Genomic organization of two families of highly repeated nuclear DNA sequences of maize selected for autonomous replicating activity in yeast

Maize nuclear DNA sequences capable of promoting the autonomous replication of plasmids in yeast were isolated by ligating Eco RI-digested fragments into yeast vectors unable to replicate autonomously. Three such autonomously replicating sequences (ARS), representing two families of highly repeated sequences within the maize genome, were isolated and characterized. Each repetitive family shows hybridization patterns on a Southern blot characteristic of a dispersed sequence. Unlike most repetitive sequences in maize, both ARS families have a constant copy number and characteristic genomic hybridization pattern in the inbred lines examined. Larger genome clones with sequence homology to the ARS-containing elements were selected from a lambda library of maize genomic DNA. There was typically only one copy of an ARS-homologous sequence on each 12–15 kb genomic fragment.     

The occurrence of families of repetitive sequences in a library of cloned cDNA from human lymphocytes

A library of cloned cDNAs representative of lymphocyte total poly(A)+ RNA was screened with total DNA probes at high clone density. 10% of the recombinants showed the presence of sequences which are repeated in the genome. Further analysis of six such isolated cDNA clones indicated that they contain different families of repetitive sequences with reiteration frequencies of between 150 and 45,000 copies per haploid genomes. Five of the six clones were found to contain single copy sequences as well as a repetitive sequence. cDNA clones containing repetitive sequences have been found to be derived from high, intermediate and low abundance classes of lymphocyte poly(A)+ RNA.     

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.     

DNA changes involving repeated sequences in senescing soybean (Glycine max) cotyledon nuclei

Age-related DNA changes in soybean ( Glycine max L. cv. Ransom) cotyledon nuclei were investigated by Feulgen cytophotometry and cloned DNA probes. The amount of nuclear DNA in 17-day-old senescing cotyledons was 23% lower than that of 5-day-old young cotyledons. In order to understand the nature of senescence-related DNA loss, fragments of repetitive DNA from young cotyledons were cloned into Escherichia coli HB101 cells by DNA recombination. The cloned DNA probed changes in specific repeated nucleotide sequences in senescing cotyledons. The colony hybridization between cloned DNA and [³²P]-labeled total DNA from 5- and 17-day cotyledons indicated loss of specific repeated sequences. The selected sequences of repeated DNA further showed a loss in their copy numbers. The study suggested that some repetitive DNA sequences were degraded selectively in the genome of senescing cotyledons.     

Use of repetitive DNA probes as physical mapping strategy in Caenorhabditis elegans.

A method for linking genomic sequences cloned in yeast artificial chromosomes (YACs) has been tested using Caenorhabditis elegans as a model system. Yeast clones carrying YACs with repeated sequences were selected from a C. elegans genomic library, total DNA was digested with restriction enzymes, transferred to nylon membranes and probed with a variety of repetitive DNA probes. YAC clones that overlap share common bands with one or more repetitive DNA probes. In 159 YAC clones tested with one restriction enzyme and six probes 28 overlapping clones were detected. The advantages and limitations of this method for construction of YAC physical maps is discussed.     

Identification of procaryotic repetitive DNA suitable for use as fingerprinting probes.

We have developed a method which enables the cloning and identification of procaryotic repetitive DNA suitable for use as DNA fingerprinting probes. The method involves shotgun cloning of restricted genomic DNA with subsequent selection of clones containing repetitive DNA by reverse-probed genomic hybridizations, in which the plasmid DNA clones are probed with labelled genomic DNA. Confirmation that the clones contained repeated sequences was by Southern hybridization, gene copy equivalence, and DNA sequencing. The sequences were used for highly specific and sensitive detection of bacteria and as target sequences for the mediation of chromosomal integration of reporter gene constructs.     

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.     

Sequence organisation in nuclear DNA from Physarum polycephalum: Genomic organisation of DNA segments containing foldback sequences

DNA clones containing foldback sequences, derived from Physarum polycephalum nuclear DNA, can be classified according to their pattern of hydridisation to Southern blots of genomic DNA. One group of DNA clones map to unique DNA loci when used as a probe to restriction digests of Physarum nuclear DNA. These cloned segments appear to contain dispersed repetitive sequence elements located at many hundreds of sites in the genome. Similar patterns of hybridisation are generated when these cloned DNA probes are annealed to DNA restriction fragments of genomic DNA obtained from a number of different Physarum strains, indicating that no detectable alteration has occurred at these genomic loci subsequent to the divergence of the strains as a result of the introduction or deletion of mobile genetic elements. However, deletion of segments of some cloned DNA fragments occurs following their propagation in Escherichia coli. A second, distinct group of clones are shown to be derived from highly methylated segments of Physarum DNA which contain very abundant repetitive sequences with regular, though complex, arrangements of restriction sites at their various genomic locations. It is suggested that these DNA segments contain clustered repetitive sequence elements. The results lead to the conclusion that foldback elements in Physarum DNA are located in segments of the genome which display markedly different patterns of sequence organisation and degree of DNA methylation.     

Characterization of four dispersed repetitive DNA sequences from Zea mays and their use in constructing contiguous DNA fragments using YAC clones.

We have isolated four repetitive DNA fragments from maize DNA. Only one of these sequences showed homology to sequences within the EMBL database, despite each having an estimated copy number of between 3 x 104 and 5 x 104 per haploid genome. Hybridization of the four repeats to maize mitotic chromosomes showed that the sequences are evenly dispersed throughout most, but not all, of the maize genome, whereas hybridization to yeast colonies containing random maize DNA fragments inserted into yeast artificial chromosomes (YACs) indicated that there was considerable clustering of the repeats at a local level. We have exploited the distribution of the repeats to produce repetitive sequence fingerprints of individual YAC clones. These fingerprints not only provide information about the occurrence and organization of the repetitive sequences within the maize genome, but they can also be used to determine the organization of overlapping maize YAC clones within a contiguous fragment (contigs). Key words : maize, repetitive DNA, YACs.     

Organization of a family of highly repetitive sequences within the human genome

Recombinant clones containing the highly repetitive human DNA sequence approximately 340 base-pairs in length obtained after EcoRI digestion (αRI-DNA) were cloned in plasmid pAT153. Two clones contained a single copy of the αRI-DNA sequence, and the third had an insert with two copies of the sequence in tandem. When radioactive recombinant DNA was hybridized to total human DNA partially digested with EcoRI, a series of multiple bands was obtained up to 22 repeats in length, demonstrating that the αRI-DNA sequences occur in tandem arrays in the genomic DNA. A reassociation analysis using isolated insert DNA from one of the recombinant clones showed that the family of sequences is repeated 22,000 times in the human genome. Clones containing the αRI-DNA sequence were also isolated from a library of human genomic DNA in bacteriophage λ. Using these clones it was shown that, in at least some cases, the repetitive element is bounded by DNA less abundant than the αRI sequence.     

The suitability of restriction fragment length polymorphisms as genetic markers in maize

Summary Strain identification in Zea mays by restriction fragment length polymorphism should be feasible due to the high degree of polymorphism found at many loci. The polymorphism in maize is apparently higher than that currently known for any other organism. Five randomly selected maize inbred lines were examined by Southern filter hybridization with probes of cloned low copy sequences. Typically, several alleles could be distinguished among the inbred lines with any one probe and an appropriately selected restriction enzyme. Despite considerable polymorphism at the DNA level, 16 RFLP markers in three inbred lines of maize were examined for six to 11 generations and found be stable. Mapping of RFLP markers in maize can be accelerated by the use of B-A translocation stocks, which enable localization of a marker to chromosome arm in one generation. The use of recombinant inbred lines in further refinement of the map is discussed.     

Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.     

Molecular characterization and chromosomal distribution of species-specific repetitive DNA sequences from Beta corolliflora, a wild relative of sugar beet.

Repetitive DNA sequences have been isolated from a Sau3AI plasmid library of tetraploid Beta corolliflora (2n = 4x = 36), a wild relative of sugar beet (B. vulgaris). The library was screened by differential hybridization with genomic DNA of B. corolliflora and B. vulgaris. When used as probes for Southern hybridization of genomic DNA, six clones were determined to represent highly repetitive DNA families present only in the B. corolliflora genome. Five other sequences were highly repetitive in B. corolliflora and low or single copy in B. vulgaris. The insert size varied between 43 bp and 448 bp. Two sequences pBC1279 and pBC1944 displayed strong homology to a previously cloned satellite DNA from B. nana. With one exception, sequences are tandemly arranged as revealed by a typical ladder pattern after genomic Southern hybridization. The chromosomal distribution of five probes was determined by fluorescence in situ hybridization (FISH) of mitotic metaphases from B. corolliflora and a triploid hybrid between B. vulgaris and B. corolliflora. Three sequences were spread along all chromosome arms of B. corolliflora while one sequence was present on only six chromosomes. The chromosome-specific sequence pBC216 was found in close vicinity to the 5S rDNA located on B. corolliflora chromosome IV. This set of species-specific sequences has the potential to be used as probes for the identification of monosomic alien addition lines and for marker-assisted gene transfer from wild beet to cultivated beet.     

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.     

The integrated forms of the S1 and S2 DNA elements of maize male sterile mitochondrial DNA are flanked by a large repeated sequence.

The mitochondrial DNA of maize was cloned using the cosmid, Homer I. Recombinants carrying sequences homologous to the S1 and S2 DNA elements of male sterile maize have been analysed. Restriction endonuclease maps for Sac II, Sma I and Bam HI have been constructed. The S1 and S2 sequences are single copy sequences occurring at unique sites; each is flanked by a 26 kb repeated sequence. The repeated sequence has been shown not to contain the mitochondrial ribosomal RNA genes.     

Maize DNA enrichment by representational difference analysis in a maize chromosome addition line of oat

 The recent recovery of maize (Zea mays L.) single-chromosome addition lines of oat (Avena sativa L.) from oat x maize crosses has provided novel source materials for the potential isolation of maize chromosome-specific sequences for use in genetic mapping and gene cloning. We report here the application of a technique, known as representational difference analysis (RDA), to selectively isolate maize sequences from a maize chromosome-3 addition line of oat. DNA fragments from the addition line and from the oat parent were prepared using BamHI digestion and primer ligation followed by PCR amplification. A subtractive hybridization technique using an excess of the oat parental DNA was employed to reduce the availability for amplification of DNA fragments from the addition lines that were in common with the ones from the oat parental line. After three rounds of hybridization and amplification, the resulting DNA fragments were cloned into a plasmid vector. A DNA library containing 400 clones was constructed by this method. In a test of 18 clones selected at random from this library, four (22%) detected maize-specific repetitive DNA sequences and nine (50%) showed strong hybridization to genomic DNA of maize but weak hybridization to genomic DNA of oat. Among these latter nine clones, three detected low-copy DNA sequences and two of them detected DNA sequences specific to chromosome 3 of maize, the chromosome retained in the source maize addition line of oat. The other eight out of the 13 clones that had strong hybridization to maize DNA detected repetitive DNA sequences or high-copy number sequences present on most or all maize chromosomes. We estimate that the maize DNA sequences were enriched from about 1.8% to over 72% of the total DNA by this procedure. Most of the isolated DNA fragments detected multiple or repeated DNA sequences in maize, indicating that the major part of the maize genome consists of repetitive DNA sequences that do not cross-hybridize to oat genomic sequences. Received: 18 November 1997 / Accepted: 12 March 1998     

Translocation and amplification of an X-chromosome DNA repeat in inbred strains of mice.

A 9-kb repetitive DNA fragment (70-38) located near the centromere of the mouse X chromosome is amplified and translocated to an autosome in different inbred strains of mice. In situ hybridization and hybrid cell studies showed that probe 70-38 is located only on the X chromosome in mouse strains A/J, AKR/J, BALB/cJ, CBA/J, C3H/HeJ, C57BL/6J, DBA/2J and SWR/J. However, in four other mouse strains the DNA sequence is found near the centromere of an autosome in addition to the X chromosome. This autosome differs among the mouse strains (chromosome 11 in C57BL/10J or ScSn, chromosome 13 in NZB/B1NJ and chromosome 17 in SJL/J and PO). In those strains where the repeated sequence is located on an autosome, it has been amplified to about 100 copies. Restriction enzyme digestion patterns suggest a common structure for 70-38 sequences in the different strains. The changes in copy number, restriction enzyme digestion patterns, and chromosomal location of 70-38 reflect a rapid genomic evolution inbred mouse strains.     

Removal of repeated sequences from hybridisation probes.

Pre-reassociation of human clone probes, containing dispersed highly repeated sequences, (e.g. Alu and KpnI families), with a large excess of sonicated total human DNA allows signal from single and low copy number components to be detected in transfer hybridisations. The signal from non-dispersed repeated sequences is reduced to single copy levels. The procedure, which is simple and quick, is illustrated using model combinations of well characterised cloned probes, and is applied to a sample of randomly chosen cosmid clones. A theoretical assessment is presented which may be useful to those wishing to use this procedure.