Codon bias differentiates between the duplicated amylase loci following gene duplication in Drosophila

We examined the pattern of synonymous substitutions in the duplicated Amylase (Amy) genes (called the Amy1- and Amy3-type genes, respectively) in the Drosophila montium species subgroup. The GC content at the third synonymous codon sites of the Amy1-type genes was higher than that of the Amy3-type genes, while the GC content in the 5'-flanking region was the same in both genes. This suggests that the difference in the GC content at third synonymous sites between the duplicated genes is not due to the temporal or regional changes in mutation bias. We inferred the direction of synonymous substitutions along branches of a phylogeny. In most lineages, there were more synonymous substitutions from G/C (G or C) to A/T (A or T) than from A/T to G/C. However, in one lineage leading to the Amy1-type genes, which is immediately after gene duplication but before speciation of the montium species, synonymous substitutions from A/T to G/C were predominant. According to a simple model of synonymous DNA evolution in which major codons are selectively advantageous within each codon family, we estimated the selection intensity for specific lineages in a phylogeny on the basis of inferred patterns of synonymous substitutions. Our result suggested that the difference in GC content at synonymous sites between the two Amy-type genes was due to the change of selection intensity immediately after gene duplication but before speciation of the montium species.     

Mismatch and blunt to protruding-end joining by DNA ligases.

A nuclear DNA ligase activity from immature chicken erythrocytes, and to a lesser extent T4-induced DNA ligase, can join cohesive-ends (3 and 5-nucleotides long) having one of the mismatches, A/A, T/T, C/C, G/G, at the middle position. The rate of ligation depends on the length and stability of the mispaired intermediate (G/G, T/T greater than A/A, C/C). When the non-complementary overhanging-ends are short (i.e. 1-nucleotide) both ligases catalyze the joining of the single-stranded protruding-end with a blunt-end. This reaction occurs at low but significant rates compared to blunt-end ligation. The chicken ligase has lower flush-end joining activity than T4 DNA ligase, but it is more permissive since it joins C/C or A/A mismatched-ends, whereas the prokaryotic ligase does not. Possible biological implications of the reactions are discussed. We have also found that BstEII easily cleaves at sites harboring a C/C or a G/G mismatch at the center of its recognition sequence, whereas AvaII (T/T or A/A), HinfI (G/G) and DdeI (G/G) do not.     

Mutagenesis of the 3' nontranslated region of Sindbis virus RNA.

A cDNA clone from which infectious RNA can be transcribed was used to construct 42 site-specific mutations in the 3' nontranslated region of the Sindbis virus genome. The majority of these mutations were made in the 3'-terminal 19-nucleotide conserved sequence element and consisted of single nucleotide substitutions or of small (1 to 8) nucleotide deletions. An attempt was made to recover mutant viruses after transfection of SP6-transcribed RNA into chicken cells. In most cases, viable virus was recovered, but almost all mutants grew more poorly than wild-type virus when tested under a number of culture conditions. In the case of mutations having only a moderate effect, the virus grew as well as the wild type but was slightly delayed in growth. Mutations having a more severe effect led to lower virus yields. In many cases, virus growth was more severely impaired in mosquito cells than in chicken cells, but the opposite phenotype was also seen, in which the mutant grew as well as or better than the wild type in mosquito cells but more poorly in chicken cells. One substitution mutant, 3NT7C, was temperature sensitive for growth in chicken cells and severely crippled for growth in mosquito cells. Insertion mutations were also constructed which displaced the 19-nucleotide element by a few nucleotides relative to the poly(A) tail. These mutations had little effect on virus growth. Deletion of large regions (31 to 293 nucleotides long) of the 3' nontranslated region outside of the 19-nucleotide element resulted in viruses which were more severely crippled in mosquito cells than in chicken cells. From these results, the following principles emerge. (i) The entire 3' nontranslated region is important for efficient virus replication, although there is considerable plasticity in this region in that most nucleotide substitutions or deletions made resulted in viable virus and, in some cases, in virus that grew quite efficiently. Replication competence was particularly sensitive to changes involving the C at position 1, the A at position 7, and a stretch of 9 U residues punctuated by a G at position 14. (ii) The panel of mutants examined collectively deleted the entire 3' nontranslated region. Only mutants in which 8 nucleotides in the 3' terminal 19 nucleotides had been deleted or in which the 3' terminal C was deleted were nonviable. Although the 3' terminal C was essential for replication, it could be displaced by at least 7 nucleotides from its 3' terminal position adjacent to the poly(A) tract.(ABSTRACT TRUNCATED AT 400 WORDS)     

Negative correlation of G+C content at silent substitution sites between orthologous human and mouse protein-coding sequences.

We conducted a genome-wide analysis of variations in guanine plus cytosine (G+C) content at the third codon position at silent substitution sites of orthologous human and mouse protein-coding nucleotide sequences. Alignments of 3776 human protein-coding DNA sequences with mouse orthologs having >50 synonymous codons were analyzed, and nucleotide substitutions were counted by comparing sequences in the alignments extracted from gap-free regions. The G+C content at silent sites in these pairs of genes showed a strong negative correlation (r = -0.93). Some gene pairs showed significant differences in G+C content at the third codon position at silent substitution sites. For example, human thymine-DNA glycosylase was A+T-rich at the silent substitution sites, while the orthologous mouse sequence was G+C-rich at the corresponding sites. In contrast, human matrix metalloproteinase 23B was G+C-rich at silent substitution sites, while the mouse ortholog was A+T-rich. We discuss possible implications of this significant negative correlation of G+C content at silent sites.     

Novel mutational spectrum induced by N-methyl-N'-nitro-N-nitrosoguanidine in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase gene in diploid human fibroblasts

The kinds and locations of mutations in the coding region of the hypoxanthine (guanine) phosphoribosyltransferase (hprt) gene of 75 independent mutants, derived from N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treated normal human fibroblasts, were characterized by direct sequencing of mRNA-polymerase chain reaction (mRNA-PCR)-amplified cDNA. Treatment of human cells with low (6 or 8 microM) or high (10 or 12 microM) doses of MNNG resulted in 35-fold or 150-fold average increases in mutation frequency, respectively. A high frequency of mutants lacking a complete exon was observed in both groups. Further characterization of half of these mutants by DNA-PCR amplification of intron-exon boundaries showed that they contained base substitutions. The kinds of base substitutions differed distinctly between these two groups. In the low dose group, a broad mutational spectrum was observed: ten out of the 31 base substitutions were A.T to G.C transitions, six contained G.C to A.T transitions, and the other 15 exhibited transversions. In contrast, the majority (84%) of base substitutions among the high dose group were G.C to A.T transitions; the others (16%) were transversions. All of the 32 G.C to A.T transitions were located on the non-transcribed strand, assuming that the causative premutational lesion was O6-methylguanine. These results indicate preferential repair of lesions located on the transcribed strand. In addition, G.C to A.T and A.T to G.C transitions preferentially occurred at positions with guanine and thymine at the adjacent 5' position, respectively.     

Neighboring base identity affects N-ethyl-N-nitrosourea-induced mutagenesis in Escherichia coli

This study investigated the influence of different neighboring base contexts on the production of base substitutions generated by N-ethyl-N-nitrosourea (ENU). A set of bacterial strains having all possible bases neighboring an ochre (TAA) nonsense mutation in the tyrA gene of Escherichia coli were employed and true reversions of the nonsense mutation were induced by two separate doses of ENU. Base substitution mutations were investigated by direct sequencing methods. These studies revealed that 1) mutations occurring at 5'-purine-T sites were produced better, on average, than mutations involving 5'-pyrimidine-T sites, and 5'-TT sites contributed the least to the formation of mutations, 2) the order of preference for A:T to G:C transitions was 5'-GT>5'-AT, 5'-CT>5'-TT, and 3) A:T to C:G transversions at the first position of the codon (GAA mutations) were produced best at 5'-AT sites, while A:T to T:A transversions at the third position (TAT mutations) occurred more often at 5'-GT sites. These findings suggest that the occurrence of a specific mutation may reflect the sequence-dependent probability of DNA damage at a particular site as well as factors involving preferential DNA repair or differential base selection by DNA polymerase.     

DNA G+C content of the third codon position and codon usage biases of human genes

The human genome, as in other eukaryotes, has a wide heterogeneity in the DNA base composition. The evolutionary basis for this heterogeneity has been unknown. A previous study of the human genome (846 genes analyzed) has shown that, in the major range of the G+C content in the third codon position (0.25-0.75), biases from the Parity Rule 2 (PR2) among the synonymous codons of the four-codon amino acids are similar except in the highest G+C range (Sueoka, N., 1999. Translation-coupled violation of Parity Rule 2 in human genes is not the cause of heterogeneity of the DNA G+C content of third codon position. Gene 238, 53-58.). PR2 is an intra-strand rule where A=T and G=C are expected when there are no biases between the two complementary strands of DNA in mutation and selection rates (substitution rates). In this study, 14,026 human genes were analyzed. In addition, the third codon positions of two-codon amino acids were analyzed. New results show the following: (a) The G+C contents of the third codon position of human genes are scattered in the G+C range of 0.22-0.96 in the third codon position. (b) The PR2 biases are similar in the range of 0.25-0.75, whereas, in the high G+C range (0.75-0.96; 13% of the genes), the PR2-bias fingerprints are different from those of the major range. (c) Unlike the PR2 biases, the G+C contents of the third codon position for both four-codon and two-codon amino acids are all correlated almost perfectly with the G+C content of the third codon position over the total G+C ranges. These results support the notion that the directional mutation pressure, rather than the directional selection pressure, is mainly responsible for the heterogeneity of the G+C content of the third codon position.     

Discovery of a new nucleotide substitution in the MC1R gene and haplotype distribution in native and non-Japanese chicken breeds

Melanocortin 1-receptor (MC1R) is one of the major genes that controls chicken plumage colour. In this study, we investigated the sequence and haplotype distribution of the MC1R gene in native Japanese chickens, along with non-Japanese chicken breeds. In total, 732 and 155 chickens from 30 Japanese and eight non-Japanese breeds respectively were used. Three synonymous and 11 non-synonymous nucleotide substitutions were detected, resulting in 15 haplotypes (H0–H14). Of these, three were newly found haplotypes (H9, H13 and H14), of which one (H9) was composed of known substitutions C69T, T212C, G274A and G636A. The second one (H13) possessed newly found non-synonymous substitution C919G, apart from the known substitutions C69T, G178A, G274A, G636A and T637C. The third one (H14) comprised a newly discovered substitution C919G in addition to the known C69T, G274A and G409A substitutions. The homozygote for this new haplotype exhibited wt like plumage despite the presence of G274A. In addition to discovering a new nucleotide substitution (C919G) and three new haplotypes, we defined the plumage colour of the bird that was homozygous for the A644C substitution (H5 haplotype) as wheaten-like for the first time; although the substitution has been already reported, its effect was not revealed. Besides detecting the new plumage colour, we also confirmed that the A427G and G274A substitutions contribute in expressing brownish and black plumage colour respectively, as reported by the previous studies. Moreover, we confirmed that the buttercup allele does not express black plumage despite possessing a G274A substitution, under the suppression effect of A644C. In contrast, the birds homozygous for the birchen allele presented solid black plumage, which was contradictory to the previous reports. In conclusion, we revealed a large diversity in the MC1R gene of native Japanese chicken breeds, along with the discovery of a new non-synonymous nucleotide substitution (C919G) and three novel haplotypes (H9, H13 and H14).     

Organisms can essentially be classified according to two codon patterns

We recently classified 23 bacteria into two types based on their complete genomes; “S-type” as represented by Staphylococcus aureus and “E-type” as represented by Escherichia coli. Classification was characterized by concentrations of Arg, Ala or Lys in the amino acid composition calculated from the complete genome. Based on these previous classifications, not only prokaryotic but also eukaryotic genome structures were investigated by amino acid compositions and nucleotide contents. Organisms consisting of 112 bacteria, 15 archaea and 18 eukaryotes were classified into two major groups by cluster analysis using GC contents at the three codon positions calculated from complete genomes. The 145 organisms were classified into “AT-type” and “GC-type” represented by high A or T (low G or C) and high G or C (low A or T) contents, respectively, at every third codon position. Reciprocal changes between G or C and A or T contents at the third codon position occurred almost synchronously in every codon among the organisms. Correlations between amino acid concentrations (Ala, Ile and Lys) and the nucleotide contents at the codon position were obtained in both “AT-type” and “GC-type” organisms, but with different regression coefficients. In certain correlations of amino acid concentrations with GC contents, eukaryotes, archaea and bacteria showed different behaviors; thus these kingdoms evolved differently. All organisms are basically classifiable into two groups having characteristic codon patterns; organisms with low GC and high AT contents at the third codon position and their derivatives, and organisms with an inverse relationship.     

The primary structure of skeletal muscle myosin heavy chain: II. Sequence of the 50 kDa fragment of subfragment-1

The complete amino acid sequence of the 50 kDa fragment of subfragment-1 from adult chicken pectoralis muscle myosin was determined. It contained 431 residues including an epsilon-N-trimethyllysine at position 346. The 431-residue sequence corresponds to the sequence of residues 206 to 639 of chicken embryonic breast muscle myosin heavy chain which was predicted from the nucleotide sequence of the cDNA by Molina et al. [Molina, M. I., Kropp, K.E., Gulick, J., & Robbins, J. (1987) J. Biol. Chem. 262, 6478-6488]. Comparing the two sequences, 23 amino acid substitutions and three deletions/insertions are recognized.     

Isolation, characterization, cDNA cloning and gene expression of an avian transthyretin. Implications for the evolution of structure and function of transthyretin in vertebrates.

A chicken liver cDNA library was constructed in bacteriophage lambda gt10. A full-length transthyretin cDNA clone was identified by screening with rat transthyretin cDNA and was sequenced. A three-dimensional model of chicken transthyretin was obtained by computer-graphics-based prediction from the derived amino acid sequence for chicken transthyretin and from the structure of human transthyretin determined by X-ray diffraction analysis [Blake, C.C.F., Geisow, M.J., Oatley, S.J., Rérat, B. & Rérat, C. (1978) J. Mol. Biol. 121, 339-356]. The similarity of the amino acid sequences of chicken and human transthyretins was 75% overall and 100% for the central channel containing the thyroxine-binding site. Also, the organization of the transthyretin gene into exons and introns and the tissue specificity of expression of the transthyretin gene were similar in chicken and mammals, despite an evolutionary distance of about 3 x 10(8) years from their common ancestor, the Cotylosaurus. By far the highest levels of transthyretin mRNA were found in choroid plexus. The data suggest a fundamental role for the cerebral expression of transthyretin in all vertebrates. It has been proposed that this role is the transport of thyroxine from the bloodstream to the brain [Schreiber, G., Aldred, A.R., Jaworowski, A., Nilsson, C., Achen, M.G. & Segal, M.B. (1990) Am. J. Physiol. 258, R338-R345].     

A mutant Escherichia coli sigma 70 subunit of RNA polymerase with altered promoter specificity

A mutation is described that alters the promoter specificity of sigma 70, the primary sigma factor of Escherichia coli RNA polymerase. In strains carrying both the mutant and wild-type sigma gene (rpoD), the mutant sigma causes a large increase in the activity of mutant P22 ant promoters with A.T or C.G instead of the wild-type, consensus G.C base-pair at position -33, the third position of the consensus -35 hexamer 5'-TTGACA-3'. There is little or no effect on the activities of the wild-type and 23 other mutant ant promoters, including one with T.A at -33. The mutant sigma also activates E. coli lac promoters with A.T or C.G, but not T.A, at the corresponding position. The rpoD mutation (rpoD-RH588) changes a CGT codon to CAT. The corresponding change in sigma 70 is Arg588----His. This residue is in a region that is conserved among most sigma factors, a region that is also homologous with the helix-turn-helix motif of DNA-binding proteins. These results suggest that this region of sigma 70 is directly involved in recognition of the -35 hexamer.     

The common cDNA and amino acid sequences of the CD14 (myeloid cell-specific leucine-rich glycoprotein) receptor

The CD14 receptor is a myeloid cell specific receptor, which plays a role in the recognition of lipopolysaccharides (endotoxins of gram-negative bacteria) and cell stimulation. To date, several sequences of the cDNA of the CD14 receptor have been described. We sought to establish whether the substitutions C(230)-->G, and G(560)-->A are polymorphic or if they result from a PCR or sequencing error. Using two mismatched PCRs, we confirmed (on 75 unrelated probands) that the substitutions are not due to common polymorphisms. The common cDNA sequence has the C in position 230 and G in position 560. This corresponds to the amino acids Ala and Cys in positions 77 and 187, respectively.     

Genomic sequence and structural organization of mouse slow skeletal muscle troponin T gene

Three muscle type-specific troponin T (TnT) genes are present in vertebrate to encode a number of protein isoforms via alternative mRNA splicing. While the genomic structures of cardiac and fast skeletal muscle TnT genes have been documented, this study cloned and characterized the slow skeletal muscle TnT (sTnT) gene. Complete nucleotide sequence and genomic organization revealed that the mouse sTnT gene spans 11.1kb and contains 14 exons, which is smaller and simpler than the fast skeletal muscle and cardiac TnT genes. Potentially representing a prototype of the TnT gene family, the 5'-region of the sTnT gene contains fewer unsplit large exons, among which two alternatively spliced exons are responsible for the NH2-terminal variation of three sTnT isoforms. The sTnT gene structure shows that the alternatively spliced central segment found in human sTnT cDNAs may be a result from splicing using an alternative acceptor site at the intron 11-exon 12 boundary. Together with the well-conserved protein structure, the highly specific expression of sTnT in slow skeletal muscles indicates a differentiated function of this member of the TnT gene family. The determination of genomic structure and alternative splicing pathways of sTnT gene lays a foundation to further understand the TnT structure-function evolution as well as contractile characteristics of different types of muscle fiber.