In situ digestion of satellite DNA of Scilla siberica

Restriction endonucleases have been used to digest DNA in fixed metaphase chromosomes of animal species. However, constitutive C-heterochromatin of plant species is resistant to these enzymes suggesting that the special structural organization of plant C-bands is an impediment to the activity of restriction endonucleases. In order to test this hypothesis, we have chosen the species Scilla siberica, whose purified satellite DNA, localised at the heterochromatic regions, is extensively digested by HaeIII. In situ treatment with HaeIII alone does not produce significant digestion of heterochromatin, but subsequent treatment with proteinase K results in extensive digestion of heterochromatic regions producing unstained gaps. These results indicate that HaeIII is able to access and cut chromosomal DNA from C-bands, but the DNA fragments remain attached to chromosomal proteins that characterize the complex structure of heterochromatin in this species. Although there are no reasons to suppose that accessibility of chromosomal DNA of S. siberica to restriction enzymes can be impeded, it would be reasonable to think from our results that some special features of heterochromatin organization in plants contribute to the formation of a complex structure that makes chromosomal DNA extraction impossible.by D. Schweizer     

High frequency DNA rearrangements associated with mouse centromeric satellite DNA

A DNA transformed mouse cell line, generated by the microinjection of a pBR322 plasmid containing the herpes thymidine kinase (tk) gene, was observed to exhibit a high frequency of DNA rearrangement at the site of exogenous DNA integration. The instability in this cell line does not appear to be mediated by the tk inserts or the immediately adjacent mouse DNA, but instead may be a consequence of the larger host environment at the chromosomal site of tk insertion. Results obtained from restriction analysis, in situ chromosome hybridizations, and cesium chloride density-gradient fractionations indicate that the tk inserts are organized as a single cluster of direct and inverted repeats embedded within pericentromeric satellite DNA. To determine the molecular identity of the flanking host sequences, one of the mouse-tk junction fragments was cloned, and subsequent restriction and sequence analyses revealed that this DNA fragment consists almost entirely of classical mouse satellite DNA. On the basis of these observations, we suggest that the instability in this cell line may reflect the endogenous instability or fluidity of satellite DNA.     

In situ binding of AT-rich repetitive DNA to the centromeric heterochromatin in polytene chromosomes of chironomids

Native highly repetitive DNA sequences have been allowed to react in situ with DNA-depleted polytene chromosomes of chironomids in cytological preparations. The double-stranded DNA can bind specifically to the centromeric heterochromatin, where these sequences have been localized previously by in situ hybridization. Various control experiments support the conception that heterochromatin-specific DNA-binding proteins are involved in the in situ binding. Dedicated to Prof. Dr. Wolfgang Beermann for his 60th birthday     

Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence

The cosmid clone, CX16-2D12, was previously localized to the centromeric region of the human X chromosome and shown to lack human X-specific satellite DNA. A 1.2 kb EcoRI fragment was subcloned from the CX16-2D12 cosmid and was named 2D12/E2. DNA sequencing revealed that this 1,205 bp fragment consisted of approximately five tandemly repeated DNA monomers of 220 bp. DNA sequence homology between the monomers of 2D12/E2 ranged from 72.8% to 78.6%. Interestingly, DNA sequence analysis of the 2D12/E2 clone displayed a change in monomer unit orientation between nucleotide positions 585–586 from a tail-to-head arrangement to a head-to-tail configuration. This may reflect the existence of at least one inversion within this repetitive DNA array in the centromeric region of the human X chromosome. The DNA consensus sequence derived from a compilation of these 220 bp monomers had approximately 62% DNA sequence similarity to the previously determined 8 satellite DNA consensus sequence. Comparison of the 2D12/E2 and 8 consensus sequences revealed a 20 bp DNA sequence that was well conserved in both DNA consensus sequences. Slot-blot analysis revealed that this repetitive DNA sequence comprises approximately 0.015% of the human genome, similar to that found with 8 satellite DNA. These observations suggest that this satellite DNA clone is derived from a subfamily of satellite DNA and is thus designated X satellite DNA. When genomic DNA from six unrelated males and two unrelated females was cut with SstI or HpaI and separated by pulsed-field gel electrophoresis, no restriction fragment length polymorphisms were observed for either X (2D12/E2) or 8 (50E4) probes. Fluorescence in situ hybridization localized the 2D12/E2 clone to the lateral sides of the primary constriction specifically on the human X chromosome.     

Isolation and characterization of salmonid telomeric and centromeric satellite DNA sequences

Satellite DNA clones with a 37 bp repeat unit were obtained from BglII-digested genomic DNA of Masu salmon (Oncorhynchus masou) and Chum salmon (O. keta). Fluorescence in situ hybridization (FISH) analysis with the isolated clones as a probe showed that these repetitive sequences were localized in the telomeric regions of chromosomes in both species. Southern and dot blot analyses suggested conservation of homologous sequences with similar repeat unit in other salmonids including the species of the genus Oncorhynchus and Salvelinus, but lack or scarcity of such sequences in the genus Hucho and Salmo. Similarly, polymerase chain reaction (PCR)-based cloning of satellite DNA referring to a reported Rainbow trout (O. mykiss) centromeric sequence was successful for the Oncorhynchus, Salvelinus and Hucho species. The obtained satellite DNA clones were localized with FISH in the centromeric regions of chromosomes of the species from these three genera. Although PCR cloning of the centromeric satellite DNA had failed in the Salmo species due to some base changes in the priming sites, dot blot hybridization analysis suggested conservation of homologous satellite DNA in the genus Salmo as in the other three genera. In the neighbor-joining tree of cloned centromeric satellite DNA sequences, the genus Oncorhynchus and Salvelinus formed adjacent clades, and the clade of the genus Hucho included the reported centromeric sequence of the genus Salmo. Conservation pattern and molecular phylogeny of the telomeric and centromeric satellite DNA sequences isolated herein support a close phylogenetic relationship between the genus Oncorhynchus and Salvelinus and between the Salmo and Hucho.     

The murine nuclear matrix protein specificcaly binds to centromeric satellite DNA

The nuclear matrix (NM) of mouse contains a protein (miSat BP) that can specifically bind to mouse centromeric minor satellite DNA as shown by gel shift assay. The ion-exchange chromatography on DEAE-Sepharose was used as the first miSat BP purification. MiSat BT was eluted by 0.2 M NaCl. Antibodies against p70, a human NM protein of 70 kDa described earlier as a protein recognizing human alphoid DNA, produce hypershift effect when added to the retardation incubation mix. Immunoblotting of NM and an active NM fraction (0.2 M NaCl) with these antibodies revealed a protein with 70 kDa in both preparations. This antigen retained in NM in situ being associated with residual DNA as shown by indirect immunofluorescent staining. In the untreated interphase nucleus most of miSat BP granules were shown to be colocalized with prekinetochores. We suggest that miSat BP is capable of recognizing the minor satellite DNA due to its structural features, but it does not belong to a group of constitutive centromeric proteins.     

Evolution of a centromeric satellite DNA and phylogeny of lacertid lizards.

1. The composition and phyletic distribution of a highly repetitive satellite DNA, isolated from Podarcis sicula, was studied. 2. This DNA was rich in adenine and thymine and displayed frequent adenine stretches. It was always located on the centromeric heterochromatin even in quite taxonomically distant species. 3. Southern blot hybridization of the Taq I satellite on various species of lacertid families showed a close affinity among Podarcis, Algyroides and Lacerta dugesii. 4. All the other taxa investigated did not seem to possess this repeated sequence.     

PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis.

Alpha satellite DNA is a tandemly repetitive DNA family found at the centromere of every human chromosome. Chromosome-specific subsets have been isolated for over half the chromosomes and have prove useful as markers for both genetic and physical mapping. We have developed specific oligonucleotide primer sets for polymerase chain reaction (PCR) amplification of alpha satellite DNA from chromosomes 3, 7, 13/21, 17, X, and Y. For each set of primers, PCR products amplified from human genomic DNA are specific for the centromere of the target chromosome(s), as shown by somatic cell hybrid mapping and by fluorescence in situ hybridization. These six subsets represent several evolutionarily related alpha satellite subfamilies, suggesting that specific primer pairs can be designed for most or all chromosomal subsets in the genome. The PCR products from chromosome 17 directly reveal the polymorphic nature of this subset, and a new DraI polymorphism is described. The PCR products from chromosome 13 are also polymorphic, allowing in informative cases genetic analysis of this centromeric subset distinguished from the highly homologous chromosome 21 subset. These primer sets should allow placement of individual centromeres on the proposed STS map of the human genome and may be useful for somatic cell hybrid characterization and for making in situ probes. In addition, the ability to amplify chromosome-specific repetitive DNA families directly will contribute to the structural and functional analysis of these abundant classes of DNA.     

An alpha satellite DNA polymorphism specific for the centromeric region of chromosome 13

Alpha satellite DNA is composed of variants of a short consensus sequence that are repeated in tandem arrays in the centromeric heterochromatin of each human chromosome. To define centromeric markers for linkage studies, we screened human genomic DNA for restriction fragment length polymorphisms using a probe detecting alphoid sequences on chromosomes 13 and 21. We describe one such DNA polymorphism. Analysis of linkage of this DNA marker to other polymorphic markers in the CEPH pedigrees demonstrates linkage to markers on the proximal long arm of chromosome 13 and defines the centromeric end of the linkage map of this chromosome.     

Functional selection and analysis of yeast centromeric DNA

A direct selection procedure has been used to isolate 11 distinct yeast genomic DNA fragments that eliminate the extreme segregation bias characteristic of autonomously replicating yeast plasmids. The selection scheme takes advantage of the fact that the cloned ochre suppressing tRNA gene, SUP11, is lethal at high copy number and therefore causes cell death when present on an ARS plasmid that lacks a cis-acting partition function. Each of the cloned DNA sequences was mapped to specific yeast chromosomes by hybridization to chromosome-sized DNA molecules separated by alternating field electrophoresis. Ten of the cloned fragments correspond to chromosomal centromeres; one fragment corresponds to the cis-acting locus required for endogenous 2 mu plasmid stability. Nucleotide sequence comparison of the ten centromere DNAs gives a new picture of conserved centromere DNA elements.     

Analysis of extrachromosomal structures containing human centromeric alphoid satellite DNA sequences in mouse cells

Yeast artificial chromosomes (YACs) spanning the centromeric region of the human Y chromosome were introduced into mouse LA-9 cells by spheroplast fusion in order to determine whether they would form mammalian artificial chromosomes. In about 50% of the cell lines generated, the YAC DNA was associated with circular extrachromosomal structures. These episomes were only present in a proportion of the cells, usually at high copy number, and were lost rapidly in the absence of selection. These observations suggest that, despite the presence of centromeric sequences, the structures were not segregating efficiently and thus were not forming artificial chromosomes. However, extrachromosomal structures containing alphoid DNA appeared cytogenetically smaller than those lacking it, as long as yeast DNA was also absent. This suggests that alphoid DNA can generate the condensed chromatin structure at the centromere. Edited by: H. F. Willard     

Detection of novel centromeric polymorphisms associated with alpha satellite DNA from human chromosome 11

Summary The pericentromeric region of human chromosomes is composed of diverse classes of repetitive DNAs, which provide a rich source of genetic variability. Here, we describe two novel centromeric polymorphisms associated with a subset of alpha satellite repetitive DNA, D11Z1, which is specific for human chromosome 11. Segregation and inheritance of the polymorphisms are demonstrated and their relative frequencies are determined. These polymorphisms may be useful genetic tools for distinguishing between individual chromosome 11 centromeres. In addition, these polymorphisms may be applied to the development of a centromerebased genetic linkage map of chromosome 11. Molecular models for the generation of these polymorphisms are discussed.     

A centromeric satellite DNA in the European plethodontid salamanders (Amphibia, Urodela).

A highly repeated satellite DNA (Hy500) located in the centromeric heterochromatin of the European plethodontid salamander Speleomantes (formerly Hydromantes) was studied. The Hy500 family represents about 1% of the Speleomantes supramontis genome and has a major repeating unit of about 500 base pairs, which may have evolved from the progressive amplification of shorter sequences. This centromeric satellite is conserved in all the Speleomantes species, which nevertheless show distinct patterns of chromosomal distribution, which are of relevance as to their phylogenetic relationships.     

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.     

Direct visualization of the genomic distribution and organization of two cervid centromeric satellite DNA families

Several repetitive DNA fragments were generated from PCR amplifications of caribou DNA using primer sequences derived from the white-tailed deer satellite II DNA clone OvDII. Two fragments, designated Rt-0.5 and Rt-0.7, were sequenced and found to have 96% sequence similarity. These caribou clones also had 85% sequence similarity with OvDII. Multiple-colored fluorescence in situ hybridization (FISH) studies with satellite I and satellite II DNA probes to caribou metaphase chromosomes and extended chromatin fibers provided direct visualization of the genomic organization of these two satellite DNA families, with the following findings: (1) Cervid satellite I DNA is confined to the centromeric regions of the acrocentric autosomes, whereas satellite II DNA is found at the centromeric regions of all chromosomes except for the Y. (2) For most acrocentric chromosomes, the satellite I signal appeared to be medially located at the primary constriction, in contrast to that of satellite II, which appeared to be oriented toward the lateral sides as two separate fluorescent dots. (3) The satellite II clone Rt-0.7 appeared to be enriched in the centromeric region of the caribou X chromosome, a pair of biarmed autosomes, and a number of other acrocentric autosomes. (4) Fiber-FISH demonstrated that the satellite I and satellite II arrays were juxtaposed. On highly extended chromatin fibers, the total length of the hybridization signals for the two satellite DNA arrays often reached 300-400 microm. The length of a given satellite II array usually reached 200 microm, corresponding to 2 x 10(3) kb of DNA in a given centromere.     

Phylogenetic and structural analysis of centromeric DNA and kinetochore proteins

Background  

Kinetochores are large multi-protein structures that assemble on centromeric DNA (CEN DNA) and mediate the binding of chromosomes to microtubules. Comprising 125 base-pairs of CEN DNA and 70 or more protein components, Saccharomyces cerevisiae kinetochores are among the best understood. In contrast, most fungal, plant and animal cells assemble kinetochores on CENs that are longer and more complex, raising the question of whether kinetochore architecture has been conserved through evolution, despite considerable divergence in CEN sequence.     

DNA amplification fingerprinting analysis of bermudagrass (Cynodon): genetic relationships between species and interspecific crosses

We have used DNA amplification fingerprinting (DAF) to study the genetic variation of bermudagrass (Cynodon) species and cultivars of interspecific crosses that exhibit leaf-blade textural characteristics ranging from coarse to fine. Arbitrary octamer primers produced complex and reproducible amplification profiles with high levels of polymorphic DNA. Phylogenetic analysis using parsimony (PAUP) and unweighted pair group cluster analysis using arithmetic means (UPGMA) grouped 13 bermudagrass cultivars into several clusters, including one containing the African-type bermudagrasses (C. transvaalensis) and another containing the common-type bermudagrasses (C. dactylon). The latter group included C. magennissii (Sunturf) and a interspecific C. transvaalensisxC. dactylon cross (Midiron), 2 cultivars that exhibited leaf textural characteristics closer to the common-types. All other C. transvaalensisxC. dactylon crosses grouped between the African and common types. An extended screen of 81 octamer primers was needed to separate cultivar Tifway from the irradiation-induced mutant Tifway II. The use of either template endonuclease digestion prior to amplification or arbitrary mini-hairpin primers increased detection of polymorphic DNA and simplified the task of distinguishing these closely related cultivars. Alternatively, the use of capillary electrophoresis (CE) resolved fingerprints adequately and detected products with high sensitivity, thereby promising to increase throughput and the detection of polymorphic DNA. When used to fingerprint samples from commercial sources, DAF identified bermudagrass plant material on the basis of unique reference profiles generated with selected primers. DAF represents an excellent technique for bermudagrass cultivar verification, seed certification, varietal protection, and for the identification of mistakes in plantings, mislabeled plant materials, and contamination or substitutions of sod fields.     

Quantitative analysis of chromosomal localization of two human cloned satellite DNA III sequences by in situ hybridization

Chromosomal location of two cloned human satellite DNA III sequences pPD9 and pPD18 has been studied in 30 individuals by in situ hybridization. Pericentromeric localization of the DNA subsets studied was found in practically all chromosomes of the set. The majority of label was observed over the pericentromeric region of chromosome 9 (38.3% for pPD18 clone and 26.2% for pPD9), the short arm of chromosome 15 (17.2% - the pPD9 clone and 10.6% - the pPD18 clone) and the distal part of the long arm of Y chromosome (19.6% - the pPD9 clone and 15.4% - the pPD18 clone). Besides significant interchromosomal differences, moderately pronounced interindividual differences were also detected in the number of grains over the regular sites of the chromosomal location. Pretreatment of slides with DA/DAPI induced differences in the results of quantitative analysis is described.     

In situ hybridization with fluoresceinated DNA.

We have used fluorescein-11-dUTP in a nick-translation format to produce fluoresceinated human nucleic acid probes. After in situ hybridization of fluoresceinated DNAs to human metaphase chromosomes, the detection sensitivity was found to be 50-100 kb. The feasibility and the increase in detection sensitivity of microscopic imaging of in situ hybridized, fluoresceinated DNA with an integrating solid state camera for rapid cosmid mapping is illustrated. Combination of fluoresceinated DNA with biotinated and digoxigeninated DNAs allowed easy performance of triple fluorescence in situ hybridization. The potential of these techniques for DNA mapping, cytogenetics and biological dosimetry is briefly discussed.