Organization of highly repeated sequences in surface-spread pachytene chromosomes of rye.

A method of preparing two-dimensional surface spreads of plant synaptonemal complexes (SCs) associated with fluorescence in situ hybridization (FISH) has been applied to analyze the location and organization of five different highly repeated DNA sequences in rye. Our observations indicate that, depending on the type of sequence, the chromatin displays different types of organization. Telomeric sequences were seen tightly associated with the SC while other repetitive DNA sequences were found to form loops that are associated with SCs only at their bases. On the contrary, the FISH signal of a centromeric satellite had a granular appearance, reflecting that the hybridization occurs only with parts of the chromatin loops.     

Subtelomeric regions of yeast chromosomes contain a 36 base-pair tandemly repeated sequence.

We have determined the nucleotide sequence of a region of DNA derived from the end of one chromosome of the yeast, Saccharomyces cerevisiae. Inspection of the sequence reveals the presence of 12 tandem direct repeats, each 36 nucleotides long and having nearly identical sequence. Each 36 base-pair repeat can be further subdivided into three tandem sub-repeats of a similar 12 base-pair sequence. Analysis of total genomic yeast DNA from several strains by Southern hybridization suggests that the number of tandem 36 base-pair repeat units may vary from approximately 8 to 25 among different telomeric regions. Differences in the number of repeats may have arisen by unequal crossing over between them. Furthermore, the finding that the pattern of bases at multiple variable positions within the repeat unit is not random suggests that these regions may undergo gene conversion events that render them homogeneous.     

Chromosomal localization of a tandemly repeated DNA sequence in Trifolium repens L

INTRoDUCTIoNlYho1iumrePensL,whiteclover,isaneconomicallyimportantplantspeciesintemperatepastures.Asbrieflyreportedby[1],ithas16pairsofchromosomes(2n=32).Asyet,nodetailedcytologicalexaminationofthisspecies,suchasC-banding,hasbeenrep0rted.Inthelastdecade,thetechnique0fC-bandinghasbeenusedt0examinehighlyrepeatedsequencesinplantchrom0s0mesandhasprovidedausefultoolf0rtheanalysis0fcyt0geneticstructureincr0pplants[2-71.Inplants,thechr0m0s0mall0calizationofhighlyrepeatedDNAsequencesbyinsituhybr…     

Isolation and identification of a non-specific tandemly repeated DNA sequence in Oryza species

A tandemly repeated DNA sequence (RRS7) was isolated from Oryza alta (CCDD). RRS7-related sequences were also found tandemly arrayed in genomes AA, BB, BBCC, CC, and EE, and a small amount of RRS7-related sequences were detected in genome FF and the Oryza species with unknown genomes. DNA sequence analysis of the 1844-bp insert of RRS7 revealed that it contained six tandemly repeated units, of which five were 155 bp in length and one was 194 bp in length and contained an imperfect internal 39-bp duplication. Southern blot analysis showed that the boundary sequence contained in RRS7 is a single-copy sequence. A 155-bp consensus sequence derived from the six monomeric repeats contained no internal repeat and showed no significant homology to other currently known sequences. The results of Southern blot and sequence analysis revealed that there are at least two subfamilies present in the RRS7 family; these are represented by the DraI site and the MspI site, respectively. Restriction digestion with two pairs of isoschizomers MboI/Sau3A and MspI/HpaII demonstrated that most of the C residues in the GATC sites and the internal C in the CCGG sites of the RRS7 family in O. Alta were methylated. The usefulness of the RRS7 family in determining the evolutionary relationship of the genome DD and other Oryza genomes is discussed.     

DNA content of heterochromatin and euchromatin in tomato (Lycopersicon esculentum) pachytene chromosomes.

Lycopersicon esculentum (tomato) has a small genome (2C = 1.90 pg of DNA) packaged in 2n = 2x = 24 small acrocentric to metacentric chromosomes. Like the chromosomes of other members of the family Solanaceae, tomato chromosomes have pericentromeric heterochromatin. To determine the fraction of the tomato genome found in euchromatin versus heterochromatin, we stained pachytene chromosomes from primary microsporocytes with Feulgen and analyzed them by densitometry and image analysis. In association with previously published synaptonemal complex karyotype data for tomato, our results indicate that 77% of the tomato microsporocyte genome is located in heterochromatin and 23% is found in euchromatin. If heterochromatin is assumed to contain few active genes, then the functional genes of the tomato must be concentrated in an effective genome of only 0.22 pg of DNA (1C = 0.95 pg x 0.23 = 0.22 pg). The physical segregation of euchromatin and heterochromatin in tomato chromosomes coupled with the small effective genome size suggests that tomato may be a more useful subject for chromosome walking and gene mapping studies than would be predicted based on its genome size alone. Key words : tomato, Lycopersicon esculentum, genome size, heterochromatin, euchromatin, pachytene chromosomes, synaptonemal complex.     

Chromosomal localization of a tandemly repeated DNA sequence in Trifilium repens L.

A karyotype of Trifolium repens constructed from mitotic cells revealed 13 pairs of metacentric and 3 pairs of submetacentric chromosomes including a pair of satellites located at the end of the short arm of chromosome 16.C-bands were identified around the centromeric regions of 8 pairs of chromosomes.A 350 bp tandemly repeated DNAsequence from T.repens labelled with digoxygenin hybridized to the proximal centromeric regions of 12 chromosome pairs.Some correlation between the distribution of the repeat sequence and the distribution of C-banding was demonstrated.     

Cloning and characterization of a tandemly repeated DNA sequence in the crane family (Gruidae)

A tandemly repeated DNA sequence possessing a unique PstI site has been characterized in several species of the crane family. The "Pst family" comprises at least 8800 monomer units 187 base pairs (bp) in length and constitutes 0.14% of the genome of the sarus crane (Grus antigone). The array is located in the centromeric heterochromatin of chromosome 2 in the two species where in situ hybridizations of a cloned monomer to metaphase chromosome spreads were carried out. DNA sequence comparisons between five monomer units from G. antigone revealed a high degree of homology between four of the individual repeats, while the fifth was somewhat divergent. The G + C content deduced from the DNA sequence makes it likely that the Pst family constitutes part of a density satellite seen in profiles of crane DNA centrifuged to equilibrium in CsCl. The common occurrence of tandem arrays such as the Pst family, with repeat lengths close to 200 bp, leads us to an hypothesis implicating nucleosomes in the evolution of such families.     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes

Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)_n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.D.P. Bazett-Jones     

Preparations of meiotic pachytene chromosomes and extended DNA fibers from cotton suitable for fluorescence in situ hybridization

Fluorescence in situ hybridization (FISH) has become one of the most important techniques applied in plant molecular cytogenetics. However, the application of this technique in cotton has lagged behind because of difficulties in chromosome preparation. The focus of this article was FISH performed not only on cotton pachytene chromosomes, but also on cotton extended DNA fibers. The cotton pollen mother cells (PMCs) instead of buds or anthers were directly digested in enzyme to completely breakdown the cell wall. Before the routine acetic acid treatment, PMCs were incubated in acetic acid and enzyme mixture to remove the cytoplasm and clear the background. The method of ice-cold Carnoy's solution spreading chromosome was adopted instead of nitrogen removed method to avoid chromosomes losing and fully stretch chromosome. With the above-improved steps, the high-quality well-differentiated pachytene chromosomes with clear background were obtained. FISH results demonstrated that a mature protocol of cotton pachytene chromosomes preparation was presented. Intact and no debris cotton nuclei were obtained by chopping from etiolation cotyledons instead of the conventional liquid nitrogen grinding method. After incubating the nuclei with nucleus lysis buffer on slide, the parallel and clear background DNA fibers were acquired along the slide. This method overcomes the twist, accumulation and fracture of DNA fibers compared with other methods. The entire process of DNA fibers preparation requires only 30 min, in contrast, it takes 3 h with routine nitrogen grinding method. The poisonous mercaptoethanol in nucleus lysis buffer is replaced by nonpoisonous dithiothreitol. PVP40 in nucleus isolation buffer is used to prevent oxidation. The probability of success in isolating nuclei for DNA fiber preparation is almost 100% tested with this method in cotton. So a rapid, safe, and efficient method for the preparation of cotton extended DNA fibers suitable for FISH was established.     

Isolation and identification of a novel tandemly repeated DNA sequence in the centromeric region of human chromosome 8

EcoRI subclones, designated as 50E1 and 50E4, were independently obtained from a cosmid clone previously mapped to the centromeric region of human chromosome 8. Southern blot hybridization analyses suggested that both subclones contain repetitive DNA sequences different from the chromosome 8 specific alphoid DNA. DNA sequence analysis of the 704 bp insert of 50E1 and the 1, 962 bp insert of 50E4 revealed that both inserts contained tandemly repeated units of 220 bp. Fluorescence in situ hybridization studies confirmed these two subclones to be specifically located on the centromeric region of chromosome 8. A 220 bp consensus sequence, derived from nine monomeric repeats, showed no significant homology to alphoid consensus sequences or to other currently known human centromeric DNA sequence. Furthermore, no significant homology was found with any other DNA sequence deposited in the EMBL or GenBank databases, indicating that this chromosome 8 specific repetitive DNA sequence is novel. From slot blot experiments it was estimated that 0.013% of the human genome comprises 1,750 of these monomeric repeats, residing on the centromeric region of chromosome 8 in tandem array(s).     

A cytomegalovirus DNA sequence containing tracts of tandemly repeated CA dinucleotides hybridizes to highly repetitive dispersed elements in mammalian cell genomes.

A single 880-base-pair region within the genome of simian cytomegalovirus strain Colburn contains sequences that hybridize intensely with both human and mouse total genome DNA probes. This sequence was also found in a second simian cytomegalovirus isolate and was retained in both plaque-purified virus subclones and in plasmid DNA clones containing the SalI P fragment. Cleaved genomic DNAs from several mammalian species all exhibited strong dispersed hybridization with the SalI-P probes, and over 70% of the lambda clones in a mouse genomic library plus several selected clones containing globin, 45S rDNA, or 5S rDNA genes all formed hybrids with SalI-P. The appropriate region of cytomegalovirus SalI-P contains relatively A + T-rich unique sequences interrupted by three stretches of the simple alternating dinucleotides, (CA)15, (CA)22, and (CA)21, which we show to be responsible for most of the cell-virus homology. We conclude that discrete, tandemly repeated (CA) dinucleotide tracts capable of forming left-handed Z-DNA helices punctuate mammalian genomes at greater than 10(5) copies per cell and that three adjacent copies of what appear to be a family of interspersed repetitive elements containing these (CA)n stretches are carried in the genomes of simian cytomegaloviruses.     

Characterisation of a highly repeated DNA sequence from Mycobacterium bovis

Abstract We report characterisation of a novel repeat sequence from a Mycobacterium bovis genomic library. The highly repeated sequence belongs to a family consisting of a 24 base pair (bp) direct repeat (DR), that appears to be organized into clusters on the chromosome. We classify the 24-bp DR into the group of prokaryotic DNA repeats known as the interspersed repetitive sequence elements. The 24-bp DR will be of potential use as a DNA fingerprinting tool in epidemiological studies of M. bovis .     

Characterization of a tandemly repeated DNA from the fleshflySarcophaga bullata

In studies on the highly repetitive DNA sequences of the flesh flySarcophaga bullata, a 279 bp tandem repeat was cloned and sequenced. A 17 bp stretch within the clone was identical to a motif repeated five times in the satellite DNA of the Bermuda land crab. Southern DNA blotting showed the tandem repeat had a high degree of conservation of MboI sites, but had divergence for EcoRI sites; thus, all repeat units were not identical. The cloned DNA localized to the quinacrine-bright centromeric heterochromatin of the C and E autosomes and to sites on the chromosomal arms. In cases of asynapsis of homologs, the probe localized to euchromatic sites on both homologs or sometimes only on one homolog. The probe also localized near, to, or at a major developmental puff (B9). We conclude that blocks of this short interspersed repetitive DNA occur throughout theSarcophaga genome in both heterochromatin and euchromatin, and also that the variable position of these sequences suggests they possess a degree of instability.     

Structure of a hypervariable tandemly repeated DNA sequence on the short arm of the human Y chromosome

The structure of a repeated DNA sequence located on the short arm of the human Y chromosome is described. Genomic mapping and cloning in lambda or cosmid vectors show that the repeated sequence consists of units 20.3 x 10(3) base-pairs long that contain the three previously described DNA sequences: Y-156, Y-190 and Y-223a. Analysis of male genomic DNA by pulsed-field gel electrophoresis shows that the units are tandemly arranged and are organized into two blocks. The major block is hypervariable in size and alleles in the range approximately 540 x 10(3) to 800 x 10(3) base-pairs were detected. The minor block is not variable in size and is approximately 60 x 10(3) base-pairs long. Analysis of rearranged Y chromosomes shows that both blocks are located on the short arm of the chromosome. Most commonly, the major block is distal to the minor block, but the opposite arrangement is also found.     

A natural meiotic DNA break site in Schizosaccharomyces pombe is a hotspot of gene conversion, highly associated with crossing over

In Schizosaccharomyces pombe, meiosis-specific DNA breaks that initiate recombination are observed at prominent but widely separated sites. We investigated the relationship between breakage and recombination at one of these sites, the mbs1 locus on chromosome I. Breaks corresponding to 10% of chromatids were mapped to four clusters spread over a 2.1-kb region. Gene conversion of markers within the clusters occurred in 11% of tetrads (3% of meiotic chromatids), making mbs1 a conversion hotspot when compared to other fission yeast markers. Approximately 80% of these conversions were associated with crossing over of flanking markers, suggesting a strong bias in meiotic break repair toward the generation of crossovers. This bias was observed in conversion events at three other loci, ade6, ade7, and ura1. A total of 50-80% of all crossovers seen in a 90-kb region flanking mbs1 occurred in a 4.8-kb interval containing the break sites. Thus, mbs1 is also a hotspot of crossing over, with breakage at mbs1 generating most of the crossovers in the 90-kb interval. Neither Rec12 (Spo11 ortholog) nor I-SceI-induced breakage at mbs1 was significantly associated with crossing over in an apparently break-free interval >25 kb away. Possible mechanisms for generating crossovers in such break-free intervals are discussed.