Marker rescue allows direct selection for recombinant plasmids in streptococci

Summary Resident deletion derivatives (Em^s or Cm^s) of the streptococcal plasmid vector pGB301 rescue antibiotic resistance genes from linearized pGB301 (Em^r, Cm^r) DNA with high frequency. Insertion of passenger DNA next to an antibiotic resistance determinant of pGB301, which is missing on the resident plasmid, forces corescue of these two plasmid domains, thus allowing direct selection for recombinant plasmids.     

A new runaway type episomal vector for mammalian cells based on a temperature-sensitive simian virus 40 and inducible erythropoietin production

A runaway vector for mammalian cells was constructed from the simian virus 40 (SV40) genome with a temperature-sensitive mutation of the large T antigen and bacterial neo ^r gene. Replication of this plasmid was repressed above 39°C and vigorous DNA propagation was observed below 33°C in simian CV-1 cells. The human erythropoietin gene was inserted downstream of the SV40 late promoter of the plasmid and the recombinant plasmid was introduced into CV-1 cells. By a temperature shift from 37 to 33°C, the plasmid copy number increased from 5 × 10² to 5 × 10³ copies per cell and the specific production rate of erythropoietin increased more than ten-fold. The bacterial-derived sequences such as the neo ^r gene and vector pUC sequences were prone to delete but the main body of the recombinant plasmid such as SV40 and the erythropoietin-coding sequences were stably maintained at either 33 or 37°C.     

Apparent gene conversion in an Escherichia coli rec+ strain is explained by multiple rounds of reciprocal crossing-over

Summary Gene conversion, the non-reciprocal transfer of sequence information between homologous DNA sequences, has been reported in lower eukaryotes, mammals and in Escherichia coli. In an E. coli rec ⁺ strain, we established a plasmid carrying two different deleted neo genes (neoDL and neoDR) in an inverted orientation and then selected for homologous recombination events that had reconstructed an intact neo ⁺ gene. We found some plasmids that had apparently experienced intramolecular gene conversion. Further evidence, however, suggests that they are products of multiple rounds of reciprocal crossing-over,apparently involving two plasmid molecules. First, most of the Neo⁺ clones contained multiple types of Neo⁺ plasmids, although the frequency of producing the neo ⁺ clones was low. Second, all the neo ⁺ clones also contained, as a minority, one particular form of dimer, which can be formed by reciprocal crossing-over between neoDL of one plasmid molecule and neoDR of another plasmid molecule. Third, in reconstruction experiments, we cloned and purified this dimer and transferred it back into the rec ⁺ cells. The dimer gave rise to clones containing multiple types of neo ⁺ recombinant monomers, including those apparent gene conversion types, and containing only few molecules of this dimer plasmid.     

Independent translation of ORFs in dicistronic operons,synthetic building blocks for polycistronic chloroplast gene expression

We designed a dicistronic plastid marker system that relies on the plastid's ability to translate polycistronic mRNAs. The identification of transplastomic clones is based on selection for antibiotic resistance encoded in the first open reading frame (ORF) and accumulation of the reporter gene product in tobacco chloroplasts encoded in the second ORF. The antibiotic resistance gene may encode spectinomycin or kanamycin resistance based on the expression of aadA or neo genes, respectively. The reporter gene used in the study is the green fluorescent protein (GFP). The mRNA level depends on the 5′‐untranslated region of the first ORF. The protein output depends on the strengths of the ribosome binding, and is proportional with the level of translatable mRNA. Because the dicistronic mRNA is not processed, we could show that protein output from the second ORF is independent from the first ORF. High‐level GFP accumulation from the second ORF facilitates identification of transplastomic events under ultraviolet light. Expression of multiple proteins from an unprocessed mRNA is an experimental design that enables predictable protein output from polycistronic mRNAs, expanding the toolkit of plant synthetic biology.     

Plasmid shuttle vector with two insertionally inactivable markers for coryneform bacteria

A new shuttle vector pCEM500 replicating inEscherichia coli and inBrevibacterium flavum was constructed. It carries two antibiotic resistance determinants (Km^r/Gm^r from plasmid pSa of Gram-negative bacteria and Sm^r/Sp^r from plasmid pCG4 ofCorynebacterium glutamicum) which are efficiently expressed in both hosts and can be inactivated by insertion of DNA fragments into the unique restriction endonuclease sites located within them. This vector was found to be stably maintained inB. flavum and can be used for transfer of the cloned genes into this amino-acid-producing coryneform bacterium.     

Bacteriophage λ DNA fragments replicate in the Paramecium macronucleus: Absence of active copy number control

We show that bacteriophage λ DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division. © 1992 Wiley-Liss, Inc.     

Construction of Broad Host Range Cloning Vectors for Gram-negative Bacteria

A new cloning vector, pMFY31, has been constructed based on the high-copy-number, broad-host-range plasmid RSF1010. The plasmid has a size of 13.2 kb and carries the Ap^r, Cm^r, and Tc^r genes. It contains unique PstI, EcoRI, HindIII, BamHI, and SalI sites, all of which are located within the antibiotic resistance genes, therefore all sites are applicable to insertional inactivation. We also constructed pMFY40, a 11.6 kb derivative of pMFY31, by the elimination of the Cm^r gene. Plasmid pMFY31 has been efficiently introduced into a Pseudomonas putida strain not only by plasmid-DNA transformation but also by conjugal co-transfer with the helper plasmid, and was maintained stably in the strain.     

Stability of Escherichia coli strains harboring recombinant plasmids for L-threonine production

Escherichia coli recombinant strains bearing the thr operon have been previously selected for threonine production and phenotypically classified according to antibiotic resistance properties (Nudel et al. 1987).Further analysis of those strains permitted the isolation and restriction mapping of two different plasmids of 13 kb and 18.6 kb. The smaller one, which expressed tetracycline resistance gave better results on threonine accumulation but it was rather unstable when grown without antibiotic pressure. Therefore, other hosts were transformed with those plasmids to improve stability.A threonine-auxotrophic strain was a better host for plasmid maintenance and expression of thr operon. Host influence in plasmid-mediated threonine production was studied in terms of specific yields (the ratios of threonine accumulated to biomass values) and of plasmid maintenance (percent of Ap^rTc^r clones after cultivation in non selective media).We also determined that semisynthetic media of defined composition were better than rich media for threonine expression, due to feed-back controls exerted by undesired catabolites accumulated in complex media.     

Identification of Tf1 integration events in S. pombe under nonselective conditions

Integration of retroviral elements into the host genome is a phenomena observed among many classes of retroviruses. Much information concerning the integration of retroviral elements has been documented based on in vitro analysis or expression of selectable markers. To identify possible Tf1 integration events within silent regions of the Schizosaccharomyces pombe genome, we focused on performing an in vivo genome-wide analysis of Tf1 integration events from the nonselective phase of the retrotransposition assay. We analyzed 1000 individual colonies streaked from four independent Tf1 transposed patches under nonselection conditions. Our analysis detected a population of G418^S/neo⁺ Tf1 integration events that would have been overlooked during the selective phase of the assay. Further RNA analysis from the G418^S/neo⁺ clones revealed 50% of clones expressing the neo selectable marker. Our data reveals Tf1's ability to insert within silent regions of S. pombe's genome.     

The Product of the cysK Gene of Bacillus stearothermophilus V Mediates Potassium Tellurite Resistance in Escherichia coli

The nucleotide sequence of a 4,539 bp fragment of Bacillus stearothermophilus V mediating tellurite resistance in Escherichia coli was determined. Four ORFs of more than 150 amino acids encoding polypeptides of 244, 258, 308, and 421 residues were found in the restriction fragment. E. coli cells harboring a recombinant plasmid containing the Te^r determinant express, when challenged with tellurite, a 32 kDa protein with an amino terminal sequence identical to the ten first residues of the 308 ORF. This ORF shows great similarity with the cysteine synthase gene (cysK) of a number of organisms. Recombinant clones carrying the active cysK gene have minimal inhibitory concentrations to K₂TeO₃ that were tenfold higher than those determined for the host strain or that of clones carrying ORFs 244, 258, and 421. Introduction of the B. stearothermophilus V cysK gene into a cysK strain of Salmonella typhimurium LT2 resulted in complementation of the mutation as well as transfer of tellurite resistance. Received: 28 March 2001 / Accepted: 17 April 2001     

Construction of a shuttle vector betweenEscherichia coli andZymomonas anaerobia

Summary A 1.7-kb cryptic plasmid was isolated fromZymomonas anaerobia and used to construct a shuttle vector inserting useful parts of pUC9, pBR322, and pRK2501.Escherichia coli was employed to clone the new plasmid designated pSR12. The 7.7-kb plasmid pSR12 reisolated from the host cells could transform competent cells ofZ.anaerobia at 2×10^–7 frequency. This shuttle vector contains two antibiotic resistance markers, Kan^r and Tet^r, as well as restriction sites such as EcoRI, PstI, and XhoI, suitable for DNA recombinations.     

Cloning and expression of the erythromycin resistance determinant from Campylobacter jejuni in Escherichia coli

The erythromycin resistance gene (Em^r) from Campylobacter jejuni ABA94 plasmid DNA was cloned into the pUC18 vector and then expressed in Escherichia coli. The location of the Em^r determinant on the chimeric plasmid was determined by restriction endonuclease mapping within a 0.8-kb EcoRI fragment. This fragment then hybridized to the 78-kb plasmid DNA but not to the 3.3-or 12.6-kb plasmid DNA of Campylobacter jejuni ABA94. Em^r in Campylobacter jejuni is therefore probably plasmid-mediated.The authors are with the Department of Genetics and Cellular Biology, University of Malaya, 59100 Kuala Lumpur, Malaysia     

Intranuclear Parasitism of the Ciliate Euplotes by a Trypanosomatid Flagellate

A trypanosomatid flagellate, Leptomonas sp., develops and multiplies in the macronucleus (only) of natural and laboratory-reared populations of the ciliate Euplotes. Up to 90% of the natural populations of Euplotes in our test pond had such nuclear infections. Laboratory infections were transmitted to this ciliate by feeding it liberated parasites. Paramecium resisted infections. All laboratory-induced infections were lethal to Euplotes, while control clones of the uninfected ciliates remained viable. This leptomonad, unlike Leptomonas karyophilus (found in Paramecium), shows no leishmanial forms in its several ciliate hosts and shows a varied pattern of locomotion.     

Transposon Mutagenesis and Excision of R′ Plasmids by Conjugative, Chimeric Plasmid pUW942 in Extra-Slow-Growing Rhizobium japonicum Strains

Transposons Tn501 (specifying mercury resistance) and Tn7 (specifying resistance to trimethoprim and streptomycin) were introduced into extra-slow-growing Rhizobium japonicum by conjugal transfer of the 82 kilobase chimeric plasmid pUW942. Mercury-resistant transconjugants were obtained at a frequency of 10⁻⁷ to 10⁻⁹. The transfer frequency of streptomycin resistance was lower than that of mercury resistance, and Tn7 was relatively unstable. pUW942 was not maintained as an autonomously replicating plasmid in R. japonicum strains. However, some of the Hg^r transconjugants from the RJ19FY, RJ17W, and RJ12S strains acquired antibiotic markers of the vector plasmid pUW942. Southern hybridization of plasmid and chromosomal DNA of R. japonicum strains with ³²P-labeled pUW942 and pAS8Rep-1, the same plasmid as pUW942 except that it does not contain Tn501, revealed the formation of cointegrates between pUW942 and the chromosome of R. japonicum. More transconjugants with only Tn501 insertions in plasmids or the chromosome were obtained in crosses with strains RJ19FY and RJ17W than with RJ12S. These retained stable Hg^r both in plant nodules and under nonselective in vitro growth conditions. One of the RJ19FY and two of the RJ12S Hg^r transconjugants with vector plasmid-chromosome cointegrates conjugally transferred plasmids of 82, 84 or 86, and 90 kilobases, respectively, into plasmidless Escherichia coli C. These plasmids strongly hybridized to pUW942 and EcoRI digests of total DNA of each respective R. japonicum strain but not to indigenous plasmid DNA of the R. japonicum strains. These R′ plasmids consisted of pUW942-specific EcoRI fragments and an additional one or two new fragments derived from the R. japonicum chromosome.     

Controlled gene expression utilising Lambda phage regulatory signals in a cyanobacterium host

Summary This study presents plasmid systems that utilize regulatory signals of bacteriophage Lambda to accomplish regulated expression of cloned genes in an A. nidulans R2 derivative strain. An operator-promoter region and the temperature-sensitive repressor gene cI857 of bacteriophage Lambda were employed. Linked to a cyanobacterial replicon, the plasmid vectors efficiently transformed Anacystis and were stably maintained within this host. The cat structural gene, encoding chloramphenicol acetyltransferase, was used to demonstrate that expression can be regulated by temperature shift. We have identified in extracts from the vector bearing Anacystis, a protein similar in size and immunology to the Lambda repressor. The systems described should allow controlled expression of adventitious genes in the cyanobacterial host.Abbreviations AP^r ampicillin resistance - Cm^r chloramphenicol resistance - CmActase chloramphenicol acetyltransferase - Km^r Kanamycine resistance - [ ] indicates plasmid carrier state     

Transfer of nodulation ability in Rhizobium using R68.45 derived plasmids

Summary Nodulation ability was transferred from Rhizobium meliloti L5.30 to the non-nodulating mutant Rhizobium trifolii 24K using plasmid R68.45. Transconjugants selected for the carbenicillin resistance (cb ^r) marker became simultaneously capable of nodulating clover and showed changes in phage sensitivity. Besides the indigenous plasmid of 90 MD (pUCS201), the nodulating transconjugants harbored the newly introduced plasmid pUCS202 (ca. 40 MD). After treatment of the transconjugants with curing agents the simultaneous loss of antibiotic resistance and ability to form nodules were associated with the disappearance of pUCS202. nod and cb ^r genes were cotransferred into R. trifolii strains by conjugation and transformation. There is genetic evidence that the nod gene(s) was integrated into R68.45.     

Molecular characterization of a gene from Saccharopolyspora erythraea (Streptomyces erythraeus) which is involved in erythromycin biosynthesis

A 7.3 kbp DNA fragment, encompassing the erythromycin (Em) resistance gene (ermE) and a portion of the gene cluster encoding the biosynthetic genes for erythromycin biosynthesis in Saccharopolyspora erythraea (formerly Streptomyces erythraeus) has been cloned in Streptomyces lividans using the plasmid vector pIJ702, and its nucleotide sequence has been determined using a modified dideoxy chain-termination procedure. In particular, we have examined the region immediately 5′ of the resistance determinant, where the tandem promoters for ermE overlap the promoters for a divergently transcribed coding sequence (ORF). Disruption of this ORF using an integrational pIJ702-based plasmid vector gave mutants which were specifically blocked in erythromycin biosynthesis, and which accumulated 3-O-α-L-mycarosylerythronolide B: this behaviour is identical to that of previously described eryC1 mutants. The eryC1-gene product, a protein of subunit M_r 39200, is therefore involved either as a structural or as a regulatory gene in the formation of the deoxyamino-sugar desosamine or in its attachment to the macro-lide ring.     

Expression of the green fluorescent protein in Paramecium tetraurelia

In this paper we describe the expression of green fluorescent protein (GFP) as a reporter in vivo to monitor transformation in Paramecium cells. This is not trivial because of the limited number of strong promoters available for heterologous expression and the very high AT content of the genomic DNA, the consequence of which is a very aberrant codon usage. Taking into account differences in codon usage we selected and modified the original GFP open reading frame (ORF) from Aequorea victoria and placed the altered ORF into the Paramecium expression vector pPXV. Injection of the linearized plasmid into the macronucleus resulted in a cytoplasmic fluorescence signal in the clonal descendants, which was proportional to the number of copies injected. Southern hybridization indicated the establishment and replication of the plasmid during vegetative growth. Expression was also monitored by Northern and Western analysis. The results indicate that the modified GFP can be used in Paramecium as a reporter for transformation as an alternative to selection with antibiotics and that it may also be used to construct and localize fusion proteins.     

Developmentally Controlled Rearrangement of Surface Protein Genes in Paramecium tetraurelia1

ABSTRACT Early research on Paramecium genetics highlighted the role of the cytoplasm on inheritance. Today this tradition continues as recent investigations of macronuclear development in Paramecium have revealed unusual cytoplasmic effects that are not easily explained within current paradigms. It is generally assumed that most programmed DNA rearrangements in ciliates are regulated by cis acting signals encoded within the germline (micronuclear) DNA, but there are increasing examples in which the old macronucleus acts through the cytoplasm (in trans) to affect the loss and rearrangement of DNA in the developing macronucleus. The remarkable specificity of this effect has forced a reevaluation of the standard view of macronuclear determination in Paramecium. This review summarizes our knowledge of the effect of the old macronucleus on the developmentally controlled rearrangements of the P. tetraurelia, stock 51A and B variable surface protein genes.