The maturation of coliphage lambda DNA in the absence of its packaging

In vivo, λ DNA cannot be cleaved at cos (matured) if proheads are not present; in vitro, however, cos cleavage readily takes place in the absence of proheads. In order to investigate this paradox, we have constructed plasmids that synthesize λ terminase in vivo upon induction. The plasmids also contain cos at the normal position, about 190 bp upstream of λ gene Nul. One of the plasmids, pFM3, produces levels of terminase comparable to those found after phage induction. If cells carrying pFM3 are thermoinduced, almost 100% of the intracellular plasmid DNA has a double-strand interruption at or near cos.

Since the only λ genes that pFM3 carries are Nul, A, W and B, this in vivo cleavage is occurring in the absence of proheads. Previous failure to observe 2 maturation with phages carrying prohead mutations may be due to exonucleolytic degradation of the unprotected DNA ends, a different DNA topology or compartmentalization, or terminase inhibition in the absence of prohead by the product of another λ gene that maps to the right of gene B.     

Efficient expression of a promoter-controlled gene: Tandem promoters of lambda PR and PL functional in enteric bacteria

We describe an expression vector that functions in enteric bacteria. The vector contains the coliphage λ promoters P_R and P_L and entire P_R and P_L operators in tandem upstream from the multiple cloning sites containing the kanamycin-resistant gene. The vector also specifies a ribosome binding site and a thermolabile repressor, cI₈₅₇, and the P_RM promoter. These promoters as well as lacUV5 and trp promoters were inserted into the EcoRI site of pKO-1 plasmid so that they drove the expression of a reporter gene, galactokinase (galK). The P_RP_L promoter showed the highest efficiency of galK expression in the Escherichia coli strain K12ΔH1Δtrp; it was strong in Klebsiella aerogenes, and weak in Serratia marcescens and Citrobacter freundii.     

A chain of interlinked genes in the ninR region of bacteriophage lambda

The 3612-bp DNA sequence of the phage lambda P-Q (ninR) region contains a series of nine open reading frames in a distinctly overlapping pattern: ATGA sequence modules occur at the boundaries of consecutive genes and are able to serve both as terminator (TGA) and (re)initiator (ATG) codons for most of the adjacent frames. Together with genes O, P, and Q, the newly detected ren and ninA through ninH constitute a series of twelve closely linked genes in the pR operon. Based upon the available evidence for several of the nin proteins, and on plasmid expression data, we conclude that at least the larger nin genes, and probably all of the newly detected open reading frames code for proteins. The nin5 deletion of 2803 bp is a frame-to-frame fusion of ren and ninH, and covers the t R2 termination signal located near its left boundary, immediately behind the ren gene. The possible significance of the observed chain of closely interlinked genes for the regulation of Q expression is discussed.     

Overproduction of the Bacillus sphaericus R modification methylase in Escherichia coli and its purification to homogeneity

A DNA fragment containing the information coding for the GGCC-specific Bacillus sphaericus R modification methylase, BspR, was inserted into plasmid vector pKK223-3 under the control of the strong and inducible tac promoter, and transformed into Escherichia coli HB101. Upon induction this strain accumulated the methylase enzyme (while cell growth was inhibited) up to several percent of total cellular protein. Homogeneous methylase could be prepared in three purification steps.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

Identification and characterization of Clostridium difficile promoter element that is functional in Escherichia coli

The promoter element involved in the expression of a previously characterized cloned clostridial antigen was isolated and characterized. A restriction fragment containing the promoter element of the Clostridium difficile insert was cloned using the promoter probe vector, pGA46. Subclones of the clostridial DNA insert in pGA46 were then analyzed by nucleotide sequencing and by S1 nuclease experiments. The clostridial promoter element exhibits a high degree of homology with typical Escherichia coli promoter elements. This sequence probably represents a unique class of clostridial promoter elements which, given their ability to function in E. coli and C. difficile, can be used in the construction of a shuttle vector capable of gene expression in E. coli and C. difficile.     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.     

Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive rDNA sequences

An attempt was made to transform Alternaria alternata protoplasts using a plasmid vector, pDH25, bearing the Escherichia coli hygromycin B (Hy) phosphotransferase gene (hph) under the control of the Aspergillus nidulans trpC promoter. Transformants arose on a selective medium containing 100 μg Hy/ml. There were two types of transformants, forming large and small colonies on the selective medium. Transformation with one μg of the vector produced an average of 4.5 large colonies and 600 small ones. In large-colony transformants, the vector often integrated into the recipient chromosome in the form of highly rearranged tandem arrays. To increase transformation efficiency, fragments of the highly repetitive ribosomal RNA gene cluster (rDNA) of A. alternata were used to construct four new vectors for homologous recombination system. Use of these vectors gave higher transformation efficiency than the original plasmid. The best vector, pDH25r1a, gave rise to large-colony transformants at a frequency 20 times higher than pDH25. Transformation events in A. alternata with pDH25r1a occured by homologous recombination as a single crossover between the plasmid-borne rDNA segment and its homologue in the chromosome, often giving rise to tandemly repeated vector DNA.     

Direct selection of recombinant plasmids in Bacillus subtilis

A system is described which permits the direct, positive selection of recombinant plasmids in Bacillus subtilis. This system relies on the plasmid pBD214 which confers chloramphenicol (Cm) resistance and carries a thy gene, and on BD393, a highly competent B. subtilis thy A thy B host. Thy⁻ strains are resistant to trimethoprim (Tmp), and Thy⁺ strains are sensitive. Inactivation of the pBD214 thy determinant by insertion of a DNA fragment permits selection of Cm^r Tmp^r clones, all of which carry recombinant plasmids. This insertional inactivation can be accomplished using the unique EcoRl, Bell, Pvull, or EcoRV sites, all of which are located within the thy gene on pBD214. Some properties of this selective system are described, and its uses for molecular cloning are discussed     

Plasmid vectors for high-efficiency expression controlled by the PL promoter of coliphage lambda

A family of plasmid cloning vectors has been constructed that make use of the leftward promoter ($\text{P}{}_{\mathrm{<mtext>L</mtext>}}$) of phage λ to provide for efficient expression of cloned genes in Escherichia coli. The promoter activity of $\text{P}{}_{\mathrm{<mtext>L</mtext>}}$ is fully repressed at low temperature by a thermolabile repressor product of the $\text{λc}\text{I}\text{1857}$ gene, and can be activated by heat induction. Examples are given (β-lactamse, tryptophan synthetase A) where, under optimal conditions, between 30 and 40% of the total protein synthesis is directed by the cloned gene under $\text{P}{}_{\mathrm{<mtext>L</mtext>}}$ control.     

Transcription termination: sequence and function of the rho-independent tL3 terminator in the major leftward operon of bacteriophage lambda

For cloning, assaying the function and sequencing terminators, we have constructed the pD12 plasmid, in which the late promotor p'R of phage lambda controls the expression of the galK gene of the pK03 plasmid of McKenney et al. (1981). The lambda tL3 terminator region was cloned in this plasmid between the promoter and the galK gene, and found to be 90-94% effective in preventing galactokinase expression in both rho+ and rho- hosts. Is is also active in vitro, both in the presence or absence of the rho factor. The termination point is located at 4320 bp to the left of the SL startpoint of the PL-RNA, just downstream of gene exo. We have sequenced 356 bp of the hitherto uncharted lambda DNA to the right of the TaqI cut, which in turn is 110 bp to the right of the b522 deletion at 63.9% lambda. The tL3 terminator has several features common to other rho-independent termination sequences, including an 81% G+C-rich region of 2X8-bp symmetry ("stem") with a 5-bp intervening "loop", partially overlapping and followed by a sequence transcribed into the pyrimidine-rich CCUUUCU-OH 3' terminus of the RNA. The termination point that follows the last U was determined by the S1 mapping technique.