Distribution and sequence homogeneity of an abundant satellite DNA in the beetle, Tenebrio molitor.

The mealworm beetle, Tenebrio molitor, contains an unusually abundant and homogeneous satellite DNA which constitutes up to 60% of its genome. The satellite DNA is shown to be present in all of the chromosomes by in situ hybridization. 18 dimers of the repeat unit were cloned and sequenced. The consensus sequence is 142 nt long and lacks any internal repeat structure. Monomers of the sequence are very similar, showing on average a 2% divergence from the calculated consensus. Variant nucleotides are scattered randomly throughout the sequence although some variants are more common than others. Neighboring repeat units are no more alike than randomly chosen ones. The results suggest that some mechanism, perhaps gene conversion, is acting to maintain the homogeneity of the satellite DNA despite its abundance and distribution on all of the chromosomes.     

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.     

Organization and dynamics of satellite and telomere DNAs in Ascaris: implications for formation and programmed breakdown of compound chromosomes

In the nematode genus Ascaris the germline genome contains considerable amounts of extra DNA, which is discarded from the somatic founder blastomeres during early cleavage. In Parascaris univalens the haploid germline genome is contained in one large compound chromosome, which consists of a euchromatic region containing the somatic genome flanked by large blocks of heterochromatin. Fluorescence in situ hybridization of fractions of the germline-limited satellite DNA revealed two highly repeated sequence families establishing the entire heterochromatin (HET blocks). The repeats, a pentanucleotide, TTGCA, and a decanucleotide, TTTGTGCGTG, constitute separate segments of the HET blocks. The blocks are polymorphic in length and, hence, in copy number of the repeats, and the arrangement of the segments. The numerous sequence variants of both repeats display a disperse distribution. The type and rate of base substitutions within both repeat units depend on position. Prior to the elimination process in presomatic cells, termed chromatin diminution, the chromosomes undergo differential mitotic condensation. Interstitial 'chromatin linkers' flanking the prospective numerous somatic chromosomes remain entirely decondensed. The somatic chromosomes are released from the plurivalent chromosomes via excision of the linkers at onset of anaphase, followed by exclusion of the akinetic linker chromatin and HET blocks from the daughter nuclei. In Ascaris suum, the germline-limited satellite, which consists of one 123 bp repeat, is scattered throughout the numerous chromosomes in small heterochromatic knobs of variable sizes, residing at chromosomal ends and/or intercalary positions. The programmed breakage, which appears to proceed in a similar manner to that in P. univalens, results in the loss of all heterochromatic knobs, accompanied by an increase in chromosome number. In both species, all germline chromosomes are capped by tracts of TTAGGC repeats. In P. univalens, such telomeric tracts also occur at the termini of the euchromatic intercalary regions. Upon diminution all telomeric tracts are discarded. De novo telomere addition occurs in all somatic cell lineages of both species. The presented data shed light on the evolutionary history of chromosome aggregation and satellite DNA formation, and putative mechanisms involved in the process of site-directed breakage to reestablish stable somatic chromosomes.     

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     

Cloning,characterization and chromosomal location of a satellite DNA from the Pacific oyster,Crassostrea gigas

We report the cloning and characterization of a high-copy-number, tandem-repeat satellite DNA sequence from the genome of the Pacific oyster, Crassostrea gigas (Cg). The monomeric unit was found to be 166 (±2) bp in length with 79–;94% homology between monomers of the array. The sequence is A+T-rich (60%) and lacks internal repetition and substructural features. The repeat was estimated to account for 1–;4% of the Cg genome. Fluorescence in situ hybridization (FISH) studies mapped the repeat to two distinct heterochromatic regions of two pairs of homologous chromosomes on Cg embryonic metaphases. Also, the number of metaphase chromosomes containing this repeat varied with the ploidy of the cell.     

Unusual Mitochondrial Genome Organization in Cytoplasmic Male Sterile Common Bean and the Nature of Cytoplasmic Reversion to Fertility

Spontaneous reversion to pollen fertility and fertility restoration by the nuclear gene Fr in cytoplasmic male sterile common bean (Phaseolus vulgaris L.) are associated with the loss of a large portion of the mitochondrial genome. To understand better the molecular events responsible for this DNA loss, we have constructed a physical map of the mitochondrial genome of a stable fertile revertant line, WPR-3, and the cytoplasmic male sterile line (CMS-Sprite) from which it was derived. This involved a cosmid clone walking strategy with comparative DNA gel blot hybridizations. Mapping data suggested that the simplest model for the structure of the CMS-Sprite genome consists of three autonomous chromosomes differing only in short, unique regions. The unique region contained on one of these chromosomes is the male sterility-associated 3-kb sequence designated pvs. Based on genomic environments surrounding repeated sequences, we predict that chromosomes can undergo intra- and intermolecular recombination. The mitochondrial genome of the revertant line appeared to contain only two of the three chromosomes; the region containing the pvs sequence was absent. Therefore, the process of spontaneous cytoplasmic reversion to fertility likely involves the disappearance of an entire mitochondrial chromosome. This model is supported by the fact that we detected no evidence of recombination, excision or deletion events within the revertant genome that could account for the loss of a large segment of mitochondrial DNA.     

Satellite DNA of the red flour beetle Tribolium castaneum--comparative study of satellites from the genus Tribolium

A highly abundant satellite DNA comprising 17% of the Tribolium castaneum (Insecta, Coleoptera) genome was cloned and sequenced. The satellite monomer is 360 bp long, has a high A+T content of 73%, and lacks significant internal substructures. The sequence variability is 3.6%, essentially due to random distribution of single-point mutations. The satellite is evenly distributed in the regions of centromeric heterochromatin of all 20 chromosomes, as shown by fluorescent in situ hybridization. Comparison of T. castaneum satellite with those from three different but congeneric species reveals the highest sequence similarity of 47.1% with the satellite from the sibling species Tribolium freemani. The phylogenetic relationships among Tribolium species deduced from satellite sequence agree with those based on karyological, chemotaxonomic, and hybridization data. This indicates a parallel in the divergence of satellites and some genetic and cytogenetic characters. Despite low mutual sequence similarity, which makes them species-specific, Tribolium satellites have a common structural characteristic: a block of about 95% A+T content, 20 to 42 bp long, flanked at one side by an inverted repeat which can potentially form a thermodynamically stable dyad structure. Since similar structural features are found in centromeric DNA of Saccharomyces cerevisiae and Chironomus pallidivittatus, their possible importance in centromere function may be inferred.      

Structure, organization, and sequence of alpha satellite DNA from human chromosome 17: evidence for evolution by unequal crossing-over and an ancestral pentamer repeat shared with the human X chromosome.

The centromeric regions of all human chromosomes are characterized by distinct subsets of a diverse tandemly repeated DNA family, alpha satellite. On human chromosome 17, the predominant form of alpha satellite is a 2.7-kilobase-pair higher-order repeat unit consisting of 16 alphoid monomers. We present the complete nucleotide sequence of the 16-monomer repeat, which is present in 500 to 1,000 copies per chromosome 17, as well as that of a less abundant 15-monomer repeat, also from chromosome 17. These repeat units were approximately 98% identical in sequence, differing by the exclusion of precisely 1 monomer from the 15-monomer repeat. Homologous unequal crossing-over is suggested as a probable mechanism by which the different repeat lengths on chromosome 17 were generated, and the putative site of such a recombination event is identified. The monomer organization of the chromosome 17 higher-order repeat unit is based, in part, on tandemly repeated pentamers. A similar pentameric suborganization has been previously demonstrated for alpha satellite of the human X chromosome. Despite the organizational similarities, substantial sequence divergence distinguishes these subsets. Hybridization experiments indicate that the chromosome 17 and X subsets are more similar to each other than to the subsets found on several other human chromosomes. We suggest that the chromosome 17 and X alpha satellite subsets may be related components of a larger alphoid subfamily which have evolved from a common ancestral repeat into the contemporary chromosome-specific subsets.     

Similarity of Structural Features and Evolution of Satellite DNAs from Palorus subdepressus (Coleoptera) and Related Species

A novel highly abundant satellite DNA comprising 20% of the genome has been characterized in Palorus subdepressus (Insecta, Coleoptera). The 72-bp-long monomer sequence is composed of two copies of T₂A₅T octanucleotide alternating with 22-nucleotide-long elements of an inverted repeat. Phylogenetic analysis revealed clustering of monomer sequence variants into two clades. Two types of variants are prevalently organized in an alternating pattern, thus showing a tendency to generate a new complex repeating unit 144 bp in length. Fluorescent in situ hybridization revealed even distribution of the satellite in the region of pericentric heterochromatin of all 20 chromosomes. P. subdepressus satellite sequence is clearly species specific, lacking similarity even with the satellite from congeneric species P. ratzeburgii. However, on the basis of similarity in predicted tertiary structure induced by intrinsic DNA curvature and in repeat length, P. subdepressus satellite can be classified into the same group with satellites from related tenebrionid species P. ratzeburgii, Tenebrio molitor, and T. obscurus. It can be reasonably inferred that repetitive sequences of different origin evolve under constraints to adopt and conserve particular features. Obtained results suggest that the higher-order structure and repeat length, but not the nucleotide sequence itself, are maintained through evolution of these species. Received: 23 April 1997 / Accepted: 11 July 1997     

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     

On the mode of evolution of alpha satellite DNA in human populations

Summary The hypothesis that highly reiterated satellite DNAs in present-day populations evolve by molecular mechanisms that create, by saltatory amplification steps, new long arrays of satellite DNA, and that such long arrays are used for homogenization purposes, has been tested both in mouse and in humans. In mouse, the data obtained are consistent with this hypothesis. This was tested in more detail on chromosomes 13 and 21 of the human genome. A Centre d'Etudes du Polymorphisme Humain family, which in some individuals exhibits strong supplementary DNA bands following TaqI restriction endonuclease digestion and conventional gel electrophoresis, was analyzed by pulse field gel electrophoresis following restriction by BamHI. The supplementary bands on chromosome 13 (18 times the basic alpha satellite DNA repeat) and on chromosome 21 (a 9.5-mer) segregated with centromeric alpha satellite DNA blocks of 5 and 5.3 megabases, respectively. These are by far the largest alpha satellite block lengths seen in all chromosome 13 and chromosome 21 centrometric sequences so far analyzed in this manner. The possibility that these supplementary alpha satellite sequences were created in single individuals by saltatory amplification steps is discussed in light of our own data and that published by others. It is proposed that deletion events and unequal cross-overs, which both occur in large satellite DNA arrays, contribute to the homogenization of size and sequence of the alpha satellite DNA on most chromosomes of humans.