Novel simple sequence repeats (SSRs) detected by ND-FISH in heterochromatin of Drosophila melanogaster

<p>Abstract</p> <p>Background</p> <p>In recent years, substantial progress has been made in understanding the organization of sequences in heterochromatin regions containing single-copy genes and transposable elements. However, the sequence and organization of tandem repeat DNA sequences, which are by far the majority fraction of <it>D. melanogaster </it>heterochromatin, are little understood.</p> <p>Results</p> <p>This paper reports that the heterochromatin, as well as containing long tandem arrays of pentanucleotide satellites (AAGAG, AAGAC, AATAT, AATAC and AACAC), is also enriched in other simple sequence repeats (SSRs) such as A, AC, AG, AAG, ACT, GATA and GACA. Non-denaturing FISH (ND-FISH) showed these SSRs to localize to the chromocentre of polytene chromosomes, and was used to map them on mitotic chromosomes. Different distributions were detected ranging from single heterochromatic clusters to complex combinations on different chromosomes. ND-FISH performed on extended DNA fibres, along with Southern blotting, showed the complex organization of these heterochromatin sequences in long tracts, and revealed subclusters of SSRs (several kilobase in length) flanked by other DNA sequences. The chromosomal characterization of C, AAC, AGG, AAT, CCG, ACG, AGC, ATC and ACC provided further detailed information on the SSR content of <it>D. melanogaster </it>at the whole genome level.</p> <p>Conclusion</p> <p>These data clearly show the variation in the abundance of different SSR motifs and reveal their non-random distribution within and between chromosomes. The greater representation of certain SSRs in <it>D. melanogaster </it>heterochromatin suggests that its complexity may be greater than previously thought.</p

Nuclear Importation of Mariner Transposases among Eukaryotes: Motif Requirements and Homo-Protein Interactions

Mariner-like elements (MLEs) are widespread transposable elements in animal genomes. They have been divided into at least five sub-families with differing host ranges. We investigated whether the ability of transposases encoded by Mos1, Himar1 and Mcmar1 to be actively imported into nuclei varies between host belonging to different eukaryotic taxa. Our findings demonstrate that nuclear importation could restrict the host range of some MLEs in certain eukaryotic lineages, depending on their expression level. We then focused on the nuclear localization signal (NLS) in these proteins, and showed that the first 175 N-terminal residues in the three transposases were required for nuclear importation. We found that two components are involved in the nuclear importation of the Mos1 transposase: an SV40 NLS-like motif (position: aa 168 to 174), and a dimerization sub-domain located within the first 80 residues. Sequence analyses revealed that the dimerization moiety is conserved among MLE transposases, but the Himar1 and Mcmar1 transposases do not contain any conserved NLS motif. This suggests that other NLS-like motifs must intervene in these proteins. Finally, we showed that the over-expression of the Mos1 transposase prevents its nuclear importation in HeLa cells, due to the assembly of transposase aggregates in the cytoplasm

Cis-by-Trans Regulatory Divergence Causes the Asymmetric Lethal Effects of an Ancestral Hybrid Incompatibility Gene

The Dobzhansky and Muller (D-M) model explains the evolution of hybrid incompatibility (HI) through the interaction between lineage-specific derived alleles at two or more loci. In agreement with the expectation that HI results from functional divergence, many protein-coding genes that contribute to incompatibilities between species show signatures of adaptive evolution, including Lhr, which encodes a heterochromatin protein whose amino acid sequence has diverged extensively between Drosophila melanogaster and D. simulans by natural selection. The lethality of D. melanogaster/D. simulans F1 hybrid sons is rescued by removing D. simulans Lhr, but not D. melanogaster Lhr, suggesting that the lethal effect results from adaptive evolution in the D. simulans lineage. It has been proposed that adaptive protein divergence in Lhr reflects antagonistic coevolution with species-specific heterochromatin sequences and that defects in LHR protein localization cause hybrid lethality. Here we present surprising results that are inconsistent with this coding-sequence-based model. Using Lhr transgenes expressed under native conditions, we find no evidence that LHR localization differs between D. melanogaster and D. simulans, nor do we find evidence that it mislocalizes in their interspecific hybrids. Rather, we demonstrate that Lhr orthologs are differentially expressed in the hybrid background, with the levels of D. simulans Lhr double that of D. melanogaster Lhr. We further show that this asymmetric expression is caused by cis-by-trans regulatory divergence of Lhr. Therefore, the non-equivalent hybrid lethal effects of Lhr orthologs can be explained by asymmetric expression of a molecular function that is shared by both orthologs and thus was presumably inherited from the ancestral allele of Lhr. We present a model whereby hybrid lethality occurs by the interaction between evolutionarily ancestral and derived alleles

RSF Governs Silent Chromatin Formation via Histone H2Av Replacement

Human remodeling and spacing factor (RSF) consists of a heterodimer of Rsf-1 and hSNF2H, a counterpart of Drosophila ISWI. RSF possesses not only chromatin remodeling activity but also chromatin assembly activity in vitro. While no other single factor can execute the same activities as RSF, the biological significance of RSF remained unknown. To investigate the in vivo function of RSF, we generated a mutant allele of Drosophila Rsf-1 (dRsf-1). The dRsf-1 mutant behaved as a dominant suppressor of position effect variegation. In dRsf-1 mutant, the levels of histone H3K9 dimethylation and histone H2A variant H2Av were significantly reduced in an euchromatic region juxtaposed with heterochromatin. Furthermore, using both genetic and biochemical approaches, we demonstrate that dRsf-1 interacts with H2Av and the H2Av-exchanging machinery Tip60 complex. These results suggest that RSF contributes to histone H2Av replacement in the pathway of silent chromatin formation

Analysis of the piggyBac transposase reveals a functional nuclear targeting signal in the 94 c-terminal residues

<p>Abstract</p> <p>Background</p> <p>The <it>piggyBac</it> transposable element is a popular tool for germ-line transgenesis of eukaryotes. Despite this, little is known about the mechanism of transposition or the transposase (TPase) itself. A thorough understanding of just how <it>piggyBac</it> works may lead to more effective use of this important mobile element. A PSORTII analysis of the TPase amino acid sequence predicts a bipartite nuclear localization signal (NLS) near the c-terminus, just upstream of a putative ZnF (ZnF).</p> <p>Results</p> <p>We fused the <it>piggyBac</it> TPase upstream of and in-frame with the enhanced yellow fluorescent protein (EYFP) in the <it>Drosophila melanogaster</it> inducible metallothionein protein. Using Drosophila Schneider 2 (S2) cells and the deep red fluorescent nuclear stain Draq5, we were able to track the pattern of <it>piggyBac</it> localization with a scanning confocal microscope 48 hours after induction with copper sulphate.</p> <p>Conclusion</p> <p>Through n and c-terminal truncations, targeted internal deletions, and specific amino acid mutations of the <it>piggyBac</it> TPase open reading frame, we found that not only is the PSORTII-predicted NLS required for the TPase to enter the nucleus of S2 cells, but there are additional requirements for negatively charged amino acids a short length upstream of this region for nuclear localization.</p

Genomic Organization of H2Av Containing Nucleosomes in Drosophila Heterochromatin

H2Av is a versatile histone variant that plays both positive and negative roles in transcription, DNA repair, and chromatin structure in Drosophila. H2Av, and its broader homolog H2A.Z, tend to be enriched toward 5′ ends of genes, and exist in both euchromatin and heterochromatin. Its organization around euchromatin genes and other features have been described in many eukaryotic model organisms. However, less is known about H2Av nucleosome organization in heterochromatin. Here we report the properties and organization of individual H2Av nucleosomes around genes and transposable elements located in Drosophila heterochromatic regions. We compare the similarity and differences with that found in euchromatic regions. Our analyses suggest that nucleosomes are intrinsically positioned on inverted repeats of DNA transposable elements such as those related to the “1360” element, but are not intrinsically positioned on retrotransposon-related elements

Mutational analysis of highly conserved aspartate residues essential to the catalytic core of the piggyBac transposase

<p>Abstract</p> <p>Background</p> <p>The <it>piggyBac </it>mobile element is quickly gaining popularity as a tool for the transgenesis of many eukaryotic organisms. By studying the transposase which catalyzes the movement of <it>piggyBac</it>, we may be able to modify this vector system to make it a more effective transgenesis tool. In a previous publication, Sarkar A, Sim C, Hong YS, Hogan JR, Fraser MJ, Robertson HM, and Collins FH have proposed the presence of the widespread 'DDE/DDD' motif for <it>piggyBac </it>at amino acid positions D268, D346, and D447.</p> <p>Results</p> <p>This study utilizes directed mutagenesis and plasmid-based mobility assays to assess the importance of these residues as the catalytic core of the <it>piggyBac </it>transposase. We have functionally analyzed individual point-mutations with respect to charge and physical size in all three proposed residues of the 'DDD' motif as well as another nearby, highly conserved aspartate at D450. All of our mutations had a significant effect on excision frequency in S2 cell cultures. We have also aligned the <it>piggyBac </it>transposase to other close family members, both functional and non-functional, in an attempt to identify the most highly conserved regions and position a number of interesting features.</p> <p>Conclusion</p> <p>We found all the designated DDD aspartates reside in clusters of amino acids that conserved among <it>piggyBac </it>family transposase members. Our results indicate that all four aspartates are necessary, to one degree or another, for excision to occur in a cellular environment, but D450 seems to have a tolerance for a glutamate substitution. All mutants tested significantly decreased excision frequency in cell cultures when compared with the wild-type transposase.</p

Integrative genetic map of repetitive DNA in the sole Solea senegalensis genome shows a Rex transposon located in a proto-sex chromosome

Repetitive sequences play an essential role in the structural and functional evolution of the genome, particularly in the sexual chromosomes. The Senegalese sole (Solea senegalensis) is a valuable flatfish in aquaculture albeit few studies have addressed the mapping and characterization of repetitive DNA families. Here we analyzed the Simple Sequence Repeats (SSRs) and Transposable elements (TEs) content from fifty-seven BAC clones (spanning 7.9 Mb) of this species, located in chromosomes by multiple fluorescence in situ hybridization (m-BAC-FISH) technique. The SSR analysis revealed an average density of 675.1 loci per Mb and a high abundance (59.69%) of dinucleotide coverage was observed, being 'AC' the most abundant. An SSR-FISH analysis using eleven probes was also carried out and seven of the 11 probes yielded positive signals. 'AC' probes were present as large clusters in almost all chromosomes, supporting the bioinformatic analysis. Regarding TEs, DNA transposons (Class II) were the most abundant. In Class I, LINE elements were the most abundant and the hAT family was the most represented in Class II. Rex/Babar subfamily, observed in two BAC clones mapping to chromosome pair 1, showed the longest match. This chromosome pair has been recently reported as a putative sexual proto-chromosome in this species, highlighting the possible role of the Rex element in the evolution of this chromosome. In the Rex1 phylogenetic tree, the Senegalese sole Rex1 retrotransposon could be associated with one of the four major ancient lineages in fish genomes, in which it is included O. latipes

Large Tandem, Higher Order Repeats and Regularly Dispersed Repeat Units Contribute Substantially to Divergence Between Human and Chimpanzee Y Chromosomes

Comparison of human and chimpanzee genomes has received much attention, because of paramount role for understanding evolutionary step distinguishing us from our closest living relative. In order to contribute to insight into Y chromosome evolutionary history, we study and compare tandems, higher order repeats (HORs), and regularly dispersed repeats in human and chimpanzee Y chromosome contigs, using robust Global Repeat Map algorithm. We find a new type of long-range acceleration, human-accelerated HOR regions. In peripheral domains of 35mer human alphoid HORs, we find riddled features with ten additional repeat monomers. In chimpanzee, we identify 30mer alphoid HOR. We construct alphoid HOR schemes showing significant human-chimpanzee difference, revealing rapid evolution after human-chimpanzee separation. We identify and analyze over 20 large repeat units, most of them reported here for the first time as: chimpanzee and human ~1.6 kb 3mer secondary repeat unit (SRU) and ~23.5 kb tertiary repeat unit (~0.55 kb primary repeat unit, PRU); human 10848, 15775, 20309, 60910, and 72140 bp PRUs; human 3mer SRU (~2.4 kb PRU); 715mer and 1123mer SRUs (5mer PRU); chimpanzee 5096, 10762, 10853, 60523 bp PRUs; and chimpanzee 64624 bp SRU (10853 bp PRU). We show that substantial human-chimpanzee differences are concentrated in large repeat structures, at the level of as much as ~70% divergence, sizably exceeding previous numerical estimates for some selected noncoding sequences. Smeared over the whole sequenced assembly (25 Mb) this gives ~14% human--chimpanzee divergence. This is significantly higher estimate of divergence between human and chimpanzee than previous estimates.Comment: 22 pages, 7 figures, 12 tables. Published in Journal of Molecular Evolutio