Three novel families of miniature inverted-repeat transposable elements are associated with genes of the yellow fever mosquito, Aedes aegypti

Three novel families of transposable elements, Wukong, Wujin, and Wuneng, are described in the yellow fever mosquito, Aedes aegypti. Their copy numbers range from 2,100 to 3,000 per haploid genome. There are high degrees of sequence similarity within each family, and many structural but not sequence similarities between families. The common structural characteristics include small size, no coding potential, terminal inverted repeats, potential to form a stable secondary structure, A+T richness, and putative 2- to 4-bp A+T-biased specific target sites. Evidence of previous mobility is presented for the Wukong elements. Elements of these three families are associated with 7 of 16 fully or partially sequenced Ae. aegypti genes. Characteristics of these mosquito elements indicate strong similarities to the miniature inverted-repeat transposable elements (MITEs) recently found to be associated with plant genes. MITE-like elements have also been reported in two species of Xenopus and in Homo sapiens. This characterization of multiple families of highly repetitive MITE-like elements in an invertebrate extends the range of these elements in eukaryotic genomes. A hypothesis is presented relating genome size and organization to the presence of highly reiterated MITE families. The association of MITE-like elements with Ae. aegypti genes shows the same bias toward noncoding regions as in plants. This association has potentially important implications for the evolution of gene regulation.

Eight novel families of miniature inverted repeat transposable elements in the African malaria mosquito, Anopheles gambiae

Eight novel families of miniature inverted repeat transposable elements (MITEs) were discovered in the African malaria mosquito, Anopheles gambiae, by using new software designed to rapidly identify MITE-like sequences based on their structural characteristics. Divergent subfamilies have been found in two families. Past mobility was demonstrated by evidence of MITE insertions that resulted in the duplication of specific TA, TAA, or 8-bp targets. Some of these MITEs share the same target duplications and similar terminal sequences with MITEs and other DNA transposons in human and other organisms. MITEs in A. gambiae range from 40 to 1340 copies per genome, much less abundant than MITEs in the yellow fever mosquito, Aedes aegypti. Statistical analyses suggest that most A. gambiae MITEs are in highly AT-rich regions, many of which are closely associated with each other. The analyses of these novel MITEs underscored interesting questions regarding their diversity, origin, evolution, and relationships to the host genomes. The discovery of diverse families of MITEs in A. gambiae has important practical implications in light of current efforts to control malaria by replacing vector mosquitoes with genetically modified refractory mosquitoes. Finally, the systematic approach to rapidly identify novel MITEs should have broad applications for the analysis of the ever-growing sequence databases of a wide range of organisms.

A computer-based systematic survey reveals the predominance of small inverted-repeat elements in wild-type rice genes.

Several recent reports indicate that mobile elements are frequently found in and flanking many wild-type plant genes. To determine the extent of this association, we performed computer-based systematic searches to identify mobile elements in the genes of two "model" plants, Oryza sativa (domesticated rice) and Arabidopsis thaliana. Whereas 32 common sequences belonging to nine putative mobile element families were found in the noncoding regions of rice genes, none were found in Arabidopsis genes. Five of the nine families (Gaijin, Castaway, Ditto, Wanderer, and Explorer) are first described in this report, while the other four were described previously (Tourist, Stowaway, p-SINE1, and Amy/LTP). Sequence similarity, structural similarity, and documentation of past mobility strongly suggests that many of the rice common sequences are bona fide mobile elements. Members of four of the new rice mobile element families are similar in some respects to members of the previously identified inverted-repeat element families, Tourist and Stowaway. Together these elements are the most prevalent type of transposons found in the rice genes surveyed and form a unique collection of inverted-repeat transposons we refer to as miniature inverted-repeat transposable elements or MITEs. The sequence and structure of MITEs are clearly distinct from short or long interspersed nuclear elements (SINEs or LINEs), the most common transposable elements associated with mammalian nuclear genes. Mobile elements, therefore, are associated with both animal and plant genes, but the identity of these elements is strikingly different.

Purification and characterization of a CENP-B homologue protein that binds to the centromeric K-type repeat DNA of Schizosaccharomyces pombe

We have purified and characterized a novel 60-kDa protein that binds to centromeric K-type repeat DNA from Schizosaccharomyces pombe. This protein was initially purified by its ability to bind to the autonomously replicating sequence 3002 DNA. Cloning of the gene encoding this protein revealed that it possesses significant homology to the mammalian centromere DNA-binding protein CENP-B and S. pombe Abp1, and this gene was designated as cbh+ (CENP-B homologue). Cbh protein specifically interacts in vitro with the K-type repeat DNA, which is essential for centromere function. The Cbh-binding consensus sequence was determined by DNase I footprinting assays as PyPuATATPyPuTA, featuring an inverted repeat of the first four nucleotides. Based on its binding activity to centromeric DNA and homology to centromere proteins, we suggest that this protein may be a functional homologue of the mammalian CENP-B in S. pombe.

Dual roles for DNA sequence identity and the mismatch repair system in the regulation of mitotic crossing-over in yeast

Sequence divergence acts as a potent barrier to homologous recombination; much of this barrier derives from an antirecombination activity exerted by mismatch repair proteins. An inverted repeat assay system with recombination substrates ranging in identity from 74% to 100% has been used to define the relationship between sequence divergence and the rate of mitotic crossing-over in yeast. To elucidate the role of the mismatch repair machinery in regulating recombination between mismatched substrates, we performed experiments in both wild-type and mismatch repair defective strains. We find that a single mismatch is sufficient to inhibit recombination between otherwise identical sequences, and that this inhibition is dependent on the mismatch repair system. Additional mismatches have a cumulative negative effect on the recombination rate. With sequence divergence of up to approximately 10%, the inhibitory effect of mismatches results mainly from antirecombination activity of the mismatch repair system. With greater levels of divergence, recombination is inefficient even in the absence of mismatch repair activity. In both wild-type and mismatch repair defective strains, an approximate log-linear relationship is observed between the recombination rate and the level of sequence divergence.

Molecular characterization of a mutable pigmentation phenotype and isolation of the first active transposable element from Sorghum bicolor

Accumulation of red phlobaphene pigments in sorghum grain pericarp is under the control of the Y gene. A mutable allele of Y, designated as y-cs (y-candystripe), produces a variegated pericarp phenotype. Using probes from the maize p1 gene that cross-hybridize with the sorghum Y gene, we isolated the y-cs allele containing a large insertion element. Our results show that the Y gene is a member of the MYB-transcription factor family. The insertion element, named Candystripe1 (Cs1), is present in the second intron of the Y gene and shares features of the CACTA superfamily of transposons. Cs1 is 23,018 bp in size and is bordered by 20-bp terminal inverted repeat sequences. It generated a 3-bp target site duplication upon insertion within the Y gene and excised from y-cs, leaving a 2-bp footprint in two cases analyzed. Reinsertion of the excised copy of Cs1 was identified by Southern hybridization in the genome of each of seven red pericarp revertant lines tested. Cs1 is the first active transposable element isolated from sorghum. Our analysis suggests that Cs1-homologous sequences are present in low copy number in sorghum and other grasses, including sudangrass, maize, rice, teosinte, and sugarcane. The low copy number and high transposition frequency of Cs1 imply that this transposon could prove to be an efficient gene isolation tool in sorghum.

Repetitive-DNA elements are similarly distributed on Caenorhabditis elegans autosomes

The positions of ≈4,800 individual miniature inverted-repeat transposable element (MITE)-like repeats from four families were mapped on the Caenorhabditis elegans chromosomes. These families represent 1–2% of the total sequence of the organism. The four MITE families (Cele1, Cele2, Cele14, and Cele42) displayed distinct chromosomal distribution profiles. For example, the Cele14 MITEs were observed clustering near the ends of the autosomes. In contrast, the Cele2 MITEs displayed an even distribution through the central autosome domains, with no evidence for clustering at the ends. Both the number of elements and the distribution patterns of each family were conserved on all five C. elegans autosomes. The distribution profiles indicate chromosomal polarity and suggest that the current genetic and physical maps of chromosomes II, III, and X are inverted with respect to the other chromosomes. The degree of conservation of both the number and distribution of these elements on the five autosomes suggests a role in defining specific chromosomal domains.

The MITE family Heartbreaker (Hbr): Molecular markers in maize

Transposable elements are ubiquitous in plant genomes, where they frequently comprise the majority of genomic DNA. The maize genome, which is believed to be structurally representative of large plant genomes, contains single genes or small gene islands interspersed with much longer blocks of retrotransposons. Given this organization, it would be desirable to identify molecular markers preferentially located in genic regions. In this report, the features of a newly described family of miniature inverted repeat transposable elements (MITEs) (called Heartbreaker), including high copy number and polymorphism, stability, and preference for genic regions, have been exploited in the development of a class of molecular markers for maize. To this end, a modification of the AFLP procedure called transposon display was used to generate and display hundreds of genomic fragments anchored in Hbr elements. An average of 52 markers were amplified for each primer combination tested. In all, 213 polymorphic fragments were reliably scored and mapped in 100 recombinant inbred lines derived from a cross between the maize inbreds B73 × Mo17. In this mapping population, Hbr markers are distributed evenly across the 10 maize chromosomes. This procedure should be of general use in the development of markers for other MITE families in maize and in other plant and animal species where MITEs have been identified.

Thermostable and site-specific DNA binding of the gene product ORF56 from the Sulfolobus islandicus plasmid pRN1, a putative archael plasmid copy control protein

There is still a lack of information on the specific characteristics of DNA-binding proteins from hyperthermophiles. Here we report on the product of the gene orf56 from plasmid pRN1 of the acidophilic and thermophilic archaeon Sulfolobus islandicus. orf56 has not been characterised yet but low sequence similarily to several eubacterial plasmid-encoded genes suggests that this 6.5 kDa protein is a sequence-specific DNA-binding protein. The DNA-binding properties of ORF56, expressed in Escherichia coli, have been investigated by EMSA experiments and by fluorescence anisotropy measurements. Recombinant ORF56 binds to double-stranded DNA, specifically to an inverted repeat located within the promoter of orf56. Binding to this site could down-regulate transcription of the orf56 gene and also of the overlapping orf904 gene, encoding the putative initiator protein of plasmid replication. By gel filtration and chemical crosslinking we have shown that ORF56 is a dimeric protein. Stoichiometric fluorescence anisotropy titrations further indicate that ORF56 binds as a tetramer to the inverted repeat of its target binding site. CD spectroscopy points to a significant increase in ordered secondary structure of ORF56 upon binding DNA. ORF56 binds without apparent cooperativity to its target DNA with a dissociation constant in the nanomolar range. Quantitative analysis of binding isotherms performed at various salt concentrations and at different temperatures indicates that approximately seven ions are released upon complex formation and that complex formation is accompanied by a change in heat capacity of –6.2 kJ/mol. Furthermore, recombinant ORF56 proved to be highly thermostable and is able to bind DNA up to 85°C.

Dynamics of mobile element activity in chalcone synthase loci in the common morning glory (Ipomoea purpurea)

Mobile element dynamics in seven alleles of the chalcone synthase D locus (CHS-D) of the common morning glory (Ipomoea purpurea) are analyzed in the context of synonymous nucleotide sequence distances for CHS-D exons. By using a nucleotide sequence of CHS-D from the sister species Ipomoea nil (Japanese morning glory [Johzuka-Hisatomi, Y., Hoshino, A., Mori, T., Habu, Y. & Iida, S. (1999) Genes Genet. Syst. 74, 141–147], it is also possible to determine the relative frequency of insertion and loss of elements within the CHS-D locus between these two species. At least four different types of transposable elements exist upstream of the coding region, or within the single intron of the CHS-D locus in I. purpurea. There are three distinct families of miniature inverted-repeat transposable elements (MITES), and some recent transpositions of Activator/Dissociation (Ac/Ds)-like elements (Tip100), of some short interspersed repetitive elements (SINEs), and of an insertion sequence (InsIpCHSD) found in the neighborhood of this locus. The data provide no compelling evidence of the transposition of the mites since the separation of I. nil and I. purpurea roughly 8 million years ago. Finally, it is shown that the number and frequency of mobile elements are highly heterogeneous among different duplicate CHS loci, suggesting that the dynamics observed at CHS-D are locus-specific.

The highly recombinogenic bz locus lies in an unusually gene-rich region of the maize genome

The bronze (bz) locus exhibits the highest rate of recombination of any gene in higher plants. To investigate the possible basis of this high rate of recombination, we have analyzed the physical organization of the region around the bz locus. Two adjacent bacterial artificial chromosome clones, comprising a 240-kb contig centered around the Bz-McC allele, were isolated, and 60 kb of contiguous DNA spanning the two bacterial artificial chromosome clones was sequenced. We find that the bz locus lies in an unusually gene-rich region of the maize genome. Ten genes, at least eight of which are shown to be transcribed, are contained in a 32-kb stretch of DNA that is uninterrupted by retrotransposons. We have isolated nearly full length cDNAs corresponding to the five proximal genes in the cluster. The average intertranscript distance between them is just 1 kb, revealing a surprisingly compact packaging of adjacent genes in this part of the genome. At least 11 small insertions, including several previously described miniature inverted repeat transposable elements, were detected in the introns and 3′ untranslated regions of genes and between genes. The gene-rich region is flanked at the proximal and distal ends by retrotransposon blocks. Thus, the maize genome appears to have scattered regions of high gene density similar to those found in other plants. The unusually high rate of intragenic recombination seen in bz may be related to the very high gene density of the region.

Transcriptional regulation of the Rhodococcus rhodochrous J1 nitA gene encoding a nitrilase.

The 1.4-kb downstream region from a nitrilase gene (nitA) of an actinomycete Rhodococcus rhodochrous J1, which is industrially in use, was found to be required for the isovaleronitrile-dependent induction of nitrilase synthesis in experiments using a Rhodococcus-Escherichia coli shuttle vector pK4 in a Rhodococcus strain. Sequence analysis of the 1.4-kb region revealed the existence of an open reading frame (nitR) of 957 bp, which would encode a protein with a molecular mass of 35,100. Deletion of the central and 3'-terminal portion of nitR resulted in the complete loss of nitrilase activity, demonstrating that nitR codes for a transcriptional positive regulator in nitA expression. The deduced amino acid sequence of nitR showed similarity to a positive regulator family including XylS from Pseudomonas putida and AraC from E. coli. By Northern blot analysis, the 1.4-kb transcripts for nitA were detected in R. rhodochrous J1 cells cultured in the presence of isovaleronitrile, but not those cultured in the absence of isovaleronitrile. The transcriptional start site for nitA was mapped to a C residue located 26 bp upstream of its translational start site. Deletion analysis to define the nitA promoter region suggested the possible participation of an inverted repeat sequence, centered on base pair -52, in induction of nitA transcription.

Initiation of (-) strand DNA synthesis from tRNA(3Lys) on lentiviral RNAs: implications of specific HIV-1 RNA-tRNA(3Lys) interactions inhibiting primer utilization by retroviral reverse transcriptases.

Initiation of minus (-) strand DNA synthesis was examined on templates containing R, U5, and primer-binding site regions of the human immunodeficiency virus type 1 (HIV-1), feline immunodeficiency virus (FIV), and equine infectious anemia virus (EIAV) genomic RNA. DNA synthesis was initiated from (i) an oligoribonucleotide complementary to the primer-binding sites, (ii) synthetic tRNA(3Lys), and (iii) natural tRNA(3Lys), by the reverse transcriptases of HIV-1, FIV, EIAV, simian immunodeficiency virus, HIV type 2 (HIV-2), Moloney murine leukemia virus, and avian myeloblastosis virus. All enzymes used an oligonucleotide on wild-type HIV-1 RNA, whereas only a limited number initiated (-) strand DNA synthesis from either tRNA(3Lys). In contrast, all enzymes supported efficient tRNA(3Lys)-primed (-) strand DNA synthesis on the genomes of FIV and EIAV. This may be in part attributable to the observation that the U5-inverted repeat stem-loop of the EIAV and FIV genomes lacks an A-rich loop shown with HIV-1 to interact with the U-rich tRNA anticodon loop. Deletion of this loop in HIV-1 RNA, or disrupting a critical loop-loop complex by tRNA(3Lys) extended by 9 nt, restored synthesis of HIV-1 (-) strand DNA from primer tRNA(3Lys) by all enzymes. Thus, divergent evolution of lentiviruses may have resulted in different mechanisms to use the same host tRNA for initiation of reverse transcription.

Molecular cloning of a rat chromosome putative recombinogenic sequence homologous to the hepatitis B virus encapsidation signal.

Previously, we reported that a 61-bp subgenomic HBV DNA sequence (designated as 15AB, nt 1855-1915) is a hot spot for genomic recombination and that a cellular protein binding to 15AB may be the putative recombinogenic protein. In the present study, we established the existence of a 15AB-like sequence in human and rat chromosomal DNA by Southern blot analysis. The 15AB-like sequence isolated from the rat chromosome demonstrated a 80.9% identity with 5'-CCAAGCTGTGCCTTGGGTGGC-3', at 1872-1892 of the hepatitis B virus genome, thought to be the essential region for recombination. Interestingly, this 15AB-like sequence also contained the pentanucleotide motifs GCTGG and CCAGC as an inverted repeat, part of the chi known hot spot for recombination in Escherichia coli. Importantly, a portion of the 15AB-like sequence is homologous (82.1%, 23/28 bp) to break point clusters of the human promyelocytic leukemia (PML) gene, characterized by a translocation [t(15;17)], and to rearranged mouse DNA for the immunoglobulin kappa light chain. Moreover, 15AB and 15AB-like sequences have striking homologies (12/15 = 80.0% and 13/15 = 86.7%, respectively) to the consensus sequence for topoisomerase II. Our present results suggest that this 15AB-like sequence in the rat genome might be a recombinogenic candidate triggering genomic instability in carcinogenesis.

Copy-choice recombination mediated by DNA polymerase III holoenzyme from Escherichia coli.

Formation of deletions by recombination between short direct repeats is thought to involve either a break-join or a copy-choice process. The key step of the latter is slippage of the replication machinery between the repeats. We report that the main replicase of Escherichia coli, DNA polymerase III holoenzyme, slips between two direct repeats of 27 bp that flank an inverted repeat of approximately equal 300bp. Slippage was detected in vitro, on a single-stranded DNA template, in a primer extension assay. It requires the presence of a short (8 bp) G+C-rich sequence at the base of a hairpin that can form by annealing of the inverted repeats. It is stimulated by (i) high salt concentration, which might stabilize the hairpin, and (ii) two proteins that ensure the processivity of the DNA polymerase III holoenzyme: the single-stranded DNA binding protein and the beta subunit of the polymerase. Slippage is rather efficient under optimal reaction conditions because it can take place on >50% of template molecules. This observation supports the copy-choice model for recombination between short direct repeats.