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PubMed1993

Cloning, expression and sequencing of Helicobacter felis urease genes.

Urease genes from Helicobacter felis were cloned and expressed in Escherichia coli cells. A genomic bank of Sau3A-digested H. felis chromosomal DNA was created using a cosmid vector. Cosmid clones were screened for urease activity following subculture on a nitrogen-limiting medium. Subcloning of DNA from an urease-positive cosmid clone led to the construction of pILL205 (9.5 kb) which conferred a urease activity of 1.2 +/- 0.5 mumole urea min-1 mg-1 bacterial protein to E. coli HB101 bacteria grown on a nitrogen-limiting medium. Random mutagenesis using a MiniTn3-Km transposable element permitted the identification of three DNA regions on pILL205 which were necessary for the expression of an urease-positive phenotype in E. coli clones. To localize the putative structural genes of H. felis on pILL205, extracts of clones harbouring the mutated copies of the plasmid were analysed by Western blotting with anti-H. felis rabbit serum. One mutant clone did not synthesize the putative UreB subunit of H. felis urease and it was postulated that the transposable element had disrupted the corresponding structural gene. By sequencing the DNA region adjacent to the transposon insertion site two open reading frames, designated ureA and ureB, were identified. The polypeptides encoded by these genes had calculated molecular masses of 26,074 and 61,663 Da, respectively, and shared 73.5% and 88.2% identity with the corresponding gene products of Helicobacter pylori urease.

Amino Acid Sequence↗
PubMed2005

The origin and evolution of mosquito APE retroposons.

The detection of horizontal transfer is important to understanding the origin and spread of transposable elements and in assessing their impact on genetic diversity. The occurrence of the phenomenon is not in doubt for two of the three major groups of elements, but is disputed for retroposons, largely on the grounds of data paucity and overreliance on divergence estimates between host species. We present here the most wide-ranging retroposon data set assembled to date for a species group, the mosquitoes. The results provide no evidence for horizontal transfer events and show conclusively that four previously reported events, involving Juan-A, Juan-C, T1, and Q, did not occur. We propose that the origin of all known mosquito retroposons can be attributed to vertical inheritance and that retroposons have therefore been a persistent source of genetic diversity in mosquito genomes since the emergence of the taxon. Furthermore, the data confirm that the unprecedented levels of retroposon diversity previously reported in Anopheles gambiae extends to at least seven other species representing five genera and all three mosquito subfamilies. Most notably, this included the L1 elements, which are not known in other insects. A number of novel well-defined monophyletic groups were also identified, particularly, JM2 and JM3 within the Jockey clade, which included sequences from seven and five mosquito species, respectively. As JM3 does not contain an Anopheles element, this represents a good example of stochastic loss and the best out of at least four found in this study. This exceptionally diverse data set when compared with the wealth of data available for the many unrelated species with which mosquitoes have intimate contact through blood feeding ought to be fertile ground for the discovery of horizontal transfer events. The absence of positive results therefore supports the view that retroposon horizontal transfer does not occur or is far more exceptional than for other types of transposable elements.

Animals↗
PubMed1990

[Mutational variability of enzyme loci in unstable Drosophila melanogaster strains].

Biochemical analysis of a number of related unstable Drosophila melanogaster strains was carried out. These strains have been shown to undergo mutational transformations caused by insertion/excision of transposable elements (Gerasimova, 1985). Activity and mobility variants of alpha-GPDH, ADH, SOD, G6PD, 6PGD and EST-6 were analysed. Two loci controlling SOD and 6PGD proved to be invariable, the loci alpha-Gpdh and Est-6 causing reduced activity of their products in the initial strain ctMR2. The direct and reversed transformations analogous to the mutational passages of morphological characters were traced in two loci controlling ADH and G6PD. The data obtained are discussed in terms of up-to-date views on the functional role of transposable elements, in relation to the genetic stability of the strains studied, under the multiple transpositions of mobile elements.

Animals↗
PubMed2003

A few-polyhedra mutant and wild-type nucleopolyhedrovirus remain as a stable polymorphism during serial coinfection in Trichoplusia ni.

Few-polyhedra (FP) mutants of nucleopolyhedroviruses (NPVs) are a well-known phenomenon during serial passage of virus in cell culture. Under these circumstances such mutants produce low yields of occlusion bodies (OBs) and poorly occlude virions, but they are selected for through advantageous rates of budded virus replication. Spontaneous insertion of transposable elements originating from host cell DNA into the viral fp25 gene has been shown to be a common cause of the phenotype. A model of NPV population genetics predicts that mutants with these characteristics might persist within stable polymorphisms in viral populations during serial passage of virus in vivo. However, this hypothesis was previously untested, and FP mutants have not been recovered from field isolates of NPVs. We isolated and characterized an FP mutant that arose during routine passage of Autographa californica multinucleocapsid NPV (AcMNPV) in cell culture and identified a transposable element within the fp25 gene. We tracked the fates of coinfecting wild-type and FP mutant AcMNPV strains through serial passage in fifth-instar Trichoplusia ni larvae. The levels of both strains remained stable during successive rounds of infection. We applied the data obtained to a model of NPV population genetics in order to derive the frequency distribution of the multiplicity of cell infection in infected insects and estimated that 4.3 baculovirus genomes per OB-producing cell would account for this equilibrium.

Animals↗
PubMed1994

Effects of autosomal inversions on meiotic exchange in distal and proximal regions of the X chromosome in a natural population of Drosophila melanogaster.

We have investigated the interchromosomal effect of the naturally-occurring paracentric inversions In(2L)t and In(3R)P on meiotic recombination in two regions of the X chromosome in Drosophila melanogaster. Previous authors have suggested that the rate of recombination at the tip of the X chromosome may be substantially higher in some natural populations than values measured in the laboratory, due to the interchromosomal effect of heterozygous autosomal inversions. This suggestion was motivated by observations that transposable elements are not as common at the tip of the X chromosome as predicted by recent research relating reduced meiotic exchange to increased element abundance in D. melanogaster. We examined the effects of heterozygous In(2L)t and In(3R)P on recombination at both the tip and base of the X chromosome on a background of isogenic major chromosomes from a natural population. Both inversions substantially increased the rate of recombination at the base; neither one affected recombination at the tip. The results suggest that the presence of inversions in the study population does not elevate rates of crossing over at the tip of the X chromosome. The relevance of these results to ideas relating transposable element abundance to recombination rates is discussed.

Animals↗
PubMed1994

A self-stabilizing Ac derivative and its potential for transposon tagging.

With the goal of developing a self-stabilizing transposon tagging system, a derivative of the maize transposable element Ac (Ds303) harbouring a deletion between bp 246 and 736 has been introduced into tomato (Lycopersicon esculentum) by Agrobacterium tumefaciens-mediated transformation. The deletion removes the major transcription start site, 84 bp of the putative Ac promoter and part of the untranslated leader. Transpositions from the T-DNA, where Ds303 was inserted between the mannopine synthase 1' promoter and the GUS gene, were observed in four independent transgenic plants analysed. After transposition the transposed Ds303 elements were stably integrated in the genome and could not transactivate a tester Ds element. However, somatic and germinal transpositions of the transposed Ds303 elements occurred in the presence of a transposase source.

Base Sequence↗
PubMed1988

Transposable and nontransposable elements similar to the I factor involved in inducer-reactive (IR) hybrid dysgenesis in Drosophila melanogaster coexist in various Drosophila species.

The I factor is a transposable element controlling inducer-reactive (IR) hybrid dysgenesis in Drosophila melanogaster, which occurs when males from the class of inducer strains are crossed with females from the class of reactive strains. Inducer strains contain several copies of the complete 5.4-kilobase (kb) I factor at various sites on the chromosomal arms; reactive strains contain no complete I factor. Incomplete and defective I elements occur at constant locations in pericentromeric heterochromatin of both types of strains. The 5.4-kb I factors transpose, whereas incomplete I elements do not transpose. The constant location of defective I elements in all strains indicates that they were in the genome before the spread of D. melanogaster throughout the world. Sequences homologous to I occur in other Drosophila species, and their distribution correlates with the phylogenetic relationships between species. We have studied the organization of I homologues in Drosophila simulans and Drosophila teissieri. These species seem to contain both transposable I elements, even though their structure may differ from that of the 5.4-kb I factors of the inducer strains of D. melanogaster, and nontransposable I elements, which are always at the same place in the genome when different stocks of the same species are compared. These results suggest that both mobile and nonmobile I elements are very old components of the Drosophilidae genome.

Animals↗
PubMed1989

Restriction map and alpha-amylase activity variation among Drosophila mutation accumulation lines.

The specific activities of alpha-amylase were measured for two sets of mutation accumulation lines, each set having originated from a different lethal-carrying second chromosome and SM1(Cy) chromosome and having been maintained by a balanced lethal system for about 300 generations. Significant variation was found to have accumulated among lines of both sets. Because of dysgenic crosses in the early generations of mutation accumulation, insertions or deletions of transposable elements in the Amy gene region were suspected of being the cause of this variation. In order to test this possibility, the structural changes in the 14 kb region of these chromosomes that includes the structural genes for alpha-amylase were investigated by restriction map analysis. We found that most part of the activity variation is due to replacements of a chromosomal region of SM1(Cy), including the structural genes for alpha-amylase, by the corresponding regions of the lethal chromosomes. One line also contained an insertion in this region but this line has an intermediate activity value. Thus, insertions of transposable elements into the Amy gene region were not found to be responsible for the new variation observed in alpha-amylase activity. If we remove those lines with structural changes from the analysis, the genetic variance of alpha-amylase specific activity among lines becomes non-significant in both sets of chromosomes.

Animals↗
PubMed2005

The Genomes of Oryza sativa: a history of duplications.

We report improved whole-genome shotgun sequences for the genomes of indica and japonica rice, both with multimegabase contiguity, or almost 1,000-fold improvement over the drafts of 2002. Tested against a nonredundant collection of 19,079 full-length cDNAs, 97.7% of the genes are aligned, without fragmentation, to the mapped super-scaffolds of one or the other genome. We introduce a gene identification procedure for plants that does not rely on similarity to known genes to remove erroneous predictions resulting from transposable elements. Using the available EST data to adjust for residual errors in the predictions, the estimated gene count is at least 38,000-40,000. Only 2%-3% of the genes are unique to any one subspecies, comparable to the amount of sequence that might still be missing. Despite this lack of variation in gene content, there is enormous variation in the intergenic regions. At least a quarter of the two sequences could not be aligned, and where they could be aligned, single nucleotide polymorphism (SNP) rates varied from as little as 3.0 SNP/kb in the coding regions to 27.6 SNP/kb in the transposable elements. A more inclusive new approach for analyzing duplication history is introduced here. It reveals an ancient whole-genome duplication, a recent segmental duplication on Chromosomes 11 and 12, and massive ongoing individual gene duplications. We find 18 distinct pairs of duplicated segments that cover 65.7% of the genome; 17 of these pairs date back to a common time before the divergence of the grasses. More important, ongoing individual gene duplications provide a never-ending source of raw material for gene genesis and are major contributors to the differences between members of the grass family.

Base Sequence↗
PubMed2004

Assessing fitness costs for transgenic Aedes aegypti expressing the GFP marker and transposase genes.

The development of transgenic mosquitoes that are refractory to the transmission of human diseases such as malaria, dengue, and yellow fever has received much interest due to the ability to transform a number of vector mosquito species with transposable elements. Transgenic strains of mosquitoes have been generated with molecular techniques that exhibit a reduced capacity to transmit pathogens. These advancements have led to questions regarding the fitness of transgenic mosquitoes and the ability of transformed mosquitoes to compete and effectively spread beneficial genes through nontransformed field populations, the core requirement of a genetically based control strategy aimed at reducing the spread of mosquito-borne human disease. Here we examine the impact of transgenesis on the fitness of Aedes aegypti, a mosquito that transmits yellow fever. Mosquitoes were altered with two types of transgene, the enhanced GFP gene and two transposase genes from the Hermes and MOS1 transposable elements. We examined the effects of these elements on the survivorship, longevity, fecundity, sex ratio, and sterility of transformed mosquitoes and compared results to the nontransformed laboratory strain. We show that demographic parameters are significantly diminished in transgenic mosquitoes relative to the untransformed laboratory strain. Reduced fitness in transgenic mosquitoes has important implications for the development and utilization of this technology for control programs based on manipulative molecular modification.

Aedes↗
PubMed1998

Transposition of the Drosophila element mariner into the chicken germ line.

The ability of the Drosophila transposable element mariner to transpose in the chicken was tested using a plasmid carrying an active mariner element injected into chick zygotes. Surviving embryos and chicks were analyzed for presence of mariner. Analysis of embryos that survived for at least 12 days of development indicated that mariner had transposed at high frequency into the chicken genome. Germline transmission of mariner from one of three surviving birds confirmed transposition. Analysis of the first-generation (G1) chicks showed that they each contained between one and three copies of mariner. Six different transposition events were represented in the G1 birds, and the transposition was catalyzed by expression of the mariner element's transposase gene. Transmission from G1 to G2 occurred at a 1:1 ratio. Mariner therefore has potential for development as a vector for transgenesis in avian species.

Animals↗
PubMed1989

A hot-spot for transposition of various Ty elements on chromosome V in Saccharomyces cerevisiae.

Ty4 is a novel transposable element in the yeast, Saccharomyces cerevisiae, which is present in only a few copies in the genome (Stucka et al. 1989). In strain C836 one of the three copies (Ty4-90) is contained in cosmid clone c90, where it resides on chromosome V. Analysis of this region reveals a "hot-spot" of transposition: in addition to Ty4-90, the locus contains a complete Ty3 element and seven singular delta, sigma and tau elements. Three tRNA genes (for His, Lys, and Ile) are located in this region, and these are closely associated with one or the other of the elements, a phenomenon commonly observed in yeast. A comparison of c90 with corresponding regions from other strains shows that the locus is highly polymorphic and that this polymorphism is explicitly associated with Ty transposition and recombination events.

Base Sequence↗
PubMed2006

oskar gene expression in the vector mosquitoes, Anopheles gambiae and Aedes aegypti.

A disease control strategy based on the introduction into mosquito populations of a gene conferring a pathogen-refractory phenotype is currently under investigation. This population replacement approach requires a drive system that will quickly spread and fix antipathogen effector genes in target populations. Modified transposable elements containing the control sequences of developmentally regulated genes may provide the basis for a gene drive system that regulates gene mobilization in a sex- and stage-restrictive manner. Screening of a Drosophila melanogaster database for genes whose products localize exclusively in the future germ cells during early embryonic development resulted in the identification of several candidate genes. The regulatory sequences of these genes could be used to drive transposition. Mosquito orthologous genes of oskar were identified based on sequence homology and characterized further. The tissue- and sex-specific expression profiles and hybridizations in situ show that oskar orthologous transcripts in Anopheles gambiae and Aedes aegypti accumulate in developing oocytes of adult females and localize to the posterior poles of early embryos. These characteristics potentiate the use of the regulatory sequences of mosquito oskar genes for the control of modified transposable elements.

Aedes↗
PubMed1987

Genetic organization of insertion element IS2 based on a revised nucleotide sequence.

We identified a transposable element resident in the chromosome of Escherichia coli K-12 strain HB101. This is an approx. 4400-bp-long transposon flanked by two copies of insertion sequence (IS) 1 element in direct orientation. One of the IS1 elements was found to be integrated into an IS2 element between IS2 bp 139 and bp 140 with the large moiety of IS2 within the transposon. The sequence of this part of IS2 differs from the published sequence of galOP-308::IS2 at a number of positions. Restriction analysis of the published allele, however, indicated that both alleles may in fact be identical. Since six of the eight differences found alter open reading frames, the revised sequence presents a new outlook for the potential genetic organization of IS2.

Base Composition↗
PubMed1999

Oculocutaneous albinism in the i6 mutant of the medaka fish is associated with a deletion in the tyrosinase gene.

Three mutant alleles (i1, i4, and i5) of the tyrosinase gene in the i locus of the medaka fish Oryzias latipes have hitherto been described, all being associated with transposable element insertion. We have recently identified another allele causing a complete albino phenotype in homozygous carriers and named it i6. Sequence comparison between the tyrosinase gene for the i6 allele (Tyr-i6) and the wild-type gene previously obtained (Tyr-i+) revealed three deletions of 8, 44, and 245 bp. The first two deletions reside in an intron and are differences in the number of tandem tetranucleotide repeats that are polymorphic even among wild-type genes, and, thus, not likely to be responsible for the i6 albino phenotype. The largest deletion spans over the last 180 bp of the second intron and the first 65 bp of the third exon. Because of this deletion, the Tyr-i6 gene lacks the branch point sequence and the acceptor site for the second intron, both being considered to be necessary for normal RNA splicing. Therefore, the 245-bp deletion is likely to be responsible for the albino phenotype. With a mutant gene of this type, unlike ones bearing transposable element insertions, the possibility of reversion mutations to the wild-type would be negligible. Therefore, fish having the i6/i6 genotype should serve as superior recipients for the tyrosinase gene in rescue experiments.

Albinism, Oculocutaneous↗
PubMed2006

Vertebrate DNA transposon as a natural mutator: the medaka fish Tol2 element contributes to genetic variation without recognizable traces.

DNA-based transposable elements, or DNA transposons, transpose in a cut-and-paste fashion, involving excision from the chromosome. If this process affects the function of a host gene and the excision rate is high, any gene associated with such an element would clearly be in a genetically "unstable" state, and there are many examples of unstable genes in various organisms. However, none have hitherto been reported in vertebrates. We here document the finding of an unstable mutant gene in the medaka fish, Oryzias latipes, a useful model animal for vertebrate genetics and evolutionary studies. In an inbred strain, excision of the Tol2 element inserted in a pigmentation gene occurs spontaneously, giving rise to different heritable phenotypes and new mutant genes that carry different excision footprint sequences. The phenotypic mutation rate is as high as 2% per gamete, representing a 1000-fold increase from spontaneous mutation rates so far determined with the same organism. With mutations caused by insertion, and then excision, of transposons, one can no longer recognize participation of transposons in their generation. Thus, the impact of DNA transposons on vertebrate genomes may be, and may have been, larger than commonly supposed.

Alleles↗
PubMed2002

R391: a conjugative integrating mosaic comprised of phage, plasmid, and transposon elements.

The conjugative, chromosomally integrating element R391 is the archetype of the IncJ class of mobile genetic elements. Originally found in a South African Providencia rettgeri strain, R391 carries antibiotic and mercury resistance traits, as well as genes involved in mutagenic DNA repair. While initially described as a plasmid, R391 has subsequently been shown to be integrated into the bacterial chromosome, employing a phage-like integration mechanism closely related to that of the SXT element from Vibrio cholerae O139. Analysis of the complete 89-kb nucleotide sequence of R391 has revealed a mosaic structure consisting of elements originating in bacteriophages and plasmids and of transposable elements. A total of 96 open reading frames were identified; of these, 30 could not be assigned a function. Sequence similarity suggests a relationship of large sections of R391 to sequences from Salmonella, in particular those corresponding to the putative conjugative transfer proteins, which are related to the IncHI1 plasmid R27. A composite transposon carrying the kanamycin resistance gene and a novel insertion element were identified. Challenging the previous assumption that IncJ elements are plasmids, no plasmid replicon was identified on R391, suggesting that they cannot replicate autonomously.

Bacteriophages↗
PubMed2006

Dual inhibitory effects of APOBEC family proteins on retrotransposition of mammalian endogenous retroviruses.

We demonstrated previously that the cytosine deaminase APOBEC3G inhibits retrotransposition of two active murine endogenous retroviruses, namely intracisternal A-particles (IAP) and MusD, in an ex vivo assay where retrotransposition was monitored by selection of neo-marked elements. Sequencing of the transposed copies further disclosed extensive editing, resulting in a high load of G-to-A mutations. Here, we asked whether this G-to-A editing was associated with an impact of APOBEC3G on viral cDNA yields. To this end, we used a specially designed quantitative PCR method to selectively measure the copy number of transposed retroelements, in the absence of G418 selection. We show that human APOBEC3G severely reduces the number of MusD and IAP transposed cDNA copies, with no effect on the level of the intermediate RNA transcripts. The magnitude of the decrease closely parallels that observed when transposed copies are assayed by selection of G418-resistant cells. Moreover, sequencing of transposed elements recovered by PCR without prior selection of the cells reveals high-level editing. Using this direct method with a series of cytosine deaminases, we further demonstrate a similar dual effect of African green monkey APOBE3G, human APOBEC3F and murine APOBEC3 on MusD retrotransposition, with a distinct extent and site specificity for each editing activity. Altogether the data demonstrate that cytosine deaminases have a protective effect against endogenous retroviruses both by reducing viral cDNA levels and by introducing mutations in the transposed copies, thus inactivating them for subsequent rounds of retrotransposition. This dual, two-step effect likely participates in the efficient defense of the cell genome against invading endogenous retroelements.

APOBEC-3G Deaminase↗