Integrative and replicative genetic transformation of Aureobasidium pullulans

A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1- (TEF1) adjacent to the 5 end of the Escherichia coli hygromycin B phosphotransferase gene (HPT). Plasmids containing this hybrid gene (TEF1p/HPT) transformed A. pullulans strain R106 to a hygromycin B-resistant (HmB^R) phenotype. A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41 bp of homologous DNA has also been shown to transform A. pullulans to HmB^R. Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA. Analyses of 23 HmB^R transformants revealed integration of the plasmid in only eight of these transformants. In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype. In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter. The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining. These plasmids were generally 7–8 kb in size. One transformant appeared to contain four plasmids ranging in size from 4 to 8 kb, suggesting rearrangement of the transforming DNA. One plasmid obtained from a HmB^R A. pullulans transformant was able to transform E. coli to ampicillin resistance. However, after recovery from E. coli, this plasmid (approximately 4 kb) was unable to transform A. pullulans to HmB^R.     

Repair of plasmid DNA used for transformation of <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> by gene conversion

Techniques for genetic manipulation of the filamentous fungus Rhizopus have been hampered due to a lack of understanding regarding the recombination and replication mechanisms that affect the fate of introduced DNA. The ability to target chromosomal integration of a plasmid has been difficult because DNA transformed into Rhizopus rarely integrates and is autonomously replicated in a high molecular weight concatenated arrangement (i.e., series or chain). Linearization of the plasmid prior to transformation at a site having homology with the genomic DNA yields the highest frequency of integration, but repair of the double-strand break by end-joining is still the predominant event. We recently attempted to circumvent replication of the plasmid by introducing frameshift mutations in pyrG, the R. oryzae orotidine-5-monophosphate decarboxylase gene used for selection of the vector. It was hypothesized that autonomous replication of the mutated plasmids would be incapable of restoring prototrophic growth, since the genomic pyrG also contained a mutation. However, homologous integration of the plasmid results in duplication of the pyrG gene, which can create a functional copy of pyrG if both the genomic and plasmid mutations are paired on the same duplicate copy. While this event was detected in one of the isolates, it represented less than 8% of the total transformants. The majority of transformants contained plasmid replicating autonomously in a concatenated arrangement. Sequence analysis showed that prototrophic growth was restored by repairing the non-functional pyrG sequence in the plasmid, while the genomic pyrG gene was unaltered. Frequent transfer of the genomic pyrG mutation to the plasmid suggests that gene conversion is likely occurring by recombination pathways involving break-induced replication or synthesis-dependent strand annealing.USDA: Names are necessary to report factually on available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by USDA implies no approval of the product to the exclusion of others that may also be suitable.     

Analysis of homologous recombination in cultured mammalian cells in transient expression and stable transformation assays

Recombination between plasmid molecules, each containing a nonoverlapping deletion mutation in the hamster adenine phosphoribosyltransferase gene, was measured after coinjection into rat cells. Using these two plasmids, as linear or circular molecules, the recombination efficiency was measured soon after injection in a transient expression assay or after selection for stable transformants. The transient assay revealed that linear molecules were a better substrate for recombination, with double strand breaks within the region of homology stimulating recombination more than breaks outside the region of homology. A 20 to 70-fold increase in the efficiency of recombination was observed when two linear molecules were coinjected as compared to two circular molecules. Linear molecules were found to not only stimulate recombination but also to facilitate stable integration of the recombinant molecule into the host genome.     

The terminal protein of a linear mitochondrial plasmid is encoded in the N-terminus of the DNA polymerase gene in white-rot fungus Pleurotus ostreatus

The gene structure and expression of the linear mitochondrial plasmids of the white-rot fungus Pleurotus ostreatus, pMLP1 and pMLP2, were analyzed. Cleavage by proteinase K and exonucleases indicated that the 5′ ends of pMLP1 and pMLP2 DNAs were associated with terminal proteins. Nucleotide sequencing of the entire pMLP1 DNA revealed that it consists of 9,879 bp with terminal inverted repeat (TIR) sequences of 381 bp. The end sequence of TIR in pMLP1 is 3′-CCCCC-5′, similar to those of Escherichia coli phage PRD1. The pMLP1 plasmid harbors two long open reading frames (ORF1 and ORF2) and at least one minor ORF (mORF1). The deduced product of ORF1 is homologous to RNA polymerases of yeast mitochondria and several bacteriophages, whereas that of ORF2 is homologous to the protein-primed DNA polymerases of family B type. The mORF1 encodes a highly basic protein, most likely a TIR-binding protein, with no apparent sequence homology in the database. Expression of the predicted gene products from pMLP1 in mitochondria was demonstrated by Western blot analysis using antibodies against various expressed regions of pMLP1 ORFs. A plasmid-free strain, generated by curing with ethidium bromide, did not express any of these gene products. Terminal proteins of 70 kDa (TP1) and 73 kDa (TP2) were identified from pMLP1 and pMLP2, respectively. Western blot analysis indicated that TP1 was generated from the N-terminal half of the full-length product of ORF2 encoding a putative DNA polymerase. Received: 9 February 2000 / Accepted: 18 July 2000     

Phylogenetic analysis of the L and HN gene of ophidian paramyxoviruses

Summary.   Two reptilian paramyxoviruses, isolated from a neotropical rattlesnake (neotropical virus, NTV, ATCC VR-1408) and a bush viper (bush viper virus, BVV, ATCC VR-1409), respectively, were analysed to determine their taxonomic position among other reptilian paramyxoviruses investigated previously by Ahne et al. [7]. A 679 bp long region of the hemagglutinin-neuraminidase (HN) gene and a 627 bp long region of the large (L) gene were reverse transcribed, amplified by polymerase chain reaction (PCR), and sequenced. The deduced amino acid sequences were compared to mammalian paramyxoviruses belonging to the genera Respirovirus and Rubulavirus. The deduced amino acid sequences revealed 58.9 to 62% homology for the partial L protein and 41% to 47.1% homology for the partial HN protein. For phylogenetic analyses, a 518 bp L gene and a 352 bp HN gene fragment were used, both generating similar trees consisting of two distinct main groups, and some intermediate isolates. BVV clustered within group “b” while NTV clustered together with the intermediate ophidian paramyxovirus isolate Crot2-OH90. Received November 6, 2000 Accepted January 23, 2001     

Expression of a yeast gene can be blocked by insertion of short yeast DNA fragments between a UAS and the TATA box

We have constructed a plasmid, pOV10, which facilitates the introduction of putative upstream activating sequences (UAS) or upstream repressing sequences (URS) from yeast genes into plasmids containing CYC1-lacZ fusions. We have observed that the insertion of yeast sequences from 155 to 195 bp between the UAS and the TATA box of a CYC1-lacZ fusion gene can block -galactosidase expression. It is suggested that this block is related to the formation of nucleosomes on the DNA.     

Highly efficient homologous integration via tandem exo-β-1,3-glucanase genes in the common mushroom,Agaricus bisporus

  Homologous integration was studied in the common mushroom, Agaricus bisporus, using a plasmid (pHAG3-1) carrying the hygromycin-resistance gene and a 3.2-kb genomic fragment from A. bisporus. Homologous integration was found in 30–60% of the transformants obtained with pHAG3-1 linearized at three different positions within the homologous sequence, generating either blunt, 5′- or 3′-protruding ends. The genomic fragment was found to contain two homologous open reading frames in tandem, which showed 60% similarity to exo-β-1,3-glucanases from Saccharomyces cerevisiae and Candida albicans. The level of the corresponding mRNA is low in the vegetative mycelium and relatively high in fruiting bodies. In the vegetative mycelium of a transformant with tandemly integrated pHAG3-1 plasmids at the homologous position, exoglucanase mRNA was strongly increased without any apparent effect on growth rate or morphology. Received: 6 February 1996 / 27 March 1996     

Genetic diversity of avian infectious bronchitis virus California variants isolated between 1988 and 2001 based on the S1 subunit of the spike glycoprotein

Summary.  Twenty-nine isolates of avian infectious bronchitis virus (IBV) recovered from commercial chicken flocks in California between 1988 and 2001 and identified as California variants by serotype and direct automated cycle sequencing of the IBV spike glycoprotein S1 subunit, were further characterized phylogenetically and by nucleotide sequence comparison. California variants were grouped according to production type of chicken, by comparison with public access sequence databases (NCBI GenBank and EMBL), or based on phylogenetic analysis. Fisher’s Exact test was used to compare mutations per year, purifying and positive selection, predictive antigenicity, and a ≥ 6 bp deletion between California variant groups. A high number of mutations at the nucleotide level (p = 0.013) and a ≥ 6 bp deletion in the nucleotide sequence (p = 0.006) was significantly associated with broiler-type chickens. However, 88% of significant comparisons at the amino acid level such as purifying and positive selection were seen in layer-type chickens. A pronounced predictive antigenicity in the HVR2 region was also associated with layer-type chickens (p = 0.001). The study indicates that IBV in California is in a phase of slow evolution with different evolutionary patterns being associated with the production type of chicken. Received March 14, 2002; accepted August 9, 2002     

R-type plasmids in mitochondria from a single source of Zea luxurians teosinte

Two linear DNA plasmids resembling the R1 and R2 plasmids that are present in the mitochondria of several South American strains of maize were found in mitochondria from a single source of Zea luxurians collected by L. Mazoti. The Mazoti mtDNA is closely related to mtDNAs of other Z. luxurians, but mitochondria derived from the other Z. luxurians sources lack the plasmids. The larger plasmid from Mazoti mitochondria, M1, was cloned and large portions of it were sequenced. Restriction mapping and sequence comparisons showed that approximately 4.9 kb is similar to the S1 plasmid of maize and an additional 2.6 kb is related to R1 sequences integrated into the main mitochondrial genome of N cytoplasm. Therefore, the M1 plasmid appears to be very similar to the R1 plasmid. The inverted repeats at the ends of the M1 plasmid are not identitical. The left end IR is similar to the S-TIRs found at the termini of the S plasmids. The right end IR more closely resembles the integrated R1 sequences, including the variant region of the TIR. Whereas the variant region contains 13 bp in the S-TIRs and 15 bp in an integrated version of R1, it is 16 bp long in M1. The region of M1 that has no homology to the S1 plasmid is expressed at very low levels in Mazoti and RU cytoplasms, but at much higher levels in CMS-S mitochondria, where part of it is present in the main mitochondrial genome.     

Genetic analysis of transformation in a microconidiating strain of Neurospora crassa

Summary We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2 ⁺) gene (Hughes et al. 1983) 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, 8268-6, 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.     

Characterization of a genomic region encoding the 32-kDa dense granule antigen of Sarcocystis muris (Apicomplexa)

Two subgenomic libraries constructed from Sarcocystis muris total DNA were screened with a cDNA probe, specific for a 32-kDa protein associated with the dense granules. Two clones reacted positively and were isolated, gDG 32/1 and gDG 32/2. Genomic clone gDG32/1 and part of clone gDG 32/2 have been sequenced. The composite nucleotide sequence of these genomic clones comprises 4.34 kb. It contains a 5′ region of 2.14 kb, a first coding region (222 bp, exon I), a noncoding region (608 bp, intron), a second coding region (660 bp, exon II), and a 3′ region of 693 bp. The upstream region shows a eukaryotic promoter structure and a consensus sequence for the 5′ and 3′ splicing sites. Thus the open reading frame (ORF DG32) coding for the 32-kDa protein of the dense granules of S. muris cyst merozoites is interrupted by an intron. To our knowledge, dg32 is the first sarcosporidian mosaic gene to be characterized. Received: 27 April 1999 / Accepted: 11 May 1999     

Identification of an ARS element and development of a high efficiency transformation system for Pichia guilliermondii

An Autonomously Replicating Sequence element adjacent to the RIB1 gene encoding GTP cyclohydrolase II of the yeast Pichia guilliermondii was identified by transformation experiments. Detailed sequence analysis unveiled two potential ARS elements located 5′ and 3′ of the RIB1 open reading frame. The chromosomal fragment containing the ARS-like sequence 3′ to the RIB1 structural gene, called PgARS, conferred high transformation frequencies of 10⁴–10⁵ transformants/μg of DNA to a pUC19-derived plasmid in P. guilliermondii. The PgARS element also conferred autonomous replication to hybrid plasmids in this host. Based on this element a series of Escherichia coli shuttle vectors for efficient transformation of the flavinogenic yeast P. guilliermondii was developed. Received: 22 September 1998 / 1 June 1999     

Transformation of Schizosaccharomyces pombe by non-homologous,unstable integration of plasmids in the genome

Summary In the fission yeast, Schizosaccharomyces pombe, transformation with recombinant plasmids always results in a high proportion of mitotically unstable transformants. This suggested that specialised (ARS) sequences might not be required for autonomous replication of plasmids in S. pombe, contrary to the situation in Saccharomyces cerevisiae. We have shown that specialised ARS sequences, analogous to those in S. cerevisiae, do exist in S. pombe, supporting the view that ARS elements are a general feature of eukaryotes. In addition, there is a further mechanism of plasmid maintenance which involves homologous and non-homologous integration into, and excision from the genome.     

Transformation of the mycoparasite Parasitella simplex to neomycin resistance

Summary The facultatively parasitic zygomycete Parasitella simplex was transformed to neomycin resistance by a vector, which had been developed primarily for transformation of its host Absidia glauca. This plasmid, pAmNEF21, contained the bacterial resistance gene for neomycin (NPTII) under the control of the promoter region from the gene for elongation factor 1 (tef) isolated from A. glauca. Both flanking regions of the marker gene contain parts of the structural tef gene. DNA isolated from two Parasitella transformants was re-transformed in E. coli and the resulting plasmids, pAt21 and pAt35, were analyzed. The restriction map and Southern blot analysis show that both plasmids are rearranged. They had lost the structural tef information and were found to contain new DNA fragments, which were identical in both cases. Southern blot analysis of the transformants indicates that the rearranged plasmids are present in the fungal transformants and that the changes are not the result of re-transformation in E. coli. Plasmids were only recovered after growth under selective conditions. Southern blot analysis and re-transformation with undigested transformant DNA shows that the plasmids are replicated autonomously.     

Mismatch-stimulated plasmid integration in yeast

Summary A single base pair mismatch (G:T or A:C) in the CYC1 gene of the integrative plasmid pAB218 stimulates up to a five-fold integration into the yeast chromosome. Analysis of chromosomal sites of plasmid integration suggests that the mismatch-stimulated integration is not targeted as would be expected if crossovers, localised in the region of the mismatch, were a necessary step in mismatch repair. Instead, the observed mismatch-stimulated plasmid integration could be due to potentially recombinogenic structures formed during mismatch repair, such as single-stranded gaps or denatured DNA regions extending around the plasmid molecule.     

Cloning, sequence analysis, and expression of Lactobacillus casei phage PL-1 lysis genes

Summary.  The genes encoding the host cell wall-lytic proteins were searched in the genome DNA of phage PL-1 active against Lactobacillus casei ATCC 27092 by comparing the amino acid sequences with those of others using a computer software of the DDBJ data base. The gene regions found were cloned into E. coli by inserting PCR-amplified DNA fragments into the EcoRI site of pUC19, and the nucleotide sequences were determined. One of the ORFs (hol) consisted of 270 bp encoding 90 amino acids. The hol product (holin) possessed a putative secretion signal, two putative transmembrane helices, and a highly charged C-terminus. Another ORF (lys) consisted of 1050 bp encoding an N-acetylmuramoyl-L-alanine amidase of 350 amino acids. The gene lys was expressed in E. coli using pCALn expression vector, and the purified gene product hydrolysed the amide linkage in the peptidoglycans of L. casei. The amino acid sequence of PL-1 amidase showed a high homology to those of Lactococcus lactis phage rlt and Listeria monocytogenes phage A511. It was suggested that the N-terminal region was involved in enzyme activity and the C-terminal region in binding the enzyme to the cell wall substrate, respectively. Received January 10, 2000 Accepted April 13, 2000     

Ric1, a Phytophthora infestans gene with homology to stress-induced genes

From a set of Phytophthora infestans cDNA clones that were randomly selected from a potato- P. infestans interaction cDNA library, a relatively high proportion (5 out of 22) appeared to be derived from the same gene. The gene was designated ric1. P. infestans contains two copies of ric1 which share 98% homology at the nucleotide-sequence level and 100% at the amino-acid level. The nucleotide sequence predicts an open reading frame of 171 bp encoding a 57 amino-acid hydrophobic-peptide with two potential membrane-spanning domains. The predicted peptide shows high homology to a peptide encoded by plant genes whose expression is specifically induced during stress conditions. Southern-blot analysis of genomic DNA of several Phytophthora species indicated that most species contain ric1 homologues. During the life cycle of P. infestans, ric1 was expressed in all developmental stages but the level of expression varied. Sporangia and germinating cysts appeared to contain only very little ric1 mRNA whereas in the mycelium and during in planta growth higher levels were detected. Subjecting the mycelium to osmotic stress or to a high pH resulted in increased ric1 expression. Received: 5 March / 17 May 1999     

Multiple-copy integration in the yeast Yarrowia lipolytica

Using an EcoRI-BglII fragment of the G unit of the rDNA of Y. lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA. These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E. coli. Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773). Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene. Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion. Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25–60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization. Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes. Multiple integrations were found both as tandem repeats and as dispersed copies. Plasmid copy number was stable in both minimum and rich media, for strains containing less than ten copies per cells. However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.     

The heterogeneity of choanomastigote-shaped trypanosomatids as analyzed by their kDNA minicircle size: taxonomic implications

The kDNA minicircle size was analyzed in 15 species of choanomastigote-shaped trypanosomatids and four main groups of species were identified: (1) “Crithidia”deanei, “C.”desouzai and “Herpetomonas”roitmani, which presented the largest molecules (≥ 3,800 bp), (2) “C.”oncopelti with minicircles of at least four different sizes within 1,300–2,650 bp, (3) C. fasciculata, C. guilhermei and C. luciliae, having at least two sizes of minicircle (2,650 bp and 3,000 bp) and (4) a heterogeneous group of species presenting minicircles of a single size, including several Crithidia spp. (having 1,600 bp or 1,700 bp minicircles) and two Proteomonas spp. presenting the smallest minicircles (about 1,500 bp). These results were compared with other observations and discussed from a taxonomic point of view. Received: 7 January 2000 / Accepted: 1 March 2000     

Endocrine Metabolic Disorders in Patients with Breast Cancer, Carriers of BRCA1 Gene Mutations

Two groups of breast cancer patients (53 ± 2 years) in clinical remission receiving no specific therapy were examined: group 1, with BRCA1 gene mutations (N = 11) and group 2, without mutations of this kind (N = 11). The two groups did not differ by insulinemia and glycemia, insulin resistance index, blood levels of thyrotropic hormone, sex hormone-binding globulin, insulin-like growth factor-1, triglycerides, or lipoproteins. In group 1, blood estradiol level was higher. Intensive glucose-induced generation of reactive oxygen species in these patients was associated with a decrease of cholesterolemia, of the C-peptide/insulin proportion, and a trend to higher urinary excretion of 4-hydroxyestrone, one of the most genotoxic catecholestrogens. BRCA1 gene mutations in breast cancer patients were associated with signs of estrogenization and a pro-genotoxic shift in the estrogen and glucose system, which could modulate the disease course and requires correction.