Integrative transformation of a zygomycete,Absidia glauca,with vectors containing repetitive DNA

Summary Experimental evidence for integration of transformed DNA into the genome of Absidia glauca, a member of the fungal class of zygomycetes is presented. According to the limited knowledge on the molecular biology of these fungi, autonomous replication of transformed plasmids seems to be the preferential mode of DNA propagation. By inserting fragments of highly repetitive DNA elements into an autonomously replicating vector conferring neomycin resistance, we were able to obtain integrative transformation events. With such plasmids we observed stable mitotic propagation of a selective marker gene (NPT II under the control of a homologous actin promoter). Analysis of DNA from transformants in Southern type experiments, as well as restriction analysis of retransformants into Escherichia coli, provide evidence that integration of foreign DNA into the genome of Absidia glauca is possible. These transformation events are often associated with the appearance of mutant phenotypes.     

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.     

Neomycin resistance as a dominantly selectable marker for transformation of the zygomycete Absidia glauca

Summary A plasmid (pAmN61) containing the NPT II structural gene (neomycin phosphotransferase) fused to the N-terminal region of a homologous actin gene was used for the transformation of Absidia glauca protoplasts. Neomycin resistant transformants could be selected for on complete medium containing 1.2 mg/ml neomycin sulfate. The physical presence of plasmid DNA in Absidia glauca was demonstrated by retransformation into Escherichia coli and by Southern blot analysis. No integration of plasmid DNA at either one of the two actin loci was observed; Southern blot experiments provide evidence that pAmN61 is autonomously replicated in Absidia glauca.     

Improving live attenuated bacterial carriers for vaccination and therapy

Live attenuated bacteria are well established as vaccines. Thus, their use as carriers for prophylactic and therapeutic macromolecules is a logical consequence. Here we describe several experimental applications of bacteria to carry heterologous macromolecules into the murine host. First, Listeria monocytogenes are described that are able to transfer eukaryotic expression plasmids into host cells for gene therapy. High multiplicities of infection are still required for efficient gene transfer and we point out some of the bottlenecks that counteract a more efficient transfer and application in vivo. Then, we describe Salmonella enterica serovar Typhimurium (S. typhimurium) as an expression plasmid transfer vehicle for oral DNA vaccination of mice. We demonstrate that the stabilization of the plasmid transformants results in an improved immune response. Stabilization was achieved by replacing the origin of replication of the original high-copy-number plasmid by a low-copy-number origin. Finally, we describe Salmonella carriers for the improved expression of heterologous proteins. We introduce a system in which the plasmid is carried as a single copy during cultivation but is amplified several fold upon infection of the host. Using the same in vivo inducible promoter for both protein expression and plasmid amplification, a substantial increase in antigen expression in vivo can be achieved. A modification of this approach is the introduction of inducible gene expression in vivo with a low-molecular-weight compound. Using P_BAD promoter and l-arabinose as inducer we were able to deliberately activate genes in the bacterial carrier. No background activity could be observed with P_BAD such that an inducible suicide gene could be introduced. This is adding an important safety feature to such live attenuated carrier bacteria.     

How heterologously expressed <Emphasis Type="Italic">Escherichia coli</Emphasis> genes contribute to understanding DNA repair processes in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

DNA-damaging agents constantly challenge cellular DNA; and efficient DNA repair is therefore essential to maintain genome stability and cell viability. Several DNA repair mechanisms have evolved and these have been shown to be highly conserved from bacteria to man. DNA repair studies were originally initiated in very simple organisms such as Escherichia coli and Saccharomyces cerevisiae, bacteria being the best understood organism to date. As a consequence, bacterial DNA repair genes encoding proteins with well characterized functions have been transferred into higher organisms in order to increase repair capacity, or to complement repair defects, in heterologous cells. While indicating the contribution of these repair functions to protection against the genotoxic effects of DNA-damaging agents, heterologous expression studies also highlighted the role of the DNA lesions that are substrates for such processes. In addition, bacterial DNA repair-like functions could be identified in higher organisms using this approach. We heterologously expressed three well characterized E. coli repair genes in S. cerevisiae cells of different genetic backgrounds: (1) the ada gene encoding O⁶-methylguanine DNA-methyltransferase, a protein involved in the repair of alkylation damage to DNA, (2) the recA gene encoding the main recombinase in E. coli and (3) the nth gene, the product of which (endonuclease III) is responsible for the repair of oxidative base damage. Here, we summarize our results and indicate the possible implications they have for a better understanding of particular DNA repair processes in S. cerevisiae.     

Structural organization of the genome of the zygomycete Absidia glauca: evidence for high repetitive DNA content

Summary Total cell DNA of Absidia glauca has a GC-content of 44.6% ± 0.5% as determined from optical melting profiles which is in good accordance with values from equilibrium centrifugation in bisbenzimide containing CsCl gradients (46.2% = 1.1%), whereas mitochondrial DNA has a GC-content of only 30%. The genome size of Absidia glauca is approximately 36,000 kb, 8.6 times that of Escherichia coli. Three kinetically different fractions could be identified in reassociation experiments: a foldback-DNA fraction, comprising approximately 10% of the total DNA, repetitive DNA (25%) and single copy DNA (65%). This relatively high amount of repetitive DNA could partly be ascribed to ribosomal DNA (13%) and a new interspersed repetitive element (rAg1) which has been cloned in pBR325.     

Cloned mitochondrial DNA from the zygomycete Absidia glauca promotes autonomous replication in Saccharomyces cerevisiae

Summary We have cloned fragments from mitochondrial and chromosomal DNA of the zygomycete Absidia glauca in Saccharomyces cerevisiae using the ARS selection vector YIp5. Though it has not been possible to select ARS elements from chromosomal DNA, we succeeded in isolating two clones of mitochondrial origin that support autonomous replication in bakers' yeast. DNA from these plasmids has been shown to hybridize with mitochondrial DNA from both mating types. Generation times of the transformed yeast strain in selective medium are around 20 h. In liquid minimal medium only 6% of the cells contain the plasmid; in complete medium a mitotic stability of 50% has been determined.     

Inter-mating type protoplast fusion in the zygomycete Absidia glauca

Summary Methods for obtaining the release, regeneration, and fusion of protoplasts from the dioecious zygomycete Absidia glauca are presented. Somatic hybridization was obtained between protoplasts from several amino acid auxotrophic mutants either belonging to the same or complementary mating type. Both kinds of mating type information are expressed within the same organism and give rise to a monoecious phenotype. Without the selective pressure on the auxotrophic markers used for the construction of the fusion strains, the ability to form zygospores is rapidly lost. However, the gene clusters for ribosomal RNA which are distinguishable between mating types are stable and maintained in the fusion strains, independent of growth conditions.     

Rational design of Salmonella recombinant vaccines

Salmonella enterica is an important pathogen of animals and humans causing a variety of infectious diseases. The large number of cases of typhoid fever due to S. enterica serovar Typhi infections gives rise to the continuous need for improved vaccines against this life-threatening infection. However, S. enterica is also an interesting organism to act as a live attenuated carrier for the presentation of recombinant heterologous antigens. Comprehensive experimental studies have been performed and a detailed knowledge of the molecular mechanisms of important virulence factors is available. This allows the rationale design of improved Salmonella carrier strains and the development of novel strategies for the expression and presentation of recombinant antigens. Here, we review recent advances in generation of live attenuated Salmonella vaccines and discuss criteria for expression strategies of heterologous antigens by Salmonella carrier strains.     

Changes in the histone/DNA ratio during the development of a multicellular organism

Changes in the histone/DNA ratio are studied in the star-fish. It is found that the development of a multicellular organism is based on the principle of nonequivalence of the sister cells which is described by the distribution of Polya. It is postulated that the development of an organism obeys the laws of mathematics. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 12, pp. 695–698, December, 1996     

Electrophoretic karyotype of the zygomycete Absidia glauca: evidence for differences between mating types

Summary Experimental conditions for the separation of chromosomes from the model zygomycete Absidia glauca by rotating field electrophoresis were established. The sexually compatible strains of the mating type pair A. glauca CBS 100.48 (+) and CBS 101.48 (-) showed considerable differences in their electrophoretic karyotype. By Southern hybridization with homologous probes we have mapped the chromosomal locations for rDNA, the repetitive element rAg1, and the genes for actin and the elongation factor EF1. For the mapping of ubiquitin information we used a heterologous probe from U. maydis. The combination of electrophoretic karyotyping and Southern mapping proved to be a useful tool for characterizing mutant genotypes, which were induced by integrative transformation.     

Cloning of a Candida utilis gene which complements leu2 mutation in Saccharomyces cerevisiae

Summary DNA fragments containing the LEU2 gene of Candida utilis have been isolated, utilizing the genome library (constructed in YRp12) of this organism. Two recombinant plasmids pZR84 and pZR32, containing the cloned LEU2 gene, were 4.24 kb and 10.4 kb, respectively, and were shown to complement leu2 mutation in Saccharomyces cerevisiae and leuB mutation in Escherichia coli. The cloned fragment in pZR84 contained one restriction site each for EcoRI and PvuII, and two for HindIII, but none for SalI, BamHI or Pstl. This cloned fragment hybridized with the total DNA from C. utilis and from Leu⁺ transformants of S. cerevisiae, but not with that from untransformed S. cerevisiae. Subcloning analyses showed that a 2.34 kb BamHI HindIII fragment of the cloned C. utilis sequence contains the region essential for the expression of the LEU2 gene.Journal Article No. 11669 from the Michigan Agricultural Experiment Station     

Development of a gene transfer system for Penicillium chrysogenum

Summary We report here the development of an endogenous gene transfer system for the industrially-important Deuteromycete Penicillium chrysogenum, utilising a recombinant plasmid designated pPC-31 to complement a tryptophan — auxotrophic strain. Transformation frequencies in the order of 300–1800 transformants per g DNA have been obtained, and Southern hybridisation analysis has demonstrated that in the majority of cases, integration is mediated by homologous recombination between pPC-31 and the host genome at the site of the resident mutant allele.     

Agrobacterium-mediated transformation as a tool for functional genomics in fungi

In the era of functional genomics, the need for tools to perform large-scale targeted and random mutagenesis is increasing. A potential tool is Agrobacterium-mediated fungal transformation. A. tumefaciens is able to transfer a part of its DNA (transferred DNA; T-DNA) to a wide variety of fungi and the number of fungi that can be transformed by Agrobacterium-mediated transformation (AMT) is still increasing. AMT has especially opened the field of molecular genetics for fungi that were difficult to transform with traditional methods or for which the traditional protocols failed to yield stable DNA integration. Because of the simplicity and efficiency of transformation via A. tumefaciens, it is relatively easy to generate a large number of stable transformants. In combination with the finding that the T-DNA integrates randomly and predominantly as a single copy, AMT is well suited to perform insertional mutagenesis in fungi. In addition, in various gene-targeting experiments, high homologous recombination frequencies were obtained, indicating that the T-DNA is also a useful substrate for targeted mutagenesis. In this review, we discuss the potential of the Agrobacterium DNA transfer system to be used as a tool for targeted and random mutagenesis in fungi.     

Knockout of the DNA ligase IV homolog gene in the sphingoid base producing yeast <Emphasis Type="Italic">Pichia ciferrii</Emphasis> significantly increases gene targeting efficiency

The yeast Pichia ciferrii produces large quantities of the sphingoid base tetraacetyl phytosphingosine (TAPS) and is an interesting platform organism for the biotechnological production of sphingolipids and ceramides. Ceramides have attracted great attention as a specialty ingredient for moisture retention and protection of the skin in the cosmetics industry. First attempts have been started to metabolically engineer P. ciferrii for improved production of TAPS and other sphingoid bases. However, rational metabolic engineering of P. ciferrii is difficult due to a low gene targeting efficiency. In eukaryotes, two major pathways coexist, which are responsible for genomic DNA integration, homologous recombination (HR) and non-homologous end joining (NHEJ). Integration via HR is targeted, while NHEJ involves ectopic (non-targeted) integration depending on a ligation step mediated by DNA ligase IV (Lig4). Here, we demonstrate a dramatical increase in gene targeting efficiency in a P. ciferrii lig4 knockout strain, deficient in NHEJ. Furthermore, a quick and easy to use freeze–thaw method was developed to transform P. ciferrii with high efficiency. Owing to the ability of targeting genomic DNA integration our results pave the way for further genetic and metabolic engineering approaches with P. ciferrii by means of knocking out or overexpressing predestinated genes.     

A large deletion in the plastid DNA of the holoparasitic flowering plant Cuscuta reflexa concerning two ribosomal proteins (rpl2, rpl23), one transfer RNA (trnI) and an ORF 2280 homologue

We have determined the nucleotide sequence of a 5.3-kb region of the plastid DNA (ptDNA) from the heterotrophic holoparasitic plant Cuscuta reflexa. The cloned area contains genes for the D1-protein (32-kDa protein; psbA), tRNA^His (trnH), ORF 740 (homologous to ORF 2280 from Nicotiana tabacum), ORF 77 (homologous to ORF 70), tRNA^Leu (trnL) and a hypothetical ORF 55 which has no homology to any known gene among higher plants. This 5.3-kb area is colinear with a 12.4-kb region of tobacco ptDNA and has therefore undergone several deletions totalling 7.1 kb. Most of the missing nucleotides belong to one large deletion in the ptDNA of C. reflexa of approximately 6.5 kb. This deletion involves two ribosomal protein genes, rpl2 and rpl23, as well as the transfer RNA for Isoleucin (trnI) and a region encoding 1540 amino-acid residues of an ORF 2280 homologue, as compared to tobacco chloroplast DNA. This is remarkable since the remaining genes, especially the psbA gene, are highly conserved in C. reflexa. Furthermore, we found that the expression of the psbA gene is in the same range as in the autotrophic Ipomoea purpurea which belongs to the same family as Cuscuta (Convolvulaceae). Here we hypothesize a total loss of rpl2 and rpl23 in the entire genome of C. reflexa. The phylogenetic position of, and the evolutionary change of ptDNA from, Cuscuta are discussed.     

MboI,ThaI and HinfI endonuclease cleavage maps of the yeast mitochondrial DNA

Summary MboI, HinfI and ThaI cleavage maps have been constructed for the region of the mitochondrial DNA from S. cerevisiae where transfer RNA genes are principally located. About 40 cleavage sites have been localized between the C and P genetic markers. The MboI map covers about 50% of the total mitochondrial genome. For constructing maps we have used a series of rho^– deletion mutants whose mitochondrial DNAs have a typical single deletion structure as judged by previous genetic and physical analyses. The mutant DNAs carry known transfer RNA genes and genetic markers and, therefore, the comparison between genetic and restriction maps has allowed us to localize individual transfer RNA genes within defined physical segments.Abbreviations bp base pairs - mtDNA mitochondrial DNA - tRNA transfer RNA - rRNA ribosomal RNA - ThaI formerly TacI     

Genetic interactions in somatic inter-mating type hybrids of the zygomycete Absidia glauca

Summary Protoplasts of auxotrophic mutants of the heterothallic zygomycete Absidia glauca were efficiently fused by electrofusion. Fusion heterokaryons between the complementary mating types, which grew prototrophically, were selected. Among 72 fusion colonies which were further analysed, 11 did not only grow prototrophically but also combined both mating types as they formed zygotes within the same mycelium. These homothallic hybrids possessed two sets of rDNA repeats originating from the complementary mating types. Those fusion colonies which gave only a (+) type mating reaction, synthesized a 15 kDa (+) type-specific surface protein; fusion colonies which mated as (-) type, did not produce this surface protein. In homothallic fusion products no expression of the (+) type-specific protein could be detected. Single spore derivatives of fusion colonies in general show the phenotype of only one fusion partner, but in some cases prototrophic recombinants were obtained. This system opens up the possibility for genetic analysis of an organism with inefficient meiotic system, lacking a natural parasexual cycle.     

Isolation and characterization of a 70 kDa protein fromMycobacterium avium

Mycobacterium aviumcomplex (MAC) is an intracellular pathogen which causes disseminated bacterial infection in immunocompromised individuals. This organism predominantly infects macrophages. Attachment of MAC to macrophages is the first step prior to invasion. We have previously shown that a 70 kDa protein ofM. avium(Ma) is one of nine monocyte-binding proteins. In the present study, we have purified this protein from sonic extracts of Ma and studied some of its properties. The N-terminal sequence of this protein was identified and found to exhibit a strong homology to the 70 kDa heat shock protein (hsp) ofM. leprae(Ml) andM. tuberculosis(Mtb). This protein was found to be present on the surface of the organism and was able to inhibit the attachment of intact Ma to human monocyte derived macrophages (MDM) up to 49% in anin vitroattachment assay using intact fluorescein isothiocyanate (FITC)-labelled Ma. Bovine serum albumin (BSA) and recombinant 70 kDa hsp from Mtb, which were used as controls, inhibited this attachment by 9.8 and 18%, respectively. These results suggest that the 70 kDa protein may have a role in the attachment of intact Ma to MDM. When tested in lymphocyte activation assays, this protein did not appear to significantly stimulate proliferation. However, it was found to stimulate the production of tumor necrosis factor (TNF)-αby MDM. This protein may be one of several Ma antigens that trigger host immune response by binding to MDM and stimulating the production of inflammatory cytokines such as TNF-αby these cells.     

<Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>: a convenient model system for the study of DNA repair in photoautotrophic eukaryotes

The green alga Chlamydomonas reinhardtii is a convenient model organism for the study of basic biological processes, including DNA repair investigations. This review is focused on the studies of DNA repair pathways in C. reinhardtii. Emphasis is given to the connection of DNA repair with other cellular functions, namely the regulation of the cell cycle. Comparison with the results of repair investigations that are already available revealed the presence of all basic repair pathways in C. reinhardtii as well as special features characteristic of this alga. Among others, the involvement of UVSE1 gene in recombinational repair and uniparental inheritance of chloroplast genome, the specific role of TRXH1 gene in strand break repair, the requirement of PHR1 gene for full activity of PHR2 gene, or encoding of two excision repair proteins by the single REX1 gene. Contrary to yeast, mammals and higher plants, C. reinhardtii does not appear to contain the ortholog of RAD6 gene, which plays an important role in DNA translesion synthesis and mutagenesis. Completed genome sequences will be a basis for molecular analyses allowing to explain the differences that have been observed in DNA repair of this alga in comparison with other model organisms.