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PubMed1997

Electrophoretic karyotype of clinical isolates of Paracoccidioides brasiliensis.

Chromosomal DNA molecules were obtained from eight clinical isolates of Paracoccidioides brasiliensis during the early staged of the yeast to mycelial transformation process and were separated by contour-clamped homogeneous electric field gel electrophoresis. In seven of the isolates, it was possible to separate five bands, albeit with two different migration patterns (A and B). In the remaining isolate only four bands were observed (pattern C). The isolated chromosomes showed molecular sizes ranging from 3.2 to 10 Mb according to Schizosaccharomyces pombe molecular size standards. The three different patterns observed had bands of 3.2, 3.8, 4.1, 5.2, 6.7, 7.2, 8.8, and 10 Mb. These data imply that at least 30 Mb of the genome are organized as chromosomal macromolecules. Due to the fact that P. brasiliensis spheroplasts are difficult to obtain, technical details are provided.

Chromosomes, Fungal↗
PubMed1997

Complementation analysis of carotenogenic mutants of Mucor circinelloides.

Mutants of the filamentous fungus Mucor circinelloides altered in the synthesis of beta-carotene (genotype car) have been isolated by direct inspection of the color shown by the colonies derived from mutagenized spores. The mutants were analyzed for the carotenoid content in darkness and light and studied with respect to complementation in heterokaryons made by spheroplast fusion. The results revealed the existence of at least four complementation groups. The carB mutants are white and accumulate phytoene. The carR mutants are red and accumulate lycopene. The carP mutants are most likely disturbed in the synthesis of phytoene: two of them are white and do not accumulate carotenoids; another carP mutant is yellowish, probably because it is leaky. There are three yellowish mutants which belong to one or more complementation groups different from carB, carR, and carP. Two white mutants obtained in a single mutagenization step failed to complement with the carR and the carP mutants. The wild-type and the carB and carR mutants tested for M. circinelloides showed similar photoinduction.

Genetic Complementation Test↗
PubMed1993

Extrachromosomal maintenance and amplification of yeast artificial chromosome DNA in mouse cells.

Linear and circular forms of a 660-kb yeast artificial chromosome (YAC) containing the human hypoxanthine phosphoribosyltransferase gene were introduced into mouse L A-9 cells by fusion with yeast spheroplasts, and in situ hybridization was used to determine the location and fate of the yeast- and YAC-derived DNA in 25 fusion cell lines. Human and yeast DNAs were observed as extrachromosomal DNA molecules, present at least 27 cell divisions after fusion, in half the cell lines. The extrachromosomal molecules replicate extrachromosomally but segregate poorly like plasmids that contain an autonomously replicating sequence element in yeast. This system may allow analysis of DNA requirements for replication and segregation in mammalian cells. The extrachromosomal elements resemble double minutes (DMs), and in five cell lines human and yeast DNAs were present as very large integrated blocks of DNA resembling homogeneously staining regions (HSRs). Thus, the fusion cell lines contain the two characteristic forms of amplified DNA observed in cancer cell lines, DMs and HSRs, indicating that these can be formed from fragments of DNA introduced by cell fusion.

Animals↗
PubMed2001

Large-insert BAC/YAC libraries for selective re-isolation of genomic regions by homologous recombination in yeast.

We constructed representative large-insert bacterial artificial chromosome (BAC) libraries of two human pathogens (Trypanosoma brucei and Giardia lamblia) using a new hybrid vector, pTARBAC1, containing a yeast artificial chromosome (YAC) cassette (a yeast selectable marker and a centromere). The cassette allows transferring of BACs into yeast for their further modification. Furthermore, the new hybrid vector provides the opportunity to re-isolate each DNA insert without construction of a new library of random clones. Digestion of a BAC DNA by an endonuclease that has no recognition site in the vector, but which deletes most of the internal insert sequence and leaves the unique flanking sequences, converts a BAC into a TAR vector, thus allowing direct gene isolation. Cotransformation of a TAR vector and genomic DNA into yeast spheroplasts, and subsequent recombination between the TAR vector's flanking ends and a specific genomic fragment, allows rescue of the fragment as a circular YAC/BAC molecule. Here we prove a new cloning strategy by re-isolation of randomly chosen genomic fragments of different size from T. brucei cloned in BACs. We conclude that genomic regions of unicellular eukaryotes can be easily re-isolated using this technique, which provides an opportunity to study evolution of these genomes and the role of genome instability in pathogenicity.

Animals↗
PubMed1999

Integration of the colicin A pore-forming domain into the cytoplasmic membrane of Escherichia coli.

The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherichia coli and apparently formed a functional channel, when generated in vivo. We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments. Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins. We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm. We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9. These two domains function independently. Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts. In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening.

Alkaline Phosphatase↗
PubMed1993

Expression and translocation of the chlamydial major outer membrane protein in Escherichia coli.

The entire gene encoding the major outer membrane protein (MOMP) from Chlamydia psittaci strain GPIC has been cloned and expressed in Escherichia coli. A tightly regulated T7 promoter is used to control expression of the protein in Escherichia coli. Upon induction of expression, the precursor (pre-MOMP) is synthesized in the cell. This is followed by the appearance of a lower molecular weight protein that comigrates with mature MOMP from chlamydial elementary bodies by both one-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis. When E. coli cells expressing MOMP are converted to spheroplasts and subjected to protease treatment, MOMP is quantitatively degraded while cytoplasmic pre-MOMP is protected from degradation. Whole cells subjected to the same protease treatment show no degradation of MOMP. Furthermore, MOMP is not detected in surface-labeling experiments using several MOMP-specific antibodies. These data indicate that pre-MOMP is translocated to the periplasmic space and processed but is not surface exposed in E. coli. Expression of MOMP in this system causes a significant reduction in cell viability. In addition, coexpression in E. coli of MOMP or a MOMP-PhoA fusion with various chaperone proteins does not alter the level of MOMP translocation.

Amino Acid Sequence↗
PubMed1997

An in vitro system for the investigation of heterologous RNA recombination.

Bacteriophage Phi6 has a genome of three segments of double-stranded RNA enclosed in a polyhedral procapsid. Purified procapsids are capable of the specific packaging of viral plus strands and the synthesis of their complementary minus strands. The genomic segments of Phi6 are capable of heterologous recombination. We have prepared an in vitro system containing purified procapsids that is capable of packaging plus strands of the genomic segments and synthesizing minus strands on these templates. The system generates heterologous recombination products when stimulated by having one of the plus strands incapable of serving as a template for minus strand synthesis. Recombinants were produced upon transfection of spheroplasts with the in vitro packaged and replicated RNA. Sites of recombination were not found to be localized in particular regions of either the donor or the recipient strands.

Bacteriophage phi 6↗
PubMed1994

Periplasmic aggregation limits the proteolytic maturation of the Escherichia coli penicillin G amidase precursor polypeptide.

The Escherichia coli penicillin G amidase (PGA), which is a key enzyme in the production of penicillin G derivatives is generated from a precursor polypeptide by an unusual internal maturation process. We observed the accumulation of the PGA precursor polypeptide in the insoluble material recovered after sonication of recombinant E. coli JM109 cells grown at 26 degrees C. The aggregated nature of the accumulated molecules was demonstrated using detergents and chaotropic agents in solubilization assays. The periplasmic location of the aggregates was shown by trypsin-accessibility experiments performed on the spheroplast fraction. Finally, we showed that addition of sucrose or glycerol in the medium strongly reduces this periplasmic aggregation and as a consequence PGA production is substantially increased. Thus, periplasmic aggregation of the PGA precursor polypeptide limits PGA production by recombinant E. coli and this limitation can be overcome by addition in the medium of a non-metabolizable sugar, such as sucrose, or of glycerol.

Biotechnology↗
PubMed1992

Targeted integrative transformation of Candida tropicalis by electroporation.

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/micrograms 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.

Candida↗
PubMed1978

Kinetic characterization of plasma membrane ATPase from Saccharomyces cerevisiae.

1. Plasma membrane preparations have been isolated from spheroplasts of Saccharomyces cerevisiae, strain R XII, via lysis and subsequent differential centrifugation. These preparations are almost devoid of mitochondrial contamination. 2. The plasma membrane ATPase is fairly stable when refrigerated, but loses activity at 8 degrees C and above. Below pH 5.6 the ATPase is irreversibly inactivated. The enzyme also splits GTP and ITP, although to a lesser extent. 3. Mg2+-ions are essential as part of the reactive substrate, MgATP, and furthermore they activate the ATPase. Optimal conditions depend on substrate concentration. When the concentration of free Mg2+ ions exceeds about 0.1 mM, competitive inhibition occurs. 4. In the range of pH 5.6-9.2 two functional groups dissociate. One, with pKb = 8.1 +/- 0.1 participated in substrate binding and another one with pKb' = 8.1 +/- 0.1 is involved in substrate splitting. 5. The experiments with group-specific inhibitors suggest that an alpha-amino group and a sulfhydryl residue are involved in substrate binding and conversion. Furthermore, imidazole, tryptophan and carboxyl residues may be important for the catalytic process.

Adenosine Triphosphatases↗
PubMed1979

Translocation of the pre-synaptic complex formed upon DNA uptake by Streptococcus sanguis and its inhibition by ethidium bromide.

Donor DNA in its initially bound, single-stranded form exists in a chromosomally-unassociated complex where it is resistant to exogenous DNase I but sensitive to micrococcal nuclease. Most of the complexes are readily recuperable from the supernatant of recipients converted into spheroplasts. Subsequent to formation of this superficially located complex, donor DNA progressively associates with the recipient chromosome into which it is eventually integrated. Treatment of recipients with ethidium bromide at various times after initial DNA binding almost immediately halts translocation of whatever donor material is not yet synapsed with the chromosome. On the other hand, donor DNA that has already synapsed experiences no difficulty in becoming genetically integrated. Some degradation occurs to DNA that fails to undergo translocation as a result of ethidium bromide treatment, the acid-soluble products appearing in the culture medium. DNA in untranslocated complexes surviving treatment is not appreciably different in single-strand length from that in untreated complexes. When these surviving complexes are isolated from a cell lysate, the contained DNA can be shown by spectrofluorometry to have bound the drug.

Chromosomes, Bacterial↗
PubMed1978

Disturbance of the machinery for the gene expression by acidic pH in the repressible acid phosphatase system of Saccharomyces cerevisiae.

When the pH of growth medium containing a limited amount of inorganic phosphate is kept below 3.0, cells of Saccharomyces cerevisiae produce repressible alkaline phosphatase but no repressible acid phosphatase. The same cells produce acid phosphatase immediately on shifting the medium pH to 4.0 or above. Like intact cells, spheroplasts prepared from cells grown at pH 3.0 or 4.5 in medium with a limited amount of inorganic phosphate in suspension begin production of acid phosphatase immediately after pH shift from below 3.0 to 4.0 whereas sheroplasts from cells grown in inorganic phosphate-rich medium showed a prolonged lag period (3 h). The enzyme formation on the pH shift was sensitive to cycloheximide. No significant differences could be detected in cellular growth or in incorporation of 3H-L-lysine or 14C-adenine between cells cultivated at pH 3.0 and 4.5. These results along with the fact that the expression of structural genes of repressible acid and alkaline phosphatases is controlled by a common genetic regulatory system, at least in part, indicate that the genetic regulatory system operates to express the structural genes even at low pH, though the expression of repressible acid phosphatase is interrupted. Coupled experiments of temperature and pH shifts with the temperature-sensitive mutants of the regulatory genes suggest that the acidic pH affects the function of the cytoplasmic products of those genes in the expression of the structural gene. Based on these observations, a revised model involving the simultaneous functioning of the regulatory factors was suggested for the genetic regulation of repressible acid phosphatase synthesis.

Acid Phosphatase↗
PubMed1991

Localization and orientation of the VirD4 protein of Agrobacterium tumefaciens in the cell membrane.

The virD4 gene of Agrobacterium tumefaciens is essential for the formation of crown galls. Analysis of the nucleotide sequence of virD4 has suggested that the N-terminal region of the encoded protein acts as a signal peptide for the transport of the VirD4 protein to the cell membrane of Agrobacterium. We have examined the localization and orientation of this protein in the cell membrane. When the nucleotides encoding the first 30 to 41 amino acids from the N-terminus of the VirD4 protein were fused to the gene for alkaline phosphatase from which the signal sequence had been removed, alkaline phosphatase activity was detectable under appropriate conditions. Immunoblotting with VirD4-specific antiserum indicated that the VirD4 protein could be recovered exclusively from the membrane fraction of Agrobacterium cells. Moreover, when the membrane fraction was separated into inner and outer membrane fractions by sucrose density-gradient centrifugation, VirD4 protein was detected in the inner-membrane fraction and in fractions that sedimented between the inner and outer membrane fractions. By contrast, the VirD4'/alkaline phosphatase fusion protein with the N-terminal sequence from VirD4 was detected only in the inner membrane fraction. Treatment of spheroplasts of Agrobacterium cells with proteinase K resulted in digestion of the VirD4 protein. These results indicate that the VirD4 protein is transported to the bacterial membrane and anchored on the inner membrane by its N-terminal region. In addition, the C-terminal portion of the VirD4 protein probably protrudes into the periplasmic space, perhaps in association with some unidentified cellular factor(s).

Alkaline Phosphatase↗
PubMed1991

High efficiency transformation of Tolypocladium geodes conidiospores to phleomycin resistance.

A convenient and efficient transformation system has been developed for the filamentous fungus Tolypocladium geodes. In contrast to most of the commonly described techniques requiring prior preparation of protoplasts or spheroplasts, this method leads to high efficiency transformation of T. geodes conidiospores following moderate lytic enzyme treatment. Competent cells so obtained are still resistant to osmotic pressure and can be stored frozen without loss of viability. The highest transformation frequency (3-5 x 10(3) transformants per microgram of DNA) was obtained with plasmid pUT737 containing the Sh ble gene conferring phleomycin resistance under the control of a strong promoter isolated from Trichoderma reesei. Southern hybridization revealed multiple integration sites of plasmid DNA into the T. geodes nuclear DNA despite the absence of homology between the transforming DNA and the recipient genome. Instability could not be detected for the phleomycin phenotype during more than five generations of mitotic growth under non-selective conditions.

Blotting, Southern↗
PubMed1988

Probing FhuA'-'PhoA fusion proteins for the study of FhuA export into the cell envelope of Escherichia coli K12.

The FhuA protein (formerly TonA) is located in the outer membrane of Escherichia coli K12. Fusions between fhuA and phoA genes were constructed. They determined proteins containing a truncated but still active alkaline phosphatase of constant size and a variable FhuA portion which ranged from 11%-90% of the mature FhuA protein. The fusion sites were nearly randomly distributed along the FhuA protein. The FhuA segments directed the secretion of the truncated alkaline phosphatase across the cytoplasmic membrane. The fusion proteins were proteolytically degraded up to the size of alkaline phosphatase and no longer reacted with anti-FhuA antibodies. The fusion proteins were more stable in lon and pep mutants lacking cytoplasmic protease and peptidases, respectively. The larger fusion proteins above a molecular weight of 64,000 dalton were predominantly found in the outer membrane fraction. They were degraded by trypsin when cells were converted to spheroplasts so that trypsin gained access to the periplasm. In contrast, FhuA protein in the outer membrane was largely resistant to trypsin. It is concluded that the larger FhuA'-'PhoA fusion proteins were associated with, but not properly integrated into, the outer membrane.

Alkaline Phosphatase↗
PubMed1988

Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanus.

A plasmid, carrying the Tn5 gene for kanamycin resistance lacking its own promoter, has successfully been used in the selection of DNA sequences of the fungus Phycomyces blakesleeanus having promoter activity in Escherichia coli. Many of these sequences were also effective in promoting resistance to kanamycin when the corresponding chimeric plasmids were introduced in the fungus via spheroplast transformation. The selected phenotype was easily propagated through vegetative spores and behaved as a stable character since it was not appreciably lost in the absence of selection.

DNA Transposable Elements↗
PubMed1985

Genetic analysis of transformation in a microconidiating strain of Neurospora crassa.

We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2+) gene (Hughes et al. 1983a) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products. The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium. About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic. Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei. The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination. No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E. coli. One transformant, fl268-6, was unstable and generated a high proportion of segregants. Plasmid pDV1001 sequences were absent in their DNA. Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character. These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth.

DNA, Fungal↗
PubMed1988

A colony procedure for transformation of Saccharomyces cerevisiae.

A rapid and simple yeast transformation procedure has been developed using colonies on agar plates. Saccharomyces cerevisiae SHY3 cells were picked up from colonies on YPD plates grown freshly or stored at 4 degrees C and incubated with M13RK9-T DNA at 30 degrees C for 1-2 h in a solution of Li+, Ca2+, Mg2+, triacetin and polyethylene glycol. About 3,500 transformants were obtained per microgram of double stranded M13RK9-T DNA. Unlike the existing spheroplast techniques, single stranded M13RK9-T DNA transformed intact cells below one-hundredth frequency of the duplex form.

Genetic Engineering↗