Highest Transformation Frequency Research Articles

High-frequency transformation of yeast by plasmids containing the cloned yeast ARG4 gene

Hybrid ColE1 plasmids, containing cloned DNA from the yeast ARG4 region [e.g., pYe(arg4)1], transform yeast arg4 mutants to ARG4(+) with a frequency of 10(-4) (about 10(3) transformants per mug of plasmid DNA) and can replicate autonomously without integrating into the yeast genome. The yeast transformants are genetically unstable when grown on nonselective medium, but can be readily grown and maintained on minimal medium lacking arginine. The existence of unintegrated replicating plasmid DNA in the yeast transformants was demonstrated by Southern gel hybridization and by transformation of Escherichia coli argH mutants with DNA preparations from yeast transformants and subsequent recovery of intact plasmid DNA from the bacterial transformants. Plasmid DNAs recovered from the E. coli-yeast-E. coli "shuttle" remain essentially unchanged, as judged by DNA restriction fragment patterns. Some plasmid mutations leading to increased efficiency of expression of the ARG4 gene in E. coli do not appear to affect expression of the cloned ARG4 gene in yeast. Appropriate derivatives of these ARG4 plasmids are of potential usefulness as vectors for cloning genes in yeast and for studying the mechanism of yeast DNA replication.

High frequency of yeast transformation by plasmids carrying part of entire 2-μm yeast plasmid

By using two chimeric plasmids containing yeast ura3 gene and 2-μm yeast DNA linked to the bacterial plasmid pCR1, yeast transformation of a high frequency has been achieved. The first plasmid is such that the 2-μm DNA part, in which the ura3 gene is incorporated, can be removed in one step and thus the 2-μm— ura3 sequence can be considered as a “transposable” block. In contrast, the second one bears the entire 2-μm plasmid and the ura3 gene is inserted in the bacterial plasmid part. As shown through hybridization experiments and genetic studies, the ura3 gene was maintained as a cytoplasmic element. Plasmids recovered from the yeast transformants were used to transform Escherichia coli. Their analysis by EcoRI showed that in many cases the vector had recombined with the endogenous 2-μm DNA of the recipient strain. The specific activity of orotidine 5′-monophosphate decarboxylase (coded by ura3) in yeast transformants was 10- to 30-fold higher than in the wild type.

High-frequency transformation of yeast: autonomous replication of hybrid DNA molecules.

A set of vector DNAs (Y vectors) useful for the cloning of DNA fragments in Saccharomyces cerevisiae (yeast) and in Escherichia coli are characterized. With these vectors, three modes of yeast transformation are defined. (i) Vectors containing yeast chromosomal DNA sequences (YIp1, YIp5) transform yeast cells at low frequency (1--10 colonies per microgram) and integrate into the genome by homologous recombination; this recombination is reversible. (ii) Hybrids containing endogenous yeast plasmid DNA sequences (YEp2, YEp6) transform yeast cells at much higher frequency (5000--20,000 colonies per microgram). Such molecules replicate autonomously with an average copy number of 5--10 covalently closed circles per yeast cell and also replicate as a chromosomally integrated structure. This DNA may be physically isolated in intact form from either yeast or E. coli and used to transform either organism at high frequency. (iii) Vectors containing a 1.4-kilobase yeast DNA fragment that includes the centromere linked trp1 gene (YRp7) transform yeast with an efficiency of 500--5000 colonies per microgram; such molecules behave as minichromosomes because they replicate autonomously but do not integrate into the genome. The uses of Y vectors for the following genetic manipulations in yeast are discussed: isolation of genes; construction of haploid strains that are merodiploid for a particular DNA sequence; and directed alterations of the yeast genome. General methods for the selection and the analysis of these events are presented.

High frequency transformation of Bacillus subtilis protoplasts by plasmid DNA.

A highly efficient method for transformation of Bacillus subtilis by plasmid DNA is reported. The procedure, which involves polyethylene glycol-induced DNA uptake by protoplasts and subsequent regeneration of the bacterial cell wall, yields up to 80% transformants with an efficiency of 4 x 10(7) transformants per microgram of supercoiled DNA. Plasmids constructed by in vitro ligation or endonuclease-generated fragments of linear plasmid DNA can also transform PEG-treated protoplasts, but at a lower frequency.

Co-transfer of human X-linked markers into murine somatic cells via isolated metaphase chromosomes

Transformation frequencies of 4 x 10(-5) were obtained in chromosome-mediated gene transfer experiments using human cell line HeLa S3 as donor and mouse cell line A9 as recipient. This high frequency of interspecific transformation was achieved by treating the recipient cells with dimethylsulfoxide in addition to other facilitators. The high frequency of transformation correlated positively with transgenome size on the basis of both co-transfer of linked markers and chromosome analysis. The syntenic human markers glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP(+) 1-oxidoreductase, EC 1.1.1.49) and phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) were sometimes transferred together with the selected X-linked prototrophic marker hypoxanthine phosphoribosyltransferase (IMP: pyrophosphate phosphoribosyltransferase, EC 2.4.2.8) into murine somatic cells. Donor human chromosome material could be demonstrated cytologically in some of the transformed cell lines. Transformants exhibited various rates of loss of the human hypoxanthine phosphoribosyltransferase marker when grown under nonselective conditions. These results reveal a broader range of possible interspecific transgenome sizes than has been recognized in the past. The largest transgenomes consist of cytologically detectable donor fragments and contain syntenic markers that are not closely linked to the selected marker.

Genetic exchange in Bacillus subtilis in soil.

Genetically labelled strains of Bacillus subtilis have been shown to exchange blocks of linked genes while growing together in soil. After eight days of incubation, 79% of unselected colony-forming units exhibited a phenotype containing markers from both parents; the parental strains were not detected after the first day of incubation. High frequencies of transformation were also obtained by adding genetically labelled deoxyribonucleic acid to single-strain soil cultures. Observed linkage of genetic markers was greater in soil transformation than in standard laboratory procedures. The results indicate that transformation may play an important role in the adaptation of the Bacilli to their natural habitat.

A survey of epithelial inclusions in the ovarian cortex of 470 patients.

The ovaries of 470 patients obtained for various indications at operation revealed 178 with 193 ovarian cysts and tumors - including 63 serous, 23 endometrial, and 27 mucinous - as well as 103 with tubal (serous), 35 with endometrial, and 2 with cervical (mucinous) epithelial inclusions. A very high frequency of transformation of mucinous epithelial inclusions into cysts and tumors contrasted with about 40% similar transformation of serous and endometrial inclusions into cysts and tumors.

Comparison of Herpes Simplex Virus Isolates Using a Quantitative Selection Assay for Transformation

Transformation potential of herpes simplex virus types 1 and 2 (HSV-1 and HSV-2) was quantitatively compared in a system selecting for biochemical transformants. The highest frequency of transformation was obtained with HSV-2 strain 333. The transformation rates for two strains of HSV-1 were lower and distinctly different from HSV-2 and from each other. The dose of inactivating ultraviolet light received by the virus and the multiplicity of infection proved to be critical components of the procedure. Clones of these biochemically transformed cells expressed HSV-specific antigens, altered cell morphology, and increased levels of thymidine kinase activity. Isolates of HSV-1 recovered from the same site 11 years apart yielded similar transforming frequencies. These results suggest that this assay should prove useful in further characterizing the biological properties of HSV.

A complex of recombination and repair genes in Haemophilus influenzae

Two transformation-defective strains of Haemophilias influenzae have been studied with respect to their ability (1) to be transformed (2) to repair DNA damage induced by ultraviolet light, X-rays and methyl methanesulfonate (3) to allow recombination of temperature-sensitive mutants of the H. influenzae phage HP1 c1, and (4) to be induced after lysogenization with wild-type phage. One of the strains was obtained following exposure of wild-type cells to DNA from the other, originally selected as a u.v.-sensitive strain. These strains have been crossed by transformation with wild type and with each other, and repair and recombination in the transformants have been investigated. The results indicate that both strains contain a defect in their recombination mechanism, since phage recombination is not detectable in either of them. The recombination defects in the two strains are different from each other by the following criteria: (1) The residual transformation frequencies in the strains are different, about 10 −6 and 10 −7 that of the wild type. (2) The recombination defect in the original strain with the higher transformation frequency, which contains a mutation we have called rec 1 −, is inseparable from u.V., X-ray, and methyl methanesulfonate sensitivity and lack of inducibility following lysogenization. The derived strain, which is u.v. resistant and inducible, has an intermediate sensitivity to methyl methanesulfonate and slight sensitivity to X-rays. (3) The rec 1 + gene can be transferred into the original Rec 1 − strain, either with wild-type DNA or with DNA from the derived recombination-defective mutant; rec 1 − can also be transferred into the wild-type strain. We have been unable to transfer the derived Rec − defect into the wild type, nor have we succeeded in transforming the derived strain carrying this recombination defect to normal recombination, either with wild-type DNA or with DNA from the Rec − 1 mutant. In spite of the fact that one of the Rec − strains was obtained following transformation of wild-type cells with DNA from the other Rec −, the two Rec − strains can be crossed to produce a Rec +. To account for this anomalous result it is postulated that the original Rec − strain contains two mutations, rec − 1 and mex −, and that the derived strain is rec + 1, mex −, the mex − mutation conferring intermediate sensitivity to methyl methanesulfonate and slight sensitivity to X-rays. It is further postulated that the derived Rec − strain contains a third altered gene, rec p 2, which was obtained through the selection procedure used to isolate the strain from the wild-type culture exposed to rec 1 − mex − rec 2 + DNA, and that the combination mex − rec 2 p causes the Rec − phenotype.

Homology in capsular transformation reactions in Pneumococcus.

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 D61, (II)capIII D15 P11, and (II)capII-11 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.

A comparison of streptococcal competence factors produced by strain Challis in chemically defined and complex media.

SUMMARY: A comparison was made between streptococcal competence factors (CF) elaborated by strain CHALLIS in a chemically defined medium and in a complex medium (defined medium containing neopeptone and horse serum). The CF preparations acted similarly in that: (i) both CFs maximally activated the normally non-transformable streptococcal strain WICKY to the competent state when the WICKY cells were incubated for 40 to 100 min. in phase 2; (ii) both CF preparations had to react with WICKY cells for about 20 min. before the cells would take up deoxyribonucleic acid (DNA); (iii) the rates of DNA uptake in WICKY populations were identical when either CF preparation was used; (iv) both CFs activated different concentrations of WICKY cells so that a fixed proportion of the total population transformed to dihydro-streptomycin resistance; and (v) upon dilution both CF preparations behaved as if they contained more than a single component. The CF preparations behaved differently in that: (i) WICKY cells activated to the competent state with CF made in the complex medium generally yielded frequencies of transformation two to eight times higher than CF prepared in the defined medium, and (ii) CF prepared in the complex medium occurred in culture filtrates at an apparently higher concentration than CF prepared in the defined medium. This observation may be one reason for the high frequencies of transformation observed with CF made in the complex medium.

Genetic transformation in Pseudomonas.

SUMMARY Intraspecific and interspecific transformation in Pseudomonas has been investigated. Two isoleucine+valine requiring mutants of Pseudomonas aeruginosa have been transformed to prototrophy. By using gelatin liqui-faction as a marker character it was observed that two strains of P. fluorescens, a phytopathogen P. mangiferaeindicae and a strain of Pseudomonas originally isolated from fish were transformable. When P. solanacearum was used as a donor, the transformation frequency of P. mangiferaeindicae was very high. DNA from fish Pseudomonas I was capable of transforming two strains of P. fluorescens and P. mangiferaeindicae in addition to fish Pseudomonas 21. When P. fluorescens was treated with the DNA from P. aeruginosa transformation frequency was lower; the capacity of pigment production, however, could not be transferred to any of the strains. The highest transformation frequency was obtained towards the end of the log phase of growth of the receptor bacteria, with a DNA concentration of about 10 μg./ml. Optimum temperature for transformation was 25–30° and agitation and starvation in diluted media increased the number of transformants. The significance of the observations has been discussed from the point of view of relationships among the strains and species studied.

High frequency of SV40 transformation of mouse cell line 3T3

Members of the casitas B-lineage lymphoma (Cbl) family (Cbl, Cbl-b and Cbl-c) of ubiquitin ligases serve as negative regulators of receptor tyrosine kinases (RTKs). An essential role of Cbl-family protein-dependent ubiquitination for efficient ligand-induced lysosomal targeting and degradation is now well-accepted. However, a more proximal role of Cbl and Cbl-b as adapters for CIN85-endophilin recruitment to mediate ligand-induced initial internalization of RTKs is supported by some studies but refuted by others. Overexpression and/or incomplete depletion of Cbl proteins in these studies is likely to have contributed to this dichotomy. To address the role of endogenous Cbl and Cbl-b in the internalization step of RTK endocytic traffic, we established Cbl/Cbl-b double-knockout (DKO) mouse embryonic fibroblasts (MEFs) and demonstrated that these cells lack the expression of both Cbl-family members as well as endophilin A, while they express CIN85. We show that ligand-induced ubiquitination of EGFR, as a prototype RTK, was abolished in DKO MEFs, and EGFR degradation was delayed. These traits were reversed by ectopic human Cbl expression. EGFR endocytosis, assessed using the internalization of 125I-labeled or fluorescent EGF, or of EGFR itself, was largely retained in Cbl/Cbl-b DKO compared to wild type MEFs. EGFR internalization was also largely intact in Cbl/Cbl-b depleted MCF-10A human mammary epithelial cell line. Inducible shRNA-mediated knockdown of CIN85 in wild type or Cbl/Cbl-b DKO MEFs had no impact on EGFR internalization. Our findings, establish that, at physiological expression levels, Cbl, Cbl-b and CIN85 are largely dispensable for EGFR internalization. Our results support the model that Cbl–CIN85–endophilin complex is not required for efficient internalization of EGFR, a prototype RTK.

Factors affecting transformation of Bacillus licheniformis.

Thorne, Curtis B. (Fort Detrick, Frederick, Md.), and Harold B. Stull. Factors affecting transformation of Bacillus licheniformis. J. Bacteriol. 91:1012-1020. 1966.-Transformation systems involving two types of transformable mutants of Bacillus licheniformis 9945A were compared. Each system required its specific growth medium, but a single transformation medium could be used for both. Cells from a culture of optimal age were not competent, at least to any great extent, but they developed competence during incubation in a transformation medium. With each system, 3 to 5% of the recipient cells were transformed upon exposure to wild-type deoxyribonucleic acid (DNA) for 2 to 3 hr. When competent cells were exposed to DNA for 30 min, 1 to 2% of them were transformed. The data are interpreted to mean that cells were heterogeneous with respect to development of competence, and when properly grown cells were incubated in transformation medium some of them gained competence, whereas others lost it. If DNA was present during the entire period, the cells were transformed as they became competent and the transformants accumulated. However, during any short period of exposure to DNA, only those cells that were competent at the time were potential transformants. The high frequencies of transformation obtained in these studies made it feasible to prepare marked strains by transforming markers into recipient cells. These experiments demonstrated that the characteristics of the two transformation systems could not be attributed to specific nutritional markers. Presumably, each of the two series of highly transformable auxotrophic mutants also carried at least one other mutation that resulted in development of competence under the specific conditions.

TRANSFORMING ACTIVITIES AND BASE COMPOSITION OF DEOXYRIBONUCLEATES FROM STRAINS OF MORAXELLA AND MIMA.

Fourteen strains of Moraxella and possibly allied organisms were studied: M. liquefaciens, M. bovis, M. nonliquefaciens, oxidase-positive and oxidasenegative strains of Mima polymorpha and Herellea vaginicola. Classical bacteriological characteristics, capacity to undergo genetic transformation (resistance to 500 μg. dihydrostreptomycin/ml.), and DNA base composition were examined. Moraxella nonliquefaciens and Mima polymorpha var. oxidans were transformable (2 strains at frequencies of 1-4%). Attempts to elicit transformation of the remaining organisms were unsuccessful. The ability of DNA preparations from M. polymorpha var. oxidans to elicit high frequencies of transformation of Moraxella nonliquefaciens, and vice versa, indicated their close relationship. Moreover, their DNA base contents were indistinguishable (about 44·4% guanine + cytosine, 6 strains). The G + C contents averaged 43·1% for 3 oxidase-negative Mima polymorpha strains and 39·8% for 2 Herellea vaginicola strains.

Transforming activities and base contents of deoxyribonucleate preparations from various Neisseriae.

SUMMARY: Genetic transformation was investigated among Neisseria spp. whose normal habitat is the nasopharynx of humans. Seven species, as characterized in Bergey's Manual (1957), were represented. Deoxyribonucleate (DNA) preparations from streptomycin-resistant mutants of N. meningitidis, N. perflava, N. flava, N. subflava, N. sicca, and N. flavescens conferred resistance upon streptomycin-susceptible parent strains of the corresponding species (intraspecific transformation) and of each other species (interspecific transformation). Ratios of interspecific to intraspecific transformation were 0.01 or higher for all possible combinations of DNA and recipient cells of the six species. On the other hand, N. catarrhalis cells, which exhibited high frequencies of intraspecific transformation, were not transformed at detectable frequencies by DNA from any of the six Neisseria species listed above. In turn, DNA from N. catarrhalis had little or no transforming activity for these other neisseriae. Possible evidence of structural differences between these DNA's was sought by analysing the base contents of transforming preparations. The bases adenine, thymine, guanine and cytosine were present in about equal proportions in the DNA's of the six Neisseria: meningitidis, perflava, flava, subflava, sicca and flavescens. In DNA preparations from two strains of N. catarrhalis, however, adenine and thymine predominated. The ratio (adenine + thymine/guanine + cytosine) was higher than 1.4 compared to 1.0 for the others.