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Agrobacterium tumefaciens is known to transfer part of its tumor-inducing (Ti) plasmid to the filamentous fungus Aspergillus awamori by illegitimate recombination with the fungal genome. Here, we show that when this Ti DNA shares homology with the A. awamori genome, integration can also occur by homologous recombination. On the basis of this finding, we have developed an efficient method for constructing recombinant mold strains free from bacterial DNA by A. tumefaciens-mediated transformation. Multiple copies of a gene can be integrated rapidly at a predetermined locus in the genome, yielding transformants free of bacterial antibiotic resistance genes or other foreign DNA. Recombinant A. awamori strains were constructed containing up to nine copies of a Fusarium solani pisi cutinase expression cassette integrated in tandem at the pyrG locus. This allowed us to study how mRNA and protein levels are affected by gene copy number, without the influence of chromosomal environmental effects. Cutinase mRNA and protein were maximal with four gene copies, indicating a limitation at the transcriptional level. This transformation system will potentially stimulate market acceptance of derived products by avoiding introduction of bacterial and other foreign DNA into the fungi.

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A rhamnogalacturonan hydrolase gene of Aspergillus aculeatus was used as a probe for the cloning of two rhamnogalacturonan hydrolase genes of Aspergillus niger. The corresponding proteins, rhamnogalacturonan hydrolases A and B, are 78 and 72% identical, respectively, with the A. aculeatus enzyme. In A. niger cultures which were shifted from growth on sucrose to growth on apple pectin as a carbon source, the expression of the rhamnogalacturonan hydrolase A gene (rhgA) was transiently induced after 3 h of growth on apple pectin. The rhamnogalacturonan hydrolase B gene was not induced by apple pectin, but the rhgB gene was derepressed after 18 h of growth on either apple pectin or sucrose. Gene fusions of the A. niger rhgA and rhgB coding regions with the strong and inducible Aspergillus awamori exlA promoter were used to obtain high-producing A. awamori transformants which were then used for the purification of the two A. niger rhamnogalacturonan hydrolases. High-performance anion-exchange chromatography of oligomeric degradation products showed that optimal degradation of an isolated highly branched pectin fraction by A. niger rhamnogalacturonan hydrolases A and B occurred at pH 3.6 and 4.1, respectively. The specific activities of rhamnogalacturonan hydrolases A and B were then 0.9 and 0.4 U/mg, respectively, which is significantly lower than the specific activity of A. aculeatus rhamnogalacturonan hydrolase (2.5 U/mg at an optimal pH of 4.5). Compared to the A enzymes, the A. niger B enzyme appears to have a different substrate specificity, since additional oligomers are formed.

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In this study, induction and repression kinetics of the expression of the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) gene under defined physiological conditions was analyzed at the mRNA and the protein levels. Induction was analyzed by pulsing D-xylose to a sucrose-limited continuous culture of an A. awamori 1,4-beta-endoxylanase A (EXLA)-overproducing strain. Directly after the D-xylose pulse, exIA mRNA was synthesized, and it reached a constant maximal level after 45 to 60 min. This level was maintained as long as D-xylose was present. The kinetics of mRNA synthesis of the genes encoding Thermomyces lanuginosa lipase (lplA) and Escherichia coli beta-glucuronidase (uidA), which were also under the control of the exlA promoter, were similar to those observed for exlA mRNA. The repression of exlA expression was analyzed by pulsing D-glucose to a D-xylose-limited continuous culture. Immediately after the glucose pulse, the exlA mRNA level declined rapidly, with a half-life of approximately 20 to 30 min, and it reached a minimal level after 60 to 90 min. The time span between mRNA synthesis and the secretion of proteins was determined for EXLA and lipase. In both cases, mRNA became visible after approximately 7.5 min. After 1 h, both proteins became detectable in the medium but the rate of secretion of EXLA was faster than that of lipase.

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A new, highly inducible fungal promoter derived from the Aspergillus awamori 1,4-beta-endoxylanase A (exlA) gene is described. Induction analysis, carried out with the wild-type strain in shake flasks, showed that exlA expression in regulated at the transcriptional level. Using a beta-glucuronidase (uidA) reporter strategy, D-xylose was shown to be an efficient inducer of the exlA promoter, whereas sucrose or maltodextrin were not. Upon D-xylose induction, the exlA promoter was threefold more efficient than the frequently used A. niger glucoamylase (glaA) promoter under maltodextrin induction. Detailed induction analyses demonstrated that induction was dependent on the presence of D-xylose in the medium. Carbon-source-limited chemostat cultures with the uidA reporter strain showed that D-xylose was also a very good inducer in a fermenter, even in the presence of sucrose.

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A synthetic derivative of the cutinase cDNA of Fusarium solani pisi was expressed in Aspergillus awamori using the A. awamori endoxylanase II (exlA) promoter and terminator. The influence of the origin of the pre-sequence and the presence of a pro-sequence on the efficiency of extracellular cutinase production was analysed in single-copy transformants containing an expression cassette integrated at the pyrG locus. Transformants containing a construct encoding a direct, inframe fusion of the xylanase pre-peptide to the mature cutinase showed a 2-fold higher cutinase production level compared to strains containing constructs with an additional cutinase pro-peptide. The effect of multicopy integration of the expression cassette on cutinase production was analysed in strains with different numbers of a cutinase construct containing its own pre-pro-sequence. The multicopy strains showed a 6-to 12-fold increased production of extracellular cutinase relative to the single-copy strains. No linear dose response relation to the number of expression cassettes present in the strains was observed. The amount of active enzyme produced by the strains correlated with the amount of cutinase-specific mRNA, suggesting that cutinase overproduction is not limited at the level of translation or secretion.

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Rhamnogalacturonase was purified from culture filtrate of Aspergillus aculeatus after growth in medium with sugar-beet pulp as carbon source. Purified protein was used to raise antibodies in mice and with the antiserum obtained a gene coding for rhamnogalacturonase (rhgA) was isolated from a lambda cDNA expression library. The cloned rhgA gene has an open-reading frame of 1320 base pairs encoding a protein of 440 amino acids with a predicted molecular mass of 45 962 Da. The protein contains a potential signal peptidase cleavage site behind Gly-18 and three potential sites for N-glycosylation. Limited homology with A. niger polygalacturonase amino acid sequences is found. A genomic clone of rhgA was isolated from a recombinant phage lambda genomic library. Comparison of the genomic and cDNA sequences revealed that the coding region of the gene is interrupted by three introns. Furthermore, amino acid sequences of four different peptides, derived from purified A. aculeatus rhamnogalacturonase, were also found in the deduced amino acid sequence of rhgA. A. aculeatus strains overexpressing rhamnogalacturonase were obtained by cotransformation using either the A. niger pyrA gene or the A. aculeatus pyrA gene as selection marker. For expression of rhamnogalacturonase in A. awamori the A. awamori pyrA gene was used as selection marker. Degradation patterns of modified hairy regions, determined by HPLC, show the recombinant rhamnogalacturonase to be active, and the enzyme was found to have a positive effect in the apple hot-mash liquefaction process.

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A copy of the cutinase cDNA from Fusarium solani pisi was constructed starting from synthetic oligonucleotides. For this construction three separate cassettes were made, which were subsequently assembled to form the cutinase gene. Heterologous expression of the synthetic cutinase gene and the subsequent secretion of the recombinant enzyme was achieved in Saccharomyces cerevisiae and Aspergillus awamori.

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A homologous gene transfer system for Aspergillus awamori for site-specific integration is described, based on two components. First, a defined A. awamori pyrG mutant strain constructed by a selection strategy for gene-replacement in fungi. Second, a vector with a homologous pyrG selection marker containing a defined mutation at a site different from that of the mutations in the pyrG gene of the defined mutant strain. Defined mutation in the A. awamori pyrG gene, isolated from a genomic library by heterologous hybridisation with the A. niger pyrG gene as a probe, were introduced by specifically altering sequences at restriction sites in the coding region of the gene. After transformation of the A. awamori wild-type strain with vectors containing these mutated pyrG genes, and selection for 5-fluoro-orotic acid resistance (5-FOAR), on the average 60% of the 5-FOAR colonies originated from replacement of the wild-type pyrG gene by the mutated pyrG allele. After transformation of a mutant strain, carrying a mutation near the 5' end of the pyrG gene with vectors containing a mutation near the 3' end of the pyrG gene, 35% of the resulting transformants contained one copy of the vector at the pyrG locus.

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Replacement of the protein L11 binding domain within Escherichia coli 23S ribosomal RNA (rRNA) by the equivalent region from yeast 26S rRNA appeared to have no effect on the growth rate of E.coli cells harbouring a plasmid carrying the mutated rrnB operon. The hybrid rRNA was correctly processed and assembled into ribosomes, which accumulated normally in polyribosomes. Of the total ribosomal population, < 25% contained wild-type, chromosomally encoded rRNA; the remainder were mutant. The hybrid ribosomes supported GTP hydrolysis dependent upon E.coli elongation factor G, although at a somewhat reduced rate compared with wild-type particles, and were sensitive to the antibiotic, thiostrepton, a potent inhibitor of ribosomal GTPase activity that binds to 23S rRNA within the L11 binding domain. That thiostrepton could indeed bind to the mutant ribosomes, although at a reduced level relative to that seen with wild-type ribosomes, was confirmed in a non-equilibrium assay. The rationale for the ability of the hybrid ribosomes to bind the antibiotic, given that yeast ribosomes do not, was provided when yeast rRNA was shown by equilibrium dialysis to bind thiostrepton only 10-fold less tightly than did E.coli rRNA. The extreme conservation of secondary, but not primary, structure in this region between E.coli and yeast rRNAs allows the hybrid ribosomes to function competently in protein synthesis and also preserves the interaction with thiostrepton.

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The LEU2 gene, coding for beta-isopropylmalate dehydrogenase, of the yeast Kluyveromyces marxianus was isolated and sequenced. An open reading frame, coding for a protein with a molecular weight of 38 kDa was found. Comparison of the deduced amino acid sequence of the LEU2 gene with the corresponding enzymes of three other yeasts and two thermophilic bacteria, revealed extensive sequence similarities. The cloned gene could complement a leuB mutation of Escherichia coli and a leu2 mutation of Saccharomyces cerevisiae. Using orthogonal field alternation gel electrophoresis, the genomic copy of the gene was found to be located at chromosome VI or VII. Analysis of the 5'-untranslated region indicated the presence of a putative binding site for the LEU3 protein, which is involved in the leucine-specific regulation of transcription. We show that the cloned gene can be used for the construction of a non-reverting K. marxianus leu2 mutant.

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Using the "tagged" rRNA gene system, which allows in vivo mutational analysis of Saccharomyces cerevisiae rRNA, we studied the role of two distinct structural elements of 26S rRNA in ribosome biogenesis and function--namely, the evolutionarily highly conserved "GTPase center" located in domain II and the eukaroyote-specific variable region V9 in domain III. Replacement of the S. cerevisiae GTPase center with its counterpart from Escherichia coli did not affect the assembly of the mutant 26S rRNA into functional (as judged by their polysomal distribution) 60S subunits, indicating that the E. coli GTPase center functions efficiently in the context of the heterologous rRNA. Removal of most of the S. cerevisiae V9 region or replacement of this segment by the equivalent segment from mouse 28S rRNA also did not affect the formation of functional 60S subunits carrying the mutant 26S rRNA. Therefore, the V9 region does not seem to play a role in the biological functioning of the yeast 60S subunits, and these subunits appear to be able to accommodate V9 regions of various size and secondary structure without apparent loss of function.

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Making use of an rDNA unit, containing oligonucleotide tags in both the 17S and 26S rRNA gene, we have analyzed the effect of various deletions in the External Transcribed Spacer (ETS) and in one of the Internal Transcribed Spacers 1 (ITS1) on the process of ribosome formation in yeast. By following the fate of the tagged transcripts of this rDNA unit in vivo by Northern hybridization we found that deleting various parts of the ETS prevents the accumulation of tagged 17S rRNA and its assembly into 40S subunits, but not the formation of 60S subunits. Deleting the central region of ITS1, including a processing site that is used in an early stage of the maturation process, was also found to prevent the accumulation of functional 49 S subunits, whereas no effect on the formation of 60S subunits was detected. The implications of these findings for yeast pre-rRNA processing are discussed.

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To define the RNA polymerase I promoter in the rDNA of Saccharomyces cerevisiae more precisely, we have constructed a series of 5'- and 3'-deletion mutants in a novel, plasmid-borne rDNA minigene, that also contains the transcriptional enhancer. Our data show that the Pol I promoter, in this context, extends from position -155 to +27, with 5'-deletions up to -134 and 3'-deletions up to -2 removing essential sequence information. To investigate the internal organization of the yeast Pol I promoter, linker scanning mutants were constructed, that traverse the Pol I promoter region and comprise between 5 and 12 clustered point mutations. Analysis of minigene transcription in yeast cells transformed with these plasmids demonstrates that the pol I promoter consists of three domains. Mutations in Domain I (from position -28 to +8) and Domain II (-70 to -51) drastically reduce promoter activity, whereas clustered point mutations in Domain III (starts at position -146 and presumably extends to position -76) appear to have less effect. Furthermore, the insertion of 4 nt between Domains I and II diminishes minigene transcription, indicating that the relative positions of these domains is essential.

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To develop a system for the analysis of eucaryotic ribosomal DNA (rDNA) mutations, we cloned a complete, transcriptionally active rDNA unit from the yeast Saccharomyces cerevisiae on a centromere-containing yeast plasmid. To distinguish the plasmid-derived ribosomal transcripts from those encoded by the rDNA locus, we inserted a tag of 18 base pairs within the first expansion segment of domain I of the 26S rRNA gene. We demonstrate that this insertion behaves as a neutral mutation since tagged 26S rRNA is normally processed and assembled into functional ribosomal subunits. This system allows us to study the effect of subsequent mutations within the tagged rDNA unit on the biosynthesis and function of the rRNA. As a first application, we wanted to ascertain whether the assembly of a 60S subunit is dependent on the presence in cis of an intact 17S rRNA gene. We found that a deletion of two-thirds of the 17S rRNA gene has no effect on the accumulation of active 60S subunits derived from the same operon. On the other hand, deletions within the second domain of the 26S rRNA gene completely abolished the accumulation of mature 26S rRNA.

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Deletions in the promoter region of the 37S pre-rRNA operon in yeast were constructed and analysed in vivo using an artificial ribosomal minigene present on an extrachromosomal yeast vector. Sequences required for correct transcription initiation were found to be located between positions -192 and +15 relative to the start; a 5'-deletion down to position -133 reduces the transcription yield of the minigene at least five-fold. To allow detection of transcription of the minigene in isolated nuclei of yeast transformed with a minigene-bearing plasmid we attempted to increase the minigene copy number. The transcription yield in vivo appeared not to be proportional to the copy number but was found to be greatly enhanced when two or three minigenes are present in tandem. alpha-Amanitin sensitivity of transcription of these minigenes in isolated nuclei proved that RNA polymerase I is responsible for their transcription.

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We constructed an artificial yeast rRNA gene and studied its transcription after introduction into a recipient yeast strain. The artificial gene comprised a fragment containing the sequence from position -207 to +128 relative to the site of initiation of Saccharomyces carlsbergensis 37S pre-rRNA, followed by a marker fragment from Spirodela oligorhiza chloroplast DNA and finally a fragment containing the sequence from position -36 to +101 relative to the 3' end of the 26S rRNA gene. The resulting construct was cloned into the yeast-Escherichia coli shuttle vector pJDB207. Both Northern blot hybridization and R-loop analysis of RNA from transformed Saccharomyces cerevisiae cells revealed a discrete transcript of the expected length. S1 nuclease mapping as well as primer extension analysis showed that the major proportion of the transcripts was initiated at exactly the same site as 37S pre-rRNA. These results show that the respective rDNA fragments contain the information for correct initiation of transcription and formation of the 3' end. A minor proportion of the transcripts was initiated at a number of sites between positions -1 and -100 upstream of the predominant start. The proportion and the pattern of these upstream starts is affected by the vector context of the artificial rRNA gene.

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A method of preparing antibodies against c mu 3 and c mu 4 domains of human IgM is described. c mu 3- and c mu 4-binding antibody fractions were isolated by affinity chromatography from IgG fractions of antisera raised against Fc5 mu and Fc mu' fragments. c mu 3 and c mu 4 fragments had been prepared from human IgM kappa (Key) by hot trypsin digestion. Haemagglutination inhibition tests showed that the c mu 4-binding fraction only reacted with c mu 4 fragments. The c mu 3-binding fraction reacted with c mu 3 fragments but showed a minor reaction with c mu 4 fragments. Immunization with Fc mu' fragments predominantly yielded antibodies against the c mu 3 domain, whereas immunization with Fc5 mu fragments yielded antibodies more directed against the c mu 4 domain. Immunization with isolated c mu 4 fragments led to the production of antibodies which reacted with the isolated c mu 4 domain but not with the c mu 4 domain within the larger structures of Fc mu' or Fc5 mu fragments.

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