Chimeric Plasmid Research Articles

Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids.

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.

Common regulatory elements control gene expression from polyoma early and late promoters in cells transformed by chimeric plasmids.

In a previous report we showed that transcripts initiating from the late promoter of integrated polyoma plasmids could be detected at significant levels when neomycin resistance (neo) coding sequences were linked to this promoter. In this report we used chimeric plasmids that contain either a limited portion of the polyoma genome or deletions within the polyoma noncoding regulatory region to determine the sequence requirements for late promoter activity in this system. We observed no absolute requirement for either the polyoma early coding region or the origin of DNA replication for Neo-r colony formation. We were therefore able to independently assess the effects of deletions in the polyoma enhancer region on gene activity in both the early and late directions. We measured the ability of cells transfected with plasmids containing deletions in this region to form colonies in either semisolid or G418-containing medium under nonreplicative conditions. Our results indicate that either the PvuII 4 fragment, which contains the simian virus 40 core enhancer sequence, or a region from nucleotides 5099 to 5142, which contains the adenovirus type 5 E1A core enhancer sequence, can be deleted without significantly affecting gene expression in either direction. However, a deletion of nucleotides 5099 to 5172 reduced activities to similar extents in both directions, and a plasmid containing a larger deletion of nucleotides 5055 to 5182 showed a further reduction in activity. Although having no effect by itself, a second origin region deletion of nucleotides 5246 to 127 when present in these mutant backgrounds caused either a further reduction or elimination, respectively, of both G418 and agar colony-forming ability, suggesting the presence of an additional common regulatory element within this region. A comparison of 5' ends of neo transcripts present in cells transformed by these plasmids suggested that the reduction in activity was due to deletion of regulatory rather than structural elements of the late promoter. Our results indicate that the noncoding region of polyoma contains multiple complementing regulatory elements that control the level of both early and late gene expression.

Isolation of cis-acting vaccinia virus DNA fragments promoting the expression of herpes simplex virus thymidine kinase by recombinant viruses.

Recombinant TK- vaccinia viruses containing the pBR322 sequence inserted in either orientation within the coding sequence of the viral thymidine kinase gene were constructed. They were characterized by genomic analysis, hybridization studies, reversion to wild-type virus by in vivo recombination, and rescue from their genomes of plasmids which contained all or parts of the pBR322 sequence. TK- cells were infected with one of these recombinant viruses and then transfected with pools of chimeric plasmids composed of a cloned herpes simplex virus thymidine kinase gene which contained upstream inserts of different vaccinia DNA fragments prepared by restriction or sonication. Recombination between homologous pBR322 sequences within infected cells generated selectable recombinant viruses in which expression of the herpes simplex virus thymidine kinase gene was promoted by the upstream vaccinia insert. These viruses were characterized by genomic analysis, hybridization, and in vivo or in vitro phosphorylation of (5-[125I]deoxycytidine as a specific assay for the expressed herpes simplex virus thymidine kinase. Vaccinia DNA inserts were isolated conveniently for transfer to bacteria by rescuing appropriate plasmids from the genome of recombinant viruses. The sequence of 100 nucleotides adjacent to the upstream region of the herpes simplex virus gene was determined in nine different inserts measuring 0.17 to 1.07 kilobase pairs.

Construction of a Gene Bank of Streptococcus lactis Plasmid pLM2001 Deoxyribonucleic Acid in Escherichia coli

Plasmid pLM2001, which is essential for the fermentation of lactose by Streptococcus lactis LM0232, was isolated and cut into eight fragments with restriction endonuclease EcoRI. The deoxyribonucleic acid fragments ranged in size from approximately .8 to 10.3 kilobases as determined by agarose gel electrophoresis. EcoRI-restricted pLM-2001 deoxyribonucleic acid was shotgun cloned into the single EcoRI site of the multifunctional Escherichia coli-Streptococcus sanguis shuttle vector, pSA4, insertionally inactivating the chloroamphenicol resistance gene. Following transformation of the deoxyribonucleic acid into Escherichia coli HB101, 242 transformants containing chimeric plasmids were detected by their resistance to tetracycline and failure to grow in the presence of chloramphenicol. Plasma deoxyribonucleic acid from 98 transformants was cleaved with EcoRI and analyzed by agarose gel electrophoresis for the presence of pLM2001 inserts. Clones were obtained, which individually contained each of the eight fragments. In addition, a series of clones containing various combinations of the fragments was obtained. The availability of a system for cloning and rapidly screening for fragments of Streptococcus lactis deoxyribonucleic acid under conditions where deletion of passenger or vector deoxyribonucleic acid does not occur will facilitate studies designed to isolate and characterize metabolically important genes carried by plasmids from the group N streptococci.

CDNA cloning of portions of the bacteriophage phi 6 genome.

Phage phi 6 has a genome consisting of three pieces of double-stranded RNA. Single-stranded RNA was prepared from phi 6 nucleocapsids by in vitro transcription with the phage RNA polymerase. These transcripts were polyadenylated and used as templates for the preparation of cDNA copies. The resulting DNA was cloned into the PstI restriction nuclease site of plasmid pBR322. Insert-bearing plasmids were annealed to phi 6 RNA to assign the inserts to their proper segments. In this way we identified inserts corresponding to the large, medium, and small segments. Two large overlapping inserts of the small segment constitute the complete complement of the segment as determined by the sequence analysis of the DNA. In vitro coupled transcription and translation showed that the small segment inserts were able to direct the synthesis of the four known genes in the small segment. Two overlapping inserts in the medium segment constitute the entire segment and were shown to direct the in vitro synthesis of two of the three known proteins of the medium segment. Several inserts bearing about one-third the complement of the large segment were also isolated, and one of these directed the synthesis of a peptide that resembles protein P1. Restriction endonuclease maps were prepared for the inserts, and by in vitro synthesis it was possible to refine the genetic map of phi 6. A chimeric plasmid was constructed that combines plasmids pUC8 and RSF1010. Inserts placed on this plasmid were transformed to Pseudomonas phaseolicola, the natural host of phage phi 6. It was possible to refine further the genetic map by complementation of nonsense mutants of phi 6 with the cDNA.

Cloning of genes specifying carbohydrate catabolism in Pseudomonas aeruginosa and Pseudomonas putida.

A 6.0-kilobase EcoRI fragment of the Pseudomonas aeruginosa PAO chromosome containing a cluster of genes specifying carbohydrate catabolism was cloned into the multicopy plasmid pRO1769. The vector contains a unique EcoRI site for cloning within a streptomycin resistance determinant and a selectable gene encoding gentamicin resistance. Mutants of P. aeruginosa PAO transformed with the chimeric plasmid pRO1816 regained the ability to grow on glucose, and the following deficiencies in enzyme or transport activities corresponding to the specific mutations were complemented: glcT1, glucose transport and periplasmic glucose-binding protein; glcK1, glucokinase; and edd-1, 6-phosphogluconate dehydratase. Two other carbohydrate catabolic markers that are cotransducible with glcT1 and edd-1 were not complemented by plasmid pRO1816: zwf-1, glucose-6-phosphate dehydrogenase; and eda-9001, 2-keto-3-deoxy-6-phosphogluconate aldolase. However, all five of these normally inducible activities were expressed at markedly elevated basal levels when transformed cells of prototrophic strain PAO1 were grown without carbohydrate inducer. Vector plasmid pRO1769 had no effect on the expression of these activities in transformed mutant or wild-type cells. Thus, the chromosomal insert in pRO1816 contains the edd and glcK structural genes, at least one gene (glcT) that is essential for expression of the glucose active transport system, and other loci that regulate the expression of the five clustered carbohydrate catabolic genes. The insert in pRO1816 also complemented the edd-1 mutation in a glucose-negative Pseudomonas putida mutant but not the eda-1 defect in another mutant. Moreover, pRO1816 caused the expression of high specific activities of glucokinase, an enzyme that is naturally lacking in these strains of Pseudomonas putida.

Construction and characterization of plasmid vectors for cloning in the entomocidal organism Bacillus sphaericus 1593

A plasmid vector for cloning in Bacillus sphaericus 1593 was constructed in B. subtilis from two parent plasmids, pBC16 and pBD64. When characterized, the 3.9-MDa chimeric plasmid pNN101 was found to consist of the MspI fragment containing the chloramphenicol acetyltransferase (CAT) gene from pBD64 inserted into an MspI site in pBC16. pNN101 was shown to replicate, express, and be stably maintained in B. sphaericus 1593 without affecting the mosquito larvicidal activity of this organism. A derivative of this plasmid, pNN302, was constructed in which a unique HindIII site was introduced into the CAT gene without loss of chloramphenicol resistance.

Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids.

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.

Construction and characterization of chimeric beta-lactamase plasmids of Neisseria gonorrhoeae with altered ability to be mobilized during conjugation.

A series of chimeric plasmids derived from the transfer factor, beta-lactamase-encoding R factor, and cryptic plasmid of Neisseria gonorrhoeae was constructed. Two of these plasmids, each lacking a 1.9-kilobase-pair (kb) HinfI fragment, could not be mobilized by the 40-kb gonococcal transfer factor to recipient strains of N. gonorrhoeae or Escherichia coli during conjugation. The proteins encoded by both the naturally occurring and chimeric plasmids were examined with an E. coli cell-free transcription-translation system and in E. coli maxicells. Six plasmid-specific proteins were identified when DNA from a naturally occurring 7.1-kb R factor was used as template. A small protein (16,000 daltons), which is apparently not encoded by the two plasmids lacking the 1.9-kb HinfI fragment, appears to be necessary for plasmid transfer during conjugation.

Transcription from the polyoma late promoter in cells stably transformed by chimeric plasmids.

We have examined the expression of chimeric plasmids containing coding sequences for the herpes simplex virus thymidine kinase (tk) gene or the Tn5 gene for neomycin resistance (neo) linked to the late promoter of polyoma DNA. Although polyoma late genes are generally not expressed in transformed cells containing only integrated viral DNA molecules, rat tk- or wild-type cells transfected with the tk- or neo-containing plasmids were capable of growing in medium containing either hypoxanthine-aminopterin-thymidine or G418, respectively, under conditions nonpermissive for extrachromosomal DNA replication, indicating that the tk or neo genes were fully expressed. Moreover, cells were capable of growth in either hypoxanthine-aminopterin-thymidine or G418, even in the absence of direct selection for this activity. Northern analysis indicated steady-state levels of tk or neo transcripts that approximated the levels of polyoma early transcripts. S1 analysis showed that these transcripts initiated within the late promoter of polyoma and that their 5' ends mapped at positions similar or identical to those utilized during late lytic infection. The effect of substitution of polyadenylation signals was examined. Although plasmids containing the polyoma early polyadenylation signal were more efficient in conferring to cells a stable G418-resistant phenotype than similar constructions using the late signal, both signals were found to be effectively utilized. This indicates that the inability to detect late transcripts in polyoma-transformed cells in the absence of free viral DNA production is not an effect of inefficient mRNA cleavage or polyadenylation. Our results suggest that late gene expression in integrated polyoma genomes is not regulated at the level of message initiation but, most likely, through posttranscriptional events.

Mutational mechanisms by which an inactive replication origin of bacteriophage M13 is turned on are similar to mechanisms of activation of ras proto-oncogenes.

M13 viral strand synthesis is initiated by nicking of the viral strand of the duplex replicative form by the M13 gene II initiator protein at a specific site within a sequence of about 40 base pairs having dyad symmetry. Efficient replication of the M13 viral strand also requires the presence of an adjacent sequence of ca. 100 base pairs. Together these sequences constitute the minimal origin for M13 viral strand synthesis. A pBR322 derivative having a 182-base-pair insert of M13 DNA contains a functional M13 viral strand origin and, when provided with M13 gene functions in trans, replicates under conditions nonpermissive for the parent plasmid. Chimeric plasmids containing deletions within the sequence flanking the viral strand origin are unable to replicate under these conditions. We isolated spontaneous mutants of M13 based on their ability to activate replication of such plasmids. The mutations found in these strains, as well as several produced by oligonucleotide-directed mutagenesis, all result in the substitution of any of at least four different amino acids for a specific glycine residue near the amino-terminal end of the initiator protein. Other studies have shown that overproduction of the wild-type initiator protein also restores replication. These alternate mechanisms are discussed in terms of their striking similarity to the mechanisms of activation of the ras proto-oncogenes which can be activated either by increased expression of the wild-type protein or by substitution of any of several amino acids for a glycine residue near the amino terminus.

Cold sensitivity in the transfer of a plasmid with a deletion hot spot into recombination deficient B. subtilis cells.

Various recombination-deficient mutants of B. subtilis, which are readily transformable by plasmid DNA at 42 degrees C cannot be transformed at 30 degrees C with chimeric plasmid derivatives that contain the deletion hot spot defined previously (Alonso and Trautner 1985a, b). Such interference was also observed in protoplast transformation and SPP1 transduction.

Molecular cloning of the delta-endotoxin gene of Bacillus thuringiensis var. israelensis

A transformant of Bacillus megaterium, VB131, was isolated which carries a 6.3-kb XbaI segment of the crystal toxin gene of Bacillus thuringiensis var. israelensis (BTI) cloned in a vector plasmid pBC16 to yield pVB131. The chimeric plasmid DNA from VB131 was introduced into a transformable Bacillus subtilis strain by competence transformation. Both the B. megaterium VB131 strain and the B. subtilis strain harboring the chimeric plasmid produced irregular, parasporal, phase-refractile, crystalline inclusions (Cry +) during sporulation. The sporulated cells as well as the isolated crystal inclusions of the pVB131-containing B. megaterium and B. subtilis strains were highly toxic to the larvae of Aedes aegypti. Also, the solubilized crystal protein preparation from VB131[pVB131] showed clear immuno cross-reaction with antiserum to the BTI crystal toxin. 32P-labeled pVB131 plasmid DNA showed specific hybridization with a 112-kb plasmid DNA of Cry + strains of BTI, and no hybridization with other plasmid or chromosomal DNA of either Cry + or Cry − variants. These results are in agreement with our previous findings (González and Carlton, 1984) that the 112-kb plasmid of BTI is associated with the production of the crystal toxin.

Isolation and expression of a constitutive variant of the chloramphenicol-inducible plasmid gene cat-86 under control of the Bacillus subtilis 168 amylase promoter

The amyR 1 region controls the regulated expression of the Bacillus subtilis 168 amylase gene amyE. When cloned into the B. subtilis promoter-cloning plasmid pPL603, amyR1 has been shown to activate expression of the promoter-indicator gene cat-86. In this chimeric plasmid, p5' αB10, cat-86 expression was maximal in stationary phase B. subtilis cells and cat-86 expression was repressible by glucose. Both these properties are similar to the regulated expression of the B. subtilis amyE gene. In addition, cat-86 expression in p5' αB10 was inducible with chloramphenicol (Cm). The inducibility phenotype of cat-86 has been shown to be independent of the promoter that is used to activate the gene, and inducibility has been suggested to result from the presence of a pair of inverted-repeat sequences that span the ribosome-binding site (RBS) for cat-86. A spontaneous deletion mutant of p5' αB10 was isolated, p5' αB 10Δ1, in which cat-86 expression was constitutive with respect to Cm, but the basic pattern of amyR 1-directed regulation of cat-86 was intact. The rightward deletion endpoint was within the upstream member of the pair of inverted repeats that immediately precede cat-86. This result is therefore consistent with the role proposed for the inverted repeats in Cm inducibility. The leftward endpoint of the deletion is within the amyR 1 region and thus allows a more precise determination of the functional domain of amyR 1.

In vivo transfer of chromosomal mutations onto multicopy plasmids by transduction with bacteriophage P1

A technique is presented by which chromosomal mutations may be efficiently transferred onto chimeric multicopy plasmids in vivo. The technique employs the transduction of plasmids using bacteriophage P1 as vector. The utility of this method was demonstrated by cloning a chromosomal ompR mutation of Escherichia coli K-12. The high-frequency transduction of the chimeric plasmid appeared to be dependent on its integration into the chromosome by homologous recombination. The results also suggest that the plasmid was transduced as part of the chromosome and resolved from its integrated state in the recipient cell, resulting in a high yield of mutant plasmid segregants.

Coordinate expression of myogenic functions and polyoma virus replication.

Polyoma virus has been extensively used to study degrees of cell differentiation (Georges et al. 1982; Tanaka et al. 1982) and tissue specificity (de Villiers et al. 1984; De Simone et al. 1985; Maione et al. 1985) by means of analyses of cell permissivity to viral growth. The viral nontranscribed regulatory region that directs early and late mRNA transcription, as well as DNA replication (Tyndall et al. 1981), has also been used as an enhancer for heterologous genes in chimeric plasmids (de Villiers and Shaffner 1981) to analyze the regulatory interactions of putative cell functions in different tissue cell lines (Khoury and Gruss 1983; Herbomel et al. 1984).

Construction of new shuttle vectors for Acetobacter.

Shuttle vector pMV301 was constructed by ligation of pMV102 found in A. aceti subsp. xylinum NBI 1002 to E. coli plasmid pACYC177. It is 6.0 kb in size, has unique restriction sites suitable for insertion of a foreign DNA fragment and confers ampicillin resistance to the Acetobacter host. This vector transforms A. aceti subsp. aceti 10-80S1 and industrial vinegar producer A. aceti subsp. xylinum NBI 1002 as well as E. coli. Various chimeric plasmids were also constructed by ligation of pMV102 to E. coli plasmids to examine the expression of drug resistance genes. In addition to the ampicillin resistance gene, resistance genes for kanamycin, chloramphenicol and tetracycline derived from E. coli plasmids were expressed in Acetobacter. Most of the constructed shuttle vectors were stably maintained in Acetobacter.

Conservation of a 29-base-pair sequence within maxicircle ARS fragments from six species of trypanosomes

The maxicircles from Trypanosoma brucei, Herpetomonas samuelpessoai, Leptomonas seymouri, and Phytomonas davidi were examined for the presence of a 29-bp sequence termed CF29 that has been found in the ars 189 sequence from the Crithidia fasciculata maxicircle and in Lt- ars 189 from the maxicircle of Leishmania tarentolae. The CF29 sequence also contains a yeast consensus ARS of(T/A)TTTATPuTTT(T/A). All of the maxicircles examined contained specific fragments that hybridized to the CF29 probe. The non-replicating yeast plasmid vector YIp5 was used to clone these CF29-containing maxicircle fragments. High-frequency transformation was observed when these chimeric plasmids were used to transform Saccharomyces cerevisiae. Autonomous replication of these transforming plasmids was verified by Southern analysis of yeast-cell extracts using pBR322 as a hybridization probe. Therefore it appears that the CF29 sequence is widely conserved in kinetoplastid protozoa and is associated with ARS sequences in the maxicircles. Hybridization of the CF29 probe to a population of P. davidi minicircles was also observed. However, the YIp5 chimeric plasmid containing this CF29-hybridizing minicircle fragment failed to transform yeast.

Cloning of the hexA mismatch-repair gene of Streptococcus pneumoniae and identification of the product

The hexA mismatch repair gene of Streptococcus pneumoniae has been cloned into multicopy plasmid vectors. The cloned hexA gene is expressed as judged from its ability to complement various chromosomal hexA − alleles. Its direction of transcription was defined and the functional limits were localized by original methods relying on homology-dependent integration of nonautonomous chimeric plasmids carrying chromosomal inserts into the chromosome. Comparison of the proteins encoded by recombinant plasmids and by restriction fragments allowed us to identify an M r 94000 protein as the probable product of the hexA gene.

Expression in Escherichia coli of streptococcal plasmid-determined erythromycin resistance directed by the cat gene promoter of pACYC 184.

The streptococcal erythromycin resistance (Emr) plasmid pSM7 (6.4 kb) and the E. coli vector pACYC184 (4.0 kb) were fused at their single EcoRI sites to form the bifunctional chimeric plasmid pSM7184 (10.4 kb) in which the Emr determinant was placed under control of the chloramphenicol acetyl transferase (cat) promoter of pACYC184. In the sense orientation (orientation I) of pSM7, the cat promoter directed expression of Emr in the E. coli host strains 294 and DB11 more efficiently than did the indigenous transcription signals of pSM7, which were functional in the opposite orientation II. In Streptococcus sanguis (Challis), the level of Emr was independent of the orientation of pSM7 in pACYC184, showing that the cat promoter was not recognized in the gram-positive host. The growth of E. coli (pSM7184I) in a defined medium containing glycerol as carbon source, or containing glucose plus extraneous cyclic 3'-5' adenosine monophosphate (cAMP) led to an Emr level which was 15-30 times higher than that of cultures grown on glucose. These results showed that under control of the cat promoter, Emr is subject to cAMP-mediated catabolite repression and provided conclusive evidence that the enhancement of Emr expression in E. coli carrying pSM7184I is controlled at the transcriptional level. Besides enabling us to determine the orientation of transcription of the Emr gene in pSM7 and related vectors, this work also made available new bifunctional cloning vehicles able to replicate in both E. coli and S. sanguis.