Role of DNA regions flanking the tryptophan promoter of Escherichia coli. I. Insertion of synthetic oligonucleotides

To examine the effect of altering the nucleotide sequence near the promoter on its activity, pKO-1 vector derivatives have been constructed which allow insertion of DNA fragments at specified sites upstream or downstream from the trp promoter. Oligonucleotides that might be expected to alter the melting properties, or have a tendency to form a distinctive nonstandard structure were introduced. These oligonucleotides had the repeating dinucleotide sequences GC, AT or AG. Sequence analysis of the inserts and studies of the relative galactokinase expression from the altered plasmids indicated that changes upstream from the trp promoter at -39 or beyond had little effect on trp promoter activity, whereas changes at +2 or farther downstream produced up to two-fold increases in gene expression, as compared to the control plasmid.     

Genetic and physical analysis of the thioredoxin (trxA) gene of Escherichia coli K-12

The trxA gene of Escherichia coli K-12 has been cloned into multicopy plasmids on DNA fragments of varying sizes. The smallest of these was a 1-kb fragment resulting from partial digestion with Sau3A (pBHK10). The complete nucleotide sequence of the trxA gene and its promoter was determined. Comparison of the DNA sequence with the published amino acid sequence revealed the inversion of two amino acid pairs and the possibility of a leader peptide 18 amino acids in length. Three-factor P1 transductional crosses and physical mapping experiments have determined a map order of ilv-trxA-uvrD-corA-metE.     

Cloning of aroG, the gene coding for phospho-2-keto-3-deoxy-heptonate aldolase(phe), in Escherichia coli K-12, and subcloning of the aroG promoter and operator in a promoter-detecting plasmid

Defective transducing phages carrying aroG, the structural gene for phenylalanine (phe)-inhibitable phospho-2-keto-heptonate aldolase (EC 4.1.2.15; previously known as 3-deoxy-D-arabinoheptulosonate-7-phosphate synthetase[phe]), have been isolated, and DNA from two of these phages has been used to construct a restriction map of the region from att lambda to aroG. A 7.6-kb PstI-HindIII fragment from one of these phages was cloned into pBR322 and shown to contain aroG. The location of aroG within the 7.6 kb was established by subcloning and Tn3 transpositional mutagenesis. A fragment carrying the aroG promoter and operator has been cloned into a high copy number promoter-cloning vector (pMC489), and the resulting aroGpo-LacZ' (alpha) fusion subcloned in a low copy number vector. Strains with this fusion on the low copy number vector exhibit negative regulation of beta-galactosidase expression by both phenylalanine and tryptophan and positive regulation by tyrosine in a tyrR+ background.     

Region- and strand-specific mutagensis of a recombinant plasmid

Techniques were developed to mutagenize a single DNA strand in a specific region of the tetracycline-resistance (tetr) gene of the plasmid pKB280 that also carries the lambda repressor gene. Separate annealings of complementary single strands gave two isomeric, circular plasmids containing a 275-nucleotide, single-stranded region (gap) in the tetr gene. One of the isomeric, gapped plasmids was mutagenized specifically with sodium bisulfite such that an estimated 98% of the molecules had suffered at least one C to U conversion in the gap. The mutagenized gap was filled in with DNA polymerase. These molecules transformed Escherichia coli strain MM294 to lambda-immunity with the same frequency as unmutagenized, gap-filled pKB280. Of the lambda-immune transformants, 32% were Tcr and 68% were Tcs. Restriction analysis of plasmids from some Tcs transformants showed losses of restriction sites within the gap and at the gap termini, but none outside the gap. No deletions were detected.     

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.     

Isolation and expression of cytochrome P450 genes in the antennae and gut of pine beetle Dendroctonus rhizophagus (Curculionidae: Scolytinae) following exposure to host monoterpenes

Bark beetles oxidize the defensive monoterpenes of their host trees both to detoxify them and convert them into components of their pheromone system. This oxidation is catalyzed by cytochrome P450 enzymes and occurs in different tissues of the insect, including the gut (i.e., the site where the beetle's pheromones are produced and accumulated) and the antennae (i.e., the olfactory organs used for perception of airborne defensive monoterpenes as well as other host-associated compounds and pheromones). We identified ten new CYP genes in the pine beetle Dendroctonus rhizophagus in either antennae or gut tissue after stimulation with the vapors of major host monoterpenes α-pinene, β-pinene and 3-carene. Five genes belong to the CYP4 family, four to the CYP6 family and one to the CYP9 family. Differential expression of almost all of the CYP genes was observed between sexes, and within these significant differences among time, stimuli, anatomical region, and their interactions were found upon exposure to host monoterpenes. Increased expression of cytochrome P450 genes suggests that they play a role in the detoxification of monoterpenes released by this insect's host trees.