Genomic integration system based on pBR322 sequences for the cyanobacterium Synechococcus sp. PCC7942: transfer of genes encoding plastocyanin and ferredoxin.

Synechococcus sp. PCC7942 recipient strains were constructed for the chromosomal integration of DNA fragments cloned in any pBR322-derived vector, which carries the ampicillin resistance (ApR) marker. The construction was based on the incorporation of specific recombination targets, the so-called 'integration platforms', into the chromosomal metF gene. These platforms consist of an incomplete bla gene (ApS) and the pBR322 ori separated from each other by a gene encoding an antibiotic (streptomycin or kanamycin) resistance (SmR or KmR). Recombination between a pBR322-derived donor plasmid and such a chromosomal platform results with high frequency in restoration of the bla gene and replacement of the chromosomal marker (SmR or KmR) by the insert of the donor plasmid. The integration into the platform depends on recombination between pBR322 ori and bla sequences only and is therefore independent of the DNA insert to be transferred. The desired recombinants are found by selection for a functional bla gene (ApR) and subsequent screening for absence of the chromosomal antibiotic marker. Gene transfer with this integration system was found to occur efficiently and reliably. Furthermore, the presence of the pBR322 ori in the platform allowed for 'plasmid rescue' of integrated sequences. The system was applied successfully for the transfer of the gene encoding plastocyanin (petE1) from Anabaena sp. PCC7937 and for the integration of an extra copy of the gene encoding ferredoxin I (petF1) from Synechococcus sp. PCC7942 itself.     

Cloning vectors that yield high levels of single-stranded DNA for rapid DNA sequencing

We have constructed chimeric plasmid vectors with the origin and intergenic region from M13 phage cloned into the PvuII ( pZ145 ) and AhaIII ( pZ150 , pZ152 ) sites of pBR322. In the absence of M13 phage, these plasmids replicate like any other ColE1-derived plasmid and confer both ampicillin and tetracycline resistance (Amp, Tet). Upon infection with M13 phage, the viral origin present on the plasmids permits phage-directed plasmid replication and results in high yields of single-stranded (ss) plasmid DNA in M13-like particles. This ssDNA, which represents only one of the plasmid strands, is useful as a substrate for rapid DNA sequence determination by the dideoxy sequencing method described by Sanger et al. (1977). Since these plasmids contain an intact pBR322, the intergenic region can be transferred onto most pBR322 derivatives to yield ss plasmid DNA without affecting the recipient plasmid for further studies. We also constructed a deletion derivative of pZ145 , plasmid pZ146 , that does not exhibit interference with the growth of the M13 helper, although this plasmid is encapsidated into phage particles. This result confirms the theory that the intergenic region consists of two domains: one domain being a segment involved in phage morphogenesis and the other being a region of functional origin which interferes with M13 replication.     

The transfer and stable integration of the HSV thymidine kinase gene into mouse cells.

Treatment of mutant mouse cells (Ltk-) deficient in thymidine kinase with Bam I restriction endonuclease-cleaved HSV-1 DNA results in the appearance of numerous surviving colonies which stably express thte tk+ phenotype. Through a series of electrophoretic fractionations in concert with transfection assays, we isolated a 3.4 kb fragment which contains the thymidine kinase gene and which alone is competent in the biochemical transformation of Ltk- cells. In this report, we have examined the distribution of tk sequences in the DNA of several transformed clones following stable gene transfer. A series of complementary experiments involving reassociation kinetics in solution and annealings with tk DNA to restriction-cleaved cellular DNA following electrophoresis and transfer to filters allow us to make the following general conclusions concerning the fate of the tk gene in all clones examined: the tk gene is present in all cells at a frequency of one copy per chromosomal complement; the tk gene is stably integrated in the DNA of all transformants; and integration is not site-specific and occurs at different loci in the DNA of all transformants examined. The existence of a single active tk gene in tk+ transformants now facilitates an analysis of the sequence organization of tk- mutant cells and provides a useful model system for studies on the transfer of cellular genes.     

Transfer of genes to Chinese hamster ovary cells by DNA-mediated transformation

We have transferred DNa to Chinese hamster ovary (CHO) cells by DNA-mediated transformation. CHO tk- cells were transformed with the clones gene for herpes simplex virus thymidine kinase (HSV-tk) and were found to have a 50-fold lower frequency of transformation than mouse Ltk- cells at the same DNA dosage. By altering the amount of tk gene and carrier DNA present, frequencies of up to 5 x 10(-5) were obtained. CHO HSV-tk+ transformants were very stable, and in several clones the HSV-tk gene copies integrated in higher-molecular-weight DNA. These cells also exhibited cotransformation for unselected markers. CHO lines were also transformed at a frequency of 10(-4) with the bacterial gene Ecogpt in a SV40-pBR322 vector. CHO tk-cells could be transformed at a frequency of 10(-7) with cellular DNA isolated from CHO tk+ cells. CHO cells offer a well-defined genetic system within which to transfer either cloned or whole cellular DNAs.     

Foreign DNA introduced by calcium phosphate is integrated into repetitive DNA elements of the mouse L cell genome.

We investigated the sites of integration of exogenous DNA fragments introduced by DNA-mediated gene transfer. Mouse Ltk- cells were transformed with the herpes simplex virus thymidine kinase gene and pBR322 DNA by the calcium phosphate precipitation method. Some of the integrated exogenous DNA sequences were recovered from the stable tk+ transformants in the form of plasmids that were capable of propagation in bacteria. Four plasmids derived from two cloned cell lines were analyzed in detail by nucleotide sequencing and hybridization techniques. These plasmids contained a total of seven cellular-exogenous DNA junctions. In all cases, there was no sequence homology between the exogenous and cellular DNA sequences adjacent to the joining sites, and no specific exogenous or cellular sequences occurred at the junctions. Rearrangement or deletion of Ltk- DNA was always associated with the integration of exogenous DNA. All of the assignable cellular sequences at the junctions were repetitive sequences. Two of these sequences were from the MIF-1 repetitive sequence family, and a third consisted of a 40-base pair simple copolymer of alternating deoxyadenosine-deoxythymidine. Our results suggest that repetitive sequences are relatively favorable sites for the integration of exogenous DNA.     

Construction and characterization of new cloning vehicles V. Mobilization and coding properties of pBR322 and several deletion derivatives including pBR327 and pBR328

A DNA sequence essential for the R64drd11 + ColK-mediated conjugal transfer of pBR322 has been located in a 540 bp HaeIII fragment (HaeIII-2) between the vegetative origin of replication and the tetracycline resistance (Tc^r) gene of this vector. The pBR322 derivatives pBR327 and pBR328 lack this DNA sequence and are not mobilized by conjugation. Two derivatives of pBR328 were constructed by re-inserting the HaeIII-2 fragment in both orientations into the chloramphenicol-resistance gene of the same vector. One orientation of the HaeIII-2 fragment permitted mobilization by conjugation while the opposite orientation prevented mobilization. Further examination of pBR322 and derivatives revealed that the region between the origin of replication and Tc^r gene also plays a role in regulating plasmid copy number.     

Stability of the transformants obtained by phage particle-mediated gene transfer

Recombinant lambda phage DNA, encapsulated in phage particles and coprecipitated with calcium phosphate, efficiently transforms cultured mammalian cells without a requirement for carrier DNA. The present paper analyzes the stability of the transformants obtained by the phage transfer method. lambda phage particles containing recombinant DNA that includes the thymidine kinase (TK) gene of herpes simplex virus type 1 as a selective marker were introduced into Ltk- cells deficient in TK activity, and TK+ transformants were selected in HAT medium. To test the stability of the TK+ phenotype of the transformants, seven individual transformant clones were isolated, cultured in HAT selective medium and then in non-selective medium for various lengths of time. After such culture, transformants were allowed to develop colonies in both selective and non-selective medium. For all seven transformant clones, the numbers of colonies obtained in the two types of medium were almost identical, irrespective of whether or not each transformant clone had been previously cultured for 15 to 50 days in non-selective medium. This result suggests that most transformants obtained by the phage transfer method maintain the TK+ phenotype stably, for at least 50 days, when grown in non-selective medium.     

Fine-resolution analysis of products of intrachromosomal homeologous recombination in mammalian cells.

Mouse Ltk- cell lines that contained a herpes simplex virus type 1 (HSV-1) thymidine kinase (tk) gene with a 16-bp insertion mutation linked to either a defective HSV-2 tk gene or a hybrid tk sequence comprised of HSV-1 and HSV-2 tk sequences were constructed. HSV-1 and HSV-2 tk genes have 81% nucleotide identity and hence are homeologous. Correction of the insertion mutant HSV-1 tk gene via recombination with the hybrid tk sequence required an exchange between homeologous tk sequences, although recombination could initiate within a region of significant sequence identity. Seven cell lines containing linked HSV-1 and HSV-1-HSV-2 hybrid tk sequences gave rise to tk+ segregants at an average rate of 10(-8) events per cell division. DNA sequencing revealed that each recombinant from these lines displayed an apparent gene conversion which involved an accurate transfer of an uninterrupted block of information between homeologous tk sequences. Conversion tract lengths ranged from 35 to >330 bp. In contrast, cell lines containing linked HSV-1 and HSV-2 tk sequences with no significant stretches of sequence identity had an overall rate of homeologous recombination of <10(-9). One such cell line produced homeologous recombinants at a rate of 10(-8). Strikingly, all homeologous recombinants from this latter cell line were due to crossovers between the HSV-1 and HSV-2 tk genes. Our results, which provide the first detailed analysis of homeologous recombination within a mammalian genome, suggest that rearrangements in mammalian genomes are regulated by the degree of sequence divergence located at the site of recombination initiation.     

Fate of cloned bacteriophage T4 DNA after phage T4 infection of clone-bearing cells

Plasmid pBR322 replication is inhibited after bacteriophage T4 infection. If no T4 DNA had been cloned into this plasmid vector, the kinetics of inhibition are similar to those observed for the inhibition of Escherichia coli chromosomal DNA. However, if T4 DNA has been cloned into pBR322, plasmid DNA synthesis is initially inhibited but then resumes approximately at the time that phage DNA replication begins. The T4 insert-dependent synthesis of pBR322 DNA is not observed if the infecting phage are deleted for the T4 DNA cloned in the plasmid. Thus, this T4 homology-dependent synthesis of plasmid DNA probably reflects recombination between plasmids and infecting phage genomes. However, this recombination-dependent synthesis of pBR322 DNA does not require the T4 gene 46 product, which is essential for T4 generalized recombination. The effect of T4 infection on the degradation of plasmid DNA is also examined. Plasmid DNA degradation, like E. coli chromosomal DNA degradation, occurs in wild-type and denB mutant infections. However, neither plasmid or chromosomal degradation can be detected in denA mutant infections by the method of DNA--DNA hybridization on nitrocellulose filters.     

Carrier DNA affects the integration of HSV-1 TK gene in the genome of L929TK- cells.

L929TK- cells were cotransfected with DNA mixtures containing tk gene of HSV-1, plasmids carrying LTR of MoMLV or RSV and carrier DNA of salmon sperm or chromosomal DNA of recipient cells. Selection of TK+ transformants was conducted in DMEM supplemented with HAT. Plasmids carrying LTR sequences of MoMLV or RSV retroviruses showed enhancing effect on the frequency of TK+ transformation. Southern blot analysis of chromosomal DNA of TK+ transformants demonstrated in clones deriving from cotransfections of tk gene and carrier DNA of L929TK- cells multiple copies of tk gene integrated into several genomic sites of host. Single copies of tk gene integrated into different sites of host genome occurred in chromosomal DNA of TK+ clones deriving from cotransfections of tk gene and carrier DNA of salmon sperm. Cells cotransfected with tk gene and plasmids carrying LTR sequences of MoMLV or RSV formed three dimensional colonies in semisolid agar medium. No effect of carrier DNA on the morphology of TK+ transformant clones was noticed.     

Single-stranded hexameric linkers: a system for in-phase insertion mutagenesis and protein engineering

An efficient method for introducing two (or four) codons into a cloned gene has been developed. Single-stranded (ss) hexameric linkers are inserted into a plasmid linearized at cohesive-end restriction sites. The resultant 6 (or 12)-bp insertion creates a new 6-bp restriction site. Plasmids containing linker insertions are enriched by using biochemical selection, or selected by using a kanamycin-resistance (KmR) cassette (biological selection). A total of 57 new linkers have been designed, and compatible KmR cassettes flanked by eleven different restriction sites have been constructed. Two-codon insertions into the tetracycline-resistance (TcR) gene of pBR322 yielded a series of new plasmid vectors. Moreover, proteins with internally duplicated domains have been constructed from beta-lactamase (ApR) insertions into the ApR gene of pBR322. Some of the resulting "gemini" proteins retained the beta-lactamase activity.     

Hybrid plasmids containing an active thymidine kinase gene of Herpes simplex virus 1.

The gene for the thymidine kinase (TK) of Herpes simplex virus type 1 (HSV-1) is located in the KpnI m and BamHI p fragments of the genome (Wigler et al., Cell 11, 223-232 (1977)). These fragments have been inserted into the EcoRI and BamHI sites, respectively, of plasmid pBR322, and propagated in E.coli. The TK gene contained in the recombinant plasmids was shown to be biologically active when introduced into TK- mouse L cells. Detailed restriction site maps of the BamHI p fragment have been constructed and the approximate location of the TK gene has been determined. Mouse cells transformed with cloned HSV-1 tk+ DNA produced HSV-1-specific thymidine kinase; superinfection with HSV-1 tk- virus increased the level of TK activity tenfold, suggesting that the BamHI p sequences present in transformed cells respond to virus-encoded regulatory gene product(s).     

Mutant of herpes simplex virus type 1 conditionally able to transform thymidine kinaseless L cells to a tk+ phenotype.

After nitrous acid mutagenesis of herpes simplex virus type 1 (HSV-1), a mutant, 1093, was isolated which, during productive infection, induced very low levels of thymidine kinase (tk). The mutant virus was found, after UV irradiation, to be unable to transform L cells lacking tk (Ltk-) to a tk+ phenotype as chararcterized by growth of the cells in a modified HAT-selective medium containing 1.6 X 10(-5) M thymidine. Cells transformed by wild-type virus grew vigorously under the same conditions. The mutant was able to transform Ltk- cells if the medium contained 10(-3) M thymidine. These transformed cells maintained their conditional character and would not grow in low concentrations of thymidine in selective medium. Therefore, this mutant is conditional on the thymidine concentration in the selection medium in its ability to transform Ltk- cells to a tk+ phenotype. The conditionally transformed cells could be supertransformed with wild-type UV-irradiated HSV-1 to a phenotype which would grow in low-thymidine selective medium. The frequency of supertransformation closely approximated the frequency of transformation of Ltk- cells by wild-type virus. Supertransformation at high frequency could not be effected by mutant 1093 or the tk- mutant B2006. These results indicate that the presence of HSV-1 genetic information in HSV-1-transformed cells does not preclude the acquisition by these cells of at least one additional HSV-1 gene, that for tk.     

Cloned mouse DNA fragments can replicate in a simian virus 40 T antigen-dependent system in vivo and in vitro.

Mouse liver DNA was cut out with BamHI and cloned into YIp5, which contained the URA3 gene of Saccharomyces cerevisiae in pBR322. Of the several plasmids isolated, two plasmids, pMU65 and pMU111, could transform S. cerevisiae from the URA- to the URA+ phenotype and could replicate autonomously within the transformant, indicating that mouse DNA fragments present in pMU65 or pMU111 contain autonomously replicating sequences (ARS) for replication in S. cerevisiae. Furthermore, to determine the correlation between ARS function in yeast cells and that in much higher organisms, we tried to challenge these plasmids with the simian virus 40 (SV40) DNA replication system. Of the two plasmids tested, the EcoRI-BglII region of pMU65 could be hybridized with a chemically synthesized 13-nucleotide fragment corresponding to the origin region of SV40 DNA. Both pMU65 (the EcoRI-BglII region cloned in pBR322) and its subclone pMU65EB could replicate semiconservatively, and initiation of DNA replication started from the EcoRI-BglII region when the replicating activity of these plasmids was tested in the in vitro SV40 DNA replication system we have established before. Furthermore, pMU65 and pMU65EB could replicate autonomously within monkey Cos cells which produce SV40 T antigen constitutively. These results show that a 2.5-kilobase fragment of the EcoRI-BglII region in pMU65 contains the ARS needed for replication in the SV40 DNA replication system.     

Production of a T-antigen-related protein in mammalian cells after stable transformation with a cloned SV40 gene fragment

A recombinant plasmid based on pBR322 has been constructed which carries the replicator proximal early region of SV40 DNA, including the viral origin of replication (ORI). It lacks a major part of the tumour antigen 3'-coding region, the large T-antigen termination codon and the polyadenylation site. The recombinant plasmid was transferred together with the herpes simplex virus thymidine kinase gene, as a selectable marker into mouse LTK- cells. Integration and expression of the cloned SV40 gene fragment in TK+ transformants could be demonstrated by DNA restriction and blot hybridization and by immunofluorescence techniques.     

Temperature-sensitive mutants of herpes simplex virus type 1 defective in lysis but not in transformation.

Twelve temperature-sensitive (ts) mutants of herpes simplex virus type 1 (HSV-1), representing seven complementation groups, were isolated subsequent to 5-bromodeoxyuridine mutagenesis. These mutants were identified by their inability to replicate in a line of monkey (CV-1) cells at 39 C. Seven of these mutants, representing six complementation groups, induced thymidine kinase (tk) and transformed Ltk- cells, a line of mouse L cells lacking tk, to a tk+ phenotype at both the permissive (34 C) and nonpermissive (39 C) temperatures. Thus, the defective cistrons in these six complementation groups, although necessary for lysis, have no essential function in this transformation system. Transformation by these 12 mutants was dependent on prior UV irradiation. Infection of cells with unirradiated virus under conditions which did not permit virus replication was not sufficient to allow cell transformation. Five mutants, representing two complementation groups, were tk- and were incapable of causing the tk--to-tk+ transformation at either 34 C of 39 C. The tk defects in these mutants are probably unrelated to the ts defects, since one of these complementation groups contains a tk+ member. Therefore, transformation of Ltk- cells to a tk+ phenotype by HSV-1 requires an active viral tk gene. One complementation group was represented by a single tk- member. The role of this cistron in transformation remains undetermined since the primary block to transformation is presumed to be the tk- phenotype. Mutants representing the seven complementation groups were unable to replicate at 39 C in two lines of HSV-1-transformed cells, indicating that the activities of resident wild-type copies of the defective cistrons, if present, could not be detected by complementation.     

Genetic transformation of somatic cells. VI. Transfer of the trait of the rate of loss of transformant phenotype by means of DNA from cells containing the thymidine kinase gene of the herpes simplex virus

Using dot-hybridization with thymidine kinase gene (tk gene) of Herpes simplex virus type 1 (HSV 1) of DNA preparations obtained from isolated metaphase chromosomes and lysate fractions of metaphase cells, which presumably contain smaller particles compared to metaphase chromosomes, it has been shown that the tk gene of HSV 1 is localized in chromosomes of cells of transformant clones unstable in TK+-phenotype. The DNA isolated from the metaphase chromosomes from cells of transformant clones is 1.5- or 2-fold more efficient in transforming TK-Chinese hamster cells than is the total high molecular weight DNA from the same cells. Upon transformation of TK- cells by the high molecular weight DNA from the tk gene of HSV 1-containing clones, varying in the rate of the loss of TK+-phenotypes, the character "rate of the loss of transformant phenotype" is transferred together with the tk gene of HSV 1 in 22% of cases. Cells of rerevertant clones, produced from TK- subclones of transformant clones, display the rate of the loss of transformant phenotype characteristic of cells of parental TK+-clones. A comparison of the results allows a conclusion that DNA sequences, determining the character "rate of the loss of transformant phenotype", are linked tightly with the transforming DNA proper containing the tk gene of HSV 1, but are not localized inside such a DNA.