Influence of the codon following the AUG initiation codon on the expression of a modified lacZ gene in Escherichia coli.

In a lacZ expression vector (pMC1403Plac), all 64 codons were introduced immediately 3' from the AUG initiation codon. The expression of the second codon variants was measured by immunoprecipitation of the plasmid-coded fusion proteins. A 15-fold difference in expression was found among the codon variants. No distinct correlation could be made with the level of tRNA corresponding to the codons and large differences were observed between synonymous codons that use the same tRNA. Therefore the effect of the second codon is likely to be due to the influence of its composing nucleotides, presumably on the structure of the ribosomal binding site. An analysis of the known sequences of a large number of Escherichia coli genes shows that the use of codons in the second position deviates strongly from the overall codon usage in E. coli. It is proposed that codon selection at the second position is not based on requirements of the gene product (a protein) but is determined by factors governing gene regulation at the initiation step of translation.     

Expression of a Saccharomyces cerevisiae photolyase gene in Escherichia coli.

A 3.3-kilobase PvuII fragment carrying the PHR1 gene of Saccharomyces cerevisiae has been cloned into an Escherichia coli expression vector and introduced into E. coli strains deficient in DNA photolyase. Complementation of the E. coli phr-1 mutation was observed, strongly suggesting that the yeast PHR1 gene encodes a DNA photolyase.     

Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae.

Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.     

Effects of GUG and AUG initiation codons on the expression of lacZ in Escherichia coli

We have replaced the ribosomal binding site (RBS) of the lacZ gene of E. coli by those of the maturation (A) gene of phage MS2 and that of the tufA gene. Both RBSs contain a GUG initiation codon. The expression with the tufA RBS is at least 25-fold higher than with the phage RBS. Changing the GUG into AUG results in a 3-fold increase in expression in both cases. In general, higher expression is accompanied by an increase of lac-specific mRNA. It is argued that this is a consequence of the more efficient translation of the mRNA.     

Sequence of the lacZ gene of Escherichia coli.

The nucleotide sequence of the lacZ gene coding for beta-galactosidase (EC 3.2.1.23) in Escherichia coli has been determined. Beta-Galactosidase is predicted to consist of 1023 residues, resulting in a protein with a mol. wt. of 116 353 per subunit. The protein sequence originally determined by Fowler and Zabin was shown to be essentially correct and in an Appendix these authors comment on the discrepancies.     

Mutational inactivation of the Saccharomyces cerevisiae RAD4 gene in Escherichia coli.

The RAD4 gene of Saccharomyces cerevisiae is required for the incision of damaged DNA during nucleotide excision repair. When plasmids containing the wild-type gene were transformed into various Escherichia coli strains, transformation frequencies were drastically reduced. Most plasmids recovered from transformants showed deletions or rearrangements. A minority of plasmids recovered from E. coli HB101 showed no evidence of deletion or rearrangement, but when they were transformed into S. cerevisiae on centromeric vectors, little or no complementation of the UV sensitivity of rad4 mutants was observed. Deliberate insertional mutagenesis of the wild-type RAD4 allele before transformation of E. coli restored transformation to normal levels. Plasmids recovered from these transformants contained an inactive rad4 allele; however, removal of the inserted DNA fragment restored normal RAD4 function. These experiments suggest that expression of the RAD4 gene is lethal to E. coli and show that lethality can be prevented by inactivation of the gene before transformation. Stationary-phase cultures of some strains of E. coli transformed with plasmids containing an inactivated RAD4 gene showed a pronounced delay in the resumption of exponential growth, suggesting that the mutant (and, by inference, possibly wild-type) Rad4 protein interferes with normal growth control in E. coli. The rad4-2, rad4-3, and rad4-4 chromosomal alleles were leaky relative to a rad4 disruption mutant. In addition, overexpression of plasmid-borne mutant rad4 alleles resulted in partial complementation of rad4 strains. These observations suggest that the Rad4 protein is relatively insensitive to mutational inactivation.     

Expression of the Escherichia coli xylose isomerase gene in Saccharomyces cerevisiae

Transformation of Saccharomyces cerevisiae by yeast expression plasmids bearing the Escherichia coli xylose isomerase gene leads to production of the protein. Western blotting (immunoblotting) experiments show that immunoreactive protein chains which comigrate with the E. coli enzyme are made in the transformant strains and that the amount produced parallels the copy number of the plasmid. When comparable amounts of immunologically cross-reactive xylose isomerase protein made in E. coli or S. cerevisiae were assayed for enzymatic activity, however, the yeast protein was at least 10(3)-fold less active.     

A codon window in mRNA downstream of the initiation codon where NGG codons give strongly reduced gene expression in Escherichia coli

The influences on gene expression by codons at positions +2, +3, +5 and +7 downstream of the initiation codon have been compared. Most of the +2 codons that are known to give low gene expression are associated with a higher expression if placed at the later positions. The NGG codons AGG, CGG, UGG and GGG, but not GGN or GNG (where N is non-G), are unique since they are associated with a very low gene expression also if located at positions +2, +3 and +5. All codons, including NGG, give a normal gene expression if placed at positions +7. The negative effect by the NGG codons is true for both the lacZ and 3A′ model genes. The low expression is suggested to originate at the translational level, although it is not the result of mRNA secondary structure or a lowered intracellular mRNA pool.     

Expression of the Escherichia coli recA gene in the yeast Saccharomyces cerevisiae

The isolation of the protein coding region of the recA gene from Escherichia coli by extensive Bal31 digestion is described. The structural recA gene was ligated into an extrachromosomally replicating yeast expression vector, downstream of the yeast alcohol-dehydrogenase gene promoter region, to produce pADHrecA plasmid. The pADHrecA plasmid was transformed into the wild-type and the repair deficient strains of Saccharomyces cerevisiae. The crude protein samples were extracted from the individual yeast transformants. A 38 kDa protein was present in all transformants containing the recA gene on plasmid. Thus the recA gene from E coli was successfully expressed in cells from a lower eukaryote.     

Construction of hybrid plasmids containing the lysA gene of Escherichia coli: studies of expression in Escherichia coli and Saccharomyces cerevisiae

Summary The lysA gene of Escherichia coli has been cloned from a transducing phage on various plasmids, present in different copy numbers in bacterial cells. Synthesis of the product of this gene, diaminopimelate (DAP)-decarboxylase, and its regulation have been studied. Expression does not follow a simple gene dosage effect, maximal expression already being obtained with a six-copy plasmid. This result suggests that either a positive or an autogenous regulatory mechanism is involved. We also used one of the hybrid plasmids to look for expression of the bacterial lysA gene in Saccharomyces cerevisiae. The results indicate that the product of the E. coli gene is not actively translated in yeast.     

Expression in Escherichia coli of the Saccharomyces cerevisiae CCT gene encoding cholinephosphate cytidylyltransferase.

The coding region of the CCT gene from the yeast Saccharomyces cerevisiae was cloned into the pUC18 expression vector. The plasmid directed the synthesis of an active cholinephosphate cytidylyltransferase in Escherichia coli, confirming that CCT is the structural gene for this enzyme. The enzyme produced in E. coli efficiently utilized cholinephosphate and N,N-dimethylethanolaminephosphate, but N-methylethanolamine-phosphate and ethanolaminephosphate were poor substrates. Consistently, disruption of the CCT locus in the wild-type yeast cells resulted in a drastic decrease in activities with respect to the former two substrates. When activity was expressed in E. coli, over 90% was recovered in the cytosol, whereas most of the activity of yeast cells was associated with membranes, suggesting that yeast cells possess a mechanism that promotes membrane association of cytidylyltransferase.     

Expression of the Bacillus subtilis levanase gene in Escherichia coli and Saccharomyces cerevisiae.

The gene coding for the inulin hydrolyzing enzyme levanase which was previously cloned from Bacillus subtilis was fused to the tac-promoter. Overexpression in Escherichia coli resulted in high amounts of intracellularly produced levanase (up to 20 U mg-1). After removal of the bacterial 5' sequences, the levanase gene was also cloned into a yeast expression vector based on the PGK-promoter. Clones containing the intact levanase gene including the bacterial signal sequence gave rise to synthesis of active levanase by Saccharomyces cerevisiae transformants. A considerable amount of levanase protein was found in the culture medium (around 0.5 U ml-1) indicating efficient secretion of B. subtilis levanase from yeast.     

Fusion of the Saccharomyces cerevisiae leu2 gene to an Escherichia coli beta-galactosidase gene.

The promoter and translation initiation region of the Saccharomyces cerevisiae leu2 gene was fused to the Escherichia coli beta-galactosidase gene. This fusion located the control region of the leu gene and orientated its direction of expression. When the fusion was placed into yeast cells, beta-galactosidase was expressed under the same regulatory pattern as the original leu2 gene product: its synthesis was repressed in the presence of leucine and threonine. Sensitive chromogenic substrates for beta-galactosidase were used to detect expression in isolated colonies growing on agar medium. Mutant yeast cells with increased beta-galactosidase activity were identified by the color of the colonies they formed. One class of mutants obtained appeared to affect ars1 plasmid maintenance, and another class appeared to affect beta-galactoside uptake.     

Expression of an Escherichia coli phr gene in the yeast Saccharomyces cerevisiae

Summary A 2 kb DNA fragment, containing the photoreactivation gene phr1 from Escherichia coli, was inserted at the BamH1 site in the tet gene of the yeast — E. coli shuttle vector pJDB207. Photoreactivation — deficient Saccharomyces cerevisiae cells transformed with this plasmid showed photoreactivation of killing after UV irradiation of the cells, while extracts of transformed cells exhibited photoreactivating activity in vitro. Far more photoreactivating enzyme molecules were found when the gene was inserted in the plasmid in the opposite orientation to the tet gene as compared with a plasmid carrying the inserted gene in the same orientation. Photoreactivating enzyme encoded by the E. coli phr1 gene and produced in transformed yeast cells has characteristics of the E. coli photoreactivating enzyme (flavoprotein) as judged from the influence of ionic strength on photoreactivating activity.     

Escherichia coli translation initiation factor 3 discriminates the initiation codon in vivo

In a genetic selection designed to isolate Escherichia coli mutations that increase expression of the IS 10 transposase gene ( tnp ), we unexpectedly obtained viable mutants defective in translation initiation factor 3 (IF3). Several lines of evidence led us to conclude that transposase expression, per se , was not increased. Rather, these mutations appear to increase expression of the tnp'–'lacZ gene fusions used in this screen, by increasing translation initiation at downstream, atypical initiation codons. To test this hypothesis we undertook a systematic analysis of start codon requirements and measured the effects of IF3 mutations on initiation from various start codons. Beginning with an efficient translation initiation site, we varied the AUG start codon to all possible codons that differed from AUG by one nucleotide. These potential start codons fall into distinct classes with regard to translation efficiency in vivo : Class I codons (AUG, GUG, and UUG) support efficient translation; Class IIA codons (CUG, AUU, AUC, AUA, and ACG) support translation at levels only 1–3% that of AUG; and Class IIB codons (AGG and AAG) permit levels of translation too low for reliable quantification. Importantly, the IF3 mutations had no effect on translation from Class I codons, but they increased translation from Class II codons 3–5-fold, and this same effect was seen in other gene contexts. Therefore, IF3 is generally able to discriminate between efficient and inefficient codons in vivo , consistent with earlier in vitro observations. We discuss these observations as they relate to IF3 autoregulation and the mechanism of IF3 function.     

Genetic complementation of the Saccharomyces cerevisiae leu2 gene by the Escherichia coli leuB gene.

The leucine operon of Escherichia coli was cloned on a plasmid possessing both E. coli and Saccharomyces cerevisiae replication origins. This plasmid, pEH25, transformed leuA, leuB, and leuD auxotrophs of E. coli to prototrophy; it also transformed leu2 auxotrophs of S. cerevisiae to prototrophy. beta-Isopropylmalate dehydrogenase was encoded by the leuB gene of E. coli and the leu2 gene of yeast. Verification that the leuB gene present on pEH26 was responsible for complementing yeast leu2 was obtained by isolating in E. coli several leuB mutations that resided on the plasmid. These mutant leuB- plasmids were no longer capable of complementing leu2 in S. cerevisiae. We conclude that S. cerevisiae is capable of transcribing at least a portion of the polycistronic leu operon of E. coli and can translate a functional protein from at least the second gene of this operon. The yeast Leu+ transformants obtained with pEH25, when cultured in minimal medium lacking leucine, grew with a doubling time three to four times longer than when cultured in medium supplemented with leucine.     

Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions.

A recA-lacZ protein fusion was constructed in vivo by using bacteriophage Mu dII301(Ap lac). The fusion contained the promoter and first 47 codons of the recA mutant, as determined by DNA sequence analysis. The fusion was cloned and used to construct a recA-lacZ operon fusion at the same site within the recA gene. These fusions were introduced into the Escherichia coli chromosome at the lambda attachment site either as complete or cryptic lambda prophages. Synthesis of beta-galactosidase from these fusions was inducible by UV radiation. As the UV dose was increased, induction became slower and persisted for a longer period of time. At low doses of UV radiation, more beta-galactosidase was produced in a uvrA mutant than in a wild-type strain; however, at high doses, no induced synthesis of beta-galactosidase occurred in a uvrA mutant. recA+ strains carrying either the protein or operon fusion on a multicopy plasmid showed reduced survival after UV irradiation. This UV sensitivity was not exhibited by strains containing a single copy of either fusion, however; hence, the fusions provide a reliable measure of recA expression.