Differential stability of trp messenger RNA synthesized originating at the trp promoter and pL promoter of lambda trp phage.

The trp operon translocated into the early region of phage λ can be transcribed under the control of two promoters, the authentic trp promoter (p_Ttrp mRNA) and the p_L promoter of the N gene (p_Ltrp mRNA) (Imamoto &; Tani, 1972; Segawa &; Imamoto, 1974). The p_Ltrp mRNA has a 5′-terminal λ N message. The functional and chemical stability of trp segments in these mRNA species have been assayed. To determine trp mRNA from λtrp, appropriate φ80trp DNAs were used as a DNA complement in DNA-RNA hybridization assays.When formation of mRNA is inhibited, the capacity to serve as template for enzyme synthesis decays at a comparable rate for p_L and p_Ttrp mRNA, and p_Ltrp mRNA seems to be translated as efficiently as is the normal p_Ttrp mRNA. In contrast to this similar functional stability, p_Ltrp mRNA shows a more than tenfold greater chemical stability than p_Ttrp mRNA. (p_Ttrp mRNA is degraded at the same rate as trp mRNA in uninfected bacteria. Bulk host mRNA also decays at its normal rate in cells infected with λtrp.)On the basis of those and more extensive experiments including the sedimentation analysis of those stabilized trp mRNA molecules, it is inferred that (1) the rate-limiting step to initiate bulk mRNA degradation is determined by a sequence located at or near the 5′ end of the messenger RNA; and (2) functional inactivation of each messenger is regulated independently of bulk chemical degradation of the message.Stabilization of the trp mRNA produced from the p_L promoter increases with time after phage infection. Thus, the stabilization requires a modification of the decay trigger, possibly by a phage-specific protein such as a nuclease or the N and/or tof gene that might bind to the mRNA.     

Construction and analysis of in vivo activity of E. coli promoter hybrids and promoter mutants that alter the -35 to -10 spacing

A series of promoter hybrids has been constructed by exchanging the ? 35 and ? 10 regions of lacUV5, tet, and trp promoters. These three promoters and the six hybrid promoters constructed from them have been inserted into a pKO plasmid which places galactokinase expression under the control of the inserted promoter. Additionally, promoter mutants were prepared which had altered the spacing between the ? 35 and ? 10 regions of the promoter. Derivatives of the tet promoter with one or two extra base pairs in this spacer region and constructions of the lac:: tet hybrid promoter with two different spacings have been inserted into the galactokinase expression plasmid. Measurements of galactokinase levels in strains harboring these plasmids permited the comparison of in vivo activities of the promoters. The strongest of the hybrid promoters (order: ? 35, ? 10) were trp:: lac and trp:: tet suggesting a high efficiency for the ? 35 region of the trp promoter. The weakest promoters were tet:: trp, lac:: trp and lac::tet indicating a weak ? 10 region for the trp promoter and the importance of ? 35 to ? 10 spacing. Analysis of activity of related promoters with differences in spacing indicated that a distance of 19 bp yields a very weak promoter, and that 18 bp is less active than the 17-bp spacing, which is the most frequently found spacing in promoters.     

Plasmids for heterologous expression in Pasteurella haemolytica

New cloning and expression vectors that replicate both in Pasteurella haemolytica and in Escherichia coli were constructed based on a native sulfonamide (Su^R) and streptomycin (Sm^R) resistant plasmid of P. haemolytica called pYFC1. Each shuttle vector includes an MCS and a selectable antibiotic resistance marker that is expressed in both organisms. Plasmid pNF2176 carries the P. haemolytica ROB-1 β-lactamase gene (blaP, Ap^R) and pNF2214 carries the Tn903 aph3 kanamycin resistance (Km^R) element. The expression vector, pNF2176, was created by placing the MCS downstream of the sulfonamide gene promoter (P_sulII) on pYFC1; this was used to clone and express the promoterless Tn9 chloramphenicol resistance gene (cat, Cm^R) in P. haemolytica (pNF2200). A promoter-probe vector (pNF2283) was constructed from pNF2200 by deleting P_sulII.     

Identification and characterization of P GCW14 : a novel, strong constitutive promoter of Pichia pastoris

The available promoters in the Pichia pastoris expression platform are still limited. We selected and identified a novel strong constitutive promoter, P _GCW14 , and tested its promoter activity using enhanced green fluorescent protein (EGFP) as a reporter. Potential promoter regions of P _GCW14 were cloned upstream of the EGFP gene and promoter activity was analyzed by measuring fluorescence intensity. P _GCW14 exhibited significantly stronger promoter activity than the classic strong constitutive promoters P _TEF1 and P _GAP under various carbon sources, suggesting that P _GCW14 is a strong and constitutive promoter. Hence, P _GCW14 can be used as a promoter for high-level expression of heterologous proteins.     

Thr region between the operator and first structural gene of the tryptophan operon of Escherichia coli may have a regulatory function

Among a collection of 34 independent mutants with internal deletions in the trp operon of Escherichia coli we found six that fail to recombine with any known point mutant in trpE, the first gene in the operon. These six deletion mutants are regulated normally by tryptophan and thus appear to have the trp operator region intact. However, four of these deletions result in alterations in the maximum level of expression of the trpC, B and A genes when compared with wild type or with an internal deletion of similar length which retains a small operatorproximal segment of trpE. Two of these deletion mutants, trpΔED1 and trpΔED12, have lower levels of the protein products of trpB and trpA than the control strains. In contrast, deletions trpΔED2 and trpΔED102 both markedly increase the levels of the trpB and trpA polypeptides. Deletion mutant trpΔED2 has 3 to 3.5 times and mutant trpΔED102 has seven to eight times as much tryptophan synthetase β₂ and α proteins as the wild-type or deletion control strains. The increase in tryptophan synthetase β₂ and α proteins seen is a consequence of an increase in the level of trp mRNA directing the synthesis of these enzymes. The rate of synthesis of trpBA mRNA is increased in trpΔAED2 about twofold, and in trpΔED102 about four- to sixfold over the control strain. The left-hand deletion end-points of both trpΔED2 and trpΔAED102 have been shown to map to the right of a known trp operator-constitutive mutation and appear to lie before the first translation start codon in trpE (M. Bronson, C. Squires &; C. Yanofsky, unpublished results). We propose that these deletions alter a region between the earliest known trpE point mutation and the trp operator which influences the maximum rate of synthesis of trp operon mRNA.     

Restriction of lambda trp bacteriophages by Escherichia coli K

trp-transducing derivatives of phage λ have been used to study Escherichia coli K specific restriction in vivo. The expression of the trp genes from unmodified phages during infection of a rec⁺, restricting host is eliminated by restriction. In a K-restricting recB,C host, where degradation of restricted phage DNA is prevented, expression of the trp genes is little affected by the presence of a single unmodified, K-restriction recognition site, even when that site is within the trpE gene. RI restriction, in contrast to K restriction, prevents trp gene expression in a recB,C host when the restriction target is between the trp genes and the relevant promoter. The presence of two K-restriction recognition sites in a λtrp phage can have a marked effect on trp gene expression. This effect can be interpreted as the result of preferential breakage between the two restriction recognition sites. We conclude that K restriction does not break susceptible DNA at, or even preferentially near, a restriction recognition sequence.     

Separation and analysis of promoter sites in bacteriophage lambda DNA by specific endonucleases

Specific endonucleases from Hemophilus influenzae, H. parainfluenzae and H. aegyptius were used to separate fragments bearing only one of the various promoters in phage λ DNA. Fragments containing these promoters were characterized by comparative analysis on polyacrylamide gels of the digestion products from λ and a variety of deletion or deletion-substitution derivatives. A single endonuclease from H. influenzae, Hin-II, is shown to cleave the early leftward and rightward promoters, p_L and p_R, at the sites of cleavage of the operators, O_L and O_R, because the corresponding cleavage sites are specifically protected by the DNA-dependent RNA polymerase. With altered p_L (mutations sex1 and sex3), the cleavage in the corresponding promoter is abolished. With X13, a mutation that presumably inactivates p_R, the cleavage in O_R still occurs.     

Mutational Analysis of the Mycobacteriophage BPs Promoter PR Reveals Context-Dependent Sequences for Mycobacterial Gene Expression

The P_R promoter of mycobacteriophage BPs directs early lytic gene expression and is under the control of the BPs repressor, gp33. Reporter gene fusions showed that P_R has modest activity in an extrachromosomal context but has activity that is barely detectable in an integrated context, even in the absence of its repressor. Mutational dissection of P_R showed that it uses a canonical −10 hexamer recognized by SigA, and mutants with mutations to the sequence 5′-TATAMT had the greatest activities. It does not contain a 5′-TGN-extended −10 sequence, although mutants with mutations creating an extended −10 sequence had substantially increased promoter activity. Mutations in the −35 hexamer also influenced promoter activity but were strongly context dependent, and similar substitutions in the −35 hexamer differentially affected promoter activity, depending on the −10 and extended −10 motifs. This warrants caution in the construction of synthetic promoters or the bioinformatic prediction of promoter activity. Combinations of mutations throughout P_R generated a calibrated series of promoters for expression of stably integrated recombinant genes in both Mycobacterium smegmatis and M. tuberculosis, with maximal promoter activity being more than 2-fold that of the strong hsp60 promoter.     

Evaluation of copper-inducible fungal laccase promoter in foreign gene expression in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

The promoter region of copper-inducible laccase gene, LCC1, from Pycnoporus coccineus was explored in the heterologous expression of foreign protein in Pichia pastoris. The promoter region (P_PPLCC1) was isolated and used to replace the methanol-inducible AOXI promoter (P_AOX1) of pPICHOLI-2, an episomal expression vector for P. pastoris, to generate a new copper-inducible expression vector. The promoter activity of P_PPLCC1 was compared with those of P_AOX1 and P_CUP1, a copper-inducible promoter of a commercial vector pPICHOLI-C, using a laccase gene as a reporter gene in P. pastoris GS115. Reporter laccase activity of the culture broth reached 182 and 43 units/L for P_PPLCC1 and P_CUP1, respectively, after induction with 0.2 mM CuSO₄ at OD₆₀₀ = 1 and culture for 120 h at 15°C in complex medium containing 1% glucose. For P_AOX1 activity, yeast cells harboring P_AOX1-laccase plasmid were cultured for 120 h at 15°C in complex medium with intermittent feeding with 1% methanol every 12 h to avoid methanol toxicity. Laccase activity of culture broth was 124 units/L. Conclusively, P_PPLCC1 is a new copper-inducible promoter that shows superior performance in terms of efficiency of laccase production compared to commercial vectors. P_PPLCC1 is additionally superior to P_AOX1 since it does not require laborious feeding with a carbon source.     

Internal promoter of the tryptophan operon of Escherichia coli is located in a structural gene

The constitutive low-efficiency promoter site (P₂) near the middle of the tryptophan operon of Escherichia coli has been mapped by analysis of short deletions internal to the trp operon. Comparison of deletions which remove this internal promoter with those which retain it show that P₂ is located within trpD, the region coding for phosphoribosyl anthranilate transferase. P₂ maps near the operator-distal end of trpD, on the operator-proximal side of two trpD point mutants. Comparisons of strains with and without the P₂ site indicate that initiations at this promoter are responsible for synthesis of 80% of the trpC, trpB and trp A polypeptides present in repressed cells.