Stable and unstable transformation by microinjection of macronucleoplasm in Paramecium

Transformation by microinjection of macronucleoplasm in Paramecium caudatum was investigated. Macronucleoplasm with three genetic markers (behavior, trichocyst, and mating type) was injected into the macronucleus. To facilitate microinjection, in most cases, paramecia were immobilized in a gelatin (7.5%) solution. The injected cells began to express a dominant gene (cnrA⁺ or cnrB⁺) of the donor 9–24 hr after injection. Expression did not require cell division suggesting injected macronucleoplasm was capable of expressing a phenotype. The amount of injected macronucleoplasm appears to correlate with the frequency of successful expression but not to correlate with the time required for expression. After a number of fissions, the injected cells produced clones which had cells expressing the phenotype of the donor. This suggests that injected macronucleoplasm was replicated and expressed in the recipient cell lines. The transformed clones were classified into two groups. In one group, transformation was stable. All cell lines derived from the injected cells expressed a phenotype similar to the heterozygote of donor and recipient cells. In the other group, transformation was unstable. During the first five to seven fissions after injection, at each division, cells produced one daughter cell which later reverted to the recipient phenotype. After this unstable period, cells no longer produced the recipient phenotype but produced the donor phenotype exclusively. Donor and recipient phenotypes were, thus, segregated in different cell lines. Observation of genetic markers and analysis by computer simulation shed light on the mode of transmission of injected macronucleoplasm. In stable transformation, injected macronucleoplasm appears to be distributed equally to daughter cells. In unstable transformation, injected macronucleoplasm is distributed only to one of the daughter cells at every division until about the fifth to seventh fission after injection and then begins to assort equally to daughter cells. The cell cycle stage at injection may influence the mode of transformation. Interspecific microinjection of macronucleoplasm from P. multimicronucleatum and P. tetraurelia to P. caudatum. resulted in the expression of foreign genes in P. caudatum. In one case, injection of macronucleoplasm of P. tetraurelia produced a stable transformant indicating replication of foreign macronucleoplasm in P. caudatum. This work reveals the mode of transformation by injected macronucleoplasm and shows the possibility of transformation among Paramecium species, which is significant in the study of the conservation of gene products and the mechanism of gene expression in different species. © 1992 Wiley-Liss, Inc.     

Transfer of anchorage independence by isolated metaphase chromosomes in hamster cells

The cellular property of being able to grow on agar (aga⁺) or to show anchorage independence has been transferred by means of metaphase chromosomes from CHO cells to BHK and other permanent transformed hamster lines unable to grow on agar. As with other genetic markers, the transferents are unstable when grown under non-selective conditions. The aga⁺ transferents are tumorigenic, providing further evidence for the association between the ability to grow in agar or anchorage independence and tumorigenicity. Evidence has been obtained in these experiments for the existence of at least two discrete events in the transformation of normal into tumorigenic cells. The ability to transfer and select for the aga⁺ marker in recipient cells indicates that tumorigenicity behaves dominantly phenotypically.     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

Homology in capsular transformation reactions in Pneumococcus

Summary Experiments were carried out to determine the relative effect of homology inside or outside of the capsular genomes of donor and recipient strains of pneumococci on the frequency of transfer of capsular markers. In one series of experiments, 3 recipient strains were transformed to CapIII⁺ by DNA from 2 donor strains. Recipient strains (III)capIII D6 ¹, (II)capIII D15 P1 ¹, and (II)capII-1 ¹ were each transformed to CapIII⁺ at different absolute frequencies dependent upon the amount of genetic information that the strain had to acquire. The chromosomal background of the donor strain carrying the CapIII capsular genome had no influence on the results, however, for each strain was transformed at the same frequency by DNA from donor strain (II)CapIII⁺ or donor strain (III)CapIII⁺. In a second series of experiments, 2 (I)CapIII-strains, a (II)CapIII-strain and a (III)CapIII-strain were transformed to heterologous type I and binary type I-III with DNA from donor strains (I)CapI⁺, (II)CapI⁺, and (III)CapI⁺. Again, the chromosomal background of the donor strain was unimportant to the results. The origin of the recipient strain, however, markedly influenced the frequency of transformation. (I)CapIII-strains were transformed to CapI⁺ at about 10 times the frequency and to CapI-III at from 18–6000 times the frequency of the other CapIII-strains. Consideration of the results leads to the conclusion that transformation of CapIII-strains to CapI⁺ and transformation of CapI-strains to CapIII⁺ are not reciprocal reactions; CapI-strains lose less information in transformation to CapIII⁺ than CapIII-strains gain in transformation to CapI⁺. In (I)CapIII-recipient strains, the residual information from the CapI capsular genome is responsible for the higher frequency of transformation to both CapI⁺ and to CapI-III. It is suggested that addition of exogenous linear DNA to a recipient chromosome to give rise to binary strains occurs when sequence homology with the recipient is limited to one end of a piece of transforming DNA. Models to explain the results (Figs. 1 through 3) are consistent with the experimental findings and are amenable to further testing.     

W-mutagenesis in competent cells of Bacillus subtilis

Summary The relative yield (N _m/N) of fluorescent mutants Ind^- after the transformation of Bacillus subtilis cells by means of UV-irradiated DNA is much higher in an uvr ^- recipient than in an uvr ⁺ strain, when compared at equal fluence, but practically identical at equal survival. Ind^- mutations are induced by UV-irradiation of separated single strands of transforming DNA. The H-strand is much more sensitive to the mutagenic action of UV light. Preliminary irradiation of competent recipient cells by moderate UV fluences increases the survival of UV-or -irradiated transforming DNA (W-reactivation) and the frequency of Ind^- mutations (W-mutagenesis). During transfection of B. subtilis cells by UV-irradiated prophage DNA isolated from lysogenic cells B. subtilis (Ø105 c ⁺) c-mutants of the phage are obtained in high yield only in conditions of W-mutagenesis, i.e. in UV-irradiated recipient cells. These data show that there is no substantial spontaneous induction of error-prone SOS-repair system in the competent cells of B. subtilis.     

A competence specific inducible protein promotes in vivo recombination in Streptococcus sanguis

Summary We describe the first example of a recombination-specific protein induced during the development of competence for transformation in Streptococcus sanguis. Elaborated in response to stimulation by competence-protein, the 51,000 Molecular Weight (MW) polypeptide is one of at least 10 new polypeptides transiently induced during the competence phase. Biochemical and genetic analyses of the parental, cipA⁺ (competence specific inducible polypeptide A), and mutant, cipA, strains have shown that the 51,000 MW polypeptide has two roles: its low level constitutive synthesis is required for repair of damage to DNA due to UV light and methylmethane sulfonate; its induced synthesis (3–6x10⁴ copies/cell) during the competence phase is essential for promoting recombination between donor single-stranded DNA and the recipient chromosome. Also, ccc plasmid donor DNA transformation, which occurs as a decreasing probability of the increasing donor plasmid MW, requires the inducible function specified by the 51,000 MW polypeptide. The MW independent low level transformation with ccc plasmids, the inheritance of plasmids by conjugation, and the stable maintenance of plasmids introduced by transformation and conjugation, respectively, are independent of the function specified by the 51,000 MW polypeptide.     

Effect of mismatched base pairs on the fate of donor DNA in transformation of Streptococcus pneumoniae

Summary Investigation of the mechanism that discriminates against mismatched base pairs in transformation of Streptococcus pneumoniae of genotype hex ⁺ was based on the use of a radioactively labeled cloned fragment of pneumococcal DNA as donor in transformation. The fate of the donor label was followed by lysis of the transformed cells and separation by agarose gel electrophoresis of DNA fragments generated by restriction endonucleases. As a result of Hex action, most of the donor DNA fragment, which was a few kilobases in length, was lost when a mismatched base pair occurred between donor and recipient DNA. This was not observed in hex ^- recipient cells. Kinetic studies of mismatch-induced donor DNA loss showed that the process is faster in strain 800, an R6 derivative, than in DP 1601, a strain of different origin. In the latter strain, the amount of donor label that becomes double stranded rises substantially, indicating extensive formation of donorrecipient heteroduplex structures, before falling to the expected level. At 30°C the process is essentially completed 15 min after entry.     

Influence of a Small Number of Mature T Cells in Donor Bone Marrow Inocula on Reconstitution of Lymphoid Tissues and Negative Selection of a T Cell Repertoire in the Recipient

Allo-chimerism and clonal elimination of self antigen (Ag) (Ia + Mls-1^a) reactive Vβ6+ T cells were analyzed and compared between allogeneic bone marrow (BM) chimeras reconstituted with BM cells which had been treated with anti-Thy-1 monoclonal antibody (mAb) plus complement (C) (T^– chimeras) and BM chimeras which had been reconstituted with BM cells pretreated with anti-Thy-1 mAb alone (T⁺ chimeras). When lethally irradiated AKR (Mls-1^a) mice were reconstituted with BM cells from B10 or B10 H-2 congenic mice, both T⁺ and T^– chimeras were entirely free of signs of graft-versus-host reaction (GVHR). However, complete replacement of the AKR lymphoid tissues by donor BM cells was accomplished at an early stage in T⁺ chimeras but not in T^– chimeras. On the other hand, clonal elimination of Vβ6⁺ T cells reactive to the recipient Ag (Mls-1^a) was abolished in T⁺ chimeras but successfully induced in T^– chimeras. The Vβ6⁺ T cells not eliminated in T⁺ chimeras showed depressed responses against Mls-1^a antigens. The findings herein demonstrate that T cells which contaminate a BM inoculum survive in recipient mice after treatment with anti-Thy-1 mAb without C in vitro followed by BMT. The surviving T cells have been estimated to represent fewer than 0.5% of the BM cells inoculated. These cells appear to accelerate the full replacement of recipient lymphoid tissues by donor cells. Furthermore, the T cells which survive in the marrow inoculum influence eventually the development of a tolerant state in the T cell repertoire of the donor.     

Conditions for quantitative transformation inAcinetobacter calcoaceticus

A transformation procedure that yielded high efficiencies was developed forAcinetobacter calcoaceticus. Strain BD413 Ura⁻ trpE was transformed to tryptophan prototrophy using highly purified DNA. Experimental parameters studied were: (i) recipient cell concentration, (ii) DNA concentration, (iii) growth phase of the recipient cell population, (iv) composition of the growth and transforming medium, and (v) time of incubation of recipient cells with donor DNA. Strain BD413 was competent for transformation throught the growth cycle, with highest competence occurring early in the exponential phase of growth. Maximal transformation efficiencies of 0.5% to 0.7% were obtained in media supporting rapid growth. Recipient cell concentrations of 1×10⁶ to 6×10⁶ cells/ml yielded the highest transformation frequencies, regardless of DNA concentration.     

Restriction of plasmid-mediated transformation in Bacillus subtilis 168.

Summary When plasmids carrying leucine genes of Bacillus subtilis 168 were isolated from a restriction and modification deficient (r^-m^-) strain and used for transformation of a restricting strain B. subtilis 168 leu recE4, the number of transformants was greatly reduced. Transformation of a rec ⁺ strain (transformation by integration of the donor DNA into the chromosome) with the plasmids was not affected irrespective of whether the recipient carried the r⁺ or r^- phenotype. These results show that the plasmid-mediated transformation is subject to the host controlled restriction and suggest that r^-m^- strains should be used for construction of recombinant DNA molecules in B. subtilis 168.     

On the ability of Salmonella typhimurium cells to form deoxycytidine nucleotides.

Summary The fate of the donor DNA after conjugation in Escherichia coli was studied through crosses with a Hfr lacZ5 donor and several F^- lacZ22 recipients. The fate of the donor allele was studied by assaying the -galactosidase activity formed by complementation between the lacZ5 allele and the lacZ22 allele. We used continuous cultures of the recipient in order to be able to study the fate of the donor DNA during many generations under constant physiological conditions. We could show that the donor DNA allele is inactivated in Rec⁺, recA171 and recB21 recipient cells. The inactivation rate depends on the nature of the recipient, Rec⁺ or recombination deficient, and especially in the case of the recombination deficient mutants on the growth rate of the recipient.     

On the role of the recipient cell during conjugation in Escherichia coli

To study the role of the E. coli recipient cell in conjugation recipient cell mutants deficient in conjugation (Con^-) were isolated. Mutants specific for F-type E. coli donor cells (ConF^-) and mutants specific deficient in conjugation with I-type donor cells (ConI^-) were isolated.Both ConF^- and ConI^- mutants were blocked in stable mating pair formation. Biochemical analysis of the mutants suggests that the outer membrane protein coded by the ompA gene and LPS are important for recipient activity in F-type conjugation while LPS is important for recipient activity in I-type conjugation.     

Expression of an excision repair gene in transformation of Bacillus subtilis.

Summary DNA of Bacillus subtilis proficient in excision repair (hcr ⁺) was introduced into Angiografinpurified competent cells of an excision repair-deficient strain UVS-1 (hcr-1). The hcr ⁺ gene was found to affect the UV-survival curve of the cells, giving rise to a UV-resistant component. However, a considerable number of colonies of the UV-resistant component consisted of cells that were not transformed to hcr ⁺ as judged by their sensitivity to mitomycin C (MC), UV, and by their ability to reactivate UV-irradiated M2 phages. This suggests that the hcr gene may be expressed without integration. The recA function of B. subtilis was necessary for expression of UV resistance to occur. When DNA-treated cells were selected for met ⁺ recombinants, the UV-resistant component was again found on the UV-survival curve and about half of the colonies of the UV-resistant component consisted of Hcr^- cells. This result was explained by an integration-segregation model for hcr ⁺ and met ⁺ genes. The effect of the hcr ⁺ gene was seen even when DNA was added after cells were irradiated with UV, although this effect was gradually diminished by delaying the time of DNA addition. A complementation effect was found between two excision repair mutations residing in two distant loci, using hcr-114 DNA as a donor and hcr-1 cells as a recipient.     

Uptake of donor lymphocytes treated with 8-methoxypsoralen and ultraviolet A light by recipient dendritic cells induces CD4CD25Foxp3 regulatory T cells and down-regulates cardiac allograft rejection

Extracorporeal photopheresis (ECP) is an effective immunomodulatory therapy and has been demonstrated to be beneficial for graft-vs-host disease and solid-organ allograft rejection. ECP involves reinfusion of a patient’s autologous peripheral blood leukocytes treated ex vivo with 8-methoxypsoralen and UVA light radiation (PUVA). Previous studies focused only on ECP treatment of recipient immune cells. Our study is the first to extend the target of ECP treatment to donor immune cells. The results of in vitro co-culture experiments demonstrate uptake of donor PUVA-treated splenic lymphocytes (PUVA-SPs) by recipient immature dendritic cells (DCs). Phagocytosis of donor PUVA-SPs does not stimulate phenotype maturation of recipient DCs. In the same co-culture system, donor PUVA-SPs enhanced production of interleukin-10 and interferon-γ by recipient DCs and impaired the subsequent capability of recipient DCs to stimulate recipient naïve T cells. Phagocytosis of donor PUVA-SP (PUVA-SP DCs) by recipient DCs shifted T-cell responses in favor of T helper 2 cells. Infusion of PUVA-SP DCs inhibited cardiac allograft rejection in an antigen-specific manner and induced CD4⁺CD25^highFoxp3⁺ regulatory T cells. In conclusion, PUVA-SP DCs simultaneously deliver the donor antigen and the regulatory signal to the transplant recipient, and thus can be used to develop a novel DC vaccine for negative immune regulation and immune tolerance induction.     

A Fast and Practical Yeast Transformation Method Mediated by Escherichia coli Based on a Trans-Kingdom Conjugal Transfer System: Just Mix Two Cultures and Wait One Hour

Trans-kingdom conjugation is a phenomenon by which DNA is transferred into a eukaryotic cell by a bacterial conjugal transfer system. Improvement in this method to facilitate the rapid co-cultivation of donor bacterial and recipient eukaryotic cell cultures could make it the simplest transformation method, requiring neither isolation of vector DNA nor preparation of competent recipient cells. To evaluate this potential advantage of trans-kingdom conjugation, we examined this simple transformation method using vector combinations, helper plasmids, and recipient Saccharomyces cerevisiae strains. Mixing donor Escherichia coli and recipient S. cerevisiae overnight cultures (50 μL each) consistently yielded on the order of 10¹ transformants using the popular experimental strain BY4742 derived from S288c and a shuttle vector for trans-kingdom conjugation. Transformation efficiency increased to the order of 10² using a high receptivity trans-kingdom conjugation strain. In addition, either increasing the amount of donor cells or pretreating the recipient cells with thiols such as dithiothreitol improved the transformation efficiency by one order of magnitude. This simple trans-kingdom conjugation-mediated transformation method could be used as a practical yeast transformation method upon enrichment of available vectors and donor E. coli strains.     

Transduction versus transformation of methicillin resistance inStaphylococcus aureus

Requirements for the transformation of methicillin resistance (Mec^r) inStaphylococcus aureus were found to differ from those of transduction in three significant respects. Production of -lactamase by the recipient strain is not a stringent requirement for Mec^r transformation as it is for transduction althoughbla ⁺ recipients exhibited a higher transformation frequency in the absence of added NaCl. Incorporation of NaCl into the methicillin-selective media markedly inhibited Mec^r transduction but increased the transformation frequency of plasmid-free recipients to the level obtained withbla ⁺ recipients. Four separately maintained clones of strain 8325-4 exhibited marked variation in recipient effectiveness for Mec^r transduction but were equally effective as recipients in transformation.     

Excision and repair of mismatched base pairs in transformation of Streptococcus pneumoniae

Summary The use of heteroduplex DNA molecules as donors in pneumococcal transformation makes it possible to follow the fate of each DNA strand. The integration efficiency of each strand depends strongly upon the single base changes it carries. The function (hex) which reduces drastically the transformation yield of markers referred to as low efficiency (LE) tends to remove either donor strand without respect to which one is introduced. In the case of high efficiency (HE) markers the reduction in the transformation yield involves the elimination of only one donor strand. For a given locus it can be either one depending upon the mutation. The reduction in transformation yield can be less drastic for HE markers than for both strands of the LE markers. These data are discussed in terms of differences in the affinity for mismatched base pairs.We have studied the transfer of information from each donor DNA strand to the recipient genome, on the basis of differences in the rates of phenotypic expression of a given marker introduced on opposite strands. Results show that, as in the case of LE markers, the information from HE markers, when introduced on the strand recognized by the hex function, is transmitted to both strands of the recipient molecule. Correction of the recipient strand to homozygosis probably accounts for this information transfer. These results, together with earlier investigations, strongly suggest that the hex function is an excision-repair system acting on donor-recipient base pair mismatches.     

Conjugal Transfer of Genetic Information in Group N Streptococci

Streptococcus lactis strains ML3 and C₂O and S. lactis subsp. diacetylactis strains DRC3, 11007, and WM₄ were found to transfer lactose-fermenting ability to LM0230, an S. lactis C2 lactose-negative (Lac⁻) derivative which is devoid of plasmid deoxyribonucleic acid (DNA). Lactose-positive streptomycin-resistant (Lac⁺ Str^r) recombinants were found when the Lac⁺ Str^s donor was mixed with Lac⁻ Str^r LM0230 in solid-surface matings. Transduction and transformation were ruled out as the mechanism of genetic exchange in strains ML3, DRC3, 11007, and WM₄, nor was reversion responsible for the high number of Lac⁺ Str^r recombinants. Furthermore, chloroform treatment of the donor prevented the appearance of recombinants, indicating that transfer of lactose-fermenting ability required viable cell-to-cell contact. Strain C₂O demonstrated transduction as well as conjugation. Transfer of plasmid DNA during conjugation for all strains was confirmed by demonstrating the presence of plasmid DNA in the transconjugants by using agarose gel electrophoresis. In some instances, a cryptic plasmid was transferred in conjunction with the lactose plasmid by using strains DRC3, 11007, and WM₄. In S. lactis C2 × LM0230 matings, the Str^r marker was transferred from LM0230 to C2, suggesting conjugal transfer of chromosomal DNA. The results confirm conjugation as another mechanism of genetic exchange occurring in dairy starter cultures.     

Measuring the rate of conjugal plasmid transfer in a bacterial population using quantitative PCR

Horizontal transfer of genes between species is an important mechanism for bacterial genome evolution. In Escherichia coli, conjugation is the transfer from a donor (F⁺) to a recipient (F⁻) cell through cell-to-cell contact. We demonstrate what we believe to be a novel qPCR method for quantifying the transfer kinetics of the F plasmid in a population by enumerating the relative abundance of genetic loci unique to the plasmid and the chromosome. This approach allows us to query the plasmid transfer rate without the need for selective culturing with unprecedented single locus resolution. We fit the results to a mass action model where the rate of plasmid growth includes the lag time of newly formed F⁺ transconjugants and the recovery time between successive conjugation events of the F⁺ donors. By assaying defined mixtures of genotypically identical donor and recipient cells at constant inoculation densities, we extract an F plasmid transfer rate of 5 × 10⁻¹⁰ (cells/mL · min)⁻¹. We confirm a plasmid/chromosome ratio of 1:1 in homogenous F⁺ populations throughout batch growth. Surprisingly, in some mixture experiments we observe an excess of F plasmid in the early saturation phase that equilibrates to a final ratio of one plasmid per chromosome.