Surrogate transformation of perennial ryegrass, Lolium perenne, using genetically modified Acremonium endophyte.

Summary Conditions have been developed for transforming protoplasts of the perennial ryegrass endophyteAcremonium strain 187BB. Unlike most other ryegrass endophytes, this strain does not produce the lolitrem B neurotoxin and is therefore suitable as a host for surrogate introduction of foreign genes into grasses. Transformation frequencies of 700–800 transformants/g DNA were obtained for both linear and circular forms of pAN7-1, a hygromycin (hph) resistant plasmid. Up to 80% of the linear transformants were stable on further culturing but only 25% of the circular transformants retained hygromycin resistance. Integration of pAN7-1 into the genome was confirmed by Southern blotting and probing of genomic digests of transformant DNA. Both single and tandemly repeated copies of the plasmid were found in the genome and both the number and sites of integration varied among the transformants. At least 13 chromosomes were identified in 187BB using contour-clamped homogeneous electric field (CHEF) gel electrophoresis. Probing of Southern blots of these gels confirmed that pAN7-1 had integrated into different chromosomes. The -glucuronidase (GUS) gene,uidA, was also introduced into 187BB by co-transformation of pNOM-2 with pAN7-1. GUS activity was detected by growing the transformants on plates containing 5-bromo-4-chloro-3-indolyl -D-glucuronic acid and by enzyme assays of mycelial extracts. Severalhph- anduidA-containing transformants were reintroduced into ryegrass seedlings and expression of GUS visualized in vivo, demonstrating that 187BB can be used as a surrogate host to introduce foreign genes into perennial ryegrass. Molecular analysis of fungal isolates from the leaf sheath confirmed that the pattern of pAN7-1 and pNOM-2 hybridizing fragments was identical to that observed in the fungus used as inoculum.     

A comprehensive characterization of single-copy T-DNA insertions in the Arabidopsis thaliana genome

T-DNA flanking sequences were isolated from 112 Arabidopsis thaliana single-copy T-DNA lines and sequence mapped to the chromosomes. Even though two T-DNA insertions mapped to a heterochromatic domain located in the pericentromeric region of chromosome I, expression of reporter genes was detected in these transgenic lines. T-DNA insertion did not seem to be biased toward any of Arabidopsis' five chromosomes. The observed distribution of T-DNA copies in intergenic sequence versus gene sequence (i.e. 5-upstream regions, coding sequences and 3-downstream regions) appeared randomly. An evaluation of T-DNA insertion frequencies within gene sequence revealed that integration into 5-upstream regions occurred more frequently than expected, whereas insertions in coding sequences (exons and introns) were found less frequently than expected based on random distribution predictions. In the majority of cases, single-copy T-DNA insertions were associated with small or large rearrangements such as deletions and/or duplications of target site sequences, deletions and/or duplications of T-DNA sequences, and gross chromosomal rearrangements such as translocations. The accuracy of integration was similarly high for both left- and right-border sequences. These results may be called upon when making detailed molecular analyses of transgenic plants or T-DNA induced mutants.     

Enhancement of α-amylase production by integrating and amplifying the α-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis

Summary The -amylase gene of Bacillus amyloliquefaciens was integrated into the genome of Bacillus subtilis by homologous recombination. In the first transformation step, several strains were obtained carrying the -amylase gene as two randomly located copies. These strains produced -amylase in the quantities comparable with that of the multicopy plasmid pKTH10, carrying the same -amylase gene. With the plasmid system, however, the rate of the -amylase synthesis was faster and the production phase shorter than those of the chromosomally encoded -amylase. The two chromosomal gene copies were further multiplied either by amplification using increasing antibiotic concentration as the selective pressure or by performing a second transformation step, identical to the first integration procedure. Both methods resulted in integration strains carrying up to eight -amylase gene copies per one genome and producing up to eightfold higher -amylase activity than the parental strains. Six out of seven transformants, studied in more detail, were stable after growth of 42 h even without antibiotic selection. The number of the DNA and mRNA copies of the -amylase gene was quantitavely determined by sandwich hybridization techniques, directly from culture medium.     

Development of a genetic transformation system for Histoplasma capsulatum: complementation of uracil auxotrophy

Summary We have developed a simple and efficient transformation system for the dimorphic fungus Histoplasma capsulatum. Mutants of H. capsulatum defective in orotidine-5-monophosphate pyrophosphorylase were transformed to prototrophy by the cloned URA5 gene of the filamentous fungus Podospora anserina. Abortive and mitotically stable transformants were obtained. The stable transformants had integrated copies of the plasmid, some in tandem head-to-tail orientation. Free plasmid identical to the transforming plasmid was present in some of the transformants. We obtained a transformation efficiency of up to 30 transformants/g DNA for plasmid pPAura5-1 (9.2 kb). pPW2001, a smaller plasmid (4.7 kb) derived from pPAura5-1, transformed H. capsulatum more efficiently (up to 155 transformants/gm DNA).     

Development of a homologous transformation system for Aspergillus niger based on the pyrG gene

Summary The development of a homologous transformation system for Aspergillus niger is described. The system is based on the use of an orotidine-5-phosphate decarboxylase deficient mutant (pyrG) and a vector, pAB4-1, which contains the functional A. niger pyrG gene as a selection marker. Transformation of the A. niger pyrG mutant with pAB4-1 resulted in the appearance of stable Pyr⁺ transformants at a frequency of 40 transformants per g of DNA. In 90% of these transformants integration had occurred at the resident pyrG locus, resulting either in replacement of the mutant allele by the wild-type allele (60%) or in insertion of one or two copies of the vector (40%). The A. niger pyrG mutant could also be transformed with the vector pDJB2 containing the pyr4 gene of Neurospora crassa, at a frequency of 2 transformants per g of DNA. Integration at the resident pyrG locus was not found with this vector. The vector pAB4-1 is also capable of transforming an Aspergillus nidulans pyrG mutant to Pyr⁺. The pyrG transformation system was used for the introduction of a non-selectable gene into A. niger.     

Targeted integrative transformation of Candida tropicalis by electroporation

Summary A method for integrative transformation of the diploid yeast Candida tropicalis by electroporation has been developed. By linearizing the transforming plasmid DNA containing the URA3 gene prior to electroporation of recipient cells, its integration was targeted to a specific locus in the genome, resulting in single or multiple tandem integrations. The optimal electroporation conditions for this yeast were established and include an electric pulse of 2.25 kV/cm for a duration of 50 ms. Using these conditions, Ura⁺ transformants were readily obtained at a high frequency (45 transformants/g DNA) as the result of targeted integration of the URA3 gene containing plasmid DNA at the chromosomal ura3 locus. The number of transformants resulting from this procedure is comparable to that achieved with a recently reported spheroplast transformation procedure for C. tropicalis; in addition, it offers the advantages of being simple, rapid and reproducible.     

High-frequency transformation ofLactobacillus casei with plasmid pHY300PLK by electroporation

To optimize the conditions for transformation ofLactobacillus casei ATCC 27092 cells with plasmid pHY300PLK, a shuttle vector forEscherichia coli andBacillus subtilis, by electroporation, we investigated the effects of the electrical parameters (voltage and resistance), the concentration of plasmid DNA, the cell age and density, the electroporation buffer, and other factors. Under optimal conditions of 2.0 kV, 100 ohm, and 25F, a transformation efficiency as high as 1.4×10⁷ transformants per g of plasmid DNA was obtained, with a survival rate of about 50%.L. casei YIT 9021, one of the PL-1 phage mutants of the ATCC 27092 strain, was also transformed with the same plasmid under optimal conditions. The transformants were confirmed to harbor the same intact plasmid molecules by agarose gel electrophoretic analysis.     

Relationship of vector insert size to homologous integration during transformation of Neurospora crassa with the cloned am (GDH) gene

Summary We used lambda and plasmid vectors containing the am ⁺ gene in an insert of from 2.7 to 9.1 kb, to transform am point mutant and deletion strains. A total of 199 transformants were examined with the potential to yield am ^– transformants by homologous recombination. When we used vectors that had 9.1 kb of homology with the chromosomal DNA, 30% of the transformants obtained were the result of homologous recombination regardless of whether the vector was a lambda molecule, a circular plasmid, or a plasmid that had been linearized prior to transformation. When vectors with up to 5.1 kb of homology were used, very few transformants (1 of 89 tested) resulted from homologous recombination. Of a sample of 29 ectopic integration events obtained by transformation with the 9.1 kb fragment cloned in a vector, 18 included a major part (usually almost all) of both arms of lambda with the entire Neurospora 9.1 kb insert between them. Four included only long arm sequence together with an adjacent segment of the insert containing the am gene. The remaining seven were the result of multiple integrations. There was no evidence of circularization of the vector prior to integration. All transformants that had multiple copies of the am gene appeared to be subject to the RIP process, which causes multiple mutations in duplicated sequences during the sexual cycle.     

Stable transfer and expression of chimeric genes in licorice (Glycyrrhiza uralensis) using an Ri plasmid binary vector

The pharmaceutically important plant, licorice (Glycyrrhiza uralenesis Fisher), was transformed with a binary vector system of an Ri plasmid, pRi15834, and a mini Ti vector, pGSGluc1, containing chimeric neo and gus genes. The transgenic state of transformed roots was confirmed by detection of agropine and mannopine and by Southern blot hybridization with T-DNA of pGSGluc1. One to three copies of T-DNA of pGSGluc1 was integrated into the genomic DNA of G. uralensis. The expression of chimeric neo and gus genes driven by TR 1 and 2 promoters, respectively, was demonstrated by enzymatic assays. Histochemical analysis showed that the chimeric TR2-gus gene was expressed specifically in phloem and pericycle tissues of the transformed licorice roots.Abbreviations NPT-II neomycin phosphotransferase II - neo NPT-II gene from Tn5 - GUS ß-glucuronidase - gus GUS gene from Escherichia coli - TR 1–2 genes 1 and 2 of TR-DNA of pTiAch5 - Rif rifampicin     

Agrobacterium-mediated transformation of Asparagus officinalis L.: molecular and genetic analysis of transgenic plants

Four long-term embryogenic lines of Asparagus officinalis were co-cultured with the hypervirulent Agrobacterium tumefaciens strain AGL1Gin carrying a uidA gene and an nptII gene. 233 embryogenic lines showing kanamycin resistance and -glucuronidase (GUS) activity were obtained. Transformation frequencies ranged from 0.8 to 12.8 transformants per gram of inoculated somatic embryos, depending on the line. Southern analysis showed that usually 1 to 4 T-DNA copies were integrated. Regenerated plants generally exhibited the same insertion pattern as the corresponding transformed embryogenic line. T₁ progeny were obtained from crosses between 6 transformed plants containing 3 or 4 T-DNA copies and untransformed plants. They were analysed for GUS activity and kanamycin resistance. In three progenies, Mendelian 1:1 segregations were observed, corresponding to one functional locus in the parent transgenic plants. Southern analysis confirmed that T-DNA copies were inserted at the same locus. Non-Mendelian segregations were observed in the other three progenies. T₂ progeny also exhibited non-Mendelian segregations. Southern analysis showed that GUS-negative and kanamycin-sensitive plants did not contain any T-DNA, and therefore inactivation of transgene expression could not be responsible for the abnormal segregations.     

Transformation of the fungal maize pathogen Cochliobolus heterostrophus using the Aspergillus nidulans amdS gene

Summary Cochliobolus heterostrophus protoplasts transformed with a plasmid carrying the Aspergillus nidulans amdS gene (Hynes et al. 1983) gave rise to colonies on a selective medium that did not support significant growth of wild type cells. The plasmid integrated at a single chromosomal locus in each transformant analyzed and the site of integration differed among transformants. Some transformants had one copy of the plasmid, others had multiple copies tandemly arranged and oriented head-to-tail. Both single and multiple copies segregated meiotically as single genes and were mitotically stable on either selective or nonselective medium. The andS gene is advantageous for transformation of genetically undeveloped fungi because it is selectable in wild type cells in organisms that lack a functional amdS gene, thus eliminating the need for induced mutations in recipient strains. Moreover, there is no background due to reversion of a counter-selected mutant allele.     

A suitable method for construction and cloning hybrid plasmids containing EcoRI-fragments of E. coli genome.

Summary A convenient procedure for the isolation of specificEcoRI-fragments ofE. coli genome and their amplification on Km-resistance plasmid vector CK 11 is described. The hybrid molecules were constructedin vitro usingEcoRI-digestion, followed by ligation. Then appropriatedE. coli strain was transformed with ligated DNA mixture and hybrid plasmids CK 11-arg ⁺, CK 11-his ⁺, CK 11-thr ⁺ and CK 11-leu ⁺ containing loci ofE. coli genome were selected by molecular cloning. The hybrid plasmids obtained consisted of oneEcoRI-fragment of initial plasmid CK 11 and one respective specific portion ofE. coli genome.     

Integration of exogenous DNA into the genome of Azotobacter vinelandii

The soil bacterium Azotobacter vinelandii was genetically transformed by chromosomal integration to ampicillin and/or tetracycline resistance using restriction endonuclease-linearized plasmids. Polyacrylamide gel electrophoresis of protein extracts from three independently isolated ampicillin resistant transformants showed the presence of a 28 Kd band which is the approximate size of the ampicillin resistance gene product (i.e., -lactamase). Moreover, with nitrocefin, a chromogenic cephalosporin, as a substrate, it was shown that all of the ampicillin resistant transformants produced functional -lactamase. DNA hybridization showed that the chromosomal DNA from transformed cells contained plasmid DNA sequences at discrete sites. Growth experiments indicated that stable A. vinelandii transformants that carry functional integrated DNA were physiologically impaired.     

Rapid sequence divergence in a heat shock locus of Drosophila

In situ hybridization of cRNA transcribed from cloned D. melanogaster heat shock sequences to D. hydei chromosomes has shown that the D. hydei locus 2–32 A corresponds to the D. melanogaster locus 87 A/C and the D. hydei locus 2–36 A to the D. melanogaster locus 95 D, while the D. hydei locus 4–81 B corresponds to the D. melanogaster locus 63 BC. No hybridization to D. hydei chromosomes was found with cRNA transcribed from a clone containing the sequences encoded by the D. melanogaster locus 87 C. Neither D. melanogaster heat shock RNA nor D. virilis heat shock RNA hybridized significantly to the D. hydei heat shock locus 2–48 B. Furthermore, D. hydei heat shock RNA did not hybridize to the cytological homologs of locus 2–48 B found in D. repleta or in D. virilis. D, hydei heat shock. RNA did hybridize to the cytological homologs of locus 2–48 B in D. neohydei and D. eohydei, both of which belong to the hydei subgroup.     

Protoplast culture and plant regeneration in Lycium barbarum L.

Protoplasts were isolated from leaves of the woody plant Lycium barbarum L. and cultured in liquid nutrient medium TM-2 at a density of 10⁴–10⁵ cells ml^-1. After ten days of culture, regenerated colonies were transferred to the agar-solidified medium TM-3, and 5–7 days later to regeneration media PRM or TM-4. Formation of shoots was observed after 30–40 days. Completely formed and rooted plants were transferred to the soil. Cytological and morphological analysis of the regenerated plants revealed relative genetic stability of this species in the process protoplast — plant. The results obtained allow us to conclude that L. barbarum can be used in the experiments on somatic hybridization or direct gene transfer.     

Introduction and maintenance of prokaryotic DNA inUstilago violacea

Summary A strain of the basidiomycete,Ustilago violacea, was transformed with a prokaryotic plasmid, pMP4-1, which confers resistance to neomycin.U. violacea transformants were selected at a frequency of 5 per g pMP4-1 DNA. Such transformants were at least 8-fold more resistant to neomycin than was the untransformed recipientU. violacea. Enzyme activity associated with the neomycin resistance gene was also found in the transformants. Southern DNA-DNA hybridization detected pMP4-1-derived sequences in both nuclear and mitochondrially-associated DNAs from transformants. The patterns of hybridization suggested integration of pMP4-1 sequences into the respective genomes. DNA from the nuclear fraction ofU. violacea transformants failed to produceE. coli transformants resistant to neomycin or to carbenicillin. In contrast, DNA from the mitochondrially-associated fraction inU. violacea transformants producedE. coli transformants resistant to neomycin. TheE. coli transformants contained a pMP4-1-derivative, pWP8, which was subsequently shown by Southern blot analysis to harborU. violacea mitochondrial DNA. Thus, a prokaryotic plasmid can be used to transform the eukaryoteU. violacea and acquire endogenous sequences from this organism.     

Cloning and expression of Candida albicans ADE2 and proteinase genes on a replicative plasmid in C. albicans and in Saccharomyces cerevisiae.

Summary A plasmid vector (denoted pRC2312) was constructed, which replicates autonomously in Escherichia coli, Saccharomyces cerevisiae and Candida albicans. It contains LEU2, URA3 and an autonomously replicating sequence (ARS) from C. albicans for selection and replication in yeasts, and bla (ampicillin resistance) and ori for selection and replication in E. coli. S. cerevisiae AH22 (Leu^–) was transformed by pRC2312 to Leu^– at a frequency of 1.41 × 10⁵ colonies per g DNA. Transformation of C. albicans SGY-243 (Ura-) to Ura⁺ with pRC2312 resulted in smaller transformant colonies at a frequency of 5.42 × 10³ per g DNA where the plasmid replicated autonomously in transformed cells, and larger transformant colonies at a frequency of 32 per g DNA, in which plasmid integrated into the genome. Plasmid copy number in yeasts was determined by a DNA hybridization method and was estimated to be 15±3 per haploid genome in S. cerevisiae and 2–3 per genome in C. albicans replicative transformants. Multiple tandem integration occurred in integrative transformants and copy number of the integrated sequence was estimated to be 7–12 per diploid genome. The C. albicans ADE2 gene was ligated into plasmid pRC2312 and the construct transformed Ade^– strains of both C. albicans and S. cerevisiae to Ade⁺. The vector pRC2312 was also used to clone a fragment of C. albicans genomic DNA containing an aspartic proteinase gene. C. albicans transformants harboring this plasmid showed a two-fold increase in aspartic proteinase activity. However S. cerevisiae transformants showed no such increase in proteinase activity, suggesting the gene was not expressed in S. cerevisiae.     

Efficient Transformation of the Astaxanthin-Producing Yeast Phaffia Rhodozyma

An efficient transformation system for the astaxanthin-producing yeast Phaffia rhodozyma was developed based on electroporation that routinely yields approximately 1000 transformants per g of plasmid DNA. The high transformation efficiency depends on vector integration in the ribosomal DNA (rDNA) and the presence of the homologous glycolytic glyceraldehyde-3-phosphate dehydrogenase (gpd) promoter and terminator to drive the expression of the transposon Tn5 encoded kanamycin resistance gene (Km^R) as a selective marker. Using this system stable transformants were obtained, carrying multiple plasmid copies. Plasmid copy number could be markedly increased by deletion of the gpd terminator from the transforming plasmid.     

Electrotransformation ofStreptococcus sanguis Challis

Plasmid DNA was introduced into noncompetent cells ofStreptococcus sanguis Challis by an electrotransformation technique. The procedure was simple and rapid, did not require elaborate pretreatment of cells, and yielded transformant colonies in 24 h. The maximum transformation efficiency attained was 2.1×10⁴ transformants per g of pVA736. Molecular rearrangements and deletions were not detected in plasmid DNA isolated from transformants.     

The biotin-dependent sodium ion pump glutaconyl-CoA decarboxylase from Fusobacterium nucleatum (subsp. nucleatum)

Membrane preparations of Fusobacterium nucleatum grown on glutamate contain glutaconyl-CoA decarboxylase at a high specific activity (13.8 nkat/mg protein). The enzyme was solubilized with 2% Triton X-100 in 0.5M NaCl and purified 63-fold to a specific activity of 870 nkat/mg by affinity chromatography on monomeric avidin-Sepharose. The activity of the decarboxylase was strictly dependent on Na⁺ (K _m=3 mM) and was stimulated up to 3-fold by phospholipids. The glutaconyl-CoA decarboxylases from the gram-positive bacteria Acidaminococcus fermentans and Clostridium symbiosum have a lower apparent K _m for Na⁺ (1 mM) and were not stimulated by phospholipids. In addition only the fusobacterial decarboxylase required sodium ion for stability and was inactivated by potassium ion. By incorporation of this purified enzyme into phospholipids an electrogenic sodium ion pump was reconstituted. The enzyme consists of four subunits, (m=65 kDa), (33 kDa), (19 kDa), and (16 kDa) with the functions of a carboxy transferase (), a carboxy lyase ( and probably ) and a biotin carrier (). The subunits are very similar to those of the glutaconyl-CoA decarboxylases from the gram-positive bacteria. With an antiserum directed against the decarboxylase from A. fermentans the - and the biotin containing subunits of the three decarboxylases and that from Peptostreptoccus asaccharolyticus could be detected on Western blots.