Complex Satellite DNA Research Articles

Testing the Drosophila maternal haploid gene for functional divergence and a role in hybrid incompatibility.

Crosses between Drosophila simulans females and Drosophila melanogaster males produce viable F1 sons and poorly viable F1 daughters. Unlike most hybrid incompatibilities, this hybrid incompatibility violates Haldane's rule, the observation that incompatibilities preferentially affect the heterogametic sex. Furthermore, it has a different genetic basis than hybrid lethality in the reciprocal cross, with the causal allele in Drosophila melanogaster being a large species-specific block of complex satellite DNA on its X chromosome known as the 359-bp satellite, rather than a protein-coding locus. The causal allele(s) in Drosophila simulans are unknown but likely involve maternally expressed genes or factors since the F1 females die during early embryogenesis. The maternal haploid (mh) gene is an intriguing candidate because it is expressed maternally and its protein product localizes to the 359-bp repeat. We found that this gene has diverged extensively between Drosophila melanogaster and Drosophila simulans. This observation led to the hypothesis that Drosophila melanogaster mh may have coevolved with the 359-bp repeat and that hybrid incompatibility thus results from the absence of a coevolved mh allele in Drosophila simulans. We tested for the functional divergence of mh by creating matched transformants of Drosophila melanogaster and Drosophila simulans orthologs in both Drosophila melanogaster and Drosophila simulans strains. Surprisingly, we find that Drosophila simulans mh fully complements the female sterile phenotype of Drosophila melanogaster mh mutations. Contrary to our hypothesis, we find no evidence that adding a Drosophila melanogaster mh gene to Drosophila simulans increases hybrid viability.

Single-molecule sequencing resolves the detailed structure of complex satellite DNA loci in Drosophila melanogaster.

Highly repetitive satellite DNA (satDNA) repeats are found in most eukaryotic genomes. SatDNAs are rapidly evolving and have roles in genome stability and chromosome segregation. Their repetitive nature poses a challenge for genome assembly and makes progress on the detailed study of satDNA structure difficult. Here, we use single-molecule sequencing long reads from Pacific Biosciences (PacBio) to determine the detailed structure of all major autosomal complex satDNA loci in Drosophila melanogaster, with a particular focus on the 260-bp and Responder satellites. We determine the optimal de novo assembly methods and parameter combinations required to produce a high-quality assembly of these previously unassembled satDNA loci and validate this assembly using molecular and computational approaches. We determined that the computationally intensive PBcR-BLASR assembly pipeline yielded better assemblies than the faster and more efficient pipelines based on the MHAP hashing algorithm, and it is essential to validate assemblies of repetitive loci. The assemblies reveal that satDNA repeats are organized into large arrays interrupted by transposable elements. The repeats in the center of the array tend to be homogenized in sequence, suggesting that gene conversion and unequal crossovers lead to repeat homogenization through concerted evolution, although the degree of unequal crossing over may differ among complex satellite loci. We find evidence for higher-order structure within satDNA arrays that suggest recent structural rearrangements. These assemblies provide a platform for the evolutionary and functional genomics of satDNAs in pericentric heterochromatin.

Genomic characterization of large heterochromatic gaps in the human genome assembly.

The largest gaps in the human genome assembly correspond to multi-megabase heterochromatic regions composed primarily of two related families of tandem repeats, Human Satellites 2 and 3 (HSat2,3). The abundance of repetitive DNA in these regions challenges standard mapping and assembly algorithms, and as a result, the sequence composition and potential biological functions of these regions remain largely unexplored. Furthermore, existing genomic tools designed to predict consensus-based descriptions of repeat families cannot be readily applied to complex satellite repeats such as HSat2,3, which lack a consistent repeat unit reference sequence. Here we present an alignment-free method to characterize complex satellites using whole-genome shotgun read datasets. Utilizing this approach, we classify HSat2,3 sequences into fourteen subfamilies and predict their chromosomal distributions, resulting in a comprehensive satellite reference database to further enable genomic studies of heterochromatic regions. We also identify 1.3 Mb of non-repetitive sequence interspersed with HSat2,3 across 17 unmapped assembly scaffolds, including eight annotated gene predictions. Finally, we apply our satellite reference database to high-throughput sequence data from 396 males to estimate array size variation of the predominant HSat3 array on the Y chromosome, confirming that satellite array sizes can vary between individuals over an order of magnitude (7 to 98 Mb) and further demonstrating that array sizes are distributed differently within distinct Y haplogroups. In summary, we present a novel framework for generating initial reference databases for unassembled genomic regions enriched with complex satellite DNA, and we further demonstrate the utility of these reference databases for studying patterns of sequence variation within human populations.

Effete, a Drosophila Chromatin-Associated Ubiquitin-Conjugating Enzyme That Affects Telomeric and Heterochromatic Position Effect Variegation

Drosophila telomeres are elongated by the transposition of telomere-specific retrotransposons rather than telomerase activity. Proximal to the terminal transposon array, Drosophila chromosomes contain several kilobases of a complex satellite DNA termed telomere-associated sequences (TASs). Reporter genes inserted into or next to the TAS are silenced through a mechanism called telomere position effect (TPE). TPE is reminiscent of the position effect variegation (PEV) induced by Drosophila constitutive heterochromatin. However, most genes that modulate PEV have no effect on TPE, and systematic searches for TPE modifiers have so far identified only a few dominant suppressors. Surprisingly, only a few of the genes required to prevent telomere fusion have been tested for their effect on TPE. Here, we show that with the exception of the effete (eff; also called UbcD1) mutant alleles, none of the tested mutations at the other telomere fusion genes affects TPE. We also found that mutations in eff, which encodes a class I ubiquitin-conjugating enzyme, act as suppressors of PEV. Thus, eff is one of the rare genes that can modulate both TPE and PEV. Immunolocalization experiments showed that Eff is a major constituent of polytene chromosomes. Eff is enriched at several euchromatic bands and interbands, the TAS regions, and the chromocenter. Our results suggest that Eff associates with different types of chromatin affecting their abilities to regulate gene expression.

Characterization and evolutionary dynamics of a complex family of satellite DNA in the leaf beetle Chrysolina carnifex (Coleoptera, Chrysomelidae)

The present study characterizes the complex satellite DNA from the specialized phytophagous beetle species Chrysolina carnifex. The satellite DNA is formed by six monomer types, partially homologous but having diverged enough to be separate on the phylogenetic trees, since each monomer type is located on a different branch, having statistically significant bootstrap values. Its analysis suggests a common evolutionary origin of all monomers from the same 211-bp sequence mainly by means of base-substitution mutations evolutionarily fixed to each monomer type and duplications and/or deletions of pre-existing segments in the 211-bp sequence. The analysis of the sequences and Southern hybridizations suggest that the monomers are organized in three types of repeats: monomers (211-bp) and higher-order repeats in the form of dimers (477-bp) or even trimers (633-bp). These repetitive units are not isolated from others, and do not present the pattern characteristic for the regular tandem arrangement of satellite DNA. In-situ hybridization with biotinylated probes corresponding to the three types of repeats showed the pericentromeric location of these sequences in all meiotic bivalents, coinciding with the heterochromatic blocks revealed by C-banding, indicating in addition that each type of repeat is neither isolated from others nor located in specific chromosomes but rather that they are intermixed in the heterochromatic regions. The presence of this repetitive DNA in C. haemoptera, C. bankii and C. americana was also tested by Southern analysis. The results show that this satellite DNA sequence is specific to the C. carnifex genome but has not been found in three other species of Chrysolina occupying similar or different host plants.

Complex satellite DNA reshuffling in the polymorphic t(1;29) Robertsonian translocation and evolutionarily derived chromosomes in cattle.

We have analysed and mapped physically the satellite I, III (subunits pvu and sau) and IV DNA sequences in cattle using in-situ hybridization. Four breeds were analysed including individuals with a chromosome number of 2n = 60 and individuals with the widespread t(1;29) in the homozygous (2n = 58) and heterozygous state (2n = 59). All three satellite DNA families were present at the centromeres of the many but not all of the autosomal acrocentric chromosomes, and essentially absent from the sex chromosomes. In the translocated t(1;29) chromosome, the satellite DNA families showed a different pattern from that simply derived by fusion of the acrocentric autosomes and loss of satellite sequences, with no variation between breeds. A model of centromeric evolution is presented involving two independent events. Knowledge of mechanisms of translocation formation within cattle is important for a functional understanding of centromere and satellites, investigation of chromosomal abnormalities, and for understanding chromosomal fusion during evolution of other bovids and genome evolution in general.

A telomeric satellite in Drosophila virilis and its sibling species.

Telomere elongation by telomerase is the most widespread mechanism among eukaryotes. However, alternative mechanisms such as homologous recombination between terminal satellite DNAs are probably used in lower dipteran insects and in some plants. Drosophila melanogaster uses the very unusual telomere elongation pathway of transposition of telomere-specific retrotransposable elements. The uniqueness of this telomere elongation mechanism raises the question of its origin. We, therefore, analyzed sequences located at telomeres of fairly distantly related Drosophila species, and in this paper we describe the characterization of complex satellite DNA sequences located at the telomeres of D. virilis and other species in the virilis group. We suggest an involvement of these DNA satellites in telomere elongation by homologous recombination similar to that found in lower dipterans. Our findings raise the possibility that telomere elongation by specific retrotransposons as found in D. melanogaster and its sibling species is a recent event in the evolution of dipteran insects.

CENP-A associated complex satellite DNA in the kinetochore of the Indian muntjac.

The centromere/kinetochore complex is a chromosomal assembly that mediates chromosome motility and mitotic regulation by interacting with microtubules of the mitotic spindle apparatus. Centromere protein A (CENP-A) is a histone H3 homolog that is concentrated in the chromatin of the inner kinetochore plate of human chromosomes. To identify DNA sequences associated with the inner kinetochore plate, we used anticentromere autoantibodies to immunoprecipitate CENP-A associated chromatin selectively from Indian muntjac fibroblasts. DNA was cloned from immunoprecipitated CENP-A- associated chromatin and characterized by DNA sequence and hybridization analyses. A novel centromeric satellite DNA sequence was identified and shown by fluorescence in situ hybridization analysis to be present at all centromeres of the Indian muntjac. This satellite DNA constitutes a 972 bp monomer repeat and shows partial homology with satellite II DNA of the white-tailed deer. Southern blot analysis of muntjac genomic DNA suggests that this satellite DNA is present in repetitive tandem arrays and contains complex internal arrangements. In conjunction with previous work showing the association of CENP-A with human alpha-satellite DNA, we conclude that the mammalian inner kinetochore plate contains a unique form of chromatin that contains CENP-A in association with complex satellite DNA.

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.

Chromatin containing CENP-A and α-satellite DNA is a major component of the inner kinetochore plate

The pathway of molecular interactions leading to kinetochore assembly on mammalian chromosomes is unknown. Kinetochores could be specified by structural features of centromeric satellite DNA [1–3] or by specific DNA sequences, analogous to budding yeast centromeres, interspersed in centromeric satellite DNA arrays [4,5]. Alternatively, kinetochores could be epigenetic structures that replicate without strict dependence on DNA sequence [6–8]. We purified kinetochore-associated chromatin from human chromosomes by immunoprecipitation of CENP-A, a centromere-specific histone H3 homologue located in the inner plate of the kinetochore [6,9,10]. Hybridization and DNA sequence analyses of cloned kinetochore DNA fragments revealed α-satellite as the predominant sequence associated with CENP-A. A major site of micrococcal nuclease digestion was identified by mapping the termini of α-satellite clones, suggesting that the inner kinetochore plate contains phased arrays of CENP-A– α-satellite nucleosomes. These experiments demonstrate for the first time that complex satellite DNA is a structural component of the kinetochore. Further, because complex satellite DNA is evolutionarily unconserved, these results suggest that molecular recognition events necessary for kinetochore formation take place at the level of DNA conformation or epigenetic mechanisms rather than DNA sequence per se.

Characterization of a complex satellite DNA in the mollusc Donax trunculus: Analysis of sequence variations and divergence

A highly repetitive sequence in the genomic DNA of the bivalve mollusc Donax trunculus (Dt) has been identified upon restriction with EcoRV. During the time-course of DNA digestion, genomic fragments resolved electrophoretically into a ladder-like banding pattern revealing a tandem arrangement of the repeated elements, thus representing satellite DNA sequences. Cloning and sequence analysis unraveled the presence of two groups of monomer units which can be considered distinctive satellite subfamilies. Each subclass is distinguishable by the presence of 17 evenly spread diagnostic nucleotides (nt). The respective consensus sequences are 155 bp in length and differ by 11%, while relevant internal substructures were not observed. The two satellite subfamilies constitute 0.23 and 0.09% of the Dt genome, corresponding to 20 000 and 7600 copies per haploid complement, respectively. Sequence mutations often appear to be shared between two or more monomer variants, indicating a high degree of homogenization as opposed to that of random mutational events. Shared mutations among variants appear either as single changes or in long stretches. This pattern may arise from gene conversion mechanisms acting at different levels, such as the spread of nt sequences of a similar length to the monomer repeat itself, and the diffusion of short tracts a few bp long. Subfamilies might have evolved from the occasional amplification and spreading of a monomer variant effected by gene conversion events

RAP1 protein activates and silences transcription of mating-type genes in yeast

The pathway of molecular interactions leading to kinetochore assembly on mammalian chromosomes is unknown. Kinetochores could be specified by structural features of centromeric satellite DNA [1], [2], [3] or by specific DNA sequences, analogous to budding yeast centromeres, interspersed in centromeric satellite DNA arrays [4], [5]. Alternatively, kinetochores could be epigenetic structures that replicate without strict dependence on DNA sequence [6], [7], [8]. We purified kinetochore-associated chromatin from human chromosomes by immunoprecipitation of CENP-A, a centromere-specific histone H3 homologue located in the inner plate of the kinetochore [6], [9], [10]. Hybridization and DNA sequence analyses of cloned kinetochore DNA fragments revealed α-satellite as the predominant sequence associated with CENP-A. A major site of micrococcal nuclease digestion was identified by mapping the termini of α-satellite clones, suggesting that the inner kinetochore plate contains phased arrays of CENP-A–α-satellite nucleosomes. These experiments demonstrate for the first time that complex satellite DNA is a structural component of the kinetochore. Further, because complex satellite DNA is evolutionarily unconserved, these results suggest that molecular recognition events necessary for kinetochore formation take place at the level of DNA conformation or epigenetic mechanisms rather than DNA sequence per se.

Physical map of the Saccharomyces cerevisiae genome at 110-kilobase resolution

A physical map of the Saccharomyces cerevisiae genome is presented. It was derived by mapping the sites for two restriction endonucleases, SfiI and NotI, each of which recognizes an 8-bp sequence. DNA-DNA hybridization probes for genetically mapped genes and probes that span particular SfiI and NotI sites were used to construct a map that contains 131 physical landmarks--32 chromosome ends, 61 SfiI sites and 38 NotI sites. These landmarks are distributed throughout the non-rDNA component of the yeast genome, which comprises 12.5 Mbp of DNA. The physical map suggests that those genes that can be detected and mapped by standard genetic methods are distributed rather uniformly over the full physical extent of the yeast genome. The map has immediate applications to the mapping of genes for which single-copy DNA-DNA hybridization probes are available.

The Complex Satellite DNA of Tropaeolum majus L.: Partial Characterization of Isolated and of Cloned Restriction Fragments

Satellite DNA (satDNA) was purified by repeated neutral CsCl, ethidium bromide/CsCl and actinomycin D/CsCl density centrifugation from total nuclear DNA of Tropeolum majus. Digestins patterns of satDNA and main band DNA as generated with 22 different restriction enzymes were partially characterized and hybridized by the Southern blot technique with Hpa II-generated satDNA fragments and a ribosomal DNA fragment. Some restriction fragments were sequenced by the chemical method, either directly, or after cloning, and possible secondary structures were determined

Preservation of a complex satellite DNA in two species of Echinoderms

The cloning and sequencing of a tandemly arrayed repetitive DNA sequence from the sea cucumber Holothuria tubulosa has been recently described (Sainz, J., Azorín, F. and Cornudella, L. 1989. Gene 80, 57-64). We have now searched the genomes of several echinoderm species for the presence of homologous repetitive elements. A close but not identical repeated sequence has been identified in a related holothuroid, H. polii. The monomeric repeat unit is 391 bp long and has a base composition of 66.8% A and T residues, lined up in tracts of 4 nt or larger. The monomeric sequence lacks any internal subrepeat organization although it displays a substantial degree of internal redundancy in the form of inverted and direct repeats. The repeated element accounts for 0.34% of the genome which corresponds to a repetition frequency of about 0.5 x 10(5) copies per haploid complement. The intra- and interspecific homologies among monomers of the satellite DNA as derived from sequence analyses are very high, averaging 97%. The results suggest that the homogeneity of the highly reiterated DNA sequence may be attributed to evolutionary conservative trends.

A domain that assumes a Z-conformation includes a specific deletion in some cloned variants of a complex satellite

Sequence analyses show that deletions of 10 and 12 bp occur at homologous sites in a domain that is rich in alternating purines and pyrimidines (Pu/Py) in B42 and EXT, two cloned variants of a complex satellite DNA. A 3-bp deletion occurs 27 bp upstream from the site of the specific deletions in B42 and RU, a third cloned satellite variant that has not suffered the 10-bp deletion. Under torsional stress, the Pu/Py-rich domain adopts a Z-conformation as shown by ( i) inhibition of cutting at a BssHII site that accounts for built2 5 of a 15-bp tract of pure Pu/Py in the domain; ( ii) binding of polyclonal and monoclonal anti-Z-DNA antibodies to the domain; and (iii) antibody stabilization and subsequent relaxation of the Z-region.

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

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).

Eukaryotic DNA diverges at a long and complex pyrimidine.purine tract that can adopt altered conformations.

A domain exhibiting major sequence divergences among three cloned repeat units of a complex satellite DNA of the Bermuda land crab contains a repetitive polypyrimidine.polypurine segment consisting of a long C.G tract embedded between runs of CCT.AGG and CGCAC.GTGCG and their variations. The domain adopts at least two types of altered conformations that are markedly affected by pH and negative superhelical density; only one is sensitive to ionic strength. Supercoil-dependent distortions in helical structure are most pronounced at points of interruption in compositional bias in this domain and a similar although less extensive, divergent domain nearby. Since the domain is the site of major sequence divergences among individual satellite repeat units, the altered conformations may be involved in site-specific recombination between repeat units, either those arranged in tandem or those scattered throughout the genome.

Sequences of three closely related variants of a complex satellite DNA diverge at specific domains.

Major differences among the sequences of the repeat units of a very complex satellite DNA are located in domains which are sensitive to S1 nuclease under torsional stress, indicating that the domains assume unusual secondary or tertiary structures. Repeat units of the satellite, which accounts for 3% of the DNA of a land crab, have been inserted into pBR322 and the primary sequences of three cloned variants determined. The variants selected for sequencing include 1) RU (2089 base pairs (bp) ), representative of the average size of repeat units of cellular satellite; 2) TRU (1674 bp), truncated at an extra EcoRI site; and 3) EXT (2639 bp), extended by a 5-fold amplification of a 142-bp segment, one copy of which is present in RU and TRU (Bonnewell, V., Fowler, R.F., and Skinner, D.M. (1983) Science 221, 862-865). It appears that every copy of the satellite may be different and that the variants do not arise from cloning accidents. Extensive domains, as long as approximately 560 bp, are greater than 95% homologous among RU, TRU, and EXT; these conserved domains are composed of DNA whose base composition and sequences do not have remarkable features. By contrast, the sequences that comprise the divergent domains are unusually rich in 1) tracts of (dG X dC) 13-23 and arrangements of similar but not identical repetitive oligonucleotides or 2) alternating purines and pyrimidines (pu/py).

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.