Release of transforming plasmid DNA from actively growing genetically engineered Escherichia coli

We studied the transforming ability of the extracellular plasmid DNA released from a genetically engineered Escherichia coli pEGFP and the culturing conditions for the release of transforming DNA. The transforming ability was evaluated by transformation of competent cells with filtrates of E. coli pEGFP cultures. The number of transformants increased with time when E. coli pEGFP cells grew exponentially in rich medium, but not in stationary phase or when inoculated in freshwater. These results suggested that crude extracellular plasmid DNA had transforming ability and this transforming DNA was mainly released by actively growing bacteria.     

A deoxyribonuclease of Diplococcus pneumoniae specific for methylated DNA.

A deoxyribonuclease specific for methylated DNA was isolated from Diplococcus pneumoniae. The enzyme, an endonuclease, degrades DNA for Escherichia coli to fragments of average molecular weight about half a million; it forms discrete fragments from phage lambda DNA. Methyl-deficient E. coli DNA is not attacked, neither is DNA from Micrococcus radiodurans, which contains no methylated adenine or cytosine. Nor is DNA from D. pneumoniae or phage T7 attacked. However, DNA from M. radiodurans, D. pneumoniae, and T7 is attacked after methylation with and E. coli extract. Methylated T7 DNA is degraded to discrete fragments. Although the genetic transforming activity of normal DNA from D. pneumoniae is not affected by the enzyme, transforming activity of methylated DNA is destroyed. The enzyme is designated endonuclease R Dpn I. Under certain conditions another enzyme of complementary specificity can be isolated. This enzyme, designated endonuclease R Dpn II, produces a similar pattern of fragments from the DNA of T7 without prior methylation of the DNA. It also degrades normal DNA for D. pneumoniae. It is suggested that this pair of enzymes plays a role in some unknown control process, which would involve a large fraction of the specific base sequences that are methylated in E. coli DNA and are present but not methylated in DNA from other sources.     

Cloning the quinic acid (aq) gene cluster from Neurospora crassa: identification of recombinant plasmids containing both qa-2+ and qa-3+

A 22.2-kb insert of Neurospora crassa DNA containing at least two of the genes from the inducible catabolic quinic acid pathway has been cloned into the cosmid vehicle pHC79 resulting in a recombinant plasmid, pMSK308. The qa-2+ locus (which encodes catabolic dehydroquinase) is functionally expressed in both Escherichia coli and qa-2 mutants of N. crassa transformed with pMSK308 plasmid DNA. Expression of the qa-3 gene (which encodes quinate dehydrogenase) is only detected upon reintroduction into N. crassa. Results were also obtained which suggested that the qa-4 gene, which maps between qa-2 and qa-3, may also be present on both pMSK308 and the previously described plasmid pVK88. Certain anomalies in the types of N. crassa transformants obtained with pMSK308 plasmid DNA were noted.     

Molecular cloning of a Neurospora crassa carotenoid biosynthetic gene (albino-3) regulated by blue light and the products of the white collar genes.

The albino-3 (al-3) gene of Neurospora crassa, which probably encodes the carotenoid biosynthetic enzyme geranylgeranyl pyrophosphate synthetase, was cloned. The N. crassa triple mutant al-3 qa-2 aro-9 was transformed to qa-2+ with mixtures of plasmids bearing N. crassa DNA inserts, and the transformants were screened for the al-3+ phenotype. One al-3+ qa-2+ transformant (AL3-1) was examined in detail and shown to contain intact vector sequences integrated into the N. crassa genome. The vector and some flanking sequences were recovered from AL3-1 after restriction, ligation, and selection of chloramphenicol-resistant transformants of Escherichia coli. The flanking sequences were subsequently used to detect the al-3-containing plasmid in the mixture of about 1,800 plasmids. Restriction fragment length polymorphism mapping was carried out to confirm the identity of the cloned fragment. The level of the al-3 mRNA was shown to be increased 15-fold in light-induced (compared with that in dark-grown) wild-type mycelia. The light-dependent increase in al-3 mRNA levels was not observed in presumed regulatory mutant (white collar) strains.     

Gene disruption by transformation in Neurospora crassa.

To establish conditions which might permit deliberate gene disruptions in Neurospora crassa, we studied transformation with linear DNA fragments. The transformation frequency observed was increased about twofold in comparison with that obtained with circular plasmid DNA. However, only a low proportion, approximately 10%, of the integration events occurred at the homologous site, whereas most integrations of transforming DNA took place in nonhomologous regions. It was also found that multiple integration events frequently occurred in individual transformants. A plasmid, designated pJP12, was constructed that contains the N. crassa am+ gene interrupted by insertion into its coding region of a DNA segment carrying a functional Neurospora qa-2+ gene. A fragment of Neurospora DNA that contains this am qa-2+ construction was obtained from plasmid pJP12 and used to transform an am+ qa-2 strain in an attempt to disrupt the resident am+ gene. After the initial qa-2+ transformants were converted to homokaryons by appropriate crosses, 10 independent transformants with an am mutant phenotype were found among 117 examined. Each of these qa-2+ am transformants showed the loss of a hybridization band in Southern blots of genomic DNA that corresponded to the normal am+ gene and the presence of a new hybridization band, consistent with an alteration in the am+ region.     

A targeted-replacement system for identification of signals for de novo methylation in Neurospora crassa.

Transformation of eukaryotic cells can be used to test potential signals for DNA methylation. This approach is not always reliable, however, because of chromosomal position effects and because integration of multiple and/or rearranged copies of transforming DNA can influence DNA methylation. We developed a robust system to evaluate the potential of DNA fragments to function as signals for de novo methylation in Neurospora crassa. The requirements of the system were (i) a location in the N. crassa genome that becomes methylated only in the presence of a bona fide methylation signal and (ii) an efficient gene replacement protocol. We report here that the am locus fulfills these requirements, and we demonstrate its utility with the identification of a 2.7-kb fragment from the psi 63 locus as a new portable signal for de novo methylation.     

Signal for DNA methylation associated with tandem duplication in Neurospora crassa.

Most cytosine residues are subject to methylation in the zeta-eta (zeta-eta) region of Neurospora crassa. The region consists of a tandem direct duplication of a 0.8-kilobase-pair element including a 5S rRNA gene. The repeated elements have diverged about 15% by the occurrence of numerous CG to TA mutations, which probably resulted from deamination of methylated cytosines. Most but not all common laboratory strains of N. crassa have methylated duplicated DNA at the zeta-eta locus. However, many strains of N. crassa and strains of N. tetrasperma, N. sitophila, and N. intermedia have one instead of two copies of the homologous DNA and it is not methylated. A cross of strains differing at the zeta-eta locus produced progeny which all had duplicated, methylated, or unique, unmethylated DNA, like the parental strains. We conclude that a signal causing unprecedented heavy DNA methylation is present in the zeta-eta region.     

Genetical and Molecular Analyses of qa-2 Transformants in NEUROSPORA CRASSA

Neurospora crassa qa-2+ transformants from five different donor DNA clones were analyzed by genetical and molecular techniques. None of the 32 transformants have the qa-2+ DNA replacing the qa-2- gene in linkage group VII. In one transformant, the qa-2+ DNA was inserted adjacent to the qa-2- gene. Thirty-one transformants have the qa-2+ inserts at sites not linked, or not closely linked, to the qa-2 locus in LG VII. Plasmid sequences were integrated along with the qa-2+ gene in 28 transformants. In the unlinked duplication-type transformants, catabolic dehydroquinase (the qa-2+ gene product) was induced at 5-100% of the wild-type-induced enzyme activity, with 24 transformants in the 5-80% range. The reduced levels of enzyme activity may be due to "position effects" of sequences adjacent to the integration site either in the N. crassa genomic DNA or in the flanking plasmid (pBR322 or pBR325) sequences. Unexpected gene conversion-like events, in which a qa-2+ gene was changed to qa-2-, were observed in tetrads from intercrosses between unlinked duplication-type transformants and in selfings of such transformants.     

Ectopic Integration of Transforming DNA Is Rare among Neurospora Transformants Selected for Gene Replacement

In a variety of organisms, DNA-mediated transformation experiments commonly produce transformants with multiple copies of the transforming DNA, including both selected and unselected molecules. Such ``cotransformants' are much more common than expected from the individual transformation frequencies, suggesting that subpopulations of cells, or nuclei, are particularly competent for transformation. We found that Neurospora crassa transformants selected for gene replacement at the am gene had not efficiently incorporated additional DNA, suggesting that nuclei that undergo transformation by homologous recombination are not highly competent at integration of DNA by illegitimate recombination. Spheroplasts were treated with DNA fragments homologous to am and with an Escherichia coli hph plasmid. Transformants were initially selected for hph (hygromycin(R)), allowed to conidiate to generate homokaryons and then selected for either Am(-) (gene replacements) or hph. Surprisingly, most am replacement strains were hygromycin(S) (124/140) and carried no extraneous DNA (116/140). Most transformants selected for hph also had ectopic copies of am DNA and/or multiple copies of hph sequences (32/35), generally at multiple sites, confirming that efficient cotransformation could occur. To test the implication that cotransformation involving gene replacement and ectopic integration is rare, we compared the yields of am replacement strains with or without prior selection for hph. The initial selection did not appreciably help (or hinder) recovery of strains with replacements.     

Assessment of the utilization of the antisense RNA strategy to identify essential genes in heterologous bacteria

We employed an antisense RNA approach to identify essential genes common in both Gram-positive and Gram-negative bacteria by cloning a random library of Streptococcus mutans chromosomal DNA into an expression vector and transforming Escherichia coli. Twelve out of 27 E. coli transformants with growth defective phenotypes contained individual structural genes of S. mutans in the antisense orientation relative to the E. coli promoter. Thirty-three percent of these transformants (4/12) corresponded to the genes (gyrA, ileS, rplE and yihA orthologs) which are essential for bacterial viability.     

Transformation of Aspergillus nidulans by the orotidine-5'-phosphate decarboxylase gene of Neurospora crassa

Relief of an auxotrophic requirement for uridine in Aspergillus nidulans strain G191 has been achieved by transformation with a segment of Neurospora crassa DNA containing the corresponding gene coding for orotidine-5'-phosphate decarboxylase. The mitotic stability of such transformants suggests that the DNA has integrated into the genome. Southern hybridisation analysis of DNA isolated from transformants revealed the presence of pBR322 sequences which have integrated into the host genome along with the N. crassa DNA.     

A eukaryotic gene encoding an endonuclease that specifically repairs DNA damaged by ultraviolet light.

Many eukaryotic organisms, including humans, remove ultraviolet (UV) damage from their genomes by the nucleotide excision repair pathway, which requires more than 10 separate protein factors. However, no nucleotide excision repair pathway has been found in the filamentous fungus Neurospora crassa. We have isolated a new eukaryotic DNA repair gene from N.crassa by its ability to complement UV-sensitive Escherichia coli cells. The gene is altered in a N.crassa mus-18 mutant and responsible for the exclusive sensitivity to UV of the mutant. Introduction of the wild-type mus-18 gene complements not only the mus-18 DNA repair defect of N.crassa, but also confers UV-resistance on various DNA repair-deficient mutants of Saccharomyces cerevisiae and a human xeroderma pigmentosum cell line. The cDNA encodes a protein of 74 kDa with no sequence similarity to other known repair enzymes. Recombinant mus-18 protein was purified from E.coli and found to be an endonuclease for UV-irradiated DNA. Both cyclobutane pyrimidine dimers and (6-4)photoproducts are cleaved at the sites immediately 5' to the damaged dipyrimidines in a magnesium-dependent, ATP-independent reaction. This mechanism, requiring a single polypeptide designated UV-induced dimer endonuclease for incision, is a substitute for the role of nucleotide excision repair of UV damage in N.crassa.     

Transformation of the entomopathogenic fungus, Metarhizium flavoviride strain CG423 to benomyl resistance

Metarhizium flavoviride strain CG423 is being developed as a mycoinsecticide against grasshoppers. This strain has been transformed to resistance to the fungicide benomyl by a polyethylene glycol (PEG)-mediated procedure using a mutant tubulin gene from Neurospora crassa . Transformation frequencies of up to 84 transformants per microgram of transforming DNA were achieved. Benomyl-resistant transformants were obtained that could tolerate greater than 30 μg ml⁻¹ benomyl. Southern blot analysis of genomic DNA reveals that the mechanism of genetic transformation of all transformants was by homologous gene replacement of the β-tubulin allele.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

Expression of the Escherichia coli beta-glucuronidase gene in Pseudocercosporella herpotrichoides.

The plant-pathogenic fungus Pseudocercosporella herpotrichoides has been successfully transformed by using two different positive selection systems in combination with the Escherichia coli gusA gene. The selectable markers used in this study were the hygromycin B phosphotransferase gene (hph) from E. coli and the gene (bml) for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa. A lower transformation rate was obtained with the bml system than with the hph system. Conversely, cotransformation frequencies, as determined with medium plates containing the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronic acid, were higher with bml than with hph as the selectable marker. The hygromycin-resistant transformants were mitotically stable, and both the selectable gene and gusA were maintained through conidiation. The vector DNA was integrated into the genome, and the number and sites of insertion varied among transformants. Enzyme assays of mycelial extracts showed that beta-glucuronidase activity was highest in transformants with a high gusA copy number. Expression of gusA during growth of the fungus on plants was easily detectable and did not affect pathogenicity. These results form the basis for construction of a versatile and sensitive reporter gene system for P. herpotrichoides.     

Cloning in Escherichia coli K-12 of a Na+-dependent transport system from a marine bacterium.

The transport of D-alanine by Escherichia coli K-12 neither requires nor is stimulated by Na+. The transport of D-alanine by the marine bacterium Alteromonas haloplanktis 214 requires Na+ specifically. Mutants of E. coli which were unable to transport D-alanine were isolated by enrichment for D-cycloserine resistance. One of the mutants was transformed with a gene bank of A. haloplanktis chromosomal DNA. Two transformants, E. coli RM1(pPM1) and E. coli RM1(pPM2) were able to transport D-alanine by a Na+-dependent mechanism. Li+ and K+ were unable to replace Na+. Both transformants contained chimeric plasmids with inserts which hybridized with A. haloplanktis but not E. coli chromosomal DNA or each other. Despite the lack of homology between the inserts, Na+-dependent D-alanine transport in the two transformants could not be distinguished either by kinetic studies or by differences in the capacity of various amino acids to compete for D-alanine uptake.