Genomic Subtraction Recovers Rye-Specific DNA Elements Enriched in the Rye Genome

Repetitive DNA sequence families have been identified in methylated relic DNAs of rye. This study sought to isolate rye genome-specific repetitive elements regardless of the level of methylation, using a genomic subtraction method. The total genomic DNAs of rye-chromosome-addition-wheat lines were cleaved to short fragments with a methylation-insensitive 4-bp cutter, MboI, and then common DNA sequences between rye and wheat were subtracted by annealing with excess wheat genomic DNA. Four classes of rye-specific repetitive elements were successfully isolated from both the methylated and non-methylated regions of the genome. Annealing of the DNA mixture at a ratio of the enzyme-restricted fragments:the sonicated fragments (1:3–1:5) was key to this success. Two classes of repetitive elements identified here belong to representative repetitive families: the tandem 350-family and the dispersed R173 family. Southern blot hybridization patterns of the two repetitive elements showed distinct fragments in methylation-insensitive EcoO109I digests, but continuous smear signals in the methylation-sensitive PstI and SalI digests, indicating that both of the known families are contained in the methylated regions. The subtelomeric tandem 350-family is organized by multimers of a 380-bp-core unit defined by the restriction enzyme EcoO109I. The other two repetitive element classes had new DNA sequences (444, 89 bp) and different core-unit sizes, as defined by methylation-sensitive enzymes. The EcoO109I recognition sites consisting of PyCCNGGPu-multi sequences existed with high frequency in the four types of rye repetitive families and might be a useful tool for studying the genomic organization and differentiation of this species.     

Cloning and genetic variability of a HindIII repetitive DNA in Acrossocheilus paradoxus (Cyprinidae).

Thirty clones of a highly repetitive HindIII fragment of DNA from seven populations of Acrossocheilus paradoxus (Cyprinidae) were isolated and sequenced. The fragment represents a tandemly repeated sequence, with a monomeric unit of 270 bp, amounting to 0.08-0.10% of the fish genome. Higher units of this monomer appear as a ladder in Southern blots. The HindIII satellite DNA family is conserved in three genera of the Cyprinidae. Variation in nucleotide sequences of this repetitive fragment, which is A+T-rich, is distributed both within individuals and among populations. High overall nucleotide divergence (dij = 0.056 +/- 0.001) was detected among clones of the HindIII satellite DNAs of Acrossocheilus paradoxus. Based on the molecular clock hypothesis, the maximum evolutionary rate was estimated to be 5.3 x 10(-7) substitutions per site per year. Lineage sorting may have contributed to the genetic heterogeneity within individuals and populations. Cladistic analyses indicated a closer phylogeographic relationship between populations of the central and south regions in Taiwan.     

Two repeated DNA sequences from the heterochromatic regions of rye (Secale cereale) chromosomes

Rye DNA sequences renaturing with a C₀t <0.02 mol·sec/l, are largely undigested by the restriction enzyme HindIII. These HindIII-spared sequences are mostly located in telomeric heterochromatin. When digested with EcoRI* and cloned into the EcoRI site of pBR 325, these sequences yielded clones of two classes when hybridized to a probe of rapidly renaturing DNA. One class contains a DNA sequence which is a major constituent of the telomeric heterochromatic blocks, while the other is a minor component of the highly repeated DNA of the genome. The major component was sequenced, its chromosomal distribution mapped using wheat-rye addition lines and its distribution in meiotic prophase nuclei determined. The minor component is present in significant amounts in wheat as well as in rye and is localized at the terminal heterochromatic regions of three rye chromosomes but not in the major blocks of heterochromatin.     

Studies of He-T DNA sequences in the pericentric regions of Drosophila chromosomes

He-T DNA is a complex set of repeated DNA sequences with sharply defined locations in the polytene chromosomes of Drosophila melanogaster. He-T sequences are found only in the chromocenter and in the terminal (telomere) band on each chromosome arm. Both of these regions appear to be heterochromatic and He-T sequences are never detected in the euchromatic arms of the chromosomes (Young et al. 1983). In the study reported here, in situ hybridization to metaphase chromosomes was used to study the association of He-T DNA with heterochromatic regions that are under-replicated in polytene chromosomes. Although the metaphase Y chromosome appears to be uniformly heterochromatic, He-T DNA hybridization is concentrated in the pericentric region of both normal and deleted Y chromosomes. He-T DNA hybridization is also concentrated in the pericentric regions of the autosomes. Much lower levels of He-T sequences were found in pericentric regions of normal X chromosomes; however compound X chromosomes, constructed by exchanges involving Y chromosomes, had large amounts of He-T DNA, presumably residual Y sequences. The apparent co-localization of He-T sequences with satellite DNAs in pericentric heterochromatin of metaphase chromosomes contrasts with the segregation of satellite DNA to alpha heterochromatin while He-T sequences hybridize to beta heterochromatin in polytene nuclei. This comparison suggests that satellite sequences do not exist as a single block within each chromosome but have interspersed regions of other sequences, including He-T DNA. If this is so, we assume that the satellite DNA blocks must associate during polytenization, leaving the interspersed sequences looped out to form beta heterochromatin. DNA from D. melanogaster has many restriction fragments with homology to He-T sequences. Some of these fragments are found only on the Y. Two of the repeated He-T family restriction fragments are found entirely on the short arm of the Y, predominantly in the pericentric region. Under conditions of moderate stringency, a subset of He-T DNA sequences cross-hybridizes with DNA from D. simulans and D. miranda. In each species, a large fraction of the cross-hybridizing sequences is on the Y chromosome.     

Genomic representation of the Hind II 1.9 kb repeated DNA.

The genomic representation and organization of sequences homologous to a cloned Hind III 1.9 kb repeated DNA fragment were studied. Approximately 80% of homologous repeated DNA was contained in a genomic Hind III cleavage band of 1.9 kb. Double digestion studies indicated that the genomic family, in the majority, followed the arrangement of the sequenced clone, with minor restriction cleavage variations compatible with a few base changes. Common restriction sites external to the 1.9 kb sequence were mapped, and hybridization of segments of the cloned sequence indicated the 1.9 kb DNA was itself not tandemly repeated. Kpn I bands which were homologous to the sequence contained specific regions of the repeat, and the molecular weight of these larger fragments could be simply explained. Mapping of common external restriction sites indicated that in some but not all cases the repeat could be organized in larger defined blocks of greater than or equal to 5.5 kb. In some instances, flanking regions adjacent to the repeat may contain common DNA elements such as other repeated DNA sequences, or possibly rearranged segments of the 1.9 kb sequence. It is suggested that although the 1.9 kb sequence is not strictly contiguous, at least some of these repeated sequences in the human genome are arranged in clustered or intercalary arrays. A region of the 1.9 kb sequence hybridized to a mouse repeated DNA, indicating homology beyond the primates.     

Most of the murine leukemia virus sequences in the DNA of NIH/swiss mice consist of two closely related proviruses, each repeated several times

The structure of the endogenous murine leukemia virus (MuLV) sequences of NIH/Swiss mice was analyzed by restriction endonuclease digestion, gel electrophoresis, and hybridization to an MuLV nucleic acid probe. Digestion of mouse DNA with certain restriction endonucleases revealed two classes of fragments. A large number of fragments (about 30) were present at a relatively low concentration, indicating that each derived from a sequence present once in the mouse genome. A smaller number of fragments (one to five) were present at a much higher concentration and must have resulted from sequences present multiple times in the mouse genome. These results indicated that the endogenous MuLV sequences represent a family of dispersed repetitive sequences. Hybridization of these same digested mouse DNAs to nucleic acid probes representing different portions of the MuLV genome allowed construction of a map of the sites where restriction endonucleases cleave the endogenous MuLV sequences. Several independent recombinant DNA clones of endogenous MuLV sequences have been isolated from C3H mice (Roblin et al., J. Virol. 43:113-126, 1982). Analysis of these sequences shows that they have the structure of MuLV proviruses. The sites at which restriction endonucleases cleave within these proviruses appeared to be similar or identical to the sites at which these nucleases cleaved within the MuLV sequences of NIH/Swiss mice. This identity was confirmed by parallel electrophoresis. We conclude that the apparently complex pattern of endogenous MuLV sequences of NIH/Swiss mice consists largely of only two kinds of provirus, each repeated multiple times at dispersed sites in the mouse genome.     

Aerobactin iron uptake sequences in plasmid ColV-K30 are flanked by inverted IS1-like elements and replication regions.

By using Southern blot hybridization procedures, we found that a specific sequence within a 16.3-kilobase HindIII restriction fragment of pColV-K30 was also present in at least three other pColV-K30 HindIII fragments. Restriction endonuclease mapping of these HindIII fragments indicated that two of these repeated sequences, identified as IS1-like, occur in reverse orientation adjacent to the ends of the aerobactin iron uptake region, also included in the 16.3-kilobase HindIII fragment. There are two distinct replication regions enclosing the IS1-flanked aerobactin genes. A pColV-K30 BamHI restriction endonuclease fragment, carrying one of these replication regions, showed homology with the F plasmid EcoRI fragment f5, which carries the F replication sequences.     

Sequences hybridizing to mRNA, oligo(dT) and dsRNA from pre-mRNA are contiguous in the cloned mouse DNA fragments.

Fragments from the DNA of mouse embryos produced by restriction endonucleases HindIII were cloned in pBR322 plasmid and examined for the ability to hybridize in situ with [32P] labeled cDNA synthesized from the polysomal poly(A)+mRNA template. Several of the selected clones were examined for the presence of specific sequences inside the cloned mouse DNA fragments by the blotting procedure of southern [1]. The data obtained indicate that the majority of the cloned mouse DNA fragments contained sequences hybridizing with cDNA, oligo(dT) and double-stranded regions from pre-mRNA. The results of hybridization experiments and double digestion with HindIII+HaeIII endonucleases provide evidence that these sequences could be contiguous in the given restriction DNA fragments.     

Distribution of repeated DNA families in the human genome

By means of restriction enzymes analysis and molecular hybridization, the distribution of repeated DNA families has been studied in the different DNA components into which the human genome can be fractionated by density gradient techniques. Three classes of DNA molecules have been analyzed: i) an homogeneous DNA component (satellite-like sequences; Q = 1.696 g/cm3, 3% of total DNA, AT repeated), ii) AT rich (Q = 1.698 g/cm3, 30% of total DNA, AT main-band) and GC rich (Q = 1.708 g/cm3, 6% of total DNA, GC main-band) DNA components. By this approach we have observed that Sau3A digestion of GC main-band gives rise to two bands of 75bp and 150bp, absent or under-represented in both AT rich DNA components. A preliminary characterization of these DNA fragments suggests that they contain one or more families of repeated sequences which fail to hybridize to EcoRI, HindIII and AluI families of repeats. In addition, we have observed that EcoRI sequences (alpha-RI DNA) are under-represented in GC main-band and show the same clustered organization in both AT rich DNA components.     

Structure and organization of the highly repeated and interspersed 1.3 kb EcoRI-Bg1II sequence family in mice.

EcoRI digestion of total mouse DNA yields a prominant 1.3 kb fragment amounting to between 1 and 2% of the mouse genome. The majority of the 1.3 kb EcoRI fragments have a single Bg1II site 800 bp from one end. This EcoRI-Bg1II sequence family shows HindIII and HaeIII sequence heterogeneity. We have cloned representatives of the EcoRI-Bg1II gene family in Charon 16A and studied their structure and organization within the genome. The cloned 1.3 kb fragments show the expected restriction enzyme patterns as well as additional heterogeneity. Representatives of the EcoRI-Bg1II sequence family were found to be interspersed throughout the mouse genome as judged by CsCl density gradient centrifugation experiments. Family members were also found to be organized in higher order repeating units. Homologous sequences were also found in other rodent species including rat and Chinese hamster. Cross hybridization between a cloned 1.3 kb mouse fragment and a cloned CHO repeated sequence is of special interest since the latter has been shown to contain sequences homologous to the Human A1uI family by nucleotide sequencing.     

Heterogeneity of the ribosomal DNA episome in strains and species of Entamoeba

Ribosomal DNA sequences in several species of the genus Entamoeba are highly repeated and display restriction fragment-length polymorphism (RFLP), which has been used to identify species and differentiate strains. However, the continuous variability of the non-transcribed repeat sequences in the ribosomal episome hinders an accurate typification. Looking for more reliable markers, we used DNA probes containing conserved sequences in the ribosomal episome — coding regions for the 16S and 5.8S rRNAs and transcribed spacers flanking the rDNA sequences, and the coding region for the 3 end of the 26S rRNA — to analyse hybridization patterns from five cloned pathogenic strains of Entamoeba histoiytica, two strains of the also pathogenic Entamoeba invadens and the non-pathogenic Laredo strain of Entamoeba moshkovskii. Our results provide reliable bases for the differentation of clones, strains and species of Entamoeba and the reconstruction of E. histolytica episomes. Differences in the number and length of rDNA-containing DNA fragments, previously observed by other investigators and confirmed by us, can be better defined by the present analysis.     

Structural organization of interspersed repetitive elements present in the DNA of Mus musculus

Two-dimensional displays of the restriction fragments from the DNA of Mus musculus revealed a complex species-specific pattern produced from nonsatellite repetitive sequences. The patterns have been used as a guide in the direct purification of a group of broadly interspersed repeated DNA sequences (characterized by a 1350-bp Eco-Bam fragment) that have been studied by molecular cloning, restriction mapping and genomic Southern blotting. These studies show that the cloned representatives originate from an abundant group of sequences that share homology with about 2% of the mouse genome. The sequences do not appear to share homology with mouse-interspersed-family-1 (MIF-1) nor with the major AT-rich satellite sequences of mouse. They appear to be part of a group of larger repetitive elements that is both broadly interspersed and heavily methylated in normal mouse tissue.     

Unusual and Strongly Structured Sequence Variation in a Complex Satellite DNA Family from the Nematode Meloidogyne chitwoodi

An AluI satellite DNA family has been isolated in the genome of the root-knot nematode Meloidogyne chitwoodi. This repeated sequence was shown to be present at approximately 11,400 copies per haploid genome, and represents about 3.5% of the total genomic DNA. Nineteen monomers were cloned and sequenced. Their length ranged from 142 to 180 bp, and their A + T content was high (from 65.7 to 79.1%), with frequent runs of As and Ts. An unexpected heterogeneity in primary structure was observed between monomers, and multiple alignment analysis showed that the 19 repeats could be unambiguously clustered in six subfamilies. A consensus sequence has been deduced for each subfamily, within which the number of positions conserved is very high, ranging from 86.7% to 98.6%. Even though blocks of conserved regions could be observed, multiple alignment of the six consensus sequences did not enable the establishment of a general unambiguous consensus sequence. Screening of the six consensus sequences for evidence of internal repeated subunits revealed a 6-bp motif (AAATTT), present in both direct and inverted orientation. This motif was found up to nine times in the consensus sequences, also with the occurrence of degenerated subrepeats. Along with the meiotic parthenogenetic mode of reproduction of this nematode, such structural features may argue for the evolution of this satellite DNA family either (1) from a common ancestral sequence by amplification followed by mechanisms of sequence divergence, or (2) through independent mutations of the ancestral sequence in isolated amphimictic nematode populations and subsequent hybridization events. Overall, our results suggest the ancient origin of this satellite DNA family, and may reflect for M. chitwoodi a phylogenetic position close to the ancestral amphimictic forms of root-knot nematodes. Received: 23 April 1997 / Accepted: 9 July 1997     

Germ line-restricted,highly repeated DNA sequences and their chromosomal localization in a Japanese hagfish (Eptatretus okinoseanus)

The various species of Japanese hagfish, namely, Eptatretus okinoseanus (types A and B), Eptatretus burgeri and Myxine garmani, are known to eliminate a fraction of their chromosomes during early embryogenesis. High molecular weight DNA from germ line cells and somatic cells of these hagfish species was isolated and digested with different restriction enzymes. The DNA fragments were separated by agarose gel electrophoresis. Digestion with BamHI and DraI generated two weak bands and one weak band, respectively, that were estimated to be about 90, and 180 bp and about 90 bp long and were limited to the germ line DNA in both types of E. okinoseanus. DNA filter hybridization experiments showed that the two BamHI fragments and the one DraI fragment were present almost exclusively in the germ line DNA of E. okinoseanus. Thus, these DNA fragments appear to be eliminated during embryogenesis. Moreover, evidence was obtained that these fragments are highly and tandemly repeated. Molecular cloning and sequence analysis revealed that the BamHI fragments are mainly composed of a family of closely related sequences that are 95 bp long (EEEo1, for Eliminated Element of E. okinoseanus 1), and the DraI fragment is composed of another family of closely related sequences that are 85 bp long (EEEo2). The two DNA families account for about 19% of the total eliminated DNA in E. okinoseanus type A. Fluorescence in situ hybridization experiments demonstrated that the two families of DNA are located on several C-band-positive, small chromosomes that are limited to germ cells in both types of E. okinoseanus.by W. Hennig     

Specific association of repetitive DNA sequences with major histocompatibility genes.

The DNA sequence organization of a 17.8-kilobase segment of porcine DNA, containing a functional major histocompatibility (MHC) gene, has been studied. The DNA flanking the MHC gene contains at least 10 distinct repetitive DNA sequence elements, each of which occurs only once within the 17.8-kilobase DNA segment. Their reiteration frequencies in the genome range from 10(2) to 10(4). The genomic organization of seven of these sequence elements has been examined; all are interspersed with other, unrelated DNA sequences. These seven repeated sequences are not generally associated in the genome. However, they appear to be nonrandomly linked in MHC-associated regions of the genome: at least two additional DNA segments containing MHC-homologous DNA also contain sequences homologous to DNA fragments bearing the seven different repeats. Of the seven sequences, four can be detected in splenic total RNA. These results suggest that these repeated elements are specifically associated with the MHC locus.     

Restriction endonuclease cleavage site map of safflower (Carthamus tinctorius L.) chloroplast DNA

Summary The restriction endonucleases SalI, PstI, KpnI and HindIII have been used to construct a physical map of safflower (Carthamus tinctorius L.) chloroplast DNA. This was accomplished by hybridizing Southern blots of single and double digested chloroplast DNA with ³²P-dCTP nick-translated SalI, KpnI and HindIII probes which were individually isolated from agarose gels. The chloroplast DNA was found to be circular and to contain approximately 151 kbp. In common with many other higher plant chloroplast DNAs a sequence of about 25 kbp is repeated in an inverted orientation. The small and large single copy regions separating the two repeated segments contain about 20 kbp and 81 kbp, respectively. The rRNA structural genes were also mapped by Southern blot hybridization and are co-linear with several other plant species.     

Analysis of the occurrence and nature of repeated DNA in an 850 kb region of Arabidopsis thaliana chromosome 4

The occurrence and nature of repeated DNA sequences has been analysed within an 850 kb YAC contig on Arabidopsis thaliana chromosome 4. Hybridization analysis with seven RFLP markers, six cosmid contigs, 29 YAC end probes and eight YAC clones showed that a least 585 kb of the 850 kb contained only low-copy sequences. One YAC end probe, EG15C8LE, hybridized to multiple genomic fragments and contained a sequence with predicted protein homology to cytochrome P450 monooxygenases. Another one, EG11B7RE, was found to be non-contiguous with the other YAC clones and contained a dispersed repetitive sequence associated with centromeric regions     

A highly repeated DNA sequence in Arabidopsis thaliana

Summary Three members of a family of highly repeated DNA sequences from Arabidopsis thaliana have been cloned and characterized. The repeat unit has an average length of 180 bp and is tandemly repeated in arrays longer than 50 kb. This family represents more than one percent of the Arabidopsis genome. Sequence comparisons with tandemly repeated DNA sequences from other Cruciferae species show several regions of homology and a similar length of the repeat unit. Homologies are also found to highly repeated sequences from other plant species. When the sequence CCGG occurs in the repeated DNA, the inner cytosine is generally methylated.     

Newly evolved highly repeated DNA sequences ofTupaia glis (Tupaiidae,Scandentia)

We have studied highly repeated DNA sequences ofTupaia glis (Tupaiidae, Scandentia) with restriction endonucleases and Southern blotting techniques. Five highly repeated DNA fragments have been isolated fromT. glis and hybridized with genomic DNAs (cleaved by different restriction enzymes) of several non-human primate species and one insectivore (E. europaeus), in order to highlight eventual differences or similarities of their highly repeated DNA sequences. Our first preliminary findings suggest that the newly isolated highly repeated DNA fragments ofT. glis are distinct from both non-human primates and insectivore, the two taxonomic groups considered most similar to the Tupaiidae.