The nucleotide sequence of the rat cytoplasmic beta-actin gene.

The nucleotide sequence of the rat beta-actin gene was determined. The gene codes for a protein identical to the bovine beta-actin. It has a large intron in the 5' untranslated region 6 nucleotides upstream from the initiator ATG, and 4 introns in the coding region at codons specifying amino acids 41/42, 121/122, 267, and 327/328. Unlike the skeletal muscle actin gene and many other actin genes, the beta-actin gene lacks the codon for Cys between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. The usage of synonymous codons in the beta-actin gene is nonrandom, and is similar to that in the rat skeletal muscle and other vertebrate actin genes, but differs from the codon usage in yeast and soybean actin genes.     

Codon bias as a factor in regulating expression via translation rate in the human genome

We study the interrelations between tRNA gene copy numbers, gene expression levels and measures of codon bias in the human genome. First, we show that isoaccepting tRNA gene copy numbers correlate positively with expression-weighted frequencies of amino acids and codons. Using expression data of more than 14,000 human genes, we show a weak positive correlation between gene expression level and frequency of optimal codons (codons with highest tRNA gene copy number). Interestingly, contrary to non-mammalian eukaryotes, codon bias tends to be high in both highly expressed genes and lowly expressed genes. We suggest that selection may act on codon bias, not only to increase elongation rate by favoring optimal codons in highly expressed genes, but also to reduce elongation rate by favoring non-optimal codons in lowly expressed genes. We also show that the frequency of optimal codons is in positive correlation with estimates of protein biosynthetic cost, and suggest another possible action of selection on codon bias: preference of optimal codons as production cost rises, to reduce the rate of amino acid misincorporation. In the analyses of this work, we introduce a new measure of frequency of optimal codons (FOP'), which is unaffected by amino acid composition and is corrected for background nucleotide content; we also introduce a new method for computing expected codon frequencies, based on the dinucleotide composition of the introns and the non-coding regions surrounding a gene.     

Selection on GGU and CGU Codons in the High Expression Genes in Bacteria

The fourfold degenerate site (FDS) in coding sequences is important for studying the effect of any selection pressure on codon usage bias (CUB) because nucleotide substitution per se is not under any such pressure at the site due to the unaltered amino acid sequence in a protein. We estimated the frequency variation of nucleotides at the FDS across the eight family boxes (FBs) defined as Um(g), the unevenness measure of a gene g. The study was made in 545 species of bacteria. In many bacteria, the Um(g) correlated strongly with Nc′—a measure of the CUB. Analysis of the strongly correlated bacteria revealed that the U-ending codons (GGU, CGU) were preferred to the G-ending codons (GGG, CGG) in Gly and Arg FBs even in the genomes with G+C % higher than 65.0. Further evidence suggested that these codons can be used as a good indicator of selection pressure on CUB in genomes with higher G+C %.     

Novel third-letter bias in Escherichia coli codons revealed by rigorous treatment of coding constraints

A novel bias in codon third-letter usage was found in Escherichia coli genes with low fractions of "optimal codons", by comparing intact sequences with control random sequences. Third-letter usage has been found to be biased according to preference in codon usage and to doublet preference from the following first letter. The present study examines third-letter usage in the context of the nucleotide sequence when these preferences are considered. In order to exclude any influence by these factors, the random sequences were generated such that the amino acid sequence, codon usage, and the doublet frequency in each gene were all preserved. Comparison of intact sequences with these randomly generated sequences reveals that third letters of codons show a strong preference for the purine/pyrimidine pattern of the next codons: purine (R) is preferred to pyrimidine (Y) at the third site when followed by an R-Y-R codon, and pyrimidine is preferred when followed by an R-R-Y, an R-Y-Y or a Y-R-Y codon. This bias is probably related to interactions of tRNA molecules in the ribosome.     

Primary structure of the tolC gene that codes for an outer membrane protein of Escherichia coli K12.

We present the nucleotide sequence of the tolC gene of Escherichia coli K12, and the amino acid sequence of the TolC protein (an outer membrane protein) as deduced from it. The mature TolC protein comprises 467 amino acid residues, and, as previously reported (1), a signal sequence of 22 amino acid residues is attached to the N-terminus. The C-terminus of the gene is followed by a stem-loop structure (8 base pair stem, 4 base loop) which may be a rho-independent termination signal. The codon usage of the gene is nonrandom; the major isoaccepting species of tRNA are preferentially utilised, or, among synonomous codons recognized by the same tRNA, those codons are used which can interact better with the anticodon (2,3). In contrast to the codon usage for other outer membrane proteins of E. coli (4) the rare arginine codons AGA and AGG are used once and twice respectively.     

The nucleotide sequence of the chick cytoplasmic beta-actin gene

The nucleotide sequence of the chick beta-actin gene was determined. The gene contains 5 introns; 4 interrupt the translated region at codons 41/42, 120/122, 267, 327/328 and a large intron occurs in the 5' untranslated region. The gene has a 97 nucleotide 5'-untranslated region and a 594 nucleotide 3'-untranslated region. A slight heterogeneity in the position of the poly A addition site exists; polyadenylation can occur at either of two positions two nucleotides apart. The gene codes for an mRNA of 1814 or 1816 nucleotides, excluding the poly(A) tail. In contrast to the chick skeletal muscle actin gene the beta-actin gene lacks the Cys codon between the initiator ATG and the codon for the N-terminal amino acid of the mature protein. In the 5' flanking DNA, 15 nucleotides downstream from the CCAAT sequence, is a tract of 25 nucleotides that is highly homologous to the sequence found in the same region of the rat beta-actin gene.     

Phosphonate biosynthesis: molecular cloning of the gene for phosphoenolpyruvate mutase from Tetrahymena pyriformis and overexpression of the gene product in Escherichia coli.

The phosphoenolpyruvate mutase gene from Tetrahymena pyriformis has been cloned and overexpressed in Escherichia coli. To our knowledge, this is the first Tetrahymena gene to be expressed in E. coli, a task made more complicated by the idiosyncratic codon usage by Tetrahymena. The N-terminal amino acid sequence of phosphoenolpyruvate mutase purified from T. pyriformis has been used to generate a precise oligonucleotide probe for the gene, using in vitro amplification from total genomic DNA by the polymerase chain reaction. Use of this precise probe and oligo(T) as primers for in vitro amplification from a T. pyriformis cDNA library has allowed the cloning of the mutase gene. A similar amplification strategy from genomic DNA yielded the genomic sequence, which contains three introns. The sequence of the DNA that encodes 10 amino acids upstream of the N-terminal sequence of the isolated protein was found by oligonucleotide hybridization to a subgenomic library. These 10 N-terminal amino acids are cleanly removed in Tetrahymena in vivo. The full mutase gene sequence codes for a protein of 300 amino acids, and it includes two amber (TAG) codons in the open reading frame. In Tetrahymena, TAG codes for glutamine. When the two amber codons are each changed to a glutamine codon (CAG) that is recognized by E. coli and the gene is placed behind a promoter driven by the T7 RNA polymerase, expression in E. coli is observed. The mutase gene also contains a large number of arginine AGA codons, a codon that is very rarely used by E. coli. Cotransformation with a plasmid carrying the dnaY gene [which encodes tRNA(Arg)(AGA)] results in more than 4-fold higher expression. The mutase then comprises about 25% of the total soluble cell protein in E. coli transformants. The mutase gene bears significant similarity to one other gene in the available data bases, that of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus, an enzyme that catalyzes a closely related transformation. Due to the large evolutionary distance between Tetrahymena and Streptomyces, this similarity can be interpreted as the first persuasive evidence that the biosynthesis of phosphonates is an ancient metabolic process.     

A content-centric organization of the genetic code

The codon table for the canonical genetic code can be rearranged in such a way that the code is divided into four quarters and two halves according to the variability of their GC and purine contents, respectively. For prokaryotic genomes, when the genomic GC content increases, their amino acid contents tend to be restricted to the GC-rich quarter and the purine-content insensitive half, where all codons are fourfold degenerate and relatively mutation-tolerant. Conversely, when the genomic GC content decreases, most of the codons retract to the AUrich quarter and the purine-content sensitive half; most of the codons not only remain encoding physicochemically diversified amino acids but also vary when transversion （between purine and pyrimidine） happens. Amino acids with sixfolddegenerate codons are distributed into all four quarters and across the two halves; their fourfold-degenerate codons are all partitioned into the purine-insensitive half in favorite of robustness against mutations. The features manifested in the rearranged codon table explain most of the intrinsic relationship between protein coding sequences （the informational content） and amino acid compositions （the functional content）. The renovated codon table is useful in predicting abundant amino acids and positioning the amino acids with related or distinct physicochemical properties.     

Does the Genetic Code Have A Eukaryotic Origin?

In the RNA world, RNA is assumed to be the dominant macromolecule performing most, if not all, core "house-keeping" functions. The ribo-cell hypothesis suggests that the genetic code and the translation machinery may both be born of the RNA world, and the introduction of DNA to ribo-cells may take over the informational role of RNA gradually, such as a mature set of genetic code and mechanism enabling stable inheritance of sequence and its variation. In this context, we modeled the genetic code in two content variables-GC and purine contents-of protein-coding sequences and measured the purine content sensitivities for each codon when the sensitivity (% usage) is plotted as a function of GC content variation. The analysis leads to a new pattern-the symmetric pattern-where the sensitivity of purine content variation shows diagonally symmetry in the codon table more significantly in the two GC content invariable quarters in addition to the two existing patterns where the table is divided into either four GC content sensitivity quarters or two amino acid diversity halves. The most insensitive codon sets are GUN (valine) and CAN (CAR for asparagine and CAY for aspartic acid) and the most biased amino acid is valine (always over-estimated) followed by alanine (always under-estimated). The unique position of valine and its codons suggests its key roles in the final recruitment of the complete codon set of the canonical table. The distinct choice may only be attributable to sequence signatures or signals of splice sites for spliceosomal introns shared by all extant eukaryotes.     

Evolution of Prokaryotic Genes by Shift of Stop Codons

De novo origin of coding sequence remains an obscure issue in molecular evolution. One of the possible paths for addition (subtraction) of DNA segments to (from) a gene is stop codon shift. Single nucleotide substitutions can destroy the existing stop codon, leading to uninterrupted translation up to the next stop codon in the gene’s reading frame, or create a premature stop codon via a nonsense mutation. Furthermore, short indels-caused frameshifts near gene’s end may lead to premature stop codons or to translation past the existing stop codon. Here, we describe the evolution of the length of coding sequence of prokaryotic genes by change of positions of stop codons. We observed cases of addition of regions of 3′UTR to genes due to mutations at the existing stop codon, and cases of subtraction of C-terminal coding segments due to nonsense mutations upstream of the stop codon. Many of the observed stop codon shifts cannot be attributed to sequencing errors or rare deleterious variants segregating within bacterial populations. The additions of regions of 3′UTR tend to occur in those genes in which they are facilitated by nearby downstream in-frame triplets which may serve as new stop codons. Conversely, subtractions of coding sequence often give rise to in-frame stop codons located nearby. The amino acid composition of the added region is significantly biased, compared to the overall amino acid composition of the genes. Our results show that in prokaryotes, shift of stop codon is an underappreciated contributor to functional evolution of gene length.     

Comparative analysis of the base biases at the gene terminal portions in seven eukaryote genomes

Adenine nucleotides have been found to appear preferentially in the regions after the initiation codons or before the termination codons of bacterial genes. Our previous experiments showed that AAA and AAT, the two most frequent second codons in Escherichia coli, significantly enhance translation efficiency. To determine whether such a characteristic feature of base frequencies exists in eukaryote genes, we performed a comparative analysis of the base biases at the gene terminal portions using the proteomes of seven eukaryotes. Here we show that the base appearance at the codon third positions of gene terminal regions is highly biased in eukaryote genomes, although the codon third positions are almost free from amino acid preference. The bias changes depending on its position in a gene, and is characteristic of each species. We also found that bias is most outstanding at the second codon, the codon after the initiation codon. NCN is preferred in every genome; in particular, GCG is strongly favored in human and plant genes. The presence of the bias implies that the base sequences at the second codon affect translation efficiency in eukaryotes as well as bacteria.     

The six genes of the Rubisco small subunit multigene family from Mesembryanthemum crystallinum,a facultative CAM plant

The nucleotide sequences of the entire gene family, comprising six genes, that encodes the Rubisco small subunit (rbcS) multigene family in Mesembryanthemum crystallinum (common ice plant), were determined. Five of the genes are arranged in a tandem array spanning 20 kb, while the sixth gene is not closely linked to this array. The mature small subunit coding regions are highly conserved and encode four distinct polypeptides of equal lengths with up to five amino acid differences distinguishing individual genes. The transit peptide coding regions are more divergent in both amino acid sequence and length, encoding five distinct peptide sequences that range from 55 to 61 amino acids in length. Each of the genes has two introns located at conserved sites within the mature peptide-coding regions. The first introns are diverse in sequence and length ranging from 122 by to 1092 bp. Five of the six second introns are highly conserved in sequence and length. Two genes, rbcS-4 and rbcS-5, are identical at the nucleotide level starting from 121 by upstream of the ATG initiation codon to 9 by downstream of the stop codon including the sequences of both introns, indicating recent gene duplication and/or gene conversion. Functionally important regulatory elements identified in rbcS promoters of other species are absent from the upstream regions of all but one of the ice plant rbcS genes. Relative expression levels were determined for the rbcS genes and indicate that they are differentially expressed in leaves.     

Analyses of the gene and amino acid sequence of thePrevotella (bacteroides) ruminicola 23 xylanase reveals unexpected homology with endoglucanases from other genera of bacteria

The DNA sequence for the xylanase gene fromPrevotella (Bacteroides) ruminicola 23 was determined. The xylanase gene encoded for a protein with a molecular weight of 65,740. An apparent leader sequence of 22 amino acids was observed. The promoter region for expression of the xylanase gene inBacteroides species was identified with a promoterless chloramphenicol acetyltransferase gene. A region of high amino acid homology was found with the proposed catalytic domain of endoglucanases from several organisms, includingButyrivibrio fibrisolvens, Ruminococcus flavefaciens, andClostridium thermocellum. The cloned xylanase was found to exhibit endoglucanase activity against carboxymethyl cellulose. Analysis of the codon usage for the xylanase gene found a bias towards G and C in the third position in 16 of 18 amino acids with degenerate codons.     

Molecular cloning and sequence analysis of the ribosomal ‘A’ protein gene from the archaebacterium,Halobacterium halobium

The ribosomal ‘A’ protein gene of Halobacterium halobium has been cloned and the nucleotide sequence of the DNA fragment containing the ‘A’ protein gene has been determined. The amino-acid sequence of the protein deduced from the nucleotide sequence was established from manual sequence analysis of the protein and structural data provided by peptides derived from cleavage of the protein with various proteinases. The ‘A’ protein consisted of 114 amino acids with a molecular weight of 11562 and was characterized mainly by a high amounts of alanine and acidic amino acid in the C-terminal half of the molecule. The coding sequence of the gene was preceded by a predicted Shine-Dalgarno sequence and two terminal codons. There was no intron or insertion sequence in the coding sequence. Following the terminal codon of the ‘A’ gene, there was a structure reminiscent of the Escherichia coli rho-independent terminator. The G + C content of the coding sequence was found to be 71%. Inspection of the codon usage for the ‘A’ gene revealed 85% preference for G or C at the third codon position.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe: highly homologous introns are inserted at the same position of the otherwise less conserved cox1 genes in Schizosaccharomyces pombe and Aspergillus nidulans.

The DNA sequence of the second intron in the mitochondrial gene for subunit 1 of cytochrome oxidase (cox1), and the 3'' part of the structural gene have been determined in Schizosaccharomyces pombe. Comparing the presumptive amino acid sequence of the 3'' regions of the cox1 genes in fungi reveals similarly large evolutionary distances between Aspergillus nidulans, Saccharomyces cerevisiae and S. pombe. The comparison of exon sequences also reveals a stretch of only low homology and of general size variation among the fungal and mammalian genes, close to the 3'' ends of the cox1 genes. The second intron in the cox1 gene of S. pombe contains an open reading frame, which is contiguous with the upstream exon and displays all characteristics common to class I introns. Three findings suggest a recent horizontal gene transfer of this intron from an Aspergillus type fungus to S. pombe. (i) The intron is inserted at exactly the same position of the cox1 gene, where an intron is also found in A. nidulans. (ii) Both introns contain the highest amino acid homology between the intronic unassigned reading frames of all fungi identified so far (70% identity over a stretch of 253 amino acids). However, in the most homologous region, a GC-rich sequence is inserted in the A. nidulans intron, flanked by two direct repeats of 5 bp. The 37-bp insert plus 5 bp of direct repeat amounts to an extra 42 bp in the A. nidulans intron. (iii) TGA codons are the preferred tryptophan codons compared with TGG in all mitochondrial protein coding sequences of fungi and mammalia.(ABSTRACT TRUNCATED AT 250 WORDS)     

杨树派间不同种的遗传密码子使用频率分析

遗传密码子的简并性特征造成了不同物种使用的密码子存在偏爱性。了解不同物种的密码子使用特点,可以为外源基因导入过程中的基因改造提供依据,从而实现外源基因的高效表达。杨树是世界上广泛栽培的重要造林树种之一,已经成为林木基因工程研究的模式植物。本研究采用高频密码子分析法,对美洲山杨P.tremuloides,毛白杨P.tomentosa,美洲黑杨P.deltoids和毛果杨P.trichocarpa 4种杨树的蛋白质编码基因序列(CDS)进行了分析,计算出了杨树同义密码子相对使用频率(RFSC),确定了4种杨树的高频率密码子,发现虽然不同种类的杨树密码子使用上有一些差别,但是偏爱密码子的差别却很小,共性的密码子占绝大多数。仅有Pro,Thr和Cys等少数几个氨基酸的偏爱密码子有差别。这种“共性”提示我们,用不同种的杨树中任何一种杨树的偏爱密码子所设计的外源基因在其他杨树中也可以使用。     

Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene.