A chimeric gene (orf256) is expressed as protein only in cytoplasmic male-sterile lines of wheat.

Mitochondria derived from Triticum timopheevi have a chimeric gene, orf256, immediately upstream from coxI. Antibodies to a peptide corresponding to a part of the encoded amino acid sequence of orf256 detect a 7 kDa protein on western blots of mitochondrial proteins from cytoplasmic male-sterile (cms) wheat (T. aestivum nucleus, T. timopheevi mitochondria) but not in mitochondrial proteins from T. aestivum, T. timopheevi, or cms plants restored to fertility by introduction of nuclear genes for fertility restoration. The 7 kDa protein appears to serve as a marker for cms wheat. Its occurrence as an integral protein of the inner membrane may indicate a cms effect through an influence on mitochondrial membrane function.

Influence of nuclear background on transcription of a chimeric gene (orf256) and coxI in fertile and cytoplasmic male sterile wheats.

Crosses between Triticum timopheevi, as maternal donor, and T. aestivum can lead to cytoplasmic male sterile (cms) plants. The T. timopheevi derived mitochondrial DNA from parental, cms, and fertility-restored lines differs from that of T. aestivum derived mtDNA in the coxI gene region. Our previous results for cms lines showed that there is an open reading frame, orf256, upstream from coxI in T. timopheevi derived mtDNA that is not present in T. aestivum DNA. The 5' flanking region and the first 33 nucleotides of the coding region of orf256 are identical to the corresponding region of T. aestivum coxI, whereas the rest of orf256, including the 3' flank, is not related to coxI. Also, the organization of orf256 and coxI on a HindIII fragment from T. timopheevi derived mtDNA are identical in T. timopheevi, cms, and fertility-restored lines. We now report that the DNA sequence of orf256 is identical in T. timopheevi, cms, and fertility-restored lines. Major transcripts in cms and fertility-restored lines encode both orf256 and coxI with 5' termini like coxI mRNA of T. aestivum, whereas parental mitochondria from T. timopheevi have major transcripts with 5' termini within the orf256 coding region. Mitochondria from cms and fertility-restored lines have the potential to produce a protein that would not be present in parental T. timopheevi or in T. aestivum.

Cytoplasmic male sterility and fertility restoration in wheat are not associated with rearrangements of mitochondrial DNA in the gene regions for cob, coxII, or coxI.

In comparing the genetic organization and exploring the molecular basis of cytoplasmic male sterility (CMS) in wheat, mitochondrial DNAs (mtDNA) from Triticum aestivum, T. timopheevi, CMS alloplasmic wheat with T. aestivum nucleus and T. timopheevi mitochondria, and fertility-restored lines were compared by hybridization analysis with specific probes for three gene regions: coxII, cob, and coxI. Minor differences between T. aestivum- and T. timopheevi-derived sources were found for gene regions for coxII and cob. For coxI, there are significant differences between T. timopheevi-derived mtDNAs and T. aestivum mtDNA extending beyond an 8 kb distance. All T. timopheevi-derived mtDNA sources have a chimeric gene region (orf256) with part of the upstream coxI gene region, including some coxI-coding region, preceding coxI. The part of orf256 that does not include any of coxI and the 3'-flanking region of CMS coxI are not found in T. aestivum mtDNA. Neither orf256 nor the CMS 3'-flanking region of coxI are found in T. timopheevi or T. aestivum chloroplastic or nuclear DNA. There do not appear to be DNA sequence differences for the three gene regions studied that are related to either CMS or fertility-restored states.

Isolation of mitochondrial DNA from cytoplasmic male sterile and maintainer lines of pearl millet, Pennisetum americanum (L.) Leeke.

Mitochondrial DNA has been isolated from paired lines of pearl millet maintainer and cytoplasmic male sterile plants. Evaluation of the DNA by agarose gel electrophoresis shows that good quality DNA of high molecular weight can be obtained from mitochondria of both maintainer and male sterile pearl millet.

Structural relationship between tRNALys2 and tRNALys4 from mouse lymphoma cells.

Mouse lymphoma cells have three major isoaccepting lysine tRNAs. Two of these isoacceptors, tRNALys2 and tRNALys4, were sequenced by rapid gel or chromatogram readout methods. They have the same primary sequence but differ in two modified nucleotides. tRNALys4 has an unmodified uridine replacing one dihydrouridine and an unidentified nucleotide, t6A*, replacing t6A. This unidentified nucleotide is not a hypomodified form of t6A. Thus, tRNALys4 is not a simple precursor of tRNALys2. Both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Also, partial sequences of minor homologs of tRNALys2 and tRNALys4 were obtained. The distinctions between tRNALys2 and tRNALys4 may be part of significant cellular roles as illustrated by the differential effects of these isoacceptors on the synthesis by lysyl-tRNA synthetase of diadenosine-5',5'''-P1,P4-tetraphosphate, a putative signal in DNA replication.

Developmentally regulated plant genes: the nucleotide sequence of a wheat gliadin genomic clone.

Gliadins, the major wheat seed storage proteins, are encoded by a multigene family. Northern blot analysis shows that gliadin genes are transcribed in endosperm tissue into two classes of poly(A)+ mRNA, 1400 bases (class I) and 1600 bases (class II) in length. Using poly(A)+ RNA from developing wheat endosperm we constructed a cDNA library from which a number of clones coding for alpha/beta and gamma gliadins were identified by hybrid-selected mRNA translation and DNA sequencing. These cDNA clones were used as probes for the isolation of genomic gliadin clones from a wheat genomic library. One such genomic clone was characterized in detail and its DNA sequence determined. It contains a gene for a 33-kd alpha/beta gliadin protein (a 20 amino acid signal peptide and a 266 amino acid mature protein) which is very rich in glutamine (33.8%) and proline (15.4%). The gene sequence does not contain introns. A typical eukaryotic promoter sequence is present at -104 (relative to the translation initiation codon) and there are two normal polyadenylation signals 77 and 134 bases downstream from the translation termination codon. The coding sequence contains some internal sequence repetition, and is highly homologous to several alpha/beta gliadin cDNA clones. Homology to a gamma-gliadin cDNA clone is low, and there is no homology with known glutenin or zein cDNA sequences.

Lysine tRNAs from rat liver: lysine tRNA sequences are highly conserved.

The two major lysine tRNAs from rat liver, tRNA2Lys and tRNA5Lys, were sequenced by rapid gel or chromatogram readout methods. The major tRNA2Lys differs from a minor form only by a base pair in positions 29 and 41; both tRNAs have an unidentified nucleotide, U**, in the third position of the anticodon. Although highly related, the major tRNA2Lys and tRNA5Lys differ in four base pairs and four unpaired nucleotides, including the first position of the anticodons, but have the same base pair in positions 29 and 41. The three tRNAs maintain a m2G-U pair in the acceptor stem. Detection of this m2G is in contrast to other reports of lysine tRNAs. Sequences of lysine tRNAs are strongly conserved in higher eukaryotes.

Perturbation of the mitochondrial lysine tRNA population by virus-induced transformation or stress of mammalian cells: functional properties and nucleotide sequence of a mitochondrially associated lysine tRNA.

Eleven isoaccepting lysine tRNAs from mammalian sources are demonstrable by RPC-5 chromatography and polyacrylamide gel electrophoresis. The appearance and amounts of these isoacceptors varies with the source and growth state of cells. One isoacceptor, tRNALys6, observed in preparations of tRNA from some virus-transformed cells in culture, has been characterized by determining functional properties, cellular location, and its nucleotide sequence. tRNALys6 responds primarily to the lysine codon AAA, but it is not used efficiently in a wheat germ translational system in vitro. Compared with lysine isoacceptors 1, 2, 4, 5a, and 5, [3H]lysine appears in vivo in tRNALys6 with a delay of about 3 h. This delay may in part be a result of a less functional tRNA, but a compartmented state of tRNALys6 also appears to be important. tRNALys6 is associated with mitoplasts prepared from KA31 fibroblasts. The nucleotide sequence of tRNALys6 was determined by rapid postlabeling procedures involving limited hydrolysis in formamide, 32P-labeling of 5' ends of fragments with polynucleotide kinase, separation of the nested set of fragments in polyacrylamide denaturing gels, release of 5'-labeled nucleotides with RNase T2, and identification of the released nucleotides by chromatography on PEI cellulose. Confirmation of the positions of major nucleotides was done by using limited digestions by RNases of tRNALys6 labeled with 32P on the 3' terminus in a gel readout procedure. The nucleotide sequence of tRNALys6 differs from that of cytoplasmic lysine tRNAs and mammalian mitochondrial lysine tRNAs. It contains U*, an unidentified modified uridine occurring in the anticodon of some mitochondrial tRNAs. tRNALys6 appears to occur in very limited amounts, or not at all, in most cells unless stressed, but when present it is associated with mitochondria, although it is probably coded in the nucleus.

Comparison of complexes containing lysyl-tRNA synthetase from normal and virus-transformed cells.

We compared the lysyl-tRNA synthetases from normal (Balb/3T3) and murine sarcoma virus-transformed (KA31) mouse fibroblasts. In agreement with several other reports of mammalian systems, the lysyl-tRNA synthetases from these cells occurred in very large postmicrosomal complexes as determined by gel filtration on agarose columns. Arginyl-, isoleucyl-, methionyl-, phenylalanyl-, and tyrosyl-tRNA synthetases also occurred as part of a large complex or complexes. Activity of glycyl- or leucyl-tRNA synthetase was not detected in a complex. The specific activities of arginyl- and methionyl-tRNA synthetases were three- and five-fold higher, respectively, in a complex from KA31 as compared with a complex from Balb/3T3. In contrast, the specific activity of lysyl-tRNA synthetase from the Balb/3T3 complex was 50% higher than that of the KA31 complex. tRNALys obtained from the complexes of Balb/3T3 and KA31 was fractionated into isoacceptors on columns of RPC-5. The relative amounts of lysine isoacceptors in total preparations of tRNA from normal whole cells and in tRNA obtained from the normal enzyme complex were the same. However, two isoacceptors were present in greater amounts and two were present in lesser amounts in the KA31 enzyme complex as compared with lysine isoacceptors in a total preparation of tRNA from KA31 cells.

Codon binding and translational properties of an isoaccepting lysine tRNA peculiar to virus-transformed Cells.

Isoaccepting lysyl-tRNAs from virus-transformed cells in culture were fractionated in the RPC-5 system into peaks 1, 2, 4, 5a, 5, and 6. tRNALys6 previously was found predominantly associated with transformed cells. The codon response of each peak was determined in an E. coli ribosomal binding assay. tRNALys1, tRNALys2, and tRNALys4 are highly specific for the 5'AAG3' codon. tRNALys5 and tRNALys5a preferentially bind in response to AAA. tRNALys6 binds in response to AAA 3-fold better than in response to AAG. The presence of thiolated nucleosides in the anticodon regions of tRNALys5a, tRNALys5, and tRNALys6 is indicated by I2-inactivation of aminoacylation ability with no effect on the other is isoacceptors. Functional abilities of the isoacceptors were compared in a wheat germ translational system with tobacco mosaic virus RNA as messenger. All of the isoacceptors function about equally well in translation except for tRNALys6, which is only 14 to 24% as effective as the other isoacceptors.

Microanalytical RPC-5 columns for rapid chromatography of aminoacyl-tRNAs.

A microanalytical reversed-phase chromatographic system for the separation of aminoacyl-tRNA's on microbore (0.3cm X 25cm) columns is described. Chromatography of lysyl-tRNA from polyoma virus transformed mouse fibroblasts revealed at least seven identifiable isoacceptors. The system has the advantages of speed, sensitivity and excellent resolution of isoaccepting tRNA's.

Precursor relationship of phenylalanine transfer ribonucleic acid from Escherichia coli treated with chloramphenicol or starved for iron, methionine, or cysteine.

When treated with chloramphenicol, Escherichia coli 15T minus produces two new species (IV and V) of transfer ribonucleic acid specific for phenylalanine in addition to the major normal species (II) and two minor normal species (I and III), which are seen as distinct components upon fractionation by chromatography on columns of benzoylated diethylaminoethyl-cellulose. Species IV is produced when cells are grown in iron-deficient medium and is, therefore, probably deficient in the 2-methylthio modification of N-6-(delta-2-isopentenyl) adenosine. A new minor species (Va) also appears under those conditions. All of the new components elute earlier than the major normal species. Addition of chloramphenicol to iron-deficient cells leads to the production of species V, and that production is blocked by rifampin, as is the production of species IV. Thus, species IV and V appear to be transcriptional products. Although E. coli 15T minus appears to be rel plus, starvation for methionine or cysteine leads to the accumulation of species IV (without addition of chloramphenicol); rifampin blocks the accumulation. Species V is still produced on addition of chloramphenicol to starved cultures. Starvation for arginine or tryptophan does not alter the chromatographic profile from the normal case. Treatment with permanganate indicates that species II and IV contain isopentenyladenosine but that species V does not. Species V appears to be deficient in both isopentenyl and methylthio modifications of adenosine and perhaps at least one other modification, because removing the isopentenyl moiety from adenosine does not convert species IV into species V, but converts it into species Va. A precursor relationship among species V, VI, and II is suggested by following the chromatographic profile of phenylalanine transfer ribonucleic acid during recovery of E. coli from treatment with chloramphenicol; the various species increase and decrease in a sequential manner.