Polymorphisms and genomic organization of repetitive DNA from centromeric regions of Arabidopsis chromosomes.

A highly abundant repetitive DNA sequence family of Arabidopsis, AtCon, is composed of 178-bp tandemly repeated units and is located at the centromeres of all five chromosome pairs. Analysis of multiple copies of AtCon showed 95% conservation of nucleotides, with some alternative bases, and revealed two boxes, 30 and 24 bp long, that are 99% conserved. Sequences at the 3' end of these boxes showed similarity to yeast CDEI and human CENP-B DNA-protein binding motifs. When oligonucleotides from less conserved regions of AtCon were hybridized in situ and visualized by using primer extension, they were detected on specific chromosomes. When used for polymerase chain reaction with genomic DNA, single primers or primer pairs oriented in the same direction showed negligible amplification, indicating a head-to-tail repeat unit organization. Most primer pairs facing in opposite directions gave several strong bands corresponding to their positions within AtCon. However, consistent with the primer extension results, some primer pairs showed no amplification, indicating that there are chromosome-specific variants of AtCon. The results are significant because they elucidate the organization, mode of amplification, dispersion, and evolution of one of the major repeated sequence families of Arabidopsis. The evidence presented here suggests that AtCon, like human alpha satellites, plays a role in Arabidopsis centromere organization and function.     

The genomic organization and localization on the chromosomes of the HvRT family of DNA repetitive sequences in barley

HvRT family of repetitive DNA sequences from barley genome appears to have complex hierarchical organization. Tandem repetition of 118-bp monomers constitutes lower level of HvRT-family organization. Amplification units of the higher level consist of several contiguous 118-bp monomers. RFLP between different species and cultivars of barley resulted from the differences in the higher-order repeat structure. Individual chromosomes of barley contain specific HvRT subfamilies. This family also possesses separate domains differing in the restriction enzyme sites density. HvRT family is presented in the genomes of H. vulgare, H. leporinum, H. murinum, H. jubatum, but is absent in the genomes of H. marinum, H. geniculatum and wheat.     

The DNA content of the individual chromosomes of rye

Summary The relative DNA content of individual chromosomes of Secale cereale L. was determined in 25 cells by microdensitometry of Feulgen stained preparations. The correlation value between relative DNA content and relative chromosome length was r=0.61 for all 328 chromosomes measured. However, the correlation coefficients calculated for individual cells as well as for mean values always approached 1. Taking into account the structure of rye chromosomes, this indicates that microdensitometric results may not be accurate when large quantities of heterochromatic DNA sequences are present in analyzed material.     

Molecular and genomic organization of clusters of repetitive DNA sequences in Caenorhabditis elegans.

Repetitive sequences in Caenorhabditis elegans are interspersed along the holocentric chromosomes. We have physically mapped some of these repetitive families and found that, although the distribution of members of each family is relatively even along the chromosomes, members of more than one family tend to cluster in some locations. We compared the sequence organization of 11 clusters located at known positions on different chromosomes in the N2 strain. These studies allow a comparison between repetitive elements belonging to the same family that are located on the same or on different chromosomes, providing an important tool in the study of genome turnover and evolution.     

Diverse patterns of the tandem repeats organization in rye chromosomes

Although the monomer size, nucleotide sequence, abundance and species distribution of tandemly organized DNA families are well characterized, little is known about the internal structure of tandem arrays, including total arrays size and the pattern of monomers distribution. Using our rye specific probes, pSc200 and pSc250, we addressed these issues for telomere associated rye heterochromatin where these families are very abundant. Fluorescence in situ hybridization (FISH) on meiotic chromosomes revealed a specific mosaic arrangement of domains for each chromosome arm where either pSc200 or pSc250 predominates without any obvious tendency in order and size of domains. DNA of rye-wheat monosomic additions studied by pulse field gel electrophoresis produced a unique overall blot hybridization display for each of the rye chromosomes. The FISH signals on DNA fibres showed multiple monomer arrangement patterns of both repetitive families as well as of the Arabidopsis-type telomere repeat. The majority of the arrays consisted of the monomers of both families in different patterns separated by spacers. The primary structure of some spacer sequences revealed scrambled regions of similarity to various known repetitive elements. This level of complexity in the long-range organization of tandem arrays has not been previously reported for any plant species. The various patterns of internal structure of the tandem arrays are likely to have resulted from evolutionary interplay, array homogenization and the generation of heterogeneity mediated by double-strand breaks and associated repair mechanisms.     

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.     

Evidence of a large-scale functional organization of mammalian chromosomes

Evidence from inbred strains of mice indicates that a quarter or more of the mammalian genome consists of chromosome regions containing clusters of functionally related genes. The intense selection pressures during inbreeding favor the coinheritance of optimal sets of alleles among these genetically linked, functionally related genes, resulting in extensive domains of linkage disequilibrium (LD) among a set of 60 genetically diverse inbred strains. Recombination that disrupts the preferred combinations of alleles reduces the ability of offspring to survive further inbreeding. LD is also seen between markers on separate chromosomes, forming networks with scale-free architecture. Combining LD data with pathway and genome annotation databases, we have been able to identify the biological functions underlying several domains and networks. Given the strong conservation of gene order among mammals, the domains and networks we find in mice probably characterize all mammals, including humans.     

Characterizing the three-dimensional organization of telomeres.

BACKGROUND: Quantitative analysis can be used in combination with fluorescence microscopy. Although the human eye is able to obtain good qualitative results, when analyzing the spatial organization of telomeres in interphase nuclei, there is a need for quantitative results based on image analysis. METHODS: We developed a tool for analyzing three-dimensional images of telomeres stained by fluorescence in situ hybridization in interphase nuclei with DNA counterstained with 4',6-diamidino-2-phenylindole. After deconvolution of the image, we segmented individual telomeres. From the location of the telomeres we derived a distribution parameter rhoT, which indicated whether the telomeres were in a disk (rhoT > 1) or not (rhoT approximately 1). We sorted mouse lymphocyte nuclei and measured rhoT. We also performed a bromodeoxyuridine synchronous cell sorting experiment on live cells and measured rhoT at several instances. RESULTS: Measuring rhoT for nuclei in G0/G1, S, and G2 produced 1.4 +/- 0.1, 1.5 +/- 0.2, and 14 +/- 2, respectively, showing a significant difference between G2 and G0/G1 or S. For the bromodeoxyuridine synchronous cell sorting experiment, we found a cell cycle dependency of rhoT and a correlation between rhoT and an observer. CONCLUSIONS: In this study we present a quantitative method to characterize the organization of telomeres using three-dimensional imaging, image processing, and image analysis.     

Molecular organization of DNA in chromosomes

Metaphase chromosomes of Chinese hamster and lampbrush chromosomes of Triturus viridescens were studied for DNA arrangement by polarized fluorescence microscopy and by linear dichroic ratio measurements. Intact chromosomes showed a small but negative polarization (?8%) of fluorescence, while the dichroic ratio was negative and quite appreciable (0.85). On treatment with trypsin or RNase the polarization value increased to about ?34% with no change in the polarization direction. The dichroic ratio on the other hand remained unchanged on such enzyme treatments. These results, along with the data of form dichroism and the change in fluorescence color following any enzyme action, suggest that basically the preexisting DNA arrangement was revealed by these enzymes. The fully enzyme-treated chromosome–dye complexes showed cross-bands. Quantitatively, the highest degree of DNA orientation across the above two chromosomes was in the range of 15–34%. This orientation fits well with a coiled coil-DNA, which is mostly longitudinal in these chromosomes. It is suggested that this longitudinal coil may have occasional defects in DNA structure. Alternatively, this coil may terminate into regions of random DNA and unsupercoiled DNA showing lateral projections, such as loops. This alternating feature of the DNA arrangement would tandemly repeat in either metaphase and lampbrush chromosomes.     

The complete DNA sequence and genomic organization of the avian adenovirus CELO.

The complete DNA sequence of the avian adenovirus chicken embryo lethal orphan (CELO) virus (FAV-1) is reported here. The genome was found to be 43,804 bp in length, approximately 8 kb longer than those of the human subgenus C adenoviruses (Ad2 and Ad5). This length is supported by pulsed-field gel electrophoresis analysis of genomes isolated from several related FAV-1 isolates (Indiana C and OTE). The genes for major viral structural proteins (Illa, penton base, hexon, pVI, and pVIII), as well as the 52,000-molecular-weight (52K) and 100K proteins and the early-region 2 genes and IVa2, are present in the expected locations in the genome. CELO virus encodes two fiber proteins and a different set of the DNA-packaging core proteins, which may be important in condensing the longer CELO virus genome. No pV or pIX genes are present. Most surprisingly, CELO virus possesses no identifiable E1, E3, and E4 regions. There is 5 kb at the left end of the CELO virus genome and 15 kb at the right end with no homology to Ad2. The sequences are rich in open reading frames, and it is likely that these encode functions that replace the missing El, E3, and E4 functions.     

Characterization of repetitive DNA in rye (Secale cereale)

Nuclear DNA of rye (Secale cereale), a plant species with a relatively large genome (i.e., 18 pg diploid), has been characterized by determination of its content in repetitive sequences, buoyant density, and thermal denaturation properties. The reassociation kinetics of rye DNA reveals the presence of 70 to 75% repeated nucleotide sequences which are grouped into highly (Cot 1) and intermediately repetitive (Cot 1–100) fractions. On sedimentation in neutral CsCl gradients, native, high molecular weight DNA forms an almost symmetrical band of density 1.702 g/cm³. The highly repetitive DNA (Cot 1), on the other hand, is separated into two distinct peaks; the minor component has a density of 1.703 g/cm³ corresponding to that of a very rapidly reassociating fraction (Cot 0.01) which comprises 10 to 12% of the rye genome. The latter DNA contains segments which are repeated 6×10⁵ to 6×10⁶ times. The major peak of the Cot 1 fraction shows a density of 1.707 g/cm³ and consists of fragments repeated about 3.7×10⁴ times. The intermediately repetitive DNA is much more heterogeneous than the Cot 1 fraction and has a low degree of repetition of the order of 8.5×10². The melting behavior of the Cot 1 fraction reveals the presence of a high degree of base pairing (i.e., 7% mismatching). When native rye DNA is resolved into fractions differing in GC content by hydroxyapatite thermal column chromatography and these fractions are analyzed for the presence of repetitive sequences, it is observed that the highly redundant DNA (Cot 1) is mostly located in the fraction denaturing between 80° and 90°C. This result suggests that highly repetitive rye DNA occurs in a portion of the genome which is neither very rich in AT nor in GC.     

Mapping of repetitive bovine DNA sequences on cattle Y chromosomes.

Three male-specific PCR products of the sequences BC1.2, lambda ES6.0, and BRY.1 were used as probes for Southern blot analyses. Each of these probes generated a complex male-specific band pattern, which showed some quantitative variations among bulls. Hybridization patterns obtained with the BC1.2 and lambda ES6.0 PCR products were interrelated. Chromosomal locations of these repeats were determined by hybridizing the tritiated PCR products in situ to male metaphase spreads. The BC1.2 and lambda ES6.0 PCR products hybridized to Yp13-->p12, whereas the BRY.1 PCR product hybridized over the entire Y chromosome. In addition, the BC1.2 and lambda ES6.0 PCR products hybridized to the distal half of the acrocentric Y chromosome of Bos indicus, indicating that the short arm of the B. taurus Y chromosome is homologous with the telomeric end of the B. indicus Y and supporting the notion that the Y chromosomes of these two species differ by a pericentric inversion.     

Arrays of repetitive DNA elements in the largest chromosomes of Toxoplasma gondii.

A novel tandemly repeated DNA structure of Toxoplasma gondii that meets the requirements assigned for satellital DNA was characterized. A DNA fragment of 1002 bp contains two different elements of repetitive DNA families named ABGTg7 and ABGTg8.2. Both repeats are members of a more complex tandem structure where ABGTg7-like monomers can be arranged either as direct tandems or flanked by other related or non-related repeats. Pulse-field gel electrophoresis analysis showed that these repeats hybridize with the largest T. gondii chromosomes. Bal31 sensitivity assays indicated that these elements are located near the telomeres and along other regions too. Five genomic lambda phages were isolated and two different completed clusters of the repeated structure were analyzed.     

Pattern of repetitive DNA of the chromosomes of Microtus agrestis

The distribution of repetitive DNA in the chromosomes of Microtus agrestis was studied with the method for demonstrating constitutive heterochromatin given by Yunis et al. (1971) and the reassociation technique described by Schnedl (1971). All autosomes can be individually recognized by means of the position of their bands. The euchromatic segment of the X1 chromosome shows the same banding pattern as the corresponding segment of X2 which consists of facultative heterochromatin. The short arms of the Y chromosome are not deeply stained with either method and therefore do not contain noticeable amounts of repetitive DNA. The relative distances between the bands remain constant during chromosome contraction in mitosis.     

Evidence for transposition of dispersed repetitive DNA families in yeast.

Dispersed repetitive DNA sequences from yeast (Saccharomyces cerevisiae) nuclear DNA have been isolated as molecular hybrids in lambdagt. Related S. cerevisiae strains show marked alterations in the size of the restriction fragments containing these repetitive DNAs. "Ty1" is one such family of repeated sequences in yeast and consists of a 5.6 kilobase (kb) sequence including a noninverted 0.25 kb sequence of another repetitious family, "delta", on each end. There are about 35 copies of Ty1 and at least 100 copies of delta (not always associated with Ty1) in the haploid genome. A few Ty1 elements are tandem and/or circular, but most are disperse and show (along with delta) some sequence divergence between repeat units. Sequence alterations involving Ty1 elements have been found during the continual propagation of a single yeast clone over the course of a month. One region with a large number of delta sequences (SUP4) also shows a high frequency of sequence alterations when different strains are compared. One of the differences between two such strains involves the presence or absence of a Ty1 element. The novel joint is at one inverted pair of delta sequences.     

The replicative organization of DNA in polytene chromosomes ofDrosophila hydei

Salivary-gland nuclei ofDrosophila hydei were pulse-labeledin vitro with³H-thymidine and studied autoradiographically in squash preparations. The distribution of radioactive label over the length of the polytene chromosomes was discontinuous in most of the labeled nuclei; in some nuclei the pattern of incorporation was continuous. Comparison of the various labeling patterns of homologous chromosome regions in different nuclei showed that specific replicating units are replicated in a specific order. By combining autoradiography with cytophotometry of Feulgen-stained chromosomes, it was possible to correlate thymidine labeling of specific bands with their DNA content. The resulting data indicate that during the S-period many or perhaps all of the replicating units in a salivary-gland nucleus start DNA synthesis simultaneously but complete it at different times. Furthermore, the data support the hypothesis that the chromomere is a unit of replication or replicon. The DNA content of haploid chromomeres was found to be about 5×10^-4 pg for the largest bands inDrosophila hydei. From the results of H³-thymidine autoradiography and Feulgen-cytophotometry on neuroblast and anlage nuclei it was concluded that during growth of the polytenic nucleus heterochromatin is for the most part excluded from duplication. The results of DNA measurements in interbands of polytene chromosomes do not agree with a multistrand structure for the haploid chromatid. A chromosome model is proposed which is in accordance with the reported results and with current views concerning the replicative organization of chromosomes.