Transfer RNA gene mapping studies on chloroplast DNA from Chlamydomonas reinhardii

Total tRNA of Chlamydomonas reinhardii was fractionated by 2-dimensional gel electrophoresis. Sixteen tRNAs specific for eleven amino acids could be identified by aminoacylation with Escherichia coli tRNA synthetases. Hybridization of these tRNAs with chloroplast restriction fragments allowed for the localization of the genes of tRNA^Tyr, tRNA^Pro, tRNA^Phe (2 genes), tRNA^Ile (2 genes) and tRNA^His (2 genes) on the chloroplast genome of C. reinhardii. The genes for tRNA^Ala (2 genes), tRNA^Asn and tRNA^Leu were mapped by using individual chloroplast tRNAs from higher plants as probes.     

Transfer RNA genes associated with the 16S and 23S rRNA genes of Euglena chloroplast DNA

Hybridization studies of Euglena chloroplast ¹²⁵I-labeled tRNAs to restriction fragments of Euglena chloroplast DNA have shown that the spacer between the 16S and 23S rRNA genes, in two and possibly all three of the ribosomal DNA units, contains genes for tRNA^Ile and tRNA^Ala, whereas a tRNA gene (for either tRNA^Trp or tRNA^Glu) is located before probably all four 16S rRNA genes present on the chloroplast DNA molecule.     

The tRNATyr multigene family of Nicotiana rustica: genome organization,sequence analyses and expression in vitro

Tobacco tRNA^Tyr genes are mainly organized as a dispersed multigene family as shown by hybridization with a tRNA^Tyr-specific probe to Southern blots of Eco RI-digested DNA. A Nicotiana genomic library was prepared by Eco RI digestion of nuclear DNA, ligation of the fragments into the vector gtWES·B and in vitro packaging. The phage library was screened with a 5-labelled synthetic oligonucleotide complementary to nucleotides 18 to 37 of cytoplasmic tobacco tRNA^Tyr. Eleven hybridizing Eco RI fragments ranging in size from 1.7 to 7.5 kb were isolated from recombinant lambda phage and subcloned into pUC19 plasmid. Four of the sequenced tRNA^Tyr genes code for the known tobacco tRNA₁ ^Tyr (GA) and seven code for tRNA₂ ^Tyr (GA). The two tRNA species differ in one nucleotide pair at the basis of the TC stem. Only one tRNA^Tyr gene (pNtY5) contains a point mutation (T54A54). Comparison of the intervening sequences reveals that they differ considerably in length and sequence. Maturation of intron-containing pre-tRNAs was studied in HeLa and wheat germ extracts. All pre-tRNAs^Tyr-with one exception-are processed and spliced in both extracts. The tRNA^Tyr gene encoded by pNtY5 is transcribed efficiently in HeLa extract but processing of the pre-tRNA is impaired.     

Physical mapping of the transfer RNA genes on lambda-h80dglytsu+36

The three Escherichia coli transfer RNA genes of the DNA of the transducing phage λ80cI857S^?t68dglyTsu⁺₃₆tyrTthrT (abbreviated λh80T), which specify the structures of tRNA^Gly₂(su⁺₃₆), tRNA^Tyr₂ and tRNA^Thr₃, have been mapped by hybridizing ferritin-labeled E. coli tRNA to heteroduplexes of λh80T DNA with the DNA of the parental phage (λh80cI857S^?t68) and examining the product in the electron microscope. The DNA of λh80T contains a piece of bacterial DNA of length 0·43 λ unit³ that replaces a piece of phage DNA of length 0·46 λ unit, proceeding left from B · P′ (the junction of bacterial DNA and phage DNA) (i.e. att⁸⁰). A cluster of three ferritin binding sites, and thus of tRNA genes, is seen at a position of 0·24 λ unit (1·1 × 10⁴ nucleotides) to the left of B· P′. The three tRNA genes of the cluster are separated by the unequal spacings of 260 (±30) and 140 (± 30) nucleotides, proceeding left from B·P′. The specific map positions have been identified by hybridization competition between ferritin-labeled whole E. coli tRNA with unlabeled purified tRNA^Tyr₂ and with unlabeled partially purified tRNA^Gly₂. The central gene of the cluster is tRNA^Tyr₂. The tRNA^Gly₂gene is probably the one furthest from B·P′. Thus, the gene order and spacings, proceeding left from B·P′, are: tRNA^Thr₃, 260 nucleotides, tRNA^Try₂, 140 nucleotides, tRNA^Gly₂.     

The genes coding for tRNATyr of Drosophila melanogaster: Localization and determination of the gene numbers

Transfer RNA^Tyr (anticodon GA) was isolated from Drosophila melanogaster by means of Sepharose 4B, RPC-5, and polyacrylamide gel electrophoresis. The tRNA was iodinated in vitro with Na¹²⁵I and hybridized in situ to salivary gland chromosomes from Drosophila. The genes of tRNA^Tyr were localized in eight regions of the genome by autoradiography. Restriction enzyme analysis of genomic DNA indicated that the haploid Drosophila genome codes for about 23 tRNA^Tyr genes. The regions 22F and 85A each contain four to five tRNA^Tyr genes, whereas the regions 28C, 41AB, 42A, 42E, and 56D each contain two to three tRNA^Tyr genes.     

Relative stabilities of RNA/DNA hybrids: effect of RNA chain length in competitive hybrization

Escherichia coli DNA and fragmented rRNA were used as a model system to study the effect of RNA fragment size in hybridization-competition experiments. Though no difference in hybridization rates was observed, the relative stabilities of the RNA/DNA hybrids were found to be largely affected by the fragment size of the RNA molecule. Intact rRNA was shown to replace shorter homologous rRNA sequences in their hybrids, the rate of the displacement being dependent on the molecular size of the RNA fragments. Hybridization-competition experiments between molecules of different lengths are expected to be complicated by the displacement reaction. The synthesis of tRNA^Tyr-like sequences transcribed in vitro on φ80psu₃⁺ bacteriophage DNA was measured by hybridization competition assays. Indirect competition with labelled E. coli tRNA^Tyr hybridization revealed that the in vitro-synthesized RNA contained significant amounts of tRNA^Tyr; these sequences could not, however, be detected by the direct competition method in which labelled in vitro-synthesized RNA competes with E. coli tRNA^Tyr for hybridization to φ80psu₃⁺ DNA. These contradictory results can be traced to the differences in size of the competing molecules in the hybridization-competition reaction. Indeed, in vitro-transcribed tRNA^Tyr-like sequences, longer than mature tRNA, were found to displace efficiently E. coli tRNA^Tyr from its hybrids with φ80psu₃⁺ DNA. These findings explain why such sequences could not be detected by direct competition with E. coli tRNA^Tyr.     

Cloning of genes for bacteriophage T5 stable RNAs

One EcoRI-generated fragment (440 basepairs) and two EcoRI/HindIII fragments (220 and 960 basepairs) from the deletion region of T5 phage have been inserted into the phage λ XIII and the plasmid pBR322 as vectors. Recombinant DNA molecules were studied by hybridization with in vivo ³²P-labeled T5 4–5 S RNAs on nitrocellulose filters. Two-dimensional polyacrylamide gel electrophoretic fractionation and fingerprint analysis of the RNAs eluted from the filters were carried out to identify RNAs coded by cloned fragments. For the accurate localization of the genes for these RNAs, RNA-DNA hybrids were treated with T1 and pancreatic RNAases, and the eluted RNA fragments stable against RNAase action were electrophoresed. It was shown that the EcoRI¹440 fragment contains the gene for tRNA 10 (tRNA^Asp), the EcoRI/HindIII¹220 fragment contains the gene for RNA III (107 bases) and parts of the genes for RNA I (107 bases) and tRNA 12 (tRNA^His), and the EcoRI/HindIII¹960 fragment contains only a part of the gene for tRNA 9 (tRNA^Gln). The arrangement of these genes on the physical map of T5 phage was as follows: -tRNA^Gln-tRNA^His-RNA III-RNA I-…-tRNA^Asp.     

Identification of the yeast DNA sequences that correspond to specific tyrosine-inserting nonsense suppressor loci

There are eight unlinked genes for yeast tyrosine transfer RNA. In previous work, nonsense suppressors have been isolated at each of the eight loci, and these loci have been genetically mapped (Hawthorne &; Leupold, 1974). It has also been demonstrated by RNA-DNA hybridization that the genes are physically located on eight different EcoRI restriction fragments (Olson et al., 1977). The purpose of the present report is to cross-correlate the set of tyrosine-inserting suppressor loci with the set of tRNA^Tyr-hybridizing restriction fragments. This cross-correlation was achieved for six of the eight loci by analyzing the meiotic and mitotic linkage between the tyrosine-inserting suppressors and the genetic determinants of naturally occurring size variants of the tRNA^Tyr-hybridizing restriction fragments.Now that individual suppressor loci have been identified with specific DNA fragments, it should be possible to analyze the phenotypes of these mutant genes in terms of their DNA sequences. The method by which these assignments were made also offers a new approach to the general problem of correlating genes with restriction fragments; it is particularly suited to organisms with powerful genetic systems in which hybridization to chromosome spreads in situ is impractical.     

Chromosomal location of a major tRNA gene cluster of Xenopus laevis

In Xenopus laevis, genes encoding tRNA^Phe, tRNA^Tyr, tRNA ₁ ^Met , tRNA^Asn, tRNA^Ala, tRNA^Leu, and tRNA^Lys are clustered within a 3.18-kb (kilobase) fragment of DNA. This fragment is tandemly repeated some 150 times in the haploid genome and its components are found outside the repeat only to a limited extent. The fragment hybridizes in situ to a single site very near the telomere on the long arm of one of the acrocentric chromosomes of the group comprising chromosomes 13–18. All the chromosomes of this group also hybridize with DNA coding for oocyte-specific 5S RNA. The tRNA gene cluster is slightly proximal to the cluster of 5S RNA genes.We respectfully dedicate this paper to Prof. H. Bauer on the occasion of his 80th birthday.     

The preparation and properties of a stable mercury derivative of transfer RNAs from Escherichia coli

Further investigations into the properties of the mercury derivative formed by the reaction of 4-thiouridine-containing tRNAs and pentafluorophenylmercury chloride have been carried out. tRNA^fMet (which contains only one 4-thiouridine residue) has been isolated by a one-step column Chromatographic procedure from unfractionated Escherichia coli tRNA and has been shown to react with the mercury compound to give a derivative which has similar properties to those previously reported for the corresponding mercury derivative of tRNA^Tyr which contains two adjacent 4-thiouridine residues. The mercury derivative of tRNA^Tyr appears to be a competitive inhibitor of tRNA^Tyr in the aminoacylation reaction (tRNA^TyrK_m = 0.42 μM, mercury derivative of tRNA^TyrK_i = 0.11 μM). The mercury derivative of Tyr-tRNA^Tyr can be made, but only by the reaction of the mercury compound with the aminoacylated tRNA.     

Mitochondrial tyrosyl transfer ribonucleic Acid in soybean seedlings

Soybean seedlings were examined for the presence of mitochondrial tRNA. Tyrosyl transfer tRNAs from whole cells, from a well characterized mitochondrial preparation, and from a snake venom phosphodiesterase-treated mitochondrial preparation, were compared by reverse phase chromatography. It was concluded that none of the three previously reported tRNA^Tyr species were mitochondrial. Rather, a fourth tRNA^Tyr species, eluting somewhat later, was of mitochondrial origin. Mitochondrial tRNA^Tyr was chromatographically similar to Escherichia coli tRNA^Tyr.     

Identification of transfer RNA suppressors in Escherichia coli. II. Duplicate genes for tRNA2Gln.

The number of gene copies for tRNA₂^Gln in λpsu⁺2 was determined by genetic and biochemical studies. The transducing phage stimulates the production of the su⁺2 (amber suppressor) and su°2 glutamine tRNAs and methionine tRNA_m. When the su⁺2 amber suppressor was converted to an ochre suppressor by single-base mutation, the phage stimulated ochre-suppressing tRNA₂^Gln, instead of the amber-suppressing tRNA₂^Gln. From the transducing phage carrying the ochre-suppressing allele, strains carrying both ochre and amber suppressors were readily obtainable. These phages stimulated both ochre-suppressing and amber-suppressing tRNA₂^Gln, but not the non-suppressing form. We conclude that the original transducing phage carries two tRNA₂^Gln genes, one su⁺2 and one su°2. The transducing phage carrying two suppressors, ochre and amber, segregates one-gene derivatives that encode only one or the other type of suppressor tRNA. These derivatives apparently arise by unequal recombination involving the two glutamine tRNA genes in the parental phage. This segregation is not accompanied by the loss of the tRNA_m^Met gene. Based on these results, it is suggested that Escherichia coli normally carries in tandem two identical genes specifying tRNA₂^Gln at 15 minutes on the bacterial chromosome. su⁺2 mutants may arise by single-base mutations in the anticodon region of either of these two, leaving the other intact. By double mutations, tRNA₂^Gln genes could also become ochre suppressors. A tRNA_m^Met gene is located near, but not between, these two tRNA₂^Gln genes.     

Nuclear tRNATyr genes are highly amplified at a single chromosomal site in the genome of Arabidopsis thaliana

Summary We have examined the organization of tRNA^Tyr genes in three ecotypes of Arabidopsis thaliana, a plant with an extremely small genome of 7 × 107 bp. Three tRNA^Tyr gene-containing EcoRI fragments of 1.5 kb and four fragments of 0.6, 1.7, 2.5 and 3.7 kb were cloned from A. thaliana cv. Columbia (Col-O) DNA and sequenced. All EcoRl fragments except those of 0.6 and 2.5 kb comprise an identical arrangement of two tRNA^Tyr genes flanked by a tRNA^Ser gene. The three tRNA genes have the same polarity and are separated by 250 and 370 bp, respectively. The tRNA^Tyr genes encode the known cytoplasmic tRNA_GA ^Tyr. Both genes contain a 12 by long intervening sequence. Densitometric evaluation of the genomic blot reveals the presence of at least 20 copies, including a few multimers, of the 1.5 kb fragment in Col-O DNA, indicating a multiple amplification of this unit. Southern blots of EcoRl-digested DNA from the other two ecotypes, cv. Landsberg (La-O) and cv. Niederzenz (Nd-O) also show 1.5 kb units as the major hybridizing bands. Several lines of evidence support the idea of a strict tandem arrangement of this 1.5 kb unit: (i) Sequence analysis of the EcoRI inserts of 2.5 and 0.6 kb reveals the loss of an EcoRI site between 1.5 kb units and the introduction of a new EcoRI site in a 1.5 kb dimer. (ii) Complete digestion of Col-O DNA with restriction enzymes which cleave only once within the 1.5 kb unit also produces predominantly 1.5 kb fragments. (iii) Partial digestion with EcoRI shows that the 1.5 kb fragments indeed arise from the regular spacing of the restriction sites. The high degree of sequence homology among the 1.5 kb units, ranging from 92% to 99%, suggests that the tRNA^Ser/tRNA^Tyr cluster evolved 1–5 million years ago, after the Brassicaceae diverged from the other flowering plants about 5–10 million years ago.     

Cytokinin-Active Ribonucleosides in Phaseolus RNA: II. DISTRIBUTION IN tRNA SPECIES FROM ETIOLATED P. VULGARIS L. SEEDLINGS

The distribution of cytokinin-active ribonucleosides in tRNA species from etiolated Phaseolus vulgaris L. seedlings has been examined. Phaseolus tRNA was fractionated by benzoylated diethylaminoethyl-cellulose and RPC-5 chromatography, and the distribution of cytokinin activity was compared with the distribution of tRNA species expected to correspond to codons beginning with U. Phaseolus tRNA^Cys, tRNA^Trp, tRNA^Tyr, a major peak of tRNA^Phe, and a large fraction of tRNA^Leu were devoid of cytokinin activity in the tobacco bioassay. Cytokinin activity was associated with all fractions containing tRNA^Ser species and with minor tRNA^Leu species. In addition, several anomalous peaks of cytokinin activity that could not be directly attributed to U group tRNA species were detected.     

Translational nonsense codon suppression as indicator for functional pre-tRNA splicing in transformed Arabidopsis hypocotyl-derived calli

The transient expression of three novel plant amber suppressors derived from a cloned Nicotiana tRNA^Ser(CGA), an Arabidopsis intron-containing tRNA^Tyr(GTA) and an Arabidopsis intron-containing tRNA^Met(CAT) gene, respectively, was studied in a homologous plant system that utilized the Agro bacterium-mediated gene transfer to Arabidopsis hypocotyl explants. This versatile system allows the detection of β-glucuronidase (GUS) activity by histochemical and enzymatic analyses. The activity of the suppressors was demonstrated by the ability to suppress a premature amber codon in a modified GUS gene. Co-transformation of Arabidopsis hypocotyls with the amber suppressor tRNA^Ser gene and the GUS reporter gene resulted in ~10% of the GUS activity found in the same tissue transformed solely with the functional control GUS gene. Amber suppressor tRNAs derived from intron-containing tRNA^Tyr or tRNA^Met genes were functional in vivo only after some additional gene manipulations. The G3:C70 base pair in the acceptor stem of tRNA^Met(CUA) had to be converted to a G3:U70 base pair, which is the major determinant for alanine tRNA identity. The inability of amber suppressor tRNA^Tyr to show any activity in vivo predominantly results from a distorted intron secondary structure of the corresponding pre-tRNA that could be cured by a single nucleotide exchange in the intervening sequence. The improved amber suppressors tRNA^Tyr and tRNA^Met were subsequently employed for studying various aspects of the plant-specific mechanism of pre-tRNA splicing as well as for demonstrating the influence of intron-dependent base modifications on suppressor activity.     

Mechanism of suppression in Drosophila: II. Enzymatic discrimination of wild-type and suppressor tyrosine transfer RNA

Previous studies had shown that two principle forms of tyrosine transfer RNA of Drosophila melanogaster were present in wild-type adult flies but that the second form was virtually absent in a suppressor mutant, su(s)². Current results are at variance with the previous ones, in that the suppressor mutant has significant amounts of the second form of tRNA^Tyr. A second chromatography system for separating these forms of tRNA^Tyr is described, RPC-5, and is compared to the system used previously, RPC-2. Both systems indicate that wild-type flies contain the two forms of tRNA^Tyr in a ratio of $\text{40}\text{60}$, the suppressor mutant in a ratio of $\text{60}\text{40}$. The difference between current and previous results can be attributed to the procedures used in the preparation of the enzyme that is used as a source of tyrosyl-tRNA ligase. The enzyme activity can be separated into two fractions on DEAE-cellulose chromatography. With suppressor tRNA as substrate, one enzyme fraction charges both forms of tRNA^Tyr but the second enzyme fraction charges the first form preferentially or nearly exclusively in some cases, as was seen in the previous experiments. With wild-type tRNA as substrate both enzyme fractions charge both forms of tRNA^Tyr. Storage results in the loss of the enzyme's ability to discriminate against the second form of tRNA^Tyr from the suppressor mutant, while the enzymatic activity is retained. We postulate that the su(s)⁺ locus produces an enzyme that modifies the second isoacceptor of tRNA^Tyr and that, when such modification fails to occur (as in the su(s)² mutant), the tRNA is unable to accept tyrosine from one form of tyrosyl-tRNA ligase. How the discrimination against the second isoacceptor by the ligase may be important metabolically is not apparent.     

In vivo identification of essential nucleotides in tRNALeu to its functions by using a constructed yeast tRNALeu knockout strain

The fidelity of protein biosynthesis requires the aminoacylation of tRNA with its cognate amino acid catalyzed by aminoacyl-tRNA synthetase with high levels of accuracy and efficiency. Crucial bases in tRNA^Leu to aminoacylation or editing functions of leucyl-tRNA synthetase have been extensively studied mainly by in vitro methods. In the present study, we constructed two Saccharomyces cerevisiae tRNA^Leu knockout strains carrying deletions of the genes for tRNA^Leu(GAG) and tRNA^Leu(UAG). Disrupting the single gene encoding tRNA^Leu(GAG) had no phenotypic consequence when compared to the wild-type strain. While disrupting the three genes for tRNA^Leu(UAG) had a lethal effect on the yeast strain, indicating that tRNA^Leu(UAG) decoding capacity could not be compensated by another tRNA^Leu isoacceptor. Using the triple tRNA knockout strain and a randomly mutated library of tRNA^Leu(UAG), a selection to identify critical tRNA^Leu elements was performed. In this way, mutations inducing in vivo decreases of tRNA levels or aminoacylation or editing ability by leucyl-tRNA synthetase were identified. Overall, the data showed that the triple tRNA knockout strain is a suitable tool for in vivo studies and identification of essential nucleotides of the tRNA.     

Nucleotide sequences of transfer RNA genes in the Pisum sativum chloroplast DNA

Summary Eight transfer RNA (tRNA) genes which were previously mapped to five regions of the Pisum sativum (pea) chloroplast DNA (ctDNA) have been sequenced. They have been identified as tRNA^Val(GAC), tRNA^Asn(GUU), tRNA^Arg(ACG), tRNA^Leu(CAA), tRNA^Tyr(GUA), tRNA^Glu(UUC), tRNA^His(GUG), and tRNA^Arg(UCU) by their anticodons and by their similarity to other previously identified tRNA genes from the chloroplast DNAs of higher plants or from E. gracilis. In addition,two other tRNA genes, tRNA^Gly (UCC) and tRNA^Ile(GAU), have been partially sequenced. The tRNA genes are compared to other known chloroplast tRNA genes from higher plants and are found to be 90–100% homologous. In addition there are similarities in the overall arrangement of the individual genes between different plants. The 5 flanking regions and the internal sequences of tRNA genes have been studied for conserved regions and consensus sequences. Two unusual features have been found: there is an apparent intron in the D-loop of the tRNA^Gly(UCC), and the tRNA^Glu(UUC) contains GATTC in its T-loop.