Henry M. Krisch

In vitro insertional mutagenesis with a selectable DNA fragment

A new method for in vitro insertional mutagenesis of genes cloned in Escherichia coli is presented. This simple procedure combines the advantages of in vitro DNA linker mutagenesis with those of in vivo transposition mutagenesis. It makes use of the omega fragment, a 2.0-kb DNA segment consisting of an antibiotic resistance gene (the Smr/Spcr gene of the R100.1 plasmid) flanked by short inverted repeats carrying transcription and translation termination signals and synthetic polylinkers. The omega fragment is inserted into a linearized plasmid by in vitro ligation, and the recombinant DNA molecules are selected by their resistance to streptomycin and spectinomycin. The omega fragment terminates RNA and protein synthesis prematurely, thus allowing the definition and mapping of both transcription and translation units. Because of the symmetrical structure of omega, the same effect is obtained with insertions in either orientation. The antibiotic resistance gene can be subsequently excised from the mutated molecules, leaving behind its flanking restriction site(s).

Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of Gram-negative bacteria

We have constructed a series of derivatives of the omega interposon [Prentki and Krisch, Gene 29 (1984) 303-313] that can be used for in vitro insertional mutagenesis. Each of these DNA fragments carries a different antibiotic or Hg2+ resistance gene (ApR, CmR, TcR, KmR or HgR) which is flanked, in inverted orientation, by transcription and translation termination signals and by synthetic polylinkers. The DNA of these interposons can be easily purified and then inserted, by in vitro ligation, into a plasmid linearized either at random by DNase I or at specific sites by restriction enzymes. Plasmid molecules which contain an interposon insertion can be identified by expression of its drug resistance. The position of the interposon can be precisely mapped by the restriction sites in the flanking polylinker. To verify their properties we have used these omega derivatives to mutagenize a broad host range plasmid which contains the entire meta-cleavage pathway of the toluene degradation plasmid pWW0 of Pseudomonas putida. Insertion of these interposons in the plasmid between the promoter and the catechol 2,3-dioxygenase (C23O) gene dramatically reduced the expression of this enzyme in Escherichia coli. We also show that when a plasmid containing an omega interposon is transferred by conjugal mobilization from E. coli to P. putida, Agrobacterium tumefaciens, Erwinia chrysanthemi, Paracoccus denitrificans or Rhizobium leguminosarum, the appropriate interposon drug resistance is usually expressed and, compared to the non-mutated plasmid, much reduced levels of C23O activity are detected. Thus, the selection and/or characterization of omega insertional mutations can be carried out in these bacterial species.

RNase E, an endoribonuclease, has a general role in the chemical decay of Escherichia coli mRNA: evidence that rne and ams are the same genetic locus

Escherichia coli RNase E is known to process RNA precursors at specific sites. We show that this endoribonuclease has a general role in E. coli mRNA turnover and affects the stability of specific transcripts. The effect of the rne mutation on functional stability of mRNA was much less pronounced than that on chemical stability, although the expression of some genes was affected. The E. coli ams (altered mRNA stability) mutation was found to have phenotypes indistinguishable from those of the rne mutation, affecting both 9S RNA and T4 gene 32 mRNA processing. The rne and ams mutations were both complemented by the same 3.7kb fragment of E. coli DNA and are probably allelic. RNase E is the first endoribonuclease Identified as having a general role in the chemical decay of E. coli mRNA.

Processing of unstable bacteriophage T4 gene 32 mRNAs into a stable species requires Escherichia coli ribonuclease E

Gene 32 from bacteriophage T4 is transcribed as precursor transcripts which are processed to a stable product. This processing of the gene 32 mRNA was observed in RNase III or P‐deficient strains of Escherichia coli. However, after infection of an RNase E‐deficient strain, the amount of processed transcript was significantly reduced while the levels of the precursor transcripts remained high. RNase E therefore appears to have an essential role in the processing of the gene 32 mRNA. We have mapped the exact 5′ end of the processed transcript by primer extension. The cleavage occurs near a stem‐loop structure at a site which shows some similarity to other known RNase E cleavage sites. The effects of the processing on the differential stability of the upstream and downstream sequences, and on gene expression, are discussed.

Regulation of the synthesis of bacteriophage T4 gene 32 protein

Abstract The synthesis of T4 gene 32 product (P32) has been followed by gel electrophoresis of infected cell lysates. In wild-type infections, its synthesis starts soon after infection and begins to diminish about the time late gene expression commences. The absence of functional P32 results in a marked increase in the amount of the non-functional P32 synthesized. For example, infections of T4 mutants which contain a nonsense mutation in gene 32 produce the nonsense fragment at more than ten times the maximum rate of synthesis of the gene product observed in wild-type infections. All of the temperature-sensitive mutants in gene 32 that were tested also overproduce this product at the non-permissive temperature. This increased synthesis of the non-functional product is recessive, since mixed infections (wild-type, gene 32 nonsense mutant) fail to overproduce the nonsense fragment. Mutations in genes required for late gene expression (genes 33 and 53) as well as some genes required for normal DNA synthesis also result in increased production of P32. The overproduction in such infections is dependent on DNA synthesis; in the absence of DNA synthesis no overproduction occurs. This contrasts with the overproduction resulting from the absence of functional P32 which is not dependent on DNA synthesis. These results are compatible with a model for the regulation of expression of gene 32 in which the synthesis of P32 is either directly or indirectly controlled by its own function. Thus, in the absence of P32 function the expression of this gene is increased as is manifest by the high rate of P32 synthesis. It is further suggested that in infections defective in late gene expression and consequently in the maturation of replicated DNA, the increased P32 production is caused by the large expansion of the DNA pool. This DNA is presumed to compete for active P32 by binding it non-specifically to single-stranded regions, thus reducing the amount of P32 free to block gene 32 expression. Similarly, the aberrant DNA synthesized following infections with mutants in genes 41, 56, 58, 60 and 30, although quantitatively less than that produced in the maturation defective infections, can probably bind large quantities of P32 to single-stranded regions resulting in increased P32 synthesis.

mRNA degradation in procaryotes.

The fast turnover of mRNA permits rapid changes in the pattern of gene expression. In procaryotes, many enzymes involved in mRNA degradation have been identified and some of these endo‐ and exo‐ribonucleases are now being intensively studied. Some of the structural features of mRNA that influence decay rates have also recently been defined. Although important components of the decay pathway are still elusive, a coherent and simple model for mRNA decay has emerged in the last few years.— Ehretsmann, C. P.; Carpousis, A. J., and Krisch, H. M. mRNA degradation in procaryotes. FASEB J. 6: 3186‐3192; 1992.

Omegon-Km: a transposable element designed for in vivo insertional mutagenesis and cloning of genes in gram-negative bacteria.

To combine the features of the omega interposons with the advantages of in vivo transposition mutagenesis, we have constructed an artificial transposon, called Omegon-Km. The Omegon-Km transposon is carried on the plasmid pJFF350 which can be conjugally mobilized into a broad range of Gram-negative bacteria. Omegon-Km is flanked, in inverted orientation, by synthetic 28-bp repeats derived from the ends of IS1. In addition, each end of Omegon-Km has the very efficient transcription and translation terminators of the omega interposon. Internally, Omegon-Km carries the selectable kanamycin (Km)-neomycin resistance gene (alph A) which is expressed well in many Gram-negative bacteria. The IS1 transposition functions are located on the donor plasmid but external to Omegon-Km. Thus, insertions of Omegon-Km are very stable because they lack the capacity for further transposition. Omegon-Km mutagenesis is performed by conjugal transfer of pJFF350 from Escherichia coli into any Gram-negative recipient strain in which this plasmid is unable to replicate. Those cells which have had a transposition event are selected by their resistance to Km. Very high frequencies of Omegon-Km transposition were observed in Pseudomonas putida. Preliminary experiments with other Gram-negative soil and water bacteria (Rhizobium leguminosarum, Paracoccus denitrificans) yielded mutants at reasonable levels. The presence of an E. coli-specific origin of replication (ori) within Omegon-Km allows the rapid and easy cloning, in E. coli, of the nucleotide sequences flanking the site of the transposition event.

A modified pBR322 vector with improved properties for the cloning, recovery, and sequencing of blunt-ended DNA fragments.

The construction of a plasmid vector which facilitates the cloning and recovery of blunt-ended DNA fragments is described. This plasmid, called pHP34, differs from pBR322 by a 10-bp insertion which introduces a unique SmaI site immediately flanked by two EcoRI sites. Blunt-ended DNA fragments cloned in the SmaI site can be recovered by digestion with EcoRI. Small cloned fragments can be chemically sequenced using a strategy which does not require their purification. The use of a plasmid related to pHP34 for in vitro mutagenesis by the insertion of a DNA linker fragment conferring an antibiotic resistance is also discussed.

Transcription and messenger RNA processing upstream of bacteriophage T4 gene 32.

SummaryBacteriophage T4 gene 32 lies at the 3′ end of a complex transcription unit which includes genes 33, 59, and several open reading frames. In the course of an infection, four major transcripts are synthesized from this unit: two overlapping polycistronic transcripts about 3800 and 2800 nucleotides in length, and two monocistronic gene 32 transcripts about 1150 and 1100 nucleotides in length. These transcripts are made at different times in infection and the polycistronic transcripts have segmental differences in stability. Messenger RNA processing yields a 1025 nucleotide monocistronic gene 32 transcript, and a 135 nucleotide transcript containing part of the gene 59 coding sequence. Processing depends on Escherichia coli encoded ribonuclease E. This pattern of transcription and processing leads to the synthesis of gene 32 mRNA throughout infection, whereas transcripts encoding the upstream genes are present only early in infection. The 3800 nucleotide polycistronic transcript initiates at a promoter that does not require T4 encoded factors for activity. However, full-length synthesis of this transcript depends on the T4 mot gene product. The region upstream of gene 32 also contains four E. coli-like promoters that are active on chimeric plasmids in uninfected cells, but inactive in bacteriophage T4. The location of these cryptic T4 promoters is intriguing in that they lie near the 5′ ends of open reading frame B, gene 59 and gene 32. They could play a role in phage development under particular conditions of growth or in bacterial hosts other than those examined here.

Sense and antisense transcription of bacteriophage T4 gene 32: Processing and stability of the mRNAs

Analysis of bacteriophage T4 gene 32 transcription has revealed a multiplicity of mRNAs. In plasmids, gene 32 is expressed primarily from a strong promoter that is shut off after phage infection. In a wild-type infection, gene 32 is initially transcribed from prereplicative polycistronic and monocistronic promoters; subsequently, a monocistronic late mRNA predominates. This transcript, as well as a post-transcriptionally processed product of the earlier mRNA, can be stable. The eventual degradation of the stable mRNAs is temporally regulated by the phage. Finally, the transcription termination region of gene 32 can function as an antisense promoter both in vitro and in vivo.