Molecular analysis of a novel tandemly organized repetitive DNA sequence in<Emphasis Type="Italic">Citrus limon</Emphasis> (L.) Burm

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 of the genome of interest. Here, we report the isolation and molecular analysis of a novel tandemly organized repetitive DNA sequence from the genome of Citrus limon. Digestion of C. limon DNA with Hinf I produced a prominent fragment of approximately 300 bp. Southern blotting revealed a ladder composed of DNA fragments that were multimers of the 300-bp Hinf I band. Thus, Hinf I digestion revealed a novel satellite, which we have called the C. limon satellite DNA 300 (CL300). Sequence analysis shows significant homology between a portion of the CL300 monomer and the transposase box of an En/Spm-like element. The CL300 satellite was also detected in grapefruit, sour orange, trifoliate orange and kumquat. These results suggest that the CL300 repeat is an ancient satellite, and we propose that a significant portion originated by amplification of a genomic region containing the En/Spm-like transposase element.     

Characterization of a novel satellite DNA sequence from Flying Dragon (Poncirus trifoliata)

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 of the genome of interest. Here, we report the isolation and the molecular analysis and methylation status of a novel tandemly organized repetitive DNA sequence from the genome of Poncirus trifoliata. Digestion of P. trifoliata DNA with Afa I produced a prominent fragment of approximately 400 bp. Southern blotting analysis of genomic DNA digested with the same enzyme revealed a ladder composed of DNA fragments that are multimers of the 400-bp Afa I band, indicating that the repetitive DNA is arrayed in tandem. This suggests that Afa I isolated a novel satellite that we have called Poncirus trifoliata satellite DNA 400 (PN400). This satellite composes 25% of the genome and it is also present in lemon, sour orange and kumquat. Analysis of the methylation status demonstrated that the cytosines in CCGG sequences in this satellite were methylated.     

A chromosome 5-specific repetitive DNA sequence in rice (Oryza sativa L)

Repetitive DNA sequences in the rice genome comprise more than half of the nuclear DNA. The isolation and characterization of these repetitive DNA sequences should lead to a better understanding of rice chromosome structure and genome organization. We report here the characterization and chromosome localization of a chromosome 5-specific repetitive DNA sequence. This repetitive DNA sequence was estimated to have at least 900 copies. DNA sequence analysis of three genomic clones which contain the repeat unit indicated that the DNA sequences have two sub-repeat units of 37 bp and 19 bp, connected by 30-to 90-bp short sequences with high similarity. RFLP mapping and physical mapping by fluorescence in situ hybridization (FISH) indicated that almost all copies of the repetitive DNA sequence are located in the centromeric heterochromatic region of the long arm of chromosome 5. The strategy for cloning such repetitive DNA sequences and their uses in rice genome research are discussed.     

A novel repetitive DNA sequence in the genus Oryza

Summary Repetitive DNA sequences in the genus Oryza (rice) represent a large fraction of the nuclear DNA. The isolation and characterization of major repetitive DNA sequences will lead to a better understanding of rice genome organization and evolution. Here we report the characterization of a novel repetitive sequence, CC-1, from the CC genome. This repetitive sequence is present as long tandem arrays with a repeat unit 194 bp in length in the CC-diploid genome but 172 bp in length in the BBCC and CCDD tetraploid genomes. This repetitive sequence is also present, though at lower copy numbers, in the AA and BB genomes, but is absent in the EE and FF genomes. Hybridization experiments revealed considerable differences both in copy numbers and in restriction fragment patterns of CC-1 both between and within rice species. The results support the hypothesis that the CC genome is more closely related to the AA genome than to the BB genome, and most distantly related to the EE and FF genomes.     

Characterization of plant satellite DNA using restriction nucleases

The structural organization of satellite DNAs of mustard Brassica nigra and lemon Citrus limon has been studied by digestion with restriction nucleases. Analysis of DNA products produced by EcoRI and Bam I shows that two satellite DNAs contain long range periodicities belonging to several repeated sequences. The periodicities in two satellite DNAs differ characteristically, however, they have been found to contain common homologous sequences. Using the restriction nuclease Bsp I, a highly periodical fractions has been found in Citrus satellite DNA, composed of Bsp I fragments ranging from 80 to 1240 basepain. The major repeat units comprise five Bsp I fragments ranging from 80 to 200 bp. These fractions characterized by a high content of 5-methyl-cytosine.     

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.     

A subtelomeric satellite DNA family isolated from the genome of the dioecious plant Silene latifolia.

In an ongoing effort to trace the evolution of the sex chromosomes of Silene latifolia, we have searched for the existence of repetitive sequences specific to these chromosomes in the genome of this species by direct isolation from low-melting agarose gels of satellite DNA bands generated by digestion with restriction enzymes. Five monomeric units belonging to a highly repetitive family isolated from Silene latifolia, the SacI family, have been cloned and characterized. The consensus sequence of the repetitive units is 313 bp in length (however, high variability exists for monomer length variants) and 52.9% in AT. Repeating units are tandemly arranged at the subtelomeric regions of the chromosomes in this species. The sequence does not possess direct or inverted sequences of significant length, but short direct repeats are scattered throughout the monomer sequence. Several short sequence motives resemble degenerate monomers of the telomere repeat sequence of plants (TTTAGGG), confirming a tight association between this subtelomeric satellite DNA and the telomere repeats. Our approach in this work confirms that SacI satellite DNA sequences are among the most abundant in the genome of S. latifolia and, on the other hand, that satellite DNA sequences specific of sex chromosomes are absent in this species. This agrees with a sex determination system less cytogenetically diverged from a bisexual state than the system present in other plant species, such as R. acetosa, or at least a lesser degree of differentiation between the sex chromosomes of S. latifolia and the autosomes.     

Restriction endonuclease cleavage of satellite DNA in intact bovine nuclei

We have analyzed the efficiency with which specific nucleotide sequences within nucleosomes are recognized and cleaved by DNA restriction endonucleases. A system amenable to this sort of analysis is the cleavage of the bovine genome with the restriction endonuclease EcoRI. Bovine satellite I comprises 7% of the genome and is tandemly repetitious with an EcoRI site at 1400 base pair (bp) intervals within this sequence. The ease with which this restriction fragment can be measured permits an analysis of the accessibility of this sequence when organized in a nucleosomal array.Initial studies indicated that satellite I sequences are organized in a nucleosomal structure in a manner analogous to that observed for total genomic DNA. We then examined the accessibility of the EcoRI cleavage sites in satellite to endonucleolytic cleavage in intact nuclei. We find that whereas virtually all the satellite I sequences from naked DNA are cleaved into discrete 1400 bp fragments, only 33% of the satellite I DNA is liberated as this fragment from intact nuclei. These data indicate that 57% of the EcoRI sites in nuclei are accessible to cleavage and that cleavage can occur within the core of at least half the nucleosomal subunits. Analysis of the products of digestion suggests a random distribution of nucleosomes about the EcoRI sites of satellite I DNA.Finally, the observation that satellite sequences can be cleaved from nuclei to 1400 bp length fragments with their associated proteins provides a method for the isolation of specific sequences as chromatin. Using sucrose gradient velocity centrifugation, we have isolated a 70% pure fraction of satellite I chromatin. Nuclease digestion of this chromatin fraction reveals the presence of nucleosomal subunits and indicates that specific sequences can be isolated in this manner without gross disorganization of their subunit structure.     

Cloning, characterization and physical mapping of three cervid satellite DNA families in the genome of the Formosan muntjac (Muntiacus reevesi micrurus)

Due to their high sequence diversity even among closely related species, satellite DNA sequences can be a useful molecular marker for phylogenetic and taxonomic analyses. To characterize the satellite DNA in the genome of a native muntjac species of Taiwan, the Formosan muntjac, satellite DNA clones representing three different cervid satellite DNA families from this species were isolated and analyzed. Genomic organization study of these satellite DNAs was also undertaken. Three Formosan muntjac satellite DNA clones were obtained and designated as FM-satI (1,391 bp), FM-satII (1,143 bp) and FM-satIV (1,103 bp), and found to share approximately 82, 81 and 98% sequence homology with the Chinese muntjac satellite I clone (C5), Indian muntjac satellite II clone (Mmv-0.7) and Chinese muntjac satellite IV clone (MR-1.0), respectively. These three satellite DNA families are organized in a pter<--FM-satII-FM-satIV-FM-satI-->qter orientation in the centromeric region with satII closely associated with the telomeric sequences. Satellite DNA sequence comparison, in combination with chromosome data concludes that the Formosan muntjac is likely a subspecies of M. reevesi, closely related to the Chinese muntjac. With the kinetochore satellite II DNA co-localizing with the telomeric sequences, the Formosan muntjac chromosomes could be truly telocentric.     

Characterization of a tandemly repeated DNA from the fleshflySarcophaga bullata

In studies on the highly repetitive DNA sequences of the flesh flySarcophaga bullata, a 279 bp tandem repeat was cloned and sequenced. A 17 bp stretch within the clone was identical to a motif repeated five times in the satellite DNA of the Bermuda land crab. Southern DNA blotting showed the tandem repeat had a high degree of conservation of MboI sites, but had divergence for EcoRI sites; thus, all repeat units were not identical. The cloned DNA localized to the quinacrine-bright centromeric heterochromatin of the C and E autosomes and to sites on the chromosomal arms. In cases of asynapsis of homologs, the probe localized to euchromatic sites on both homologs or sometimes only on one homolog. The probe also localized near, to, or at a major developmental puff (B9). We conclude that blocks of this short interspersed repetitive DNA occur throughout theSarcophaga genome in both heterochromatin and euchromatin, and also that the variable position of these sequences suggests they possess a degree of instability.     

Organization and evolution of a higher plant alphoid-like satellite DNA sequence

Two recombinant plasmids containing, respectively, three and eight tandem repeats of a 177 base-pair (bp) element from radish nuclear DNA have been isolated. These plasmids were used as probes to investigate the organization and the copy number of this element within the genome. This sequence is present in congruent to 0.6 million copies. Restriction analysis provides evidence for sequence heterogeneity and reveals the occurrence of non-overlapping subfamilies. Nine units were sequenced and found to be remarkably conserved. However, sequences in the two clones clearly belong to two distinct subgroups. Our data suggest that these sequences evolved in a concerted manner and that homogenization mechanisms such as gene conversions certainly took place. The 177 bp sequence is made from three 60 bp blocks that are derived from a common ancestor. Exchanges between the three blocks probably occurred before they became fixed as a patchwork of short sequences, the 177 bp element. This unit of 177 bp was then amplified in several steps. The presence of such a repeated sequence can be detected in other Cruciferae when hybridizations are carried out under low stringency conditions. Direct comparison with a previously published mustard satellite DNA sequence indicates a similar organization and a 75% homology. Homology was also found with shorter regions (congruent to 60 bp) of broad bean and corn satellite DNA. Finally, homology was also found with several animal alphoid sequences, suggesting that this family also occurs in the plant genomes.     

Varied truncation and clustering characterize some short repeats identified in micronucleus-specific DNA of Tetrahymena thermophila

There are over 6000 internally eliminated DNA sequences (IESs) in the Tetrahymena genome that are deleted in a programmed fashion during the development of a polyploid, somatic macronucleus from a diploid germline micronucleus. Recently, based on several results, a homology and small RNA-based mechanism has been proposed for the efficient elimination of IES elements. Since the RNAi machinery is proposed to be intimately involved in silencing potentially harmful repeats such as transposons and viruses, characterization of repeats and the conditions for their developmental elimination from the somatic genome is warranted. Three short (500–600 bp) repeat families, members of which had been experimentally identified in IESs, that is, in micronucleus-specific DNA, are examined here using the Tetrahymena genome database. Members of all three families display varied degrees of truncation and are represented in macronuclear sequences. A 200 bp segment of one of the families can appear in the genome on its own, or as part of a 600 bp repeat detected experimentally, or in association with an unrelated 1 kb sequence to form a 1.2 kb repeat that is also frequently truncated. The 1 kb sequence contains a 300 bp section similar to a repeat associated with a non-long terminal repeat-like element and is often found accompanied by several more copies of this shorter repeat. These observations indicate that transposition may have had a role in the evolution of the short repeat families.     

PlantSat: a specialized database for plant satellite repeats

MOTIVATION: Tandemly organized repetitive sequences (satellite DNA) are widespread in complex eukaryotic genomes. In plants, satellite repeats often represent a substantial part of nuclear DNA but only a little is known about the molecular mechanisms of their amplification and their possible role(s) in genome evolution and function. Unfortunately, addressing these questions via characterization of general sequence properties of known satellite repeats has been hindered by a difficulty in obtaining a complete and unbiased set of sequence data for this analysis. This is mainly due to the presence of multiple entries of homologous sequences and of single entries that contain more than one repeated unit (monomer) in the public databases. RESULTS: We have established a computer database specialized for plant satellite repeats (PlantSat) that integrates sequence data available from various resources with supplementary information including repeat consensus sequences, abundances, and chromosomal localizations. The sequences are stored as individual repeat monomers grouped into families, which simplifies their computer analysis and makes it more accurate. Using this feature, we have performed a basic sequence analysis of the whole set of plant satellite repeats with respect to their monomer length and nucleotide composition. The analysis revealed several preferred length ranges of the monomers (approximately 165 bp and its multiples) and an over-representation of the AA/TT dinucleotide in the repeats. We have also detected an enrichment of satellite DNA sequences for the motif CAAAA that is supposed to be involved in breakage-reunion of repeated sequences.     

Characterization of MboI repeat DNA sequence of Anopheles stephensi.

MboI repeat fragment of mosquito Anopheles stephensi has been isolated by molecular cloning. The restriction map and entire nucleotide sequence of the 433bp insert has been determined. Hybridization of this repeat DNA with restriction enzyme digest of mosquito DNA does not show an interspersed pattern but suggests that this repeat may be tandemly repeated at one major site and a few minor sites in the genome of Anopheles stephensi. The hybridization pattern also indicates that this repeat family comprises of many similar but non-identical sequences. An open reading frame encoding 66 amino acids with an initiation and two tandem termination codons has been identified. This putative 66 amino acid polypeptide sequence has significant homology to a small region of RNA tumour viral envelope protein.     

Satellite DNA sequences in the New World primateCebus apella (Platyrrhini,Primates)

Two satellite DNAs, designated CapA and CapB, were isolated from the neotropical primate,Cebus apella. The satellites exhibit nonoverlapping distributions onC. apella chromosomes. CapA is a major component of interstitial regions of constitutive heterochromatin, a very large block of heterochromatin comprising most of the long arm of chromosome 11, and some telomeres. The CapA monomer has a length of about 1500 bp and appears recently to have undergone an amplification episode in theC. apella genome. CapA-like sequences are probably present in members of the family Cebidae (to whichC. apella belongs), but not in members of the family Callitrichidae (marmosets). CapB sequences can be detected at the centromeres of manyC. apella chromosomes, and similar sequences are present in all neotropical primates. The 342 bp CapB monomer shares 60%–64% sequence identity with several alpha satellite sequences of human origin. Because of its structure, sequence, and location, it appears that CapB is the New World primate homolog of Old World primate alpha satellite DNA.     

Pericentric satellite DNA sequences in Pipistrellus pipistrellus (Vespertilionidae; Chiroptera)

This paper reports the molecular and cytogenetic characterization of a HindIII family of satellite DNA in the bat species Pipistrellus pipistrellus. This satellite is organized in tandem repeats of 418 bp monomer units, and represents approximately 3% of the whole genome. The consensus sequence from five cloned monomer units has an A-T content of 62.20%. We have found differences in the ladder pattern of bands between two populations of the same species. These differences are probably because of the absence of the target sites for the HindIII enzyme in most monomer units of one population, but not in the other. Fluorescent in situ hybridization (FISH) localized the satellite DNA in the pericentromeric regions of all autosomes and the X chromosome, but it was absent from the Y chromosome. Digestion of genomic DNAs with HpaII and its isoschizomer MspI demonstrated that these repetitive DNA sequences are not methylated. Other bat species were tested for the presence of this repetitive DNA. It was absent in five Vespertilionidae and one Rhinolophidae species, indicating that it could be a species/genus specific, repetitive DNA family.     

A petite mitochondrial DNA segment arising in exceptionally high frequency in a mit- mutant of Saccharomyces cerevisiae

In cultures of the mit- mutant strain Mb12 of Saccharomyces cerevisiae (carrying a mutation in the oli2 gene), 70% of the cells are petite mutants. More than 80% of the petites from Mb12 contain a particular mtDNA segment, denoted BB5, that is 880 bp long and carries a single MboI site. Thus, in cultures of Mb12, about 56% of the cells are petites containing the defective BB5 mtDNA genome, and only 30% are mit- cells containing parental Mb12 mtDNA. The BB5 mtDNA segment is also found in petites arising from the wild-type strain J69-1B (from which Mb12 was derived), but in this case mtDNA from only five out of 24 petites produced an 880 bp band after MboI digestion. Since J69-1B cultures carry a petite frequency of about 5%, approximately 1% of cells in J69-1B cultures contain the BB5 mtDNA segment. The difference between Mb12 and J69-1B cultures is reflected in the MboI digestion patterns of the respective mtDNAs. While Mb12 mtDNA contains a grossly superstoicheiometric 880 bp MboI fragment, the corresponding fragment in J69-1B mtDNA cannot be seen on stained gels, but can be readily visualized in Southern blots hybridized to a 32P-labelled DNA probe obtained from the 880 bp MboI fragment. The BB5 mtDNA segment was shown to contain the ori1 sequence (one of several very similar sequences in wild-type mtDNA thought to act as origins of replication of mtDNA) which confers the genetic property of very high suppressiveness on petites carrying this mtDNA. The efficient replication of BB5 mtDNA may contribute to its abundance in Mb12 cultures. Nevertheless, other factors must operate to influence the abundance of the BB5 mtDNA segment in cultures of different strains, the most important of which is likely to be the rate of excision of this mtDNA segment from the parental mtDNA genome.     

A Musca domestica satellite sequence detects individual polymorphic regions in insect genome.

A recombinant plasmid, pHMd.24, was constructed which contains an array of a 24 bp sequence from the genome of the Musca domestica. This sequence hybridizes to sites occurring at various genomic locations in different housefly strains. The pHMd.24 insert has a structural organisation similar to satellite DNA sequences and hybridizes to homologous repeated sequences representing a small fraction of the M. domestica genome. Interestingly, this clone reveals polymorphic fragments from DNA isolated from single houseflies, thus generating an insect DNA fingerprint. In addition, the segregation of the fragments detected by hybridisation in a M. domestica pedigree indicates a transmission that follows mendelian inheritance.     

Identification and chromosomal location of a new tandemly repeated DNA in maize.

Two clones of a new family of tandemly repeated DNA sequences have been isolated from a maize random genomic DNA library. MR68 is 410 bp, representing a monomeric unit and MR77 is 1222 bp, containing three units. The copy number was estimated to be about 3000 per 1C maize genome. Its methylation pattern was also determined. Fluorescent in situ hybridization (FISH) indicates that the sequence is located on the subtelomeric region of the long arm of chromosomes 3 and 6, as well as on the satellite of chromosome 6.     

Histone gene clusters of the newt notophthalmus are separated by long tracts of satellite DNA

The genomic organization of the histone genes of the newt Notophthalmus viridescens is described. Genes for the five proteins are clustered on a 9.0 kb segment of cloned DNA which is part of a homogeneous family of sequences containing 600–800 members per haploid genome. The 9.0 kb histone gene clusters are not adjacent in the genome, but are separated from neighboring clusters by up to 50 kb or more of cluster spacer sequences; some or all of these spacer sequences are members of a predominantly centromeric satellite DNA with a 225 bp repeating unit.