UAA Codons Research Articles

DNA sequence analysis of the complete mitochondrial genome of the green alga Scenedesmus obliquus: evidence for UAG being a leucine and UCA being a non-sense codon

The complete DNA sequence of the mitochondrial genome of the chlorophyceen alga Scenedesmus obliquus was determined. The circular genome of 42 781 bp contains a basic set of 13 mitochondrial genes, which are conserved among plant or algal chondriomes. In addition, two scrambled rRNA and 27 tRNA genes are present, together with four intronic sequences (group I and II) and five open reading frames (ORFs), which show no significant homology to other ORFs from organellar genomes. The comparison with deduced amino acid sequences from 13 conserved mitochondrial genes gives rise to the conclusion that two deviations from the standard genetic code must be present in S. obliquus mitochondria: (i) UAG codes for leucine as was already found in some other algal mitochondria; (ii) UCA is a stop codon, which seems unique for mitochondrial genomes. This was supported by our finding that a tRNA-Leu gene possesses a UCA anticodon and by a missing tRNA-serine, able to decode the UCA codon. Consistent with these data is the absence of any UCA codon from conserved mitochondrial ORFs. This codon occurs only close to the end of all ORFs, while UAA or UGA codons are found at some distance from any conserved ORF. Codon changes by RNA editing can be excluded, since RT–PCR analysis does not reveal any evidence for post-transcriptional RNA modifications of the primary transcript.

The trimethylamine methyltransferase gene and multiple dimethylamine methyltransferase genes of Methanosarcina barkeri contain in-frame and read-through amber codons.

Three different methyltransferases initiate methanogenesis from trimethylamine (TMA), dimethylamine (DMA) or monomethylamine (MMA) by methylating different cognate corrinoid proteins that are subsequently used to methylate coenzyme M (CoM). Here, genes encoding the DMA and TMA methyltransferases are characterized for the first time. A single copy of mttB, the TMA methyltransferase gene, was cotranscribed with a copy of the DMA methyltransferase gene, mtbB1. However, two other nearly identical copies of mtbB1, designated mtbB2 and mtbB3, were also found in the genome. A 6.8-kb transcript was detected with probes to mttB and mtbB1, as well as to mtbC and mttC, encoding the cognate corrinoid proteins for DMA:CoM and TMA:CoM methyl transfer, respectively, and with probes to mttP, encoding a putative membrane protein which might function as a methylamine permease. These results indicate that these genes, found on the chromosome in the order mtbC, mttB, mttC, mttP, and mtbB1, form a single transcriptional unit. A transcriptional start site was detected 303 or 304 bp upstream of the translational start of mtbC. The MMA, DMA, and TMA methyltransferases are not homologs; however, like the MMA methyltransferase gene, the genes encoding the DMA and TMA methyltransferases each contain a single in-frame amber codon. Each of the three DMA methyltransferase gene copies from Methanosarcina barkeri contained an amber codon at the same position, followed by a downstream UAA or UGA codon. The C-terminal residues of DMA methyltransferase purified from TMA-grown cells matched the residues predicted for the gene products of mtbB1, mtbB2, or mtbB3 if termination occurred at the UAA or UGA codon rather than the in-frame amber codon. The mttB gene from Methanosarcina thermophila contained a UAG codon at the same position as the M. barkeri mttB gene. The UAG codon is also present in mttB transcripts. Thus, the genes encoding the three types of methyltransferases that initiate methanogenesis from methylamine contain in-frame amber codons that are suppressed during expression of the characterized methyltransferases.

The tRNA N2, N2-dimethylguanosine-26 methyltransferase encoded by gene trm1 increases efficiency of suppression of an ochre codon in Schizosaccharomyces pombe

In the majority of eukaryotic tRNAs, the guanosine at position 26 is modified by a dimethyl group, but so far a function of this modification has not been detected. We isolated the Schizosaccharomyces pombe gene, trm1, encoding the tRNA N2, N2-dimethylguanosine-26 methyltransferase. Strains having the gene deleted completely lack N2, N2-dimethylguanosine. In strains carrying the weak ochre tRNA suppressor sup3-i, deletion of trm1 abolishes suppression indicating that the trm1 deletion acts as an antisuppressor mutation. The result suggests that in vivo N2, N2-dimethylguanosine-26 increases the capacity of the sup3-i serine tRNA to translate the UAA ( ochre) codon.

Polypeptide release factor eRF1 from Tetrahymena thermophila: cDNA cloning, purification and complex formation with yeast eRF3

The first cDNA for the translational release factor eRF1 of ciliates was cloned from Tetrahymena thermophila. The coding frame contained one UAG and nine UAA codons that are reassigned for glutamine in Tetrahymena. The deduced protein sequence is 57% identical to human eRF1. The recombinant Tetrahymena eRF1 purified from a yeast expression system was able to bind to yeast eRF3 as do other yeast or mammalian eRF1s as a prerequisite step for protein termination. The recombinant Tetrahymena eRF1, nevertheless, failed to catalyze polypeptide termination in vitro with rat or Artemia ribosomes, at least in part, due to less efficient binding to the heterologous ribosomes. Stop codon specificity and phylogenetic significance of Tetrahymena eRF1 are discussed from the conservative protein feature.

Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are found.

A small spliceosomal-type intron occurs in a ribosomal protein gene of the microsporidian Encephalitozoon cuniculi

Among unicellular eukaryotes, microsporidia are obligately intracellular amitochondrial parasites that are considered to be of very ancient origin as deduced from the prokaryotic features of their ribosomes [1–3], and rRNA [4] and EF1a /EF2 phylogenies [5]. Microsporidia are also characterized by small nuclear genomes ranging from 19.6 Mb in Glugea atherinae [6] to only 2.9 Mb in Encephalitozoon cuniculi [7], and thus partially spanning the prokaryotic range. Sequencing data on the E. cuniculi genome have supplied information on noncoding regions but have not allowed identification of introns [8]. However, the relatively small sample of genes sequenced in microsporidia may be insufficiently representative for detection of potential intervening sequences. During screening of a partial plasmid library from E. cuniculi genomic DNA, we isolated a clone (c141) coding for a homolog of a large subunit ribosomal protein (L27a or L29 in yeast) gene known to contain usually one or more introns. Blast alignments first revealed that a single reading frame accounts for the entire putative L27a coding region with a termination codon UAA. However, an AUG start codon could not be placed in the position expected on the basis of the conserved character of the L27a amino-terminal region. A thorough examination of this region identified an AT-rich (61%) intervening sequence, with a size of only 28 bp, that creates a frameshift and extends from just after an AUG codon (Fig. 1A). This intron harbors consensus spliceosomal boundaries (5%-GT...AG-3%) with a 5% region identical to the consensus of higher eukaryotes (GTAAGT). In the mould Neurospora crassa, one Abbre6iations: NLS, nuclear localization signal. * Corresponding author. Tel.: +33 473 407457; fax: +33 473 407455; e-mail: vivares@cicsun.univ-bpclermont.fr 1 Note: Nucleotide sequence data reported in this paper are available in the EMBL, GenBank and DDJB data bases under the accession number AF054829.

A Nonsense Mutation in the Exon 2 of the 3-Hydroxy- 3-methylglutaryl Coenzyme A Lyase (HL) Gene Producing Three Mature mRNAs Is the Main Cause of 3-Hydroxy-3-methylglutaric Aciduria in European Mediterranean Patients

3-Hydroxy-3-methylglutaric aciduria is a rare recessive monogenic disorder that affects ketogenesis and the catabolism ofl-leucine. We report the biochemical and molecular characterization of a mutation in the 3-hydroxy-3-methylglutaryl coenzyme A lyase gene in four new probands, three Spanish and one Turkish, affected by 3-hydroxy-3-methylglutaric aciduria, all homozygous for the nonsense mutation Glu37Ter, which was reported by our group in two probands of Portuguese and Moroccan origin (15). In addition to the aberrant mRNAs found in the two previous probands, a novel species of mature HL mRNA was observed in the patients studied here, since a new cDNA, skipped in exons 2 and 3, was obtained from the mRNAs by reverse-transcription PCR (RT-PCR). Thus, three mRNA species were produced in aberrant splicings as a result of this nonsense mutation: (i) one of the expected size that contains the premature stop codon UAA, (ii) another with a deletion of 84 bp corresponding to the whole of exon 2, and (iii) a new species found now, with a deletion of 192 bp corresponding to skipping of the whole of exons 2 and 3, whose translation product led to the loss of seven amino acids in the leader peptide and 57 amino acids in the terminal domain of the mature enzyme. The association of a nonsense mutation with the skipping of the exon that contains it, plus the following exon, is an unusual finding not seen previously in HL deficiencies. The mutation described here shows the highest incidence (>37%) of total HL deficiencies reported.

The tRNATyr-isoacceptors and their genes in the ciliate Tetrahymena thermophila: cytoplasmic tRNATyr has a QPsiA anticodon and is coded by multiple intron-containing genes.

In the ciliated protozoa Tetrahymena thermophila introns have been detected in rRNA and mRNAs until now. We have isolated and sequenced seven tRNATyr genes from the T.thermophila nuclear genome. All of these genes contain introns of identical length and sequence. The 11 bp long intervening sequences are located 1 nt 3' to the anticodon as found in other eukaryotic nuclear tRNA genes. Tetrahymena tRNATyr genes are efficiently transcribed in HeLa cell nuclear extract. Moreover, processing and splicing occurred in HeLa as well as in wheat germ extracts, supporting the notion that Tetrahymena tRNATyr introns can be classified as authentic tRNA introns. We have also isolated cytoplasmic tRNATyr from Tetrahymena cells. This tRNATyr isoacceptor has a QPsiA anticodon and is not a UAG suppressor as shown in in vitro translation studies. Since UAG and UAA codons are used as glutamine codons in Tetrahymena macronuclear DNA, the presence of a strong natural UAG suppressor such as tRNATyr with GPsiA anticodon should cause misreading of the glutamine as tyrosine codons and the absence of the latter had thus been predicted. Furthermore we have studied the organization of tRNATyr genes in the genome of T.thermophila and have found two types of tRNATyr gene arrangement. A minimum of 12 tRNATyr genes are present as single copies in genomic DNA HindIII restriction fragments ranging in size from 0.6 to 7 kb. Additionally one cluster of tRNATyr genes consisting of six members has been detected in a 2.3 kb HindIII fragment.

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.

An RNA-binding protein recognizes a mammalian selenocysteine insertion sequence element required for cotranslational incorporation of selenocysteine.

In mammalian selenoprotein mRNAs, the recognition of UGA as selenocysteine requires selenocysteine insertion sequence (SECIS) elements that are contained in a stable stem-loop structure in the 3' untranslated region (UTR). In this study, we investigated the SECIS elements and cellular proteins required for selenocysteine insertion in rat phospholipid hydroperoxide glutathione peroxidase (PhGPx). We developed a translational readthrough assay for selenoprotein biosynthesis by using the gene for luciferase as a reporter. Insertion of a UGA or UAA codon into the coding region of luciferase abolished luciferase activity. However, activity was restored to the UGA mutant, but not to the UAA mutant, upon insertion of the PhGPx 3' UTR. The 3' UTR of rat glutathione peroxidase (GPx) also allowed translational readthrough, whereas the PhGPx and GPx antisense 3' UTRs did not. Deletion of two conserved SECIS elements in the PhGPx 3' UTR (AUGA in the 5' stem or AAAAC in the terminal loop) abolished readthrough activity. UV cross-linking studies identified a 120-kDa protein in rat testis that binds specifically to the sense strands of the PhGPx and GPx 3' UTRs. Direct cross-linking and competition experiments with deletion mutant RNAs demonstrated that binding of the 120-kDa protein requires the AUGA SECIS element but not AAAAC. Point mutations in the AUGA motif that abolished protein binding also prevented readthrough of the UGA codon. Our results suggest that the 120-kDa protein is a significant component of the mechanism of selenocysteine incorporation in mammalian cells.

UGA suppression by tRNACmCATrp occurs in diverse virus RNAs due to a limited influence of the codon context.

We have recently identified chloroplast and cytoplasmic tRNACmCATrp as the first natural UGA suppressor tRNAs in plants. The interaction of these tRNAs with UGA involves a Cm: A mismatch at the first anticodon position. We show here that tRNACmCATrp is incapable of misreading UAA and UAG codons in vitro, implying that unconventional base pairs are not tolerated in the middle anticodon position. Furthermore, we demonstrate that the ability of tRNACmCATrp to promote UGA read-through depends on a quite simple codon context. Part of the sequence surrounding the leaky UGA stop codon in tobacco rattle virus RNA-1 was subcloned into a zein reporter gene and read-through efficiency was measured by translation of RNA transcripts in wheat germ extract. A number of mutations in the codons adjacent to the UGA were introduced by site-directed mutagenesis. It was found that single nucleotide exchanges at either side of the UGA had little effect on read-through efficiency. A pronounced influence on suppression by tRNACmCATrp was seen only if 2 or 3 nt at the 3'-side of the UGA codon had been simultaneously replaced. As a consequence of the flexible codon context accepted by tRNACmCATrp, this tRNA is able to misread the UGA in a number of plant and animal viral RNAs that use translational read-through for expression of some of their genes.

Depletion in the levels of the release factor eRF1 causes a reduction in the efficiency of translation termination in yeast.

In Saccharomyces cerevisiae, translation termination is mediated by a complex of two proteins, eRF1 and eRF3, encoded by the SUP45 and SUP35 genes, respectively. Mutations in the SUP45 gene were selected which enhanced suppression by the weak ochre (UAA) suppressor tRNA(Ser) SUQ5. In each of four such allosuppressor alleles examined, an in-frame ochre (TAA) mutation was present in the SUP45 coding region; therefore each allele encoded both a truncated eRF1 protein and a full-length eRF1 polypeptide containing a serine missense substitution at the premature UAA codon. The full-length eRF1 generated by UAA read-through was present at sub-wild-type levels. In an suq5+ (i.e. non-suppressor) background none of the truncated eRF1 polypeptides were able to support cell viability, with the loss of only 27 amino acids from the C-terminus being lethal. The reduced eRF1 levels in these sup45 mutants did not lead to a proportional reduction in the levels of ribosome-bound eRF3, indicating that eRF3 can bind the ribosome independently of eRF1. A serine codon inserted in place of the premature stop codon at codon 46 in the sup45-22 allele did not generate an allosuppressor phenotype, thereby ruling out this "missense' mutation as the cause of the allosuppressor phenotype. These data indicate that the cellular levels of eRF1 are important for ensuring efficient translation termination in yeast.

The complete nucleotide sequence and genome organization of bean common mosaic virus (NL3 strain)

The nucleotide sequences of 3 cDNA clones corresponding to entire RNA genome of bean common mosaic virus NL3 strain have been determined. The RNA is 9612 nucleotides long, excluding a 3′-terminal poly(A) tail. A putative start codon located at nucleotide positions 170–172 initiates one large open reading frame that is terminated with a UAA codon at position 9368–9370. The predicted polyprotein has 3066 amino acids and an M r of 340.3 kDa. The positions of putative protein cleavage sites have been determined by analogy to consensus sequences in other potyviruses. The nucleotides sequences of the non-translated regions and the predicted amino acid sequences of BCMV NL3 were compared with those of other potyviruses. Comparison of the BCMV NL3 proteins with those of other potyviruses indicated a similar genomic organization, and high percentage of amino acid sequence identity in the cylindrical inclusion protein, nuclear inclusion ‘b’ protein and coat protein. BCMV NL3 displays the highest amino acid sequence identity with soybean mosaic virus.

Genetic code deviations in the ciliates: evidence for multiple and independent events.

In several species of ciliates, the universal stop codons UAA and UAG are translated into glutamine, while in the euplotids, the glutamine codon usage is normal, but UGA appears to be translated as cysteine. Because the emerging position of this monophyletic group in the eukaryotic lineage is relatively late, this deviant genetic code represents a derived state of the universal code. The question is therefore raised as to how these changes arose within the evolutionary pathways of the phylum. Here, we have investigated the presence of stop codons in alpha tubulin and/or phosphoglycerate kinase gene coding sequences from diverse species of ciliates scattered over the phylogenetic tree constructed from 28S rRNA sequences. In our data set, when deviations occur they correspond to in frame UAA and UAG coding for glutamine. By combining these new data with those previously reported, we show that (i) utilization of UAA and UAG codons occurs to different extents between, but also within, the different classes of ciliates and (ii) the resulting phylogenetic pattern of deviations from the universal code cannot be accounted for by a scenario involving a single transition to the unusual code. Thus, contrary to expectations, deviations from the universal genetic code have arisen independently several times within the phylum.

Nonsense suppressor and antisuppressor mutations at the 1409-1491 base pair in the decoding region of Escherichia coli 16S rRNA.

Using a genetic selection for suppressors of a UGA nonsense mutation in trpA, we have isolated a G to A transition mutation at position 1491 in the decoding region of 16S rRNA. This suppressor displayed no codon specificity, suppressing UGA, UAG and UAA nonsense mutations and +1 and -1 frameshift mutations in lacZ. Subsequent examination of a series of mutations at G1491 and its base-pairing partner C1409 revealed various effects on nonsense suppression and frameshifting. Mutations that prevented Watson-Crick base pairing between these residues were observed to increase misreading and frameshifting. However, double mutations that retained pairing potential produced an antisuppressor or hyperaccurate phenotype. Previous studies of antibiotic resistance mutations and antibiotic and tRNA footprints have placed G1491 and C1409 near the site of codon-anticodon pairing. The results of this study demonstrate that the nature of the interaction of these two residues influences the fidelity of tRNA selection.

Genome Characterization and Identification of Viral-Associated dsDNA Component of Banana Bunchy Top Virus

The single-stranded (ss) and double-stranded (ds) viral-associated DNAs (VADs) of banana bunchy top virus (BBTV) were characterized. The ssVADs were shown to be the viral genomic DNA and the dsVADs are the likely replication forms. Both dsVADs and ssVADs contain DNA species that cross-hybridize to each other. Under the electron microscope, both circular and linear DNAs were observed from these VAD species. Libraries were constructed from VADs to further characterize them. Two overlapping VAD clones were isolated and their combined sequence indicated that their corresponding VAD, named as BBTV component 2, is circular with a size of 1095 nt. The largest ORF spans from nucleotides 82 to 390, which is sandwiched by a putative TATA box, 54 nt upstream from the first ATG codon, and a AATAAA motif, 7 nt downstream from the UAA codon. Although the sequence of this VAD is different from the previously reported BBTV component 1 (Harding et al., J. Gen. Virol. 74, 323-328, 1993) and the genome of coconut foliar decay virus (Rohde et al., Virology 176, 648-651, 1990), stretches of highly homologous sequences were found between BBTV component 2 and the other two viral genomes by computer analysis. The sequence of BBTV component 1 is highly homologous to one of the clones, pBTs26, in the VAD library. Nevertheless, our libraries still contain many clones which hybridized with ss viral genomic DNAs but not with pBTs26 and BBTV component 2, indicating that the viral genome consists of more than two components.

Mutations to nonsense codons in human genetic disease: implications for gene therapy by nonsense suppressor tRNAs.

Nonsense suppressor tRNAs have been suggested as potential agents for human somatic gene therapy. Recent work from this laboratory has described significant effects of 3' codon context on the efficiency of human nonsense suppressors. A rapid increase in the number of reports of human diseases caused by nonsense codons, prompted us to determine how the spectrum of mutation to either UAG, UAA or UGA codons and their respective 3' contexts, might effect the efficiency of human suppressor tRNAs employed for purposes of gene therapy. This paper presents a survey of 179 events of mutations to nonsense codons which cause human germline or somatic disease. The analysis revealed a ratio of approximately 1:2:3 for mutation to UAA, UAG and UGA respectively. This pattern is similar, but not identical, to that of naturally occurring stop codons. The 3' contexts of new mutations to stop were also analysed. Once again, the pattern was similar to the contexts surrounding natural termination signals. These results imply there will be little difference in the sensitivity of nonsense mutations and natural stop codons to suppression by nonsense suppressor tRNAs. Analysis of the codons altered by nonsense mutations suggests that efforts to design human UAG suppressor tRNAs charged with Trp, Gln, and Glu; UAA suppressors charged with Gln and Glu, and UGA suppressors which insert Arg, would be an essential step in the development of suppressor tRNAs as agents of human somatic gene therapy.

Apolipoprotein B mRNA Editing: A Key Controlling Element Targeting Fats to Proper Tissue

Apolipoprotein B (apo B) circulates in two distinct forms referred to as apo B100 and apo B48. Apo B48 is colinear with the amino-terminal half of apo B100 and arises as a result of a post-transcriptional modification, termed apo B mRNA editing. This process changes a single cytidine nucleotide in apo B100 mRNA thereby altering a CAA codon, encoding glutamine in apo B100, to a UAA codon, which specifies an in-frame stop codon in apo B48. The functional consequences of apo B mRNA editing include the divergent catabolism of plasma lipoproteins expressing either apo B100 or B48, and also the ability to generate the hybrid lipoprotein, Lp(a). These differences arise because the requisite regions of apo B for interaction either with the low-density lipoprotein receptor or with apolipoprotein (a) are contained within the carboxyl terminus of apo B100. Apo B mRNA editing is regulated by species, tissue and cell-specific factors, one of which has been recently cloned. The further characterization of apo B mRNA editing, the first example of a mammalian gene regulated by post-transcriptional nucleotide alteration, will be important for an understanding of lipoprotein assembly.

Apolipoprotein B mRNA editing in 12 different mammalian species: hepatic expression is reflected in low concentrations of apoB-containing plasma lipoproteins

Two different isoproteins are encoded by the apolipoprotein (apo) B gene, apoB-48 and apoB-100. ApoB-48, core component of intestinally derived chylomicrons, has an accelerated plasma turnover as compared with the full-length protein apoB-100. A posttranscriptional modification of the apoB mRNA by conversion of cytidine into uridine at nucleotide position 6666 changes the genomically encoded glutamine codon CAA at amino acid residue 2153 into a translational stop codon UAA. This mRNA editing explains the formation of the truncated isoform apoB-48. In the present investigation editing of apoB mRNA in liver and intestine from 12 different mammalian species was measured by a quantitative primer extension analysis of reverse-transcribed and polymerase chain reaction- (PCR) amplified apoB mRNA in order to determine whether i) editing of apoB mRNA is generally restricted to the intestine or may also be found in the liver of other species than rodents, and ii) hepatic expression of apoB mRNA editing influences lipoprotein concentrations in plasma. Intestinal apoB mRNA was edited at high levels in all species, 40% in sheep, 73% in horse, 82% in pig, 84% in dog, 84% in cat, 87% in guinea pig, 88% in rat, 89% in mouse, and > 90% in human, monkey, cow, and rabbit. In liver apoB mRNA was edited to 18% in dog, to 43% in horse, to 62% in rat, and to 70% in mouse. Low levels of editing below 1% were detected in liver of rabbit and guinea pig. In contrast, hepatic apoB mRNA from human, monkey, pig, cow, sheep, and cat liver was not edited. The results of the primer extension analysis were confirmed by cloning and sequencing of the PCR products from dog, horse, cat, guinea pig, sheep, and cow for all of which the apoB cDNA sequence had not been established by previous investigations. Primer extension analysis of apoB mRNA from dog intestine and dog liver indicated C/U editing at C6655 in addition to C6666. Cloning and sequencing of apoB cDNA from dog liver and intestine confirmed additional C/U editing at C6655 which changes ACA for threonine at amino acid residue 2149 into AUA for isoleucine. Synthesis and secretion of apoB-48-containing lipoproteins from liver was demonstrated by pulse labeling of freshly isolated horse hepatocytes and immunoprecipitation with apoB-specific antibodies or density gradient ultracentrifugation. The concentrations of VLDL, LDL, and HDL in all species were determined after fractionation by density gradient ultracentrifugation.(ABSTRACT TRUNCATED AT 400 WORDS)

Termination of translation in bacteria may be modulated via specific interaction between peptide chain release factor 2 and the last peptidyl-tRNA(Ser/Phe).

The 5' context of 671 Escherichia coli stop codons UGA and UAA has been compared with the context of stop-like codons (UAC, UAU and CAA for UAA; UGG, UGC, UGU and CGA for UGA). We have observed highly significant deviations from the expected nucleotide distribution: adenine is over-represented whereas pyrimidines are under-represented in position -2 upstream from UAA. Uridine is over-represented in position -3 upstream from UGA. Lysine codons are preferable immediately prior to UAA. A complete set of codons for serine and the phenylalanine UUC codon are preferable immediately 5' to UGA. This non-random codon distribution before stop codons could be considered as a molecular device for modulation of translation termination. We have found that certain fragment of E. coli release factor 2 (RF2) (amino acids 93-114) is similar to the amino acid sequences of seryl-tRNA synthetase (positions 10-19 and 80-93) and of beta (small) subunit (positions 72-94) of phenylalanyl-tRNA synthetase from E. coli. Three-dimensional structure of E. coli seryl-tRNA synthetase is known [1]: Its N-terminus represents an antiparallel alpha-helical coiled-coil domain and contains a region homologous to RF2. On the basis of the above-mentioned results we assume that a specific interaction between RF2 and the last peptidyl-tRNA(Ser/Phe) occurs during polypeptide chain termination in prokaryotic ribosomes.