Genetic studies on heterochromatin in Drosophila melanogaster and their implications for the functions of satellite DNA

In Drosophila melanogaster the centromeric heterochromatin of all chromosomes consists almost entirely of several different satellite DNA sequences. In view of this we have examined by genetic means the meiotic consequences of X chromosomes with partial deletions of their heterochromatin, and have found that the amount and position of recombination on each heterochromatically deleted X is substantially different from that of a normal X. It appears that the amount of heterochromatin is important in modifying the centromere effect on recombination. — In all the deleted Xs tested, chromosome segregation is not appreciably altered from that of a nondeleted control chromosome. Thus satellite DNA does not appear to be an important factor in determining the regular segregation of sex chromosomes in Drosophila. Additionally, since X chromosomes with massive satellite DNA deficiencies are able to participate in a chromocenter within salivary gland nuclei, a major role of satellite DNA in chromocenter formation in this tissue is also quite unlikely. — In order to examine the mechanisms by which the amount of satellite DNA is increased or decreased in vivo, we have measured cytologically the frequency of spontaneous sister chromatid exchanges in a ring Y chromosome which is entirely heterochromatic and consists almost exclusively of satellite DNA. In larval neuroblast cells the frequency of spontaneous SCE in this Y is approximately 0.3% per cell division. Since there is no meiotic recombination in D. melanogaster males and since meiotic recombination in the female does not occur in heterochromatin, our results provide a minimum estimate of the in vivo frequency of SCE in C-banded heterochromatin (which is predominantly simple sequence DNA), without the usual complications of substituted base analogs, incorporated radioactive label or substantial genetic content. — We emphasise that: (a) satellite DNA is not implicated in any major way in recognition processes such as meiotic homologue recognition or chromocenter formation in salivaries, (b) there is likely to be continuous variation in the amount of satellite DNA between individuals of a species; and (c) the amount of satellite DNA can have a crucial functional role in the meiotic recombination system.     

Human gamma X satellite DNA: an X chromosome specific centromeric DNA sequence

The cosmid clone, CX16-2D12, was previously localized to the centromeric region of the human X chromosome and shown to lack human X-specific satellite DNA. A 1.2 kb EcoRI fragment was subcloned from the CX16-2D12 cosmid and was named 2D12/E2. DNA sequencing revealed that this 1,205 bp fragment consisted of approximately five tandemly repeated DNA monomers of 220 bp. DNA sequence homology between the monomers of 2D12/E2 ranged from 72.8% to 78.6%. Interestingly, DNA sequence analysis of the 2D12/E2 clone displayed a change in monomer unit orientation between nucleotide positions 585–586 from a tail-to-head arrangement to a head-to-tail configuration. This may reflect the existence of at least one inversion within this repetitive DNA array in the centromeric region of the human X chromosome. The DNA consensus sequence derived from a compilation of these 220 bp monomers had approximately 62% DNA sequence similarity to the previously determined 8 satellite DNA consensus sequence. Comparison of the 2D12/E2 and 8 consensus sequences revealed a 20 bp DNA sequence that was well conserved in both DNA consensus sequences. Slot-blot analysis revealed that this repetitive DNA sequence comprises approximately 0.015% of the human genome, similar to that found with 8 satellite DNA. These observations suggest that this satellite DNA clone is derived from a subfamily of satellite DNA and is thus designated X satellite DNA. When genomic DNA from six unrelated males and two unrelated females was cut with SstI or HpaI and separated by pulsed-field gel electrophoresis, no restriction fragment length polymorphisms were observed for either X (2D12/E2) or 8 (50E4) probes. Fluorescence in situ hybridization localized the 2D12/E2 clone to the lateral sides of the primary constriction specifically on the human X chromosome.     

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     

A new family of satellite DNA sequences as a major component of centromeric heterochromatin in owls (Strigiformes)

We isolated a new family of satellite DNA sequences from Hae III- and Eco RI-digested genomic DNA of the Blakistons fish owl ( Ketupa blakistoni). The repetitive sequences were organized in tandem arrays of the 174 bp element, and localized to the centromeric regions of all macrochromosomes, including the Z and W chromosomes, and microchromosomes. This hybridization pattern was consistent with the distribution of C-band-positive centromeric heterochromatin, and the satellite DNA sequences occupied 10% of the total genome as a major component of centromeric heterochromatin. The sequences were homogenized between macro- and microchromosomes in this species, and therefore intraspecific divergence of the nucleotide sequences was low. The 174 bp element cross-hybridized to the genomic DNA of six other Strigidae species, but not to that of the Tytonidae, suggesting that the satellite DNA sequences are conserved in the same family but fairly divergent between the different families in the Strigiformes. Secondly, the centromeric satellite DNAs were cloned from eight Strigidae species, and the nucleotide sequences of 41 monomer fragments were compared within and between species. Molecular phylogenetic relationships of the nucleotide sequences were highly correlated with both the taxonomy based on morphological traits and the phylogenetic tree constructed by DNA-DNA hybridization. These results suggest that the satellite DNA sequence has evolved by concerted evolution in the Strigidae and that it is a good taxonomic and phylogenetic marker to examine genetic diversity between Strigiformes species.An erratum to this article can be found at Communicated by Y. Hiraoka     

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).     

Karyotypic evolution of a novel cervid satellite DNA family isolated by microdissection from the Indian muntjac Y-chromosome

A minilibrary was constructed from DOP-PCR products using microdissected Y-chromosomes of Indian muntjac as DNA templates. Two microclones designated as IM-Y4-52 and IM-Y5-7 were obtained from negative screening of all three cervid satellite DNAs (satellites I, II, and IV). These two microclones were 295 and 382 bp in size, respectively, and shared 70% sequence homology. Southern blot analysis showed that the IM-Y4-52 clone was repetitive in nature with an 0.32-kb register in HaeIII digest. Sequence comparison revealed no similarities to DNA sequences deposited in the GenBank database, suggesting that the microclone sequences were from a novel satellite DNA family designated as cervid satellite V. A subclone of an Indian muntjac BAC clone which screened positive for IM-Y4-52 had a 3,325-bp insert containing six intact monomers, four deleted monomers, and two partial monomers. The consensus sequence of the monomer was 328 bp in length and shared more than 80% sequence homology with every intact monomer. A zoo blot study using IM-Y4-52 as a probe showed that the strong hybridization with EcoRI digested male genomic DNA of Indian muntjac, Formosan muntjac, Chinese muntjac, sambar deer, and Chinese water deer. Female genomic DNA of Indian muntjac, Chinese water deer, and Formosan muntjac also showed positive hybridization patterns. Satellite V was found to specifically localize to the Y heterochromatin region of the muntjacs, sambar deer, and Chinese water deer and to chromosome 3 of Indian muntjac and the X-chromosome of Chinese water deer.Y.-C. Li and Y.-M. Cheng contributed equally to this work.     

Meiotic chromosome behaviour reflects levels of sequence divergence inSus scrofa domestica satellite DNA

We present a general model for the evolution of chromosome-specific satellite DNA subfamilies.Sus scrofa domestica has a bimodal karyotype with two autosomal subsets of 12 meta-/submetacentric (Mc) and 6 acrocentric (Ac) chromosome types (Mc and Ac subgenomes). We show that the centromeric heterochromatin is characterised by two distinct satellite DNA families designed Mc1 and Ac2. Mc1 is a diverse satellite family of the Mc subgenome of which certain members with a 100 bp repeat unit are found to occur at the pericentromeric regions of each Mc autosome, while others are chromosome-specific, e.g. clone Mc pAv1.5, a higher order repeat variant, which hybridises specifically to chromosome 1. Ac2 is a homogeneous satellite occurring at the subterminal pericentromeric regions of all Ac autosomes. DNA sequence analyses showed that all clones investigated are built up from a 14 bp repeat unit which is highly conserved. In situ hybridisation to meiotic pachytene nuclei revealed a distinct spatial arrangement of the Ac2 centromeric satellite.     

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.     

DNA regions flanking the major Arabidopsis thaliana satellite are principally enriched in Athila retroelement sequences

An analysis of Arabidopsis thaliana heterochromatic regions revealed that genomic sequences immediately flanking the major 180 bp satellite are essentially made of middle repetitive sequences and that most of these sequences correspond to defective Athila retroelements. Using YAC and clones, we evaluated the distribution of Athila elements in the Arabidopsis genome and showed that, despite the presence of numerous euchromatic copies, these elements are especially concentrated in or near heterochromatic regions. Sequencing of the various DNA transitions between satellite and Athila repeats provides strong evidence that most of the heterochromatic elements retrotransposed directly into 180 bp satellite clusters.     

Isolation of an autonomously replicating sequence (ARS) from satellite DNA of Drosophila melanogaster

Summary Autonomously replicating sequences (ARSs) were cloned from the 1.688 satellite DNA of D. melanogaster using YIp5, consisting of pBR322 and the yeast ura3 gene, as the cloning vector and YNN27, a Ura^– yeast strain as the recipient. Three out of six clones contained an ARS and the average frequncy of the occurrence of ARS was thus calculated to be approximately one per 14 kbp of the satellite DNA. A 500 bp ARS fragment (BgHS500) was obtained from one of the resultant clones (pYDS57). BgHS500 does not hybridize with the major repeating unit (370 bp) but it does with the minor unique sequence of the satellite. The sequence of BgHS500 was determined and found to be rich in AT and to contain the sequence, 5AAAACATAAAA3, a sequence common to yeast ARSs. However, a smaller fragment (150 bp) isolated from BgHS500 and containing the 11 bp sequence did not exhibit the characteristics of an ARS. The average copy number in the transformants of pBgHS500, a recombinant molecule of BgHS500 and YIp5, ranged from 0.05–0.5, while that of the parent plasmid, pYDS57, was about 2–10. On the basis of these results, it is postulated that the sequence 5AAAACATAAAA3 may possibly consiitute the core of ARSs and certain other sequences may also be necessary to insure that the ARS consistently undergoes at least one complete replication in each cell cycle. The role of ARSs in the genome of D. melanogaster is discussed.     

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).     

The location of DNA homologous to human satellite III DNA in the chromosomes of chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and orang utan (Pongo pygmaeus)

Radioactive RNA with sequences complementary to human DNA satellite III was hybridised in situ to metaphase chromosomes of the chimpanzee (Pan troglodytes), the gorilla (Gorilla gorilla) and the orangutan (Pongo pygmaeus). A quantitative analysis of the radioactivity, and hence of the chromosomal distribution of human DNA satellite III equivalent sequences in the great apes, was undertaken, and the results compared with interspecies chromosome homologies based upon Giemsa banding patterns. In some instances DNA with sequence homology to human satellite III is present on the equivalent (homologous) chromosomes in identical positions in two or more species although quantitative differences are observed. In other cases there appears to be no correspondence between satellite DNA location and chromosome homology determined by banding patterns. These results differ from those found for most transcribed DNA sequences where the same sequence is located on homologous chromosomes in each species.     

Prototype sequence of bovine 1.720 satellite DNA

Bovine 1.720 satellite DNA (density in CsCl, 1.720 g/cm³) consists of a tandem array of 46 base-pair-repeat units without a detectable higher-order periodicity. About 80% of the satellite DNA is cleaved by AluI into a 46 base-pair fragment which has been isolated and sequenced. The sequence determined exhibits a very high homology to the 23 base-pair prototype sequence of bovine 1.706 satellite DNA (Pech et al., 1979) indicating a common origin of the two satellites. The 46 base-pairrepeat unit of the 1.720 satellite is composed of two related 23 base-pair sequences both of which are largely self-complementary. The entire 1.720 satellite DNA can be considered to be an imperfect palindrome.     

Detection of satellite DNA in Palorus ratzeburgii: Analysis of curvature profiles and comparison with Tenebrio molitor satellite DNA

Very abundant and homogenous satellite DNA has been found in the flour beetle Palorus ratzeburgii, representing 40% of its genome. Sequencing of 14 randomly cloned satelite monomers revealed a conserved monomer length of 142 bp and an average A+T content of 68%. Sequence variation analysis showed that base substitutions, appearing with a frequency of 2.3%, are predominant differences among satellite monomers. The satellite sequence is unique without significant direct repeats and with only two potentially stable inverted repeats. After electrophoresis of satellite monomers on native polyacrylamide gel retarded mobilities characteristic for curved DNA molecules are observed. The curvature profiles and DNA helix axis trajectory are calculated on the basis of three different algorithms. These calculations predict that P ratzeburgii satellite DNA forms a left-handed solenoid superstructure. Comparison of described features with other satellite DNAs reveals some striking similarities with satellite DNA from related species Tenebrio molitor, which belongs to the same family of Tenebrionidae. Both satellites are very abundant and homogenous with the same, highly conserved monomer length, although there is no homology at the nucleotide level. Their monomers, as well as multimers, exhibit very similar retarded electrophoretic mobilities. The calculated curvature profiles predict two bend centers in monomers of each satellite, resulting in a model of left-handed solenoid superstructures of similar appearance.     

The cyclization of mouse satellite DNA

Sheared fragments of mouse satellite DNA can form rings and other circular structures by several techniques. Folded rings are formed if the sheared fragments are simply annealed, indicating that shearing produces single-chain terminals, and that the repetitious sequence is shorter than the exposed ends. The occurrence of folded rings can be sharply reduced by prior treatment with single-chain specific endonuclease, and significantly increased if the fragments are treated with exonuclease III. Denaturation of satellite DNA followed by reassociation of the single chains results in the formation of slipped rings. These characteristics of the DNA lead to the conclusion that the sequences of the mouse satellite DNA are arranged in a tandemly repetitious manner.-About 20% of the DNA fragments from the main band cyclize after partial exonuclease III degradation, but not before this treatment. This indicates that a large fraction of the main band DNA is tandemly repetitious, but that the length of the repetitious sequence is on the average longer than the single-chain terminals produced by shearing.Reed Pyeritz is the recipient of a NSF predoctoral fellowship. C. S. Lee is a Fellow of the Jane Coffin Childs Memorial Fund for Medical Research. This investigation has been supported by grants from the National Institutes of Health (AI08186), the National Science Foundation (GB-8611), and the Jane Coffin Childs Memorial Fund for Medical Research.     

Structural evolution of the Drosophila 5S ribosomal genes

We compare the 5S gene structure from nine Drosophila species. New sequence data (5S genes of D. melanogaster, D. mauritiana, D. sechellia, D. yakuba, D. erecta, D. orena, and D. takahashii) and already-published data (5S genes of D. melanogaster, D. simulans, and D. teissieri) are used in these comparisons. We show that four regions within the Drosophila 5S genes display distinct rates of evolution: the coding region (120 bp), the 5-flanking region (54–55 bp), the 3-flanking region (21–22 bp), and the internal spacer (149–206 bp). Intra- and interspecific heterogeneity is due mainly to insertions and deletions of 6–17-bp oligomers. These small rearrangements could be generated by fork slippages during replication and could produce rapid sequence divergence in a limited number of steps. Correspondence to: M. Wegnez     

DNA/DNA Hybridization studies among six endemic Hawaiian Drosophila species

The nucleotide-sequence homology and divergence of six endemic Hawaiian Drosophila species have been studied by means of DNA/DNA hybridization experiments, using the fractionated unique and moderately repetitive sequences. The data indicate that the extent of homology between the nonrepetitive DNA sequences of D. crucigera, D. pilimana, D. engyochracea, D. silvarentis and D. picticornis confirms the taxonomic relationship established through cytological evidence. The average rate of divergence of nonrepetitive DNA sequences among Hawaiian Drosophila species is approximately 2.75 to 3.75% per million years. This value is much higher than that found among other species when divergence time is estimated on an absolute time base.     

Concerted evolution,a slow process for ant satellite DNA: study of the satellite DNA in the <Emphasis Type="Italic">Aphaenogaster</Emphasis> genus (Hymenoptera,Formicidae)

A family of satellite DNA is analyzed in seven ant species from the genus Aphaenogaster. This satellite DNA is organized as tandemly repeated sequences with a consensus sequence of 160 bp in length. The sampled sequences show a high similarity and belong to the same family of satellite DNA. However in Aphaenogaster spinosa, two types of repeat clearly differentiated have been found. Phylogenetic analyses using satellite DNA show that sequences do not cluster in a species-specific way, with one exception. Concretely, the second type of repeats of A. spinosa (APSP-II) which constitutes a new satellite DNA subfamily. The obtained results with satellite DNA are compared with those obtained using mitochondrial and nuclear DNA to determine the correlation between evolution of satellite DNA and phylogenetic relationships among the analyzed ants. The high interspecific similarity for the satellite DNA seems not to be in concordance with the concerted evolution pattern, commonly accepted to explain the evolution of satellite DNA. However, the accumulated data suggest that evolution of satellite DNA in ants follows the concerted evolution pattern but that this process is slow in relation with other organisms, probably due to the eusociality and haplodiploidy of these insects.     

Telomeric satellite DNA functions in regulating recombination

Molecular and cytogenetical analyses of three sibling species of Australian grasshopper, Atractomorpha australis, A. species-1 and A. similis, resolves one of the long standing problems of highly repeated DNA. In this system satellite DNA functions in regulating the level and position of recombination, irrespective of whether the repeated DNA is located in telomeric or centric regions. — Even though the three species do not differ in their euchromatic genome sizes, their relative DNA contents are 1.00/1.10/ 1.41, the difference in genome size being due solely to visible centric or telomeric blocks of heterochromatin. — Antibiotic analytical and preparative ultracentrifugation, in situ hybridization and renaturation kinetic analyses reveal that a large cryptic satellite of A. similis constitutes the heterochromatic telomeric blocks of nearly all autosomes and that the DNA of this satellite is highly repeated. — Comparison of these grasshopper data with published literature of heterochromatic rearrangements in Drosophila and with heterochromatin distribution and recombination patterns in diploid plant species reveals that in every case heterochromatin is implicated in some form of alteration in the meiotic recombination system.     

Structural variability in the genome of the Thermoproteus tenax virus TTV1

Summary Six variants of the TTV1 genome, including the primary isolate, have been characterized. DNA sequence comparison of wild-type virus (WT) and one of the variants (VT3) showed that differences are due to insertions and deletions that were confined to contiguous portions of two distinctClaI fragments. Seven similar short DNA sequences (30–102 bp) were involved in the variation. The deletions and insertions of these short DNA sequences occurred in every case adjacent to the 8 by consensus sequence 5-ACXCCTAC-3 which formed the 5 flank of the segments involved.