Organization of UV-induced,repair replicated DNA in the cellular genome of cultured human and mouse cells

The distribution of UV-induced repair-replicated DNA patches among reiterated and unique murine and human DNA has been studied by molecular reassociation. DNA-DNA renaturation was employed to fractionate labeled repair-replicated and normal cellular DNA sequences according to their reiteration frequencies. Results indicate that repair replicated DNA patches are distributed uniformly within highly repeated, moderately repeated and single copy DNA sequences. This could be due to the random localization of UV-induced lesions and repairs in the cultured murine and human cells.     

Organization of repetitive DNA sequences in the genome of the echinoderm Holothuria tubulosa.

The abundance of repetitive DNA in the haploid sea cucumber genome has been determined by screening a Holothuria genomic DNA library for clones containing repeated sequences using reverse genome hybridization. Analysis by in situ plaque hybridization of a set of 1132 clones has revealed the presence of repetitive DNA sequences in about 38.1% of the clones screened. The distribution of the reiterated DNA has been further analyzed by restriction endonuclease digestion of seven randomly selected repetitive clones. The repeated sequences have a fairly uniform distribution of lengths with an average length value of 7.3 kb. Analysis of the measurements suggests that the repetitive sequences are interspersed among longer single copy sequences with an average spacing interval of about 47.3 kb indicating that the repetitive and single copy DNA in the Holothuria genome are arranged in a long-period interspersion pattern.     

A possible difference in the mechanism of replication of moderately repeated nucleotide sequences in Chinese hamster ovary cells

Nascent DNA that was pulse-labelled with [³H]thymidine for 8 min in asynchronous Chinese hamster ovary cells contained a higher proportion of moderately repeated nucleotide sequences than did either nascent DNA pulse-labelled for 60 min or ¹⁴C-labelled parental DNA. The thymine pool equilibration time for moderately repeated sequences, which was found to be about one-half that for highly repeated and unique sequences, accounts for the high proportion of moderately repeated sequences labelled during short pulses and suggests that these sequences are replicated by a different mechanism than are other sequences. In synchronous cells, this difference in thymidine labelling was characteristic of early S and late S but not of mid S cells, suggesting that a higher proportion of moderately repeated nucleotide sequences is replicated in early and late S phase than in mid S.     

The Evolution of Restricted Recombination and the Accumulation of Repeated DNA Sequences

We suggest hypotheses to account for two major features of chromosomal organization in higher eukaryotes. The first of these is the general restriction of crossing over in the neighborhood of centromeres and telomeres. We propose that this is a consequence of selection for reduced rates of unequal exchange between repeated DNA sequences for which the copy number is subject to stabilizing selection: microtubule binding sites, in the case of centromeres, and the short repeated sequences needed for terminal replication of a linear DNA molecule, in the case of telomeres. An association between proximal crossing over and nondisjunction would also favor the restriction of crossing over near the centromere. The second feature is the association between highly repeated DNA sequences of no obvious functional significance and regions of restricted crossing over. We show that highly repeated sequences are likely to persist longest (over evolutionary time) when crossing over is infrequent. This is because unequal exchange among repeated sequences generates single copy sequences, and a population that becomes fixed for a single copy sequence by drift remains in this state indefinitely (in the absence of gene amplification processes). Increased rates of exchange thus speed up the process of stochastic loss of repeated sequences.     

The organization of the main component DNA of a crustacean genome with a paucity of middle repetitive sequences

The frequency classes and organization of the main component (mc) DNA of a crustacean, the land crab, Gecarcinus lateralis, have been characterized. The reassociation kinetics of 380 nucleotide long mcDNA fragments show that approximately 50% contain sequences repeated more than 800 times. Present in few, if any, copies are sequences repeated from 2 to 800 times. The remainder of the DNA reassociates as single copy sequences with a rate constant consistent with the organism's genome size. The reassociation kinetics of highly sheared DNA fragments of every true crab studied (Vaughn, 1975; Christie et al., 1976) are similar to each other and different from those of other invertebrate DNAs (Goldberg et al., 1975). Each of these genomes has a paucity of sequences repeated from 10 to 800 times and an abundance of highly repeated sequences. To determine if sequences repeated more than 800 times are interspersed with single copy sequences, we examined the arrangement of repetitive and non-repetitive sequences in mcDNA. The reassociation and melting properties of partially duplex mcDNA fragments of increasing lengths show that at least 75% of the DNA is organized in an interspersed pattern. In this pattern, single copy sequences with an average length of 800–900 nucleotides are interspersed with repetitive sequences. S1 nuclease digestion of reassociated 3100 nucleotide fragments indicates that 44% of the mcDNA is repetitive and that one-third of the repetitive sequences (average length=285 nucleotides) are interspersed with single copy sequences. We conclude that repetitive sequencies are interspersed with most of the single copy sequences in an interspersion pattern similar to that of Xenopus rahter than to that of another arthropod, Drosophila.Operated by Union Carbide Corporation for the Energy Research and Development Administration     

Highly repeated DNA of the baboon: organization of sequences homologous to highly repeated DNA of the African green monkey.

In the African green monkey genome, 20% of the total DNA consists of a highly reiterated DNA sequence that occurs largely in long tandem arrays of a repeat unit that is 172 base-pairs in length. The DNA of the baboon contains sequences homologous to this repeat unit. However, in the baboon genome, these sequences comprise roughly 6% of the total DNA and alternate in a regular fashion with a DNA segment that may be distantly related to the monkey repeat unit. The sequences in the baboon that are homologous to the monkey repeat unit are contained within a 340 base-pair repeat unit of the highly repeated DNA fraction of the baboon. The extent of nucleotide divergence of the homologous repeated sequences between the two species is estimated to be about 10%.     

Organization of nucleotide sequences in maize DNA

Nucleotide sequence organization in the genome of maize has been studied using renaturation kinetics of DNA and S-1 nuclease digestion of the renatured products. Approximately 40% of the genome consists of single copy sequences, and 15% of these sequences are interspersed between repeated sequences and are approximately 1100 nucleotide pairs long. About 54% of the genome consists of repeated sequences. Six per cent of the genome consists of foldback sequences. These sequences are distributed through at least 44% of the genome. It was found using renaturation kinetics that the sum of foldback and highly repeated DNA fractions of Dobrudzhanko maize and inbred lines differ in the amount of DNA composing the fractions. Comparison of the DNA of the Dobrudzhanko maize and inbred lines by the method of DNA-DNA hybridization indicates strong differences in the amount of polynucleotide homologies between the Dobrudzhanko maize and the D1 inbred line on one hand and the A619 inbred line on the other hand.     

Properties of some monkey DNA sequences obtained by a procedure that enriches for DNA replication origins.

Twelve clones of monkey DNA obtained by a procedure that enriches 10(3)- to 10(4)-fold for nascent sequences activated early in S phase (G. Kaufmann, M. Zannis-Hadjopoulos, and R. G. Martin, Mol. Cell. Biol. 5:721-727, 1985) have been examined. Only 2 of the 12 ors sequences (origin-enriched sequences) are unique (ors1 and ors8). Three contain the highly reiterated Alu family (ors3, ors9, and ors11). One contains the highly reiterated alpha-satellite family (ors12), but none contain the Kpn family. Those remaining contain middle repetitive sequences. Two examples of the same middle repetitive sequence were found (ors2 and ors6). Three of the middle repetitive sequences (the ors2-ors6 pair, ors5, and ors10) are moderately dispersed; one (ors4) is highly dispersed. The last, ors7, has been mapped to the bona fide replication origin of the D loop of mitochondrial DNA. Of the nine ors sequences tested, half possess snapback (intrachain reannealing) properties.     

Organization and heterogeneity of sequences within a repeating unit of human Y chromosome deoxyribonucleic acid.

Fragments of 3.4 kilobases (kb) are released from DNA of human males, but not DNA of human females, by cleavage with restriction endonucleases HaeIII, EcoRI, or EcoRII. Most, if not all, reiterated DNA which is specific for the Y chromosome (it-Y DNA) is present within these male-specific 3.4-kb molecules. Although such 3.4-kb molecules are themselves localized to the Y chromosome, this is not true for all sequences within them. At least two distinguishable types of reiterated sequences are found within each 3.4-kb molecule. One type consists of at least two families which are highly reiterated and are not confined to the Y chromosome. The other type is composed of an estimated minimum of 39 families, each moderately reiterated and localized to the Y chromosome. Y-specific and non-Y-specific sequences are interspersed with one another in the same 3.4-kb molecule. In the average 3.4-kb molecule, three 800 nucleotide lengths of Y-specific sequences alternate with four 250 nucleotide lengths of non-Y-specific sequences. Since the total number of families of Y-specific sequences, calculated on the basis of reiteration frequency and total abundance in a male genome, greatly exceeds the number of Y -specific sequences present in a single 3.4-kb molecule, it necessarily follows that the population of these 3.4-kb molecules is heterogeneous.     

A ubiquitous family of repeated DNA sequences in the human genome

Renatured DNA from human and many other eukaryotes is known to contain 300-nucleotide duplex regions formed from renatured repeated sequences. These short repeated DNA sequences are widely believed to be interspersed with single copy DNA sequences. In this work we show that at least half of these 300-nucleotide duplexes share a cleavage site for the restriction enzyme AluI. This site is located 170 nucleotides from one end. This Alu family of repeated sequences makes up at least 3% of the genome and is present in several hundred thousand copies.Inverted repeated sequences are also known to contain a short 300-nucleotide duplex region. We find that at least half of the 300-nucleotide duplex regions in inverted repeated sequences also have an AluI restriction site located 170 nucleotides from one end.By driven renaturation techniques, the Alu family is shown to be distributed over a minimum range of 30% to 60% of the genome. (The breadth of this range reflects the presence of inverted repeated sequences which, in part, include the Alu family.) These findings imply that the interspersion pattern of repeated and single copy sequences in human DNA is largely dominated by one family of repeated sequences.     

Organisation and properties of repeated DNA sequences in rice genome

Reassociation of high molecular weight rice DNA has revealed the occurrence of long stretches of repeated DNA which are not interrupted by single copy DNA even at a fragment length as high as 20 kilo base pairs (kbp). Majority of these repeated sequences are unusually G + C rich and show significant variations in their thermal stability. Homology studies indicate that short repeats may have evolved from long repeats in total repetitive DNA while they may be of different origin in highly repetitive DNA fraction. Restriction enzyme analysis shows the occurrence of Ava I and EcoR V repeat families.     

A molecular and cytogenetic survey of major repeated DNA sequences in tomato (Lycopersicon esculentum)

Summary The major families of repeated DNA sequences in the genome of tomato (Lycopersicon esculentum) were isolated from a sheared DNA library. One thousand clones, representing one million base pairs, or 0.15% of the genome, were surveyed for repeated DNA sequences by hybridization to total nuclear DNA. Four major repeat classes were identified and characterized with respect to copy number, chromosomal localization by in situ hybridization, and evolution in the family Solanaceae. The most highly repeated sequence, with approximately 77000 copies, consists of a 162 bp tandemly repeated satellite DNA. This repeat is clustered at or near the telomeres of most chromosomes and also at the centromeres and interstitial sites of a few chromosomes. Another family of tandemly repeated sequences consists of the genes coding for the 45 S ribosomal RNA. The 9.1 kb repeating unit in L. esculentum was estimated to be present in approximately 2300 copies. The single locus, previously mapped using restriction fragment length polymorphisms, was shown by in situ hybridization as a very intense signal at the end of chromosome 2. The third family of repeated sequences was interspersed throughout nearly all chromosomes with an average of 133 kb between elements. The total copy number in the genome is approximately 4200. The fourth class consists of another interspersed repeat showing clustering at or near the centromeres in several chromosomes. This repeat had a copy number of approximately 2100. Sequences homologous to the 45 S ribosomal DNA showed cross-hybridization to DNA from all solanaceous species examined including potato, Datura, Petunia, tobacco and pepper. In contrast, with the exception of one class of interspersed repeats which is present in potato, all other repetitive sequences appear to be limited to the crossing-range of tomato. These results, along with those from a companion paper (Zamir and Tanksley 1988), indicate that tomato possesses few highly repetitive DNA sequences and those that do exist are evolving at a rate higher than most other genomic sequences.     

Contrasting patterns of DNA sequence arrangement in Apis mellifera (Honeybee) and Musca domestica (housefly)

We have examined the organization of the repeated and single copy DNA sequences in the genomes of two insects, the honeybee (Apis mellifera) and the housefly (Musca domestica). Analysis of the reassociation kinetics of honeybee DNA fragments 330 and 2,200 nucleotides long shows that approximately 90% of both size fragments is composed entirely of non-repeated sequences. Thus honeybee DNA contains few or no repeated sequences interspersed with nonrepeated sequences at a distance of less than a few thousand nucleotides. On the other hand, the reassociation kinetics of housefly DNA fragments 250 and 2,000 nucleotides long indicates that less than 15% of the longer fragments are composed entirely of single copy sequences. A large fraction of the housefly DNA therefore contains repeated sequences spaced less than a few thousand nucleotides apart. Reassociated repetitive DNA from the housefly was treated with S1 nuclease and sized on agarose A-50. The S1 resistant sequences have a bimodal distribution of lengths. Thirty-three percent is greater than 1,500 nucleotide pairs, and 67% has an average size about 300 nucleotide pairs. The genome of the housefly appears to have at least 70% of its DNA arranged as short repeats interspersed with single copy sequences in a pattern qualitatively similar to that of most eukaryotic genomes.     

Nuclear DNA and salmonid phylogenetics

There are many unresolved problems in salmonid systematics, both at the interspecific and sub-specific levels. Some of the major systematic problems in the subfamily Salmoninae are briefly reviewed along with the available molecular methods for their analysis. Nuclear DNA markers available for use in molecular systematics include localized and dispersed highly repetitive DNA sequences, moderately repetitive sequences such as the ribosomal RNA genes (rDNA), and single copy DNA sequences. Both coding and non-coding sequences can be examined in the rDNA and single copy DNA. The rDNA is especially suitable for use in phylogenetic analysis, since different regions evolve at different rates and can be used for comparisons at different taxonomic levels. Comparison of restriction maps of the entire rDNA repeating unit in 17 salmonid species from Hucho. Sahelinus, Salmo and Oncorhynchus has shown that the transcribed spacer regions are the most informative for interspecific comparisons and that the intergenic spacer has potential for use in intraspecific comparisons. Our current approach is to amplify selected regions from each of these spacers for analysis by DNA sequencing. DNA sequence analysis of the internal transcribed spacers should be very informative in elucidating interspecific relationships in Salvelinus and Oncorhynchus . Analysis of a hypervariable region in the intergenic spacer has potential for identification of geographically separated stocks. The relative utility of different types of nuclear DNA sequences for identification of stocks and subspecies is examined.     

The 5-methylcytosine content of highly repeated sequences in human DNA.

Previously, we found much tissue- or cell-specificity in the levels of 5-methylcytosine (m5C) in the total human genome as well as in DNA fractions resolved by reassociation kinetics. We now report that there were even greater differences in the m5C content of the highly repeated, tandem EcoRI family of DNA sequences from different human organs or cell populations. The ratio of m5C levels in this DNA fraction from brain, placenta, and sperm was 2.0:1.2:1.0. At a HhaI site in this repeat family, sperm DNA was 5-10 fold less methylated than somatic DNAs. In contrast, the highly repeated Alu family, which is approximately 5% of the genome, had almost the same high m5C content in brain and placenta despite marked tissue-specific differences in m5C levels of the single copy sequences with which these repeats are interspersed. These data show that very different degrees of change in methylation levels of various highly repeated DNA sequences accompany differentiation.     

Changes in the methylated sequences and molecular population of wheat DNA during germination

The fractions of unique (Cot less than 405), moderately (Cot=0.13--405) and highly reiterated (Cot less than 0--0.13) sequences were isolated from DNA of wheat seeds and 3 day old seedlings, and GC content, amount of 5-methylcytosine and its distribution among various pyrimidine isostichs in the fractions isolated were studied. Different in Cot value DNA fractions from seeds or from seedlings are similar in GC content and in all other characteristics studied. Seed DNA differs from DNA of seedlings in the content of pyrimidine isostichs from the respective fractions of reiterated sequences. Pronounced differences in the amount of pyridmidine clusters with various base composition in the corresponding fractions of DNA from seeds and seedlings were found. These differences in the frequencies of respective pyrimidine clusters from DNA of seeds and seedlings may be considered as being a result of changes in the molecular population of wheat DNA on germination. The seed and seedling DNA differ significantly in the 5-methylcytosine content in the respective pyrimidine isostichs isolated from unique sequences. In the seedling DNA some other nucleotide sequences are to be methylated as compared to DNA of dormat seeds. Thus, on germination some changes occur in DNA methylation as well as in the genome organization.     

Highly repeated DNA families in the rat

We have analyzed the repeated DNA fraction of the rat by characterizing approximately 500 repeat DNA-containing clones using hybridization to a variety of rodent nucleic acids. To facilitate this analysis we devised a method whereby the cloned DNA is transferred to nitrocellulose paper by blotting directly out of colonies of the bacterial clones. In addition to identifying repeated sequences of potential interest (e.g. those transcribed in a tissue-specific manner, or those that are highly conserved in non-rat genomes), we found that, in contrast to what is revealed by the reassociation of rat DNA (e.g. Pearson, W. R., Wu, J. R., and Bonner, J. (1978) Biochemistry 17, 51-59), the rat genome contains a number of different highly repeated (greater than 50,000 copies) sequences. We distinguished the different highly repeated sequences both by their hybridization to different nucleic acids as well as by DNA sequence determination. The highly repeated sequences shared three characteristics that distinguished each of them from the 100,000-member rat satellite I family: (i) they were recovered less often in the cloned repeat DNA library than expected from their copy number in the rat genome; (ii) they reannealed abnormally slowly for their copy number even though they are not significantly divergent; and (iii) they are transcribed in one or more rat tissues. The implications of these findings for the organization of repeated sequences in the rat genome are discussed.     

Variation among alfalfa somaclones in copy number of repeated DNA sequences.

Repeated DNA sequences of alfalfa (Medicago sativa L.) somaclonal variants were analyzed to determine if changes in copy number had occurred during tissue culture. DNA clones containing highly repeated nuclear sequences from the diploid line HG2 (2x = 16) were slot blotted and probed with labeled DNAs from HG2 and several somaclones of HG2. Two DNA clones that differed visually in hybridization intensity among the plant DNAs and one clone that had constant hybridization intensity were selected and used as probes on Southern blots and slot blots containing equal quantities of DNAs from HG2 and 15 régénérants. Statistically significant differences were detected in the copy number of two anonymous DNA sequences initially selected as variable and in the copy number of sequences homologous to pea ribosomal DNA. Based on Southern blot analysis, these sequences appeared to be arranged as tandem repeats. The cloned sequence initially selected as stable did not vary significantly in copy number and it appeared to be arranged as a dispersed repeat. Both increases and decreases in copy number of repeated sequences were observed in plants from successive regeneration cycles. Results from this study indicate that specific repeated nuclear DNA sequences have changed copy number in plants regenerated from tissue culture.