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.     

The primary structure of bovine satellite 1.715.

The primary structure of the 1402 bp repeat unit of bovine satellite 1.715 has been determined using a dimer inserted at the SalI site of plasmid pBR322 and cloned in E. coli. In contrast with bovine satellites 1.706, 1.720b and 1.711a, the 1.715 satellite has a complex sequence with no obvious internal short prototype repeat. The sequence consists however of repeats ranging in length from 6 to 13 nucleotides. In addition, the hexanucleotide, AGATGA, present in the prototype sequences of satellites 1.706, 1.720b and 1.711a, is found in satellite 1.715 in repeats as long as, or longer than, 8 nucleotides, establishing a homology link among those satellites on one hand and satellite 1.715 (and the related satellite 1.711b) on the other. In turn, this suggests a common evolutionary origin. A comparison of the maps for 15 restriction enzymes of cloned and uncloned satellite indicates very little sequence divergence among the repeat units of the latter, most of the differences being due to methylation.     

Apparent relatedness of the main component of ovine 1.714 satellite DNA to bovine 1.715 satellite DNA

The nucleotide sequence of the principal component of ovine 1.714 g/cm3 satellite DNA was determined from a monomeric fragment inserted at the BamHI site of pBR322 and cloned in Escherichia coli strain RR1. The 816-bp tandemly repeated sequence contains a number of small repeated sequences dispersed within it, one group of which forms a pentameric tandem repeat of a 13-bp segment (positions 548-612). A 20-bp region (60-79) shows an 85% homology with the reverse-complement of the sequence from 455 through 474. There are two regions of 67 bp (75-141) and 59 bp (755-813) which show greater than 70% homology with regions of bovine 1.715 g/cm3 satellite DNA (1402 bp; positions 1218-1284 and 1079-1137, respectively) while a 31-bp region (ovine 62-92, bovine 133-163) shows 80% homology. Quasi-correlation coefficients (Qr) were determined using the triplet numbers of the sheep satellite versus all sequences in the National Biomedical Research Foundation and EMBL nucleotide sequence data bases. Qr equals 0.85 for ovine 1.714 g/cm3 satellite versus bovine 1.715 g/cm3 satellite. The next highest Qr for a bovine satellite segment was 0.58. Thus, the ovine 1.714 g/cm3 and bovine 1.715 g/cm3 satellite appear demonstrably related. Taking into account that sheep and cattle diverged 18-20 million years ago, this suggests that the material may be functional and that its function is related to its sequence.     

Methylation of satellite DNA

The lower amount of 5 methylcytosine in DNA from bull sperm relative to DNA of other bovine tissues is a result of the absence of this minor base from several of the satellite DNAs in sperm. This applies particularly to the 1.715, 1.711b and 1.709 satellites and less so to the 1.706 and 1.711a satellites. Mouse sperm DNA is also partially undermethylated.     

The independent evolution of two closely related satellite DNA elements in rats (Rattus).

We have identified and determined the sequence and organization of a new rat satellite DNA in Rattus rattus, the roof rat. This satellite DNA, which we call R. rattus satellite I', consists of tandem arrays of a 185 base pair (bp) repeat unit that we call a'. a' is 86% homologous to a 185 bp portion of the 370 bp repeat unit of the previously described rat satellite [Pech et al. (1979) Nucleic Acids Res. 7, 417-432] present in the common laboratory rat species, R. norvegicus. We can thereby distinguish two 185 bp portions of the satellite I 370 bp repeat unit: "a" (homologous to a') and "b". Satellite I has the structure (a,b)n, and satellite I' has the structure (a')n. Like a, a' is only about 60% homologous to b and fails to hybridize to b. Although R. norvegicus and R. rattus contain about the same total concentration of satellite sequences, R. norvegicus contains essentially only the a,b type (satellite I), whereas R. rattus contains a' type (satellite I') and lesser amounts of the a,b type (satellite I). The a,b type (satellite I) in R. rattus is very similar to that in R. norvegicus as judged both by hybridization and by the presence of all but one of the major restriction enzyme sites that characterize the R. norvegicus satellite I. In R. rattus the a' and a,b repeat units are not detectably present in the same tandem array. All of the sequence differences between a' (R. rattus) and a (R. norvegicus) can be accounted for by simple base substitutions, and the implication of this and other features of rat satellite DNA structure for satellite DNA evolution are discussed.     

The muntjak satellite IA sequence is composed of 31-base-pair internal repeats that are highly homologous to the 31-base-pair subrepeats of the bovine satellite 1.715

The nucleotide sequence of a cloned Muntjak satellite IA repeat unit (Muntiacus muntjak vaginalis) was determined. The repeat is 807 base pairs (bp) long. By introducing minor deletions and insertions, the whole sequence of the satellite can be arranged in 27 subrepeats of 31 bp length. Although diverged relative to each other, all subrepeats show a homology of more than 53% with the common consensus sequence. In 29 out of the 31 bp the consensus sequence of the Muntjak satellite subrepeat is identical to the 31-bp subrepeat of the bovine satellite 1.715. This suggests that both satellites are derived from a common ancestral sequence. The results have interesting implications for the evolution of the two satellites.     

Patchwork structure of a bovine satellite DNA

According to a previous restriction nuclease analysis, bovine 1.706 satellite DNA (density 1.706 g/cm3 in CsCl) is organized in an unusual structure of superimposed long- and short-range repeats (Streeck and Zachau, 1978). We have now determined the nucleotide sequence of this satellite DNA in both cloned fragments and fragments from the total satellite DNA. Each long-range repeat unit (about 2350 bp) is divided into four segments. Each segment consists of different variants of a basic 23 bp sequence which is itself composed of a dodecanucleotide and a related undecanucleotide. A total of 2400 nucleotides have been sequenced. Detailed analysis of the sequence divergence reveals that both the overall extent of divergence and the frequency of base changes at individual positions of the 23 bp repeats are characteristically different in the various segments. Preferentially methylated sites and a high incidence of symmetry elements are found. In two of the four segments, 22 of 23 bp of the prototype sequence are included in six overlapping elements of dyad symmetry and in a palindrome. A scheme for the evolution of the satellite DNA from a basic dodecanucleotide is proposed which is based on the different degrees of divergence for the various repeats superimposed in this satellite DNA.     

Characterization and chromosomal distribution of satellite DNA sequences of the water buffalo (Bubalus bubalis).

Satellite DNA sequences were isolated from the water buffalo (Bubalus bubalis) after digestion with two restriction endonucleases, BamHI and StuI. These satellite DNAs of the water buffalo were classified into two types by sequence analysis: one had an approximately 1,400 bp tandem repeat unit with 79% similarity to the bovine satellite I DNA; the other had an approximately 700 bp tandem repeat unit with 81% similarity to the bovine satellite II DNA. The chromosomal distribution of the satellite DNAs were examined in the river-type and the swamp-type buffaloes with direct R-banding fluorescence in situ hybridization. Both the buffalo satellite DNAs were localized to the centromeric regions of all chromosomes in the two types of buffaloes. The hybridization signals with the buffalo satellite I DNA on the acrocentric autosomes and X chromosome were much stronger than that on the biarmed autosomes and Y chromosome, which corresponded to the distribution of C-band-positive centromeric heterochromatin. This centromere-specific satellite DNA also existed in the interstitial region of the long arm of chromosome 1 of the swamp-type buffalo, which was the junction of the telomere-centromere tandem fusion that divided the karyotype in the two types of buffaloes. The intensity of the hybridization signals with buffalo satellite II DNA was almost the same over all the chromosomes, including the Y chromosome, and no additional hybridization signal was found in noncentromeric sites.     

Different repeat lengths in rat satellite I DNA containing chromatin and bulk chromatin.

The nucleosome repeat structure of a rat liver chromatin component containing the satellite I DNA (repeat length 370 bp) was investigated. Digestion experiments with micrococcal nuclease, DNAase II, and the Ca2+/Mg2+-dependent endogenous nuclease of rat liver nuclei revealed a repeat unit of 185 nucleotide pairs which is shorter by approximately 10 bp than the repeat unit of the bulk chromatin of this cell type. The difference seems not to be related to the histone composition which was found to be similar in the two types of chromatin.     

The spread of sequence variants in Rattus satellite DNAs

The genus Rattus has two related families of satellite DNA: Satellite I consists of tandem arrays of a 370 base pair repeat unit which is a dimer of two 185 base pair portions (a, b) which are about 60% homologous. Satellite I' consists of tandem arrays of a 185 base pair repeat unit (a') which is about 85% homologous to a and 60% homologous to b. R. norvegicus contains only satellite I but R. rattus contains both satellites I and I'. We examined certain aspects of satellite DNA evolution by comparing the spacing at which variant repeat units of each satellite have spread among non-variant repeat units in these two species. With but one exception, in R. rattus, 15 different variant repeat units have spread among non-variant repeat units of satellite I, with a spacing equal to the length of the (a,b) dimer. Similarly, fourteen different variant repeat units of the monomeric satellite I' have mixed among non-variant repeat units with a spacing equal to the length of the (a') monomer. These results suggest that a mechanism involving homologous interaction among satellite sequences could account for the spread of variant family members. We also found that a sequence variant present in certain portions of the dimeric repeat unit of satellite I is more efficiently amplified (or less efficiently corrected) than variants occurring in other regions. This was not true for the monomeric repeat unit of satellite 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).     

Concerted evolution of primate alpha satellite DNA. Evidence for an ancestral sequence shared by gorilla and human X chromosome alpha satellite

To understand evolutionary events in the formation of higher-order repeat units in alpha satellite DNA, we have examined gorilla sequences homologous to human X chromosome alpha satellite. In humans, alpha satellite on the X chromosome is organized as a tandemly repeated, 2.0 x 10(3) base-pairs (bp) higher-order repeat unit, operationally defined by the restriction enzyme BamHI. Each higher-order repeat unit is composed of 12 tandem approximately 171 base-pair monomer units that have been classified into five distinct sequence homology groups. BamHI-digested gorilla genomic DNA hybridized with the cloned human 2 x 10(3) bp X alpha satellite repeat reveals three bands of sizes approximately 3.2 x 10(3), 2.7 x 10(3) and 2 x 10(3) bp. Multiple copies of all three repeat lengths have been isolated and mapped to the centromeric region of the gorilla X chromosome by fluorescence in situ hybridization. Long-range restriction mapping using pulsed-field gel electrophoresis shows that the 2.7 x 10(3) and 3.2 x 10(3) bp repeat arrays exist as separate but likely neighboring arrays on the gorilla X, each ranging in size from approximately 200 x 10(3) to 500 x 10(3) bp, considerably smaller than the approximately 2000 x 10(3) to 4000 x 10(3) bp array found on human X chromosomes. Nucleotide sequence analysis has revealed that monomers within all three gorilla repeat units can be classified into the same five sequence homology groups as monomers located within the higher-order repeat unit on the human X chromosome, suggesting that the formation of the five distinct monomer types predates the divergence of the lineages of contemporary humans and gorillas. The order of 12 monomers within the 2 x 10(3) and 2.7 x 10(3) bp repeat units from the gorilla X chromosome is identical with that of the 2 x 10(3) bp repeat unit from the human X chromosome, suggesting an ancestral linear arrangement and supporting hypotheses about events largely restricted to single chromosome types in the formation of alpha satellite higher-order repeat units.     

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.     

The 1360 bp long basic repeat unit of calf satellite I DNA contains GC rich nucleus of about 140 bp.

Fine melting profiles of calf satellite I DNA and its fragments obtained after digestion with endoR.EcoRI and endoR.AluI nucleases were investigated. It is shown that the 1360 bp basic repeat unit of calf satellite I DNA contains an about 140 bp long GC rich nucleus. It is localized on the 600 bp restriction fragment obtained after digestion of 1360 bp fragment with endoR.AluI nuclease. The main part of satellite I DNA melts as loops between such GC rich nuclei which strongly influence the melting properties of this satellite. There exist significant differences between the thermal stabilities of fragments containing many nuclei, one nucleus and those in which such nucleus is absent.     

A satellite DNA element specific for roe deer (Capreolus capreolus)

An abundant tandem repetitive DNA segment (CCsatIII) with a repeat unit of 2.2 kb has been found in the genome of roe deer (Capreolus capreolus). It accounts for approximately 5%–10% of the genome and is only present in the two species of the genus Capreolus. The sequence has no similarity or common motifes with other deer satellite DNAs and there is no internal repeat structure. A 93 bp fragment is homologous to a bovine repeat. Fluorescent in situ hybridisation showed a predominant centromeric staining of most chromosomes accompanied by a weak interstitial staining of the same chromosomes. On Southern blots, CCsatIII probes do not discriminate between the closely related Capreolus species. Received: 16 June 1997; in revised form: 5 December 1997 / Accepted: 8 December 1997     

Sequence of the cryptic satellite DNA from the plant Sinapis alba

A satellite DNA with a buoyant density equal to that of main band DNA in neutral cesium chloride (‘cryptic satellite’) can be isolated from the DNA of mustard (Sinapis alba) nuclei by Ag⁺/Cs₂SO₄ density gradient centrifugation. This satellite is cleaved into 172 bp repeat units by HinfI, AluI or HaeIII. The HinfI fragments have been further cleaved by AluI, and seven AluI subfragments have been sequenced. As a result two versions of a basic 172 HinfI repeat have been found, one (A + B) with an additional HinfI site. These two sequences (A + B and C) are the most frequent versions of the basic repeat of mustard satellite DNA. The basic 172 bp unit does not contain subrepeats or palindromic sequences. It is not similar (at a criterion of 15 common bases) with any known satellite sequence. It is not unusually highly methylated in the native state.     

Structure and properties of a highly repetitive DNA sequence in sheep

Cleavage of sheep DNA with the restriction endonuclease EcoR I yields three discrete size classes (370, 435 and 800bp) of highly repetitive DNA. The 435bp long fragment was cloned and its nucleotide sequence determined. All three classes of repetitive DNA are related to each other as seen by cross-hybridisation. They are tandemly arranged in the genome and in situ hybridisation to sheep lymphocyte chromosomes show their location mainly in the centromere region of all chromosomes. The primary sequence of the repetitive DNA shows a close structural similarity to the bovine 1.715 satellite DNA, however only poor cross-hybridisation between the sheep and cattle repetitive DNA could be shown.     

Insertion and amplification of a DNA sequence in satellite DNA of Cucumis sativus L. (cucumber)

Summary Another satellite DNA repeat (type IV) in the genome of Cucumis sativus (cucumber) was found and investigated with respect to DNA sequence, methylation, and evolution. This satellite shows a repeat length of 360 bp and a GC-content of 47%. The repeats of type IV are highly conserved among each other. Evidence for CG and CNG methylation is presented. By comparison to the previously described satellites (type I/II and type III) from cucumber, it is evident that this repeat is created by an insertion of a 180 bp DNA sequence similar to type I–III into another DNA sequence (or vice versa), and subsequent amplification forming a new satellite repeat. The different satellites of the type I/II, type III, and the 180 bp insert of type IV show a sequence homology of 60%–70%, indicating that the complex satellite DNA of cucumber is originated from a common progenitor by mutation, additional insertion, and amplification events. Copies of a sequence similar to a part of type IV are present in the genome of the related species Cucumis melo (melon).     

Organization of the 378 bp satellite repeat in terminal heterochromatin of Allium fistulosum

Telomeres, DNA-protein structures, are important elements of the eukaryotic chromosome. Telomeric regions of the majority of higher plants contain heptanucleotides TTTAGGG arranged into a tandem repeat. However, some taxa have no such repeats. These are some species of lilies (Lilium) and onions (Allium). For example, terminal regions of chromosomes of Spanish onion (Allium fistulosum) contain satellite DNA whose unit repeats are 380 bp in length, and the short arm of its chromosome 8 contains rDNA repeats. This study deals with the terminal heterochromatin and organization of the satellite repeat in A. fistulosum. Fluorescent in situ hybridization (FISH) was used to locate the satellite DNA on chromosomes and on extended DNA of A. fistulosum. Nonsatellite DNA was found in the structure of telomeric repeat. Polymerase chain reaction (PCR) and Southern hybridization were used for analysis of terminal heterochromatin. Various rearrangements were found in the satellite repeat. The roles of retrotransposones and microsatellites in the formation of terminal heterochromatin are discussed.     

Nucleotide sequence of mouse satellite DNA.

The nucleotide sequence of uncloned mouse satellite DNA has been determined by analyzing Sau96I restriction fragments that correspond to the repeat unit of the satellite DNA. An unambiguous sequence of 234 bp has been obtained. The sequence of the first 250 bases from dimeric satellite fragments present in Sau96I limit digests corresponds almost exactly to two tandemly arranged monomer sequences including a complete Sau96I site in the center. This is in agreement with the hypothesis that a low level of divergence which cannot be detected in sequence analyses of uncloned DNA is responsible for the appearance of dimeric fragments. Most of the sequence of the 5% fraction of Sau96 monomers that are susceptible to TaqI has also been determined and has been found to agree completely with the prototype sequence. The monomer sequence is internally repetitious being composed of eight diverged subrepeats. The divergence pattern has interesting implications for theories on the evolution of mouse satellite DNA.