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Transfer RNAs (tRNAs) are noncoding RNAs involved in protein biosynthesis and have noncanonical roles in cellular metabolism, such as RNA silencing and the generation of transposable elements. Extensive tRNA gene duplications, modifications to mature tRNAs, and complex secondary and tertiary structures impede tRNA sequencing. As such, a comparative genomic analysis of complete tRNA sets is an alternative to understanding the evolutionary processes that gave rise to the extant tRNA sets. Although the tRNA gene (tDNA) structure and distribution in prokaryotes and eukaryotes, specifically in vertebrates, yeasts, and flies, are well understood, there is little information regarding plants. A detailed and comprehensive analysis and annotation of tDNAs from the genomes of 44 eudicots, 20 monocots, and five other non-eudicot and non-monocot species belonging to the Ceratophyllaceae and the ANA (Amborellales, Nymphaeales, and Austrobaileyales) clade will provide a global picture of plant tDNA structure and organization. Plant genomes exhibit varying numbers of nuclear tDNAs, with only the monocots showing a strong correlation between nuclear tDNA numbers and genome sizes. In contrast, organellar tDNA numbers varied little among the different lineages. A high degree of tDNA duplication in eudicots was detected, whereby most eudicot nuclear genomes (91%) and only a modest percentage of monocot (65%) and ANA nuclear genomes (25%) contained at least one tDNA cluster. Clusters of tRNATyr–tRNASer and tRNAIle genes were found in eudicot and monocot genomes, respectively, while both eudicot and monocot genomes showed clusters of tRNAPro genes. All plant genomes had intron-containing tRNAeMet and tRNATyr genes with modest sequence conservation and a strictly conserved tRNAAla-AGC species. Regulatory elements found upstream (TATA-box and CAA motifs) and downstream (poly(T) signals) of the tDNAs were present in only a fraction of the detected tDNAs. A and B boxes within the tDNA coding region show varying consensus sequences depending on the tRNA isotype and lineage. The chloroplast genomes, but not the mitogenomes, possess relatively conserved tRNA gene organization. These findings reveal differences and patterns acquired by plant genomes throughout evolution and can serve as a foundation for further studies on plant tRNA gene function and regulation. [ABSTRACT FROM AUTHOR]

The diversity of phage-related sequences (PRSs) and their site-specific integration into the genomes of nonpathogenic, agriculturally valuable, nitrogen-fixing root nodule bacteria, such as Sinorhizobium meliloti, were evaluated in this study. A total of 314 PRSs, ranging in size from 3.24 kb to 88.98 kb, were identified in the genomes of 27 S. meliloti strains. The amount of genetic information foreign to S. meliloti accumulated in all identified PRSs was 6.30 Mb. However, more than 53% of this information was contained in prophages (Phs) and genomic islands (GIs) integrated into genes encoding tRNAs (tRNA genes) located on the chromosomes of the rhizobial strains studied. It was found that phiLM21-like Phs were predominantly abundant in the genomes of S. meliloti strains of distant geographical origin, whereas RR1-A- and 16-3-like Phs were much less common. In addition, GIs predominantly contained fragments of phages infecting bacteria of distant taxa, while rhizobiophage-like sequences were unique. A site-specific integration analysis revealed that not all tRNA genes in S. meliloti are integration sites, but among those in which integration occurred, there were "hot spots" of integration into which either Phs or GIs were predominantly inserted. For the first time, it is shown that at these integration "hot spots", not only is the homology of attP and attB strictly preserved, but integrases in PRSs similar to those of phages infecting the Proteobacteria genera Azospirillum or Pseudomonas are also present. The data presented greatly expand the understanding of the fate of phage-related sequences in host bacterial genomes and also raise new questions about the role of phages in bacterial–phage coevolution. [ABSTRACT FROM AUTHOR]

Transfer RNAs (tRNAs) are noncoding RNAs involved in protein biosynthesis and have noncanonical roles in cellular metabolism, such as RNA silencing and the generation of transposable elements. Extensive tRNA gene duplications, modifications to mature tRNAs, and complex secondary and tertiary structures impede tRNA sequencing. As such, a comparative genomic analysis of complete tRNA sets is an alternative to understanding the evolutionary processes that gave rise to the extant tRNA sets. Although the tRNA gene (tDNA) structure and distribution in prokaryotes and eukaryotes, specifically in vertebrates, yeasts, and flies, are well understood, there is little information regarding plants. A detailed and comprehensive analysis and annotation of tDNAs from the genomes of 44 eudicots, 20 monocots, and five other non-eudicot and non-monocot species belonging to the Ceratophyllaceae and the ANA (Amborellales, Nymphaeales, and Austrobaileyales) clade will provide a global picture of plant tDNA structure and organization. Plant genomes exhibit varying numbers of nuclear tDNAs, with only the monocots showing a strong correlation between nuclear tDNA numbers and genome sizes. In contrast, organellar tDNA numbers varied little among the different lineages. A high degree of tDNA duplication in eudicots was detected, whereby most eudicot nuclear genomes (91%) and only a modest percentage of monocot (65%) and ANA nuclear genomes (25%) contained at least one tDNA cluster. Clusters of tRNATyr–tRNASer and tRNAIle genes were found in eudicot and monocot genomes, respectively, while both eudicot and monocot genomes showed clusters of tRNAPro genes. All plant genomes had intron-containing tRNAeMet and tRNATyr genes with modest sequence conservation and a strictly conserved tRNAAla-AGC species. Regulatory elements found upstream (TATA-box and CAA motifs) and downstream (poly(T) signals) of the tDNAs were present in only a fraction of the detected tDNAs. A and B boxes within the tDNA coding region show varying consensus sequences depending on the tRNA isotype and lineage. The chloroplast genomes, but not the mitogenomes, possess relatively conserved tRNA gene organization. These findings reveal differences and patterns acquired by plant genomes throughout evolution and can serve as a foundation for further studies on plant tRNA gene function and regulation.

Protein arginine methyltransferase 1 (PRMT1) is a major type I arginine methyltransferase that catalyzes the formation of monomethyl and asymmetric dimethylarginine in protein substrates. It was first identified to asymmetrically methylate histone H4 at the third arginine residue forming the H4R3me2a active histone mark. However, several protein substrates are now identified as being methylated by PRMT1. As a result of its association with diverse classes of substrates, PRMT1 regulates several biological processes like chromatin dynamics, transcription, RNA processing, and signal transduction. The review provides an overview of PRMT1 structure, biochemical features, specificity, regulation, and role in cellular functions. We discuss the genomic distribution of PRMT1 and its association with tRNA genes. Further, we explore the different substrates of PRMT1 involved in splicing. In the end, we discuss the proteins that interact with PRMT1 and their downstream effects in diseased states. [ABSTRACT FROM AUTHOR]

Coding sequence evolution is influenced by both natural selection and neutral evolutionary forces. In many species, the effects of mutation bias, codon usage, and GC-biased gene conversion (gBGC) on gene sequence evolution have not been detailed. Quantification of how these forces shape substitution patterns is therefore necessary to understand the strength and direction of natural selection. Here, we used comparative genomics to investigate the association between base composition and codon usage bias on gene sequence evolution in butterflies and moths (Lepidoptera), including an in-depth analysis of underlying patterns and processes in one species, Leptidea sinapis. The data revealed significant G/C to A/T substitution bias at third codon position with some variation in the strength among different butterfly lineages. However, the substitution bias was lower than expected from previously estimated mutation rate ratios, partly due to the influence of gBGC. We found that A/T-ending codons were overrepresented in most species, but there was a positive association between the magnitude of codon usage bias and GC-content in third codon positions. In addition, the tRNA-gene population in L. sinapis showed higher GC-content at third codon positions compared to coding sequences in general and less overrepresentation of A/T-ending codons. There was an inverse relationship between synonymous substitutions and codon usage bias indicating selection on synonymous sites. We conclude that the evolutionary rate in Lepidoptera is affected by a complex interaction between underlying G/C -> A/T mutation bias and partly counteracting fixation biases, predominantly conferred by overall purifying selection, gBGC, and selection on codon usage. [ABSTRACT FROM AUTHOR]

Simple Summary: Diabetes has a high mortality rate. Diabetes mellitus is a state of hyperglycemia or high glucose levels caused by the inability of the body to produce insulin, insulin resistance, or both. One of the causes of diabetes is the occurrence of mutations in mitochondrial genome genes and the loss of transfer RNA modification. Transfer RNA genes are part of the mitochondrial DNA genome that act as adapters and are key for protein synthesis. In this review, we discuss the structure of transfer RNA, mutations associated with and their relation to various diseases, as well as mutations associated with type 2 diabetes mellitus. In addition, methods that have been used to identify mutations and ideas for treatment are discussed. Transfer RNA (tRNA) genes in the mitochondrial DNA genome play an important role in protein synthesis. The 22 tRNA genes carry the amino acid that corresponds to that codon but changes in the genetic code often occur such as gene mutations that impact the formation of adenosine triphosphate (ATP). Insulin secretion does not occur because the mitochondria cannot work optimally. tRNA mutation may also be caused by insulin resistance. In addition, the loss of tRNA modification can cause pancreatic β cell dysfunction. Therefore, both can be indirectly associated with diabetes mellitus because diabetes mellitus, especially type 2, is caused by insulin resistance and the body cannot produce insulin. In this review, we will discuss tRNA in detail, several diseases related to tRNA mutations, how tRNA mutations can lead to type 2 diabetes mellitus, and one example of a point mutation that occurs in tRNA. [ABSTRACT FROM AUTHOR]

Saccharomyces cerevisiae has 6 synonymous tRNACCATrp genes encoding the identical sequence, including their intronic region. They are supposed to express tRNACCATrp in the same quality and quantity. Here, we generated single to quintuple deletion strains with all the possible combinations of the synonymous tRNACCATrp genes to analyze whether those individual genes equally contribute cell viability and tRNA production. The quintuple deletion strains that only harbor tW(CCA)J, tW(CCA)M, or tW(CCA)P were viable but almost lethal while the other quintuple deletions showed moderately impaired growth. These growth differences were not obvious among the quadruple deletion strains, which expressed almost one third of mature tRNACCATrp in the wild type. Therefore, no dosage compensation operates for tRNACCATrp amount, and growth variations among the quintuple deletion strains may not simply reflect differences in tRNACCATrp shortage. Yeast may retain the redundancy of tRNACCATrp genes for a noncanonical function(s) beyond the supply of the tRNA to translation. [ABSTRACT FROM AUTHOR]

Abstract Background Mycobacteria occupy various ecological niches and can be isolated from soil, tap water and ground water. Several cause diseases in humans and animals. To get deeper insight into our understanding of mycobacterial evolution focusing on tRNA and non-coding (nc)RNA, we conducted a comparative genome analysis of Mycobacterium mucogenicum (Mmuc) and Mycobacterium neoaurum (Mneo) clade members. Results Genome sizes for Mmuc- and Mneo-clade members vary between 5.4 and 6.5 Mbps with the complete Mmuc T (type strain) genome encompassing 6.1 Mbp. The number of tRNA genes range between 46 and 79 (including one pseudo tRNA gene) with 39 tRNA genes common among the members of these clades, while additional tRNA genes were probably acquired through horizontal gene transfer. Selected tRNAs and ncRNAs (RNase P RNA, tmRNA, 4.5S RNA, Ms1 RNA and 6C RNA) are expressed, and the levels for several of these are higher in stationary phase compared to exponentially growing cells. The rare tRNAIleTAT isoacceptor and two for mycobacteria novel ncRNAs: the Lactobacillales-derived GOLLD RNA and a homolog to the antisense Salmonella typhimurium phage Sar RNA, were shown to be present and expressed in certain Mmuc-clade members. Conclusions Phages, IS elements, horizontally transferred tRNA gene clusters, and phage-derived ncRNAs appears to have influenced the evolution of the Mmuc- and Mneo-clades. While the number of predicted coding sequences correlates with genome size, the number of tRNA coding genes does not. The majority of the tRNA genes in mycobacteria are transcribed mainly from single genes and the levels of certain ncRNAs, including RNase P RNA (essential for the processing of tRNAs), are higher at stationary phase compared to exponentially growing cells. We provide supporting evidence that Ms1 RNA represents a mycobacterial 6S RNA variant. The evolutionary routes for the ncRNAs RNase P RNA, tmRNA and Ms1 RNA are different from that of the core genes.

Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability., (© 2024 The Author(s). Biotechnology Journal published by Wiley‐VCH GmbH.)

Abstract Background Among viruses, bacteriophages are a group of special interest due to their capacity of infecting bacteria that are important for biotechnology and human health. Composting is a microbial-driven process in which complex organic matter is converted into humus-like substances. In thermophilic composting, the degradation activity is carried out primarily by bacteria and little is known about the presence and role of bacteriophages in this process. Results Using Pseudomonas aeruginosa as host, we isolated three new phages from a composting operation at the Sao Paulo Zoo Park (Brazil). One of the isolated phages is similar to Pseudomonas phage Ab18 and belongs to the Siphoviridae YuA-like viral genus. The other two isolated phages are similar to each other and present genomes sharing low similarity with phage genomes in public databases; we therefore hypothesize that they belong to a new genus in the Podoviridae family. Detailed genomic descriptions and comparisons of the three phages are presented, as well as two new clusters of phage genomes in the Viral Orthologous Clusters database of large DNA viruses. We found sequences encoding homing endonucleases that disrupt a putative ribonucleotide reductase gene and an RNA polymerase subunit 2 gene in two of the phages. These findings provide insights about the evolution of two-subunits RNA polymerases and the possible role of homing endonucleases in this process. Infection tests on 30 different strains of bacteria reveal a narrow host range for the three phages, restricted to P. aeruginosa PA14 and three other P. aeruginosa clinical isolates. Biofilm dissolution assays suggest that these phages could be promising antimicrobial agents against P. aeruginosa PA14 infections. Analyses on composting metagenomic and metatranscriptomic data indicate association between abundance variations in both phage and host populations in the environment. Conclusion The results about the newly discovered and described phages contribute to the understanding of tailed bacteriophage diversity, evolution, and role in the complex composting environment.

Synonymous codon use is non-random. Codons most used in highly transcribed genes, often called optimal codons, typically have high gene counts of matching tRNA genes (tRNA abundance) and promote accurate and/or efficient translation. Non-optimal codons, those least used in highly expressed genes, may also affect translation. In multicellular organisms, codon optimality may vary among tissues. At present, however, tissue specificity of codon use remains poorly understood. Here, we studied codon usage of genes highly transcribed in germ line (testis and ovary) and somatic tissues (gonadectomized males and females) of the beetle Tribolium castaneum. The results demonstrate that: (i) the majority of optimal codons were organism-wide, the same in all tissues, and had numerous matching tRNA gene copies (Opt-codon↑tRNAs), consistent with translational selection; (ii) some optimal codons varied among tissues, suggesting tissue-specific tRNA populations; (iii) wobble tRNA were required for translation of certain optimal codons (Opt-codonwobble), possibly allowing precise translation and/or protein folding; and (iv) remarkably, some non-optimal codons had abundant tRNA genes (Nonopt-codon↑tRNAs), and genes using those codons were tightly linked to ribosomal and stress-response functions. Thus, Nonopt-codon↑tRNAs codons may regulate translation of specific genes. Together, the evidence suggests that codon use and tRNA genes regulate multiple translational processes in T. castaneum. [ABSTRACT FROM AUTHOR]

Background: Among viruses, bacteriophages are a group of special interest due to their capacity of infecting bacteria that are important for biotechnology and human health. Composting is a microbial-driven process in which complex organic matter is converted into humus-like substances. In thermophilic composting, the degradation activity is carried out primarily by bacteria and little is known about the presence and role of bacteriophages in this process. Results: Using Pseudomonas aeruginosa as host, we isolated three new phages from a composting operation at the Sao Paulo Zoo Park (Brazil). One of the isolated phages is similar to Pseudomonas phage Ab18 and belongs to the Siphoviridae YuA-like viral genus. The other two isolated phages are similar to each other and present genomes sharing low similarity with phage genomes in public databases; we therefore hypothesize that they belong to a new genus in the Podoviridae family. Detailed genomic descriptions and comparisons of the three phages are presented, as well as two new clusters of phage genomes in the Viral Orthologous Clusters database of large DNA viruses. We found sequences encoding homing endonucleases that disrupt a putative ribonucleotide reductase gene and an RNA polymerase subunit 2 gene in two of the phages. These findings provide insights about the evolution of two-subunits RNA polymerases and the possible role of homing endonucleases in this process. Infection tests on 30 different strains of bacteria reveal a narrow host range for the three phages, restricted to P. aeruginosa PA14 and three other P. aeruginosa clinical isolates. Biofilm dissolution assays suggest that these phages could be promising antimicrobial agents against P. aeruginosa PA14 infections. Analyses on composting metagenomic and metatranscriptomic data indicate association between abundance variations in both phage and host populations in the environment. Conclusion: The results about the newly discovered and described phages contribute to the understanding of tailed bacteriophage diversity, evolution, and role in the complex composting environment. [ABSTRACT FROM AUTHOR]

The Gram-positive bacterium Bacillus megaterium was systematically developed for the plasmid-based production of recombinant proteins at the gram-per-liter scale. The amount of protein produced per cell was found strongly correlated to the codon usage of the heterologous gene of interest in comparison to the codon usage of B. megaterium. For analyzing the influence of rare codons on the translational efficiency and protein production in B. megaterium, a test system using the gene for the green fluorescent protein (GFP) as reporter was established. For this purpose, four consecutive identical codons were introduced into the 5′ end of gfp and the resulting variations in GFP formation were quantified. Introduction of the rare codons GCC, CGG, and ACC for alanine, arginine, and threonine reduced GFP production 2.1-, 3.3-, and 1.7-fold in comparison to the favored codons GCU, CGU, and ACA, respectively. Coexpression of the corresponding rare codon tRNA (rc tRNA) genes improved GFP production 4.2-, 2.7-, and 1.7-fold, respectively. The system was applied to the production of a formate dehydrogenase (FDH) from Mycobacterium vaccae and an extracellular hydrolase (TFH) from Thermobifida fusca. Coexpression of one to three different rc tRNA genes resulted in an up to 18-fold increased protein production. Interestingly, rc tRNA gene coexpression also elevated the production of M. vaccae FDH and T. fusca TFH from codon optimized genes, indicating a general positive effect by rc tRNA gene overexpression on the protein production in B. megaterium. Thus, the basis for a B. megaterium enhanced production strain coexpressing rc tRNA genes was laid. [ABSTRACT FROM AUTHOR]

In the effort to produce proteins coded by diverse genomes, structural genomics projects often must express genes containing codons that are rare in the production strain. To address this problem, genes expressing tRNAs corresponding to those codons are typically coexpressed from a second plasmid in the host strain, or from genes incorporated into production plasmids. Here we describe the modification of a series of LIC pMCSG vectors currently used in the high-throughput (HTP) production of proteins to include crucial tRNA genes covering rare codons for Arg (AGG/AGA) and Ile (AUA). We also present variants of these new vectors that allow analysis of ligand binding or co-expression of multiple proteins introduced through two independent LIC steps. Additionally, to accommodate the cloning of multiple large proteins, the size of the plasmids was reduced by approximately one kilobase through the removal of non-essential DNA from the base vector. Production of proteins from core vectors of this series validated the desired enhanced capabilities: higher yields of proteins expressed from genes with rare codons occurred in most cases, biotinylated derivatives enabled detailed automated ligand binding analysis, and multiple proteins introduced by dual LIC cloning were expressed successfully and in near balanced stoichiometry, allowing tandem purification of interacting proteins. [ABSTRACT FROM AUTHOR]

The mitochondrial (mt) genome of the sea anemone Metridium senile contains genes for only two transfer RNAs (tRNAs), tRNAf-Met and tRNATrp. Experiments were conducted to seek evidence for the occurrence of functional tRNAs corresponding to these genes and for the participation of nuclear DNA-encoded tRNAs in mt-protein synthesis. RNA sequences corresponding to the two mt-tRNA genes were located in mitochondria and it was shown that 3′-CC (and possibly A, but no other nucleotide) is added post-transcriptionally to the 3′ end of at least 50 % of mt-tRNAf-Met molecules and to a small fraction of the mt-tRNATrp molecules. Using specific oligonucleotide primers based on expected nuclear DNA-encoded tRNAs in a series of RACE experiments, we located the nuclear genes for tRNAGln, tRNAIle, tRNAi-Met, tRNAVal and tRNAThr. Data from Northern blot analyses indicated that mtDNA-encoded tRNAf-Met is limited to mitochondria but that nuclear DNA-encoded tRNAVal and tRNAi-Met are present in the cytoplasm and in mitochondria. These data provide direct evidence that in M. senile, mature, functional tRNAs are transcribed from the mtDNA-encoded tRNAf-Met and tRNATrp genes, and are consistent with the interpretation that both nuclear DNA-encoded tRNAVal and tRNAi-Met are utilized in mitochondrial and cytosolic protein synthesis. [ABSTRACT FROM AUTHOR]

Recent findings provide evidence that tDNAs function as chromatin insulators from yeast to humans. TFIIIC, a transcription factor that interacts with the B-box in tDNAs as well as thousands of ETC sites in the genome, is responsible for insulator function. Though tDNAs are capable of enhancer-blocking and barrier activities for which insulators are defined, new insights into the relationship between insulators and chromatin structure suggest that TFIIIC serves a complex role in genome organization. We review the role of tRNA genes and TFIIIC as chromatin insulators, and highlight recent findings that have broadened our understanding of insulators in genome biology. [ABSTRACT FROM AUTHOR]

A typical eukaryotic genome harbors a rich variety of repetitive elements. The most abundant are retrotransposons, mobile retroelements that utilize reverse transcriptase and an RNA intermediate to relocate to a new location within the cellular genomes. A vast majority of the repetitive mammalian genome content has originated from the retrotransposition of SINE (100-300 bp short interspersed nuclear elements that are derived from the structural 7SL RNA or tRNA), LINE (7kb long interspersed nuclear element), and LTR (2-3 kb long terminal repeats) transposable element superfamilies. Broadly labeled as 'evolutionary junkyard' or 'fossils', this enigmatic 'dark matter' of the genome possesses many yet to be discovered properties. [ABSTRACT FROM AUTHOR]

The article presents a study which analyzes the mitochondrial tRNA genes and flanking regions in umbilical cord tissue from uninfected infants who receive AZT-based therapies for prophylaxis of HIV-1. It utilized the sensitive vertical denaturing gradient gel electrophoresis (DGGE) method. It disinfected biological specimens and isolate the DNA. Furthermore, the human mtDNA was amplified by PCR then heteroduplex formation and photoinduced crosslinking followed. It also involved DNA sequencing and estimation of degree of heteroplasmy or mutant fraction. Lastly, human TK6 B-lymphoblastoid cells were cultured and DGGE-based screening for mtDNA sequence variants was done. It did not establish the link of increased detection of sequence changes at two polymorphic sites in AZT-exposed infants.

We determined the complete nucleotide sequence of the chloroplast genome of Selaginella uncinata, a lycophyte belonging to the basal lineage of the vascular plants. The circular double-stranded DNA is 144,170 bp, with an inverted repeat of 25,578 bp separated by a large single copy region (LSC) of 77,706 bp and a small single copy region (SSC) of 40,886 bp. We assigned 81 protein-coding genes including four pseudogenes, four rRNA genes and only 12 tRNA genes. Four genes, rps15, rps16, rpl32 and ycf10, found in most chloroplast genomes in land plants were not present in S. uncinata. While gene order and arrangement of the chloroplast genome of another lycophyte, Hupertzia lucidula, are almost the same as those of bryophytes, those of S. uncinata differ considerably from the typical structure of bryophytes with respect to the presence of a unique 20 kb inversion within the LSC, transposition of two segments from the LSC to the SSC and many gene losses. Thus, the organization of the S. uncinata chloroplast genome provides a new insight into the evolution of lycophytes, which were separated from euphyllophytes approximately 400 million years ago. [ABSTRACT FROM AUTHOR]

Abstract: The TATA box is a key upstream control element for basal tRNA gene transcription by RNA polymerase III in some eukaryotes, such as the fission yeast (Schizosaccharomyces pombe) and higher plants, but not in others such as the budding yeast (Saccharomyces cerevisiae). To gain information on this differential TATA box requirement, we examined side-by-side the in vitro transcription properties of TATA-containing and TATA-mutated plant and S. cerevisiae tDNAs in homologous in vitro transcription systems from both organisms and in a hybrid system in which yeast TBP was replaced by its plant homologue. The data support the general conclusion that specific features of the plant transcription machinery, rather than upstream region architecture per se, are responsible for the much stronger TATA box dependence of the plant system. In both systems, however, a strong influence of the TATA box on transcription start site selection was observed. This was particularly striking in the case of plant tDNAs, where TATA-rich upstream regions were found to favour the use of alternative initiation sites. Replacement of yeast TBP with its plant counterpart did not confer any general TATA box responsiveness to the yeast transcription machinery. Interactions involving components other than TBP are thus responsible for the strong TATA box requirement of plant tDNA transcription. [Copyright &y& Elsevier]

Abstract: Promoter regions regulating the transcription of all cp-like tRNA genes encoded by the sunflower chondriome have been identified. Some of these genes are part of clusters where the first gene is a typical mitochondrial isoform. Promoters regulating the transcription of single cp-like tRNA genes have a variable structure whereas those regulating the transcription of native genes or clusters with typical mitochondrial genes in the first position conform to a similar common structure. The variability of promoter regions described in this paper could be the result of modifications of regions having, at the moment of the cpDNA insertion event, only minimal structural features as promoters. [Copyright &y& Elsevier]

Transcription of eukaryotic tRNA genes relies on the TFIIIC-dependent recruitment of TFIIIB on a ∼50 bp region upstream of the transcription start site (TSS). TFIIIC specifically interacts with highly conserved, intragenic promoter elements, while the contacts between TFIIIB and the upstream DNA have long been considered as largely non-specific. Through a computer search procedure designed to detect shared, yet degenerate sequence features, we have identified a conserved sequence pattern upstream of Saccharomyces cerevisiae tDNAs. This pattern consists of four regions in which particular sequences are over-represented. The most downstream of these regions surrounds the TSS, while the other three districts of sequence conservation (appearing as a centrally located TATA-like sequence flanked by T-rich elements on both sides) are located across the DNA region known to interact with TFIIIB. Upstream regions whose sequence conforms to this pattern were found to potentiate tRNA gene transcription, both in vitro and in vivo, by enhancing TFIIIB binding. A conserved pattern of DNA bendability was also revealed, with peaks of bending propensity centered on the TATA-like and the TSS regions. Sequence analysis of other eukaryotic genomes further revealed the widespread occurrence of conserved sequence patterns upstream of tDNAs, with striking lineage-specific differences in the number and sequence of conserved motifs. Our data strongly support the notion that tRNA gene transcription in eukaryotes is modulated by composite TFIIIB binding sites that may confer responsiveness to variation in TFIIIB activity and/or concentration. [Copyright &y& Elsevier]

All known lambdoid prophages of Escherichia coli have the same orientation with respect to direction of chromosomal replication. This includes 12 prophages that are replicated in one direction and five in the other. Among candidate explanations, the most amenable to experimental study is an effect on dif site function in assuring chromosomal segregation. This is but one of numerous examples of strand bias in the E. coli genome, all of which may interact with one another. [Copyright &y& Elsevier]

The nucleotide sequence of an 8 kbp region of pea (Pisum sativum L.) chloroplast DNA containing the rRNA operon and putative promoter sites has been determined and compared to the corresponding sequences from maize, tobacco and the liverwort Marchantia polymorpha. The chloroplast DNA species of all vascular plants investigated, with the exception of a few legumes including pea, and of Marchantia contain an inverted repeat with an rRNA operon. The pea rRNA operon is the first sequenced rRNA operon from a plant with only one copy of the rRNA genes per molecule of chloroplast DNA. The organization of the operon is the same as for maize, tobacco and Marchantia, i.e. tRNA-Val gene/16S rRNA gene/spacer with intron-containing genes for tRNA-Ile and tRNA-Ala/23S rRNA gene/4.5S rRNA gene/5S rRNA gene. Current evidence suggests that the tRNA-Val gene may not be contranscribed with the other genes. For pea 16S, 23S, 4.5S and 5S rRNA have 1488, 2813, 105 and 121 nucleotides, respectively. The homologies of the entire operon (the tRNA-Val gene-5S rRNA region) to those from tobacco, maize and Marchantia are 88, 82 and 79%, respectively. The corresponding homologies for tobacco/maize, tobacco/Marchantia and maize/Marchantia have similar values. The 16S and 23S rRNA genes from pea are more than 90% homologous to those from the 3 other species. We conclude that the fact that pea only has one set of rRNA genes per molecule of chloroplast DNA is apparently not correlated with any significant difference between the pea operon and the rRNA operons from tobacco, maize and Marchantia. [ABSTRACT FROM AUTHOR]

22 tRNA genes corresponding to 17 tRNA species were localized on the master circle of Petunia hybrida mitochondrial (mt) DNA. Genes for trnN, trnM, trnS-GGA, trnW and trnH are of the 'chloroplast-like' type and presumably originate from promiscuous chloroplast (cp) DNA sequences inserted into the petunia mitochondrial genome. A comparison of the mt tRNAs or tRNA genes population present in two monocotyledonous plants (wheat and maize) and two dicotyledonous plants (petunia and potato) show slight differences in the genetic origin of individual tRNAs. The organization of the petunia mt tRNA genes as well as the number of tRNA gene copies, compared to other plant species, is discussed. [ABSTRACT FROM AUTHOR]

A genomic bank of nuclear DNA (nDNA) from the higher plant Phaseolus vulgaris, constructed using the lambda EMBL-4 vector, has been screened for the presence of tRNA genes. One of the many positive recombinants was found to hybridise several times stronger than the other positives, and has been shown to contain several tRNA genes. We report the structure of two nuclear tRNA genes for tRNA, namely tRNA(UGG) and tRNA(AGG), and that of a 'pseudogene' for tRNA. This 'pseudogene', despite showing 95% homology with the other tRNA species presented here, has several features which are likely to affect its transcription or its functioning as a tRNA. [ABSTRACT FROM AUTHOR]

Isolated chloroplasts from Pisum sativum were found to contain at least 32 tRNA species. Hybridization of in vitro labeled, identified, chloroplast tRNAs to Pisum chloroplast DNA fragments revealed the locations of the tRNA genes on the circular chloroplast genome. Comparison of this gene map to the maps of Vicia faba and Phaseolus vulgaris showed that the chloroplast genomes of Pisum and Phaseolus are otherwise more closely related than either genome is to the chloroplast genome of Vicia. Furthermore, the results suggest how possible recombination events could be involved in the evolution of these three closely related, but divergent, chloroplast genomes. [ABSTRACT FROM AUTHOR]

The location and nucleotide sequence of tobacco chloroplast genes for tRNA (GGA), tRNA (UGU), tRNA (UAA) and tRNA (GAA) ( trnS-GGA, trnT-UGU, trnL-UAA and trnF-GAA, respectively) have been determined. These genes are located in the 10 kbp BamHI fragment which lies in the middle of the large single-copy region of the chloroplast DNA. The gene order is trnS- trnT- trnL- trnF. The trnS, trnL and trnF are encoded on the same strand while the trnT on the opposite strand. The trnL contains a 503 bp intron like maize and broad bean trnL-UAAs. [ABSTRACT FROM AUTHOR]

Only three tRNA genes are present within a sequenced 12.35 kbp region of the 15.8 kbp mtDNA of Chlamydomonas reinhardtii, a unicellular green alga. The corresponding tRNAs, whose anticodons are specific for TGG (Trp), CAA/G (Gln) and ATG (Met) codons, all display conventional secondary structures. The tRNA gene encodes an elongator rather than initiator species. The standard genetic code is used in C. reinhardtii mitochondria, but codon distribution is highly biased: in a collection of six identified protein coding genes, nine codons (including TGA) are not used at all, while four other sense codons occur very infrequently. In spite of the absence of certain codons, a minimum of 23 tRNAs (assuming separate initiator and elongator tRNAs are used) is needed to translate the C. reinhardtii mitochondrial genetic code. It appears unlikely that this minimal tRNA set is encoded by C. reinhardtii mtDNA. [ABSTRACT FROM AUTHOR]

We have determined the DNA sequences of regions involved in two of the three inversions known to have occurred during the evolution of wheat chloroplast DNA. This establishes the extent of the second largest of the three inversions. Examination of these sequences suggests that although short repeated sequences are present, the endpoints of the second and third inversions are not associated with repeated sequences as long as those associated with the first inversion. However the endpoints of all three inversions are all adjacent to at least one tRNA gene, and there is evidence that three of the tRNA genes have been subjected to partial duplication, possibly at the time of inversion. This suggests that tRNA genes might be involved with rearrangements of chloroplast DNA, as has also been postulated for mitochondrial DNA. [ABSTRACT FROM AUTHOR]

We report the nucleotide sequence of three tRNA genes from maize mitochondria. The genes are located in two BamHI fragments, 3.55 and 5.7 kb long, adjacent to the S2 sequence in the maize mitochondrial genome. On the 3.55 kb BamHI fragment, we have characterized a tRNA (GCA) gene. A strong sequence homology of this tRNA (GCA) gene with its chloroplast counterpart in wheat suggests that it may be part of a chloroplast DNA insertion into the mitochondrial genome. This gene has been found to be transcribed in the mitochondrion. Two tRNA genes are located on the 5.7 kb BamHI fragment, separated from each other by 250 bp. One is a mitochondrial tRNA (GCU) gene. The other, a non-transcribed tRNA Phe-like gene, is interrupted by a 49 base-pair inserted DNA sequence in the variable loop and has a Leu (UAA) anticodon. [ABSTRACT FROM AUTHOR]

We have used bean mitochondrial (mt) and chloroplast (cp) tRNA as probes to locate the corresponding genes on the mt and cp genomes of wheat and we have determined the nucleotide sequences of the wheat mt and cp tRNATrp genes and of the flanking regions. Sequence comparisons show that the wheat mt and cp tRNA genes are 97% homologous. On the wheat cp DNA, a tRNAgene was found 139 by upstream of the cp tRNA gene. On the wheat mt DNA, a sequence of 23 nucleotides completely homologous with the 3' end of this cp tRNA gene was found 136 by upstream of the rut tRNA gene, but there is only 38% homology between cp and mt wheat genomes in the intergenic regions. The overall organization of this region in the chloroplast genome (a tRNA gene separated by about 140 by from a tRNA gene) is also found in the mitochondrial genome, suggesting that this mitochondrial fragment might have originated from a chloroplast DNA insertion. A comparison of the genes and of the intergenic regions located between the tRNA gene and the tRNA (or partial tRNA) gene shows that there is an almost complete conservation of these sequences in the mitochondrial DNA of wheat and maize, whereas wheat mt and cp intergenic regions show more sequence divergence. Wheat mt tRNA gene is encoded by the main mt genome (accounted for by the master chromosome) but, in the case of maize mitochondria, this gene was found to be encoded by the 2.3 kb linear plasmid, indicating that this plasmid is not dispensable in maize mitochondria. [ABSTRACT FROM AUTHOR]

We have isolated and characterized a Ty element from a yeast cosmid library which exhibits several unsual features: it is flanked by non-homologous delta elements and directly associated with a singular delta element. A tRNA(Glu3) gene and tRNA(Cys) gene are found in conjunction with this element, located in opposite orientation on either end of it. The sequence information now available for several Ty elements has been used in a detailed comparative analysis to determine conserved features among the Ty elements, preferably between class I elements and a class II element. Highly conserved sequence motifs appear to be located at the borders of particular segments that correspond to the putative protein domains of the Tys. Furthermore, we include a comparison of the best-conserved amino acid homologies for these putative proteins of Ty elements, transposable elements from other organisms and several retroviral proviruses to confirm their close structural resemblance. [ABSTRACT FROM AUTHOR]

Of S. cerevisiae UAA specific suppressors three leucine-insertors have been remaining unmapped. Mapping of them was attempted by using a set of eight strains that contained a total of 60 markers distributing over the entire genome; the markers were about 30 cM apart and they covered about 70% of the genome. Two supressors, SUP28 and SUP33, were localized on the right arm of chromosome XIV and on the left arm of chromosome XI, respectively. The other suppressor, SUP32, was not mapped, but its location was confined to a few regions in the genome. The present result and the previously available informations clearly show that the UAA suppressor loci are dispersedly distributing ovet the entire S. cerevisiae genome. [ABSTRACT FROM AUTHOR]

Continuing the analysis of rat liver mitochondria) DNA, we have determined the sequences of two segments containing several tRNA genes. One of these segments also comprises the L-strand origin of replication. The characteristics of the 15 rat liver mitochondrial tRNA structures known at present are compared with those from other mammalian mitochondria. [ABSTRACT FROM AUTHOR]

The full-length 5218-bp sequence of the mobile genetic element Tdd-3 from Dictyostelium discoideum is described. Tdd-3 encodes two overlapping open reading frames (ORFs) flanked by non-redundant, untranslated regions. The deduced amino acid sequence of ORF2 is homologous to reverse transcriptases (RTs) encoded by the class of poly(A) retrotransposons. ORF2 also encodes a putative protein domain related to the family of apurinic/apyrimidinic (AP) endonucleases, whose retroelement-encoded homologs have recently been proposed to represent the integrase function of poly(A) retrotransposons. Comparison of several genomic Tdd-3 copies revealed that element insertion is orientation specific and occurs about 100 bp downstream of tRNA genes in the D. discoideum genome. These properties of Tdd-3 suggest that the element is a tRNA gene-associated poly(A) retroelement present in the D. discoideum genome. Analysis of several cloned cDNAs derived from Tdd-3-specific plus strand RNAs indicate that the element is transcribed and polyadenylated during the growth of D. discoideum cells. [ABSTRACT FROM AUTHOR]

Mature tRNAs are remarkably similar in all cells. However, the primary transcripts from tRNA genes can vary considerably due to differences in gene organization. RNase P must be able to recognize the elements that are common to all tRNA precursors to accurately remove the 5'-leader sequences. [ABSTRACT FROM AUTHOR]

Bean nuclear genes for tRNAPro, tRNAThrand tRNALeu were isolated. Expression of the tRNAPro genes was demonstrated in vivo and sequence analysis suggested amplification of the tRNAPro gene copy number through duplication of a gene cluster at the same locus of the bean genome. The two tRNAThr genes isolated were actively transcribed and their transcripts processed in a HeLa cell system. In vivo expression tests of these genes and aminoacylation assays of the corresponding in vitro transcripts showed the presence of identity determinants in the anticodon of plant tRNAThr. The tRNALeu gene was not expressed due to deviation from the consensus in the internal B-box promoter. The same sequence deviation also prevented aminoacylation of the corresponding in vitro transcript. This tRNALeu however exists in plants and is synthesized from another gene with a consensus B-box promoter. Plant mitochondria import from the cytosol a number of nucleus-encoded tRNAs, including tRNALeu and tRNAThr. From the available sequence data, we could not identify any conserved structural motif characteristic for the nucleus-encoded tRNAs imported into plant mitochondria, either in the tRNAs, or in the gene flanking sequences. These results suggest that recognition of tRNAs for import is idiosyncratic and likely to depend on protein/RNA interactions that are specific to each tRNA or each isoacceptor group. [ABSTRACT FROM AUTHOR]

The New World monkeys are divided into two main groups, Callitrichidae and Cebidae. Callimico goeldii shares traits with both the Cebidae and the Callitrichidae. Recent morphological phyletic studies generally place Callimico as the most basal member of the Callitrichidae. In contrast, genetic studies (immunological, restriction fragment, and sequence data) have consistently placed Callimico somewhere within the Callitrichidae, not basal to this clade. A DNA sequence data set from the terminal 236 codons of the mitochondrial ND4 gene and the tRNAHis, tRNASer, and tRNALeu genes was generated to clarify the position of Callimico. The sequences of 887 base pairs were analyzed by maximum-parsimony, neighbor-joining, and maximum-likelihood methods. The results of these various methods are generally congruent and place Callimico within the Callitrichidae between the marmosets ( Callithrix and Cebuella) and the tamarins ( Saguinus and Leontopithecus). Combined analyses of all suitable nuclear and mitochondrial gene sequences confirm the position of Callimico between the marmosets and the tamarins. As available molecular evidence indicates that Callimico is more closely related to the marmosets than to the tamarins, a reconsideration of the morphological evidence in light of the consensus tree from DNA sequence analyses is warranted. The marmosets and tamarins share four morphological characters (loss of the third molar, loss of the hypocone, reduced body size, reproductive twinning). Dwarfism may have evolved repeatedly among the Callitrichidae. It is well-known that the loss of a character can occur many times independently. The reproduction of marmosets and tamarins is extremely specialized and it is difficult to imagine that this complex and unique twinning system evolved separately in marmosets and tamarins. However, it is possible that a secondary reversal to single offspring took place in Callimico. [ABSTRACT FROM AUTHOR]

By cloning and sequencing 3.4 kilobases of snow goose mtDNA we found that the ND5 gene is followed by the genes for cytochrome b, tRNA, tRNA, ND6, tRNA, the control region, tRNA, and srRNA. This order is identical to that of chicken, quail, and duck mtDNA but differs from that of mammals and a frog ( Xenopus). The mean extent of difference due to base substitution between goose and chicken is generally closer to the same comparison between rat and mouse but less than that between human and cow. For one of the nine regions compared (tRNA), the bird differences appear to be anomalous, possibly implicating altered functional constraints. Within the control region, several short sequences common to mammals are also conserved in the birds. Comparison of the goose control region with that of quail and chicken suggests that a sequence element with similarity to CSB-1 duplicated once prior to the divergence of goose and chicken and again on the lineage leading to chicken. Between goose (or duck) and chicken there are four times more transversions at the third positions of fourfold-degenerate codons in mitochondrial than in nuclear genes. [ABSTRACT FROM AUTHOR]

The nucleotide sequences of the mitochondrial origin of light-strand replication and the five tRNA genes surrounding it were determined for three marsupials. The region was found to be rearranged, leaving only the tRNA gene at the same position as in placental mammals and Xenopus. Distribution of the same rearranged genotype among two marsupial families indicates that the events causing the rearrangements took place in an early marsupial ancestor. The putative mitochondrial light-strand origin of replication in marsupials contains a hairpin structure similar to other vertebrate origins and, in addition, extensive flanking sequences that are not found in other vertebrates. Sequence comparisons among the marsupials as well as placentals indicate that the tRNA gene has been evolving under more constraints than the other tRNA genes. [ABSTRACT FROM AUTHOR]

The advent of direct sequencing via the polymerase chain reaction (PCR) has opened up the possibility of molecular studies on museum specimens. Here we analyze genetic variation in populations over time by applying PCR to DNA extracted from museum specimens sampled from populations of one species over the last 78 years. Included in this study were 43 museum specimens of the Panamint kangaroo rat Dipodomys panamintinus from localities representing each of three geographically distinct subspecies. These specimens were originally collected and prepared as dried skins in 1911, 1917, or 1937. For each specimen, a 225-bp segment of the mitochondrial genome was sequenced. These mitochondrial DNA sequences were compared to those of 63 specimens collected at the same localities in 1988. The three subspecies were nearly completely distinct. Only 2 of the 106 individuals shared mitochondrial types between subspecies. For all three localities, the diversity levels were maintained between the two temporal samples. The concordance observed between the two temporally separate phylogenies supports the use of museum specimens for phylogenetic inference. This study demonstrates the accuracy and routine nature of the use of museum specimens in the analysis of mitochondrial sequence variation in natural populations and, importantly, that a temporal aspect can now be added to such studies. [ABSTRACT FROM AUTHOR]

A novel group II intron has been identified in the pea ( Pisum sativum) mitochondrial genome. The gene harbouring this intron is identified as rps10 (encoding protein S10 of the small ribosomal subunit) by similarity to its known homologues in bacteria and in the mitochondrion of the liverwort Marchantia polymorpha. The rps10 gene is transcribed in pea, the intron is removed, and RNA editing in the rps10 reading frame increases similarity to its homologue in the M. polymorpha mitochondrion. Contrary to the situation in bacteria and Marchantia, rps10 is not part of a ribosomal-protein gene cluster in pea. It is flanked upstream by the genes trnF and trnP, encoding phenylalanine-and proline-accepting tRNAs, and downstream by cox1, encoding subunit 1 of the cytochrome-c-oxidase. Southern hybridization shows that sequences homologous to rps10 exist in potato mitochondria but not in mitochondria of Oenothera berteriana and Arabidopsis thaliana. The pea rps10 intron is homologous to introns in rrn26 and cox3 in the Marchantia mitochondrial genome, while the Marchantia rps10 gene lacks an intron. [ABSTRACT FROM AUTHOR]