Demonstration of remarkable sequence divergence in variants of a complex satellite DNA by molecular cloning

Repeat units of a complex G + C-rich satellite of the Bermuda land crab have been cloned by insertion into either the PstI or EcoRI site of pBR322 or the EcoRI site of pUC9. While most of the recombinants contained inserts of approx. 2.1 kb, the average size of repeat units seen in cellular satellite digests, several inserts were markedly different in size. Two domains that account for major sequence differences among the satellite variants and that may be 'hotspots' for sequence modification have been subcloned to permit characterization of their secondary and tertiary structures independent of the influence of the other unusual sequences present. One of these domains is striking in its content of simple repeats; one strand is highly biased in pyrimidines which may permit the formation of unusual secondary and/or tertiary conformations. The other subcloned domain is rich in Pu/Py; preliminary data indicate a transition from B----Z DNA in this region.     

Comparative structure and evolution of goat and sheep satellite I DNAs.

The satellite I DNAs of domestic goat (Capra hircus) and domestic sheep (Ovis aries) have been studied using molecular hybridisation and restriction enzyme analysis. Both satellite DNAs are composed of repeat units of 820 base pairs in length, but their restriction maps, although similar, differ in certain respects. Thus the majority of sheep satellite I repeat units have two EcoRI sites and one AluI site, whereas the majority of goat satellite I repeat units have one EcoRI site and two AluI sites. The sheep satellite I repeat units with the two EcoRI sites are much more homogeneous than the repeats forming the remainder of the satellite, as judged by the difference in the melting temperatures of native and reassociated molecules. DNAs from species of wild sheep and goats were screened for the presence of these repeat units, and they appear to have been amplified during the radiation of the Ovis genus. Goat satellite I is composed of a single sequence type which has changed through base substitution until the sequence now shows considerable heterogeneity. It is proposed that the major sequence types of these two satellite DNAs were amplified by different saltatory replication events at different times in the evolution of the group.     

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'.     

Sequence and sequence variation within the 1.688 g/cm3 satellite DNA of Drosophila melanogaster.

We have determined the complete nucleotide sequence of the monomer repeating unit of the 1.688 g/cm³ satellite DNA from Drosophila melanogaster. This satellite DNA, which makes up 4% of the Drosophila genome and is located primarily on the sex chromosomes, has a repeat unit 359 base-pairs in length. This complex sequence is unrelated to the other three major satellite DNAs present in this species, each of which contains a very short repeated sequence only 5 to 10 base-pairs long. The repeated sequence is more similar to the complex repeating units found in satellites of mammalian origin in that it contains runs of adenylate and thymidylate residues. We have determined the nature of the sequence variations in this DNA by restriction nuclease cleavage and by direct sequence determination of (1) individual monomer units cloned in hybrid plasmids, (2) mixtures of adjacent monomers from a cloned segment of this satellite DNA, (3) mixtures of monomer units isolated by restriction nuclease cleavage of total 1.688 g/cm³ satellite DNA. Both direct sequence determination and restriction nuclease cleavage indicate that certain positions in the repeat can be highly variable with up to 50% of certain restriction sites having altered recognition sequences. Despite the high degree of variation at certain sites, most positions in the sequence are highly conserved. Sequence analysis of a mixture of 15 adjacent monomer units detected only nine variable positions out of 359 base-pairs. Total satellite DNA showed only four additional positions. While some variability would have been missed due to the sequencing methods used, we conclude that the variation from one repeat to the next is not random and that most of the satellite repeat is conserved. This conservation may reflect functional aspects of the repeated DNA, since we have shown earlier that part of this sequence serves as a binding site for a sequence-specific DNA binding protein isolated from Drosophila embryos (Hsieh &; Brutlag, 1979).     

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.     

Interhomologue sequence variation of alpha satellite DNA from human chromosome 17: Evidence for concerted evolution along haplotypic lineages

Alpha satellite DNA is a family of tandemly repeated DNA found at the centromeres of all primate chromosomes. Different human chromosomes 17 in the population are characterized by distinct alpha satellite haplotypes, distinguished by the presence of variant repeat forms that have precise monomeric deletions. Pairwise comparisons of sequence diversity between variant repeat units from each haplotype show that they are closely related in sequence. Direct sequencing of PCR-amplified alpha satellite reveals heterogeneous positions between the repeat units on a chromosome as two bands at the same position on a sequencing ladder. No variation was detected in the sequence and location of these heterogeneous positions between chromosomes 17 from the same haplotype, but distinct patterns of variation were detected between chromosomes from different haplotypes. Subsequent sequence analysis of individual repeats from each haplotype confirmed the presence of extensive haplotype-specific sequence variation. Phylogenetic inference yielded a tree that suggests these chromosome 17 repeat units evolve principally along haplotypic lineages. These studies allow insight into the relative rates and/or timing of genetic turnover processes that lead to the homogenization of tandem DNA families. Correspondence to: H.F. Willard     

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.     

Two Types of Alpha Satellite DNA in Distinct Chromosomal Locations in Azara's Owl Monkey

Alpha satellite DNA is a repetitive sequence known to be a major DNA component of centromeres in primates (order Primates). New World monkeys form one major taxon (parvorder Platyrrhini) of primates, and their alpha satellite DNA is known to comprise repeat units of around 340 bp. In one species (Azara''s owl monkey Aotus azarae) of this taxon, we identified two types of alpha satellite DNA consisting of 185- and 344-bp repeat units that we designated as OwlAlp1 and OwlAlp2, respectively. OwlAlp2 exhibits similarity throughout its entire sequence to the alpha satellite DNA of other New World monkeys. The chromosomal locations of the two types of sequence are markedly distinct: OwlAlp1 was observed at the centromeric constrictions, whereas OwlAlp2 was found in the pericentric regions. From these results, we inferred that OwlAlp1 was derived from OwlAlp2 and rapidly replaced OwlAlp2 as the principal alpha satellite DNA on a short time scale at the speciation level. A less likely alternative explanation is also discussed.     

Deca-satellite: A highly polymorphic satellite that joins α-satellite in the African green monkey genome

Three different cloned segments of African green monkey DNA that contain α-satellite sequences linked to a previously undescribed, distinct monkey satellite (called deca-satellite) are described here. The cloned segments were derived from a monkey DNA library in λCharon4A that was constructed to select for junctions between α-satellite and other DNA sequences.The structure of the deca-satellite and of a junction between deca-satellite and α-satellite were studied by subcloning appropriate fragments of the original cloned segments and by sequence analysis. Deca-satellite has a ten base-pair repeat unit: the consensus sequence of the repeat units is 5′ A-A-A-C-C-G-G-N-T-C. Sequences homologous to the deca-satellite are in the middle repeated class of genomic DNA. Analysis of the organization of deca-satellite sequences by digestion of total DNA with various restriction endonucleases and hybridization with a cloned deca-satellite probe revealed extensive polymorphism in the genomes of different individual monkeys but not among the tissues of one organism. These observations indicate that the arrangement of deca-satellite sequences is continually changing.An unusual α-satellite repeat unit occurs at a junction between the α-satellite and deca-satellite. It resembles the major baboon α-satellite more closely than it does monkey α-satellite and thereby provides evidence in favor of the “library” hypothesis for satellite evolution.     

Sequence and evolution of related bovine and caprine satellite DNAs: Identification of a short DNA sequence potentially involved in satellite DNA amplification

The satellite II DNAs of the domestic ox Bos taurus and sheep Ovis aries have been sequenced, and that of the domestic goat Capra hircus partially sequenced. All three are related, and consist of repeat units of about 700 base-pairs. There is no evidence of internal repetition within these repeat units. When matched for maximum homology, the goat and sheep sequences show 83% homology, whereas the ox and sheep sequences share only 70% homology. Factors contributing to the uncertainty of the exact homology between these sequences are discussed, but the results are nevertheless consistent with their progenitor sequence being present in the common ancestor of cattle and sheep. Goat satellite II DNA is shown to contain another, unrelated, tandemly repeated sequence, which is composed of 22 base-pair repeat units. Both this sequence and a region of ox satellite II share good homology with the 11 base-pair progenitor sequence of ox 1.706 g/cm³ satellite DNA. It is suggested that this shared sequence could play a role in bovine satellite DNA amplification.     

PCR amplification of tandemly repeated DNA: analysis of intra- and interchromosomal sequence variation and homologous unequal crossing-over in human alpha satellite DNA.

Tandemly repeated DNA can comprise several percent of total genomic DNA in complex organisms and, in some instances, may play a role in chromosome structure or function. Alpha satellite DNA is the major family of tandemly repeated DNA found at the centromeres of all human and primate chromosomes. Each centromere is characterized by a large contiguous array of up to several thousand kb which can contain several thousand highly homogeneous repeat units. By using a novel application of the polymerase chain reaction (repPCR), we are able to amplify a representative sampling of multiple repetitive units simultaneously, allowing rapid analysis of chromosomal subsets. Direct sequence analysis of repPCR amplified alpha satellite from chromosomes 17 and X reveals positions of sequence heterogeneity as two bands at a single nucleotide position on a sequencing ladder. The use of TdT in the sequencing reactions greatly reduces the background associated with polymerase pauses and stops, allowing visualization of heterogeneous bases found in as little as 10% of the repeat units. Confirmation of these heterogeneous positions was obtained by comparison to the sequence of multiple individual cloned copies obtained both by PCR and non-PCR based methods. PCR amplification of alpha satellite can also reveal multiple repeat units which differ in size. Analysis of repPCR products from chromosome 17 and X allows rapid determination of the molecular basis of these repeat unit length variants, which appear to be a result of unequal crossing-over. The application of repPCR to the study of tandemly repeated DNA should allow in-depth analysis of intra- and interchromosomal variation and unequal crossing-over, thus providing insight into the biology and genetics of these large families of DNA.     

Mutation and Recombination in Cattle Satellite DNA: A Feedback Model for the Evolution of Satellite DNA Repeats

The cattle genome contains several distinct centromeric satellites with interrelated evolutionary histories. We compared these satellites in Bovini species that diverged 0.2 to about 5 Myr ago. Quantification of hybridization signals by phosphor imaging revealed a large variation in the relative amounts of the major satellites. In the genome of water buffalo this has led to the complete deletion of satellite III. Comparative sequencing and PCR-RFLP analysis of satellites IV, 1.711a, and 1.711b from the related Bos and Bison species revealed heterogeneities in 0.5 to 2% of the positions, again with variations in the relative amounts of sequence variants. Restriction patterns generated by double digestions suggested a recombination of sequence variants. Our results are compatible with a model of the life history of satellites during which homogeneity of interacting repeat units is both cause and consequence of the rapid turnover of satellite DNA. Initially, a positive feedback loop leads to a rapid saltatory amplification of homogeneous repeat units. In the second phase, mutations inhibit the interaction of repeat units and coexisting sequence variants amplify independently. Homogenization by the spreading of one of the variants is prevented by recombination and the satellite is eventually outcompeted by another, more homogeneous tandem repeat sequence. Received: 21 July 2000 / Accepted: 30 October 2000     

Key-string segmentation algorithm and higher-order repeat 16mer (54 copies) in human alpha satellite DNA in chromosome 7

A new key-string segmentation algorithm for identification of alpha satellite DNAs and higher-order repeat (HOR) units was introduced and exemplified. Starting with an initial key string, we determine the dominant key string and HOR. Our key-string algorithm was used to scan the recent GenBank data for human alpha satellite DNA sequence AC017075.8 (193 277 bp) from the centromeric region of chromosome 7. The sequence was computationally segmented into one HOR domain (super-repeat domain) and two non-HOR domains. Dominant key-string GTTTCT provided segmentation in terms of alpha monomers. The HOR is tandemly repeated in 54 copies in the super-repeat (HOR) domain. Five insertions and three deletions in the HOR structure associated with a dominant key string were identified. Concensus HOR was constructed. Divergence of individual HOR copies from concensus amounts to 0.7% on the average, while divergence between 16 monomer variants within each HOR is on the average 20%. In the front and back domain, 199 monomer variants were identified that are not organized in HOR and diverge by 20-40%.     

Sobo, a recently amplified satellite repeat of potato, and its implications for the origin of tandemly repeated sequences

Highly repetitive satellite DNA sequences are main components of heterochromatin in higher eukaryotic genomes. It is well known that satellite repeats can expand and contract dramatically, which may result in significant genome size variation among genetically related species. The origin of satellite repeats, however, is elusive. Here we report a satellite repeat, Sobo, from a diploid potato species, Solanum bulbocastanum. The Sobo repeat is mapped to a single location in the pericentromeric region of chromosome 7. This single Sobo locus spans approximately 360 kb of a 4.7-kb monomer. Sequence analysis revealed that the major part of the Sobo monomer shares significant sequence similarity with the long terminal repeats (LTRs) of a retrotransposon. The Sobo repeat was not detected in other Solanum species and is absent in some S. bulbocastanum accessions. Sobo monomers are highly homogenized and share >99% sequence identity. These results suggest that the Sobo repeat is a recently emerged satellite and possibly originated by a sudden amplification of a genomic region including the LTR of a retrotransposon and its flanking genomic sequences.     

The spectrin super-family

The review is focused on recent data on the primary sequences of erythroid and non-erythroid spectrins. As in other fields, the techniques of molecular genetics have allowed great advances in our knowledge of the structure and the genetic story of these molecules. Comparison of alpha-chains sequences of the non-erythroid (fodrin) and erythroid spectrin demonstrated that the fodrin alpha-genes are strictly conserved across species, while the mammalian spectrin genes have diverged rapidly. Spectrin and fodrin alpha-chains are largely composed of homologous 106-amino-acid repeat units. Spectrin alpha-chain is lacking a 37 amino-acid sequence which bears the calmodulin-binding site of the fodrin alpha-chain. The highest degree of homology between the spectrin alpha-chain and the fodrin alpha-chain lies in a central atypical segment unrelated to the canonical repeat sequence. This region is closely related to the N-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. Like the spectrin alpha-chain, the major central part of the spectrin beta-chain is made up of repeat units of 106 amino-acids. The N-terminal domain of the beta-chain, and especially the actin binding site, is the region of greatest homology among members of the spectrin super-family, including Drosophila spectrin beta-chain, dystrophin and alpha-actinin. The C-terminal extremity of the erythroid beta-chain is also of great interest, since tissue-specific differential processing of 3'beta-spectrin gene pre-mRNA generates a beta spectrin-isoform with a unique C-terminus in human skeletal muscle.     

Nonrandom localization of recombination events in human alpha satellite repeat unit variants: implications for higher-order structural characteristics within centromeric heterochromatin.

Tandemly repeated DNA families appear to undergo concerted evolution, such that repeat units within a species have a higher degree of sequence similarity than repeat units from even closely related species. While intraspecies homogenization of repeat units can be explained satisfactorily by repeated rounds of genetic exchange processes such as unequal crossing over and/or gene conversion, the parameters controlling these processes remain largely unknown. Alpha satellite DNA is a noncoding tandemly repeated DNA family found at the centromeres of all human and primate chromosomes. We have used sequence analysis to investigate the molecular basis of 13 variant alpha satellite repeat units, allowing comparison of multiple independent recombination events in closely related DNA sequences. The distribution of these events within the 171-bp monomer is nonrandom and clusters in a distinct 20- to 25-bp region, suggesting possible effects of primary sequence and/or chromatin structure. The position of these recombination events may be associated with the location within the higher-order repeat unit of the binding site for the centromere-specific protein CENP-B. These studies have implications for the molecular nature of genetic recombination, mechanisms of concerted evolution, and higher-order structure of centromeric heterochromatin.     

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.     

Repetitive sequences originating from the centromere constitute large-scale heterochromatin in the telomere region in the siamang, a small ape

Chromosomes of the siamang Symphalangus syndactylus (a small ape) carry large-scale heterochromatic structures at their ends. These structures look similar, by chromosome C-banding, to chromosome-end heterochromatin found in chimpanzee, bonobo and gorilla (African great apes), of which a major component is tandem repeats of 32-bp-long, AT-rich units. In the present study, we identified repetitive sequences that are a major component of the siamang heterochromatin. Their repeat units are 171 bp in length, and exhibit sequence similarity to alpha satellite DNA, a major component of the centromeres in primates. Thus, the large-scale heterochromatic structures have different origins between the great apes and the small ape. The presence of alpha satellite DNA in the telomere region has previously been reported in the white-cheeked gibbon Nomascus leucogenys, another small ape species. There is, however, a difference in the size of the telomere-region alpha satellite DNA, which is far larger in the siamang. It is not known whether the sequences of these two species (of different genera) have a common origin because the phylogenetic relationship of genera within the small ape family is still not clear. Possible evolutionary scenarios are discussed.     

Amino acid sequence of a novel integrin beta 4 subunit and primary expression of the mRNA in epithelial cells.

Using the polymerase chain reaction, we have isolated cDNA clones that encode a new integrin beta subunit--beta 4. Its cDNA, which is 5676 bp in length, has one long coding sequence (5256 bp), a polyadenylation signal and a poly(A) tail. The deduced sequence of 1752 amino acids is unique among the integrin beta subunits. It contains a putative signal sequence as well as a transmembrane domain that divides the molecule into an extracellular domain at the N-terminal side and a cytoplasmic domain at the C-terminal side. The extracellular domain exhibits a 4-fold repeat of cysteine-rich motif similar to those of other integrin beta subunits. Certain features of the extracellular domain, however, are unique to the beta 4 subunit sequence. Of the 56 conserved cysteine residues found within the extracellular domain of other mature beta subunits, eight such residues are deleted from the beta 4 subunit sequence. The cytoplasmic domain is much larger (approximately 1000 amino acids) than those of other beta subunits (approximately 50 amino acids) and has no significant homology with them. A protein homology search revealed that the beta 4 subunit cytoplasmic domain has four repeating units that are homologous to the type III repetition exhibited by fibronectin. The beta 4 subunit mRNA was expressed primarily in epithelial cells. The restricted expression and the new structural features distinguish the integrin beta 4 subunit from other integrin beta subunits.     

A single nucleotide change within a plant virus satellite RNA alters the host specificity of disease induction

Some RNA plant viruses contain satellite RNAs which are dependent upon their associated virus for replication and encapsidation. Some cucumber mosaic virus satellite RNAs induce chlorosis on any of several host plants, including either tobacco or tomato. The exchange of sequence domains between cDNA clones of chlorosis-inducing and non-pathogenic satellite RNAs delimited the chlorosis domain for both tobacco and tomato plants to the same region. Site-directed mutagenesis of one nucleotide (149) within this domain changed the host plant specificity for a chlorotic response to satellite RNA infection from tomato to tobacco. Within the chlorosis domain, three conserved nucleotides are either deleted or altered in all satellite RNAs that do not induce chlorosis. Deletion of one of these nucleotides (153) did not affect satellite RNA replication but rendered it non-pathogenic. Thus, two single nucleotides have been identified which play central roles in those interactions between the virus, its satellite RNA and the host plant, and together result in a specific disease state.