Automatic classification within families of transposable elements: Application to the mariner Family

The higher levels of the classification of transposable elements (TEs) from Classes to Superfamilies or Families, is regularly updated, but the lower levels (below the Family) have received little investigation. In particular, this applies to the Families that include a large number of copies. In this article we propose an automatic classification of DNA sequences. This procedure is based on an aggregation process using a pairwise matrix of distances, allowing us to define several groups characterized by a sphere with a central sequence and a radius. This method was tested on the mariner Family, because this is probably one of the most extensively studied Families. Several Subfamilies had already been defined from phylogenetic analyses based on multiple alignments of complete or partial amino-acid sequences of the transposase. The classification obtained here from DNA sequences of 935 items matches the phylogenies of the transposase. The rate of error from a posteriori re-assignment is relatively low.     

Population dynamics of miniature inverted-repeat transposable elements (MITEs) in Medicago truncatula

Miniature inverted-repeat transposable elements (MITEs) are small and high copy number transposons, related to and mobilized by some class II autonomous elements. New MITE families can be identified by computer-based mining of sequenced genomes. We describe four MITE families related to MtPH transposons mined de novo in the genome of Medicago truncatula, together with one previously described family MITRAV. Different levels of their intra-family sequence diversity and insertion polymorphism indicate that they were active at different evolutionary periods. MetMIT1 and MITRAV families were uniform in sequence and produced highly polymorphic insertion sites in 26 ecotypes representing a M. truncatula core collection. A subset of insertions was present only in the reference genome of A17 ‘Jemalong’, suggesting that the two families might have been active in the course of domestication. In contrast, all investigated insertions of the MetMIT2 family were fixed, showing that it was not active after M. truncatula speciation. MetMIT1 elements were divided into three clusters, i.e. (I) relatively heterogenous copies fixed in the genome of M. truncatula, (II) uniform but also mostly fixed, and (III) uniform and polymorphic among the investigated accessions. It might reflect the evolutionary history of the MetMIT1 family, showing multiple bursts of activity. A number of MetMIT1 and MITRAV insertions were present within 1 kb upstream or downstream the ORF. A high proportion of insertions proximal to coding regions was unique to A17 ‘Jemalong’.     

FB-NOF is a non-autonomous transposable element,expressed in Drosophila melanogaster and present only in the melanogaster group

Most foldback elements are defective due to the lack of coding sequences but some are associated with coding sequences and may represent the entire element. This is the case of the NOF sequences found in the FB of Drosophila melanogaster, formerly considered as an autonomous TE and currently proposed as part of the so-called FB-NOF element, the transposon that would be complete and fully functional. NOF is always associated with FB and never seen apart from the FB inverted repeats (IR). This is the reason why the FB-NOF composite element can be considered the complete element. At least one of its ORFs encodes a protein that has always been considered its transposase, but no detailed studies have been carried out to verify this.     

Genome-wide analysis of simple sequence repeats in the model medicinal mushroom Ganoderma lucidum

Simple sequence repeats (SSRs) or microsatellites are one of the most popular sources of genetic markers and play a significant role in gene function and genome organization. We identified SSRs in the genome of Ganoderma lucidum and analyzed their frequency and distribution in different genomic regions. We also compared the SSRs in G. lucidum with six other Agaricomycetes genomes: Coprinopsis cinerea, Laccaria bicolor, Phanerochaete chrysosporium, Postia placenta, Schizophyllum commune and Serpula lacrymans. Based on our search criteria, the total number of SSRs found ranged from 1206 to 6104 and covered from 0.04% to 0.15% of the fungal genomes. The SSR abundance was not correlated with the genome size, and mono- to tri-nucleotide repeats outnumbered other SSR categories in all of the species examined. In G. lucidum, a repertoire of 2674 SSRs was detected, with mono-nucleotides being the most abundant. SSRs were found in all genomic regions and were more abundant in non-coding regions than coding regions. The highest SSR relative abundance was found in introns (108 SSRs/Mb), followed by intergenic regions (84 SSRs/Mb). A total of 684 SSRs were found in the protein-coding sequences (CDSs) of 588 gene models, with 81.4% of them being tri- or hexa-nucleotides. After scanning for InterPro domains, 280 of these genes were successfully annotated, and 215 of them could be assigned to Gene Ontology (GO) terms. SSRs were also identified in 28 bioactive compound synthesis-related gene models, including one 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), three polysaccharide biosynthesis genes and 24 cytochrome P450 monooxygenases (CYPs). Primers were designed for the identified SSR loci, providing the basis for the future development of SSR markers of this medicinal fungus.     

Accord insertion in the 5′ flanking region of CYP6G1 confers nicotine resistance in Drosophila melanogaster

What has driven the sweep of the Accord retrotransposon insertion allele of CYP6G1 in the natural populations of Drosophila melanogaster is unknown. Previous studies on the DDT selection hypothesis produced conflicting data. To reexamine the DDT selection hypothesis and search for alternative explanations, we conducted a series of correlation and genetic linkage experiments with eight D. melanogaster natural populations collected from California (CM1, CM2, CM3, and CM7) and Africa (AM2, AM3, AM4, AM7). Diagnostic PCR showed that CM1, CM2, CM7, and AM3 have the Accord insertion in the CYP6G1 locus, whereas the other four strains do not. RT-PCR analysis exhibits a 100% correlation between Accord insertion and CYP6G1 overexpression. However, among the four strains with Accord-mediated CYP6G1 overexpression only CM1 and CM7 are resistant to DDT, and the other two strains (CM2 and AM3), like the four Accord-free strains, are susceptible to DDT. By contrast, all the four strains with Accord-mediated CYP6G1 overexpression are resistant to nicotine, a plant allelochemical. Genetic crosses between DDT resistant and susceptible Accord-insertion strains, as well as crosses between Accord-insertion and Accord-free strains demonstrated that Accord insertion and CYP6G1 overexpression are genetically linked to nicotine resistance rather than DDT resistance. These results suggest that naturally-occurring allelochemicals such as nicotine are the initial driving force for the worldwide prevalence of the Accord insertion allele of CYP6G1 in D. melanogaster natural populations.     

The genomic architecture of the PROS1 gene underlying large tandem duplication mutation that causes thrombophilia from hereditary protein S deficiency

Hereditary protein S deficiency from a mutation in the PROS1 gene causes a genetic predisposition to develop venous thromboembolic disorders in humans. Recently, the acknowledgment of the clinical significance of large copy number mutations in protein S deficiency has increased. In this study, the authors investigated the genomic architecture of PROS1 in order to understand the microscopic sequence environment leading to large intragenic copy number mutations in the gene. The study subjects were 3 unrelated male patients with hereditary protein S deficiency from a tandem duplication mutation involving exons 5–10 of PROS1. Breakpoint analyses revealed 10-bp microhomology sequences in the intervening sequence (IVS)-4 and IVS-10 at the duplication junction without additional sequence changes, suggesting a single replication-based event as the potential molecular mechanism of rearrangement and founder effect in the mutant alleles. Further analyses on nucleotide sequences flanking the microhomology sequence revealed the presence of a repeat element (LTR-ERV1) and quadruplex-forming G-rich sequences in IVS-4. The results from genotyping multi-allelic short tandem repeats supported founder effect in the identical mutations in the 3 unrelated patients. In conclusion, we identified unique genomic architectures in the intervening sequences of PROS1 that underlie a large intragenic tandem duplication mutation leading to inherited thrombophilia.     

Molecular organization of heterochromatin in malaria mosquitoes of the Anopheles maculipennis subgroup

Although heterochromatin makes up a significant portion of the malaria mosquito genome, its organization, function, and evolution are poorly understood. Sibling species of the Anopheles maculipennis subgroup, the European malaria mosquitoes, are characterized by striking differences in the morphology of pericentric heterochromatin; however, the molecular basis for the rapid evolutionary transformation of heterochromatin is not known. This study reports an initial survey of the molecular organization of the pericentric heterochromatin in nonmodel species from the A. maculipennis subgroup. Molecular identity and chromosomal localization were established for short DNA fragments obtained by microdissection from the pericentric diffuse β-heterochromatin of A. atroparvus. Among 102 sequenced clones of the Atr2R library, twenty had sequence similarity to transposable elements (TEs) from the Anopheles gambiae and Aedes aegypti genomes. At least six protein-coding single-copy genes from A. gambiae and four single-copy genes from Drosophila melanogaster were homologous to eight clones from the library. Most of these conserved genes were heterochromatic in A. gambiae but euchromatic in D. melanogaster. The remaining 74 clones were characterized as noncoding repetitive DNA. Comparative chromosome mapping of twelve clones in the sibling species A. atroparvus and A. messeae demonstrated that the noncoding repetitive sequences and the TEs have undergone independent chromosome-specific and species-specific gains and losses in the morphologically different pericentric heterochromatic regions, in accordance with the “library model.”     

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.     

Transposition burst of mariner-like elements in the sequenced genome of Rhodnius prolixus

Transposable elements (TEs) are widespread in insect's genomes. However, there are wide differences in the proportion of the total DNA content occupied by these repetitive sequences in different species. We have analyzed the TEs present in R. prolixus (vector of the Chagas disease) and showed that 3.0% of this genome is occupied by Class II TEs, belonging mainly to the Tc1-mariner superfamily (1.65%) and MITEs (1.84%). Interestingly, most of this genomic content is due to the expansion of two subfamilies belonging to: irritans himar, a well characterized subfamily of mariners, and prolixus1, one of the two novel subfamilies here described. The high amount of sequences in these subfamilies suggests that bursts of transposition occurred during the life cycle of this family. In an attempt to characterize these elements, we performed an in silico analysis of the sequences corresponding to the DDD/E domain of the transposase gene. We performed an evolutionary analysis including network and Bayesian coalescent-based methods in order to infer the dynamics of the amplification, as well as to estimate the time of the bursts identified in these subfamilies. Given our data, we hypothesized that the TE expansions occurred around the time of speciation of R. prolixus around 1.4 mya. This suggestion lays on the “Transposon Model” of TE evolution, in which the members of a TE population that are replicative active are present at multiple loci in the genome, but their replicative potential varies, and of the “Life Cycle Model” that states that when present-day TEs have been involved in amplification bursts, they share an ancestral copy that dates back to this initial amplification.