Birth of a retroposon: the Twin SINE family from the vector mosquito Culex pipiens may have originated from a dimeric tRNA precursor.

SINEs are short interspersed repetitive elements found in many eukaryotic genomes and are believed to propagate by retroposition. Almost all SINEs reported to date have a composite structure made of a 5' tRNA-related region followed by a tRNA-unrelated region. Here, we describe a new type of tRNA-derived SINEs from the genome of the mosquito Culex pipiens. These elements, called TWINs, are approximately 220 bp long and reiterated at approximately 500 copies per haploid genome. TWINs have a unique structure compared with other tRNA-SINEs described so far. They consist of two tRNA(Arg)-related regions separated by a 39-bp spacer. Other tRNA-unrelated sequences include a 5-bp leader preceding the left tRNA-like unit and a short trailer located downstream of the right tRNA-like region. This 3' trailer is a 10-bp sequence that is ended by a TTTT motif and followed by a polyA tract of variable length. The right tRNA-like unit also contains a 16-bp sequence which is absent in the left one and appears to be located in the ancestral anticodon stem precisely at a position expected for a nuclear tRNA intron. According to this singular structure, we hypothesize that the TWIN: SINE family originated from an unprocessed polymerase III transcript containing two tRNA sequences. We suggest that some peculiar properties acquired by this dicistronic transcript, such as a polyA tail and a 3' stem-loop secondary structure, promote its retroposition by increasing its chances of being recognized by a reverse transcriptase encoded elsewhere in the C. pipiens genome.

Recent amplification of miniature inverted-repeat transposable elements in the vector mosquito Culex pipiens: characterization of the Mimo family.

We describe a new family of repetitive elements, named Mimo, from the mosquito Culex pipiens. Structural characteristics of these elements fit well with those of miniature inverted-repeat transposable elements (MITEs), which are ubiquitous and highly abundant in plant genomes. The occurrence of Mimo in C. pipiens provides new evidence that MITEs are not restricted to plant genomes, but may be widespread in arthropods as well. The copy number of Mimo elements in C. pipiens ( approximately 1000 copies in a 540Mb genome) supports the hypothesis that there is a positive correlation between genome size and the magnitude of MITE proliferation. In contrast to most MITE families described so far, members of the Mimo family share a high sequence conservation, which may reflect a recent amplification history in this species. In addition, we found that Mimo elements are a frequent nest for other MITE-like elements, suggesting that multiple and successive MITE transposition events have occurred very recently in the C. pipiens genome. Despite evidence for recent mobility of these MITEs, no element has been found to encode a protein; therefore, we do not know how they have transposed and have spread in the genome. However, some sequence similarities in terminal inverted-repeats suggest a possible filiation of some of these mosquito MITEs with pogo-like DNA transposons.

Evidence that a family of miniature inverted-repeat transposable elements (MITEs) from the Arabidopsis thaliana genome has arisen from a pogo-like DNA transposon.

Sequence similarities exist between terminal inverted repeats (TIRs) of some miniature inverted-repeat transposable element (MITE) families isolated from a wide range of organisms, including plants, insects, and humans, and TIRs of DNA transposons from the pogo family. We present here evidence that one of these MITE families, previously described for Arabidopsis thaliana, is derived from a larger element encoding a putative transposase. We have named this novel class II transposon Lemi1. We show that its putative product is related to transposases of the Tc1/mariner superfamily, being closer to the pogo family. A similar truncated element was found in a tomato DNA sequence, indicating an ancient origin and/or horizontal transfer for this family of elements. These results are reminiscent of those recently reported for the human genome, where other members of the pogo family, named Tiggers, are believed to be responsible for the generation of abundant MITE-like elements in an early primate ancestor. These results further suggest that some MITE families, which are highly reiterated in plant, insect, and human genomes, could have arisen from a similar mechanism, implicating pogo-like elements.

CM-gag, a transposable-like element reiterated in the genome of Culex pipiens mosquitoes, contains only a gag gene.

CM-gag elements constitute an homogeneous family of sequences that are reiterated in the genome of Culex pipiens strains from different continents. Apparently complete 1.75 kb CM-gag copies are flanked by target-site duplications and have a polyadenylation signal near their 3' end. They potentially contain a unique gene encoding a putative protein that displays homologies with nucleic acid binding proteins and the gag polypeptide of retroviruses and retrotransposons, but that does not encode a reverse transcriptase. CM-gag elements are similar in their genetic organization to the telomeric transposable sequences Het-A from Drosophila melanogaster, but Southern-hybridization patterns indicate that the former are more probably dispersed in various areas of the mosquito genome. The homogeneity of CM-gag copies that are distributed worldwide suggests that they have most probably been amplified recently. Furthermore, selective constraints against amino acid changes have been acting on these sequences, suggesting that they need to encode the gag-like protein to be incorporated into the chromosomes.

Characterization of the unlinked 16S rDNA and 23S-5S rRNA operon of Wolbachia pipientis, a prokaryotic parasite of insect gonads.

The rRNA-encoding genes (rDNAs) have been cloned and characterized from Wolbachia pipientis (Wp), the gonadial bacteria-like parasite of the mosquito Culex pipiens (Cp) and the moth Ephestia cautella (Ec). In Wp from both insect species the rDNAs are organized in a way which appears to be very unusual. The rRNAs are encoded by two unlinked transcription units, each present in a single copy per genome. One contains the 16S rDNA only, while the other is an operon encoding both the 23S and 5S rDNAs. Each transcription unit contains two putative upstream promoters, and downstream a Rho-independent terminator. The 16S rDNA, as well as the 23S-5S rRNA operon are not linked to any tRNA-encoding sequence and lack the antitermination boxes which are usually present immediately downstream from eubacterial promoters of rDNAs. Wp infecting Ec and Cp are highly similar taking as criteria the rDNAs and their flanking sequences. However, it clearly appears that each insect species harbours a different and specific Wp strain, or even subspecies. Phylogenetic relationships deduced from the complete sequences of their rDNAs undoubtedly confirm that Wp from Cp and Ec belong to the alpha-group of Proteobacteria, and are closely related to the Rickettsia.

Mosquito transposable elements.

Most of the transposons so far characterized from mosquito genomes are retroelements which seem to be distributed worldwide. The Juan transposons constitute a family of non-LTR retroelements, or LINE-retroposons, which are dispersed in the genomes of several mosquito species. Three different Juan subfamilies have been characterized, each being amplified in the genomes of many strains, if not all, of a given mosquito species. These subfamilies have been designated respectively Juan-C in Culex pipiens, Juan-Ct in Culex tarsalis and Juan-A in Aedes aegypti. A large number of the Juan retroposons which are amplified in the mosquito genomes are apparently full-length copies and potentially encode the enzymes necessary for their transposition, a nucleic acid binding protein and a reverse transcriptase. However, these complete Juan copies seem to be most frequently transcriptionally silent in insects reared under laboratory conditions. A few of them are transcribed in C. pipiens cells grown in vitro, but from an external promoter, the Juan-C specific RNA being fused to an upstream RNA sequence. Therefore, the transcription of Juan retroposons seems to depend on external promoters which are most frequently inactive. The occurrence and distribution of Juan retroposon subfamilies among mosquito species do not reflect the phylogeny of these species. Furthermore, complete Juan-C and Juan-A copies which are reiterated in strains collected from regions covering different continents are nearly identical. Juan-C copies belonging to geographically different C. pipiens strains display low levels of divergence between their nucleotide sequences and many of the mutations which have occurred among these copies do not alter their coding potential. These results indicate that the Juan retroposons occur as homogeneous subfamilies distributed worldwide and that selective constraints against amino acid change have been acting recently on these elements, despite the fact that they are now highly repeated through mosquito genomes. Therefore, Juan transposons have most probably been recently amplified in mosquito genomes. Each subfamily may have been amplified from one master element present in a unique population which has since spread worldwide. Alternatively, this amplification may have arisen in many mosquito populations, but from highly conserved master elements submitted to selection pressures. Horizontal transfers between species may also have contributed to the spread of these transposons.

Characterization and genetic organization of full-length copies of a LINE retroposon family dispersed in the genome of Culex pipiens mosquitoes.

Many full-length copies of a long interspersed repetitive element family, designated Juan-C, are reiterated in the genome of Culex pipiens mosquitoes. The complete Juan-C elements have a length of 4.48 kb. They are terminated at one end with an adenosine-rich sequence preceded with an AATAAA polyadenylation signal, lack terminal repeats and cause duplication of the host DNA at the site of their integration. Full-length Juan-C copies display two long open reading frames potentially encoding two proteins. The first one includes a domain typical of nucleic-acid-binding proteins, while the second resembles reverse transcriptases. Therefore, Juan-C elements are similar to LINE retroposons in their overall genetic organization and can probably be transposed by reverse transcription of an RNA intermediate. Juan-C elements are most similar in their sequence and coding potential to the Juan-A elements which are reiterated in mosquito species belonging to the genus Aedes. They also display homologies with some Drosophila LINEs such as Jockey, suggesting that all these elements have arisen from a common precursor. Nearly identical full-length Juan-C copies are amplified in C. pipiens strains from different continents. This finding that Juan-C retroposons reiterated in different strains form an homogeneous family is interpreted to indicate that these elements have spread recently in the C. pipiens species.

Characterization of a LINE retroposon dispersed in the genome of three non-sibling Aedes mosquito species.

A family of long interspersed repetitive elements (LINEs) dispersed in the genome of Aedes mosquitoes is described. Basically, full-length copies of the element designated Juan-A are dispersed in the genome of A. aegypti, but some elements are truncated or deleted. Complete Juan-A elements are 4.7 kb long, and their overall genetic organization is similar to that of LINEs from other species in which this class of nonviral retrotransposons has been described. Juan-A elements are terminated at the 3' end by an adenosine(A)-rich sequence and are flanked by target-site duplications. They display two long open reading frames potentially encoding two polypeptides. The first one contains Cys-rich motifs typical of nucleic-acid-binding proteins, while the other shows homology to the reverse transcriptases. These features are characteristic of LINE retroposons and indicate that Juan-A elements can be transposed by reverse transcription of an RNA intermediate. Furthermore, Juan-A retroposons display significant homologies with the Drosophila LINEs Jockey and F, suggesting that all these elements have arisen from a common precursor. The full-length Juan-A copies which are amplified in the genomes of various strains belonging to the three non-sibling species, A. aegypti, A. albopictus and A. polynesiensis, form an internally homogeneous family. These data are interpreted to indicate that active Juan-A retroposons underwent a recent amplification in the strains analyzed. Furthermore, they suggest that these elements have spread by horizontal transfer between the three non-sibling species.

Sequence of a truncated LINE-like retroposon dispersed in the genome of Culex mosquitoes.

The Juan elements belong to a family of reiterated sequences scattered through the genome of Culex mosquitoes. The nucleotide sequence of three Juan copies has been determined. The results show that the Juan elements are truncated retroposon-like elements belonging to LINEs.

Characterization of amplification core and esterase B1 gene responsible for insecticide resistance in Culex.

Organophosphorus insecticide (OP) resistance in several Culex species is associated with increased esterase activity resulting from amplification of the corresponding structural gene. In Culex pipiens quinquefasciatus, high levels of OP resistance (approximately 800 times) are due to the esterase B1 gene, which is amplified at least 250-fold. This gene has now been sequenced, and the structure of the amplification unit (amplicon) encompassing the structural gene has been partially characterized. The inferred amino acid sequence of the enzyme revealed regions of strong homology with other eukaryotic serine-esterases, such as cholinesterases, which are the target of OPs. The amplicon covers at least 30 kilobases and contains a constant and highly conserved "core" of 25 kilobases. This core carries a single copy of the esterase gene (2.8 kilobases) as well as other sequences that are present as single or low number copies in the genomes of mosquitoes lacking overproduction of the esterase B1 protein. In the amplicon, the esterase gene is framed by two DNA sequences that are repeated in other parts of the genome of resistant mosquitoes and found in the genome of susceptible mosquitoes but not near the esterase B1 gene. It is suggested that these repetitive sequences may have a role in the amplification process.

Detection of organophosphate detoxifying esterases by dot-blot immunoassay in Culex mosquitoes.

A dot-blot immunoassay, using antiserum raised against esterase B1 responsible for organophosphate (OP) resistance in Culex quinquefasciatus, was used to study different laboratory strains and field collections of this species, as well as of Cx. pipiens, Cx. tarsalis, Anopheles albimanus and An. stephensi. The frequency of esterase B1 positive individuals revealed by this test was in agreement with the data obtained by other methods, indicating the potential of this method for the detection and monitoring of resistance due to increased detoxification by esterases.

Overproduction of detoxifying esterases in organophosphate-resistant Culex mosquitoes and their presence in other insects.

Antisera raised against the denatured polypeptide of two organophosphate-detoxifying esterases (B1 and A1) of Culex mosquitoes were used in an immunoblot method to quantify esterase production in resistant versus susceptible strains and to detect the presence of immunologically related proteins in other insects. It was demonstrated that esterase B1 of Culex quinquefasciatus and esterase A1 of Culex pipiens are overproduced in resistant strains by factors of at least 500-fold and 70-fold, respectively, as compared with the corresponding susceptible strains. These factors approximate the levels of resistance to the organophosphate chlorpyrifos determined by bioassay--i.e., about 800-fold and 100-fold, respectively. Antiesterase B1 antiserum was found to react with other type B esterases (B2 of C. quinquefasciatus and B3 of Culex tarsalis) but not with type A esterases (A1 of C. pipiens, A2 of C. quinquefasciatus, or A3 of C. tarsalis); similarly, antiesterase A1 antiserum was found to react with other type A esterases (A2 and A3) but not with type B esterases (B1, B2, and B3). Proteins immunologically related to esterase B1 were detected in Aedes aegypti L., Myzus persicae Sultzer, and Musca domestica L., although they were not overproduced in the organophosphate-resistant strains of these species. In none of these species were proteins immunologically related to esterase A1 found.

Amplification of an esterase gene is responsible for insecticide resistance in a California Culex mosquito.

An esterase gene from the mosquito Culex quinquefasciatus that is responsible for resistance to a variety of organophosphorus (OP) insecticides was cloned in lambda gt11 phage. This gene was used to investigate the genetic mechanism of the high production of the esterase B1 it encodes in OP-resistant Culex quinquefasciatus Say (Tem-R strain) from California. Adults of the Tem-R strain were found to possess at least 250 times more copies of the gene than adults of a susceptible strain (S-Lab). The finding that selection by pesticides may result in the amplification of genes encoding detoxifying enzymes in whole, normally developed, reproducing insects emphasizes the biological importance of this mechanism and opens new areas of investigation in pesticide resistance management.

Characterization of the virus encoded subunit of turnip yellow mosaic virus RNA replicase.

An antiserum raised against TYMV-RNA encoded protein P115 partially inhibits TYMV RNA replicase activity, demonstrating that this protein is involved in TYMV RNA synthesis. The detection of protein P115 by an antibody linked polymerase assay demonstrates that protein P115 is indeed a subunit of the TYMV RNA replicase, the enzyme known to synthesize viral RNA in infected Chinese cabbage. The use of translation products of other tymoviruses indicates that the serological relationship between the virus-encoded replicase subunits of these viruses and protein P115 is very weak at the best.

Probable insensitivity of mollicutes to rifampin and characterization of spiroplasmal DNA-dependent RNA polymerase.

The effect of rifampin on five mollicutes (Spiroplasma citri, Spiroplasma melliferum, Spiroplasma apis, Acholeplasma laidlawii, and Mycoplasma mycoides) was compared with that on Escherichia coli. We found that, in contrast to wild-type E. coli, mollicutes were insensitive to rifampin. DNA-dependent RNA polymerases from S. melliferum and S. apis were purified to the stage where the enzymes were dependent on the addition of exogenous templates for activity. The enzymes were then tested for their sensitivity to rifampin. Spiroplasmal enzymes were at least 1,000 times less sensitive to rifampin than the corresponding E. coli enzyme. This result provides a molecular basis for the resistance of mollicutes to rifampin. The RNA polymerase of S. melliferum was further purified and its subunit composition was investigated. The RNA polymerase has one small and two large subunits. The structure of S. melliferum RNA polymerase therefore resembles that of the eubacterial enzymes in spite of its insensitivity to rifampin.

Gene for spiralin, the major membrane protein of the helical mollicute Spiroplasma citri: cloning and expression in Escherichia coli.

A library of cloned Spiroplasma citri genomic sequences was constructed by incorporating HindIII digestion fragments into the plasmid vector pBR328. Immunological screening allowed the identification of a recombinant plasmid containing the gene for spiralin, the major membrane protein of S. citri. The spiralin produced by the Escherichia coli transformant was characterized by immunological detection with monoclonal antibody after Western blotting of two-dimensional (isoelectric focusing and sodium dodecyl sulfate-polyacrylamide) electrophoresis gels and by partial proteolytic mapping. The gene for spiralin occurred within a 6.5-kilobase-pair cloned DNA fragment. Spiralin in E. coli was produced regardless of the orientation of the insert within the pBR328 vector. A spiroplasmal DNA sequence which acted as a promoter in E. coli was cloned along with the structural spiralin gene which is expressed in E. coli from that sequence.

Expression of the Spiroplasma citri spiralin gene in Escherichia coli. Use of the recombinant plasmid carrying this gene as a molecular probe.

Expression in Escherichia coli of the cloned Spiroplasma citri spiralin gene results in the expression of a 30.5-kDa protein serologically related to spiralin. A protein with the same properties is also present in minor amounts in S. citri cells, suggesting that spiralin is first produced as a preprotein containing a signal polypeptide, which is removed during further processing. Hybridization experiments have demonstrated that pES1, the recombinant plasmid carrying the spiralin gene, can be used as a molecular probe, allowing the detection of S. citri DNA in infected plants and in insect cell cultures.

Spiroplasmas and the transfer of genetic material by transformation and transfection.

Two plasmids, pMH1 with 7 kbp and pM41 with 8 kbp were purified from Spiroplasma citri strains MH and M4 respectively. On the basis of guanine + cytosine content and restriction enzyme mapping, the two plasmids are different. The linearized pMH1 plasmid was introduced into Escherichia coli plasmid vector pBR328 and could be cloned in E. coli. Using radioactive probes specific for each plasmid, we found that pM41 was present in three additional S. citri strains and in three other spiroplasmas not belonging to the S. citri species. pMH1 was found as a free 7-kbp plasmid only in the S. citri strain MH. However, the pMH1 probe hybridized strongly with high molecular weight DNA of several S. citri strains and strains of spiroplasmas other than S. citri. The major membrane protein of S. citri, spiralin, is strongly antigenic and rabbit antibodies against whole S. citri cells strongly react with spiralin. Thus, the enzyme-linked immunosorbent assay (ELISA) has been used to screen E. coli clones that were transformed with HindIII-generated S. citri DNA fragments inserted into the HindIII site of pBR328. One E. coli transformant strongly reacted in ELISA with S. citri polyclonal antiserum. The same transformant also gave a positive reaction with monospecific antiserum against spiralin. These results demonstrate that a gene from S. citri, the spiralin gene, could be expressed in a bacterium. The isometric virus SV4, infecting honeybee spiroplasmas of Group I-2, was shown to possess circular single-stranded DNA of molecular weight 1.7 X 10(6) Da. Transfection of spiroplasma G1 with purified DNA of SV4 was achieved. These experiments open the way to the introduction of foreign genes into spiroplasmas.

Turnip yellow mosaic virus RNA-replicase contains host and virus-encoded subunits.

The enzyme (RNA-replicase) involved in the synthesis of viral RNA has been purified from turnip yellow mosaic virus (TYMV)-infected chinese cabbage leaves. The RNA-replicaset contains two major subunits: one of apparent molecular weight 115,000 (115K) and the other of 45K. We have raised antisera against the purified TYMV-RNA-replicase and have demonstrated by immunoaffinity chromatography and immunoblotting that the 115K polypeptide is coded by the viral RNA but that the 45K protein is of host origin. Furthermore the TYMV RNA-replicase is clearly different from the RNA-dependent RNA polymerase that occurs in healthy as well as in infected plants.