A possible structure for calf satellite DNA I.

Calf satellite DNA I (p = 1.715) has been hydrolysed by a number or restriction endonucleases. It consists of a repeating unit of 1460 nucleotide pairs within which the sites of Eco R II Mbo I, Sac I, Alu I, Ava II and Hha I were localised in comparison with those of Eco R I and Hind II. The distribution of the Hpa II, Sac I, Hha I, Hinf I and Mbo II sites within calf satellite DNA I, as well as that of several restriction endonuclease sites within calf satellite DNA III (p = 1.705) allowed me to define subsatellite fractions. Furthermore, some of the sites of the CpG containing restriction enzymes Hpa II and Hha I are lacking. The possible implications of these results are discussed.     

Divergence, differential methylation and interspersion of melon satellite DNA sequences.

Melon (Cucumis melo) satellite DNA consists of two components, Q and S, each with a buoyant density in CsCl of 1.707 g/ml, but differing by 9 degrees C in "melting" temperature. These physical properties appear to be in contradiction, since both depend on G + C content. In order to resolve this anomaly, base compositions were directly determined for isolated fractions. the low-"melting" component S contains 41.8% G + C, with 6% of C present as 5-methylcytosine, whereas Q DNA contains 54% G + C, with 41% of C methylated. Analyses of restriction site loss agreed well with the direct determinations of methylation and divergence, and indicated some clustering of methylated sites in Q DNA. Analysis of restricted main-band DNA by hydridization with RNA complementary to Q satellite DNA ("Southern transfer") showed satellite Q tandem arrays interspersed in DNA of main-band density. Sequence divergence and extent of methylation did not appear to depend on whether a repeat array was present as satellite or interspersed in main-band DNA. Hydridization in situ indicated considerable heterogeneity in the genomic proportion of the Q-DNA sequences in melon fruit nuclei, implying over- and under-representation consistent with extensive unequal recombination in satellite Q tandem arrays. The cucumber, Cucumis sativus, contains less than 8% as much Q-homologous DNA per genome as the melon, suggesting rapid evolutionary gain or loss of these tandem repeat sequences.     

Methylation of satellite sequences in mouse spermatogenic and somatic DNAs.

The distribution of 5-methyl cytosine (5-MeC) residues in a highly repetitive sequence, mouse major satellite, was examined in germinal versus somatic DNAs by digestion with the methylation sensitive isoschizomers Msp I and Hpa II and Southern blot analysis, using a cloned satellite probe. DNA from liver, brain, and a mouse fibroblast cell line, C3H 10T1/2, yielded a multimeric hybridization pattern after digestion with Msp I (and control Eco RI) but were resistant to digestion with Hpa II, reflecting a high level of methylation of the satellite sequences. In contrast, DNA from mature sperm was undermethylated at these same sequences as indicated by the ability of Hpa II to generate a multimeric pattern. DNAs from purified populations of testis cells in different stages of spermatogenesis were examined to determine when during germ cell differentiation the undermethylation was established. As early as in primitive type A, type A, and type B spermatogonia, an undermethylation of satellite sequences was observed. This suggest that this highly specific undermethylation of germ cell satellite DNA occurs very early in the germ cell lineage, prior to entry into meiosis.     

The complexity of satellite deoxyribonucleic acid in a higher plant.

Purified satellite DNA from melon (Cucumis melo) was shown to contain at least two components from thermal-denaturation and renaturation studies. Two components were separated after partial renaturation, a fast-renaturing fraction similar in complexity to mouse satellite DNA, and one with 6000 times greater complexity. Both components renatured very accurately, indicating a minimum of sequence divergence. Centrifugation of the purified satellite DNA in Ag+/Cs2SO4 gradients resolved two major and several minor fractions. The two major fractions were only slightly enriched for fast- or slow-renaturing sequences.     

Methylation of somatic vs germ cell DNAs analyzed by restriction endonuclease digestions.

Bacterial restriction endonucleases containing the dinucleotide CpG in their cleavage sequences were used to compare the methylation patterns of primarily repeated DNA sequences in (1) bovine somatic cell native DNAs vs bovine sperm cell native DNA and (2) native vs renatured bovine liver and sperm cell DNAs. The restriction patterns of sperm native DNA differ markedly from those of somatic cell native DNAs when using Hpa II, Hha I, and Ava I but not when using the enzymes Eco RI and Msp I. Digestion patterns of germ cell renatured DNA differed significantly from those of germ cell native DNA when using Hpa II but not when using Msp I or Eco RI. The results may not be due to artifacts of renaturation of the DNAs. The results are consistent with the concept that germ cell DNA may be strand asymmetrically hemimethylated. The data also suggest that methylation of the 5'-cytosine in the sequence CCGG renders this site insensitive to cleavage by Msp I.     

Recent amplification of an alpha satellite DNA in humans.

A repeat sequence 682 base pairs (bp) long produced by cleavage of human DNA with Xba I restriction enzyme is composed of four tandemly arranged subunits with lengths of 171, 170, 171, and 170 bp each. The sequence organization of the 682 bp Xba I repeat bears a striking resemblance to other complex satellite DNAs of primates, including the Eco RI human alpha satellite family which also occurs as a 170 bp repeat. The Eco RI tetramer and the 682 bp Xba I repeat show a sequence divergence of 21%. The 682 bp Xba I repeat sequence is restricted to humans and is only distantly related to the previously reported 340 bp Xba human repeated DNA sequence. These finding are consistent with the concept of occasional amplifications of members or groups of members of alpha satellite DNA during human evolution. Amplifications apparently occurred after humans, apes and gibbons diverged from Old World monkeys (Eco RI satellite), after humans and apes diverged from gibbons (340 bp Xba I satellite) and after humans diverged from the great apes (682 bp Xba I satellite).     

Sensitivity of chromosomal and plasmid E. coli DNA to restriction endonuclease Eco RII]

It was shown that E. coli C, E. coli MRE 600 DNA, and also plasmid DNA of Col E1, RSF 2124 from E. coli K-12, and plasmid DNA from E. coli MRE 600 were completely resistant against restriction endonuclease R. Eco RII. Plasmid DNAs of Col E1, RSF 2124 amplificated for 4 hours in the presence of chloramphenicol are sensitive to R. Eco RII but after 16-hour amplification in the presence of chloramphenicol these DNAs acquire complete resistance against R. Eco RII. These data point to the slower rate of modification of DNA in vivo by DC-methylases of Eco RII type in comparison with DNA methylase Eco RII.     

Distinction between Nuclear Satellite DNAs and Chloroplast DNA in Higher Plants

Triton X-100 solubilized chloroplast DNA but not nuclear DNA from a mixture of chloroplasts and nuclei. The buoyant density of chloroplast DNA was different from that of the satellite DNA in all of the species examined (Phaseolus coccineus, Cucumis sativus, Cucumis melo, Antirrhinum majus, Vicia faba, Oenothera fruiticosa youngii). Chloroplast DNA constituted between 4.3% and 0.25% of the total leaf DNA in these species, and was present as 5 to 20 copies in each chloroplast.     

Genetic variability in a family of satellite DNAs from tilapia (Pisces: Cichlidae).

We have cloned and sequenced members of a family of satellite DNAs from three genera of the tilapiine tribe of fishes: Oreochromis, Sarotherodon, and Tilapia. The satellite DNAs, visualized as intensely staining bands following electrophoretic separation of EcoRI-digested genomic DNA, consist of three size variants differentially distributed in the various tilapiine species. The sizes of the monomers are approximately 237 bp (type I), 230 bp (type II), and 209 bp (type III). Several cloned monomers were sequenced from Oreochromis niloticus (type III), Oreochromis placidus (types I and II), Sarotherodon galilaeus (type I), Tilapia zillii (type I), and Tilapia rendalli (type I). Comparison of derived consensus sequences for the monomer units of the satellite DNAs revealed sequence identities within and between species that ranged from 89 to 96%. The type II and type III size variants appear to have arisen by deletions of 9 and 29 bp, respectively, within different regions of the type I satellite. Hybridization of a cloned monomer satellite from O. niloticus (type III) to PalI digests of genomic DNA from all three genera detected polymorphic, high molecular weight restriction fragments that produced fingerprint-like patterns. The complexity of these DNA fingerprints varied from one species to another, suggesting a markedly different genomic organization for these polymorphic satellite DNAs.     

Chromosomal distribution and organization of three cervid satellite DNAs in Chinese water deer (Hydropotes inermis)

The species-specific profile and centromeric heterochromatin localization of satellite DNA in mammalian genomes imply that satellite DNA may play an important role in mammalian karyotype evolution and speciation. A satellite III DNA family, CCsatIII was thought to be specific to roe deer (Capreolus capreolus). In this study, however, this satellite DNA family was found also to exist in Chinese water deer (Hydropotes inermis) by PCR-Southern screening. A satellite III DNA element of this species was then generated from PCR-cloning by amplifying this satellite element using primer sequences from the roe deer satellite III clone (CCsatIII). The newly generated satellite III DNA along with previously obtained satellite I and II DNA clones were used as probes for FISH studies to investigate the genomic distribution and organization of these three satellite DNA families in centromeric heterochromatin regions of Chinese water deer chromosomes. Satellite I and II DNA were observed in the pericentric/centric regions of all chromosomes, whereas satellite III was distributed on 38 out of 70 chromosomes. The distribution and orientation of satellite DNAs I, II and III in the centromeric heterochromatin regions of the genome were further classified into four different types. The existence of a Capreolus-like satellite III in Chinese water deer implies that satellite III is not specific to the genus Capreolus (Buntjer et al., 1998) and supports the molecular phylogeny classification of Randi et al. (1998) which suggests that Chinese water deer and roe deer are closely related.     

Physical and gene mapping of chloroplast DNA from Atriplex triangularis and Cucumis sativa.

A rapid and simple method for constructing restriction maps of large DNAs (100-200 kb) is presented. The utility of this method is illustrated by mapping the Sal I, Sac I, and Hpa I sites of the 152 kb Atriplex triangularis chloroplast genome, and the Sal I and Pvu II sites of the 155 kb Cucumis sativa chloroplast genome. These two chloroplast DNAs are very similar in organization; both feature the near-universal chloroplast DNA inverted repeat sequence of 22-25 kb. The positions of four different genes have been localized on these chloroplast DNAs. In both genomes the 16S and 23S ribosomal RNAs are encoded by duplicate genes situated at one end of the inverted repeat, while genes for the large subunit of ribulose-1,5-bisphosphate carboxylase and a 32 kilodalton photosystem II polypeptide are separated by 55 kb of DNA within the large single copy region. The physical and genetic organization of these DNAs is compared to that of spinach chloroplast DNA.     

On the possibility of localizing in situ Mus musculus and Drosophila virilus satellite DNAs by Alu I and Eco RI restriction endonucleases

Mus musculus and Drosophila virilus metaphase chromosomes have been treated with Alu I or Eco RI restriction endonucleases and, to ascertain possible selective digestion, subsequently stained with the DNA-specific dye ethidium bromide. The correlation between our findings and previously known cytological and biochemical data allowed us to show that Alu I is an enzyme capable of cytologically detecting repetitive DNAs, while Eco RI is unable to do so. This would be a consequence of the fact that Alu I extensively digests and extracts all chromosomal DNA except that localized in those regions where the presence of satellite DNAs has previously demonstrated. Eco RI, on the contrary, is not capable of doing so and its activity seems to be obstructed by alcohol:acid fixation procedures.     

Sequence analysis of bovine satellite I DNA (1.715 gm/cm3).

The 1402 bp Eco RI repeating unit of bovine satellite I DNA (rho CsCl = 1.715 gm/cm3) has been cloned in pBR322. The sequence of this cloned repeat has been determined and is greater than 97% homologous to the sequence reported for another clone of satellite I (48) and for uncloned satellite I DNA (49). The internal sequence structure of the Eco RI repeat contains imperfect direct and inverted repeats of a variety of lengths and frequencies. The most outstanding repeat structures center on the hexanucleotide CTCGAG which, at a stringency of greater than 80% sequence homology, occurs at 26 locations within the RI repeat. Two of these 6 bp units are found within the 31 bp consensus sequence of a repeating structure which spans the entire length of the 1402 bp repeat (49). The 31 bp consensus sequence contains an internal dodecanucleotide repeat, as do the consensus sequences of the repeat units determined for 3 other bovine satellite DNAs (rho CsCl = 1.706, 1.711a, 1.720 gm/cm3). Based on this evidence, we present a model for the evolutionary relationship between satellite I and the other bovine satellites.     

A family of moderately repetitive sequences in mouse DNA.

When mouse DNA is digested to completion with restriction endonuclease Eco R1, a distinct band of 1.3 kb segments comprising about 0.5-3% of the genome is observed upon agarose gel electrophoresis. This DNA is not tandemly repeated in the genome and is not derived from mouse satellite DNA. Restriction endonuclease analysis suggested that the 1.3 kb segments are heterogeneous. Specific sequences were selected from the 1.3 kb segments and amplified by cloning in plasmid pBR322. Southern transfer experiments indicated that three separately cloned mouse DNA inserts hybridized predominantly to the Eco R1 1.3 kb band and to the conspicuous subsegments generated by secondary restriction endonuclease cleavage of the sucrose gradient purified 1.3 kb segments. Segments were also excised by Hha I (Hha I segments) from the chimeric plasmids containing mouse DNA inserts and subjected to restriction endonuclease and cross-hybridization analysis. It was found that the three Hha I segments were different, although two of them exhibited partial sequence homology. Cot analysis indicated that each of the Hha I segments are repeated about 10(4) times in the mouse genome. These findings indicate that a family of related but non-identical, moderately repetitive DNA sequences, rather than a single homogeneous repeat, is present in the 1.3 kb Eco R1 band.     

Sequence arrangement in satellite DNA from the muskmelon

Two fractions of a satellite DNA from the muskmelon (Cucumis melo L.) isolated as a unimodal peak from CsCl gradients, differ in melting properties and complexity as estimated by reassociation kinetics. At 49.8 C, all of the low melting fraction was denatured and all of the high melting fraction was native. There were almost no partially denatured molecules detected in the electron microscope at this temperature. This observation provides direct evidence that the two fractions are not closely linked. We conclude that satellite I, the high t_m, low complexity fraction, exists as a 600-nucleotide sequence in blocks of at least 67 tandem repeats. Since the complexity of the low melting fraction, satellite II, is greater than the size of the molecules in our assay, we can only say that the minimum size of each unit of satellite II is 2.5 × 10⁷ daltons. It is unlikely that any spacer sequences are interspersed with either satellite.     

DNA cloning and hybridization in deer species supporting the chromosome field theory

The Cervidae show the largest variation in chromosome number found within any mammalian family. The eight species of deer which are the subject of this study vary in chromosome number from 2n = 70 to 2n = 6. Three species of Bovidae are also included since they belong to a closely related family. Digestion of nuclear DNAs with the restriction endonucleases Hae III, Hpa II, Msp I, Eco RI, Xba I, Pst I and Bam HI reveals that there is a series of highly repetitive sequences forming similar band patterns in the different species. There are two bands (1100 and 550 base pairs) which are common to all species although the two families separated more than 40 million years ago. To obtain information on the degree of homology among these conserved sequences we isolated a Bam HI restriction fragment of approximately 770 base pairs from red deer DNA. This sequence was 32P labeled and hybridized by the Southern blot technique with DNAs cleaved with Bam HI, Eco RI, Hpa II and Msp I. Moreover, the same sequence was cloned in the plasmid vector pBR322 nick translated with 32P and hybridized with the DNAs of 8 species of Cervidae and 3 of Bovidae. The same cloned probe was labeled with 3H and hybridized in situ with the metaphase chromosomes of red deer (2n = 68) and Muntiacus muntjak (2n = 7 male). Homologies are still present between the highly repetitive sequences of the 8 species of Cervidae despite the drastic reorganization that led to extreme chromosome numbers. Moreover, the cloned DNA sequence was found to occupy the same position, in the proximal regions of the arms, in both red deer (2n = 68) and M. muntjak (2n = 7 male) chromosomes. The ribosomal RNA genes and the centromeres in these species have also maintained their main territory despite the drastic chromosome reorganization. These results are experimental confirmation of the chromosome field theory which predicted that each DNA sequence has an optimal territory within the centromere-telomere field and tends to occupy this same territory following chromosome reorganization.     

Variation in satellite DNA from some higher plants

Pure satellite DNAs were prepared as minor components after centrifugation to equilibrium on CsCl gradients. A single satellite DNA band was isolated from flax (Linum usitatissimum) DNA and two bands were resolved in cucumber (Cucumis sativus) DNA. These apparently homogeneous components of the plant genomes were further analyzed by thermal denaturation and renaturation. The flax satellite DNA appeared homogeneous on thermal denaturation but was shown to contain several components of renaturation. The two cucumber satellite DNAs were different from each other, but both showed at least two components in denaturation and renaturation analyses. Renaturation in the three satellites, particularly in flax, was inaccurate, indicating a considerable degree of sequence divergence. Although each satellite contained quite large amounts of simple repetitious sequences, a residual heterogeneous DNA fraction was always present. It is considered that this was too large a portion of the satellite DNA to be due to organelle or ribosomal DNA in cucumber. The latter possibility is precluded in flax, where the satellite is completely resolved in buoyant density from both organelle and ribosomal DNA.     

Satellite DNA relationships in man and the primates

We have investigated the genomes of a series of primates to identify the presence of sequences related to human satellite DNAs I, II and III by restriction enzyme digestion and hybridisation with probes of these satellite DNAs. Where we have found such related sequences we have examined the extent to which they have diverged by measuring the stability of the hybrids. DNA satellite III is the oldest sequence being common to species which have diverged some 24 million years ago. In contrast DNA satellites I and II are of much more recent origin. Our results permit us to draw conclusions about the way these sequences have evolved, and how the evolution of repeated DNA sequences may be related to the evolution of the primate lineage.     

Nucleic acid renaturation and restriction endonuclease cleavage analyses show that the DNAs of a transforming and a nontransforming strain of Epstein-Barr virus share approximately 90% of their nucleotide sequences.

Viral DNA molecules were purified from a nontransforming and a transforming strain of Epstein-Barr virus. Each viral DNA was labeled in vitro and renatured in the presence of an excess of either one or the other unlabeled viral DNA. Both viral DNAs were also digested with the Eco R1 restriction endonuclease and subsequently labeled by using avian myeloblastosis virus DNA polymerase to repair either the EcoR1 nuclease-generated single-stranded ends of the DNAs or their single-stranded ends produced by a second digestion with exonuclease III after the first EcoR1 nuclease digestion. The results of these experiments support three general conclusions: (i) the DNAs of these two strains of Epstein-Barr virus share approximately 90% of their nucleotide sequences; (ii) both viral DNA populations are reasonably homogenous; and (iii) both DNAs contain repetitions or inverted repetitions of some of their nucleotide sequences.