Isolation and characterization of repetitive DNA sequences from Panax ginseng

Repetitive sequences constitute a significant component of most eukaryotic genomes, and the isolation and characterization of repetitive DNA sequences provide an insight into the organization and evolution of the genome of interest. We report the isolation and characterization of the major classes of repetitive sequences from the genome of Panax ginseng. The isolation of repetitive DNA from P. ginseng was achieved by the reannealing of chemically hydrolyzed (200 bp-1 kb fragments) and heat-denatured genomic DNA to low C(o)t value. The low C(o)t fraction was cloned, and fifty-five P. ginseng clones were identified that contained repetitive sequences. Sequence analysis revealed that the fraction includes repetitive telomeric sequences, species-specific satellite sequences, chloroplast DNA fragments and sequences that are homologous to retrotransposons. Two of the retrotransposon-like sequences are homologous to Ty1/ copia-type retroelements of Zea mays, and six cloned sequences are homologous to various regions of the del retrotransposon of Lilium henryi. The del retrotransposon-like sequences and several novel repetitive DNA sequences from P. ginseng were used to differentiate P. ginseng from P. quinquefolius, and should be useful for evolutionary studies of these disjunct species.     

Isolation and characterization of genome-specific DNA sequences in Triticeae species

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), each representing more than 1 % of the genomes. These repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae. speltoides was a 763 by EcoRI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomerec chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280 bp, which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing-over or reciprocal translocation. In situ hybridization showed that the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 by DraI fragment from H. chilense, also tandemly repeated but more variable; loss of the DraI site resulting in a ladder pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H. chilense and hence was able to identify the origin of chromosomes in a H. chilense x Secale africanum hybrid by in situ hybridization. It has a high A + T content (66%), small internal duplications, and a 50 by degenerate inverted repeat. We speculate that it has dispersed by retrotransposition in association with other sequences carrying coding domains. The organization and evolution of such sequences are important in understanding long-range genome organization and the types of change that can occur on evolutionary and plant breeding timescales. Genome-specific sequences are also useful as markers for alien chromatin in plant breeding.     

Isolation and characterization of salmonid telomeric and centromeric satellite DNA sequences

Satellite DNA clones with a 37 bp repeat unit were obtained from BglII-digested genomic DNA of Masu salmon (Oncorhynchus masou) and Chum salmon (O. keta). Fluorescence in situ hybridization (FISH) analysis with the isolated clones as a probe showed that these repetitive sequences were localized in the telomeric regions of chromosomes in both species. Southern and dot blot analyses suggested conservation of homologous sequences with similar repeat unit in other salmonids including the species of the genus Oncorhynchus and Salvelinus, but lack or scarcity of such sequences in the genus Hucho and Salmo. Similarly, polymerase chain reaction (PCR)-based cloning of satellite DNA referring to a reported Rainbow trout (O. mykiss) centromeric sequence was successful for the Oncorhynchus, Salvelinus and Hucho species. The obtained satellite DNA clones were localized with FISH in the centromeric regions of chromosomes of the species from these three genera. Although PCR cloning of the centromeric satellite DNA had failed in the Salmo species due to some base changes in the priming sites, dot blot hybridization analysis suggested conservation of homologous satellite DNA in the genus Salmo as in the other three genera. In the neighbor-joining tree of cloned centromeric satellite DNA sequences, the genus Oncorhynchus and Salvelinus formed adjacent clades, and the clade of the genus Hucho included the reported centromeric sequence of the genus Salmo. Conservation pattern and molecular phylogeny of the telomeric and centromeric satellite DNA sequences isolated herein support a close phylogenetic relationship between the genus Oncorhynchus and Salvelinus and between the Salmo and Hucho.     

Isolation and characterization of cloned DNA sequences containing ribosomal protein genes of Drosophila melanogaster.

Ribosomal (r) proteins encoded by polyadenylated RNA were specifically precipitated in vitro from polysomes by using antibodies raised against characterized Drosophila melanogaster r proteins. The immuno-purified mRNA in the polysome complex was used to prepare cDNA with which to probe a D. melanogaster genomic library. Selected recombinant phages were used to hybrid select mRNAs, which were analyzed by in vitro translation. Three clones containing the genes for r proteins 7/8, S18, and L12 were positively identified by electrophoresis of the translation products in one-dimensional and two-dimensional polyacrylamide gels. Sequences encoding r proteins S18 and L12 were found to be present in the genome in single copies. In contrast, the polynucleotide containing the region encoding 7/8 may be repeated or may contain or be flanked by short repeated sequences. The sizes of mRNAs that hybridized to the recombinant clone containing 7/8 were significantly larger than would be expected from the molecular weight of protein 7/8, implying that there were unusually long 5' and 3' noncoding sequences. The mRNAs for r proteins S18 and L12 were however, only about 10% larger. In situ hybridizations to salivary gland polytene chromosomes, using the recombinant phage, revealed that the recombinant clone containing the gene for r protein 7/8 hybridized to 5D on the X chromosome; the recombinant clone containing the gene for S18 hybridized to 15B on the same chromosome, and the recombinant phage containing the gene for L12 hybridized to 62E on chromosome 3L. It is of interest that the genomic locations of all three r protein clones were within the chromosomal intervals known to contain the Minute mutations [M(1)0, M(1)30, and M(3)LS2]. Although each clone contained sequences specifying two to four proteins, none had more than one identifiable r protein gene, suggesting that different D. melanogaster r protein genes may not be closely linked.     

Isolation and characterization of sequences from mouse chromosomal DNA with ARS function in yeasts.

Fragments of chromosomal DNA from a variety of eucaryotes can act as ARSs (autonomously replicating sequence) in yeasts. ARSs enable plasmids to be maintained in extrachromosomal form, presumably because they function as initiation sites for DNA replication. We isolated eight different sequences from mouse chromosomal DNA which function as ARSs in Saccharomyces cerevisiae (bakers' yeast). Although the replication efficiency of the different mouse ARSs in yeasts appears to vary widely, about one-half of them functions as well as the yeast chromosomal sequence ARS1. Moreover, five of the ARSs also promote self replication of plasmids in Schizosaccharomyces pombe (fission yeast). Each of the ARSs was cloned into plasmids suitable for transformation of mouse tissue culture cells. Plasmids were introduced into thymidine kinase (TK)-deficient mouse L cells by the calcium phosphate precipitation technique in the absence of carrier DNA. In some experiments, the ARS plasmid contained the herpes simplex virus type 1 TK gene; in other experiments (cotransformations), the TK gene was carried on a separate plasmid used in the same transformation. In contrast to their behavior in yeasts, none of the ARS plasmids displayed a significant increase in transformation frequency in mouse cells compared with control plasmids. Moreover, only 1 of over 100 cell lines contained the original plasmid in extrachromosomal form. The majority of cell lines produced by transformation with an ARS TK plasmid contained multiple copies of plasmid integrated into chromosomal DNA. In most cases, results with plasmids used in cotransformations were similar to those for plasmids carrying TK. However, cell lines produced by cotransformations with plasmids containing any one of three of the ARSs (m24, m25, or m26) often contained extrachromosomal DNAs.     

Isolation and characterization of DNA sequences from flow-sorted human chromosome 22 libraries

Two flow-sorted chromosome 22 libraries were used to isolate DNA sequences specific for chromosome 22. 45-phage DNAs were probed against human genomic DNA. 12 of them showed unique or low-copy character. Using digested DNA from rodent-human hybrid cell lines, 3 of the 12 recombinants were assigned unique to chromosome 22 and regionally mapped. 1 clone mapped to 22pter-q11, 1 clone to 22q12-qter and 1 clone, for which in situ hybridization was performed, to 22q13.1. 2 low-copy probes, 1 of them displaying polymorphisms in MspI and TaqI digests of individual DNAs, must have similar sequences on 22 and additional chromosomes. Furthermore, a highly repetitive DNA representing a compound locus of some hundred kilobases on chromosome 22 was isolated. These 6 probes may provide useful tools for studying the structure and function of this small chromosome involved in a relatively high number of inherited and acquired diseases.     

Isolation and characterization of Listeria monocytogenes-specific nucleotide sequences.

Subtracter probe hybridization was used to screen a partial genomic library of a clinical isolate of Listeria monocytogenes. Three clones that hybridized with genomic DNA from 174 strains of L. monocytogenes but not with genomic DNA from 32 strains representing other Listeria spp. were recovered. These data establish the utility of subtracter probe hybridization for recovering L. monocytogenes-specific sequences.     

Isolation and characterization of genome-specific DNA sequences in Triticeae species

Two contrasting genome-specific DNA sequences were isolated from Aegilops speltoides (wild goat grass) and Hordeum chilense (wild barley), each representing more than 1 % of the genomes. These repetitive DNA fragments were identified as being genome-specific before cloning by genomic Southern hybridization (using total genomic DNA as a probe), and hence extensive screening of clones was not required. For each fragment, up to six recombinant plasmid clones were screened and about half were genome-specific. Clone pAesKB52 from Ae. speltoides was a 763 by EcoRI fragment, physically organized in simple tandem repeats and shown to localize to sub-telomerec chromosome regions of species with the Triticeae S-genome by in situ hybridization to chromosomes. The sequence data showed an internal duplication of some 280 bp, which presumably occurred before sequence amplification and dispersion, perhaps by unequal crossing-over or reciprocal translocation. In situ hybridization showed that the sequence distribution varied between closely related (S-genome) species. Clone pHcKB6 was a 339 by DraI fragment from H. chilense, also tandemly repeated but more variable; loss of the DraI site resulting in a ladder pattern in Southern blots which had little background smear. In situ hybridization showed that the tandem repeats were present as small clusters dispersed along all chromosome arms except at a few discrete regions including the centromeres and telomeres. The clone hybridized essentially specifically to the H-genome of H. chilense and hence was able to identify the origin of chromosomes in a H. chilense x Secale africanum hybrid by in situ hybridization. It has a high A + T content (66%), small internal duplications, and a 50 by degenerate inverted repeat. We speculate that it has dispersed by retrotransposition in association with other sequences carrying coding domains. The organization and evolution of such sequences are important in understanding long-range genome organization and the types of change that can occur on evolutionary and plant breeding timescales. Genome-specific sequences are also useful as markers for alien chromatin in plant breeding.     

Isolation and characterization of two middle repetitive DNA sequences of nuclear tobacco genome

Summary Two DNA sequences, R8.1 and R8.3, representing two distinct classes of tobacco genomic repeated DNA, were cloned and characterized by Southern blot analysis. Both R8.1 and R8.3 were found to be homologous to the Nicotiana tomentosiformis component of the allotetraploid Nicotiana tabacum genome, and each of them represents about 0.3% of nuclear DNA. The R8.1 and R8.3 differ in the mode of distribution in chromosomes, as revealed by in situ DNA/DNA hybridization.     

Isolation and characterization of genomic mouse DNA clones containing sequences homologous to tRNAs and 5S rRNA.

We have cloned and characterized three fragments of Balb/c mouse DNA which hybridize to mouse cell tRNAs. Fractionation of the tRNAs which hybridize to these clones reveals that two of the clones, lambda Mt-4A and lambda Mt-6A hybridize to only one or two tRNAs, while one clone, lambda Mt-4B, hybridizes to at least seven tRNAs. Two of the tRNAs were identified as tRNAProCCG and tRNAGlyGGA, and others have been identified as tRNAs which are selectively encapsidated into virions of murine leukemia virus and avian reticuloendotheliosis virus. The DNA sequences of putative genes for tRNAProCCG and tRNAGlyGGA, plus flanking regions, were determined. A clone of Balb/c mouse DNA which selectively hybridized to 5S rRNA was also isolated and partially characterized.     

Isolation and characterization of Y chromosome DNA probes.

A sorted, cloned Y chromosome phage library was screened for unique Y chromosome sequences. Of the thousands of plaques screened, 13 did not hybridize to radiolabeled 46,XX total chromosomal DNA. Three plaques were characterized further. Clone Y1 hybridized to multiple restriction enzyme fragments in both male and female DNA with more intense bands in male DNA. Clone Y2, also found in female and male DNA, is probably located in the pseudosutosomal region because extra copies of either the X or Y chromosomes increased Y2 restriction enzyme fragment intensity in total cellular DNA. Clone Y5 was male specific in three of four restriction enzyme digests although in the fourth a light hybridizing band was observed in both male and female DNA. Clone Y5 was sublocalized to band Yq 11.22 by hybridization to a panel of cellular DNA from patients with Y chromosome rearrangements. Clone Y5 can be used to test for retention of the proximally long arm Y suggested to cause gonadal cancer in carrier females. The long series of GA repeats in Y5, anticipated to be polymorphic, may provide a sensitive means to follow Y chromosome variation in human populations.     

Isolation and characterization of DNA from archaeological bone.

DNA was extracted from human and animal bones recovered from archaeological sites and mitochondrial DNA sequences were amplified from the extracts using the polymerase chain reaction. Evidence is presented that the amplified sequences are authentic and do not represent contamination by extraneous DNA. The results show that significant amounts of genetic information can survive for long periods in bone, and have important implications for evolutionary genetics, anthropology and forensic science.     

Isolation and characterization of five rice telomere-associated sequences

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Isolation of repeated and self-complementary sequences from E. coli DNA

We have used the single-strand specific nuclease from Neurospora crassa and chromatography on methylated albumin-kieselguhr to purify and characterize repeated and self-complementary sequences from Escherichia coli DNA. Approximately 0.5% of the genome renatures spontaneously at zero time and another 2% renatures somewhat more rapidly than the total DNA. The early renaturing DNA has a base composition and a Tm similar to the total DNA and contains on the average 100 base pairs; the self-complementary DNA also has a base composition like E. coli but contains a mean of 170 base pairs. No evidence was obtained for the presence of a highly redundant sequence.     

Computer analysis of DNA and protein sequences.

Some recent trends in the development of theoretical methods for DNA and protein sequence analysis are reviewed, with particular emphasis on the design of new databases, motif searches, sequence alignment algorithms and applications of neural networks.     

Isolation of repetitive DNA sequences from human chromosome 21.

We have developed a method for the isolation of phage from the human genomic library that carry repetitive DNA sequences highly represented on specific human chromosomes. We have used this technique to select recombinants carrying inserts concentrated on chromosome 21. Five clones, representing two families of sequences, have been characterized. Members of each family show cross-homology, but the two families show no homology with each other. In all but one case, the clones do not contain members of the human Alu repeat family. Single chromosome-concentrated repetitive sequences should prove to be useful in studies of the origin, evolution, and function of repetitive DNA and in regional chromosome mapping.     

Structure of nuclear pre-mRNA. VIII. Isolation and characterization of complementary sequences.

After the annealing of pre-mRNA at high Cot values, up to 20% of the material becomes resistant to the action of ribonuclease. This has been attributed to the presence of self-complementary sequences (scRNA) in the pre-mRNA. The most important properties of scRNA, which was isolated in preparative amounts, have been studied. The approximate dimensions of the complementary segments are 45-50 nucleotides. Hybridization experiments have shown that scRNA is transcribed from repeated segments of the genome. It may be postulated that the rapidly hybridizing pre-mRNA fraction consists mainly of self-complementary sequences.