同义密码子用语的位置依赖

研究了在大肠杆菌编码区不同位置上的同底密码子用语,发现许多氨基酸的密码子用语在转译起始区有显著的变化,仅有少数氨基酸在转译区有较弱的变化,由于密码子用语与基因表达关系密切。这些结果与实验发现的编码区5‘端密码子用对表达的重要性是一致的。更进一步的结果还暗示了哪些密码子在特定位置的使用可能会影响基因表达。     

Organization and codon usage of the streptomycin operon in Micrococcus luteus, a bacterium with a high genomic G + C content.

The DNA sequence of the Micrococcus luteus str operon, which includes genes for ribosomal proteins S12 (str or rpsL) and S7 (rpsG) and elongation factors (EF) G (fus) and Tu (tuf), has been determined and compared with the corresponding sequence of Escherichia coli to estimate the effect of high genomic G + C content (74%) of M. luteus on the codon usage pattern. The gene organization in this operon and the deduced amino acid sequence of each corresponding protein are well conserved between the two species. The mean G + C content of the M. luteus str operon is 67%, which is much higher than that of E. coli (51%). The codon usage pattern of M. luteus is very different from that of E. coli and extremely biased to the use of G and C in silent positions. About 95% (1,309 of 1,382) of codons have G or C at the third position. Codon GUG is used for initiation of S12, EF-G, and EF-Tu, and AUG is used only in S7, whereas GUG initiates only one of the EF-Tu's in E. coli. UGA is the predominant termination codon in M. luteus, in contrast to UAA in E. coli.     

Gene synthesis, bacterial expression and purification of the Rickettsia prowazekii ATP/ADP translocase.

The Rickettsia prowazekii ATP/ADP translocase (Tlc) is the first member of a new family of ATP/ADP exchangers that includes both prokaryotic and eukaryotic proteins. We optimized the codon usage for expression of tlc in Escherichia coli by means of gene synthesis, expressed the synthetic gene in E. coli, and purified a modified Tlc that contained a C-terminal tag of 10 consecutive histidine residues by immobilized metal affinity chromatography. Although codon usage in R. prowazekii is very different from E. coli, the optimization of the codon usage by itself was insufficient to improve expression. However, the change of the cloning vector from pET11a to pT7-5 led to a 3-10-fold increase in the specific ATP transport rate by cells expressing the synthetic construct. The authenticity of the purified protein was confirmed by N-terminal amino acid sequencing and a matrix assisted laser desorption/ionization mass spectrometry.     

Heterologous gene expression in Bacteroides fragilis.

Bacteroides fragilis and other gastrointestinal tract Bacteroides are unusual gram-negative eubacteria in that genes from other gram-negative eubacteria are not expressed when introduced into these organisms. To analyze gene expression in Bacteroides, expression vector and promoter probe (detection) vector systems were developed. The essential feature of the expression vector was the incorporation of a Bacteroides insertion sequence element, IS4351, which possesses promoter activity directed outward from its ends. Genes inserted into the multiple cloning site downstream from an IS4351 DNA fragment were readily expressed in B. fragilis. The chloramphenicol acetyltransferase (cat) structural gene from Tn9 was tested and conferred chloramphenicol resistance on B. fragilis. Both chloramphenicol resistance and CAT activity were shown to be dependent on the IS4351 promoters. Similar results were obtained with the Escherichia coli beta-glucuronidase gene (uidA) but activity was just 30% of the levels seen with cat. Two tetracycline resistance determinants, tetM from Streptococcus agalactiae and tetC from E. coli, also were examined. tetC did not result in detectable tetracycline resistance but the gram-positive tetM gene conferred high-level resistance to tetracycline and minocycline in Bacteroides hosts. Based on the cat results, promoter probe vectors containing the promoterless cat gene were constructed. These vectors were used to clone random B. fragilis promoters from partial genomic libraries and the recombinants displayed a range of CAT activities and chloramphenicol MICs in B. fragilis hosts. In addition, known E. coli promoters (Ptet, Ptac, Ptrc, Psyn, and P1P2rrnB) were tested for activity in B. fragilis. No chloramphenicol resistance or CAT activity was observed in B. fragilis with these promoters.     

Expression of tetanus toxin fragment C in E. coli: high level expression by removing rare codons.

Tetanus toxin fragment C had been previously expressed in Escherichia coli at 3-4% cell protein. The codon bias for tetanus toxin in Clostridium tetani is very different from that of highly expressed homologous genes in E. coli, resulting in the presence of many rare E. coli codons in the sequence encoding fragment C. We have replaced the coding sequence by sequence optimized for codon usage in E. coli, and show that the expression of fragment C is increased. Although the level of mRNA also increased this appeared to be a secondary consequence of more efficient translation. Complete sequence replacement increased expression to approximately 11-14% cell protein but only after the promoter strength had been improved.     

Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli

A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism.     

Overproduction from a cellulase gene with a high guanosine-plus-cytosine content in Escherichia coli.

A recombinant exoglucanase was expressed in Escherichia coli to a level that exceeded 20% of total cellular protein. To obtain this level of overproduction, the exoglucanase gene coding sequence was fused to a synthetic ribosome-binding site, an initiating ATG, and placed under the control of the leftward promoter of bacteriophage lambda contained on the runaway replication plasmid vector pCP3 (E. Remaut, H. Tsao, and W. Fiers, Gene 22:103-113, 1983). With the exception of an inserted asparagine adjacent to the initiating ATG, the highly expressed exoglucanase is identical to the native exoglucanase. The overproduced exoglucanase can be isolated easily in an enriched form as insoluble aggregates, and exoglucanase activity can be recovered by solubilization of the aggregates in 6 M urea or 5 M guanidine hydrochloride. Since the codon usage of the exoglucanase gene is so markedly different from that of E. coli genes, the overproduction of the exoglucanase in E. coli indicates that codon usage may not be a major barrier to heterospecific gene expression in this organism.     

Complete nucleotide sequence and genetic organization of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens

The complete nucleotide sequence of the bacteriocinogenic plasmid, pIP404, from Clostridium perfringens has been determined. The plasmid genome comprises 10,207 bp and has a dA + dT content of 75%. Functions have been tentatively assigned to 6 of the 10 open reading frames and an origin-like region of repeated sequence identified. The codon usage of this extremely dA + dT rich plasmid is highly unusual and displays a pronounced preference for codons with the lowest dG + dC content. Only one of the genes from pIP404 was expressed at a significant level in Escherichia coli, suggesting that the atypical codon usage could represent a major obstacle to heterologous gene expression.     

RNA primary sequence or secondary structure in the translational initiation region controls expression of two variant interferon-beta genes in Escherichia coli

Efficient expression in Escherichia coli (E. coli) of the human interferon-beta gene (IFN-beta) gene and of a chemically synthesized IFN-beta gene variant (506 base pairs; synIFN-beta) adapted to the E. coli codon usage, both fused to the E. coli atpE ribosome-binding site, is controlled either by primary sequence or by mRNA secondary-structure in the translational initiation region. High level expression of the natural human atpE/IFN-beta gene fusion is governed by the nucleotide composition preceding the initiator codon AUG. A single U----C exchange in the -2 or -1 position preceding the initiator codon AUG reduces the translational efficiency from 18% of total cellular protein to only 8% or 4%, respectively, while both U----C substitutions reduce IFN-beta expression below 1%. These sequence alterations interfere with efficient ribosome binding as revealed by toeprinting. They provide further evidence for the influence of the anticodon-flanking regions of tRNA(fMet) upon the initiation rate of translation. In contrast, translation of the synthetic variant atpE/synIFN-beta gene fusion is controlled by a moderately stable stem-loop structure (delta G = -4 kcal/mol; 37 degrees C) located within the coding region and overlapping the 30 S ribosomal subunit attachment site. That the stability of the hairpin interferes with the initiation of translation is inferred from site-directed mutagenesis and toeprint analyses. mRNA half-life in these variants is positively correlated with the rate of translation and involves two major endonucleolytic cleavage site 5'-upstream of the Shine-Dalgarno region.     

Nucleotide sequence of the gene ereA encoding the erythromycin esterase in Escherichia coli

We have cloned and determined the nucleotide sequence of the gene ereA of plasmid pIP1100 which confers high-level resistance to erythromycin (Em) in Escherichia coli. The gene was defined by initiation and termination codons and by in vitro insertion-inactivation into an open reading frame (ORF) of 1032 bp corresponding to a product with an Mr of 37 765. However, the enzyme, an Em esterase, displayed an apparent Mr of 43 000 upon electrophoresis of a minicell extract on the SDS-polyacrylamide gels. The G + C content (50.5%) of the gene ereA and the preferential codon usage in its ORF suggest that this resistance determinant should be indigenous to E. coli.     

Nucleotide sequence of the structural gene for tryptophanase of Escherichia coli K-12.

The tryptophanase structural gene, tnaA, of Escherichia coli K-12 was cloned and sequenced. The size, amino acid composition, and sequence of the protein predicted from the nucleotide sequence agree with protein structure data previously acquired by others for the tryptophanase of E. coli B. Physiological data indicated that the region controlling expression of tnaA was present in the cloned segment. Sequence data suggested that a second structural gene of unknown function was located distal to tnaA and may be in the same operon. The pattern of codon usage in tnaA was intermediate between codon usage in four of the ribosomal protein structural genes and the structural genes for three of the tryptophan biosynthetic proteins.     

Nucleotide sequence of a novel kanamycin resistance gene, aphA-7, from Campylobacter jejuni and comparison to other kanamycin phosphotransferase genes

A novel kanamycin phosphotransferase gene, aphA-7, was cloned from a 14-kb plasmid obtained from a strain of Campylobacter jejuni and the nucleotide sequence of the gene was determined. The presumed open reading frame of the aphA-7 structural gene was 753 bp in length and encoded a protein of 251 amino acids with a calculated weight of 29,691 Da. A 29-kDa protein was demonstrated in Escherichia coli maxicells containing the cloned aphA-7 gene. A ribosomal binding site corresponding to 5 of 8 bases of the 3' end of the E. coli 16S rRNA was 8 bp upstream of the start codon. Sequences corresponding to the -35 and -10 regions of the consensus promoter sequences of E. coli were upstream of the presumed initiation codon of the gene. The DNA sequence was most closely related to the aphA-3 gene from Streptococcus faecalis, showing 55.4% sequence similarity. There was 45.6% identity at the amino acid level between the aphA-3 and the aphA-7 proteins. Of the three conserved regions noted previously in phosphotransferase genes, the aphA-7 amino acid sequence was identical to the six conserved amino acids in motif 3, but differed in one of the five conserved amino acids in motif 1 (if gaps are permitted) and 3 of the 10 conserved residues in motif 2. The 32.8% G + C ratio in the open reading frame of the aphA-7 kanamycin resistance gene, which is similar to that of the C. jejuni chromosome, suggests that the aphA-7 may be indigenous to Campylobacters.     

Identification of a gene encoding an immuno-reactive membrane protein from Campylobacter jejuni

A gene encoding a putative membrane protein has been identified from Campylobacter jejuni NCTC 11168 following an immuno-screen of a lambda ZAP II genomic DNA library with antiserum raised against glycine-extractable proteins. The nucleotide sequence of the entire genomic insert revealed six open reading frames, all but one of which have sequence homologues in the complete genome sequence of Helicobacter pylori. The gene encoding the immuno-reactive protein was further identified by independent expression of these reading frames in Escherichia coli. The gene encodes an integral membrane protein, expression of which in E. coli results in a profound filamentous phenotype.     

Mutational analysis of cat-86 gene expression controlled by lactococcal promoters in Lactococcus lactis subsp. lactis and Escherichia coli.

Promoters were cloned from the chromosomal DNA of Lactococcus lactis subsp. lactis NP4510 by using promoter-probe vector pGKV210. N-Methyl-N'-nitro-N-nitrosoguanidine-induced mutagenesis of L. lactis-(pBV413), with low-level expression of the cat-86 gene, resulted in enhanced expression. Subcloning and sequencing of the mutated plasmid designated pBV415 revealed that the mutation is located within the PstI-HindIII fragment containing the coding sequence of the cat-86 gene (the 10th CTG codon was replaced by a TTG; both code for leucine). A set of otherwise identical plasmids with four combinations of CTG and TTG codons at the 10th and 46th positions in the cat-86 gene were constructed by site-directed mutagenesis. These plasmids containing cat-86 derivatives displayed a significant variation in cat expression in L. lactis and E. coli. The data suggest that cat expression is dependent on the secondary structure of the cat mRNA. New cat-86 derivatives described here can be used in lactococci, in which they provide additional flexibility for promoter cloning.     

Gene expression in Deinococcus radiodurans.

We previously reported that the Escherichia coli drug-resistance determinants aphA (kanamycin-resistance) and cat (chloramphenicol-resistance) could be introduced to Deinococcus radiodurans by transformation methods that produce duplication insertion. However, both determinants appeared to require dramatic chromosomal amplification for expression of resistance. Additional studies described here, confirming this requirement for extensive amplification, led us to the use of promoter-probe plasmids in which the E. coli promoter has been deleted, leaving only coding sequences for the marker gene. We find that the insertion of D. radiodurans sequences immediately upstream from the promoterless drug-resistance determinant produces drug-resistant transformants without significant chromosomal amplification. Furthermore, a series of stable E. coli-D. radiodurans shuttle plasmids was devised by inserting fragments of D. radiodurans plasmid pUE10 in an E. coli plasmid directly upstream from a promoterless cat gene. These constructions replicated in D. radiodurans by virtue of the pUE10 replicon and expressed the cat determinant because of D. radiodurans promoter sequences in the pUE10 fragment. Of three such constructions, none expressed the cat gene in E. coli. Similar results were obtained using a promoterless tet gene. Translational fusions were made between D. radiodurans genes and E. coli 5'-truncated lacZ. Three fusions that produced high levels of beta Gal in D. radiodurans were introduced into E. coli, but beta Gal was produced in only one. The results demonstrate that the E. coli genes cat, tet and lacZ can be efficiently expressed in D. radiodurans if a D. radiodurans promoter is provided, and that D. radiodurans promoters often do not function as promoters in E. coli.