anticodon and Chromosome-Deletion

With some exceptions, mitochondria within the class Insecta have the same gene content, and generally, a similar gene order allowing the proposal of an ancestral gene order. The principal exceptions are several orders within the Hemipteroid assemblage including the order Thysanoptera, a sister group of the order Hemiptera. Within the Hemiptera, there are available a number of completely sequenced mitochondrial genomes that have a gene order similar to that of the proposed ancestor. None, however, are available from the suborder Sternorryncha that includes whiteflies, psyllids and aphids.. We have determined the complete nucleotide sequence of the mitochondrial genomes of six species of whiteflies, one psyllid and one aphid. Two species of whiteflies, one psyllid and one aphid have mitochondrial genomes with a gene order very similar to that of the proposed insect ancestor. The remaining four species of whiteflies had variations in the gene order. In all cases, there was the excision of a DNA fragment encoding for cytochrome oxidase subunit III(COIII)-tRNAgly-NADH dehydrogenase subunit 3(ND3)-tRNAala-tRNAarg-tRNAasn from the ancestral position between genes for ATP synthase subunit 6 and NADH dehydrogenase subunit 5. Based on the position in which all or part of this fragment was inserted, the mitochondria could be subdivided into four different gene arrangement types. PCR amplification spanning from COIII to genes outside the inserted region and sequence determination of the resulting fragments, indicated that different whitefly species could be placed into one of these arrangement types. A phylogenetic analysis of 19 whitefly species based on genes for mitochondrial cytochrome b, NADH dehydrogenase subunit 1, and 16S ribosomal DNA as well as cospeciating endosymbiont 16S and 23S ribosomal DNA indicated a clustering of species that corresponded to the gene arrangement types.. In whiteflies, the region of the mitochondrial genome consisting of genes encoding for COIII-tRNAgly-ND3-tRNAala-tRNAarg-tRNAasn can be transposed from its ancestral position to four different locations on the mitochondrial genome. Related species within clusters established by phylogenetic analysis of host and endosymbiont genes have the same mitochondrial gene arrangement indicating a transposition in the ancestor of these clusters.

Topics: Animals; Anticodon; Aphids; Chromosome Deletion; DNA, Mitochondrial; Electron Transport Complex IV; Evolution, Molecular; Gene Order; Genes, Insect; Genome; Hemiptera; Mitochondria; NADH Dehydrogenase; Polymerase Chain Reaction; Protein Subunits; Recombination, Genetic; RNA, Transfer, Ala; RNA, Transfer, Arg; RNA, Transfer, Asn; RNA, Transfer, Gly; RNA, Untranslated; Sequence Analysis, DNA

16S ribosomal RNA contains three highly conserved single-stranded regions. Centrally located in one of these regions is the C1400 residue. Zero-length cross-linking of this residue to the anticodon of ribosome-bound tRNA showed that it was at or near the ribosomal decoding site [Ehresmann, C., Ehresmann, B., Millon, R., Ebel, J-P., Nurse, K., & Ofengand, J. (1984) Biochemistry 23, 429-437]. To assess the functional significance of sequence conservation of rRNA in the vicinity of this functionally important site, a series of site-directed mutations in this region were constructed and the effects of these mutations on the partial reactions of protein synthesis determined. Mutation of C1400 or C1402 to any other base only moderately affected a set of in vitro protein synthesis partial reactions. However, any base change from the normal G1401 residue blocked all of the tested ribosomal functions. This was also true for the deletion of G1401. Deletion of C1400 or C1402 had more complex effects. Whereas subunit association was hardly affected, 30S initiation complex formation was blocked by deletion of C1400 but much less so by deletion of C1402. Alternatively, tRNA binding to the ribosomal A site was more strongly affected by deletion of C1402 than by deletion of C1400. P site binding was inhibited by either deletion. HPLC analysis of the in vitro reconstituted mutant ribosomes showed that none of the functional effects were due to the absence or gross reduction in amount of any ribosomal protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Anticodon; Base Sequence; Chromosome Deletion; Escherichia coli; Guanine; Magnesium; Models, Structural; Molecular Sequence Data; Mutagenesis, Insertional; Nucleic Acid Conformation; Ribosomes; RNA, Messenger; RNA, Ribosomal, 16S; RNA, Transfer, Amino Acyl; RNA, Transfer, Met; Sequence Homology, Nucleic Acid

The monomeric form of the class I Escherichia coli methionine tRNA synthetase has a distinct carboxyl-terminal domain with a segment that interacts with the anticodon of methionine tRNA. This interaction is a major determinant of the specificity and efficiency of aminoacylation. The end of this carboxyl-terminal domain interacts with the amino-terminal Rossman fold that forms the site for amino acid activation. Thus, the carboxyl-terminal end may have evolved in part to integrate anticodon recognition with amino acid activation. We show here that internal deletions that disrupt the anticodon interaction have no effect on the kinetic parameters for amino acid activation. Moreover, an internally deleted enzyme can aminoacylate an RNA microhelix, which is based on the acceptor stem of methionine tRNA, with the same efficiency as the native protein. These results suggest that, in this enzyme, amino acid activation and acceptor helix aminoacylation are functionally integrated and are independent of the anticodon-binding site.

Topics: Acylation; Adenosine Triphosphate; Anticodon; Base Sequence; Binding Sites; Chromosome Deletion; Escherichia coli; Kinetics; Methionine; Methionine-tRNA Ligase; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Oligodeoxyribonucleotides; Protein Conformation

Deletions in cDNA clones covering the 3' 201 nucleotides of brome mosaic virus RNA 3 were produced by S1 nuclease treatment of cloned DNA linearized at several different restriction sites. Transcription of these clones yielded RNAs containing structural alterations in the 3'-terminal tRNA-like structure that is involved in aminoacylation and replication. Replicase template activity, but not aminoacylation activity, was especially sensitive to deletions in arm C, which contains a tyrosyl anticodon. Deletions in arm B were detrimental to aminoacylation, but the proportion of replicase template activity lost depended on the site of the deletion. Removal of arm D had little effect on aminoacylation and, in some instances, resulted in a 2-fold stimulation of replicase template activity.

Topics: Amino Acyl-tRNA Synthetases; Anticodon; Chromosome Deletion; Mosaic Viruses; RNA-Dependent RNA Polymerase; RNA, Transfer; RNA, Viral; Structure-Activity Relationship; Templates, Genetic; Tyrosine-tRNA Ligase; Virus Replication

Beginning with a missense suppressor tRNA and a nonsense suppressor tRNA, both in Escherichia coli and each containing an extra nucleotide in the anticodon loop, we generated new suppressors in vivo by spontaneous deletion of specific nucleotides from the anticodon loop. In one experiment, the new suppressor was generated by a double mutational event, base substitution and nucleotide deletion. A novel ochre suppressor is also described. It is very efficient in nonsense suppression but has no ms2i6 modification of the A residue on the 3' side of the anticodon. The results have important implications for tRNA structure-function relationships, tRNA recognition by tRNA-modifying enzymes, mechanisms of deletion mutation, and tRNA evolution.

Topics: Anticodon; Base Sequence; Chromosome Deletion; Chromosomes, Bacterial; Escherichia coli; Genotype; Mutation; Nucleic Acid Conformation; RNA, Transfer, Amino Acyl; Suppression, Genetic

Precise deletion of the intervening sequence of a yeast tRNATyr ochre suppressor gene (SUP6) significantly reduced its suppressor activity relative to that of the unaltered gene. This is probably the result of the absence of the pseudouridine modification, normally present at the middle anticodon position of mature suppressor tRNA, in tRNA synthesized in vivo from the deleted gene.

Topics: Anticodon; Base Sequence; Chromosome Deletion; Genes; Nucleic Acid Conformation; Plasmids; Pseudouridine; RNA, Transfer, Amino Acyl; Saccharomyces cerevisiae; Suppression, Genetic