Third Codon Position Research Articles

Structure and expression of a barley acidic ?-glucanase gene

A barley acidic beta-1,3-glucanase gene was recovered from a barley genomic library by homology with a partial cDNA of barley basic beta-1,3-glucanase isoenzyme GII. The gene, Abg2, is homologous to the PR2 family of pathogenesis-related beta-1,3-glucanase genes. The ABG2 protein has 81% amino acid similarity to barley basic beta-1,3-glucanase GII. The ABG2 protein is encoded as a preprotein of 336 amino acids including a 28 amino acid signal peptide. A 299 bp intron occurs within codon 25. The mature ABG2 protein has a predicted mass of 32,642 Da and a calculated isoelectric point of 4.9. The second exon of the Abg2 gene shows a strong preference for G + C in the third position of degenerate codons. The Abg2 gene was functionally expressed in Escherichia coli. Abg2 mRNA is constitutively expressed in barley root; leaf expression of Abg2 mRNA is induced by mercuric chloride and infection by Erysiphe graminis f. sp. hordei. Southern blot analysis indicates that Abg2 is a member of a small gene family.

Undiscriminating Codon Reading with Adenosine in the Wobble Position

To investigate the reading properties of adenosine in the wobble position we have used site-directed mutagenesis of the Escherichia coli glycine tRNA 1(CCC) gene to substitute the nucleotide A in the wobble position of the corresponding tRNA. The effect of this change on the ability of the tRNA to discriminate between the nucleotides in the third position of the glycine codons has been investigated. We have compared the ability of the mutant glycine tRNA 1(UCC) and glycine tRNA 1(ACC) as well as the mycoplasma glycine tRNA(UCC) to read the glycine codons. The results showed that glycine tRNA 1(ACC) unlike glycine tRNA 1(UCC) did not fully discriminate between the glycine codons. These experiments were carried out using a new in vitro protein synthesizing system that allows us to monitor the reading of all four glycine codons. In the present paper we give a detailed description of this new in vitro system.

Unusually biased nucleotide sequences on sense strands of Flavobacterium sp. genes produce nonstop frames on the corresponding antisense strands.

From investigation of eight Flavobacterium sp. genes encoding enzyme proteins, it was found that six genes had nonstop frames (NSFs) on the antisense strands, and base sequences of the genes are mainly composed of repeating triplet sequence(s), 5'-GNC-3' (where G and C are guanine and cytosine, and N is either of the four bases), in the reading frames. Thus, we concluded that the biased nucleotide sequences on the sense strands produce NSFs on the corresponding antisense strands. Furthermore, from the precise alignments of both nucleotide and amino acid sequences of two related Flavobacterium sp. genes, nyIB and nyIB', it was found that base replacements might have occurred symmetrically in the codons. That is, transversions between G and C were observed at high frequencies at the first and third positions of codons, but not at the second positions. At the first position, AG base transitions were observed much more than similar CT transitions, whereas CT transitions were found at the third positions at a relatively high frequency. These suggest that symmetrical base replacements in codons might be the main contribution to evolution in Flavobacterium sp. genes.

Synonymous codon usage in Zea mays L. nuclear genes is varied by levels of C and G-ending codons.

A multivariate statistical method called correspondence analysis was used to examine the codon usage of one-hundred-and-one nuclear genes of maize (Zea mays L.). Forty percent of the variation in codon usage was due to bias toward G or C-ending versus A or U-ending codons. Differences in levels of G-ending codons showed the weakest correlation with the major codon usage bias. The bias toward C or U versus A or G in the silent third nucleotide position of synonymous codons accounted for approximately 10% of the variation in codon usage. The G+C content of the silent third nucleotide position of coding regions was not strongly correlated with G+C content of introns. Codon usage was strongly biased toward codons ending in G or C for a number of highly expressed genes including most light-regulated chloroplast proteins, ABA-induced proteins, histones, and anthocyanin biosynthetic enzymes. Codon usage of genes encoding storage proteins and regulatory proteins, such as transposases, kinases, phosphatases and transcription factors, was more random than that of genes encoding cytosolic enzymes with similar bias toward G or C-ending codons. Codon usage in maize may reflect both regional bias on nucleotide composition and selection on the silent third nucleotide position.

The rate and pattern of nucleotide substitution in Drosophila mitochondrial DNA.

The nucleotide sequences of a segment of mitochondrial DNA (mtDNA) have been determined for nine species or subspecies of the subgenus Drosophila of the genus Drosophila. This segment contains two complete protein-coding genes (i.e., NADH dehydrogenase subunit 1 and cytochrome b) and a transfer RNA gene (tRNA(ser)). The G+C content at third-codon positions for the two protein-coding genes was 1.5 times higher than that in the D. melanogaster species group, which belongs to the subgenus Sophophora. However, there was a substantial difference between the nucleotide frequencies of G and C. The number of nucleotide substitutions per silent site was more than three times higher than that for nuclear DNA, although it was only 60% of that for mammalian mtDNA. Both parametric and nonparametric analyses revealed a strong transition-transversion bias in nucleotide substitution, as was observed in mammalian mtDNA. Moreover, the rate of substitution of A and T for G and C is higher than that for the opposite direction. This bias seems to be responsible for the extremely A+T-rich base composition of Drosophila mtDNA. It is also noted that the rate of transitional change between A and G is higher than that between T and C.

Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.

Nucleotide sequences of the HLA-DRw12 and DRw8 B1 chains from an Australian aborigine

To gain a more detailed understanding of the molecular structure of the HLA genes in Australian aborigines, the polymorphic first-domain sequences of the DR B alleles were determined in an aborigine who was tissue typed as HLA-DRw8 and a probable DRw12; DRw52; DQw1,7. Both peripheral blood leukocytes and a lymphoblastoid cell line were reactive with the majority of DRw12-specific sera, but also with half of the DRw11-specific sera. With the use of primers specific for the conserved regions flanking the first domain, the polymerase chain reaction technique was used to amplify first-strand synthesis products prepared from the cell line. Two distinct DRB1 sequences were obtained. One was virtually identical to the reported DRw8, Dw8.3 sequence present in an Asian haplotype, differing only by a single silent nucleotide substitution at the third position of codon 36 (A to G). A second DRB allele was closely related to two recently published and nearly identical sequences for DRw12, with amino acid differences at positions 67 and 85 of the first domain. DRB RFLP studies on this cell line using the Taq I restriction enzyme indicated bands previously described for the DRw8 and DRw12 haplotypes.

Equal G and C contents in histone genes indicate selection pressures on mRNA secondary structure.

Protein-specific versus taxon-specific patterns of nucleotide frequencies were studied in histone genes. The third positions of codons have a (well-known) taxon-specific G+C level and a histone type-specific G/C ratio. This ratio counterbalances the G/C ratio in the first and second positions so that the overall G and C levels in the coding region become approximately equal. The compensation of the G/C ratio indicates a selection pressure at the mRNA level rather than a selection pressure or mutation bias at the DNA level or a selection pressure on codon usage. The structure of histone mRNAs is compatible with the hypothesis that the G/C compensation is due to selection pressures on mRNA secondary structure. Nevertheless, no specific motifs seem to have been selected, and the free energy of the secondary structures is only slightly lower than that expected on the basis of nucleotide frequencies.

Three patterns of mitochondrial DNA nucleotide divergence in the meadow vole, Microtus pennsylvanicus.

The DNA sequence was determined for the cytochrome c oxidase II (COII), tRNALys, and ATPase 8 genes from the mitochondrial genome of the meadow vole, Microtus pennsylvanicus. When compared to other rodents, three different patterns of evolutionary divergence were found. Nucleotide variation in tRNALys is concentrated in the T psi C loop. Nucleotide variation in the COII gene in three genera of rodents (Microtus, Mus, Rattus) consists predominantly of transitions in the third base positions of codons. The predicted amino acid sequence in highly conserved (greater than 92% similarity). Analysis of the ATPase 8 gene among four genera (Microtus, Cricetulus, Mus, Rattus) revealed more detectable transversions than transitions, many fixed first and second position mutations, and considerable amino acid divergence. The rate of nucleotide substitution at nonsynonymous sites in the ATPase 8 gene is 10 times the rate in the COII gene. In contrast, the estimated absolute mutation rate as determined by analysis of nucleotide substitutions at fourfold degenerate sites probably is the same for the two genes. The primary sequences of the ATPase 8 and COII peptides are constrained differently, but each peptide is conserved in terms of predicted secondary-level configuration.

Isolation, sequence and molecular karyotype analysis of the actin gene of Cryptosporidium parvum

Actin is an ubiquitous and highly conserved microfilament protein which is hypothesized to play a mechanical force-generating role in the unusual gliding motility of sporozoan zoites and in their active penetration of host cells. We have identified and isolated an actin gene from a Cryptosporidium parvum genomic DNA library using a chicken β-actin cDNA as an hybridization probe. The nucleotide sequences of two overlapping recombinant clones were identical and the amino acid sequence deduced from the single open reading frame was 85% identical to the P. falciparum actin I and human γ-actin proteins. The predicted 42 106-Da Cryptosporidium actin contains 376 amino acids and is encoded by a single-copy gene which contains no introns. The nucleic acid coding sequence is 72% biased to the use of A or T in the third position of codons. Chromosome-sized DNA released from intact C. parvum oocysts was resolved by OFAGE into 5 discrete ethidium bromide-staining DNAs ranging in size from 900 to 1400 kb; the cloned C. parvum actin gene hybridized to a single chromosomal DNA of approximately 1200 kb.

Molecular basis of a unique African variant (A-IV 5) of human apolipoprotein A-IV and its significance in lipid metabolism.

Human apolipoprotein A-IV (apoA-IV) exhibits a genetically determined structural polymorphism amenable to analysis by isoelectric focusing and immunoblotting techniques. We have determined the allele frequency and molecular basis of a unique ApoA-IV*5 allele which is widely distributed among blacks but is absent in other populations. The frequency of the ApoA-IV*5 allele in blacks (N = 308) was estimated to be 3.2%. In comparison to the common ApoA-IV*1 allele, analysis of coding and non-coding sequences of the ApoA-IV*5 allele revealed an in-frame insertion of 12 nucleotides near the carboxyl terminal region of the mature protein. The insertion involves an exact duplication of the second of the four repeats and codes for 4 amino acids glutamic acid (GAA), glutamine (CAG), glutamine (CAG), and glutamine (CAG) and is responsible for the charge shift of the the apoA-IV 5 isoform slightly toward the anode as compared to the wild type apoA-IV 1 isoform on the isoelectric focusing gel. This in-frame insertion occurs in a region which is highly conserved among rat, mouse, and humans. In addition to the 12 nucleotide insertion, the four individuals sequenced for the ApoA-IV*5 allele also revealed a same-sense mutation by replacing G to T at the third position of codon 316. Our preliminary data suggest that this unique black allele marker may be of potentially significance in studies of human lipid metabolism and in microevolution.

Cloning and molecular characterization of the acetamidase-encoding gene ( amdS) from Aspergillus oryzae

We have isolated an acetamidase-encoding gene ( amdS) from Aspergillus oryzae by heterologous hybridization using the corresponding Aspergillus nidulans gene as a probe. The gene is located on a 3.5-kb SacI fragment and its nucleotide (nt) sequence was determined. Compared with the A. nidulans amdS gene, the coding region of A. oryzae gene consists of seven exons interrupted by six introns and encodes 545 amino acid (aa) residues. The deduced aa sequence has a high degree of homology with that of the A. nidulans acetamidase protein. Three introns (IVS-1,IVS-2, and IVS-4) exist at the same positions as those of A. nidulans amdS, whilst three additional introns (IVS-3, IVS-5, and IVS-6) are also present. There is no preference in its codon usage (G + C content in the third position of codons is 51%). Gene disruption experiments demonstrate that the resulting mutants show significantly reduced growth on acetamide-containing medium, indicating that the A. oryzae amdS gene encodes a functional acetamidase that is required for acetamide utilization. Transcriptional analysis by Northern blot reveals a 1.8-kb transcript in RNA extracted from mycelium grown in medium containing acetamide or acetate plus β-alanine as the sole carbon and nitrogen sources.

Nucleotide sequence and transcriptional start point of the kan gene encoding an aminoglycoside 3- N-acetyltransferase from Streptomyces griseus SS-1198PR

We determined the nucleotide sequence of a 1794-bp BglII- BamHI fragment containing the kan gene (Km R) which encodes a novel aminoglycoside 3- N-acetyltransferase of Streptomyces griseus SS-1198PR. The sequence was found to contain four open reading frames (ORF) in the direction of transcription determined by low resolution S1 nuclease mapping. Except for the largest one, these ORF are unlikely to be the coding sequence of kan, because of their inability to confer kanamycin resistance. The largest ORF could encode a 30.8-kDa protein consisting of 284 amino acids (aa) with 93.3 mol% G + C in the third position of codons, and is preceded by potential ribosome-binding sites. Thus, the largest ORF was likely to represent the kan gene. The transcription start point of kan was located 84 bp upstream from the start codon. The −10 region showed similarity to the Escherichia coli consensus promoter motif, while no sequence similarity was found in the −35 region. The deduced aa sequence of kan showed 85% similarity with that of the aacC7 gene of Streptomyces rimosus forma paromomycinus [Lopez-Cabrera et al., J. Bacteriol. 171 (1989) 321–328] suggesting that these genes are derived from a common ancestral gene.

Stomach lysozyme gene of the langur monkey: Tests for convergence and positive selection

Genomic blotting and enzymatic amplification show that the genome of the langur monkey (like that of other primates) contains only a single gene for lysozyme c, in contrast to another group of foregut fermenters, the ruminants, which have a multigene family encoding this protein. Therefore, the langur stomach lysozyme gene has probably evolved recently (i.e., within the period of monkey evolution) from a conventional primate lysozyme. The sequences of cDNAs for the stomach lysozyme of langur and the conventional lysozymes of three other Old World monkeys were determined. Identification of the promoter for the stomach gene and comparison to the human gene, which is expressed conventionally in macrophages, show that both lysozyme genes use the same promoter. This suggests that the difference in expression patterns is due to change(s) in enhancer or silencer regulatory elements. With the cDNA sequences the hypothesis that the langur stomach lysozyme has converged in amino acid sequence upon the stomach lysozymes of ruminants is tested. Consistent with the convergence hypothesis, only those sites that specify amino acids in the mature lysozyme are shared uniquely with ruminant lysozyme genes. None of the silent sites at third positions of codons or in noncoding regions support a link between the langur and ruminants. Statistical analysis based on silent sites rules out the possibility of horizontal transfer of a stomach lysozyme gene between the langur and ruminant lineages and supports the close relationship of the langur lysozyme gene to that of other monkeys.

Secondary structure as a constraint on the evolution of a plant viral satellite RNA

The genetic variability and evolution of the satellite RNA (satRNA) of cucumber mosaic virus (CMV) was analyzed. Twenty-five CMV-satRNAs compared clustered into three main groups, and no correlation was found between genetic proximity and other characteristics (pathogenicity, geographical origin) of the satRNAs. Values for the number of nucleotide substitutions per site between any two satRNAs suggest that divergence is checked by functional constraints. The analysis of mutations relative to an ancestral sequence, and the number of substitutions per site at first, second and third positions of codons in putative open reading frames, show that the variation of CMV-satRNAs does not follow a pattern typical of coding sequences, and indicates that preservation of the sequence of encoded products is not a constraint to evolution. On the other hand, when the observed variation was analyzed relative to a secondary structure model proposed for CMV-satRNAs, several lines of evidence indicated that the maintenance of the secondary structure is a constraint to evolution: the number of substitutions per site, the number of point insertions and deletions and the number of base substitutions that would disrupt base-pairing were significantly higher for unpaired than for base-paired positions. Also, compensatory mutations at base-paired positions occurred more frequently than expected from random. The results suggest that CMV-satRNAs are non-coding, functional RNAs whose biology would be determined by their direct interaction with components of the host and/or the helper virus.

Focusing in on P53 in hepatocellular carcinoma

Human hepatocellular carcinomas (HCC) from patients in Qidong, an area of high incidence in China, in which both hepatitis B virus and aflatoxin B1 are risk factors, were analysed for mutations in p53, a putative tumour-suppressor gene. Eight of the 16 HCC had a point mutation at the third base position of codon 249. The G→T transversion in seven HCC DNA samples and the G→C transversion in the other HCC are consistent with mutations caused by aflatoxin B1 in mutagenesis experiments. No mutations were found in exons 5, 6, 8 or the remainder of exon 7. These results contrast with p53 mutations previously reported in carcinomas and sarcomas of human lung, colon, oesophagus and breast; these are primarily scattered over four of the five evolutionarily conserved domains, which include codon 249. We suggest that the mutant p53 protein may be responsible for a selective clonal expansion of hepatocytes during carcinogenesis. Bressac B, Kew M, Wands J, Ozturk M. Selective G to T mutations of p53 gene in hepatocellular carcinoma from southern Africa. Nature 1991;350:429-431. Hepatocellular carcinoma (HCC) is a prevalent cancer in sub-Saharan Africa and eastern Asia. Hepatitis B virus and aflatoxins are risk factors for HCC, but the molecular mechanism of human hepatocellular carcinogenesis is largely unknown. Abnormalities in the structure and expression of the tumour-suppressor gene p53 are frequent in HCC cell lines, and allelic losses from chromosome 17p have been found in HCCs from China and Japan. Here we report on allelic deletions from chromosome 17p and mutations of the p53 gene found in 50% of primary HCCs from southern Africa. Four of five mutations detected were G→T substitutions, with clustering at codon 249. This mutation specificity could reflect exposure to a specific carcinogen, one candidate being aflatoxin B1, a food contaminant in Africa, which is both a mutagen that induces G to T substitution and a liver-specific carcinogen.

Comparative analysis of ribosomal protein L5 sequences from bacteria of the genus Thermus

The genes for the ribosomal 5S rRNA binding protein L5 have been cloned from three extremely thermophilic eubacteria, Thermus flavus, Thermus thermophilus HB8 and Thermus aquaticus (Jahn et al, submitted). Genes for protein L5 from the three Thermus strains display 95% G/C in third positions of codons. Amino acid sequences deduced from the DNA sequence were shown to be identical for T flavus and T thermophilus, although the corresponding DNA sequences differed by two T to C transitions in the T thermophilus gene. Protein L5 sequences from T flavus and T thermophilus are 95% homologous to L5 from T aquaticus and 56.5% homologous to the corresponding E coli sequence. The lowest degrees of homology were found between the T flavus/T thermophilus L5 proteins and those of yeast L16 (27.5%), Halobacterium marismortui (34.0%) and Methanococcus vannielii (36.6%). From sequence comparison it becomes clear that thermostability of Thermus L5 proteins is achieved by an increase in hydrophobic interactions and/or by restriction of steric flexibility due to the introduction of amino acids with branched aliphatic side chains such as leucine. Alignment of the nine protein sequences equivalent to Thermus L5 proteins led to identification of a conserved internal segment, rich in acidic amino acids, which shows homology to subsequences of E coli L18 and L25. The occurrence of conserved sequence elements in 5S rRNA binding proteins and ribosomal proteins in general is discussed in terms of evolution and function.

Analysis of the spc ribosomal protein operon of Thermus aquaticus

The gene region of Thermus aquaticus corresponding to the distal portion of the S10 operon and to the 5'-portion of the Escherichia coli spc operon was cloned, using the E. coli gene for the ribosomal protein L5 as hybridization probe. The gene arrangement was found to be identical to E. coli, i.e. S17, L14, L24, L5, S14, S8 and L6. Stop and start regions of contiguous cistrons overlap, except for the S14-S8 intergenic region, whose size (67 bases) even exceeds the corresponding spacer regions in E. coli and Bacillus subtilis. A G + C content of 94% in third positions of codons was found in the ribosomal protein genes of T. aquaticus analyzed here. The stop codon of gene S17 (the last gene of the S10 operon in E. coli) and the start codon of gene L14 (the first gene of the spc operon in E. coli) overlap in T. aquaticus, thus leaving no space to accommodate an intergenic promoter preceding spc-operon-encoded genes in T. aquaticus. A possible promoter, localized within the S17 coding region, yielded only weak resistance (20 micrograms/ml) to chloramphenicol in E. coli and therefore could be largely excluded as the main promoter for spc-operon-encoded genes. We failed to detect a structure resembling the protein S8 translational repressor site, located at the beginning of the L5 gene in E. coli, in the corresponding region or any other region in the cloned T. aquaticus spc DNA.

Evolution of Higher-Plant Glutamine Synthetase Genes: Tissue Specificity as a Criterion for Predicting Orthology

Evolutionary relationships among members of the nuclear multigene family encoding isozymes of glutamine synthetase (GS) in flowering plants were studied by using parsimony and distance methods. Analyses using subsets of the data indicated that the consistency index is not a sufficient measure of phylogenetic data quality and that assumptions about the information content of third-codon positions and transitions may be misleading. Tissue-specific expression patterns of GS isozymes were not uniformly useful as predictors of orthology. Chloroplast and cytosolic genes were shown to form two groups of orthologous sequences. Within the cytosolic group, however, genes expressed in the nodule did not form a single orthologous group, suggesting that a regulatory shift has occurred in the pea-alfalfa lineage. Organismal relationships inferred from the limited sampling are generally in concert with current phylogenetic hypotheses.

Mutational hot spot in the p53 gene in human hepatocellular carcinomas

Human hepatocellular carcinomas (HCC) from patients in Qidong, an area of high incidence in China, in which both hepatitis B virus and aflatoxin B1 are risk factors, were analysed for mutations in p53, a putative tumour-suppressor gene. Eight of the 16 HCC had a point mutation at the third base position of codon 249. The G----T transversion in seven HCC DNA samples and the G----C transversion in the other HCC are consistent with mutations caused by aflatoxin B1 in mutagenesis experiments. No mutations were found in exons 5,6,8 or the remainder of exon 7. These results contrast with p53 mutations previously reported in carcinomas and sarcomas of human lung, colon, oesophagus and breast; these are primarily scattered over four of the five evolutionarily conserved domains, which include codon 249 (refs 4-9). We suggest that the mutant p53 protein may be responsible for a selective clonal expansion of hepatocytes during carcinogenesis.