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T-DNA of the Agrobacterium Ti and Ri plasmids.

The study of T-DNA transmission from Agrobacterium Ti or Ri plasmid into the genomes of higher plant cells has revealed much about the consequences of transformation. It is now clear that the transformed phenotype is caused by hormonal changes produced directly or indirectly by T-DNA genes. The opine synthases are enzymes encoded in T-DNA that function in the plant cell. Our level of understanding of T-DNA-encoded functions is already sufficient to reveal clear and feasible ways to exploit T-DNA as a gene vector. What remains to challenge the crown gall investigator are many questions of fundamental importance: What is the mechanism of the seemingly illegitimate recombination between T-DNA and plant DNA, and is this process catalyzed by bacterial or host plant enzymes, or both? Do T-DNA genes encode enzymes that catalyze biosynthesis of auxin- and cytokinin-active substances? What gene in T-DNA confers immunity to A. tumefaciens, and what is its mode of action? Does T-DNA insert into random or specific sites in the host plant genome? Did T-DNA derive from plant genetic information or has prokaryotic DNA arrived at functional eukaryotic gene structure by convergent evolution? Although there is keen interest in T-DNA as a vector for genetic engineering, it holds equal interest as a unique interface between the biology of prokaryotes and eukaryotes.

Arginine↗

Construction and identification of recombinant plasmids carrying cDNAs coding for ovine alpha S1-, alpha S2-, beta-, kappa-casein and beta-lactoglobulin. Nucleotide sequence of alpha S1-casein cDNA.

An ovine mammary cDNA library has been constructed from total poly(A)+ RNA isolated from the mammary gland of a lactating ewe, using a classical procedure. Blunt-ended double-stranded cDNAs prepared with reverse transcriptase and nuclease S1 were tailed with dCTP, inserted into the dGMP-tailed PstI site of plasmid pBR322 and cloned in E. coli. Five series of homologous clones representing abundant messenger RNAs (strong hybridization with a single-stranded cDNA probe generated from total poly(A)+ RNA) were selected using each time a different predominant cloned ds-cDNA as probe, then identified by positive hybridization-translation of the cognate mRNA and subsequent immunoprecipitation and electrophoresis of the protein. The lengths of alpha s1-, alpha s2-, beta-, kappa-casein and beta-lactoglobulin mRNAs are in the range of 1.2, 1.1, 1.25, 1.0 and 0.85 kb, respectively, as determined by Northern blotting analysis. Five homologous mRNAs of similar sizes were identified in the porcine species by dot blot hybridization and Northern analyses. The nucleotide sequence of alpha s1-casein mRNA was determined by sequencing, according to Maxam and Gilbert, both a 1080 bp long cloned ds-cDNA and a ss-cDNA (268 nucleotides) generated by 5' extension of a 5' terminal truncated radiolabeled fragment (83 bp) of the relevant ds-cDNA, used as primer for reverse transcription. The 3' non coding region (431 nucleotides, excluding the poly(A) tail) represents 70% of the length of the coding region (618 nucleotides) flanked by a 61 nucleotide 5' region. Comparison of sequences of ovine and bovine, rat and guinea-pig alpha s1-casein mRNAs has revealed a greater homology in the 3' and especially 5' non coding regions. In the reading frame, the conserved regions are essentially those corresponding to the signal peptide and phosphopeptide domains. The derived 206 amino acid sequence of ovine pre-alpha s1-casein differs from that of its bovine counterpart (genetic variant B) by 24 amino acid substitutions and a deletion of 8 amino acid residues occurring in the polypeptide chain of the mature protein. Such a variation (84% homology only) in two phylogenetically closely related species indicates a high rate of evolution of alpha s1-casein.

Amino Acid Sequence↗

Insights on evolution of virulence and resistance from the complete genome analysis of an early methicillin-resistant Staphylococcus aureus strain and a biofilm-producing methicillin-resistant Staphylococcus epidermidis strain.

Staphylococcus aureus is an opportunistic pathogen and the major causative agent of numerous hospital- and community-acquired infections. Staphylococcus epidermidis has emerged as a causative agent of infections often associated with implanted medical devices. We have sequenced the approximately 2.8-Mb genome of S. aureus COL, an early methicillin-resistant isolate, and the approximately 2.6-Mb genome of S. epidermidis RP62a, a methicillin-resistant biofilm isolate. Comparative analysis of these and other staphylococcal genomes was used to explore the evolution of virulence and resistance between these two species. The S. aureus and S. epidermidis genomes are syntenic throughout their lengths and share a core set of 1,681 open reading frames. Genome islands in nonsyntenic regions are the primary source of variations in pathogenicity and resistance. Gene transfer between staphylococci and low-GC-content gram-positive bacteria appears to have shaped their virulence and resistance profiles. Integrated plasmids in S. epidermidis carry genes encoding resistance to cadmium and species-specific LPXTG surface proteins. A novel genome island encodes multiple phenol-soluble modulins, a potential S. epidermidis virulence factor. S. epidermidis contains the cap operon, encoding the polyglutamate capsule, a major virulence factor in Bacillus anthracis. Additional phenotypic differences are likely the result of single nucleotide polymorphisms, which are most numerous in cell envelope proteins. Overall differences in pathogenicity can be attributed to genome islands in S. aureus which encode enterotoxins, exotoxins, leukocidins, and leukotoxins not found in S. epidermidis.

Biofilms↗

Coexpression of eukaryotic tRNASer and yeast seryl-tRNA synthetase leads to functional amber suppression in Escherichia coli.

In order to gain insight into the conservation of determinants for tRNA identity between organisms, Schizosaccharomyces pombe and human amber suppressor serine tRNA genes have been examined for functional expression in Escherichia coli. The primary transcripts, which originated from E. coli plasmid promoters, were processed into mature tRNAs, but they were poorly aminoacylated in E. coli and thus were nonfunctional as suppressors in vivo. However, coexpression of cloned Saccharomyces cerevisiae seryl-tRNA synthetase led to efficient suppression in E. coli. This shows that some, but not all, determinants specifying the tRNASer identity are conserved in evolution.

Acylation↗

Microbes, enzymes and genes involved in dichloromethane utilization.

Dichloromethane (DCM) is efficiently utilized as a carbon and energy source by aerobic, Gram-negative, facultative methylotrophic bacteria. It also serves as a sole carbon and energy source for a nitrate-respiring Hyphomicrobium sp. and for a strictly anaerobic co-culture of a DCM-fermenting bacterium and an acetogen. The first step of DCM utilization by methylotrophs is catalyzed by DCM dehalogenase which, in a glutathione-dependent substitution reaction, forms inorganic chloride and S-chloromethyl glutathione. This unstable intermediate decomposes to glutathione, inorganic chloride and formaldehyde, a central metabolite of methylotrophic growth. Genetic studies on DCM utilization are beginning to shed some light on questions pertaining to the evolution of DCM dehalogenases and on the regulation of DCM dehalogenase expression. DCM dehalogenase belongs to the glutathione S-transferase supergene family. Analysis of the amino acid sequences of two bacterial DCM dehalogenases reveals 56% identity, and comparison of these sequences to those of glutathione S-transferases indicates a closer relationship to class Theta eukaryotic glutathione S-transferases than to a number of bacterial glutathione S-transferases whose sequences have recently become available. dcmA, the structural gene of the highly substrate-inducible DCM dehalogenase, is carried in most DCM utilizing methylotrophs on large plasmids. In Methylobacterium sp. DM4 its expression is governed by dcmR, a regulatory gene located upstream of dcmA, dcmR encodes a trans-acting factor which negatively controls DCM dehalogenase formation at the transcriptional level. Our working model thus assumes that the dcmR product is a repressor which, in the absence of DCM, binds to the promoter region of dcmA and thereby inhibits initiation of transcription.

Amino Acid Sequence↗

Survey of multicopy single-stranded DNAs and reverse transcriptase genes among natural isolates of Myxococcus xanthus.

Twenty different isolates of the soil bacterium Myxococcus xanthus were examined for the presence of multicopy single-stranded DNA (msDNA)-producing retroelements, or retrons. Each strain was analyzed by ethidium bromide staining for msDNA, 32P labeling of the msDNA molecule by the reverse transcriptase (RT) extension method, and DNA hybridization experiments with probes derived from two retrons, Mx162 and Mx65, previously cloned from M. xanthus DZF1. These analyses revealed that all M. xanthus strains contain an msDNA very similar to Mx162 msDNA, and 13 strains also contain a second smaller msDNA very similar to Mx65 msDNA. In addition, the strains contained retron-encoded genes msr and msd, which code for msDNA, and a gene for RT responsible for the synthesis of msDNA. These genes show greater than 80% nucleotide sequence similarity to retrons Mx162 or Mx65. The near-ubiquitous occurrence of msDNA retrons among M. xanthus strains and their homogeneous nature are in marked contrast to the highly diverse but rarely occurring msDNA-producing elements of Escherichia coli. The possible origin and evolution of RT and retron elements is discussed in view of these findings.

Bacteria, Aerobic↗

A novel IL-1 receptor, cloned from B cells by mammalian expression, is expressed in many cell types.

cDNA clones corresponding to an Mr approximately 80,000 receptor (type I receptor) for interleukin-1 (IL-1) have been isolated previously by mammalian expression. Here, we report the use of an improved expression cloning method to isolate human and murine cDNA clones encoding a second type (Mr approximately 60,000) of IL-1 receptor (type II receptor). The mature type II IL-1 receptor consists of (i) a ligand binding portion comprised of three immunoglobulin-like domains; (ii) a single transmembrane region; and (iii) a short cytoplasmic domain of 29 amino acids. This last contrasts with the approximately 215 amino acid cytoplasmic domain of the type I receptor, and suggests that the two IL-1 receptors may interact with different signal transduction pathways. The type II receptor is expressed in a number of different tissues, including both B and T lymphocytes, and can be induced in several cell types by treatment with phorbol ester. Both IL-1 receptors appear to be well conserved in evolution, and map to the same chromosomal location. Like the type I receptor, the human type II IL-1 receptor can bind all three forms of IL-1 (IL-1 alpha, IL-1 beta and IL-1ra). Vaccinia virus contains an open reading frame bearing strong resemblance to the type II IL-1 receptor.

Amino Acid Sequence↗

Identification of TINO: a new evolutionarily conserved BCL-2 AU-rich element RNA-binding protein.

Modulation of mRNA stability by regulatory cis-acting AU-rich elements (AREs) and ARE-binding proteins is an important posttranscriptional mechanism of gene expression control. We previously demonstrated that the 3'-untranslated region of BCL-2 mRNA contains an ARE that accounts for rapid BCL-2 down-regulation in response to apoptotic stimuli. We also demonstrated that the BCL-2 ARE core interacts with a number of ARE-binding proteins, one of which is AU-rich factor 1/heterogeneous nuclear ribonucleoprotein D, known for its interaction with mRNA elements of others genes. In an attempt to search for other BCL-2 mRNA-binding proteins, we used the yeast RNA three-hybrid system assay and identified a novel human protein that interacts with BCL-2 ARE. We refer to it as TINO. The predicted protein sequence of TINO reveals two amino-terminal heterogeneous nuclear ribonucleoprotein K homology motifs for nucleic acid binding and a carboxyl-terminal RING domain, endowed with a putative E3 ubiquitin-protein ligase activity. In addition the novel protein is evolutionarily conserved; the two following orthologous proteins have been identified with protein-protein BLAST: posterior end mark-3 (PEM-3) of Ciona savignyi and muscle excess protein-3 (MEX-3) of Caenorhabditis elegans. Upon binding, TINO destabilizes a chimeric reporter construct containing the BCL-2 ARE sequence, revealing a negative regulatory action on BCL-2 gene expression at the posttranscriptional level.

3' Untranslated Regions↗

Convergent evolution of TEM-26, a beta-lactamase with extended-spectrum activity.

TEM-26, an extended-spectrum beta-lactamase has been characterized in clinical isolates of Klebsiella pneumoniae and Escherichia coli derived from patients on the Paediatric Oncology Unit of St James's University Hospital, Leeds. The nucleotide sequence of this beta-lactamase gene (blaTEM26b) was determined, and compared with the nucleotide sequences of other TEM-type beta-lactamases. The blaTEM26b gene was found to differ from blaTEM12b by a single nucleotide. This difference causes the substitution of glutamic acid in blaTEM12b for lysine in blaTEM26b at position 102 in the predicted amino acid sequence. The blaTEM12b gene was first described in an isolate of Klebsiella oxytoca from a patient nursed on the same unit that yielded the strains that carry blaTEM26b. However, the blaTEM26b gene differs at no less than six nucleotides from the nucleotide sequence encoding the TEM-26 beta-lactamase that was first described in isolates from cancer patients nursed in the Children's Hospital, Stanford, California, USA. This indicates that the genes encoding TEM-26 have evolved from different progenitors.

Amino Acid Sequence↗

FAM20: an evolutionarily conserved family of secreted proteins expressed in hematopoietic cells.

BACKGROUND: Hematopoiesis is a complex developmental process controlled by a large number of factors that regulate stem cell renewal, lineage commitment and differentiation. Secreted proteins, including the hematopoietic growth factors, play critical roles in these processes and have important biological and clinical significance. We have employed representational difference analysis to identify genes that are differentially expressed during experimentally induced myeloid differentiation in the murine EML hematopoietic stem cell line. RESULTS: One identified clone encoded a previously unidentified protein of 541 amino acids that contains an amino terminal signal sequence but no other characterized domains. This protein is a member of family of related proteins that has been named family with sequence similarity 20 (FAM20) with three members (FAM20A, FAM20B and FAM20C) in mammals. Evolutionary comparisons revealed the existence of a single FAM20 gene in the simple vertebrate Ciona intestinalis and the invertebrate worm Caenorhabditis elegans and two genes in two insect species, Drosophila melanogaster and Anopheles gambiae. Six FAM20 family members were identified in the genome of the pufferfish, Fugu rubripes and five members in the zebrafish, Danio rerio. The mouse Fam20a protein was ectopically expressed in a mammalian cell line and found to be a bona fide secreted protein and efficient secretion was dependent on the integrity of the signal sequence. Expression analysis revealed that the Fam20a gene was indeed differentially expressed during hematopoietic differentiation and that the other two family members (Fam20b and Fam20c) were also expressed during hematcpoiesis but that their mRNA levels did not vary significantly. Likewise FAM20A was expressed in more limited set of human tissues than the other two family members. CONCLUSIONS: The FAM20 family represents a new family of secreted proteins with potential functions in regulating differentiation and function of hematopoietic and other tissues. The Fam20a mRNA was only expressed during early stages of hematopoietic development and may play a role in lineage commitment or proliferation. The expansion in gene number in different species suggests that the family has evolved as a result of several gene duplication events that have occurred in both vertebrates and invertebrates.

Amino Acid Sequence↗

Chimeric pneumococcal cell wall lytic enzymes reveal important physiological and evolutionary traits.

Two novel chimeric pneumococcal cell wall lytic enzymes, named LC7 and CL7, have been constructed by in vitro recombination of the lytA gene encoding the major autolysin (LYTA amidase) of Streptococcus pneumoniae, a choline-dependent enzyme, and the cpl7 gene encoding the CPL7 lysozyme of phage Cp-7, a choline-independent enzyme. In remarkable contrast with previous chimeric constructions, we fused here two genes that lack nucleotide homology. The CL7 enzyme, which contains the N-terminal domain of CPL7 and C-terminal domain of LYTA, exhibited a choline-dependent lysozyme activity. This experimental rearrangement of domains might mimic the process that have generated the choline-dependent CPL1 lysozyme of phage Cp-1 during evolution, providing additional support to the modular theory of protein evolution. The LC7 enzyme, built up by fusion of the N-terminal domain of LYTA and the C-terminal domain of CPL7, exhibited an amidase activity capable of degrading ethanolamine-containing cell walls. The chimeric amidase behaved as an autolytic enzyme when it was cloned and expressed in S. pneumoniae. The chimeric enzymes provided new insights on the mechanisms involved in regulation of the host pneumococcal autolysins and on the participation of these enzymes in the process of cell separation. Furthermore, our experimental approach confirmed the basic role of the C-terminal domains in substrate recognition and revealed the influence of these domains on the optimal pH for catalytic activity.

Base Sequence↗

Heterologous expression of the highly conserved acidic ribosomal phosphoproteins from Dictyostelium (changed from Dictyosteliumm) discoideum in Saccharomyces cerevisiae.

The genes encoding the acidic ribosomal phosphoproteins DdP1 and DdP2 from Dictyostelium discoideum have been cloned into yeast plasmid vectors under the control of the inducible GAL1 promoter. These constructions have been used to transform S. cerevisiae strains D45 and D67 lacking the equivalent ribosomal components. The D. discoideum genes are properly transcribed when cells are grown in the presence of the inducer galactose and the mRNAs incorporated into polysomes. However, the heterologous ribosomal proteins are not able to rescue the growth deficiency in S. cerevisiae caused by the absence of their own ribosomal proteins. When the heterologous proteins are analyzed using specific antibodies, only protein DdP1 is found in the ribosomes of the transformed S. cerevisiae D67 strain. No other heterologous protein is found in any other transformed strain, suggesting that the heterologous acidic ribosomal components are rapidly degraded when they are not bound to the ribosomes. The results indicate that D. discoideum DdP1 protein is able to interact with the yeast ribosome, though the interaction is functionally inefficient. Protein DdP2, in spite of having a higher sequence similarity to its yeast counterparts, is completely inactive in S. cerevisiae. Since the P proteins from both organisms have extensive amino acid sequence similarity ranging from 60% to 70%, these results warns about establishing a direct relationship between the extent of amino acid sequence similarity and the capacity of heterologous proteins to be functional in host species. Moreover, our data suggest that evolution affected the interaction of the acidic proteins with the ribosome rather than the structural features responsible for their primary functions.

Amino Acid Sequence↗

A potential disruptive technology in vaccine development: gene-based vaccines and their application to infectious diseases.

Vaccine development requires an amalgamation of disparate disciplines and has unique economic and regulatory drivers. Non-viral gene-based delivery systems, such as formulated plasmid DNA, are new and potentially disruptive technologies capable of providing 'cheaper, simpler, and more convenient-to-use' vaccines. Typically and somewhat ironically, disruptive technologies have poorer product performance, at least in the near-term, compared with the existing conventional technologies. Because successful product development requires that the product's performance must meet or exceed the efficacy threshold for a desired application, the appropriate selection of the initial product applications for a disruptive technology is critical for its successful evolution. In this regard, the near-term successes of gene-based vaccines will likely be for protection against bacterial toxins and acute viral and bacterial infections. Recent breakthroughs, however, herald increasing rather than languishing performance improvements in the efficacy of gene-based vaccines. Whether gene-based vaccines ultimately succeed in eliciting protective immunity in humans to persistent intracellular pathogens, such as HIV, malaria and tuberculosis, for which the conventional vaccine technologies have failed, remains to be determined. A success against any one of the persistent intracellular pathogens would be sufficient proof that gene-based vaccines represent a disruptive technology against which future vaccine technologies will be measured.

Communicable Disease Control↗

Novel RNA polymerization reaction catalyzed by a group I ribozyme.

We have converted a bacterial tRNA precursor containing a 205 nt self-splicing group I intron into a RNA enzyme that catalyzes polymerization of an external RNA substrate. The reaction involves transesterification steps analogous to both the forward and reverse exon ligation steps of group I splicing; as such it depends entirely on 3' splice site reactions. The RNA substrate is a 20 nt analogue of the ligated exons (E1.E2), whose 3' end resembles the 3' terminus of the intron RNA enzyme (IVS). The splice junction of the substrate is attacked by the 3' end of the intron, then the molecule displaces the original 3' terminal guanosine so that the new 3' terminus is brought into the active site and used as the attacking nucleophile in the next reaction. Polymerization occurs via a series of covalent enzyme-linked intermediates of the structure IVS.(E2)n, where n = 1 to > or = 18. The 5' exon accumulates during the course of the reaction and can attack the covalent intermediates to produce elongation products of structure E1.(E2)n, regenerating the intron RNA enzyme in unchanged form. In this manner, the enzyme converts 20 nt oligoribonucleotides into polyribonucleotides up to at least 180 nt by 10 nt increments. These results have significant implications for the evolution of RNA-based self-replicating systems.

Adenosine Triphosphate↗

Variable assortment of prophages provides a transferable repertoire of pathogenic determinants in Salmonella.

Gene transfer between separate lineages of a bacterial pathogen can promote recombinational divergence and the emergence of new pathogenic variants. Temperate bacteriophages, by virtue of their ability to carry foreign DNA, are potential key players in this process. Our previous work has shown that representative strains of Salmonella typhimurium (LT2, ATCC14028 and SL1344) are lysogenic for two temperate bacteriophages: Gifsy-1 and Gifsy-2. Several lines of evidence suggested that both elements carry genes that contribute to Salmonella virulence. One such gene, on the Gifsy-2 prophage, codes for the [Cu, Zn] superoxide dismutase SodCI. Other putative pathogenicity determinants were uncovered more recently. These include genes for known or presumptive type III-translocated proteins and a locus, duplicated on both prophages, showing sequence similarity to a gene involved in Salmonella enteropathogenesis (pipA). In addition to Gifsy-1 and Gifsy-2, each of the above strains was found to harbour a specific set of prophages also carrying putative pathogenicity determinants. A phage released from strain LT2 and identified as phage Fels-1 carries the nanH gene and a novel sodC gene, which was named sodCIII. Strain ATCC14028 releases a lambdoid phage, named Gifsy-3, which contains the phoP/phoQ-activated pagJ gene and the gene for the secreted leucine-rich repeat protein SspH1. Finally, a phage specifically released from strain SL1344 was identified as SopEPhi. Most phage-associated loci transferred efficiently between Salmonella strains of the same or different serovars. Overall, these results suggest that lysogenic conversion is a major mechanism driving the evolution of Salmonella bacteria.

Animals↗

Homologous recombination at the border: insertion-deletions and the trapping of foreign DNA in Streptococcus pneumoniae.

Integration of foreign DNA was observed in the Gram-positive human pathogen Streptococcus pneumoniae (pneumococcus) after transformation with DNA from a recombinant Escherichia coli bacteriophage lamda carrying a pneumococcal insert. Segments of lamda DNA replaced chromosomal sequences adjacent to the region homologous with the pneumococcal insert, whence the name insertion-deletion. Here we report that a pneumococcal insert was absolutely required for insertion-deletion formation, but could be as short as 153 bp; that the sizes of foreign DNA insertions (289-2,474 bp) and concomitant chromosomal deletions (45-1,485 bp) were not obviously correlated; that novel joints clustered preferentially within segments of high GC content; and that the crossovers in 29 independent novel joints were located 1 bp from the border or within short (3-10 nt long) stretches of identity (microhomology) between resident and foreign DNA. The data are consistent with a model in which the insert serving as a homologous recombination anchor favors interaction and subsequent illegitimate recombination events at microhomologies between foreign and resident sequences. The potential of homology- directed illegitimate recombination for genome evolution was illustrated by the trapping of functional heterologous genes.

Bacterial Proteins↗

Loss of a primordial identity element for a mammalian mitochondrial aminoacylation system.

In mammalian mitochondria the translational machinery is of dual origin with tRNAs encoded by a simplified and rapidly evolving mitochondrial (mt) genome and aminoacyl-tRNA synthetases (aaRS) coded by the nuclear genome, and imported. Mt-tRNAs are atypical with biased sequences, size variations in loops and stems, and absence of residues forming classical tertiary interactions, whereas synthetases appear typical. This raises questions about identity elements in mt-tRNAs and adaptation of their cognate mt-aaRSs. We have explored here the human mt-aspartate system in which a prokaryotic-type AspRS, highly similar to the Escherichia coli enzyme, recognizes a bizarre tRNA(Asp). Analysis of human mt-tRNA(Asp) transcripts confirms the identity role of the GUC anticodon as in other aspartylation systems but reveals the non-involvement of position 73. This position is otherwise known as the site of a universally conserved major aspartate identity element, G73, also known as a primordial identity signal. In mt-tRNA(Asp), position 73 can be occupied by any of the four nucleotides without affecting aspartylation. Sequence alignments of various AspRSs allowed placing Gly-269 at a position occupied by Asp-220, the residue contacting G73 in the crystallographic structure of E. coli AspRS-tRNA(Asp) complex. Replacing this glycine by an aspartate renders human mt-AspRS more discriminative to G73. Restriction in the aspartylation identity set, driven by a rapid mutagenic rate of the mt-genome, suggests a reverse evolution of the mt-tRNA(Asp) identity elements in regard to its bacterial ancestor.

Acylation↗

Autonomous transposition of the tobacco retrotransposon Tto1 in rice.

The complete nucleotide sequence of the tobacco retrotransposon Tto1, one of the few active retrotransposons of plants, was determined. The sequence analysis suggests that Tto1 carries all functions required for autonomous transposition through reverse transcription. Gene organization and the nature of the transcription product suggest that Tto1 uses a gene expression mechanism different from those employed by retroviruses and most retrotransposons to regulate Gag and Pol stoichiometry. Tto1 was introduced into rice to study its autonomous transposition in heterologous hosts. Transcription and transposition of Tto1 were observed in rice cells. To probe the autonomous transposition through reverse transcription, a modified Tto1 retrotransposon in which part of a reverse transcriptase gene was replaced with an intron-containing hygromycin resistance gene was constructed and introduced into rice cells. Loss of the intron was observed only when intact Tto1 was cotransfected. These results indicate that Tto1 can transpose autonomously through reverse transcription and that the host factors required for transposition are conserved among monocots (class Magnoliopsida; rice) and dicots (class Liliopsida; tobacco), which diverged approximately 200 million years ago. These findings are discussed in relation to the regulation and evolution of retrotransposons and the possible use of Tto1 as a molecular genetic tool.

Base Sequence↗