Demonstration of IS<Emphasis Type="Italic">711</Emphasis> transposition in <Emphasis Type="Italic">Brucella ovis</Emphasis> and <Emphasis Type="Italic">Brucella pinnipedialis</Emphasis>

Background  

The Brucella genome contains an insertion sequence (IS) element called IS711 or IS6501, which is specific to the genus. The copy number of IS711 varies in the genome of the different Brucella species, ranging from 7 in B. abortus, B. melitensis and B. suis to more than 30 in B. ovis and in Brucella strains isolated from marine mammals. At present, there is no experimental evidence of transposition of IS711, but the occurrence of this element with a high copy number in some species, and the isolation of Brucella strains with "ectopic" copies of IS711 suggested that this IS could still transpose.     

Diversification of the barley and wheat <Emphasis Type="Italic">blt101</Emphasis>/<Emphasis Type="Italic">wpi6</Emphasis> promoters by the <Emphasis Type="Italic">Xumet</Emphasis> element without affecting stress responsiveness

BLT101-family plasma membrane proteins are found in a wide range of organisms from bacteria to nematodes and are involved in the regulation of cellular cation concentration under stress conditions. A comparison of the promoter regions of barley blt101 and its wheat ortholog, wpi6, revealed highly conserved nucleotide sequences between both genes and a unique insertion of a Xumet element in the blt101 promoter. The Xumet insertion occurred between a putative abscisic acid-responsive element (ABRE) and the dehydration-responsive element/c-repeat (DRE/CRT) within the blt101 promoter. However, blt101 and wpi6 were induced similarly in response to ABA, drought and low temperature, suggesting that the insertion does not affect promoter functions. The Xumet insertion in the blt101/wpi6 promoter region was detected in five barley cultivars, but absent in two wheat cultivars tested, suggesting that the insertion is barley-specific. Genomic Southern blot analysis revealed a large number of Xumet sequences interspersed in the barley genome, whereas only one or very few copies are present in the wheat genome. The data suggested that an expansion in copy number of Xumet elements occurred in the barley genome through evolution.     

Dynamics of <Emphasis Type="Italic">Vulmar/VulMITE</Emphasis> group of transposable elements in <Emphasis Type="Italic">Chenopodiaceae</Emphasis> subfamily <Emphasis Type="Italic">Betoideae</Emphasis>

Transposable elements are important factors driving plant genome evolution. Upon their mobilization, novel insertion polymorphisms are being created. We investigated differences in copy number and insertion polymorphism of a group of Mariner-like transposable elements Vulmar and related VulMITE miniature inverted-repeat transposable elements (MITEs) in species representing subfamily Betoideae. Insertion sites of these elements were identified using a modified transposon display protocol, allowing amplification of longer fragments representing regions flanking insertion sites. Subsequently, a subset of TD fragments was converted into insertion site-based polymorphism (ISBP) markers. The investigated group of transposable elements was the most abundant in accessions representing the section Beta, showing intraspecific insertion polymorphisms likely resulting from their recent activity. In contrast, no unique insertions were observed for species of the genus Beta section Corollinae, while a set of section-specific insertions was observed in the genus Patellifolia, however, only two of them were polymorphic between P. procumbens and P. webbiana. We hypothesize that Vulmar and VulMITE elements were inactivated in the section Corollinae, while they remained active in the section Beta and the genus Patellifolia. The ISBP markers generally confirmed the insertion patterns observed with TD markers, including presence of distinct subsets of TE insertions specific to Beta and Patellifolia.     

The Tnt1 family member Retrosol copy number and structure disclose retrotransposon diversification in different <Emphasis Type="Italic">Solanum</Emphasis> species

Eukaryotic genome expansion/retraction caused by LTR-retrotransposon activity is dependent on the expression of full length copies to trigger efficient transposition and recombination-driven events. The Tnt1 family of retrotransposons has served as a model to evaluate the diversity among closely related elements within Solanaceae species and found that members of the family vary mainly in their U3 region of the long terminal repeats (LTRs). Recovery of a full length genomic copy of Retrosol was performed through a PCR-based approach from wild potato, Solanum oplocense. Further characterization focusing on both LTR sequences of the amplified copy allowed estimating an approximate insertion time at 2 million years ago thus supporting the occurrence of transposition cycles after genus divergence. Copy number of Tnt1-like elements in Solanum species were determined through genomic quantitative PCR whereby results sustain that Retrosol in Solanum species is a low copy number retrotransposon (1–4 copies) while Retrolyc1 has an intermediate copy number (38 copies) in S. peruvianum. Comparative analysis of retrotransposon content revealed no correlation between genome size or ploidy level and Retrosol copy number. The tetraploid cultivated potato with a cellular genome size of 1,715 Mbp harbours similar copy number per monoploid genome than other diploid Solanum species (613–884 Mbp). Conversely, S. peruvianum genome (1,125 Mbp) has a higher copy number. These results point towards a lineage specific dynamic flux regarding the history of amplification/activity of Tnt1-like elements in the genome of Solanum species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Genomic amplification of the <Emphasis Type="Italic">Gret1</Emphasis> retroelement in white-fruited accessions of wild <Emphasis Type="Italic">Vitis</Emphasis> and interspecific hybrids

Retrotransposons are retrovirus-related mobile sequences that have the potential to replicate via RNA intermediates and increase the genome size by insertion into new sites. The retroelement, Gret1, has been identified as playing a key role in generating fruit color variation in cultivated grape (Vitis vinifera L.) due to its insertion into the promoter of VvMybA1. Fruit color variation is an important distinguishing feature of cultivated grapes and virtually no fruit color variation is observed in wild grape species. The presence and relative copy number of Gret1 was assessed using quantitative PCR on 22 different Vitis species, only four of which (plus interspecific hybrids) are known to contain white accessions. Gret1 copy number was observed to vary by species as well as by color within species and was significantly higher in white-fruited accessions across all taxa tested. Additionally, genomic regions surrounding Gret1 insertion were sequenced in white V. vinifera, hybrid, V. labrusca, V. aestivalis, and V. riparia accessions.     

Functional conservation of the<Emphasis Type="Italic"> Sex-lethal</Emphasis> sex determining promoter,<Emphasis Type="Italic"> Sxl-Pe</Emphasis>, in<Emphasis Type="Italic"> Drosophila virilis</Emphasis>

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.     

Regularory elements of the <Emphasis Type="Italic">copia</Emphasis> retrotransposon determine different levels of expression in different organs of males and females of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Using a model system in which the expression of the reporter gene lacZ is under the control of five deleted variants of the copia retrotransposon regulatory region, which includes the 5′-long terminal repeat (LTR) and the 5′-untranslated region (5′-UTR), their contribution to the control of retrotransposon activity in different organs of males and females of Drosophila melanogaster was analyzed. The whole regulatory region provides expression of the reporter gene at the embryonic stage, and in larvae and adult flies only in generative organs. The 5′-end of LTR harbors a positive regulator that determines expression of the retrotransposon in organs of all types. The 3′-end of LTR harbors a negative regulator, which is sex- and time-specific: it represses copia expression in generative organs of males at all stages of development, and only at the imaginal stage in somatic tissues, without any effect on the expression of the retrotransposon in females. 5′-UTR contains a negative regulator of copia expression: it decreases the expression in embryos and generative organs and blocks it in somatic tissues. It may be suggested that a complex set of regulatory elements was formed in the course of the evolution of the retrotransposon, which made it possible to maintain a certain level of its expression in different types of cells and tissues and at different stages of development and, thus, to limit the harm caused to the host and provide the possibility for the retrotransposon to exist in the host genome over many generations.     

Transposon display supports transpositional activity of <Emphasis Type="Italic">P</Emphasis> elements in species of the <Emphasis Type="Italic">saltans</Emphasis> group of <Emphasis Type="Italic">Drosophila</Emphasis>

Mobilization of two P element subfamilies (canonical and O-type) from Drosophila sturtevanti and D. saltans was evaluated for copy number and transposition activity using the transposon display (TD) technique. Pairwise distances between strains regarding the insertion polymorphism profile were estimated. Amplification of the P element based on copy number estimates was highly variable among the strains (D. sturtevanti, canonical 20.11, O-type 9.00; D. saltans, canonical 16.4, O-type 12.60 insertions, on average). The larger values obtained by TD compared to our previous data by Southern blotting support the higher sensitivity of TD over Southern analysis for estimating transposable element copy numbers. The higher numbers of the canonical P element and the greater divergence in its distribution within the genome of D. sturtevanti (24.8%) compared to the O-type (16.7%), as well as the greater divergence in the distribution of the canonical P element, between the D. sturtevanti (24.8%) and the D. saltans (18.3%) strains, suggest that the canonical element occupies more sites within the D. sturtevanti genome, most probably due to recent transposition activity. These data corroborate the hypothesis that the O-type is the oldest subfamily of P elements in the saltans group and suggest that the canonical P element is or has been transpositionally active until more recently in D. sturtevanti.     

Genetic variability in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.) and in the <Emphasis Type="Italic">Helianthus</Emphasis> genus as assessed by retrotransposon-based molecular markers

The inter-retrotransposon amplified polymorphism (IRAP) protocol was applied for the first time within the genus Helianthus to assess intraspecific variability based on retrotransposon sequences among 36 wild accessions and 26 cultivars of Helianthus annuus L., and interspecific variability among 39 species of Helianthus. Two groups of LTRs, one belonging to a Copia-like retroelement and the other to a putative retrotransposon of unknown nature (SURE) have been isolated, sequenced and primers were designed to obtain IRAP fingerprints. The number of polymorphic bands in H. annuus wild accessions is as high as in Helianthus species. If we assume that a polymorphic band can be related to a retrotransposon insertion, this result suggests that retrotransposon activity continued after Helianthus speciation. Calculation of similarity indices from binary matrices (Shannon’s and Jaccard’s indices) show that variability is reduced among domesticated H. annuus. On the contrary, similarity indices among Helianthus species were as large as those observed among wild H. annuus accessions, probably related to their scattered geographic distribution. Principal component analysis of IRAP fingerprints allows the distinction between perennial and annual Helianthus species especially when the SURE element is concerned.     

DNA content of mitotically-active condensed chromosomes of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

Two-wavelength Feulgen microspectrophotometry was used to determine the DNA content of mitotically-active ganglionic cells of first-and thirdinstar larvae of Drosophila melanogaster. The measurements revealed that the DNA values differ, on the average, by a factor of approximately two, with the metaphase cells of the first-instar larvae having about four times the haploid amount of the spermatozoon, and the metaphase cells of the third-instar larvae having about eight times the haploid amount. The increase from 4C to 8C in the course of development without any pronounced modification of the heterochromatic—euchromatic ratio is interpreted as evidence of an increase in the number of chromosomal strands. It is suggested, accordingly, that these mitotically-active chromosomes are multistranded or polynemie.     

Identification of a Nitroalkane Oxidase Gene: <Emphasis Type="Italic">naoA</Emphasis> Related to the Growth of <Emphasis Type="Italic">Streptomyces ansochromogenes</Emphasis>

naoA, encoding a nitroalkane oxidase that can catalyze toxic nitroalkanes to their corresponding aldehydes or ketones and hydrogen peroxide, was cloned from Streptomyces ansochromogenes, but its function related to the growth of Streptomyces is unknown. naoA was disrupted by the insertion of a kanamycin-resistance gene; the resulting strain can grow earlier than a wild-type strain under the same conditions. It was shown that naoA disruption accelerated growth of the naoA-disruption mutant, which could restore its phenotype and morphology as a wild-type strain by complementation of a single copy number of naoA inserted into the chromosome. The introduction of an extra copy of naoA into the wild-type strain resulted in delayed growth. The result suggested that naoA is an important gene related to the growth of S. ansochromogenes.