Repetitive Component Research Articles

Properties of a DNA-Binding Protein from Rat Nuclear Scaffold Fraction

Our previous work [Hibino et al. (1992) Biochem. Biophys. Res. Commun. 184, 853-858] has shown that a highly repetitive component in rat nuclear DNA forms a sequence-directed bend to have the binding affinity for the nuclear scaffold protein, P130. In the present experiment, the mobility shift DNA-binding assay suggested that the formation of the repetitive component-P130 complex is based on some cooperative mode of interaction. The DNase I footprint analysis revealed that the major binding region of this protein in the DNA is located near the center of the 370-bp XmnI repeat which has a strongly bent overall structure. These results imply that a nuclear scaffold protein such as P130 binds to sequence-directed bend(s) in a highly repetitive DNA to play an important role in construction of a higher-order chromatin structure.

Synaptic responses to concerted genomic evolution in Lathyrus. I. Intergenomic effects

C0 autotetraploids have been derived from seven diploid (x = 7) species of Lathyrus, varying in genome size ( 11–20 pg DNA /2C) and content of moderately repetitive DNA (5–12 pg/2C). Pairing patterns at metaphase-I in 942 pollen mother cells (PMCs), each with a minimum of 28 chiasmata, were compared with a model relating pairing autonomy to genomic organization. Autotetraploids of one species, L. aphaca, form on average one multivalent more than those of the other six (5.2 vs. 3.7–4.2/PMC). With the exception of L. aphaca, intergenomic differences in bivalent frequency and incidence of synaptic exchange correlate with the size of the genome, particularly that of the moderately repetitive component. Such nucleotypic correlations appear to reflect losses in numbers of autonomous synaptic sites, concomitant with concerted reductions in the size of the genome. They also indicate that autonomous synapsis may be governed by moderately repetitive sequences.

Minimum variance control for repetitive disturbances with application to active noise control

Using minimum variance control theory, a controller is designed to reduce noise that contains a repetitive component of measurable time-varying frequency. The particular application for which the controller is designed is the active cancellation of both road and engine noise in a vehicle passenger compartment. The controller is appropriate for this case because engine noise can be shown to be expressed as a summation of harmonically related sine waves whose frequency is determined by the engine rotational speed. An example is presented where a controller is developed for active noise control in a station-wagon with road noise caused by striking a tyre and engine noise represented by the noise spectrum of a diesel engine. Computer simulations show that the developed controller significantly reduces the noise from both sources.

Affinity of a highly repetitive bent DNA for nuclear scaffold proteins from rat liver

A highly repetitive component in rat nuclear DNA was isolated by HindIII digestion and cloned. A self-ligated tandem dimer of the 370-bp cloned component was digested with each of DraI, HindIII and XmnI. The resulting 370-bp restriction fragments exhibited anomalously slow gel electrophoretic mobilities. Of them, the XmnI fragment had the slowest mobility. This suggested that sequence-directed bending of the helix axis is the strongest in this fragment. Moreover, the 370-bp restriction fragments had selective affinities for the nuclear scaffold proteins, P123 and P130. The affinity levels of XmnI fragment were higher than those of DraI or HindIII fragment. These results implied that the affinity of a highly repetitive component for the nuclear scaffold proteins depends on the degree of sequence-directed bending of the helix axis.

Cis-Diaminedichloroplatinum(II) complexes reversibly bound to water-soluble polyaspartamide carriers for chemotherapeutic applications. I. Platinum coordination to preformed, carrier-attached ethylenediamine ligands

Copolyaspartamides3–5, bearing the ethylenediamine (en) ligand as a repetitive side-chain component in addition to various hydrosolubilizing groups, are synthesized from polysuccinimide1 by a two-step aminolytic ring opening reaction. The completely water-soluble polyamides, possessing inherent viscosities of 5–20 ml g−1, are used as polymeric carriers for the “anchoring” of the biomedically important diaminedichloroplatinum(II) coordination complex. Conjugate formation is brought about by treatment of the carriers with the tetrachloroplatinate(II) dianion in aqueous solution at pH 6.0 ± 0.5. The resulting, water-soluble conjugates3-Pt-5-Pt, purified by dialysis (12,000–14,000 molecular mass cutoff) and isolated in the solid state by freeze-drying from aqueous solution, possess platinum contents in the typical range of 15–25%. The metal is bound as acis-dichloro-en-Pt(II) complex to the polymer main chain via a short spacer segment incorporating a biofissionable amide link suitable forin vivo release of the complex. The conjugates are of interest as water-soluble macromolecular platinum coordination compounds for potential use in the chemotherapy of cancerous diseases.

Base sequences of highly repetitive components in nuclear DNAs from rat liver and rat-ascites hepatoma

A 370-bp highly repetitive component in each of the nuclear DNAs from rat liver (RL) and rat-ascites hepatoma (AH) was isolated by HindIII digestion and cloned in pUC9. Ten of the resulting clones were arbitrarily selected and sequenced. Heterogeneity of size was found in 7 of the RL clones (366–369 bp), but in only 2 of the AH clones (369 bp). The sequence homology was 64.6% among the RL clones; 80.3% among the AH clones. The base compositions were AT-rich, ranging from 61.1% to 64.7%. Many A and/or T runs consisting of 2–5 bases were interspersed throughout each sequence. The restriction sites reported previously [12], EcoRI, HaeIII, HindIII, HinfI and HphI sites, were confirmed in almost all of the clones. In the present experiment, 12 kinds of the sites were further found in both RL and AH clones.

Digital control of repetitive errors in disk drive systems

Tracking errors in disk drive systems have a significant repetitive component that is not explicitly taken into account in conventional servo controllers. A modified prototype repetitive controller is applied as a plug-in module to a Winchester disk drive with a preexisting analog feedback controller to demonstrate its efficacy in the reduction of this periodic component. A review of the Winchester disk drive system is included.< <ETX xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">&gt;</ETX>

Highly repetitive DNA in the Baleen whale generaBalaenoptera andMegaptera

Three non-cross-hybridizing highly repetitive DNA components were studied in six whalebone and two toothed whale species. In one of the components, composition and repeat lengths have been preserved through the evolution of all cetaceans. Another component, primarily a whalebone whale characteristic, also has slow evolution. In the family Balaenopteridae the length of the repeat is about 420 bp. The component occurs in terminal chromosome positions. About half the component is composed of subrepeats having the motif TTAGGG, the same as has been described by other workers in the telomeres ofTrypanosoma brucei. Among the whalebone whales the third highly repetitive component was found only in the family Balaenopteridae. It is conceivably younger than the other two components. No common monomeric unit was identified, and the hybridization patterns were species specific, indicating that it evolves considerably faster than the other two components.

Evaluating quantitative variation in the genome of Zea mays.

Genomic diversity within the species Zea mays has been examined by measuring the variation in the repetitive component of the nuclear genome among North American inbred lines and varieties. This was done by preparing a set of clones of repetitive maize sequences that differ in function, molecular arrangement and multiplicity and then using these as probes for quantitative hybridization to DNA from various maize genotypes. The comparison showed that the majority of repeated sequences are markedly variable in copy number among the ten maize strains tested. The clone sample contained the rDNA and 5S genes, the major repeat of the chromosome knobs, sequences functioning as origins of DNA replication in yeast (ARS sequences) and randomly cloned sequences of unknown function and chromosomal location. The sequences ranged in reiteration frequency from 200 to greater than 10(5) copies and included both tandemly arrayed and dispersed repeats. The copy numbers were measured by hybridizing labeled cloned sequences to aliquots of high molecular weight genomic DNA that were applied to nitrocellulose filters through a slotted template (slot blotting). The hybridization signal on an autoradiogram occurred in a narrow band that could be scored reliably with a densitometer. This provided a rapid method of determining the abundance of particular repeated sequences in individual plants and plant populations. Using this technique, we found that the copy number of repeated sequences of all types generally varied among the strains by two- to threefold, although at least one sequence showed no detectable variation. In contrast to the variability found between strains, individuals within an inbred line or variety were found to be indistinguishable in terms of specific sequence multiplicity. Each genotype has a different pattern of copy numbers for the set of repeated sequence clones, and this pattern is characteristic of all individuals of a particular genotype. The data also show that the copy number of each sequence varies independently. No strains had uniformly high or low copy numbers for the entire set of probes.

Structure and function of repetitive DNA in eukaryotes

Prokaryotes possess relatively small genomes consisting predominantly oflow-copy numberDNA sequences. The genome sizes ofdifferent species vary by less than an order of magnitude (Kingsbury, 1969). In contrast, eukaryotic genomes are generally much larger than their prokaryotic counterparts, and a far greater proportion of this DNA (about 30-40%) is repeated (Britten & Kohne, 1968; Laird, 1971). This repetitive component takes on several guises, and for the purpose of previous discussions it has often been useful to classify these sequences according to their various characteristic properties: structure, distribution and reiteration frequency (Jelinek & Schmid, 1982). Recent applications ofDNA cloning methods and more powerful analytical techniques to study specific sequence families has provided a different perspective on the apparent diversity of structure and organization of eukaryotic repetitive DNA. Does this new information provide us with clues as to what the functions of these sequences might be? An unresolved, and possibly related, question centres on the considerable variation seen in the haploid nuclear DNA contents, or 'C-values', of eukaryotes (Britten & Davidson, 1969). These differences are found in many different phyla encompassing amphibians (Straus, 1971), plants (Rothfels et al., 1966; Rees & Jones, 1967), insects (Keyl, 1965) and rodents (Mazrimas & Hatch, 1972). C-value variation can, on occasion, be especially dramatic; it is sometimes observed in organisms of the same genus that have virtually identical morphology and karyotype. This is the essential element of the so-called 'C-value paradox'; nearly identical species must express about the same number of genes despite significant differences in their C-values. What is the nature of all this extra non-coding DNA? It does not, as one might have anticipated, all correspond to additional repetitive elements; it also includes a considerable increase in the amount of 'single-copy' DNA. Such considerations led some to conceive radical theories of genetic organization in eukaryotes to provide a function for this extra DNA (Callan, 1967; Britten & Davidson, 1969; Thomas, 1971) which, in their simplest forms, have proved to be incorrect. A more recent theory, based on the involvement of dispersed repetitive sequences, envisages that some of these elements may have a nuclear role in selecting which mRNA molecules enter the cell cytoplasm (Davidson & Britten, 1979). A recent study may offer some support for such an idea (Sutcliffe et al., 1984), but others disagree with the interpretation of these data (Owens et al., 1985). In any event, such proposals may provide a function for a minority of the non-coding sequences in eukaryotic genomes, but what of the remainder? Attention has periodically focused on -the persuasively presented alternative argument that the bulk of the 'extra' DNA in eukaryotes might be 'selfish' and have no function (Doolittle & Sapienza, 1980; Orgel & Crick, 1980). This Review will re-examine the idea in the light of recent studies of the structure of specific eukaryotic repetitive DNA elements.

Characteristics of a conserved 1,579-bp highly repetitive component in the killer whale, Orcinus orca.

A tandemly organized, highly repetitive DNA component of the killer whale was sequenced. The length of the repeat was 1,579 bp. This unit, which characterizes all delphinids, shows stringent hybridization homology with a 1,740-bp repeat that is characteristic of all other cetacean families. The 1,579-bp component comprises approximately 15% of the killer-whale genome, in which it is repeated 4-5 X 10(5) times. Computer analysis of the sequence showed no linear repetition within the component. This indicates that the 1,579-bp unit has not evolved by amplification of shorter repeats. Several inverted repeats of substantial length were found in the 1,579-bp unit. The most conspicuous of these was a 72-bp sequence that deviated from matching in only three positions. The 72-bp sequence occurs within an open reading frame 330 bp in length. Transcriptional activity was registered in the cloned repeat in a cell-free system. The length of the transcript was approximately 340 nucleotides. The chromosomal localization of the 1,579-bp repeat was determined by in situ hybridization. The repeat was present in eight of 21 autosomal pairs and was found in almost all C-band-positive (constitutive heterochromatin) regions of the karyotype.

On the bubble lattice generation in nonuniform pulse fields

The dynamic equilibrium bubble domain lattices created by nonuniform magnetic field pulses were investigated in the region of pulse amplitudes where the domain structure behaved in the most regular way. The lattice formation was found possible in a rather narrow range of the bias field values (withinHe<H<Hcol) and in the area of the sample betweens2- ands1-boundaries. Detailed parameters on these boundaries (position, field and field gradient) were measured and calculated as functions of the bias field and the pulse field amplitude. The boundarys2 was understood as being the position in the sample where the total stray + applied field during the field pulses equalsHe. A next magnetostatic calculation of the domain structure in the nonuniform field should give an appropriate theoretical formula fors1. High-speed photography has been used to record the immediate patterns of the domain structure both during a single field pulse and between the pulses. The obtained pictures made it possible to calculate the magnetic stray fields and stray field gradients when a simplified dipole model of the observed pattern was used. The attraction towards the wire has been compared with the repulsion due to the stray fields and their difference in the case of the equilibrium bubble lattice has been compared with the material coercivity term. The coercivity of the material was found to have very small influence on the translation of the domains during the process of the formation and spreading of the bubble lattice, which was attributed to the repetitive pulse component of the applied field.

Characterization of X-chromosome specific satellite DNA of Muntiacus muntjak vaginalis.

A highly repeated DNA (designated satellite IA) was isolated from cultured cells of Muntiacus muntjak vaginalis and its organization analyzed by the use of restriction nucleases and hybridization experiments with cloned DNA-fragments. Several restriction nucleases cleave the satellite IA DNA into a series of fragments, which are multiples of a basic repeat unit of 800 bp. Sequences homologous to the satellite IA DNA were also found in a second highly repetitive DNA component of Muntiacus muntjak vaginalis (satellite IB). Its organization is more complex than the one of satellite IA and does not conform to a simple periodicity of a basic repeat unit.--Hybridization in situ revealed, that both satellites are confined in their entirety to the X-chromosome, where they are located at both arms close to the centromere. No satellite DNA was found at the Y1-chromosome, which is considered to be homologous to the long arm of the X-chromosome. These results have interesting implications for the evolution of the X-chromosome.

Genome size and DNA complexity of Plasmodium falciparum

Plasmodium falciparum DNA was prepared from cells cultured in vitro in human erythrocytes. The P. falciparum DNA was mixed with a tritium-labeled Escherichia coli DNA standard, and the kinetics of reassociation were measured using hydroxyapatite chromatography. It was found that the P. falciparum genome size is equal to 3.8·10 8 nucleotide pairs, and that a repetitive component is present which contains about 10% of the DNA. The average repetition frequency in this component is 95 copies of each sequence.

Mosquito infectivity is directly related to the proportion of repetitive DNA in Plasmodium berghei

A strain of Plasmodium berghei (NK 65) was followed during syringe transmission in mice for over 120 passages after the last complete cycle, while the following parameters were monitored: (a) capacity to infect mosquitoes, inducing oocyst formation; (b) presence in the peripheral blood of morphologically identifiable gametocytes; (c) presence of a repetitive component in the DNA extracted from intraerythrocytic population. The suggestion of a possible role of this component in gametogenesis came from an earlier work (Dore, E., Birago, C., Frontali, C. and Battaglia, P.A. (1980) Mol. Biochem. Parasitol. 1, 199–208). Present results confirm the correlation between proportion of repetitive DNA and infectivity towards mosquitoes with a correlation coefficient r = 0.92 −0.07 +0.04. A parallel decrease of the two quantities is observed in the course of syringe transmission. A limited number of cloned lines, derived from strain NK 65 at different times during syringe transmission, shared the infectivity properties of the parent strain at the moment of cloning, thus confirming that in the infective stage single asexual parasites from the schizogenic cycle are able to originate the whole cycle. The above arguments and results suggest that differentiation into active gametocytes involves amplification of a portion of the genome.

Chromosome rearrangement between the Indian muntjac and Chinese muntjac is accompanied by a delection of middle repetitive DNA.

The organizations of the genomes of two related species of Asian deer, the Indian (2n = 6 female, 7 male) and Chinese muntjac (2n = 46), were compared at the cytogenetic and molecular levels. These dramatically different karyotypes preserve little apparent G-banding homology. The difference in chromosome number is coincident with a 22% reduction in haploid DNA content from 2.7 to 2.1 pg in the Chinese and Indian muntjac, respectively. The kinetics of reassociation of the Indian muntjac (equivalent Cot = 4285 M-1. s-1) and Chinese muntjac DNA (equivalent Cot - 4362 M-1.s-1) in 2.4 M tetraethylammonium chloride suggests conservation in amount of "single-copy" DNA. Two middle repetitive DNA sequence classes differ in both amount and in degree of repetition between the two species. A middle repetitive frequency component (935-fold repeated) represents 13% of the Indian muntjac DNA. A similar component (644-fold repeated) represents 17% of the Chinese muntjac DNA. Low repetition DNA sequence components (repeated 5- and 50-fold) represent 30 and 40% of the Indian and Chinese muntjac DNAs, respectively. These differences quantitatively account for the 0.6 pg haploid DNA content variation between species. The deletion of middle repetitive DNA has not substantively altered the distribution of restriction endonuclease DNA base composition classes as defined by buoyant density in cesium chloride. These results represent the first time that middle repetitive DNA has been directly implicated in a chromosome rearrangement within the vertebrates.

Complexity and content of the DNA and RNA in Trypanosoma cruzi

The content and sequence complexity of the nuclear DNA and messenger RNA for epimastigotes of Trypanosoma cruzi were determined. From analysis of nuclear DNA reassociation studies and microspectrofluorometric measurements of laser induced fluorescence of cellular DNA, T. cruzi is found to be a diploid organism with a nuclear DNA content of 2.5 × 10 8 nucleotide pairs (2.8 × 10 −13 g) and a kinetoplast DNA content of 4.9 × 10 7 nucleotide pairs (5.4 × 10 −14 g). Reassociation kinetics of nuclear DNA of average length 0.4 kb reveals three kinetic components: a moderately repetitive component with a reiteration frequency of 5.1 × 10 3 present in 9% of the fragments, a lowly repetitive component with a reiteration frequency of 32 present in 51% of the fragments, and a single-copy component present in 23% of the fragments. By saturation hybridization of total polysomal RNA to 3H-labeled single-copy DNA, it was determined that 68% of the single-copy DNA was represented in the epimastigote polysomal RNA. This corresponds to ca. 12 000 different mRNA species. Of these, ca. 9000 are present as poly(A) +-RNA, while the remaining 3000 appear not to be polyadenylated. Kinetic analysis of the poly(A) +-RNA population indicates it is composed of at least three classes of RNAs of different abundancy levels: two sequences which occur ca. 3000 times per cell, ca. 750 sequences which occur about 20 times per cell, and ca. 15 500 sequences which occur 1–2 times per cell.

Genome organization and characterization of the repetitive and inverted repeat DNA sequences in Neurospora crassa.

Regulatory circuits have been defined in Neurospora cmsa within which multiple unlinked genes are controlled in a positive fashion by major regulatory genes. It has been suggested that repetitive DNA sequences situated adjacent to structural genes may serve as regulatory sites. Experiments are presented which examine the amount and organization of the repetitive sequences of Neurospora. The genome consists of 2.7 X 10’ base pairs and is composed of 2% foldback, 8% repetitive, and 90% single copy sequences. Our results indicate that the repeated DNA sequences are organized in stretches of 10,000 base pairs or longer. Experiments using pure ribosomal DNA isolated from cloned recombinant DNA demonstrated that rDNA represents 7% of total nuclear DNA and 88% of the repetitive component. The rDNA sequence is repeated about 185 times per nucleus. Since the ribosomal DNA units are believed to be tandemly repeated many times, their clustering can largely account for the long repetitive stretches and their lack of interspersion with single copy DNA. These results argue that repetitive DNA sequences identified by renaturation experiments do not serve a regulatory function in Neurospora. About 350 foci of inverted repeat, or foldback, DNA sequences occur in the genome and are interspersed with single copy DNA. The relatively small number of foldback foci suggest that inverted repeats also cannot be the primary recognition elements for control of gene expression in Neurospora. We suggest that very short repeated sequences, undetected by renaturation experiments, may serve as recognition sites for eukaryotic gene regulation.

CHARACTER VARIABILITY AND EVOLUTIONARY RATE IN MENIDIA.

Johnson and Mickevich (1977) claimed to demonstrate an association between within-population variability and evolutionary rate for both allozyme loci and morphological characters in the atherinid fish genus Menidia. Their paper is extremely interesting for at least two reasons. First, it purports to demonstrate an evolutionary phenomenon exhibited by both electrophoretic and morphological characters; second, if their conclusions are correct it should be possible to estimate the relative likelihood of future evolutionary change for different characters within a lineage based solely upon measurements of present-day variability of those characters within that lineage. Johnson and Mickevich used two data sets: 24 allozyme loci sampled from 22 populations in five species of Menidia, and 22 morphological characters from a partly different set of 22 For the 16 populations that were represented in both of these data sets, phylogenetic trees constructed separately from the two different types of data provide . . nearly perfect congruence. The differences between the two trees are slight rearrangements within species. ... (Mickevich and Johnson, 1976). Two measures of evolutionary change were used: phenetic distance, or range of divergence, and patristic distance, the amount of change over a phylogenetic tree. For the allozyme loci, these quantities were based upon frequency differences between populations for each allele at a locus; the sum over these alleles was used as the measure of evolutionary change for the locus. By subtracting the phenetic from the patristic distance, an estimate of the repetitive portion of evolutionary change was also obtained. This represents reversals and parallel and convergent evolution, all of which contribute to the patristic but not to the phenetic distance. For both data sets, this repetitive component gives the highest correlation with intrapopulation variability, and the phenetic gives the lowest. Within-population variability was measured by the information statistic, H, for the allozyme data. For the morphometric characters, within-population variability was measured by the average standard deviation (divided by the grand mean) and measures of evolutionary change were computed between population means (also divided by the grand mean for that character). The point that I hope to make in this note is that there were several potential biases in the analyses performed by Johnson and Mickevich. First, they did not adequately correct for the possible contribution of sampling variance to their correlations. This bias applies to both the morphological and the allozyme data and is especially important for the repetitive component of the patristic distance. Second, their measures of variability and evolutionary change for the allozyme loci are not entirely free to vary independently, and a correlation between them, for certain frequency ranges, is unsurprising. This, too, is especially important for the repetitive component. Third, their measures of variability and change for the allozyme loci are both the result of summation processes across the number of alleles per locus. A correlation could result because different loci have different numbers of alleles. When this effect is removed, the correlation is primarily a function of the repetitive component for essentially invariant loci. Fourth, the gap-coding used for the morphological characters could introduce circular logic by giving relatively less-variable characters more weight in the construction of the phylogenetic tree. Since tree construction techniques minimize repetitive evolution, this weighting could result in a correlation between variability and apparent repetitive evolution. I do not claim that Johnson and Mickevich's conclusions are incorrect; only that they have presented no convincing evidence for those conclusions. Hopefully, future attempts to examine the relationship between variability and evolutionary rate will take some of these potential sources of bias into consideration. Are the correlations artifacts of sampling error? Sokal (1976) observed that in the absence of amonggroup differences the range of divergence among sample means should be correlated with the withingroup standard deviation. This is because the sampling variance of the mean is then proportional to the within-group variance. Johnson and Mickevich attempted to correct for this effect in the morphological data by omitting two characters that show no statistically significant differences . . between any pair of populations. Even though this procedure should reduce the bias, the fact that the amonggroup variance is partly composed of the withingroup variance (Sokal and Rohlf, 1969, p. 193) should still cause some correlation between the withinand among-group variability. A more appropriate method of analysis, at least for the phenetic component, would be to estimate variance components at the two levels as was done by Sokal (1976). Dr. Johnson informs me that he believes the in-

Interspersion of highly repetitive DNA with single copy DNA in the genome of the red crab, Geryon quinquedens

Kinetic analysis of the reassociation of 420 nucleotide (NT) long fragments has shown that essentially all of the repetitive sequences of the DNA of the red crab Geryon quinquedens are highly repetitive. There are negligible amounts of low and intermediate repetitive DNAs. Though atypical of most eukaryotes, this pattern has been observed in all other brachyurans (true crabs) studied (1,2). The major repetitive component is subdivided into short runs of 300 NT and longer runs of greater than 1200 NT while the minor component has an average sequence length of 400 NT. Both components reassociate at rates commonly observed for satellite DNAs. Unique among eukaryotes the organization of the genome includes single copy DNA contiguous to short runs (approximately 300 NT) of both repetitive components. Although patent satellites are not present, subsets of the repetitive DNA have been isolated by either restriction endonuclease digestion or by centrifugation in Ag+ or Hg2+/Cs2SO4 density gradients.