Comparative analysis of micro and macro B chromosomes in the Korean field mouse Apodemus peninsulae (Rodentia, Murinae) performed by chromosome microdissection and FISH

Comparative analysis of micro B and macro B chromosomes of the Korean field mouse Apodemus peninsulae, collected in populations from Siberia and the Russian Far East, was performed with Giemsa, DAPI, Ag-NOR staining and chromosome painting with whole and partial chromosome probes generated by microdissection and DOP-PCR. DNA composition of micro B chromosomes was different from that of macro B chromosomes. All analyzed micro B chromosomes contained clusters of DNA repeats associated with regions characterized by an uncondensed state in mitosis. Giemsa and DAPI staining did not reveal these regions. Their presence in micro B chromosomes led to their special morphology and underestimation in size. DNA repeat clusters homologous to DNA of micro B chromosome arms were also revealed in telomeric regions of some macro B chromosomes of specimens captured in Siberian regions. Neither active NORs nor clusters of ribosomal DNA were found in the uncondensed regions of micro B chromosomes. Possible evolutionary pathways for the origin of macro and micro B chromosomes are discussed.     

The delayed quinacrine mustard fluorescence from the C-blocks of Apodemus argenteus is due to the introduction of nicks into the DNA

"Delayed QM-fluorescence" refers to the unusual kinetics of fluorescence from most of the C-heterochromatic regions of the chromosomes of the small Japanese field mouse Apodemus argenteus. When stained with quinacrine mustard (QM-stained), these C-heterochromatic regions emit weak fluorescence immediately after exposure to blue light (BL); they emit bright fluorescence within a few minutes; and the intensity of the fluorescence gradually decreases after maximum fluorescence has been recorded. To elucidate the mechanism of this phenomenon, we used acridine orange staining (AO-staining) and a modified version of the in situ nick-translation method. Focusing on the large C-heterochromatic region (C-block) of the X chromosome, we noted that AO-stained C-blocks emitted greenish fluorescence, while QM-stained and BL-exposed (QM-BL-processed) C-blocks emitted reddish fluorescence upon AO-staining after removal of QM. These findings suggested that the C-block DNA of A. argenteus might undergo a structural change, such as strand breaks, during QM-BL processing. Application of the modified in situ nick-translation method revealed the generation of an appreciable number of nicks in the C-block DNA by QM-BL processing. No such nick formation was observed in the C-blocks of three other mammalian species: Apodemus peninsulae, Microtus montebelli, and Urotrichus talpoides. Our findings support the hypothesis that nick formation due to exposure to BL might play a primary role in inducing delayed QM-fluorescence in the C-blocks of A. argenteus. On the basis of the present and earlier findings, we propose a probable mechanism for delayed QM-fluorescence in A. argenteus chromosomes.     

Molecular cytogenetic analysis of the highly repetitive DNA in the genome of Apodemus argenteus, with comments on the phylogenetic relationships in the genus Apodemus.

The DNA of Apodemus argenteus was digested with DraI, and the resultant DraI fragment of highly repetitive DNA was isolated and analyzed by DNA filter hybridization, cloning, sequencing, and fluorescence in situ hybridization (FISH). Southern blot hybridization and nucleotide sequencing revealed that most of the DraI fragment consisted of a 230-bp repeating unit and contained no sex-chromosome-specific nucleotide sequences. The DraI fragment included the CENP-B box-like sequence, with a strong homology to the human CENP-B box sequence. FISH revealed that the DraI fragment was specific to all pericentromeric C-band-positive regions, as well as to the C-block of the X chromosome. No hybridization signals were obtained from A. speciosus, A. peninsulae peninsulae, A.p. giliacus, A. agrarius, A. sylvaticus, A. semotus, or Mus musculus when the DraI fragment was used as probe. Peptide nucleic acid (PNA)-FISH using the CENP-B box-like sequence in the DraI fragments as probe suggested that this nucleotide sequence was also specific to all pericentromeric C-heterochromatic regions of A. argenteus chromosomes. Zoo-blot hybridization using DraI-digested genomic DNA from three species of Apodemus (namely, A. argenteus, A. speciosus, and A. peninsulae) and from Mus musculus strongly suggested that the consensus DraI fragment contained nucleotide sequences that were species-specific for A. argenteus. These results also suggest that A. argenteus is phylogenetically distant from other Apodemus species examined, as well as the possibility that the DraI fragment might be related directly to the delayed quinacrine mustard fluorescence of many pericentromeric C-heterochromatic regions of the chromosomes in A. argenteus.     

Similarities in the chromosomal distribution of AG and AC repeats within and between Drosophila, human and barley chromosomes

Two simple sequence repeats (SSRs), AG and AC, were mapped directly in the metaphase chromosomes of man and barley (Hordeum vulgare L.), and in the metaphase and polytene chromosomes of Drosophila melanogaster. To this end, synthetic oligonucleotides corresponding to (AG)(12) and (AC)(8) were labelled by the random primer technique and used as probes in fluorescent in situ hybridisation (FISH) under high stringency and strict washing conditions. The distribution and intensity of the signals for the repeat sequences were found to be characteristic of the chromosomes and genomes of the three species analysed. The AC repeat sites were uniformly dispersed along the euchromatic segments of all three genomes; in fact, they were largely excluded from the heterochromatin. The Drosophila genome showed a high density of AC sequences on the X chromosome in both mitotic and polytene nuclei. In contrast, the AG repeats were associated with the euchromatic regions of the polytene chromosomes (and in high density on the X chromosome), but were only seen in specific heterochromatic regions in the mitotic chromosomes of all three species. In Drosophila, the AG repeats were exclusively distributed on the tips of the Y chromosome and near the centromere on both arms of chromosome 2. In barley and man, AG repeats were associated with the centromeres (of all chromosomes) and nucleolar organizer regions, respectively. The conserved chromosome distribution of AC within and between these three phylogenetically distant species, and the association of AG in specific chromosome regions with structural or functional properties, suggests that long clusters of these repeats may have some, as yet unknown, role.     

Populational chromosomal polymorphism the Asian forest mouse Apodemus peninsulae]

29 individuals of Apodemus peninsulae, an Asian forest mouse from the Suputin State Nature Reserve (Maritime Territory) were studied karyologically. Diploid chromosome sets of the animals studied contained from 48 to 52 chromosomes. The difference in the diploid chromosome numbers is caused by the presence in some individuals of supernumerary metacentric chromosomes (up to 4). The set is always represented by 48 acrocentric chromosomes, gradually decreasing in size. The supernumerary metacentric chromosomes vary in size from medium-sized to the smallest in the set. The character of intrapopulational chromosome polymorphism of continental and island populations of Apodemus peninsulae is discussed. Attempts are made to explain the polymorphism with respect to supernumerary chromosomes as the result of some viral diseases.     

Origin of an apparent B chromosome by mutation,chromosome fragmentation and specific DNA sequence amplification

The present study documents the de novo origin of an apparent B chromosome in Plantago lagopus. The origin was associated with mutation (aneuploidy), chromosome fragmentation, specific DNA sequence amplification, addition of telomeric repeats, and centromeric misdivision. It originated in the progeny of trisome 2, from the excision of 5S rDNA and 18S, 5.8S, 25S rDNA sequences located on chromosome 2, and within a few generations acquired many characteristics of an apparent B chromosome. The B chromosome has preferential transmission through the male (41%, P<0.025) and female gametes (42%, P<0.01) but does not affect plant phenotype. The B chromosome is completely heterochromatic, has a functional centromere and does not pair at meiosis with any A chromosomes of the standard complement. Fluorescence in situ hybridization analysis showed that it arose from massive amplification of 5S rDNA sequences, has 18S, 5.8S, 25S rDNA sequences at the ends of both arms and telomeric repeats at both termini. Ag-NOR-banding and determination of the maximum number of nucleoli in interphase cells indicate that the nucleolar organizer regions at the ends of both arms of the B chromosome are active in organizing nucleoli. RNA blot analysis showed that the 5S rDNA sequences are not transcribed. To our knowledge, this is the first report that fully documents one of the mechanisms by which B chromosomes may arise in nature.     

慈姑45S rDNA和端粒序列检测及核型分析

以45S r DNA和拟南芥型端粒序列为探针对慈姑(Sagittaria trifolia L.)有丝分裂中期染色体进行单色和双色荧光原位杂交分析,并用银染方法检测慈姑45S r DNA位点的表达,最后结合染色体测量数据和45S r DNA杂交信号建立慈姑的核型。结果显示,慈姑的单倍基因组总长度为76.9±1.38μm,最长染色体为11.55±0.10μm,最短染色体为4.54±0.27μm;慈姑的核型公式为:2n=22=2m+2sm+14st+4t,核型不对称性参数CI、A1、A2、As K(%)、AI分别为19.86±11.06、0.72、0.27、78.82、15.29,核型属于Stebbins类型中的3B型。慈姑具有3对45S r DNA位点,分别位于第8、9、10号染色体的短臂末端。拟南芥型端粒序列的杂交信号出现在慈姑每一条染色体的长、短臂末端。银染检测到6个Ag-NOR和6个核仁,表明3对45S r DNA位点在间期核都有表达。本研究结果为药食兼用植物慈姑提供了分子细胞遗传学基础资料。     

Regulation of the subcellular shuttling of Sgo1 between centromeres and chromosome arms by Aurora B-mediated phosphorylation

A minor fraction of cohesin complexes at chromosome arms is not removed by the prophase pathway, and maintained until metaphase and enriched at centromeres. Sgo1 localizes to chromosome arms from prophase to metaphase, and is indispensable for removing cohesin complexes from chromosome arms. However, it has not been established how the chromosome arm localization of Sgo1 leads to the establishment of cohesion on chromosomes. Here, we report that Aurora B kinase interacts with and phosphorylates Sgo1 in vitro and in vivo. Furthermore, the phosphorylation of Sgol by Aurora B kinase regulated the distribution of Sgo1 between centromeres and chromosome arms, and the expression of Aurora B kinase-dead mutants of Sgo1 caused mislocalization from centromeres to chromosome arms. These results suggest Aurora B kinase directly regulates the subcellular distribution of Sgo1 to facilitate the accurate separation of mitotic chromosomes     

Cytogenetics for the model system Arabidopsis thaliana

A detailed karyotype of Arabidopsis thaliana is presented using meiotic pachytene cells in combination with fluorescence in situ hybridization. The lengths of the five pachytene bivalents varied between 50 and 80 μm, which is 20–25 times longer than mitotic metaphase chromosomes. The analysis confirms that the two longest chromosomes (1 and 5) are metacentric and the two shortest chromosomes (2 and 4) are acrocentric and carry NORs subterminally in their short arms, while chromosome 3 is submetacentric and medium sized. Detailed mapping of the centromere position further revealed that the length variation between the pachytene bivalents comes from the short arms. Individual chromosomes were unambiguously identified by their combinations of relative lengths, arm-ratios, presence of NOR knobs and FISH signals with a 5S rDNA probe and chromosome specific DNA probes. Polymorphisms were found among six ecotypes with respect to the number and map positions of 5S rDNA loci. All ecotypes contain 5S rDNA in the short arms of chromosomes 4 and 5. Three different patterns were observed regarding the presence and position of a 5S rDNA locus on chromosome 3. Repetitive DNA clones enabled us to subdivide the pericentromeric heterochromatin into a central domain, characterized by pAL1 and 106B repeats, which accommodate the functional centromere and two flanking domains, characterized by the 17 A20 repeat sequences. The upper flanking domains of chromosomes 4 and 5, and in some ecotypes also chromosome 3, contain a 5S rDNA locus. The detection of unique cosmids and YAC sequences demonstrates that detailed physical mapping of Arabidopsis chromosomes by cytogenetic techniques is feasible. Together with the presented karyotype this makes Arabidopsis a model system for detailed cytogenetic mapping.     

Chiasma formation in the short arm of the nucleolar chromosome of the tomato

Summary A translocation heterozygote in tomato (Lycopersicon esculentum) is shown to have a cyclical type of interchange between the long arms of chromosomes 1, 2 (nucleolar) and 3. A study of chromosome association in this plant at metaphase I has indicated that in 21% of the cells a ring of six chromosomes is present. Since an open ring hexavalent can occur only if there is chiasma formation in all the translocated segments and in all the short arms of the three chromosomes, it is concluded that there is considerable frequency of chiasma formation in the short arm of the nucleolar chromosome. This conclusion contradicts the previous observations that chiasma formation is either absent or very rare in the entirely dark staining chromatic, sometimes referred to as heterochromatic, short arm of the nucleolar chromosome.Part of this investigation was carried out at the Department of Genetics, Agricultural University, Wageningen, when the author was serving a contract between the EURATOM-I.T.A.L. and the Agricultural University.     

Increase in the number of the B-chromosomes and variants of their system in mouse Apodemus peninsulae in Mountain Altai population over 26 years

A new evolutionary genetic phenomenon - increase in the number of B-chromosomes (almost threefold) accompanied by change in B chromosome morphotypes - was discovered in the Artybash population of East Asian mouse Apodemus peninsulae (Mountain Altai) over a historically short period (22 years), comparable with a small number of mouse generations (ten generations). The process of increase in the number of A. peninsulae B-chromosomes in Mountain Altai has been monitored over 26 years (1980, 1986, 1988, 1990, 2002, and 2006). A concept of a new type of genomic mutations - mutations in the number of B-chromosomes-has been substantiated. The phenomenon of genomic mutations in pro-B-chromosomes has been discovered only in the Mountain Altai populations. This region differs from the remaining A. peninsulae dwelling localities by pollution with unspent liquid propellant (UDMH, heptyl). It is assumed that the variants of mouse B chromosome system in the studied A. peninsulae populations in the overall habitation area over long-term periods are likely to remain relatively stable and that their variation is controlled by homeostatic processes. Disturbance of these processes, in particular, due to anthropogenic load, can disrupt the cyclic pattern of increase and decrease in the number of A. peninsulae B-chromosomes in individual years.     

Construction and characterization of plasmid libraries enriched in sequences from single human chromosomes

Plasmid libraries enriched in sequences from single chromosome types have been constructed for all human chromosomes. This was accomplished by transferring inserts from the Charon 21A phage libraries constructed by the National Laboratory Gene Library Project into Bluescribe plasmids. Insert material freed by complete digestion of the phage libraries with HindIII or EcoRI was cloned into the corresponding sites in Bluescribe plasmids. The sizes of the Bluescribe library inserts determined by gel electrophoresis range from near 0 to approximately 6 kb. Fluorescence in situ hybridization (FISH) with the plasmid libraries showed that all hybridize along both arms of the expected (target) chromosome type with varying intensity. However, the plasmid libraries for chromosomes 1, 4, 9, 11, 16, 18, and 20 hybridize weakly or not at all near the centromeres of the target chromosome types. The libraries for chromosomes 13, 14, 15, 21, and 22 cross-hybridize near the centromeres of all members of this group and hybridize weakly to the short arms of the target chromosomes. FISH with each library allows specific staining of the target chromosome type in metaphase spreads. The signals resulting from FISH with libraries for chromosomes 1, 4, 8, 9, 13, 14, 17, 18, 21, and Y are sufficiently intense to permit analysis in interphase nuclei. Examples of the use of these libraries for translocation detection, marker chromosome characterization, and interphase aneuploidy analysis are presented.     

Multicolor fluorescence in situ hybridization with combinatorial labeling probes enables a detailed karyotype analysis of Larix principis-rupprechtii

The chromosomes (2n = 2x = 24) of Larix principis-rupprechtii are composed of six pairs of large metacentrics and six pairs of medium-sized submetacentrics. The identification of homologous pairs is hampered by their high degree of similarity at the morphological level in each group. As one of the most extensively used methods in molecular cytogenetics producing chromosome landmarks, fluorescence in situ hybridization (FISH) has significantly facilitated karyotype construction, especially in species with morphologically similar chromosomes. This study developed a simple but effective use of combinatorial labeling probes to distinguish chromosomes of Larix principis-rupprechtii by multicolor FISH. Three highly repetitive sequences in Larix were selected: 25S rDNA hybridized at all of the secondary constrictions of two pairs of metacentrics and the largest pair of submetacentrics; 5S rDNA hybridized at subtelomeric sites of one pair of metacentrics that also harboured 25S rDNA on different arms; LPD family sequences are tandem repeats hybridized at proximal regions of 22 chromosomes. The three different probes were labeled with only two different labels, hybridized to metaphase chromosomes of Larix principis-rupprechtii, simultaneously visualized, and unequivocally distinguished in a single FISH experiment. These multicolor FISH marks largely improved the karyotype analysis of Larix principis-rupprechtii.     

Molecular and cytological characterization of repetitive DNA sequences in Brassica

Summary We isolated three different repetitive DNA sequences from B. campestris and determined their nucleotide sequences. In order to analyze organization of these repetitive sequences in Brassica, Southern blot hybridization and in situ hybridization with metaphase chromosomes were performed. The sequence cloned in the plasmid pCS1 represented a middle repetitive sequence present only in B. campestris and not detected in closely related B. Oleracea. This sequence was localized at centromeric regions of six specific chromosomes of B. campestris. The second plasmid, pBT4, contained a part of the 25S ribosomal RNA gene, and its copy number was estimated to be 1,590 and 1,300 per haploid genome for B. campestris and B. oleracea, respectively. In situ hybridization with this sequence showed a clear signal at the NOR region found in the second largest chromosome of B. Campestris. The third plasmid, pBT11, contained a 175-bp insert that belongs to a major family of tandem repeats found in all the Brassica species. This sequence was detected at centromeric regions of all the B. campestris chromosomes. Our study indicates that in situ hybridization with various types of repetitive sequences should give important information on the evolution of repetitive DNA in Brassica species.     

Chromosome bin map of expressed sequence tags in homoeologous group 1 of hexaploid wheat and homoeology with rice and Arabidopsis

A total of 944 expressed sequence tags (ESTs) generated 2212 EST loci mapped to homoeologous group 1 chromosomes in hexaploid wheat (Triticum aestivum L.). EST deletion maps and the consensus map of group 1 chromosomes were constructed to show EST distribution. EST loci were unevenly distributed among chromosomes 1A, 1B, and 1D with 660, 826, and 726, respectively. The number of EST loci was greater on the long arms than on the short arms for all three chromosomes. The distribution of ESTs along chromosome arms was nonrandom with EST clusters occurring in the distal regions of short arms and middle regions of long arms. Duplications of group 1 ESTs in other homoeologous groups occurred at a rate of 35.5%. Seventy-five percent of wheat chromosome 1 ESTs had significant matches with rice sequences (E < or = e(-10)), where large regions of conservation occurred between wheat consensus chromosome 1 and rice chromosome 5 and between the proximal portion of the long arm of wheat consensus chromosome 1 and rice chromosome 10. Only 9.5% of group 1 ESTs showed significant matches to Arabidopsis genome sequences. The results presented are useful for gene mapping and evolutionary and comparative genomics of grasses.     

B chromosome morphotypes of Apodemus peninsulae (Rodentia) from the Russian Far East

Polymorphism of B chromosome morphotypes in the natural populations of the Korean field mouse Apodemus peninsulae (n = 367) has been studied in five regions of the Russian Far East: Magadan and Amur regions, the Jewish Autonomous region, Khabarovsk and Primorskii regions. Six groups of B chromosome morphotypes have been described on the size and morphological parameters. On the basis of their combinations 78 cell clones have been revealed, 23 of which are found in the animals with a stable karyotype and 76--in mosaics. The mosaics have also more diverse and unique combinations of B chromosome morphotypes. No differences between the geographic and local populations of mice on the studied characteristics have been found. Homogeneous structure of DNA in the B chromosomes of this species at the territory of the Russian Far East, as shown previously, allowed us to combine the numerical and dimensional data for each clone by introduction conditional "mass quantity" of B chromosomes (mB index). The leading role of natural selection in production of "critical" mass of supernumerary chromosomes in individuals with a stable karyotype and the weakening of its role in mosaics has been suggested.     

Characterisation of repeated sequences from microdissected B chromosomes of Crepis capillaris

The B chromosome of Crepis capillaris was isolated from the standard chromosomes by microdissection, and the chromosomal DNA amplified using the degenerate oligonucleotide-primed polymerase chain reaction (DOP-PCR). The PCR product was cloned and a Bspecific library created and characterised. Southern and in situ hybridisation analyses of the DOP-PCR product from microdissected B chromosomes confirmed that the B chromosome is composed mainly of sequences also present in the A chromosomes but lacks the main repeated DNA families located in the A-chromosomal heterochromatin. From 100 clones analysed, 12% of the generated B-chromosomal library was shown to be composed of dispersed repeats located in both the A and B chromosomes. No B-specific repeated sequence was detected. One of the most abundant repeated DNAs within the library, the family B134, was further characterised. Repeating units show a sequence similarity range from 69% to 90% and are characterised by their richness in (CA)n repeats. In situ hybridisation revealed that members of this family are dispersed throughout the A and B chromosomes but are more concentrated in the pericentromeric heterochromatin of the B, indicating that the molecular organization of B heterochromatin is different from that of the A chromosomes. Compared with the A chromosomes, the Bs contain about 20,000 copies per micron more of the B134 sequence. This indicates that B134 was amplified on the B chromosome after its origin. The B134 sequences in the B chromosomes have also diverged from those on the A chromosomes. Although the DNA composition of A and B chromosomes is similar, Bs are evolving separately from A chromosomes at the molecular level.     

Integrated cytogenetic map of mitotic metaphase chromosome 9 of maize: resolution,sensitivity, and banding paint development

To study the correlation of the sequence positions on the physical DNA finger print contig (FPC) map and cytogenetic maps of pachytene and somatic maize chromosomes, sequences located along the chromosome 9 FPC map approximately every 10 Mb were selected to place on maize chromosomes using fluorescent in situ hybridization (FISH). The probes were produced as pooled polymerase chain reaction products based on sequences of genetic markers or repeat-free portions of mapped bacterial artificial chromosome (BAC) clones. Fifteen probes were visualized on chromosome 9. The cytological positions of most sequences correspond on the pachytene, somatic, and FPC maps except some probes at the pericentromeric regions. Because of unequal condensation of mitotic metaphase chromosomes, being lower at pericentromeric regions and higher in the arms, probe positions are displaced to the distal ends of both arms. The axial resolution of FISH on somatic chromosome 9 varied from 3.3 to 8.2 Mb, which is 12-30 times lower than on pachytene chromosomes. The probe collection can be used as chromosomal landmarks or as a "banding paint" for the physical mapping of sequences including transgenes and BAC clones and for studying chromosomal rearrangements.     

Human laminin B1 chain. A multidomain protein with gene (LAMB1) locus in the q22 region of chromosome 7

We report the isolation and characterization of six overlapping cDNA clones that provide the first and complete amino acid sequence of the human laminin B1 chain. The cDNA clones cover 5613 nucleotides with 5358 nucleotides in an open reading frame encoding 1786 amino acids, including a 21-residue signal peptide-like sequence. Sequence analysis demonstrated the presence of two types of internal homology repeats that were found in clusters within the polypeptide chain. The type A repeats contain about 50 amino acids of which 8 are cysteine. These repeats are present in two clusters toward the NH2-terminal end of the chain and are separated from each other by about 220 amino acids. The two clusters contain five and eight consecutive repeats each. There are two copies of consecutive type B repeats of about 40 amino acids close to the COOH-terminal end. Computer analysis of the amino acid sequence of the B1 chain revealed the presence of structurally distinct domains that contain cysteine-rich repeats, globular regions, and helical structures. Using somatic cell hybrid methodology and in situ hybridization to metaphase chromosomes it was established that the human laminin B1 gene (LAMB1) is located in the q22 region of chromosome 7.     

B chromosomes of the Podisma sapporensis Shir. (Orthoptera, Acrididae) analysed by chromosome microdissection and FISH

The analysis of the distribution of repetitive DNA of the B chromosomes of Podisma sapporensis in the A and B chromosomes of the natural populations and in A chromosomes of three other species of the Podismini grasshoppers were made. DNA-libraries of the B chromosome and the euchromatic segment of the A chromosome of P. sapporensis were generated by meiotic chromosome microdissection followed by degenerated oligonucleotide primed polymerase chain reaction (DOP-PCR). Paints based on these DNA-libraries were used for FISH analysis to detect localization of homologous sequences in A and B chromosomes of P. sapporensis from different natural populations. On the basis of the FISH analysis the authors suggest that evolution of the B chromosomes in Podisma sapporensis was associated mainly with the insertions of "alien DNA sequences" into ancestral A chromosome and their further amplification. The number of initial sites of amplifications differed in the different Bs, the distance between these sites also varying. Karyotype evolution in P. sapporensis was associated partly with the insertion of "alien DNA sequences" into pericentromeric chromosomal regions. Insertion into the small short arms of the acrocentric chromosomes followed, with the DNA amplification leading to the formation of the additional C-heterochromatic arms or euchromatic-like regions of different size.