A transformation system for the hypotrichous ciliate Stylonychia mytilus

We describe a transformation system for the ciliate Stylonychia mytilus. The neomycin resistance gene from Escherichia coli transposon Tn5, which codes for the enzyme phosphotransferase and confers resistance to the antibiotic G 418, was ligated into macronuclear `gene-size' DNA molecules. Using this recombinant DNA for transformation experiments we show that the gene is replicated and expressed in transformed cells.     

The structure and expression of maize genes encoding the major heat shock protein, hsp70

We have isolated and sequenced two maize genomic clones that are homologous to the Drosophila hsp70 gene. One of the maize hsp70 clones contains the entire hsp70 coding region and 81 nucleotides of the 5' nontranslated sequence. The predicted amino acid sequence for this maize protein is 68% homologous to the hsp70 of Drosophila. The second maize hsp70 clone contains only part of the coding sequence and 1.1 kb of the 5' flanking sequence. This 5' flanking sequence contains two sequences homologous to the consensus heat-shock-element sequence. Both maize genes are thermally inducible and each contains an intron in the same position as that of the heat-shock-cognate gene, hsc1, of Drosophila. The presence of an intron in the maize genes is a distinguishing feature in that no other thermally inducible hsp70 genes described to date contain an intron. We have constructed a hybrid hsp70 gene containing the entire hsp70 coding sequence with an intron, and 1.1 kb of the 5' flanking sequence. We demonstrate that this hybrid gene is thermally inducible in a transgenic petunia plant and that the gene is expressed from its own promoter.     

Gene inactivation in the plant pathogen Glomerella cingulata: three strategies for the disruption of the pectin lyase gene pnIA

The feasibility of performing routine transformation-mediated mutagenesis in Glomerella cingulata was analysed by adopting three one-step gene disruption strategies targeted at the pectin lyase gene pnIA. The efficiencies of disruption following transformation with gene replacement- or gene truncation-disruption vectors were compared. To effect replacement-disruption, G. cingulata was transformed with a vector carrying DNA from the pnlA locus in which the majority of the coding sequence had been replaced by the gene for hygromycin B resistance. Two of the five transformants investigated contained an inactivated pnlA gene (pnlA ^? );both also contained ectopically integrated vector sequences. The efficacy of gene disruption by transformation with two gene truncation-disruption vectors was also assessed. Both vectors carried a 5′and 3′truncated copy of the pnlA coding sequence, adjacent to the gene for hygromycin B resistance. The promoter sequences controlling the selectable marker differed in the two vectors. In one vector the homologous G. cingulata gpdA promoter controlled hygromycin B phosphotransferase expression (homologous truncation vector), whereas in the second vector promoter elements were from the Aspergillus nidulans gpdA gene (heterologous truncation vector). Following transformation with the homologous truncation vector, nine transformants were analysed by Southern hybridisation; no transformants contained a disrupted pnlA gene. Of nineteen heterologous truncation vector transformants, three contained a disrupted pnlA gene; Southern analysis revealed single integrations of vector sequence at pnlA in two of these transformants. pnlA mRNA was not detected by Northern hybridisation in pnlA-transformants. pnlA-transformants failed to produce a PNLA protein with a pI identical to one normally detected in wild-type isolates by silver and activity staining of isoelectric focussing gels. Pathogenesis on Capsicum and apple was unaffected by disruption of the pnlA gene, indicating that the corresponding gene product, PNLA, is not essential for pathogenicity. Gene disruption is a feasible method for selectively mutating defined loci in G. cingulata for functional analysis of the corresponding gene products.     

Reactivation of thyroglobulin gene expression in transformed thyroid cells by 5-azacytidine

Transformed rat thyroid cells fail to express thyroglobulin. Cells transformed with a Kirsten murine sarcoma virus carrying a temperature-sensitive ras allele lose their transformation phenotype when shifted to the nonpermissive (39 degrees C) temperature. The thyroglobulin promoter, however, remains inactive. Similarly, transfection of these cells with a thyroglobulin promoter fused to a neomycin resistance reporter gene does not produce clones resistant to G418. Treatment of the transfected cells with the DNA demethylating agent 5-azacytidine reactivates the thyroglobulin promoter and yields stable G418-resistant clones. We show that thyroglobulin promoter activity is correlated with the presence of a thyroid-specific nuclear factor, TgTF1. TgTF1 cannot be detected in transformed cells but reappears after treatment with 5-azacytidine at 39 degrees C. Restoration of Ras activity at 33 degrees C leads to the rapid loss of TgTF1 and G418 resistance.     

Efficient selection for high-expression transfectants with a novel eukaryotic vector.

We have developed a new expression vector which allows efficient selection for transfectants that express foreign genes at high levels. The vector is composed of a ubiquitously strong promoter based on the beta-actin promoter, a 69% subregion of the bovine papilloma virus genome, and a mutant neomycin phosphotransferase II-encoding gene driven by a weak promoter, which confers only marginal resistance to G418. Thus, high concentrations of G418 (approx. 800 micrograms/ml) effectively select for transfectants containing a high vector copy number (greater than 300). We tested this system by producing human interleukin-2 (IL-2) in L cells and Chinese hamster ovary (CHO) cells, and the results showed that high concentrations of G418 efficiently yielded L cell and CHO cell transfectants stably producing IL-2 at levels comparable with those previously attained using gene amplification. The vector sequences were found to have integrated into the host chromosome, and were stably maintained in the transfectants for several months.     

Expression of hIGF-I in the silk glands of transgenic silkworms and in transformed silkworm cells

To express human insulin-like growth factor-I (hIGF-I) in transformed Bombyx mori cultured cells and silk glands, the transgenic vector pigA3GFP-hIGF-ie-neo was constructed with a neomycin resistance gene driven by the baculovirus ie-1 promoter, and with the hIGF-I gene under the control of the silkworm sericin promoter Ser-1. The stably transformed BmN cells expressing hIGF-I were selected by using the antibiotic G418 at a final concentration of 700—800 μg/mL after the BmN cells were transfected with the piggyBac vector and the helper plasmid. The specific band of hIGF-I was detected in the transformed cells by Western blot. The expression level of hIGF-I, determined by ELISA, was about 7800 pg in 5×10⁵ cells. Analysis of the chromosomal insertion sites by inverse PCR showed that exogenous DNA could be inserted into the cell genome randomly or at TTAA target sequence specifically for piggyBac element transposition. The transgenic vector pigA3GFP-hIGF-ie-neo was transferred into the eggs using sperm-mediated gene transfer. Finally, two transgenic silkworms were obtained after screening for the neo and gfp genes and verified by PCR and dot hybridization. The expression level of hIGF-I determined by ELISA was about 2440 pg/g of silk gland of the transgenic silkworms of the G1 generation.     

Evaluation of selectable markers for rice transformation

A variety of expression systems and selection régimes have been developed to transform plants such as tobacco, petunia, and tomato. We investigated several of these to determine whether the promoters and selectable markers used in dicotyledonous plants are suitable for selecting transformed rice callus. We compared transient expression driven by constitutive and regulated promoters in rice (Oryza sativa) protoplasts and found that the 2′ promoter of the octopine T-DNA is approximately 3 to 4 times more efficient than the CAMV 35S promoter, 10 times more efficient than the nos promoter and the 1′ promoter, and more than 100 times better than two other regulated plant promoters. Similar results were obtained in tobacco (Nicotiana tabacum) protoplasts with the exception that the nos promoter was expressed nearly 10 times better in rice. Further studies demonstrated that rice callus growth is sensitive to low concentrations of methotrexate, phosphinothricin, and bleomycin, and to moderate concentrations of G418 and hygromycin, but is only partially inhibited by relatively high concentrations of kanamycin. Finally, we tested the ability of stably introduced resistance genes to protect callus against some of the selective agents. Genes that inactivated phosphinothricin or G418 permitted transformed calli to grow almost unimpeded on toxic concentrations of these selective agents. However, a gene conferring resistance to methotrexate could not be used to select for activily growing transformants. Southern analysis of the transformed cell lines demonstrated that 50% of the transformants contained a single plasmid copy and that nearly all integrated copies showed rearrangements. These results on the use of selectable markers in rice should facilitate efforts to obtain transformants of this important grain.     

Efficient transformation of Dictyostelium discoideum amoebae.

We have transformed Dictyostelium discoideum amoebae by using derivatives of a plasmid, pAG60, which was designed for transformation of mammalian cells. The plasmid carries the promoter region of the herpes simplex virus type 1 thymidine kinase gene linked to the bacterial gene kan, which codes for the enzyme aminoglycoside 3'-phosphotransferase. kan is derived from the Tn5 transposon. Expression of the phosphotransferase permits direct selection of transformed cells by their resistance to the antibiotic G-418. pAG60 is incapable of transforming D. discoideum but is made transformation proficient by cloning D. discoideum sequences into the tetracycline resistance gene. The majority of transformed cells grow and develop normally and differentiate to give G-418-resistant spores. These transformants are unstable and rapidly lose their G-418-resistance during growth in the absence of antibiotic selection. Southern blots show that these unstable G-418-resistant transformants carry the pBR322 and kan sequences of pAG60. The pAG60-D. discoideum recombinant plasmids used for transformation were constructed in a way that might make them mutagenic. We have isolated several developmental mutants after transformation of D. discoideum with libraries of pAG60-D. discoideum recombinant plasmids. These mutants are G-418 resistant and carry pAG60 in their nuclear DNA. We recovered a pAG60-D. discoideum recombinant plasmid from several developmental mutants. This plasmid transforms D. discoideum at an elevated frequency and integrates into the nuclear genome. We speculate that integration can result in insertional inactivation of genes that are essential for differentiation but not for growth. Mutagenic transformation occurred only if the transforming plasmid had homology with D. discoideum nuclear DNA. A mammalian cell transformation vector, pSV2-neo, carried no D. discoideum sequences and was able to transform. However, pSV2-neo transformation was not mutagenic. These results suggest that direct inactivation and recovery of genes that are essential for differentiation of D. discoideum will be possible.     

Efficient germ-line transformation of the economically important pest species Lucilia cuprina and Lucilia sericata (Diptera, Calliphoridae)

The green blowfly species Lucilia cuprina and Lucilia sericata are economically important pests for the sheep industries of Australia and New Zealand. L. cuprina has long been considered a good target for a genetic pest management program. In addition, L. sericata maggots are used in the cleaning of wounds and necrotic tissue of patients suffering from ulcers that are difficult to treat by other methods. Development of efficient transgenesis methods would greatly facilitate the development of strains ideal for genetic control programs or could potentially improve “maggot therapy”. We have previously reported the germ-line transformation of L. cuprina and the design of a “female killing system” that could potentially be applied to this species. However, the efficiency of transformation obtained was low and transformed lines were difficult to detect due to the low expression of the EGFP marker used. Here we describe an efficient and reliable method for germ-line transformation of L. cuprina using new piggyBac vector and helper plasmids containing the strong promoter from the L. cuprina hsp83 gene to drive expression of the transposase and fluorescent protein marker gene. We also report, for the first time, the germ-line transformation of L. sericata using the new piggyBac vector/helper combination.     

Pleiotropic mutants of NIH 3T3 cells with altered regulation in the expression of both type I collagen and fibronectin

Transformation of NIH 3T3 fibroblasts by v-mos causes a decrease in the levels of type I collagen RNA. In NIH 3T3 cells that have been made resistant to G418 by transfection with a plasmid in which the mouse alpha 2(I) collagen promoter is linked to the neo gene, subsequent v-mos transformation causes a loss of G418 resistance. After mutagenesis of these v-mos-transformed cells, G418-resistant colonies were selected. Two of these G418-resistant mutants showed an increased expression of the neo gene and of the endogenous type I collagen and fibronectin genes, without changes in their levels of v-mos RNA or in their ability to induce tumors. The mutations might alter cellular trans-acting factors that either directly or indirectly control the expression of the type I collagen and fibronectin genes in transformed cells.     

Transient expression of a Drosophila melanogaster hsp 70 promoter/lacZ construct injected into larvae of two species of predatory mites (Acari: Phytoseiidae)

The Drosophila melanogaster heat shock 70 promoter (hsp70) was used to regulate expression of the Escherichia coli -galactosidase gene (lacZ) in transiently-transformed predatory mite larvae. A construct containing the hsp70 promoter upstream of the D. melanogaster alcohol dehydrogenase (adh) translational start site and Escherichia coli lacZ gene fusion (adh/lacZ) was injected into larvae of Metaseiulus occidentalis and Amblyseius finlandicus. LacZ expression was compared to expression of a similar construct lacking any upstream regulatory sequence. Expression from the hsp70 promoter was strong and heat shock-dependent in both species. The Drosophila hsp70 promoter therefore appears useful for regulating expression of exogenous DNA in both phytoseiid species and may be broadly applicable in the Phytoseiidae. Furthermore, the lacZ gene is a useful gene for analysis of expression in both species. Larval microinjection provides a method of assessing transient expression and of examining native regulatory sequences in these two phytoseiids and will likely be useful in other phytoseiid mites with only minor modifications.     

A shuttle vector which facilitates the expression of transfected genes in Trypanosoma cruzi and Leishmania.

A Trypanosoma cruzi expression vector has been constructed using sequences derived from the flanking regions of the glyceraldehyde 3-phosphate dehydrogenase (gGAPDH) genes. The neomycin phosphotransferase (neor) gene was incorporated as a selectable marker. Using electroporation we have introduced this vector into both T. cruzi and Leishmania cells and conferred G418 resistance. Transformation is mediated by large extrachromosomal circular elements composed of head-to-tail tandem repeats of the vector. The transformed phenotype is stable for at least 6 months in the absence of G418 and can be maintained during passage through the T. cruzi life-cycle. Foreign genes inserted into an expression site within the vector (pTEX) can be expressed at high levels in transformed cells. To our knowledge this paper describes the first trypanosome shuttle vector and the first vector which functions in both trypanosomes and Leishmania.