Sequences of the 1.672 g/cm3 satellite DNA of Drosophila melanogaster.

The 1.672 g/cm³ satellite DNA of Drosophila melanogaster was purified by successive equilibrium centrifugations in a CsCl gradient, an actinomycin $\text{D}\text{CsCl}$ gradient, and a netropsin sulfate/CsCl gradient. The resulting DNA was homogeneous by the physical criteria of thermal denaturation, renaturation kinetics and equilibrium banding in each of the gradients listed above. In addition, the complementary strands could be separated in an alkaline CsCl gradient. Despite this rigorous purification procedure, nucleotide sequence analysis indicates the presence of two different DNA species in this satellite, poly $\text{A-A-T-A-T}\text{T-T-A-T-A}$ and poly$\text{A-A-T-A-T-A-T}\text{T-T-A-T-A-T-A}$. Further physical, chemical and template properties of the isolated complementary strands demonstrate that these two repeating sequences are not interspersed with each other. This result has biological significance since sequences of this particular satellite are known to be located primarily on two different chromosomes, Y and 2. These results further suggest that the sequence heterogeneity observed in satellite DNA of higher eukaryotes may result from mixtures of very closely related but molecularly homogeneous repeated sequences each restricted to a particular chromosome or chromosomal region.     

Pericentromeric regions containing 1.688 satellite DNA sequences show anti-kinetochore antibody staining in prometaphase chromosomes of Drosophila melanogaster

A striking characteristic of the centromeric heterochromatin of Drosophila melanogaster is that each chromosome carries different satellite DNA sequences. Here we show that while the major component of the 1.688 satellite DNA family expands across the centromere of the X chromosome the rest of the minor variants are located at pericentromeric positions in the large autosomes. Immunostaining of prometaphase chromosomes with the kinetocore-specific anti-BUB1 antibody reveals the transient presence of this centromeric protein in all the regions containing the 1.688 satellite.     

Population genetics and phylogenetics of DNA sequence variation at multiple loci within the Drosophila melanogaster species complex

Two regions of the genome, a 1-kbp portion of the zeste locus and a 1.1- kbp portion of the yolk protein 2 locus, were sequenced in six individuals from each of four species: Drosophila melanogaster, D. simulans, D. mauritiana, and D. sechellia. The species and strains were the same as those of a previous study of a 1.9-kbp region of the period locus. No evidence was found for recent balancing or directional selection or for the accumulation of selected differences between species. Yolk protein 2 has a high level of amino acid replacement variation and a low level of synonymous variation, while zeste has the opposite pattern. This contrast is consistent with information on gene function and patterns of codon bias. Polymorphism levels are consistent with a ranking of effective population sizes, from low to high, in the following order: D. sechellia, D. melanogaster, D.mauritiana, and D. simulans. The apparent species relationships are very similar to those suggested by the period locus study. In particular, D. simulans appears to be a large population that is still segregating variation that arose before the separation of D. mauritiana and D. sechellia. It is estimated that the separation of ancestral D. melanogaster from the other species occurred 2.5-3.4 Mya. The separations of D. sechellia and D. mauritiana from ancestral D. simulans appear to have occurred 0.58- 0.86 Mya, with D. mauritiana having diverged from ancestral D. simulans 0.1 Myr more recently than D. sechellia.      

Prod is a novel DNA-binding protein that binds to the 1.686 g/cm(3) 10 bp satellite repeat of Drosophila melanogaster

The proliferation disrupter (prod) gene of Drosophila melanogaster encodes a novel protein associated with centromeric chromosomal regions that is required for chromatin condensation and cell viability. We have examined the binding of the Prod protein to DNA in vitro. Co-immunoprecipitation experiments demonstrate that Prod is a DNA-binding protein that specifically recognizes the 10 bp AGAATAACAT satellite repeat of D.melanogaster. Footprinting experiments show that the protein interacts with a 5–8 bp target sequence in each 10 bp repeat and suggest that it can mediate condensation of this satellite into a superhelix. Gel retardation experiments indicate that Prod does not have a well defined DNA-binding domain and it binds the satellite in a co-operative manner, probably forming Prod multimers. Since Prod localizes to both heterochromatin and euchromatin in vivo, we discuss the possibility that the ability of pre-existing euchromatic proteins to bind DNA in a co-operative manner, might be a prerequisite of satellite compaction and satellite amplification, thereby providing a basic factor in heterochromatin evolution.     

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.     

Mapping and positioning DNA-binding proteins along genomic DNA. Structure of D. melanogaster ribosomal 'Alu-repeats' and 1.688 satellite chromatin.

Chromatin structure of so-called 'Alu-repeat' in D. melanogaster ribosomal non-transcribed spacer that contains sequences homologous to the promoter of ribosomal genes has been studied. Using the 'protein image' hybridization assay based on UV-light-induced DNA-protein crosslinking and 2-D gel retardation electrophoresis, two proteins of the molecular mass of 50 kD (rABP50) and 70 kD (rABP70), associated with 'Alu-repeat' DNA have been found. Exo III mapping of crosslinking sites and DNase I footprinting have provided a detailed map of H1, rABP50 and rABP70 contacts within the 'Alu-repeat' and H1 and a non-histone protein contacts on satellite DNA. These data indicate precise positioning of non-histone proteins, histone H1 and nucleosomes within genomic regions studied and account for the presence of unusual 240 bp long nucleosomal particles in 'Alu-repeats'. The same approach can be adapted for successive mapping and positioning proteins on genomic DNA.     

Cleavage patterns of Drosophila melanogaster satellite DNA by restriction enzymes.

The five satellite DNAs of Drosophila melanogaster have been isolated by the combined use of different equilibrium density gradients and hydrolyzed by seven different restriction enzymes; Hae III, Hind II + Hind III, Hinf, Hpa II, EcoR I and EcoR II. The 1.705 satellite is not hydrolyzed by any of the enzymes tested. Hae III is the only restriction enzyme that cuts the 1.672 and 1.686 satellites. The cleavage products from either of these reactions has a heterogeneous size distribution. Part of the 1.688 satellite is cut by Hae III and by Hinf into three discrete fragments with M.W. that are multiples of 2.3 X 10(5) daltons (approximately 350 base pairs). In addition, two minor bands are detected in the 1.688-Hinf products. The mole ratios of the trimer, dimer and monomer are: 1:6.30 : 63.6 for 1.688-Hae III and 1 : 22.0 : 403 for 1.688-Hinf. Circular mitochondrial DNA (rho = 1.680) is cut into discrete fragments by all of the enzymes tested and molecular weights of these fragments have been determined.     

Cloning and characterization of a complex satellite DNA from Drosophila melanogaster.

The sequence organization of the 1.688 satellite DNA (density 1.688 g/cm³ in CsCl) has been investigated, and this satellite has been found to differ from the other D. melanogaster satellite DNAs in having a much greater sequence complexity. Purification of 1.688 satellite DNA by successive equilibrium density centrifugations yielded a fraction 77% pure. Segments of satellite DNA were isolated by molecular cloning in the plasmid vector pSC101. One recombinant plasmid contained a segment of 1.688 satellite DNA 5.8 kilobase pairs in size and was stable during propagation in E. coli. Recognition sites for restriction enzymes from Haemophilus aegyptius (Hae III), Haemophilus influenzae f (Hinf) and Arthrobacter luteus (Alu I) were mapped in the satellite DNA of this hybrid plasmid. The spacing of Hae III, Hinf and two Alu I sites at regular intervals of about 365 base pairs is strong evidence that the sequence complexity of this satellite DNA is 365 base pairs. Further evidence comes from the finding that both gradient-purified and cloned 1.688 satellite DNA renature with their Hae III sites in register. The Hae III and Hinf sites in gradient-purified satellite DNA have been shown by Manteuil, Hamer and Thomas (1975) and Shen, Wiesehahn and Hearst (1976) to be distributed at intervals of 365 base pairs and integral multiples thereof. These investigators proposed that some of the sites in an otherwise regular array have been randomly inactivated. Cloned satellite DNA provided a hybridization probe for sensitive studies of the arrangement of these recognition sites in gradient-purified satellite DNA. Some regions of satellite DNA were found to contain many fewer recognition sites than expected from the proposed models. These findings suggest that different regions of 1.688 satellite DNA may exhibit different arrangements of Hae III and Hinf recognition sites.     

DNA sequence of the white locus of Drosophila melanogaster

The DNA sequence of the white locus of Drosophila melanogaster is presented. This 14,100 base-pair sequence includes the region of the locus required for wild-type levels of expression and control of expression. We also report the sequence of a complementary DNA clone which established the position of the 3' end of the white RNA on this genomic sequence. The probable exon-intron structure of the gene has been predicted from the DNA sequence of the regions known to be represented in the RNA. The amino acid sequence of the protein which would be produced by translation of this RNA suggests that the white locus gene product may be a membrane protein. The DNA sequence rearrangements associated with seven insertion mutants (white-dominant-zeste-like (wDZL), white-spotted (wsp), white-honey (wh), white-zeste-mottled (wzm), white-apricot (wa), white-buff (wbf) and white-hd81b11 (whd81b11)), one deletion mutant (white-spotted 4 (wsp4)) and one internal duplication mutant (white-ivory (wi)) have been determined and positioned on the wild-type sequence. The positions of these insertions and those of previously characterized insertions associated with six other mutations suggest that some insertions within an intron may still allow the production of correctly spliced RNA, but affect the amount, and correspondingly the expression of the w locus.     

1.688 g/cm3 satellite‐related repeats: a missing link to dosage compensation and speciation

Despite the important progress that has been made on dosage compensation (DC), a critical link in our understanding of the X chromosome recognition mechanisms is still missing. Recent studies in Drosophila indicate that the missing link could be a family of DNA repeats populating the euchromatin of the X chromosome. In this opinion article, I discuss how these findings add a new fresh twist on the DC problem. In the following sections, I first summarize our understanding of DC in Drosophila and integrate these recent discoveries into our knowledge of the X chromosome recognition problem. Next, I introduce a model according to which, 1.688 g/cm³ satellite‐related (SR) repeats would be the primary recognition elements for the dosage compensation complex. Contrary to the current belief, I suggest that the DC system in Drosophila is not conserved and static, but it is continuously co‐evolving with the target SR repeats. The potential role of the SR repeats in hybrid incompatibilities and speciation is also discussed.     

One of the copia genes is adjacent to satellite DNA in Drosophila melanogaster.

A method for purifying sequences adjacent to satellite DNA in the heterochromatin of D. melanogaster is described. A cloned DNA segment containing part of a copia gene adjacent to 1.688 g/cm3 satellite DNA has been isolated. The copia genes compose a repeated gene family which codes for abundant cytoplasmic poly(a)-containing RNA (Young and Hogness, 1977; Finnegan et al., 1978). We have identified two major poly (A)-containing RNA species [5.2 and 2.1 kilobases (kb)] produced by the copia gene family. The cloned segment contains copia sequences homologous to the 5' end of RNA within 0.65 kb of the 1.688 satellite DNA sequences. Seven different cloned copia genes from elsewhere in the genome have also been isolated, and a 5.2 kb region present in five of the clones was identified as copia by heteroduplex analysis. In addition, three ususual copies of copia were found: a "partial" copy of the gene (3.7 kb) which has one endpoint in common with the 5.2 kb unit; a copia gene flanked on one side by a 1.6 kb sequence and on the other by the same 1.6 kb sequence in the inverted orientation; and a copia gene flanked only on one side by the same sequence.     

African sequence variation accounts for most of the sequence polymorphism in non-African Drosophila melanogaster

We compared the sequence polymorphism of 12 genomic fragments in six geographically dispersed African populations to one European Drosophila melanogaster population. On the basis of one African and one European population half of these fragments have strongly reduced levels of variability outside of Africa. Despite this striking difference in European variation, we detected no significant difference in African variation between the two fragment classes. The joint analysis of all African populations indicated that all high-frequency European alleles are of African origin. We observed a negative Tajima's D in all African populations, with three populations deviating significantly from neutral equilibrium. Low, but statistically significant, population differentiation was observed among the African populations. Our results imply that the population structure and demographic past of African D. melanogaster populations need to be considered for the inference of footprints of selection in non-African populations.     

Sequence arrangement of the rDNA of Drosophila melanogaster.

The sequence arrangement of genes coding for stable rRNA species and of the interspersed spacers on long single strands of rDNA purified from total chromosomal DNA of Drosophila melanogaster has been determined by a study of the structure of rRNA:DNA hybrids which were mounted for electron microscope observation by the gene 32-ethidium bromide technique. One repeat unit contains the following sequences in the order given. First, an 18 S gene of length 2.13 +/- 0.17 kb. Second, an internal transcribed spacer (Spl) of length 1.58 +/- 0.15 kb. A short sequence coding for the 5.8S and perhaps the 2S rRNA species is located within this spacer. Third, the 28S gene with a length of 4.36 +/- 0.23 kb. About 55% of the 28S genes are unbroken or continuous (C genes). However, about 45% of the 28S genes contain an insertion of an additional segment of DNA that is not complementary to rRNA (l genes). The insertion occurs at a reproducible point 2.99 +/- 0.26 kb from the junction with Spl. The insertions are heterogeneous in length and occur in three broad size classes: 1.42 +/- 0.47, 3.97 +/- 0.55, and 6.59 +/- 0.62 kb. Fourth, an external spacer between the 28S gene and the next 18S gene which is presumably mainly nontranscribed and which has a heterogeneous length distribution with a mean length and standard deviation of 5.67 +/- 1.92 kb. Short inverted repeat stems (100-400 nucleotide pairs) occur at the base of the insertion. It is known from other studies that I genes occur only on the X chromosome. The present study shows that the I and C genes on the X chromosomes are approximately randomly assorted. The sequence arrangement on the plasmid pDm103 containing one repeat of rDNA (Glover et al., 1975) has been determined by similar methods. The I gene on this plasmid contains an inverted repeat stem. The occurrence of inverted repeat sequences flanking the insertion supports the speculation that these sequences are translocatable elements similar to procaryotic translocons.     

Analysis of copia sequence variation within and between Drosophila species

The sequences of the 5' long-terminal repeat (LTR) and adjacent leader regions of 27 full-length copia elements isolated from natural populations of Drosophila melanogaster, D. simulans, and D. mauritiana are presented. Phylogenetic analyses indicate that although D. melanogaster copia elements are distinct from those of D. simulans and D. mauritiana, the elements of these latter two species are not distinguishable from one another. LTRs and adjacent 5' leader regions of elements isolated from D. simulans and D. mauritiana are structurally similar to one another and carry substantial deletional variation mapping to regions previously identified as being of potential importance for copia expression.