed. The results indicate that the mtDNA is 16,868 bp in size, encodes 13 protein genes, 22 tRNA genes, and 2 rRNA genes, with an overall H-strand base composition of 31.2% A, 25.4% C, 15.5% G and 27.9% T. The sequence of the control region (CR) located between tRNA-Pro and tRNA-Phe is 1422 bp in size, consists of 8.43% of the whole genome, GC content is 51.9% and has a 6bp tandem repeat and two 10bp tandem repeats identified by using the Tandem Repeats Finder. U. thibetanus mupinensis mitochondrial genome shares high similarity with those of three other Ursidae: U. americanus (91.46%), U. arctos (89.25%) and U. maritimus (87.66%).     

Apparent relatedness of the main component of ovine 1.714 satellite DNA to bovine 1.715 satellite DNA

The nucleotide sequence of the principal component of ovine 1.714 g/cm3 satellite DNA was determined from a monomeric fragment inserted at the BamHI site of pBR322 and cloned in Escherichia coli strain RR1. The 816-bp tandemly repeated sequence contains a number of small repeated sequences dispersed within it, one group of which forms a pentameric tandem repeat of a 13-bp segment (positions 548-612). A 20-bp region (60-79) shows an 85% homology with the reverse-complement of the sequence from 455 through 474. There are two regions of 67 bp (75-141) and 59 bp (755-813) which show greater than 70% homology with regions of bovine 1.715 g/cm3 satellite DNA (1402 bp; positions 1218-1284 and 1079-1137, respectively) while a 31-bp region (ovine 62-92, bovine 133-163) shows 80% homology. Quasi-correlation coefficients (Qr) were determined using the triplet numbers of the sheep satellite versus all sequences in the National Biomedical Research Foundation and EMBL nucleotide sequence data bases. Qr equals 0.85 for ovine 1.714 g/cm3 satellite versus bovine 1.715 g/cm3 satellite. The next highest Qr for a bovine satellite segment was 0.58. Thus, the ovine 1.714 g/cm3 and bovine 1.715 g/cm3 satellite appear demonstrably related. Taking into account that sheep and cattle diverged 18-20 million years ago, this suggests that the material may be functional and that its function is related to its sequence.     

Study of T-DNA integration loci in tobacco transgenic plants

From the total DNA of 17 transgenic tobacco plants the DNA fragments containing T-DNA/plant DNA junctions were amplified using inverse polymerase chain reaction. Comparison of the nucleotide sequences of 34 fragments with the GENEBANK sequences revealed homology with vector sequences outside T-DNA in 10 cases and no homology with the known nucleotide sequences in most clones. The AT-content varied from 51 up to 72% that is close to the total percentage of AT pairs in tobacco genome. Alignment of the sequences truncated during embedding of the left and the right borders has shown that for the left border significant clusterization (10 bp region) of truncation sites was observed, and five sequences had identical sites of truncation (+23 T) that showed the preferable use of this nucleotide. Nine created nucleotide sequences were homologous to the repeating sequences in tobacco genome. The percentage of homology varied from 70 up to 90%. The identified repeats belong to different types.     

Structural analysis and complete physical map of Arabidopsis thaliana chromosome 5 including centromeric and telomeric regions.

Previously, we have reported a fine physical map of Arabidopsis thaliana chromosome 5, except for the centromeric and telomeric regions, by ordering clones from YAC, P1, TAC, and BAC libraries of the genome consisting of the two contigs of upper arm and lower arm, 11.6 M bases and 14.2 M bases, respectively. Here, the remaining centromeric and telomeric regions of chromosome 5 are completely characterized by the ordering of clones and PCR amplifications. Chromosome 5 of Arabidopsis thaliana ecotype Columbia is about 28.4 M bases long. The centromeric region is estimated at about 2 M bases long between two 5S-rDNA clusters. The 180-bp repeat region mainly consists of blocks of 180-bp tandem family and various type retroelements dispersed over a 500-kb region. The telomeric regions of chromosome 5 are characterized by PCR cloning, sequencing and hybridization. The telomere repeats at both ends are about 2.5-kb long and interestingly, telomere-associated repeats (approximately 700 bp) are found near both ends of chromosome 5.     

Genome-wide analysis of tandem repeats in Tribolium castaneum genome reveals abundant and highly dynamic tandem repeat families with satellite DNA features in euchromatic chromosomal arms

Although satellite DNAs are well-explored components of heterochromatin and centromeres, little is known about emergence, dispersal and possible impact of comparably structured tandem repeats (TRs) on the genome-wide scale. Our bioinformatics analysis of assembled Tribolium castaneum genome disclosed significant contribution of TRs in euchromatic chromosomal arms and clear predominance of satellite DNA-typical 170 bp monomers in arrays of ≥5 repeats. By applying different experimental approaches, we revealed that the nine most prominent TR families Cast1–Cast9 extracted from the assembly comprise ∼4.3% of the entire genome and reside almost exclusively in euchromatic regions. Among them, seven families that build ∼3.9% of the genome are based on ∼170 and ∼340 bp long monomers. Results of phylogenetic analyses of 2500 monomers originating from these families show high-sequence dynamics, evident by extensive exchanges between arrays on non-homologous chromosomes. In addition, our analysis shows that concerted evolution acts more efficiently on longer than on shorter arrays. Efficient genome-wide distribution of nine TR families implies the role of transposition only in expansion of the most dispersed family, and involvement of other mechanisms is anticipated. Despite similarities in sequence features, FISH experiments indicate high-level compartmentalization of centromeric and euchromatic tandem repeats.     

Cross-hybridizing snake satellite, Drosophila, and mouse DNA sequences may have arisen independently

Previous reports have interpreted hybridization between snake satellite DNA and DNA clones from a variety of distant taxonomic groups as evidence for evolutionary conservation, which implies common ancestry (homology) and/or convergence (analogy) to produce the cross- hybridizing sequences. We have isolated 11 clones from a genomic library of Drosophila melanogaster, using a cloned 2.5-kb snake satellite probe of known nucleotide sequence. We have also analysed published sequence data from snakes, mice, and Drosophila. These data show that (1) all of the cross-hybridization between the snake, fly, and mouse clones can be accounted for by the presence of either of two tandem repeats, [GATA]n and [GACA]n and (2) these tandem repeats are organized differently among the different species. We find no evidence that these sequences are homologous apart from the existence of the simple repeat itself, although their divergence from a common ancestral sequence cannot be ruled out. The sequences contain a variety of homogeneous clusters of tandem repeats of CATA, GA, TA, and CA, as well as GATA and GACA. We suggest that these motifs may have arisen by a self-accelerating process involving slipped-strand mispairing of DNA. Homogeneity of the clusters might simply be the result of a rate of accumulation of tandem repeats that exceeds that of other mutations.      

Construction of tandem repeats of DNA fragments by a polymerase chain reaction-based method

We describe a new application of megaprimer polymerase chain reaction (PCR) for constructing a tandemly repeated DNA sequence using the drought responsive element (DRE) from Arabidopsis thaliana as an example. The key feature in the procedure was PCR primers with partial complementarity but differing melting temperatures (T(m)). The reverse primer had a higher T(m), a 3' end complementary to the DRE sequence and a 5' region complementary to the forward primer. The initial cycles of the PCR were conducted at a lower primer annealing temperature to generate products that served as megaprimers in the later cycles conducted at a higher temperature to prevent annealing of the forward primer. The region of overlap between the megaprimers was extended for generating products with a variable copy number (one to four copies) of tandem DRE sequence repeats (71?bp). The PCR product with four tandem repeats (4× DRE) was used as a template to generate tandem repeats with higher copies (copy number large than four) or demonstrated to bind DRE-binding protein in an yeast one-hybrid assay using promotorless reporter genes (HIS and lacZ). This PCR protocol has numerous applications for generating DNA fragments of repeated sequences.     

Hypervariable 3′ UTR region of plant LTR-retrotransposons as a source of novel satellite repeats

The repetitive sequence PisTR-A has an unusual organization in the pea (Pisum sativum) genome, being present both as short dispersed repeats as well as long arrays of tandemly arranged satellite DNA. Cloning, sequencing and FISH analysis of both PisTR-A variants revealed that the former occurs in the genome embedded within the sequence of Ty3/gypsy-like Ogre elements, whereas the latter forms homogenized arrays of satellite repeats at several genomic loci. The Ogre elements carry the PisTR-A sequences in their 3′ untranslated region (UTR) separating the gag-pol region from the 3′ LTR. This region was found to be highly variable among pea Ogre elements, and includes a number of other tandem repeats along with or instead of PisTR-A. Bioinformatic analysis of LTR-retrotransposons mined from available plant genomic sequence data revealed that the frequent occurrence of variable tandem repeats within 3′ UTRs is a typical feature of the Tat lineage of plant retrotransposons. Comparison of these repeats to known plant satellite sequences uncovered two other instances of satellites with sequence similarity to a Tat-like retrotransposon 3′ UTR regions. These observations suggest that some retrotransposons may significantly contribute to satellite DNA evolution by generating a library of short repeat arrays that can subsequently be dispersed through the genome and eventually further amplified and homogenized into novel satellite repeats.