Characterization of a new family of tobacco highly repetitive DNA, GRS, specific for theNicotiana tomentosiformis genomic component

Members of a new family of highly repetitive DNA sequences called GRS were isolated fromNicotiana tabacum L. genomic DNA and characterized. Cloned, sequenced monomeric units (180–182 bp) of GRS exhibit properties characteristic of molecules that possess a stable curvature. The GRS family represents about 0.15% of total genomic DNA (104 copies per haploid genome) and could be derived from eitherNicotiana tomentosiformis orNicotiana otophora, two possible ancestors of the T genome of the amphidiploidN. tabacum. Sequence homology between the HRS60 (Koukalováet al. 1989) and the GRS family has been estimated to be 57%.In situ hybridization was used to localize GRS on mitotic chromosomes. Hybridization signals were obtained on five pairs of chromosomes at intercalary sites of the longer chromosome arms. The majority of GRS sequences appeared to be organized in tandem arrays and a minority were found to be dispersed through the genome in short clusters, interspersed with other types of DNA repeats, including 25S rDNA sequences. Several loci containing both GRS and HRS60 were also found. Such hybrid loci may indicate intergenomic transfer of the DNA in the amphidiploidN. tabacum. GRS sequences, like HRS60 (Fajkuset al. 1992), were found to specify the location of nucleosomes. The position of the nucleosome core has been mapped with respect to a conservedMbol site in the GRS sequence and an oligo A/T tract is a major centre of the DNA curvature.

[1]  R. Matyášek,et al.  A BamHI family of highly repeated DNA sequences of Nicotiana tabacum , 1989, Theoretical and Applied Genetics.

[2]  F. Fittler,et al.  Eight different highly specific nucleosome phases on alpha-satellite DNA in the African green monkey , 1983, Nucleic Acids Res..

[3]  F. Solymosy,et al.  Nucleotide sequence of a 25S rRNA gene from tomato. , 1989, Nucleic acids research.

[4]  H. Zachau,et al.  Non-random arrangement of nucleosomes in satellite I containing chromatin of rat liver. , 1980, Nucleic acids research.

[5]  S. Elgin,et al.  Regulating genes by packaging domains: bits of heterochromatin in euchromatin? , 1993, Trends in genetics : TIG.

[6]  M. Bennett,et al.  Characterization of the Nicotiana tabacum L. genome by molecular cytogenetics , 1993, Molecular and General Genetics MGG.

[7]  J. Fajkus,et al.  Isolation and characterization of two middle repetitive DNA sequences of nuclear tobacco genome , 1991, Theoretical and Applied Genetics.

[8]  T. H. Goodspeed.  The genus nicotiana : origins, relationships and evolution of its species in the light of their distribution, morphology and cytogenetics , 1957 .

[9]  B. Vyskot,et al.  5-Azacytidine-induced hypomethylation of tobacco HRS60 tandem DNA repeats in tissue culture , 1991, Planta.

[10]  W. Peacock,et al.  Highly repeated DNA sequence limited to knob heterochromatin in maize. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Heslop-Harrison.  The Molecular Cytogenetics of Plants , 1991 .

[12]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[13]  J. Fajkus,et al.  Changes in chromatin structure due to hypomethylation induced with 5‐azacytidine or DL‐ethionine , 1992, FEBS letters.

[14]  P. Vogt,et al.  Potential genetic functions of tandem repeated DNA sequence blocks in the human genome are based on a highly conserved “chromatin folding code” , 1990, Human Genetics.

[15]  M. Radic,et al.  Curvature of mouse satellite DNA and condensation of heterochromatin , 1987, Cell.

[16]  R. Flavell.  Repeated Sequences and Genome Architecture , 1983 .

[17]  B. Hamkalo,et al.  Mouse satellite DNA, centromere structure, and sister chromatid pairing , 1986, The Journal of cell biology.

[18]  B. Vyskot,et al.  Differential sensitivity of CG and CCG DNA sequences to ethionine‐induced hypomethylation of the Nicotiana tabacum genome , 1992, FEBS letters.

[19]  G. Bernardi,et al.  Compositional bimodality of the nuclear genome of tobacco. , 1991, Nucleic acids research.

[20]  T. Lockett.  A bacteriophage lambda DNA purification procedure suitable for the analysis of DNA from either large or multiple small lysates. , 1990, Analytical biochemistry.

[21]  B. Koukalová,et al.  The distribution of tobacco HRS60 DNA repeated sequences in species of the genus Nicotiana , 1993 .

[22]  P. Hagerman,et al.  Cytosine methylation can induce local distortions in the structure of duplex DNA. , 1992, Biochemistry.

[23]  J. Devereux,et al.  A comprehensive set of sequence analysis programs for the VAX , 1984, Nucleic Acids Res..

[24]  R. Goldberg,et al.  Tobacco single-copy DNA is highly homologous to sequences present in the genomes of its diploid progenitors , 1985, Molecular and General Genetics MGG.

[25]  B. Vyskot,et al.  Maintenance of the induced hypomethylated state of tobacco nuclear repetitive DNA sequences in the course of protoplast and plant regeneration , 1994, Planta.

[26]  R. B. Flavell,et al.  Genome size and the proportion of repeated nucleotide sequence DNA in plants , 1974, Biochemical Genetics.

[27]  C. Shaw.  Plant molecular biology: a practical approach. , 1988 .

[28]  J. Heslop-Harrison.  Nuclear architecture in plants. , 1992, Current opinion in genetics & development.

[29]  J. Reich,et al.  Isolation and sequencing of HRS60dim1, a dimeric member of the HRS60-family of a tobacco DNA repeat. , 1989, Nucleic acids research.