Full length L1 retroposons contain tRNA-like sequences near the 5' termini--hypothesis on the replication mechanism of retroposons

Retrotransposons replicate via a complex mechanism which depends on, among other things, the presence of long terminal repeats (LTRs) and a tRNA binding site just 3' of the 5' LTR. The LINES 1 (L1) family of sequences, which similar to retrotransposons in many other properties, represents a new class of retroposon which does not possess LTRs. However, we show here that the repetitive 5' motif associated with murine L1 elements contains a tRNA-like sequence in a location analogous to the position of the retro-transposon tRNA binding site. Although the repetition of such a 5' motif has only been found associated with murine L1 elements, we have found an analogous tRNA-like sequence near the 5' ends of the L1 elements from each of the other analyzed species for which the L1 family has been characterized, that is rat (L1Rr), human (L1Hs), drosophila (I element) and trypanosome (INGI). The conservation of this tRNA-like sequence near the 5' terminus of L1 elements from such diverse species suggests that it plays a functional role in the life of the L1 class of retroposon.     

The trans-spliceosomal U4 RNA from the monogenetic trypanosomatid Leptomonas collosoma. Cloning and identification of a transcribed trna-like element that controls its expression

U4 small nuclear RNA is essential for trans-splicing. Here we report the cloning of U4 snRNA gene from Leptomonas collosoma and analysis of elements controlling its expression. The trypanosome U4 RNA is the smallest known, it carries an Sm-like site, and has the potential for extensive intermolecular base pairing with the U6 RNA. Sequence analysis of the U4 locus indicates the presence of a tRNA-like element 86 base pairs upstream of the gene that is divergently transcribed to yield a stable small tRNA-like RNA. Two additional tRNA genes, tRNA(Pro) and tRNA(Gly), were found upstream of this element. By stable expression of a tagged U4 RNA, we demonstrate that the tRNA-like gene, but not the upstream tRNA genes, is essential for U4 expression and that the B box but not the A Box of the tRNA-like gene is crucial for expression in vivo. Mapping the 2'-O-methyl groups on U4 and U6 small nuclear RNAs suggests the presence of modifications in canonical positions. However, the number of modified nucleotides is fewer than in mammalian homologues. The U4 genomic organization including both tRNA-like and tRNA genes may represent a relic whereby trypanosomatids "hired" tRNA genes to provide extragenic promoter elements. The close proximity of tRNA genes to the tRNA-like molecule in the U4 locus further suggests that the tRNA-like gene may have evolved from a tRNA member of this cluster.     

Phylogenetic analysis of tmRNA secondary structure.

The bacterial tmRNA acts with dual tRNA-like and mRNA-like character to tag incomplete translation products for degradation. Comparative analysis of 17 tmRNA genes (including eight new sequences) has allowed us to deduce conserved features of the tmRNA secondary structure. Except in a segment that includes the first codon of the tag reading frame, tmRNA is highly structured, with four pseudoknots and a total of 11 conserved base pairing regions. The previously identified tRNA minihelix structure is connected by a long base paired region to a large structured domain composed of a pseudoknot, followed by the tag reading frame and a string of three rather similar pseudoknots. The conservation of numerous structural elements among diverse eubacterial species indicates that these elements have important function beyond simply forming an endonuclease-resistant link between the reading frame and the tRNA-like domain.     

Analysis of the hypervariable region of the Salmonella enterica genome associated with tRNA(leuX)

The divergence of Salmonella enterica and Escherichia coli is estimated to have occurred approximately 140 million years ago. Despite this evolutionary distance, the genomes of these two species still share extensive synteny and homology. However, there are significant differences between the two species in terms of genes putatively acquired via various horizontal transfer events. Here we report on the composition and distribution across the Salmonella genus of a chromosomal region designated SPI-10 in Salmonella enterica serovar Typhi and located adjacent to tRNA(leuX). We find that across the Salmonella genus the tRNA(leuX) region is a hypervariable hot spot for horizontal gene transfer; different isolates from the same S. enterica serovar can exhibit significant variation in this region. Many P4 phage, plasmid, and transposable element-associated genes are found adjacent to tRNA(leuX) in both Salmonella and E. coli, suggesting that these mobile genetic elements have played a major role in driving the variability of this region.     

Bioinformatics analysis of plant orthologous introns: identification of an intronic tRNA-like sequence

Orthologous introns have identical positions relative to the coding sequence in orthologous genes of different species. By analyzing the complete genomes of five plants we generated a database of 40,512 orthologous intron groups of dicotyledonous plants, 28,519 orthologous intron groups of angiosperms, and 15,726 of land plants (moss and angiosperms). Multiple sequence alignments of each orthologous intron group were obtained using the Mafft algorithm. The number of conserved regions in plant introns appeared to be hundreds of times fewer than that in mammals or vertebrates. Approximately three quarters of conserved intronic regions among angiosperms and dicots, in particular, correspond to alternatively-spliced exonic sequences. We registered only a handful of conserved intronic ncRNAs of flowering plants. However, the most evolutionarily conserved intronic region, which is ubiquitous for all plants examined in this study, including moss, possessed multiple structural features of tRNAs, which caused us to classify it as a putative tRNA-like ncRNA. Intronic sequences encoding tRNA-like structures are not unique to plants. Bioinformatics examination of the presence of tRNA inside introns revealed an unusually long-term association of four glycine tRNAs inside the Vac14 gene of fish, amniotes, and mammals.     

Rodent type 2 Alu family,rat identifier sequence,rabbit C family,and bovine or goat 73-bp repeat may have evolved from tRNA genes

Summary Close structural resemblances between several mammalian highly or moderately repetitive families and some specific tRNAs were detected. The rodent type 2 Alu family, rat identifier (ID) sequences, rabbit C family, and bovine or goat 73-bp repeat are most homologous with lysine tRNA₅, phenylalanine tRNA, glycine tRNA, and glycine tRNA, respectively. The homologies extend to secondary structures, and the homologous nucleotides are located on nearly the same secondary structures. The repetitive families mentioned have a common structural organization, with a tRNA-like sequence devoid of an aminoacyl stem region. These features suggest that these repetitive families may be generated by nonhomologous recombination between a tRNA gene and a tRNA-unrelated block.     

A pseudo-tRNA modulates antibiotic resistance in Bacillus cereus

Bacterial genomic islands are often flanked by tRNA genes, which act as sites for the integration of foreign DNA into the host chromosome. For example, Bacillus cereus ATCC14579 contains a pathogenicity island flanked by a predicted pseudo-tRNA, tRNA(Other), which does not function in translation. Deletion of tRNA(Other) led to significant changes in cell wall morphology and antibiotic resistance and was accompanied by changes in the expression of numerous genes involved in oxidative stress responses, several of which contain significant complementarities to sequences surrounding tRNA(Other). This suggested that tRNA(Other) might be expressed as part of a larger RNA, and RACE analysis subsequently confirmed the existence of several RNA species that significantly extend both the 3' and 5'-ends of tRNA(Other). tRNA(Other) expression levels were found to be responsive to changes in extracellular iron concentration, consistent with the presence of three putative ferric uptake regulator (Fur) binding sites in the 5' leader region of one of these larger RNAs. Taken together with previous data, this study now suggests that tRNA(Other) may function by providing a tRNA-like structural element within a larger regulatory RNA. These findings illustrate that while integration of genomic islands often leaves tRNA genes intact and functional, in other instances inactivation may generate tRNA-like elements that are then recruited to other functions in the cell.     

Pstl repeat: a family of short interspersed nucleotide element (SINE)-like sequences in the genomes of cattle, goat, and buffalo.

The PstI family of elements are short, highly repetitive DNA sequences interspersed throughout the genome of the Bovidae. We have cloned and sequenced some members of the PstI family from cattle, goat, and buffalo. These elements are approximately 500 bp, have a copy number of 2 x 10(5) - 4 x 10(5), and comprise about 4% of the haploid genome. Studies of nucleotide sequence homology indicate that the buffalo and goat PstI repeats (type II) are similar types of short interspersed nucleotide element (SINE) sequences, but the cattle PstI repeat (type I) is considerably more divergent. Additionally, the goat PstI sequence showed significant sequence homology with bovine serine tRNA, and is therefore likely derived from serine tRNA. Interestingly, Southern hybridization suggests that both types of SINEs (I and II) are present in all the species of Bovidae. Dendrogram analysis indicates that cattle PstI SINE is similar to bovine Alu-like SINEs. Goat and buffalo SINEs formed a separate cluster, suggesting that these two types of SINEs evolved separately in the genome of the Bovidae.     

Nucleotide sequences of yeast genes for tRNA(2), tRNA(2) and tRNA(1): homology blocks occur in the vicinity of different tRNA genes

Three members of a collection of pBR322-yeast DNA recombinant plasmids containing yeast tRNA genes have been analyzed and sequenced. Each plasmid carries a single tRNA gene: pY44, tRNA^Ser₂; pY41, tRNA^Arg₂; pY7, tRNA^Val₁. All three genes are intronless and terminate in a cluster of Ts in the non-coding strand. The sequence information here and previously determined sequences allow an extensive comparison of the regions flanking several yeast tRNA genes. This analysis has revealed novel features in tRNA gene arrangement. Blocks of homology in the flanking regions were found between the tRNA genes of an isoacceptor family but, more interestingly, also between genes coding for tRNAs of different amino-acid specificities. Particularly, three examples are discussed in which sequence elements in the neighborhood of different tRNA genes have been conserved to a high degree and over long distances.     

Nucleotide sequence analysis of zein mRNAs from maize endosperm

A comparison of the DNA and protein sequences of a group of zein cDNA clones reveals that they share extensive sequence homology and probably originated from a common ancestral gene. A comparison of clones corresponding to Mr 22,000 polypeptides shows they are 92% homologous, while five clones corresponding to the Mr 19,000 zeins vary in homology from 75 to 95%. The clones corresponding to the Mr 22,000 proteins are 60-65% homologous to clones encoding the Mr 19,000 zein proteins. A clone corresponding to the Mr 15,000 zein has little homology to either the Mr 22,000 or 19,000 zeins. Clones corresponding to both the Mr 22,000 and 19,000 zeins have two putative polyadenylation signals. S1 nuclease mapping indicates that the first polyadenylation signal following the stop codon is utilized by the Mr 22,000 sequences, while primarily the second polyadenylation signal is utilized by the Mr 19,000 sequences.     

A novel abundant family of retroposed elements (DAS-SINEs) in the nine-banded armadillo (Dasypus novemcinctus)

About half of the mammalian genome is composed of retroposons. Long interspersed elements (LINEs) and short interspersed elements (SINEs) are the most abundant repetitive elements and account for about 21% and 13% of the human genome, respectively. SINEs have been detected in all major mammalian lineages, except for the South American order Xenarthra, also termed Edentata (armadillos, anteaters, and sloths). Investigating this order, we discovered a novel high-copy-number family of tRNA derived SINEs in the nine-banded armadillo Dasypus novemcinctus, a species that successfully crossed the Central American land bridge to North America in the Pliocene. A specific computer algorithm was developed, and we detected and extracted 687 specific SINEs from databases. Termed DAS-SINEs, we further divided them into six distinct subfamilies. We extracted tRNA(Ala)-derived monomers, two types of dimers, and three subfamilies of chimeric fusion products of a tRNA(Ala) domain and an approximately 180-nt sequence of thus far unidentified origin. Comparisons of secondary structures of the DAS-SINEs' tRNA domains suggest selective pressure to maintain a tRNA-like D-arm structure in the respective founder RNAs, as shown by compensatory mutations. By analysis of subfamily-specific genetic variability, comparison of the proportion of direct repeats, and analysis of self-integrations as well as key events of dimerization and deletions or insertions, we were able to delineate the evolutionary history of the DAS-SINE subfamilies.     

Evolution of tRNA recognition systems and tRNA gene sequences

The aminoacylation of tRNAs by the aminoacyl-tRNA synthetases recapitulates the genetic code by dictating the association between amino acids and tRNA anticodons. The sequences of tRNAs were analyzed to investigate the nature of primordial recognition systems and to make inferences about the evolution of tRNA gene sequences and the evolution of the genetic code. Evidence is presented that primordial synthetases recognized acceptor stem nucleotides prior to the establishment of the three major phylogenetic lineages. However, acceptor stem sequences probably did not achieve a level of sequence diversity sufficient to faithfully specify the anticodon assignments of all 20 amino acids. This putative bottleneck in the evolution of the genetic code may have been alleviated by the advent of anticodon recognition. A phylogenetic analysis of tRNA gene sequences from the deep Archaea revealed groups that are united by sequence motifs which are located within a region of the tRNA that is involved in determining its tertiary structure. An association between the third anticodon nucleotide (N36) and these sequence motifs suggests that a tRNA-like structure existed close to the time that amino acid-anticodon assignments were being established. The sequence analysis also revealed that tRNA genes may evolve by anticodon mutations that recruit tRNAs from one isoaccepting group to another. Thus tRNA gene evolution may not always be monophyletic with respect to each isoaccepting group.Based on a presentation made at a workshop— Aminoacyl-tRNA Synthetases and the Evolution of the Genetic Code—held at Berkeley, CA, July 17–20, 1994 Correspondence to: M.E. Saks     

Roles of phi X174 type primosome- and G4 type primase-dependent primings in initiation of lagging and leading strand syntheses of DNA replication

Eleven single strand initiation sequences (ssi) were isolated from various plasmid genomes using a plaque-morphology assay. Out of seven ssi that require dnaB and dnaC functions for replication in a crude in vitro system, six use a phi X174 type priming mechanism, and a phi X174 type primosome is assembled at these sequences from the purified proteins, n'(priA), n(priB), n"(priC), dnaT, dnaB, dnaC, and primase. The same ssi potentiate dATPase activity of n' protein, and thus represent new n' protein recognition sequences (n'-pas). Based on sequence homology, two structural groups are evident. Two sequences show a strong homology with the phi X174 site, whereas three share extensive homology with the previously characterized n'-pas of ColE1, ssiA(ColE1). All the n'-pas have a potential to form stem and loop structures, although sequence homology between the two classes is absent. In addition to the phi X174 type priming, three ssi do not require either dnaB or dnaC function for replication, and use a G4 type priming, requiring only SSB and primase. The 5' ends of primer RNA synthesized by primase are localized within the vicinity of one of the three blocks of highly conserved nucleotide sequences. Deletions of parts of these conserved sequences result in loss of priming activity, suggesting that they are important for priming on the G4 type ssi, which are termed G site. The general significance of these two types of priming in initiation of lagging or leading strand synthesis as well as various modes of initiation at origins of replication are proposed.