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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]

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]

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]

The colourless, nonphotosynthetic protist Astasia longa is phylogenetically related to Euglena gracilis. The 73-kb plastid DNA (ptDNA) of A. longa is about half the size of most chloroplast DNAs (cpDNAs). More than 38 kb of the Astasia ptDNA sequence has been determined. No genes for photosynthetic function have been found except for rbcL. Identified genes include rpoB, tufA, and genes coding for three rRNAs, 17 tRNAs, and 13 ribosomal proteins. Not only is the nucleotide sequence of these genes highly conserved between A. longa and E. gracilis, but a number of these genes are clustered in a similar fashion and have introns in the same positions in both species. The results further support the idea that photosynthetic genes normally encoded in cpDNA have been preferentially lost in Astasia, but that the chloroplast genes coding for components of the plastid translational apparatus have been maintained. This apparatus might be needed for the expression of rbcL and also for that of still unidentified nonphotosynthetic genes of Astasia ptDNA. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, transcription of the mitochondrial genome starts at multiple initiation sites and is followed by the processing of multigenic transcripts at the 5′ and 3′ termini of tRNA sequences and in some intergenic regions. We have used a comparative approach to investigate the structure and function of the latter processing sites. We present here an analysis of the transcripts of a cluster of tRNA genes from the mitochondrial genome of Kluyveromyces lactis. The gene order of this cluster is the same as that of the cluster in S. cerevisiae but the sequence of the intergenic regions is different. A detailed analysis of transcripts has been performed using S1 mapping and primer extension techniques. The results can be summarized as follows: (1) transcription of the cluster very probably starts at initiation sites having the nonanucleotide sequence TTATAAGTA (which acts as a promoter in S. cerevisiae) and yields polygenic transcripts; (2) processing of these transcripts seems to occur through an ordered pathway of endonucleolytic events in which some tRNA sequences are preferentially excised and some endonucleolytic cuts occur more readily than others; (3) in two intergenic regions, strong signals indicate the existence of processing events. The sequences around these sites are similar in sequence and localization to S. cerevisiae intergenic processing sites, indicating a possible functional importance in maintaining a conserved order of tRNA genes in different species of yeasts. [ABSTRACT FROM AUTHOR]

To isolate the gene encoding the amino-acid biosynthetic enzyme acetolactate synthase (ALS) from the red alga Porphyra umbilicalis, PCR experiments were carried out using P. umbilicalis DNA as the template and degenerate oligonucleotides representing conserved regions of ALS amino-acid sequences. Interestingly, the PCR product (0.9 kb) hybridized exclusively to the plastid DNA of this red alga. DNA sequencing of two contiguous EcoRI plastid DNA clones revealed a 590 aminoacid open reading frame with 55 to 61% identity to cyanobacterial ALS sequences. A second gene ( argB) encoding another amino-acid biosynthetic enzyme, Nacetylglutamate kinase, was identified upstream of, and on the opposite strand to the gene encoding ALS ( ilvB). This is the first molecular characterization of a gene for an arginine biosynthetic enzyme from any plant. In addition, two tRNA genes, trnT(GGU) and trnY(GUA), were detected downstream from ilvB while four tRNA genes, trnfM(CAU), trnA(GGC), trnA(GGC), trnS(GCU) and trnD(GUC), were found downstream from argB. trnA(GGC) is not found in the chloroplast genomes of land plants. [ABSTRACT FROM AUTHOR]

RNA Polymerase III transcription factors from the cellular slime mold Dictyostelium discoideum were characterized, based on their stable binding to isolated tRNA genes. Different protein complexes are sequestered on DNA fragments containing tRNA genes depending on the conditions by which the nuclei were extracted. Binding specificity was determined through competition assays using competitor tRNA genes from the same gene family, from different gene families and from truncated tRNA genes. The complex with the highest multiformity of interdependent proteins is able to assemble with low affinity on a B-block-free tDNA template, whereas most lower molecular weight complexes require the presence of an intact B-block promoter element in order to assemble. [ABSTRACT FROM AUTHOR]

A 3454 base pair (bp) sequence of the large inverted repeat (IR) of chloroplast DNA (cpDNA) from the unicellular green alga Chlorella ellipsoidea has been determined. The sequence includes: (1) the boundaries between the IR and the large single copy (LSC) and the small single copy (SSC) regions, (2) the gene for psbA and (3) an approximately 1.0 kbp region between psbA and the rRNA genes which contains a variety of short dispersed repeats. The total size of the Chlorella IR was determined to be 15243 bp. The junction between the IR and the small single copy region is located close to the putative promoter of the rRNA operon (906 bp upstream of the-35 sequence on each IR). The junction between the IR and the large single copy region is also just upstream of the putative psbA promoter, 218 bp upstream from the ATG initiation codon. A few sets of unique sequences were found repeatedly around both junctions. Some of the sequences flanking the IR-LSC junction suggest a unidirectional and serial expansion of the IR within the genome. The psbA gene is located close to the LSC-side junction and codes for a protein of 352 amino acid residues. A highly conserved C-terminal Gly is absent. Unlike the psbA of Chlamydomonas species, which contains 2-4 large introns, the gene of Chlorella has no introns. The overall gene organization of the Chlorella IR is very different from that of higher plants, but a similar gene cluster of rrn-psbA is also found in the IR of Chlamydomonas species and in a single copy region of some chlorophyll a/c-containing algae, indicating a common evolutionary lineage of these cpDNAs. The origin and evolution of the IR structure are discussed in the light of these observations. [ABSTRACT FROM AUTHOR]

The nucleotide sequences of tRNA (GUU) and tRNA (GUA) genes from tomato mitochondria and their flanking regions have been determined. The tomato mitochondrial tRNA gene is located 2.1 kb downstream from the tRNA gene reported previously (Izuchi and Sugita 1989) and shows a nearly complete identity with the corresponding chloroplast gene. The tRNA gene, which shows only 73% homology with the corresponding chloroplast gene, has to be considered a 'native' mitochondrial tRNA gene and is 535 bp from the 'chloroplast-like' tRNA gene on the same strand. Northern hybridization analysis revealed that the three tRNA genes are transcribed in tomato mitochondria. Southern hybridization analysis of tomato, sugar beet, rice and wheat mitochondrial DNAs, with oligonucleotide probes for mitochondrial or chloroplast tRNA genes, demonstrated that the mitochondrial tRNA gene found in tomato is present in dicot plants but not in monocots. On the other hand, a chloroplast-like tRNA gene exists in monocot plants. [ABSTRACT FROM AUTHOR]

The genes coding for tRNA, tRNA, tRNA, tRNA, tRNA and tRNA have been identified in Oenothera mitochondrial DNA. Sequence analysis of these genes and their surrounding sequences are presented and compared with other known tRNA genes from plant mitochondria. All six deduced tRNA sequences can be folded into the classical cloverleaf structure model. Only the tRNA gene shows high homology with the corresponding chloroplast gene and thus appears to be derived from a transfer event of chloroplast sequences into the mitochondrial genome. The sequences surrounding this gene, however, show little similarity with the chloroplast genome. The other five deduced tRNAs display a much lower similarity with their chloroplast counterparts and thus appear to be genuine mitochondrial tRNAs. These tRNAs are highly conserved between monocots and dicots with maximally three nucleotides differing between the Oenothera sequences and their wheat homologues. A purine-rich sequence is found upstream of each tRNA gene in Oenothera, similar to wheat mitochondrial tRNA genes, that could be involved in transcription signalling. [ABSTRACT FROM AUTHOR]

Ty4 is a novel transposable element in the yeast, Saccharomyces cerevisiae, which is present in only a few copies in the genome (Stucka et al. 1989). In strain C836 one of the three copies (Ty4-90) is contained in cosmid clone c90, where it resides on chromosome V. Analysis of this region reveals a 'hot-spot' of transposition: in addition to Ty4-90, the locus contains a complete Ty3 element and seven singular delta, sigma and tau elements. Three tRNA genes (for His, Lys, and Ile) are located in this region, and these are closely associated with one or the other of the elements, a phenomenon commonly observed in yeast. A comparison of c90 with corresponding regions from other strains shows that the locus is highly polymorphic and that this polymorphism is explicitly associated with Ty transposition and recombination events. [ABSTRACT FROM AUTHOR]

The nucleotide sequences of three tRNA genes and their flanking regions from the maize mitochondrial genome is reported. These genes, which are located in the same region of the genome between the 14-kb inverted repeats, are transcribed in the mitochondria and code for tRNA (anticodon UUU) tRNA (CAU) and tRNA (GUA). The very high homology shown by the tRNA gene with its chloroplast counterpart indicates that it probably originates from a chloroplast DNA insertion. The analysis of the upstream regions of these genes showed that the tRNA and the tRNA genes possess the consensus sequence AAGAANRR, which could act as a promoter sequence in higher plant mitochondria. [ABSTRACT FROM AUTHOR]