Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)

We have used a plasmid containing the argB gene to transform an Aspergillus nidulansargB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and ～3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A.?nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants. Received: 6 March 1998 / Accepted: 25 May 1998     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53 269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80° C; ammonia-dependent reaction, 90° C). Received: 30 October 1996 / Accepted: 13 January 1997     

Short latency vestibular evoked potentials in the Japanese quail (Coturnix coturnix japonica)

Short-latency vestibular-evoked potentials to pulsed linear acceleration were characterized in the quail. Responses occurred within 8 ms following the onset of stimuli and were composed of a series of positive and negative peaks. The latencies and amplitudes of the first four peaks were quantitatively characterized. Mean latencies at 1.0 g ms⁻¹ ranged from 1265 ± 208 μs (P1, N = 18) to 4802 ± 441 μs (N4, N = 13). Amplitudes ranged from 3.72 ± 1.51 μV (P1/N1, N = 18) to 1.49 ± 0.77 μV (P3/N3, N = 16). Latency-intensity (LI) slopes ranged from −38.7 ± 7.3 μs dB⁻¹ (P1, N = 18) to −71.6 ± 21.9 μs dB⁻¹ (N3, N = 15) and amplitude-intensity (AI) slopes ranged from 0.20 ± 0.08 μV dB⁻¹ (P1/N1, N = 18) to 0.07 ± 0.04 μV dB⁻¹ (P3/N3, N = 11). The mean response threshold across all animals was −21.83 ± 3.34 dB re: 1.0 g ms⁻¹ (N = 18). Responses remained after cochlear extirpation showing that they could not depend critically on cochlear activity. Responses were eliminated by destruction of the vestibular end organs, thus showing that responses depended critically and specifically on the vestibular system. The results demonstrate that the responses are vestibular and the findings provide a scientific basis for using vestibular responses to evaluate vestibular function through ontogeny and senescence in the quail. Accepted: 18 January 1997     

A physical assay for meiotic recombination in Coprinus cinereus

We have used the polymerase chain reaction (PCR) method to monitor meiotic recombination in the basidiomycete Coprinus cinereus. We used DNA-mediated transformation to recover strains with modifications of the trp1 locus. The modifications were designed to introduce unique PCR priming sites separated by a homologous 2.4 kb region in which crossing over could occur. We showed that exchange occurred in this region at the frequency expected for a typical region of this genome (2.4 kb should correspond to a genetic length of 0.08 cM). We also detected products resulting from crossing over in DNAs extracted from cells in meiotic prophase. The assay should be useful for monitoring exchange in mutants that cannot complete meiosis. Received: 5 September 1996 / Accepted: 1 December 1996     

Restriction enzyme-mediated DNA integration in Coprinus cinereus

Restriction enzyme-mediated DNA integration (REMI) has recently received attention as a new technique for the generation of mutants by transformation in fungi. Here we analyse this method in the basidiomycete Coprinus cinereus using the homologous pab1 gene as a selectable marker and the restriction enzymes BamHI, EcoRI and PstI. Addition of restriction enzymes to transformation mixtures results in an earlier appearance of transformants and influences transformation rates in an enzyme- and concentration-dependent manner. Low concentrations of restriction enzyme result in increased numbers of transformants compared to no addition of enzymes. Transformation rates decrease with higher enzyme concentrations. If protoplasts are made from cells stored in the cold, the transformation rates drop drastically even in the presence of low amounts of enzyme. In several transformants, plasmid integration directly correlated with the action of restriction enzyme at random chromosomal restriction sites. In some cases, restriction enzymes appear to reduce the number of integration events per transformant. Simultaneously, mutation rates can be enhanced due to the presence of restriction enzymes. Although restriction enzymes clearly promote plasmid integration into the host genome they also have cytotoxic and possibly mutagenic effects that result from processes other than plasmid integration. In consequence, for any given enzyme used in REMI mutagenesis, the enzyme concentration that gives the highest number of transformants must be defined experimentally. Such optimal transformation conditions should give the highest probability of obtaining mutations caused by a single restriction enzyme-mediated integration of the selection marker. Received: 2 September 1996 / Accepted: 7 April 1997     

Identification of plant volatiles activating single receptor neurons in the pine weevil (Hylobius abietis)

Naturally produced plant volatiles, eliciting responses of single olfactory receptor neurons in the pine weevil, have been identified by gas chromatography linked with mass spectrometry. The receptor neurons (n = 72) were classified in 30 types, according to the compound which elicited the strongest response in each neuron, 20 of which compounds were identified. Most potent for 14 types of neurons (n = 50) were monoterpenes, including bicyclic (e.g. α-pinene, camphor and myrtenal) for 8 types (n = 32), monocyclic (limonene, carvone, α-terpinene) for 3 types (n = 12) and acyclic (e.g. β-myrcene and linalool) for 3 types (n = 6). Other compounds eliciting strongest responses of a neuron were five sesquiterpenes, including α-copaene and a farnesene-isomer, and an anethole type which has no biosynthetic relationship with terpenes. Within one type, receptor neurons with quite selective responses to the most potent compound as well as neurons with additional responses to several, structurally similar compounds were found, indicating that the neurons may have the same functional types of membrane receptors, but different sensitivities. Response spectra of neurons within the bicyclic-, mono-cyclic and acyclic types showed more overlapping than across the neuron types. Minimal overlapping response spectra was found between monoterpene and sesquiterpene neurons. The results suggest that this structure-activity relationship is significant for encoding plant odour information in the pipe weevil. Accepted: 6 January 1997     

Cercosporin-deficient mutants by plasmid tagging in the asexual fungus<Emphasis Type="Italic"> Cercospora nicotianae</Emphasis>

We have successfully adapted plasmid insertion and restriction enzyme-mediated integration (REMI) to produce cercosporin toxin-deficient mutants in the asexual phytopathogenic fungus Cercospora nicotianae. The use of pre-linearized plasmid or restriction enzymes in the transformation procedure significantly decreased the transformation frequency, but promoted a complicated and undefined mode of plasmid integration that leads to mutations in the C. nicotianae genome. Vector DNA generally integrated in multiple copies, and no increase in single-copy insertion was observed when enzymes were added to the transformation mixture. Out of 1873 transformants tested, 39 putative cercosporin toxin biosynthesis ( ctb) mutants were recovered that showed altered levels of cercosporin production. Seven ctb mutants were recovered using pre-linearized plasmids without the addition of enzymes, and these were considered to be non-REMI mutants. The correlation between a specific insertion and a mutant phenotype was confirmed using rescued plasmids as gene disruption vectors in the wild-type strain. Six out of fifteen rescued plasmids tested yielded cercosporin-deficient transformants when re-introduced into the wild-type strain, suggesting a link between the insertion site and the cercosporin-deficient phenotype. Sequence analysis of a fragment flanking the insert site recovered from one insertion mutant showed it to be disrupted in sequences with high homology to the acyl transferase domain of polyketide synthases from other fungi. Disruption of this polyketide synthase gene ( CTB1) using a rescued plasmid resulted in mutants that were defective in cercosporin production. Thus, we provide the first molecular evidence that cercosporin is synthesized via a polyketide pathway as previously hypothesized.Communicated by E. Cerdá-Olmedo     

Genetic evidence for 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) as a negative effector of cytochrome terminal oxidase cbb 3 production in Rhizobium etli

A Rhizobium etli Tn5mob-induced mutant (CFN035) exhibits an enhanced capacity to oxidize N,N,N′,N′, tetramethyl-p -phenylenediamine (TMPD), a presumptive indicator of elevated cytochrome c terminal oxidase activity. Sequencing of the mutated gene in CFN035 revealed that it codes for the amidophosphoribosyl transferase enzyme (PurF) that catalyzes the first step in the purine biosynthetic pathway. Two c-type cytochromes with molecular weights of 32 and 27 kDa were produced in strain CFN035, which also produced a novel CO-reactive cytochrome (absorbance trough at 553 nm), in contrast to strain CE3 which produced a single 32 kDa c-type protein and did not produce the 553 nm CO-reactive cytochrome. A wild-type R. etli strain that expresses the Bradyrhizobium japonicum fixNOQP genes, which code for the symbiotic cytochrome terminal oxidase cbb ₃, produced similar absorbance spectra (a trough at 553 nm in CO-difference spectra) and two c -type proteins similar in size to those of strain CFN035, suggesting that CFN035 also produces the cbb ₃ terminal oxidase. The expression of a R. etli fixN-lacZ gene fusion was measured in several R. etli mutants affected in different steps of the purine biosynthetic pathway. Our analysis showed that purF, purD, purQ, purL, purY, purK and purE mutants expressed three-fold higher levels of the fixNOQP operon than the wild-type strain. The derepressed expression of fixN was not observed in a purH mutant. The purH gene product catalyzes the conversion of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) to 5-formaminoimidazole-4-carboxamide ribonucleotide (FAICAR) and inosine. Supplementation with AICA riboside lowered the levels of fixN expression in the purF mutants. These data are consistent with the possibility that AICAR, or a closely related metabolite, is a negative effector of the production of the symbiotic terminal oxidase cbb ₃ in R. etli. Received: 21 November 1996 / Accepted: 22 January 1997     

The influence of either no fluid or carbohydrate-electrolyte fluid ingestion and the environment (thermoneutral versus hot and humid) on running economy after prolonged, high-intensity exercise

This study investigated the effects on running economy (RE) of ingesting either no fluid or an electrolyte solution with or without 6% carbohydrate (counterbalanced design) during 60-min running bouts at 80% maximal oxygen consumption (V˙O_2max). Tests were undertaken in either a thermoneutral (22–23°C; 56–62% relative humidity, RH) or a hot and humid natural environment (Singapore: 25–35°C; 66–77% RH). The subjects were 15 young adult male Singaporeans [V˙O_2max = 55.5 (4.4 SD) ml kg⁻¹ min⁻¹]. The RE was measured at 3 m s⁻¹ [65 (6)% V˙O_2max] before (RE1) and after each prolonged run (RE2). Fluids were administered every 2 min, at an individual rate determined from prior tests, to maintain body mass (group mean = 17.4 ml min⁻¹). The V˙O₂ during RE2 was higher (P < 0.05) than that during the RE1 test for all treatments, with no differences between treatments (ANOVA). The mean increase in V˙O₂ from RE1 to RE2 ranged from 3.4 to 4.7 ml kg⁻¹ min⁻¹ across treatments. In conclusion, the deterioration in RE at 3 m s⁻¹ (65% V˙O_2max) after 60 min of running at 80% V˙O_2max appears to occur independently of whether fluid is ingested and regardless of whether the fluid contains carbohydrates or electrolytes, in both a thermoneutral and in a hot, humid environment. Accepted: 30 October 1997     

Cold tolerance and dehydration in Enchytraeidae from Svalbard

When cooled in contact with moisture, eight species of arctic Enchytraeidae from Svalbard were killed by freezing within minutes or hours at −3 and −5 °C; an exception was Enchytraeus kincaidi which survived for up to 2 days. When the temperature approached 0 °C the enchytraeids apparently tried to escape from the moist soil. The supercooling capacity of the enchytraeids was relatively low, with mean supercooling points of −5 to −8 °C. In contrast, specimens of several species were extracted from soil cores that had been frozen in their intact state at −15 °C for up to 71 days. Compared to freezing in a moist environment, higher survival rates were obtained during cooling at freezing temperatures in dry soil. Survival was recorded in species kept at −3 °C for up to 35 days, and in some species kept at −6 °C for up to 17 days. Slow warming greatly increased survival rates at −6 °C . The results strongly suggest that arctic enchytraeids avoid freezing by dehydration at subzero temperatures. In agreement with this, weight losses of up to ca. 42% of fresh weight were recorded in Mesenchytraeus spp. and of up to 55% in Enchytraeus kincaidi at water vapour pressures above ice at −3 to −6 °C. All specimens survived dehydration under these conditions. Accepted: 12 December 1997     

The effect of gender on left ventricular function immediately after the wingate test

The effect of gender on left ventricular systolic function and exercise haemodynamics in healthy young subjects was studied during 30-s all-out sudden strenuous dynamic exercise. A group of 22 men [19.3 (SD 1) years] 20 women [19.1 (SD 1) years] volunteered to participate in this study. Two-dimensional direct M-mode and Doppler echocardiograph studies were performed with the subject in the sitting position. The Doppler examination of flow was located with continuous-wave, interrogating ascending aorta measurements. The subjects completed the study without showing any electrocardiograph abnormalities. An interaction effect with stroke volume (P < 0.05) was characterized by a decrease in the men and an increase of stroke volume in the women. Cardiac output rose significantly (P < 0.05) up to 14.5 (SD 6) l · min⁻¹) for the men and 12.1 (SD 4) l · min⁻¹ for the women compared to the rest values [5.8 (SD 0.4) and 4.7 (SD 0.5) l · min⁻¹, respectively]. Flow velocity integral and acceleration time differed significantly between the two groups at rest (P < 0.05). During exercise these differences showed an interaction effect (P < 0.05). These results would indicate that normal men and women respond to sudden strenuous exercise by reducing their left ventricular systolic function, with a significantly greater decrease in women (P < 0.05). The gender differences in the haemodynamic responses during the present study, may, as suggested by others, be attributable to differences in energy metabolism. In addition, changes in Doppler parameters of aortic flow, haemodynamics and blood pressure responses during sudden strenuous exercise differed markedly from those seen before with endurance exercise. Accepted: 8 January 1997     

Screening and identification of yeast sequences that cause growth inhibition when overexpressed

To isolate genes that negatively regulate cell growth, we constructed a galactose-inducible expression library with partially digested Saccharomyces cerevisiae genomic DNA fragments inserted downstream of the GAL10 promoter. In all, 240 000 yeast transformants were screened for lethality on galactose medium. From 17 such transformants identified, 16 nonoverlapping DNA sequences were obtained. Restriction mapping and determination of DNA sequences adjacent to the GAL10 promoter indicated that the inserts encoded part or all of the URA2, RBP1, TPK3, SAC7, BOI1, and BNI1 genes, and also open reading frames (ORFs) from chromosomes IV, V, IX, XI, and XIII. Some of the identified sequences lacked the amino-terminal sequences of the ORFs, suggesting that truncated forms of the proteins might be necessary for growth inhibition. The sequence of the pGA108 insert was highly homologous to the telomeric X-element and contained an ARS consensus sequence, suggesting a possible growth inhibitory effect of an RNA molecule. Overexpression of the BNI1ΔN and BOI1ΔN genes, which lacked amino-terminal sequences, was associated with phenotypes similar to those of mutants defective in bud formation. Overexpression of the GIN4 and GIN12 sequences induced elongated buds and a G2/M arrest-like phenotype, respectively. The phenotypes induced by the overexpression of our cloned sequences could result from either a dominant-positive or a dominant-negative effect and, unexpectedly, in one case from an effect of an RNA. Received: 3 June 1996 / Accepted: 1 October 1996     

Stem respiration of ponderosa pines grown in contrasting climates: implications for global climate change

We examined the effects of climate and allocation patterns on stem respiration in ponderosa pine (Pinus ponderosa) growing on identical substrate in the cool, moist Sierra Nevada mountains and the warm, dry, Great Basin Desert. These environments are representative of current climatic conditions and those predicted to accompany a doubling of atmospheric CO₂, respectively, throughout the range of many western north American conifers. A previous study found that trees growing in the desert allocate proportionally more biomass to sapwood and less to leaf area than montane trees. We tested the hypothesis that respiration rates of sapwood are lower in desert trees than in montane trees due to reduced stem maintenance respiration (physiological acclimation) or reduced construction cost of stem tissue (structural acclimation). Maintenance respiration per unit sapwood volume at 15°C did not differ between populations (desert: 6.39 ± 1.14 SE μmol m⁻³ s⁻¹, montane: 6.54 ± 1.13 SE μmol m⁻³ s⁻¹, P = 0.71) and declined with increasing stem diameter (P = 0.001). The temperature coefficient of respiration (Q ₁₀) varied seasonally within both environments (P = 0.05). Construction cost of stem sapwood was the same in both environments (desert: 1.46 ± 0.009 SE g glucose g⁻¹ sapwood, montane: 1.48 ± 0.009 SE glucose g⁻¹ sapwood, P = 0.14). Annual construction respiration calculated from construction cost, percent carbon and relative growth rate was greater in montane populations due to higher growth rates. These data provide no evidence of respiratory acclimation by desert trees. Estimated yearly stem maintenance respiration was greater in large desert trees than in large montane trees because of higher temperatures in the desert and because of increased allocation of biomass to sapwood. By analogy, these data suggest that under predicted increases in temperature and aridity, potential increases in aboveground carbon gain due to enhanced photosynthetic rates may be partially offset by increases in maintenance respiration in large trees growing in CO₂-enriched atmospheres. Received: 4 November 1996 / Accepted: 23 January 1997     

Homeostatic responses in the gut of Porcellio scaber (Isopoda: Oniscidea) optimize litter degradation

In order to examine the influence of differences in food conditions on gut characteristics in Porcellio scaber, pH-manipulated and microbially inoculated leaf litter from three different tree species were offered. Microbial activity was clearly influenced by the pH levels of the leaves. Analyses of the pH levels in the gut indicated the ability of P. scaber to buffer the pH value in the intestinal tract to about 5.5–6.0 in the anterior hindgut, and to about 6.0–6.5 in the posterior hindgut. The pH levels of the gut sections remained in this range, within a range of food pH from 4.0 to 7.5, no matter what kind of leaves the animals were fed. Homeostatic responses to changes in food pH guarantee optimized digestion of leaf litter. However, when the pH level of the litter dropped below 3.5, P. scaber was not able to maintain the pH conditions in the gut. Furthermore, microorganisms colonizing the litter biased the pH level in the anterior hindgut where digestive processes mainly take place. These results indicate a decline of litter quality with regard to the nutrition of terrestrial isopods, caused by acidification and consequently reduced microbial activity. Accepted: 19 July 1997     

A germline restricted, highly repetitive DNA sequence in Paramyxineatami : an interspecifically conserved, but somatically eliminated, element

In some species of hagfish, the phenomenon of chromosome elimination occurs during embryogenesis. However, only two repetitive DNA families are known to be represented in chromosomes that are eliminated from somatic cells of the Japanese hagfish Eptatretus okinoseanus. Using molecular analyses, another germ line-restricted, highly repetitive DNA family has been detected in another Japanese hagfish, Paramyxine atami. The repeat unit of this family, which is 83 bp long, has been designated “EEPa1”, for Eliminated Element of P. atami 1. DNA filter hybridization using EEPa1 as a probe revealed that this family is shared among several species and is conserved in the germline DNA. Although eliminated, repetitive DNA that is shared interspecifically has not been reported in hagfish species, cases of chromatin diminution and chromosome elimination processes have been described previously in other organisms.The patterns and intensities of hybridization signals suggest that members of the repetitive DNA family defined by EEPa1 have undergone concerted molecular evolution. Received: 7 January 1997 / Accepted: 13 May 1997     

红曲霉不同转化方法比较

为了研究红曲霉聚酮体途径,考察和比较了4种不同的转化方法以建立有效的红曲霉遗传转化系统。以潮霉素作为抗性筛选标记,pBC-Hygro作为转化载体,用基于原生质体的传统转化和电击转化、基于萌发孢子的电击转化以及REMI技术转化红曲霉。发现基于萌发孢子的电击转化由于转化率极低而不适于红曲霉转化。基于原生质体的传统转化和电击转化尽管每微克DNA分别能获得135个转化子和125个转化子,但因转化子稳定性差也适合红曲霉转化的转化。应用REMI技术,转化率提高约20倍,每微克DNA 2500个转化子,70%～75%的转化子的稳定,非常适合于红曲霉的转化。      

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked. Received: 4 December 1996 / Accepted: 21 January 1997     

Analysis of eryBI , eryBIII and eryBVII from the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea

The gene cluster (ery) governing the biosynthesis of the macrolide antibiotic erythromycin A by Saccharopolyspora erythraea contains, in addition to the eryA genes encoding the polyketide synthase, two regions containing genes for later steps in the pathway. The region 5′ of eryA that lies between the known genes ermE (encoding the erythromycin resistance methyltransferase) and eryBIII (encoding a putative S-adenosylmethionine-dependent methyltransferase), and that contains the gene eryBI (orf2), has now been sequenced. The inferred product of the eryBI gene shows striking sequence similarity to authentic β-glucosidases. Specific mutants were created in eryBI, and the resulting strains were found to synthesise erythromycin A, showing that this gene, despite its position in the biosynthetic gene cluster, is not essential for erythromycin biosynthesis. A␣mutant in eryBIII and a double mutant in eryBI and eryBIII were obtained and the analysis of novel erythromycins produced by these strains confirmed the proposed function of EryBIII as a C-methyltransferase. Also, a chromosomal mutant was constructed for the previously sequenced ORF19 and shown to accumulate erythronolide B, as expected for an eryB mutant and consistent with its proposed role as an epimerase in dTDP-mycarose biosynthesis. Received: 13 August 1997 / Accepted: 27 November 1997