Nonneutral evolution of tandem repeats in the mitochondrial DNA control region of lagomorphs

The mitochondrial DNA of the European rabbit (Oryctolagus cuniculus) contains a tandem array of 153-bp repeats in the vicinity of the replication origin of the H-stand. Variation among molecules in the number of these repeats results in inter- and intraindividual length polymorphism (heteroplasmy). Generally, in an individual, one predominant molecular type is observed, the others representing a low percentage of the mtDNA content. At the tissue level, we observe a particular distribution of this polymorphism in the gonads compared with liver, kidneys, or brain, implying a relationship between the differentiation status of the cells and the types of new mtDNA molecules which appear and accumulate during lifetime. Similar tandem repeats were also found in the mtDNA noncoding region of European hares (Lepus europaeus), a cottontail (Sylvilagus floridanus), and a pika (Ochotona rufescens). The lengths and the sequences of these units evolve rapidly and in a concerted way, but the number of repeats is maintained in a narrow range, and an internal 20-bp segment is highly conserved. Constraints restrict the evolution of the primary sequence of these repeated units, the number of which is probably controlled by a stabilizing selection.      

Distributions of dimeric tandem repeats in non-coding and coding DNA sequences

We study the length distribution functions for the 16 possible distinct dimeric tandem repeats in DNA sequences of diverse taxonomic partitions of GenBank (known human and mouse genomes, and complete genomes of Caenorhabditis elegans and yeast). For coding DNA, we find that all 16 distribution functions are exponential. For non-coding DNA, the distribution functions for most of the dimeric repeats have surprisingly long tails, that fit a power-law function. We hypothesize that: (i) the exponential distributions of dimeric repeats in protein coding sequences indicate strong evolutionary pressure against tandem repeat expansion in coding DNA sequences; and (ii) long tails in the distributions of dimers in non-coding DNA may be a result of various mutational mechanisms. These long, non-exponential tails in the distribution of dimeric repeats in non-coding DNA are hypothesized to be due to the higher tolerance of non-coding DNA to mutations. By comparing genomes of various phylogenetic types of organisms, we find that the shapes of the distributions are not universal, but rather depend on the specific class of species and the type of a dimer.     

Independent occurrences of multiple repeats in the control region of mitochondrial DNA of white-tailed deer

Deer in the genera Mazama and Odocoileus generally have two copies of a 75-base-pair (bp) repeat in the left domain of the control region of the mitochondrial DNA (mtDNA). Phylogenetic analyses further suggest an ancient origin for the duplication supporting a previously stated contention that this event occurred before the separation of Mazama and Odocoileus. However, white-tailed deer (Odocoileus virginianus) had three or four copies of a 75-bp repeat in the control region of their mtDNA in 7.8% of the individuals analyzed, and all of these animals were from the coastal plain of the southeastern United States. When copy 3 is present, it is very similar in sequence to copy 2, but variation suggests that copy 3 probably evolved multiple times from copy 2. The pattern of phylogenetic clustering of the haplotypes from across the coastal plain also suggests that phenotypes with three or four copies of the repeat have originated multiple times. The 44 observed haplotypes showed strong spatial subdivision across the area with subpopulations frequently showing complete shifts in haplotype frequencies from others taken from nearby areas. Many of the subpopulations right along the coast or on adjacent barrier islands have a limited number of haplotypes as would occur in populations undergoing drift because of small numbers of breeding females and limited female dispersal.     

Evolutionary dynamics of tandem repeats in the mitochondrial DNA control region of the minnow Cyprinella spiloptera

Length variation due to tandem repeats is now recognized as a common feature of animal mitochondrial DNA; however, the evolutionary dynamics of repeated sequences are not well understood. Using phylogenetic analysis, predictions of three models of repeat evolution were tested for arrays of 260-bp repeats in the cyprinid fish Cyprinella spiloptera. Variation at different nucleotide positions in individual repeats supported different models of repeat evolution. One set of characters included several nucleotide variants found in all copies from a limited number of individuals, while the other set included an 8- bp deletion found in a limited number of copies in all individuals. The deletion and an associated nucleotide change appear to be the result of a deterministic, rather than stochastic, mutation process. Parallel origins of repeat arrays in different mitochondrial lineages, possibly coupled with a homogenization mechanism, best explain the distribution of nucleotide variation.      

Browsing repeats in genomes: Pygram and an application to non-coding region analysis

Background  

A large number of studies on genome sequences have revealed the major role played by repeated sequences in the structure, function, dynamics and evolution of genomes. In-depth repeat analysis requires specialized methods, including visualization techniques, to achieve optimum exploratory power.     

Inverted terminal repeats in rabbit poxvirus and vaccinia virus DNA.

In both rabbit poxvirus and vaccinia virus DNA have demonstrated an identical distribution of eight HinfI. The length of the terminal repeats was found to be 3.4 to 3.6 megadaltons (Mdaltons) for rabbit poxvirus DNA and 7.4 to 8.0 Mdaltons for vaccinia virus DNA. Maps of the HinfI restriction sites within isolated EcoRI end fragments of rabbit poxvirus and vaccinia virus DNA PHAVE DEMONSTRATED AN IDENTICAL DISTRIBUTION OF EIGHT HinfI sites in an internal part (approximately 2 Mdaltons) of the EcoRI end fragments of the two genomes.     

Expansion/contraction of mammalian mitochondrial DNA repeats in Escherichia coli mimics the mitochondrial heteroplasmy.

Length polymorphism due to tandem repeats is a common feature in animal mitochondrial DNA. The rabbit mitochondrial genome contains a 20 bp repeat domain, which generates a general heteroplasmic state. The observed polymorphic patterns suggest a dynamic equilibrium between gain and loss of units that maintains the copy number in the range 3-19 repeat units. In the apparent absence of recombination, slipped-strand mispairing during replication appears to be the primary cause of additions and deletions. To investigate this hypothesis we have set up a plasmid assay in Escherichia coli. A variable number of repeat units was inserted into a plasmid in both orientations relative to the colE1 origin of replication. Our data show that (i) a minimum unit number (>3) is necessary to generate length polymorphs, (ii) the number of events increases with the length tract, (iii) an excess of additions over deletions is found when the copy number is less than 10 and the trend is reversed when it is over 10, (iv) the frequency of deletions-additions is dependent on the orientation, (v) the polymorphism patterns are different according to the orientation. The length polymorphic pattern generated in the bacteria, in one orientation, mimics that observed in the mitochondria, suggesting that slipped mispairing between repeated sequences during DNA replication is responsible for the mitochondrial heteroplasmic state.     

DNA of Epstein-Barr virus. V. Direct repeats of the ends of Epstein-Barr virus DNA.

Previous data indicated that Epstein-Barr virus DNA is terminated at both ends by direct or inverted repeats of from 1 to 12 copies of a 3 X 10(5)-dalton sequence. Thus, restriction endonuclease fragments which include either terminus vary in size by 3 X 10(5)-dalton increments (D. Given and E. Kieff, J. Virol. 28:524--542, 1978; S. D. Hayward and E. Kieff, J. Virol. 23:421--429, 1977). Furthermore, defined fragments containing either terminus hybridize to each other (Given and Kieff, J. Virol. 28:524--542, 1978). The 5' ends of the DNA are susceptible to lambda exonuclease digestion (Hayward and Kieff, J. Virol. 23:421--429, 1977). To determine whether the terminal DNA is a direct or inverted repeat, the structures formed after denaturation and reannealing of the DNA from one terminus and after annealing of lambda exonuclease-treated DNA were examined in the electron microscope. The data were as follows. (i) No inverted repeats were detected within the SalI D or EcoRI D terminal fragments of Epstein-Barr virus DNA. The absence of "hairpin- or pan-handle-like" structures in denatured and partially reannealed preparations of the SalI D or EcoRI D fragment and the absence of repetitive hairpin- or pan-handle-like structures in the free 5' tails of DNA treated with lambda exonuclease indicate that there is no inverted repeat within the 3 X 10(5)-dalton terminal reiteration. (ii) Denatured SalI D or EcoRI D fragments reanneal to form circles ranging in size from 3 X 10(5) to 2.5 X 1O(6) daltons, indicating the presence of multiple direct repeats within this terminus. (iii) Lambda exonuclease treatment of the DNA extracted from virus that had accumulated in the extracellular fluid resulted in asynchronous digestion of ends and extensive internal digestion, probably a consequence of nicks and gaps in the DNA. Most full-length molecules, after 5 min of lambda exonuclease digestion, annealed to form circles, indicating that there exists a direct repeat at both ends of the DNA. (iv) The finding of several circularized molecules with small, largely double-strand circles at the juncture of the ends indicates that the direct repeat at both ends is directly repeated within each end. Hybridization between the direct repeats at the termini is likely to be the mechanism by which Epstein-Barr virus DNA circularizes within infected cells (T. Lindahl, A. Adams, G. Bjursell, G. W. Bornkamm, C. Kaschka-Dierich, and U. Jehn, J. Mol. Biol. 102:511-530, 1976).     

Quantitative analysis of the intra- and inter-individual variability of the normal urinary proteome

Urine is a readily and noninvasively obtainable body fluid. Mass spectrometry (MS)-based proteomics has shown that urine contains thousands of proteins. Urine is a potential source of biomarkers for diseases of proximal and distal tissues but it is thought to be more variable than the more commonly used plasma. By LC-MS/MS analysis on an LTQ-Orbitrap without prefractionation we characterized the urinary proteome of seven normal human donors over three consecutive days. Label-free quantification of triplicate single runs covered the urinary proteome to a depth of more than 600 proteins. The median coefficient of variation (cv) of technical replicates was 0.18. Interday variability was markedly higher with a cv of 0.48 and the overall variation of the urinary proteome between individuals was 0.66. Thus technical variability in our data was 7.5%, whereas intrapersonal variability contributed 45.5% and interpersonal variability contributed 47.1% to total variability. Determination of the normal fluctuation of individual urinary proteins should be useful in establishing significance thresholds in biomarker studies. Our data also allowed definition of a common and abundant set of 500 proteins that were readily detectable in all studied individuals. This core urinary proteome has a high proportion of secreted, membrane, and relatively high-molecular weight proteins.     

Direct repeats of the F plasmid incC region express F incompatibility

The nucleotide sequence of the incompatibility region incC, located at 45.8--46.4 kb on the F plasmid map, was determined. This region consists of 543 bp and contains sufficient information to code for only two small polypeptides of 34 and 30 amino acids each. Deletion of the ATG start codons for these two polypeptides has no effect on expression of incC incompatibility. A prominent feature of this sequence is the presence of five 22 bp direct repeats. A 58 bp segment of the incC region that contains two of these direct repeats was inserted into plasmid pACYC184, which is compatible with the F plasmid. The pACYC184 plasmid containing the direct-repeat sequences now expresses incompatibility with the F'lac plasmid and replication-proficient derivatives of the mini-F plasmid.     

Stable heterogeneity of mitochondrial DNA in grande and petite strains of S. cerevisiae.

Several instances of mitochondrial DNA heterogeneity in grande and petite strains of Saccharomyces cerevisiae were examined. We have detected heterogeneity in the mtDNA from some of the progeny strains of a cross between two grande strains (D273-10B, MH41-7B) which differ in genome size and restriction cleavage pattern of their mtDNA. The progeny strains transmit restriction fragments characteristic of both parental strains from homologous regions of the mitochondrial genome, and this sequence heterogeneity is not eliminated by additional subcloning. Sequence diversity is more common in the mtDNA of petite than of grande strains of yeast. We have examined subclones of one petite strain to identify the origin of this variability. Many of the submolar restriction fragments persist in independent subclones of this petite after 15 and 30 cell divisions; some submolar fragments disappear, and some new fragments appear. We conclude that the observed sequence heterogeneity is due to molecular heterogeneity, i.e., to differences in the multiple copies of the petite mitochondrial genome, as well as to clonal heterogeneity. It is likely that tandem repeats on the same mtDNA molecule also differ, i.e., that there is intramolecular heterogeneity, and that this accounts for the stability of the heterogeneity. Continuing deletion is probably responsible for the appearance of “new” fragments in petite subclones.