FB elements can promote exon shuffling: a promoter-less white allele can be reactivated by FB mediated transposition in Drosophila melanogaster

Foldback (FB) elements are transposable elements found in many eukaryotic genomes; they are thought to contribute significantly to genome plasticity. In Drosophila melanogaster, FBs have been shown to be involved in the transposition of large chromosomal regions and in the genetic instability of some alleles of the white gene. In this report we show that FB mediated transposition of w ^67C23, a mutation that deletes the promoter of the white gene and its first exon, containing the start codon, can restore expression of the white gene. We have characterized three independent events in which a 14-kb fragment from the w ^67C23 locus was transposed into an intron region in three different genes. In each case a local promoter drives the expression of white, producing a chimeric mRNA. These findings suggest that, on an evolutionary timescale, FB elements may contribute to the creation of new genes via exon shuffling.Electronic Supplementary Material Supplementary material is available in the online version of this article at Communicated by G. P. Georgiev     

Interactions between WHITE Genes Carried by a Large Transposing Element and the ZESTE Allele in DROSOPHILA MELANOGASTER

TE146, a large transposing element of Drosophila melanogaster, carries two copies of the white and roughest genes in tandem. In consequence, z¹ w ^11E4; TE146(Z)/+ flies have a zeste (lemon-yellow) eye color. However, one in 10³ TE146 chromosomes mutates to a red-eyed form. The majority of these "spontaneous red" (SR) derivatives of TE146 have only one copy of the white gene and are, cytologically, two- to three-banded elements, rather than six-banded as their progenitor. The SR forms of TE146 are also unstable and give zeste-colored forms with a frequency of about one in 10⁴. One such "spontaneous zeste" (SZ) derivative carries duplicated white genes as an inverted, rather than a tandem, repeat. The genetic instability of this inverted repeat form of TE146 is different from that of the original tandem repeat form. In particular, the inverted repeat form frequently produces derivatives with internal rearrangements of the TE and gives a much lower frequency of SR forms. In addition, two novel features of the interaction between w⁺ alleles in a zeste background have been found. First, copies of w ⁺ can become insensitive to suppression by zeste even when paired. Second, an inversion breakpoint may disrupt the pairing between two adjacent w⁺ alleles, necessary for their suppression by zeste, without physically separating them.     

Isolation of a hybrid plasmid with homologous sequences to a transposing element of Drosophila melanogaster

In Drosophila several transposing elements that contain the white locus are known. Transpositions of one such element, which carries both the white-apricot (w^a) and the neighboring roughest (rst⁺) genes, have been isolated at more than 120 sites scattered over the entire genome (Ising and Ramel 1976). We have isolated a recombinant plasmid (61F4) containing sequences that appear to be present on this transposing element (TE). In nontransposed stocks, 61F4 hybridizes to approximately 40 sites in the polytene chromosomes including the nucleolus, the chromocenter and chromosome section 3C (that is, the white-apricot roughest region). In six different tranpositions tested, the genetic map position of the TE corresponds to one site of in situ hybridization of 61F4, indicating that the TE contains homologous sequences. The sites of in situ hybridization correlate with the w^a allele or alleles derived from w^a but not with w⁺ and other w alleles tested, nor with an X-ray-induced revertant of w^a. Thus w^a strains appear to carry additional DNA sequences homologous to 61F4, close to or within the w gene. The recombinant plasmid 61F4 carries 7.3 kb of Drosophila DNA inserted into pSF2124. It contains a segment homologous to a member of the copia gene family (Finnegan et al. 1978). Since copia appears to be a highly mobile element (Strobel, Dunsmuir and Rubin 1979), the association of copia sequences with the w^a-rst⁺ transposing element suggests that copia sequences may be responsible for the transposition of this element.     

The genetic analysis of a large transposing element of Drosophila melanogaster

A member of Ising's family of large transposing elements (TEs) has inserted into, or very near, the crinkled (ck, 2–50) locus. This TE (TE36) carries functional alleles of both the white and roughest loci, and causes a hypomorphic mutation of ck. The TE is visible in polytene chromosomes as a two-banded insertion between 35B9 and 35C1. These bands show homology to foldback (FB) elements by in situ hybridization. All spontaneous losses of TE36 remain mutant for ck and retain sequences homologous to FB at the site of TE's insertion. TE36 carries only one functional copy of w ⁺, by the criterion that z w, TE36/ + flies are wild-type for eye color but z w; TE36/TE36 flies are zeste. This white⁺ gene is dosage compensated since w/Y; TE36/+ males have twice as much eye pigment as w/w; TE36/ + females. A form of the TE that has four polytene chromosome bands and expresses twice as much pigment as TE36 has been recovered. However, its white genes are not suppressed by zeste.     

Cloning of DNA sequences from the white locus of D. melanogaster by a novel and general method

We describe the isolation of a cloned DNA segment carrying unique sequences from the white locus of Drosophila melanogaster. Sequences within the cloned segment are shown to hybridize in situ to the white locus region on the polytene chromosomes of both wild-type strains and strains carrying chromosomal rearrangements whose breakpoints bracket the white locus. We further show that two small deficiency mutations, deleting white locus genetic elements but not those of complementation groups contiguous to white, delete the genomic sequences corresponding to a portion of the cloned segment. The strategy we have employed to isolate this cloned segment exploits the existence of an allele at the white locus containing a copy of a previously cloned transposable, reiterated DNA sequence element. We describe a simple, rapid method for retrieving cloned segments carrying a copy of the transposable element together with contiguous sequences corresponding to this allele. The strategy described is potentially general and we discuss its application to the cloning of the DNA sequences of other genes in Drosophila, including those identified only by genetic analysis and for which no RNA product is known.     

Molecular cloning of the white locus region of Drosophila melanogaster using a large transposable element

We report the molecular cloning of a chromosome segment including the white locus of Drosophila melanogaster. This region was isolated using a deficiency extending from the previously cloned heat-shock puff sequences at 87A7 to a large transposable element containing the loci white and roughest.FB-NOF, a 7.5 kb element with partial homology to a family of inverted repeat sequences (Potter et al., 1980), is found very near the deficiency breakpoint, and is followed by DNA originating from the white locus region. Sequences totalling ˜60 kb surrounding this initial entry point were obtained by the cloning of successively overlapping fragments from a wild-type strain. Several rearrangement breakpoints have been mapped relative to the cloned DNA; these define the limits of the white locus and further differentiate the “white proximal region”, thought to function in gene regulation, from the remainder of the locus. Insertion of the dispersed repetitive element copia into the white locus is observed in strains carrying the white-apricot allele. Analysis of several white-apricot revertants suggests that copia insertion is responsible for the apricot eye color phenotype.     

Cloning and characterization of Tc1 family-derived PPTN related transposons from ridged-eye flounder (Pleuronichthys cornutus) and inshore hagfish (Eptatretus burgeri)

The Tc1 transposable element is the most widespread family among animal transposon and these elements consist of an inverted repeat (IR) sequence flanking a transposase gene that belongs to Class II type transposon, which is highly conserved in the genome of the nematode C. elegans. In order to characterize Tc1-like transposable elements from several fishes, PPTN (Tc1-like transposon was isolated from Pleuronectes platessa, marine flatfish species) IR primer-specific amplified elements were cloned from the genomic DNA of several fishes. Transposable elements were found in ridged-eye flounder (Pleuronichthys cornutus) and inshore hagfish (Eptatretus burgeri) and named as PCTN and EBTN, respectively. Amino acid sequence alignment and phylogenetic analysis confirmed that the PPTN-like transposons belonged to the Tc1 superfamily of transposons, but they comprised a unique clade of Tc1-like transposons. The IR-PCR analysis using MMTS-IR and PPTN-IR specific primers from Paralichthys olivaceus (Paralichthyidae), Paraplagusia japonica (Cynoglossidae), P. yokohamae (Pleuronectidae) and Pagurus cornutus (Pleuronectidae) (within the same order, Pleuronectiformes but different families) exhibited mutually exclusive distribution of Tc1 family-derived PPTN and MMTS-like transposons in these fish genomes. These results indicate that Tc1 family-derived PPTN and MMTS related Tc1-like transposable elements have uniquely evolved in piscine genome, and can be used as phylogenetic markers for the distribution of subfamilies of Tc1-like transposon and the involvement of horizontal and vertical transmission in the evolution of fish genome.     

Regulation of purine biosynthetic genes expression inSalmonella typhimurium IV Oc mutation site ofpurG and its function analysis

Salmonella typhimurium 5 phosphoribosylformylglycinamide (FGAR) amidotransferase encoded bypurG gene catalyzes the conversion of FGAR to formylglycinamide ribonucleotide (FGAM) in the presence of glu- tamine and ATP for thede novo purine nucleotide biosynthesis.purG gene is negatively regulated by a repressor-operator system. The O⁺ purG and O^c purG were cloned respectivelyin vivo. Restriction enzymes analysis of preliminary clones pLBG-1 (O⁺) and pLBG-2 (O^c) were carried out. The hybrid plasmids pLB1933 (O⁺) and pLB1927 (O^c) containing 5′ control region ofpurG were constructed and the DNA sequences were determined respectively, DNA sequences data showed that O^c mutation ofpurG occurred at the 3rd position of 16 bp PUR box in the 5′ control region (G→A). Gel retardation experiment indicated that the repressor bound well with O⁺ PUR box, but not with O^c PUR box. The result strongly supported the idea that PUR box is the binding region of repressor protein and the 3rd position base G of PUR box is essential for the binding function with repressor protein.     

A preliminary genetic analysis of TE146, a very large transposing element of Drosophila melanogaster

TE146 is a transposing element (TE) consisting of six polytene chromosome bands that has inserted into the no-ocelli (noc 250) locus. This member of Ising's TE family carries two copies of the white and roughest loci. TE146 is lost from noc with a spontaneous frequency of approximately 1 in 22000 chromosomes. All spontaneous losses are accompanied by the reversion of the noc mutation associated with the TE. The TE is associated with fold-back (FB) sequences. The losses of TE146 retain fold-back homology at noc. Of 26 -ray-induced losses of TE146, 16 are gross deletions, removing loci neighboring noc and ten are not. The non-deleted -ray-induced losses are either noc ^– and rst ⁺ or noc ⁺ and rst ^–. The white⁺ genes of TE146 are dosage compensated since w/Y; TE146/+ and w/w; TE146/+ flies are sexually dimorphic for eye color. These w ⁺ genes are also suppressed by zeste since z w; TE146/+ flies have zeste-colored eyes.     

Molecular lesions associated with white gene mutations induced by I-R hybrid dysgenesis in Drosophila melanogaster

We have identified molecular lesions associated with six mutations, w^IR2 and w^IR4-8, of the white gene of Drosophila melanogaster. These mutations arose in flies subject to I-R hybrid dysgenesis. Four of the mutations give rise to coloured eyes and are associated with insertions of 5.4-kb elements indistinguishable from the I factor controlling I-R dysgenesis. The insertion associated with w^IR4 is at a site which, within the resolution of these experiments, is identical to that of two previously studied I factors. This appears to be a hot-spot for I factor insertion. We have compared the sites of these insertions with sequences complementary to white gene mRNA identified by Pirrotta and Bröckl. The hot-spot is in the fourth intron. The insertion carried by w^IR5 is either within, or just beyond, the last exon. The insertion carried by w^IR6 is near the junction of the first exon and first intron. The w^IR2 mutation is a derivative of w¹. It contains an insertion of I factor DNA within, or immediately adjacent to, the F-like element associated with w¹, and results in restoration of some eye colour. This insertion is just upstream of the start of the white mRNA. Mutations w^IR7 and w^IR8 are deletions removing mRNA coding sequences. Both determine a bleached white phenotype.     

Genome-Wide Characterization of Nonreference Transposons Reveals Evolutionary Propensities of Transposons in Soybean

Preferential accumulation of transposable elements (TEs), particularly long terminal repeat retrotransposons (LTR-RTs), in recombination-suppressed pericentromeric regions seems to be a general pattern of TE distribution in flowering plants. However, whether such a pattern was formed primarily by preferential TE insertions into pericentromeric regions or by selection against TE insertions into euchromatin remains obscure. We recently investigated TE insertions in 31 resequenced wild and cultivated soybean (Glycine max) genomes and detected 34,154 unique nonreference TE insertions mappable to the reference genome. Our data revealed consistent distribution patterns of the nonreference LTR-RT insertions and those present in the reference genome, whereas the distribution patterns of the nonreference DNA TE insertions and the accumulated ones were significantly different. The densities of the nonreference LTR-RT insertions were found to negatively correlate with the rates of local genetic recombination, but no significant correlation between the densities of nonreference DNA TE insertions and the rates of local genetic recombination was detected. These observations suggest that distinct insertional preferences were primary factors that resulted in different levels of effectiveness of purifying selection, perhaps as an effect of local genomic features, such as recombination rates and gene densities that reshaped the distribution patterns of LTR-RTs and DNA TEs in soybean.     

EXO70A1-Mediated Vesicle Trafficking Is Critical for Tracheary Element Development in Arabidopsis

Exocysts are highly conserved octameric complexes that play an essential role in the tethering of Golgi-derived vesicles to target membranes in eukaryotic organisms. Genes encoding the EXO70 subunit are highly duplicated in plants. Based on expression analyses, we proposed previously that individual EXO70 members may provide the exocyst with functional specificity to regulate cell type– or cargo-specific exocytosis, although direct evidence is not available. Here, we show that, as a gene expressed primarily during tracheary element (TE) development, EXO70A1 regulates vesicle trafficking in TE differentiation in Arabidopsis thaliana. Mutations of EXO70A1 led to aberrant xylem development, producing dwarfed and nearly sterile plants with very low fertility, reduced cell expansion, and decreased water potential and hydraulic transport. Grafting of a mutant shoot onto wild-type rootstock rescued most of these aboveground phenotypes, while grafting of a wild-type shoot to the mutant rootstock did not rescue the short root hair phenotype, consistent with the role of TEs in hydraulic transport from roots to shoots. Histological analyses revealed an altered pattern of secondary cell wall thickening and accumulation of large membrane-bound compartments specifically in developing TEs of the mutant. We thus propose that EXO70A1 functions in vesicle trafficking in TEs to regulate patterned secondary cell wall thickening.     

Proteomic Analysis of Microtubule Interacting Proteins over the Course of Xylem Tracheary Element Formation in Arabidopsis

Plant vascular cells, or tracheary elements (TEs), rely on circumferential secondary cell wall thickenings to maintain sap flow. The patterns in which TE thickenings are organized vary according to the underlying microtubule bundles that guide wall deposition. To identify microtubule interacting proteins present at defined stages of TE differentiation, we exploited the synchronous differentiation of TEs in Arabidopsis thaliana suspension cultures. Quantitative proteomic analysis of microtubule pull-downs, using ratiometric ¹⁴N/¹⁵N labeling, revealed 605 proteins exhibiting differential accumulation during TE differentiation. Microtubule interacting proteins associated with membrane trafficking, protein synthesis, DNA/RNA binding, and signal transduction peaked during secondary cell wall formation, while proteins associated with stress peaked when approaching TE cell death. In particular, CELLULOSE SYNTHASE-INTERACTING PROTEIN1, already associated with primary wall synthesis, was enriched during secondary cell wall formation. RNAi knockdown of genes encoding several of the identified proteins showed that secondary wall formation depends on the coordinated presence of microtubule interacting proteins with nonoverlapping functions: cell wall thickness, cell wall homogeneity, and the pattern and cortical location of the wall are dependent on different proteins. Altogether, proteins linking microtubules to a range of metabolic compartments vary specifically during TE differentiation and regulate different aspects of wall patterning.     

A transposon as a cytogenetic marker in Drosophila melanogaster

Summary We have analyzed the behavior of a transposing element (TE) in Drosophila melanogaster. The TE carries the structural genes white (w ^a or w ^aR=white apricot reversed) and roughest (rst ⁺), which corresponds to the bands 3C2-6 and a genetic distance of approximately 0.7 map units. Due to the large size, TE can often be visualized in the polytene chromosomes as extra bands at the site of the transposon. We have identified over 100 different transpositions, most of which are situated in the large autosomes; genetic and cytological information is presented for 41 of these positions. Excision of TE may occur once in 1,000 chromosomes, while insertion in a new position is more rare, about once in 10,000 animals or less. The structure of TE itself is variable: regions within it may be lost, genes located adjacent to the site of insertion may transpose with the TE (hitch-hiking genes) or the TE may be duplicated.Possible mechanisms for transposition of the TE and its relation to dispersed gene families are discussed. Paro et al. (1983) have studied the end segments of the TE and isolated so-called FB elements (FB-NOF), which are responsible for its ability to transpose.A careful analysis of the many insertion points for TE will result in a more accurate correlation between the genetical and cytological maps for the two large autosomes of Drosophila melanogaster.