Transgenerational transmission of bovine satellite DNA in transgenic mice

Genetical, cytogenetical and molecular analysis was made for 5 generations of mice transgenic for bovine satellite DNA (Sat). In all cases transgenic mice were generated by crosses of transgenic males and females with normal (CBA x C57B1) mice. No abnormalities in the founder development were noticed. A normal (near 50 %) ratio of transgenic and nontransgenic offsprings was observed in blastocysts. However, profound differences occurred in the rate of transgene bearing offsprings, depending on the sex of grandparents rather than of parents. The grandfather Sat transmission resulted in the appearance of 0-52.4 % transgenic grandchildren, whereas the grandmother transmission ended in the theoretically expected rate. This means that stabilization of transsatellite took place upon the female germ line transmission (a positive grandmother effect). It is essential that in hemizygous transsatellite mice Sat integration led to the occurrence of mammary tumors, inflammation of uterine horns, and infringement of mother care of transgenic females. Simultaneous FISH and G-banding showed Sat to be localized in the internal region of chromosome 12 near Pax 9 and Brms 11 genes. Commonly, these genes are implicated in tumorigenesis as their expression decreases. Thus, a kind of silencing effect of these genes' expression may be supposed.     

The characteristics of chromosomal distribution and of the variability of the multiply repeating DNA fraction of the genome in Bos taurus L.]

The uniform distribution of satellite DNA II and IV has been revealed using in situ hybridization and differential staining in centromeric regions of autosomes. The sex chromosomes have not found such nucleotide blocks. There is only minor satellite IV block inside Y chromosome short arm. The Y chromosome has got some (TG)n enriched blocks distributed also among other parts of genome and one copy of sequences like human ZFY gene. The high repetitive fraction of bovine genomic DNA have not revealed RFLP. However, the difference has been found by blot hybridization between genomic organization of satellite IV in cattle and yak chromosomal DNA. Non-Mendelian distribution of some such nucleotide blocks has been obtained for interspecies crosses of cattle and yak.     

Analysis of alphoid DNA variation and kinetochore size in human chromosome 21: evidence against pathological significance of alphoid satellite DNA diminutions.

Centromeric alpha satellite DNA sequences are linked to the kinetochore CENP-B proteins and therefore may be involved in the centromeric function. The high heterogeneity of size of the alphoid blocks raises the question of whether small amount of alphoid DNA or "deletion" of this block may have a pathological significance in the human centromere. In the present study, we analysed the correlation between size variations of alphoid DNA and kinetochore sizes in human chromosome 21 by molecular cytogenetic and immunochemical techniques. FISH analyses of alpha satellite DNA sizes in chromosome 21 homologues correlated well with the variation of their physical size as determined by pulsed field gel electrophoresis (PFGE). By contrast, the immunostaining study of the same homologous chromosomes with antikinetochore antibodies suggested that there is no positive correlation between the alpha satellite DNA block and kinetochore sizes. FISH analysis of chromosome 21-specific alphoid DNA and immunostaining of kinetochore extended interphase chromatin fibers indicate that centromeric kinetochore-specific proteins bind to restricted areas of centromeric DNA arrays. Thus, probably, restricted regions of centromeric DNA play an important role in kinetochore formation, centromeric function and abnormal chromosome segregation leading to non-disjunction.     

Modeling heterochromatin regions in transgenic mice

Transgenic mice carrying bovine satellite DNA IV were obtained. The size of the transgene integrated into the mouse genome was approximately 390 kb (about 100 transgene copies) as determined by a semiquantitative PCR. Restriction analysis with isoschizomeric restrictases HpaII and MspI, showed that the alien DNA was methylated. In the genome of a transgenic founder male, two integration sites for satellite DNA IV were revealed by in situ hybridization and in situ PCR. These sites are situated on two different chromosomes: in pericentromeric heterochromatin and within a chromosomal arm. In transgenic mice, de novo formation of heterochromatin regions (C-block and the CMA3 disk within the centromeric heterochromatin of another chromosome) was revealed by C-banding and staining with chromomycin A3. This formation is not characteristic of mice, because their chromosomes normally contain no interstitial C-blocks or sequences intensely stained by chromomycin A3.     

Locations of a Human Satellite DNA in Human Chromosomes

USING techniques for DNA/RNA or DNA/DNA hybridization in situ, Pardue and Gall¹ and Jones² made several significant discoveries on the chromosomal locations of the mouse satellite DNA: (1) this fraction of DNA is found in all chromosomes except the Y, (2) the cytological location of the satellite DNA is limited to the centromeric region of each chromosome and is probably absent in other regions and (3) the centromeric regions of all mouse chromosomes are hetero-chromatic.     

Replication of yeast DNA and novel chromosome formation in mouse cells.

To determine whether yeast DNA can replicate or segregate in mammalian cells, we have transferred genomic DNA from the yeast Saccharomyces cerevisiae into mouse cells. Most of the lines contained stably integrated yeast DNA. However, in two of the lines, the yeast DNA was maintained as numerous small extrachromosomal elements which were still present after 26 cell divisions in selection but which were lost rapidly out of selection. This indicates that, although yeast DNA can replicate in mouse cells, the yeast centromere does not function to give segregation. In one cell line we observed a large novel chromosome consisting almost entirely of yeast DNA. This chromosome segregates well and contains mouse centromeric minor satellite DNA and variable amounts of major satellite DNA which probably comprise the functional centromere. The yeast DNA in the novel chromosome has a compacted chromatin structure which may be responsible for the efficient formation of anaphase bridges. Furthermore, yeast DNA integrated into mouse chromosomes forms constrictions at the point of integration. These features have previously been presumed to be hallmarks of centromeric function in transfection assays aimed at identifying putative centromeric DNA. Hence our results suggest caution be exercised in the interpretation of such assays.     

Instability of repetitive units of foreign centromeric satellite DNA in transgenic mice and transfected cells

Cytologically detectable instability of centromeric satellite DNA may cause hereditary disorders in human. To study the mechanisms of such instability, two transgenic mouse lines and 11 clones of transfected F9 mouse embryonic teratocarcinoma cells were obtained with the 3.8-kb repetitive unit (Sat) of Bos taurus satellite DNA IV. Intergeneration and somatic instability of exogenous satellite DNA (satDNA) was observed in transgenic mice and transfected cells as a change in nucleotide sequence of an internal Sat region approximately 1000 bp in size. Since Sat was in the hemizygous state in both cases by the experimental protocol, the instability was attributed to intra-allelic processes. Intergeneration instability probably took place in the premeiotic period of gametogenesis or in early embryo development and led to prenatal death of transgenic embryos after at least one generation. No direct or inverse correlation was observed between methylation and instability of Sat. The results testify that submicroscopic changes in highly repetitive noncoding DNA sequences may already affect the genome function in higher eukaryotes.     

Characterization and chromosomal distribution of satellite DNA sequences of the water buffalo (Bubalus bubalis).

Satellite DNA sequences were isolated from the water buffalo (Bubalus bubalis) after digestion with two restriction endonucleases, BamHI and StuI. These satellite DNAs of the water buffalo were classified into two types by sequence analysis: one had an approximately 1,400 bp tandem repeat unit with 79% similarity to the bovine satellite I DNA; the other had an approximately 700 bp tandem repeat unit with 81% similarity to the bovine satellite II DNA. The chromosomal distribution of the satellite DNAs were examined in the river-type and the swamp-type buffaloes with direct R-banding fluorescence in situ hybridization. Both the buffalo satellite DNAs were localized to the centromeric regions of all chromosomes in the two types of buffaloes. The hybridization signals with the buffalo satellite I DNA on the acrocentric autosomes and X chromosome were much stronger than that on the biarmed autosomes and Y chromosome, which corresponded to the distribution of C-band-positive centromeric heterochromatin. This centromere-specific satellite DNA also existed in the interstitial region of the long arm of chromosome 1 of the swamp-type buffalo, which was the junction of the telomere-centromere tandem fusion that divided the karyotype in the two types of buffaloes. The intensity of the hybridization signals with buffalo satellite II DNA was almost the same over all the chromosomes, including the Y chromosome, and no additional hybridization signal was found in noncentromeric sites.     

Instability of Repetitive Units of Foreign Centromeric Satellite DNA in Transgenic Mice and Transfected Cells

Cytologically detectable instability of centromeric satellite DNA may cause hereditary disorders in human. To study the mechanisms of such instability, two transgenic mouse lines and 11 clones of transfected F9 mouse embryonic teratocarcinoma cells were obtained with the 3.8-kb repetitive unit (Sat) of Bos taurus satellite DNA IV. Intergeneration and somatic instability of exogenous satellite DNA (satDNA) was observed in transgenic mice and transfected cells as a change in nucleotide sequence of an internal Sat region approximately 1000 bp in size. Since Sat was in the hemizygous state in both cases by the experimental protocol, the instability was attributed to intra-allelic processes. Intergeneration instability probably took place in the premeiotic period of gametogenesis or in early embryo development and led to prenatal death of transgenic embryos after at least one generation. No direct or inverse correlation was observed between methylation and instability of Sat. The results testify that submicroscopic changes in highly repetitive noncoding DNA sequences may already affect the genome function in higher eukaryotes.     

Retrotransposon-related DNA sequences in the centromeres of grass chromosomes.

Several distinct DNA fragments were subcloned from a sorghum (Sorghum bicolor) bacterial artificial chromosome clone 13I16 that was derived from a centromere. Three fragments showed significant sequence identity to either Ty3/gypsy- or Ty1/copia-like retrotransposons. Fluorescence in situ hybridization (FISH) analysis revealed that the Ty1/copia-related DNA sequences are not specific to the centromeric regions. However, the Ty3/gypsy-related sequences were present exclusively in the centromeres of all sorghum chromosomes. FISH and gel-blot hybridization showed that these sequences are also conserved in the centromeric regions of all species within Gramineae. Thus, we report a new retrotransposon that is conserved in specific chromosomal regions of distantly related eukaryotic species. We propose that the Ty3/gypsy-like retrotransposons in the grass centromeres may be ancient insertions and are likely to have been amplified during centromere evolution. The possible role of centromeric retrotransposons in plant centromere function is discussed.     

Whole-genome fractionation rapidly purifies DNA from centromeric regions

The condensed centromeric regions of higher eukaryotic chromosomes contain satellite sequences, transposons and retroelements, as well as transcribed genes that perform a variety of functions. These chromosomal domains nucleate kinetochores, mediate sister chromatid cohesion and inhibit recombination, yet their characterization has often lagged behind that of chromosome arms. Here, we describe a whole-genome fractionation technique that rapidly identifies bacterial artificial chromosome (BAC) clones derived from plant centromeric regions. This approach, which relies on hybridization of methylated genomic DNA, revealed BACs that correspond to the genetically mapped and sequenced Arabidopsis thaliana centromeric regions. Extending this method to other species in the Brassicaceae family identified centromere-linked clones and provided genome-wide estimates of methylated DNA abundance. Sequencing these clones will elucidate the changes that occur during plant centromere evolution. This genomic fractionation technique could identify centromeric DNA in genomes with similar methylation and repetitive DNA content, including those from crops and mammals.     

An integrated molecular cytogenetic map of Cucumis sativus L. chromosome 2

Background

Integration of molecular, genetic and cytological maps is still a challenge for most plant species. Recent progress in molecular and cytogenetic studies created a basis for developing integrated maps in cucumber (Cucumis sativus L.).

Results

In this study, eleven fosmid clones and three plasmids containing 45S rDNA, the centromeric satellite repeat Type III and the pericentriomeric repeat CsRP1 sequences respectively were hybridized to cucumber metaphase chromosomes to assign their cytological location on chromosome 2. Moreover, an integrated molecular cytogenetic map of cucumber chromosomes 2 was constructed by fluorescence in situ hybridization (FISH) mapping of 11 fosmid clones together with the cucumber centromere-specific Type III sequence on meiotic pachytene chromosomes. The cytogenetic map was fully integrated with genetic linkage map since each fosmid clone was anchored by a genetically mapped simple sequence repeat marker (SSR). The relationship between the genetic and physical distances along chromosome was analyzed.

Conclusions

Recombination was not evenly distributed along the physical length of chromosome 2. Suppression of recombination was found in centromeric and pericentromeric regions. Our results also indicated that the molecular markers composing the linkage map for chromosome 2 provided excellent coverage of the chromosome.     

Characterization of a New HpaI Centromeric Satellite DNA in Salmo salar

A highly repeated HpaI DNA family was revealed in Atlantic salmon (Salmo salar) and analyzed by Southern blotting and fluorescence in situ hybridization (FISH). In this report, we describe the nucleotide sequence, genomic structure and chromosomal localization of this HpaI repeat. This novel satellite appeared tandemly arrayed and located at centromeric areas of three acrocentric chromosome pairs as evidenced by FISH. The sequence was characterized by a high AT content (63%), a short consensus motif (A/T)(G/C)AAA(T/C) similar to other centromeric satellites motifs, and by short AT enriched stretches. The presence of this sequence in other salmonid species was also tested by Southern blot hybridization and used to analyze its evolution within this group.     

A degenerate alpha satellite probe, detecting a centromeric deletion on chromosome 21 in an apparently normal human male, shows limitations of the use of satellite DNA probes for interphase ploidy analysis.

A degenerate alpha satellite DNA probe specific for a repeated sequence on human chromosomes 13 and 21 was synthesized using the polymerase chain reaction (PCR). Fluorescence in situ hybridization (FISH) with this probe to normal metaphase spreads revealed strong probe binding to the centromeric regions of human chromosomes 13 and 21 with negligible cross-hybridization with other chromosomes. FISH to normal interphase cell nuclei showed four distinct domains of probe binding. However, hybridization with probe to interphase and metaphase preparations from one apparently normal human male resulted in only three major binding domains. Metaphase chromosome analysis revealed a centromeric deletion on one chromosome 21 that caused greatly reduced probe binding. The result suggest caution in the interpretation of interphase ploidy studies performed with chromosome-specific alphoid DNA probes.     

Chromosome localization and orientation of the simple sequence repeat of human satellite I DNA

The predominant chromosomal locations of human satellite I DNA were detected using fluorescent in situ hybridization (FISH). Synthetic deoxyoligonucleotides designed from consensus sequences of the simple sequence repeats of satellite 1 were used as probes. The most abundant satellite I repeat, the-A-B-A-B-A-form, is located at the pericentromeric regions of chromosomes 3, 4, 13, 14, 15, 21, and 22. The less abundant-B-B-B-form was not detected on chromosome 4, but was present at all the other locations. A variation of FISH that allows strand-specific hybridization of single-stranded probes (CO-FISH) determined that the human satellite I sequences are predominantly arranged in head-to-tail fashtion along the DNA strand.     

Biological effects of a bifunctional DNA cross-linker. II. Generation of micronuclei and attached micronuclear-like structures.

Madin-Darby bovine kidney (MDBK) cells were treated with the bifunctional DNA cross-linker, L-7, to examine the generation of micronuclei and other nuclear abnormalities. The preceding paper demonstrates that L-7 treatment induces the formation of triradial and quadriradial chromosomes in MDBK cells. These chromosomes are believed to result from interduplex DNA cross-links formed between G-C rich centromeric satellite DNA regions on non-sister chromatids. Treatment produces a majority of centromere-positive micronuclei. In addition, many daughter cells remain attached by chromatin bridges which are sometimes beaded with micronuclei. Up to 15% of cell nuclei become lobular and fused with numerous micronuclear-like structures attached to their membranes. These attached structures are classified as attached micronuclear-like structures (AMNLS). Fluorescence in situ hybridization (FISH) using a centromeric satellite sequence was performed on treated cells. Hybridization reveals that intercellular bridges are composed of centromeric sequences and initiate at centromeric foci in daughter cells. Furthermore, the majority of junctions between AMNLS and nuclei contain an enhancement of centromeric signal. The frequency of AMNLS appears dependent on the concentration of L-7 and the duration of treatment. Similar results were found for the generation of cross-linked chromosome products in the previous paper. We suggest that AMNLS result from the abnormal mitotic segregation of cross-linked chromosome products.     

Characterisation of pericentrometric and sticky intercalary heterochromatin in Ornithogalum longibracteatum (Hyacinthaceae)

The hexaploid liliaceous plant Ornithogalum longibracteatum (2n=6x=54) has a heterochromatin-rich bimodal karyotype with large (L) and small (S) chromosomes. The composition and subgenomic distribution of heterochromatin was studied using molecular and cytological methods. The major component of centromeric heterochromatin in all chromosomes is Satl, an abundant satellite DNA with a basic repeat unit of 155 bp and an average A+T content (54%). The major component of the large blocks of intercalary heterochromatin in L chromosomes is Sat2, an abundant satellite DNA with a basic repeat unit of 115 bp and a high A+T content (76%). Additionally, traces of Sat2 can be detected at the centromeric regions of S chromosomes, while minor amounts of Satl are discernible in intercalary heterochromatin of L chromosomes. The chromosomal localisation pattern of Sat2 is consistent with the fluorescent staining pattern obtained with the A+T-specific DNA ligand 4'-6-diamidino-2-phenylindole (DAPI). A+T-rich intercalary heterochromatin is sticky and tends to associate ectopically during mitosis. Sister chromatid exchange clustering was found at the junctions between euchromatin and heterochromatin and at the centromeres. The pattern of mitosis-specific phosphorylation of histone H3 was not uniform along the length of the chromosomes. In all L and S chromosomes, from early prophase to ana-/telophase, there is hyperphosphorylation of histone H3 in the pericentromeric chromatin and a slightly elevated phosphorylated histone H3 level at the intercalary heterochromatin of L chromosomes. Consequently, the overall phosphorylated histone H3 metaphase labelling resembles the distribution of Satl in the karyotype of O. longibracteatum.     

Satellite DNA I in chromatin loops of rat pachytene chromosomes and in spermatids

Biotinylated rat satellite DNA I probe p93-50 was used to visualize the chromatin of surface-spread rat pachytene chromosomes. Fluorescein isothiocyanate (FITC)-conjugated avidin produces a beaded fluorescence pattern along the chromatin loops that insert in the centromeric region of the synaptonemal complex (SC), the paired cores of homologous chromosomes. The number of fluorescent beads ranges from zero for centromeres without satellite DNA I homologous to probe p 93-50, to several hundred for satellite-rich centromeric regions. For the chromosomes that can be identified, the relative amount of satellite DNA is chromosome specific. No satellite DNA I was detected at the non-centromeric ends of the chromosomes or interstitially. DNase-digested nuclei or isolated SCs did not have detectable amounts of satellite DNA in the centromeric regions of the chromosomes or in the residual SCs. The fate of the satellite DNA was followed during spermiogenesis. In the round spermatid the centromeric regions, which appear to be attached to the nuclear envelope, are still distinct and have converging loops of fluorescent chromatin. At later stages there are fewer but still bright fluorescent patches. Satellite DNA I is still detectable in the mature sperm head. These results demonstrate the organization of satellite DNA I in the chromatin loops at the centromeric regions, and they forecast the analysis of chromosome organization in unprecedented detail with a variety of probes in surface spreads of meiotic prophase chromosomes.     

An approach for quantitative assessment of fluorescence in situ hybridization (FISH) signals for applied human molecular cytogenetics.

A number of applied molecular cytogenetic studies require the quantitative assessment of fluorescence in situ hybridization (FISH) signals (for example, interphase FISH analysis of aneuploidy by chromosome enumeration DNA probes; analysis of somatic pairing of homologous chromosomes in interphase nuclei; identification of chromosomal heteromorphism after FISH with satellite DNA probes for differentiation of parental origin of homologous chromosome, etc.). We have performed a pilot study to develop a simple technique for quantitative assessment of FISH signals by means of the digital capturing of microscopic images and the intensity measuring of hybridization signals using Scion Image software, commonly used for quantification of electrophoresis gels. We have tested this approach by quantitative analysis of FISH signals after application of chromosome-specific DNA probes for aneuploidy scoring in interphase nuclei in cells of different human tissues. This approach allowed us to exclude or confirm a low-level mosaic form of aneuploidy by quantification of FISH signals (for example, discrimination of pseudo-monosomy and artifact signals due to over-position of hybridization signals). Quantification of FISH signals was also used for analysis of somatic pairing of homologous chromosomes in nuclei of postmortem brain tissues after FISH with "classical" satellite DNA probes for chromosomes 1, 9, and 16. This approach has shown a relatively high efficiency for the quantitative registration of chromosomal heteromorphism due to variations of centromeric alphoid DNA in homologous parental chromosomes. We propose this approach to be efficient and to be considered as a useful tool in addition to visual FISH signal analysis for applied molecular cytogenetic studies.     

Precise karyotyping of carrot mitotic chromosomes using multicolour-FISH with repetitive DNA

Carrot (Daucus carota L.) chromosomes are small and uniform in shape and length. Here, mitotic chromosomes were subjected to multicolour fluorescence in situ hybridization (mFISH) with probes derived from conserved plant repetitive DNA (18-25S and 5S rDNA, telomeres), a carrot-specific centromeric repeat (Cent-Dc), carrot-specific repetitive elements (DCREs), and miniature inverted-repeat transposable elements (MITEs). A set of major chromosomal landmarks comprising rDNA and telomeric and centromeric sequences in combination with chromosomal measurements enabled discrimination of carrot chromosomes. In addition, reproducible and unique FISH patterns generated by three carrot genome-specific repeats (DCRE22, DCRE16, and DCRE9) and two transposon families (DcSto and Krak) in combination with telomeric and centromeric reference probes allowed identification of chromosome pairs and construction of detailed carrot karyotypes. Hybridization patterns for DCREs were observed as pericentromeric and interstitial dotted tracks (DCRE22), signals in pericentromeric regions (DCRE16), or scattered signals (DCRE9) along chromosomes similar to those observed for both MITE families.