Rectification of tandemly repetitious DNA. I. Influences on rate

To account for observed patterns of uniformity and heterogeneity in tandemly repetitious DNA, earlier workers have proposed three intracellular mechanisms: the rolling circle model, gene conversion between unequally aligned sister chromatids, and unequal sister chromatid exchange (SCE). All three are forms of rectification, which is the random replacement of a specific number, β of repeating units by replicates of a group (α in number) of repeating units that are present elsewhere in the segment. For all three forms of rectification: (1) the average level of uniformity, C, in the segment increases with each rectification event by an amount given by $\text{ΔC=}\text{β(α+β)}\text{αn(n?1)}\text{(1?C)}$, in which n is the number of repeating units per segment. In gene conversion and unequal SCE, α = β (2) ΔC is affected by periodicity and clustering. (3) Mutation rate, β and the frequency of rectification events can vary with n so as to offset largely the presence of n(n?1) in the denominator. This allows rectification to operate even with very large numbers of small repeating units.     

DNA Base composition and repetitious DNA in several conifers

Coniferous DNA was analyzed by ultraoentrifugation and thermal denaturation and renaturation to determine base composition and the presence of repetitive sequences. The percent quanine plus cytosine values among species were constant and independent of DNA quantity per cell, whereas the proportion of repetitive DNA per genome was greatest in those species that had the highest DNA content.     

Distribution of repetitious sequences in chick nuclear DNA

By an improved method of hydroxylapatite chromatography, the reassociated sequences of chick nuclear DNA were isolated, and their base composition analysed. By increasing the amount of reassociation, the G + C content of the renatured sequences decreased progressively to reach a mean value corresponding to that of the total DNA. In order to study the distribution of the families, or group of families having different amount of reassociation, DNA was fractionated by CsC1 density gradient centrifugation. Fractions having different G + C content were obtained, and their reassociation rates analysed. At high C(o)t value of renaturation (C(o)t=50) the amount of reassociated sequences included in the high or in the low buoyant density DNA fractions was approximately the same, but their G + C content was as expected different. At lower C(o)t values of renaturation (between C(o)t of 0.2 and the C(o)t of 10), the results indicated an heterogeneity of the repeated sequences in the A + T rich DNA fractions, as compared to the G + C rich ones.     

Distribution of repetitious DNA in randomly growing and synchronized Chinese hamster cells.

Early spermatogenic cells from the testes of 10-, 13-, 15-, 18-, 20- and 25-day-old rats were purified by sedimentation at unit gravity. Cell dissociation was accomplished in 5 mM EDTA or 0.1% trypsin in Ca-Mg-free phosphate-buffered saline (pH 7.35). Dissociation with trypsin resulted in more viable cells than with EDTA, while EDTA was more efficient for the dissociation of spermatogonia. The differential effects of the two dissociation media were particularly evident in cell preparations from the 10-day-old animal. Maximum purity of different cell types was obtained in different aged animals (spermatogonia, 98%, 10 days; preleptotene spermatocytes, 98%, 10 days; leptotene spermatocytes, 75%, 13 days; zygotene spermatocytes, 68%, 18 days; pachytene spermatocytes, 75%, 25 days). Purity of particular types was correlated with the age of the animal. Earlier stages were purified to a greater extent in younger animals and later stages to a greater extent in older animals. Later stages exhibited increasing sedimentation at unit gravity in correlation with the increase in cell size as differentiation proceeded to pachytene spermatocyte. Two early germinal cell types, spermatogonia and preleptotene spermatocytes, were greatly purified with this technique.     

Identification and chromosomal location of a new tandemly repeated DNA in maize.

Two clones of a new family of tandemly repeated DNA sequences have been isolated from a maize random genomic DNA library. MR68 is 410 bp, representing a monomeric unit and MR77 is 1222 bp, containing three units. The copy number was estimated to be about 3000 per 1C maize genome. Its methylation pattern was also determined. Fluorescent in situ hybridization (FISH) indicates that the sequence is located on the subtelomeric region of the long arm of chromosomes 3 and 6, as well as on the satellite of chromosome 6.     

Oligonucleotides in heterogeneous nuclear RNA: similarity of inverted repeats and RNA from repetitious DNA sites.

A comparison has been made by oligonucleotide analysis of three fractions of HeLa cell hnRNA: (1) the "snap-back" fraction (ds-hnRNA, 5% of the total); (2) the fraction that self-anneals during prolonged incubation (25% of total); and (3) the fraction that hybridizes most rapidly to an excess of HeLa cell DNA (rep-hnRNA, 10% of the total). T1 fingerprints of each of these hnRNA fractions were similar to one another and featured the largest T1 oligonucleotides of known sequence previously isolated from ds-hnRNA (Robertson, H.D., et al. (1977) J. Mol. Biol. 115, 571--590; Jelinek, W. (1977 J. Mol. Biol. 115, 591--602). When hybridized to DNA either in solution or immobilized on filters, the isolated ds-hnRNA and the rep-hnRNA fractions showed similar hybridization kinetics in the COt range of "intermediate" repetitive DNA sequences; the ds-hnRNA and the rep-hnRNA also self-annealed to equal extents in the absence of any DNA. DNA of all buoyant density classes contained the T1 oligonucleotides diagnostic of the ds-hnRNA and the rep-hnRNA. While hnRNA is rich in inverted repeated sequences, cytoplasmic mRNA contains far fewer such sequences.     

Localizing triplet periodicity in DNA and cDNA sequences

Background  

The protein-coding regions (coding exons) of a DNA sequence exhibit a triplet periodicity (TP) due to fact that coding exons contain a series of three nucleotide codons that encode specific amino acid residues. Such periodicity is usually not observed in introns and intergenic regions. If a DNA sequence is divided into small segments and a Fourier Transform is applied on each segment, a strong peak at frequency 1/3 is typically observed in the Fourier spectrum of coding segments, but not in non-coding regions. This property has been used in identifying the locations of protein-coding genes in unannotated sequence. The method is fast and requires no training. However, the need to compute the Fourier Transform across a segment (window) of arbitrary size affects the accuracy with which one can localize TP boundaries. Here, we report a technique that provides higher-resolution identification of these boundaries, and use the technique to explore the biological correlates of TP regions in the genome of the model organism C. elegans.     

Sequence periodicity in nucleosomal DNA and intrinsic curvature

Background

Most eukaryotic DNA contained in the nucleus is packaged by wrapping DNA around histone octamers. Histones are ubiquitous and bind most regions of chromosomal DNA. In order to achieve smooth wrapping of the DNA around the histone octamer, the DNA duplex should be able to deform and should possess intrinsic curvature. The deformability of DNA is a result of the non-parallelness of base pair stacks. The stacking interaction between base pairs is sequence dependent. The higher the stacking energy the more rigid the DNA helix, thus it is natural to expect that sequences that are involved in wrapping around the histone octamer should be unstacked and possess intrinsic curvature. Intrinsic curvature has been shown to be dictated by the periodic recurrence of certain dinucleotides. Several genome-wide studies directed towards mapping of nucleosome positions have revealed periodicity associated with certain stretches of sequences. In the current study, these sequences have been analyzed with a view to understand their sequence-dependent structures.

Results

Higher order DNA structures and the distribution of molecular bend loci associated with 146 base nucleosome core DNA sequence from C. elegans and chicken have been analyzed using the theoretical model for DNA curvature. The curvature dispersion calculated by cyclically permuting the sequences revealed that the molecular bend loci were delocalized throughout the nucleosome core region and had varying degrees of intrinsic curvature.

Conclusions

The higher order structures associated with nucleosomes of C.elegans and chicken calculated from the sequences revealed heterogeneity with respect to the deviation of the DNA axis. The results points to the possibility of context dependent curvature of varying degrees to be associated with nucleosomal DNA.

     

Generation and maintenance of tandemly repeated extrachromosomal plasmid DNA in Chlamydomonas chloroplasts

Unusual chloroplast transformants of Chlamydomonas reinhardtii that contain 2000 copies of a mutant version of the chloroplast atpB gene, maintained as an extrachromosomal tandem repeat, have recently been described. In this paper studies have been undertaken to (i) address possible mechanisms for generating and maintaining the amplified DNA and (ii) determine whether it is possible to use chloroplast gene amplification to overexpress chloroplast or foreign genes. Data presented here indicate that high copy number transformants harbor characteristic rearrangements in both copies of the chloroplast genome large inverted repeat. These rearrangements appear to be a consequence of, or required for, maintenance of the amplified DNA. In an attempt to mimic the apparently autonomous replication of extrachromosomal DNA in the chloroplast, transformation was carried out with a plasmid that lacked homology with the chloroplast genome or with the same plasmid carrying a putative chloroplast DNA replication origin ( oriA ). Transformants were recovered only with the plasmid containing oriA , and all transformants contained an integrated plasmid copy at oriA , suggesting that establishment or maintenance of the extrachromosomal tandem repeat requires conditions that were not replicated in this experiment. To determine whether other genes could be maintained at high copy number in the chloroplast, plasmids carrying the wild-type atpB gene or the bacterial aadA gene were introduced into a high copy number transformant. Surprisingly, the copy number of the plasmid tandem repeat declined rapidly after the secondary transformation events, even when strong selective pressure for the introduced gene was applied. Thus, chloroplast transformation can either create or destabilize high copy number tandem repeats.     

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.     

Identification and chromosomal location of tandemly repeated DNA sequences in Allium cepa

A 314-bp tandemly repeated DNA sequence, named pAc074, was characterized in Allium cepa by fluorescence in situ hybridization (FISH) analyses using random amplified fragment as probe. The nucleotide sequences of the clone pAc074 is partially homologous to the satellite DNA sequences, ACSAT1, ACSAT2, and ACSAT3, of A. cepa with 81%, 81% and 78% similarity, respectively. Our sequential C-banding and FISH with pAc074 probe also clearly showed a close relation between Cheterochromatin at telomeric region and pAc074 sequences on all the chromosomes except on chromosome 6. On the long arm of chromosome 7, pAc074 sequences appeared as interstitial band which did not correspond to C-heterochromatin bands. Instead, the C-heterochromatin bands corresponded with the 5S rDNA signals. This is the first evidence of simultaneous banding of the 5S rDNA and C-band in A. cepa.     

The helical periodicity of DNA on the nucleosome

The precise number of base pairs per turn of the DNA double helix in the nucleosome core particle has been the subject of controversy. In this paper the positions of nuclease cutting sites are analysed in three dimensions. Using this midpoint of the DNA on the nucleosome dyad as origin, the cutting site locations measured along a strand of DNA are mapped onto models of the nucleosome core containing DNA of different helical periodicities. It is found that a helical periodicity of 10.5 base pairs per turn leads to cutting site positions which are sterically inaccessible. In contrast, a periodicity of 10.0 base pairs per turn leads to cutting site positions which are not only sterically sound, but which fall into a pattern such as would be expected when the access of the nuclease to the DNA is restricted by the presence of the histone core on one side and of the adjacent superhelical turn of DNA on the other. As proposed earlier by us (1), a value for the helical periodicity close to 10 base pairs per turn on the nucleosome, taken together with a periodicity close to 10.5 for DNA in solution - a value now established - resolves the so-called linkage number paradox.     

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.     

Structural instability of human tandemly repeated DNA sequences cloned in yeast artificial chromosome vectors.

The suitability of yeast artificial chromosome vectors (YACs) for cloning human Y chromosome tandemly repeated DNA sequences has been investigated. Clones containing DYZ3 or DYZ5 sequences were found in libraries at about the frequency anticipated on the basis of their abundance in the genome, but clones containing DYZ1 sequences were under-represented and the three clones examined contained junctions between DYZ1 and DYZ2. One DYZ3 clone was quite stable and had a long-range structure corresponding to genomic DNA. All other clones had long-range structures which either did not correspond to genomic DNA, or were too unstable to allow a simple comparison. The effects of the transformation process and host genotype on YAC structural stability were investigated. Gross structural rearrangements were often associated with re-transformation of yeast by a YAC. rad1-deficient yeast strains showed levels of instability similar to wild-type for all YAC clones tested. In rad52-deficient strains, DYZ5 containing YACs were as unstable as in the wild-type host, but DYZ1/DYZ2 or DYZ3 containing YACs were more stable. Thus the use of rad52 hosts for future library construction is recommended, but some sequences will still be unstable.