Generating lineage-specific markers to study Drosophila development.

To generate cell- and tissue-specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P-element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P-element transposase gene or by partial promoters from the even-skipped, fushi-tarazu, or engrailed genes. The tissue-specific patterns of beta-galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P-elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development.     

Identification of active transposon dTok, a member of the hAT family, in rice

Recent completion of the sequencing of the rice genome has revealed that it contains >40% repetitive sequences, most of which are related to inactive transposable elements. During the molecular analysis of the floral organ number 1/multiple pistil 2 (fon1/mp2) mutant, we identified an active transposable element dTok0 that was inserted at the kinase domain of FON1, a homolog of CLAVATA1. Insertion of the element into FON1 generated an 8 bp duplication of its target sites, which is one of the major characteristics of the hAT family of transposons. The dTok0 element was actively transposed out of the FON1 gene, leaving 5-8 bp footprints. Reinsertion into a new location was observed at a low frequency. Analysis of the genome sequence showed that the rice cultivar 'Nipponbare' contains 25 copies of dTok elements; similar numbers were present in all the Oryza species examined. Because dTok0 does not encode a transposase, enzyme activity should be provided in trans. We identified a putative autonomous transposon, Tok1 that contains an intact open reading frame of the Ac-like transposase.     

A family of Tc1-like transposons from the genomes of fishes and frogs: evidence for horizontal transmission.

Tc1-like transposons are very widely distributed within the genomes of animal species. They consist of an inverted repeat sequence flanking a transposase gene with homology to the mobile DNA element, Tc1 of the nematode Caenorhabditis elegans. These elements seem particularly to infest the genomes of fish and amphibian species where they can account for 1% of the total genome. However, all vertebrate Tc1-like elements isolated so far are non-functional in that they contain multiple frameshifts within their transposase coding regions. Here I describe a Tc1-like transposon (PPTN) from the genome of a marine flatfish species (Pleuronectes platessa) which bears conserved inverted repeats flanking an apparently intact transposase gene. Closely related, although degenerate, Tc1-like transposons were also isolated from the genomes of Atlantic salmon (SSTN, Salmo salar) and frog (RTTN, Rana temporaria). Consensual nucleic acid sequences were derived by comparing several individual isolates from each species and conceptual amino acid sequences were thence derived for their transposases. Phylogenetic analysis of these sequences with previously isolated Tc1-like transposases shows that the elements from plaice, salmon and frog comprise a new subfamily of Tc1-like transposons. Each member is distinct in that it is not found in the genomes of the other species tested. Plaice genomes contain about 300 copies of PPTN, salmon 1200 copies of SSTN and frog genomes about 500 copies of RTTN. The presence of these closely related elements in the genomes of fish and frog species, representing evolutionary lines, which diverged more than 400 million years ago, is not consistent with a vertical transmission model for their distributions.     

MMTS, a new subfamily of Tc1-like transposons

A novel Tc1-like transposable element has been identified as a new DNA transposon in the mud loach, Misgurnus mizolepis. The M. mizolepis Tc1-like transposon (MMTS) is comprised of inverted terminal repeats and a single gene that codes Tc1-like transposase. The deduced amino acid sequence of the transposase-encoding region of MMTS transposon contains motifs including DDE motif, which was previously recognized in other Tc1-like transposons. However, putative MMTS transposase has only 34-37% identity with well-known Tc1, PPTN, and S elements at the amino acid level. In dot-hybridization analysis used to measure the copy numbers of the MMTS transposon in genomes of the mud loach, it was shown that the MMTS transposon is present at about 3.36 x 104 copies per 2 x 109 bp, and accounts for approximately 0.027% of the mud loach genome. Here, we also describe novel MMTS-like transposons from the genomes of carp-like fishes, flatfish species, and cichlid fishes, which bear conserved inverted repeats flanking an apparently intact transposase gene. Additionally, BLAST searches and phylogenetic analysis indicated that MMTS-like transposons evolved uniquely in fishes, and comprise a new subfamily of Tc1-like transposons, with only modest similarity to Drosophila melanogaster (foldback element FB4, HB2, HB1), Xenopus laevis, Xenopus tropicalis, and Anopheles gambiae (Frisky).     

Homologs of Drosophila P transposons were mobile in zebrafish but have been domesticated in a common ancestor of chicken and human

A substantial fraction of vertebrate and invertebrate genomes is composed of mobile elements and their derivatives. One of the most intensively studied transposon families, the P elements of Drosophila, was thought to exist exclusively in the genomes of dipteran insects. Based on the data provided by the human genome project, in 2001 our group has identified a P element-homologous sequence in the human genome. This P element-homologous human gene, named Phsa, is 19,533 nucleotides long, comprises six exons and five introns, and encodes a protein of still unknown function with a length of 903 amino acid residues. The N-terminal THAP domain of the putative Phsa protein shows similarities to the site-specific DNA-binding domain of the Drosophila P element transposase. In the present study, FISH analysis and the screening of a human lambda genomic library revealed a single copy of Phsa located on the long arm of chromosome 4, upstream of a gene coding for the hypothetical protein DKFZp686L1814. The same gene arrangement was found for the homologous gene Pgga in the genome of chicken, thus, displaying Pgga at orthologous position on the long arm of chromosome 4. The single-copy gene status and the absence of terminal inverted repeats and target-site duplications indicate that Phsa and Pgga constitute domesticated stationary sequences. In contrast, a considerable number of P-homologous sequences with terminal inverted repeats and intact target-site duplications could be identified in zebrafish, strongly indicating that Pdre elements were mobile within the zebrafish genome. Pdre elements are the first P-like transposons identified in a vertebrate species. With respect to Phsa, gene expression studies showed that Phsa is expressed in a broad range of human tissues, suggesting that the putative Phsa protein plays a not yet understood but essential role in a specific metabolic pathway. We demonstrate that P-homologous DNA sequences occur in the genomes of 21 analyzed vertebrates but only as rudiments in the rodents. Finally, the evolutionary history of P element-homologous vertebrate sequences is discussed in the context of the "molecular domestication" hypothesis versus the "source gene hypothesis."     

Two independent duplications forming the Cyp307a genes in Drosophila

The conserved relationship between orthologs of many cytochrome P450 genes involved in ecdysone synthesis is not reflected in the evolution of the Drosophila Cyp307a genes. In Drosophila melanogaster Cyp307a1 (spook) and Cyp307a2 (spookier) both play essential roles in ecdysone synthesis and may possess biochemically redundant functions. Using phylogenetic analyses we show that the Drosophila Cyp307a genes were formed from two independent duplication events depicting a complicated evolutionary scenario. An initial duplication, from a Cyp307a2 ancestral gene produced the Cyp307a1 gene that has been maintained only in the Sophophoran subgenus. A second duplication in the Drosophila subgenus formed an additional paralog, Cyp307a3. Microsynteny is conserved for Cyp307a2 throughout the Drosophila species, but is not conserved between Cyp307a1 and Cyp307a3. These are located in different genomic positions in the Sophophora and Drosophila subgenera, respectively. Cyp307a3 appears to encode a functional gene product and is expressed in a different spatial and temporal manner to Cyp307a1. This suggests some level of functional divergence between the Cyp307a paralogs in different Drosophila species.     

An insertion sequence unique to Frankia strain ArI5

At the genetic level, understanding of symbiotic nitrogen fixation by Frankia is limited to nif functions that are highly conserved among all organisms. The genetics and biochemistry of nodulation are largely unexplored because of a complete lack of genetic tools. In other bacteria, mobile genetic elements such as insertion sequences (IS) and transposons are commonly used to create mutations and insert new genetic material. We have characterized a 4 kbp segment of DNA from Frankia strain ArI5 that has the hallmarks of a mobile genetic element, inverted repeats flanking a gene encoding a transposase. There are at least six copies of this element in strain ArI5 but none in either strain CcI3 or CpI1. The inverted repeats are 17 nt long and separated by 2156 bp. Within that region are two, overlapping ORFs that each encode a transposase. RT-PCR analysis of RNA from Frankia ArI5 cells conclusively demonstrates the expression of one transposase gene and suggests that both may be transcribed. Numerous attempts to clone the intact IS in E. coli were unsuccessful suggesting that the element may be unstable in this context. A clone containing the complete IS was constructed in E. coli then modified by insertion of the kanamycin (KAN) resistance gene from Tn5. A fragment of DNA including the inverted repeats, transposase genes and KAN gene, was transferred to the suicide vector pJBSD1. The construct, pFRISK, was transformed into E. coli to search for transposition events.     

Tracing the origin of the compensasome: evolutionary history of DEAH helicase and MYST acetyltransferase gene families

Dosage compensation in Drosophila is mediated by a complex of proteins and RNAs called the "compensasome." Two of the genes that encode proteins of the complex, maleless (mle) and males-absent-on-the-first (mof), respectively, belong to the DEAH helicase and MYST acetyltransferase gene families. We performed comprehensive phylogenetic and structural analyses to determine the evolutionary histories of these two gene families and thus to better understand the origin of the compensasome. All of the members of the DEAH and MYST families of the completely sequenced Saccharomyces cerevisiae and Caenorhabditis elegans genomes, as well as those so far (June 2000) found in Drosophila melanogaster (for which the euchromatic part of the genome has also been fully sequenced) and Homo sapiens, were analyzed. We describe a total of 39 DEAH helicases in these four species. Almost all of them can be grouped in just three main branches. The first branch includes the yeast PRP2, PRP16, PRP22, and PRP43 splicing factors and their orthologs in animal species. Each PRP gene has a single ortholog in metazoans. The second branch includes just four genes, found in yeast (Ecm16) and Drosophila (kurz) and their orthologs in humans and Caenorhabditis. The third branch includes (1) a single yeast gene (YLR419w); (2) six Drosophila genes, including maleless and spindle-E/homeless; (3) four human genes, among them the ortholog of maleless, which encodes RNA helicase A; and (4) three C. elegans genes, including orthologs of maleless and spindle-E. Thus, this branch has largely expanded in metazoans. We also show that, for the whole DEAH family, only MLE and its metazoan orthologs have acquired new protein domains since the fungi/animals split. We found a total of 17 MYST family proteins in the four analyzed species. We determined putative orthologs of mof in both C. elegans and H. sapiens, and we show that the most likely ortholog in yeast is the Sas2 gene. Moreover, a paralog of mof exists in Drosophila. All of these results, together with those found for a third member of the compensasome, msl-3, suggest that this complex emerged after the fungi/animals split and that it may be present in mammalian species. Both gene duplication and the acquisition of new protein modules may have played important roles in the origin of the compensasome.     

En/Spm-like transposons in Poaceae species: transposase sequence variability and chromosomal distribution

Belonging to Class II of transposable elements, En/Spm transposons are widespread in a variety of distantly related plant species. Here, we report on the sequence conservation of the transposase region from sequence analyses of En/Spm-like transposons from Poaceae species, namely Zingeria biebersteiniana, Zingeria trichopoda, Triticum monococcum, Triticum urartu, Hordeum spontaneum, and Aegilops speltoides. The transposase region of En/Spm-like transposons was cloned, sequenced, and compared with equivalent regions of Oryza and Arabidopsis from the gene bank database. Southern blot analysis indicated that the En/Spm transposon was present in low (Hordeum spontaneum, Triticum monococcum, Triticum urartu) through medium (Zingeria bieberstiana, Zingeria trichopoda) to relatively high (Aegilops speltoides) copy numbers in Poaceae species. A cytogenetic analysis of the chromosomal distribution of En/Spm transposons revealed the concurence of the chromosomal localization of the En/Spm clusters with mobile clusters of rDNA. An analysis of En/Spm-like transposase amino acid sequences was carried out to investigate sequence divergence between 5 genera--Triticum, Aegilops, Zingeria, Oryza and Arabidopsis. A distance matrix was generated; apparently, En/Spm-like transposase sequences shared the highest sequence homology intra-generically and, as expected, these sequences were significantly diverged from those of O. sativa and A. thaliana. A sequence comparison of En/Spm-like transposase coding regions defined that the intra-genomic complex of En/Spm-like transposons could be viewed as relatively independent, vertically transmitted, and permanently active systems inside higher plant genomes. The sequence data from this article was deposited in the EMBL/GenBank Data Libraries under the accession nos. AY707995-AY707996-AY707997-AY707998-AY707999-AY708000-AY708001-AY708002-AY708003-AY708004-AY708005-AY708005-AY265312.