Agrobacterium-mediated insertional mutagenesis in the mycorrhizal fungus Laccaria bicolor.

Agrobacterium-mediated gene transfer (AMT) is extensively employed as a tool in fungal functional genomics and accordingly, in previous studies we used AMT on a dikaryotic strain of the ectomycorrhizal basidiomycete Laccaria bicolor. The interest in this fungus derives from its capacity to establish a symbiosis with tree roots, thereby playing a major role in nutrient cycling of forest ecosystems. The ectomycorrhizal symbiosis is a highly complex interaction involving many genes from both partners. To advance in the functional characterization of fungal genes, AMT was used on a monokaryotic L. bicolor. A collection of over 1200 transgenic strains was produced, of which 200 randomly selected strains were analyzed for their genomic T-DNA insertion patterns. By means of insertional mutagenesis, a number of transgenic strains were obtained displaying differential growth features. Moreover, mating with a compatible strain resulted in dikaryons that retained altered phenotypic features of the transgenic monokaryon. The analysis of the T-DNA integration pattern revealed mostly similar results to those reported in earlier studies, confirming the usefulness of AMT on different genetic backgrounds of L. bicolor. Taken together, our studies display the great versatility and potentiality of AMT as a tool for the genetic characterization of L. bicolor.

T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus.

The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.

T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus / Transferencia de T-DNA de Agrobacterium tumefaciens al hongo ectomicorrícico Pisolithus microcarpus

The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.

El hongo ectomicorrícico modelo Pisolithus microcarpus aislamiento 441 fue transformado utilizando Agrobacterium tumefaciens LBA 1100 y AGL-1. El marcador de selección fue el gen Shble de Streptoallotecius hidustanus, el cual confiere resistencia a fleomicina, bajo el control del promotor y terminador del gen gpd de Schizophyllum commune. La transformación resultó en clones resistentes a fleomicina comprobándose por PCR la presencia del transgen. La transferencia génica mediada por Agrobacterium podría permitir el desarrollo de la tecnología de interferencia por ARN en P. microcarpus.

T-DNA transfer from Agrobacterium tumefaciens to the ectomycorrhizal fungus Pisolithus microcarpus.

The model ectomycorrhizal fungus Pisolithus microcarpus isolate 441 was transformed by using Agrobacterium tumefaciens LBA1100 and AGL-1. The selection marker was the Shble gene of Streptoallotecius hidustanus, conferring resistance to phleomycin, under the control of the gpd gene promoter and terminator of Schizophyllum commune. Transformation resulted in phleomycin resistant clones which were confirmed by PCR to contain the resistance cassette. A. tumefaciens-mediated gene transfer would allow the development of RNA interference technology in P. microcarpus.

Characterization of small GTPases Cdc42 and Rac and the relationship between Cdc42 and actin cytoskeleton in vegetative and ectomycorrhizal hyphae of Suillus bovinus.

This work reports the isolation and molecular characterization of CDC42 and RAC1 cDNAs from the ectomycorrhiza forming filamentous homobasidiomycete Suillus bovinus. Previously, no RAC gene was described from filamentous fungi and no CDC42 gene was described from homobasidiomycetes. Southern hybridization with SbCDC42 and SbRAC1 cDNAs indicated that the S. bovinus genome contains only one CDC42 and one RAC1 gene. The predicted amino acid sequence of SbRaclp is 77% identical with the Rac1B protein of chick, whereas SbCdc42p is most identical with Schizosaccharomyces pombe Cdc42p, showing 88% identity. In the predicted amino acid sequences of SbRaclp and SbCdc42p, the five guanine nucleotide binding regions, switch I and II, and the effector domain are highly identical to those known in other small GTPases. These domain structures suggest that in S. bovinus, SbRac1p and SbCdc42p function as molecular switches regulating the organization of actin cytoskeleton, similar to yeasts and mammals. SbRAC1 and SbCDC42 were expressed in vegetative and ectomycorrhizal hyphae, and SbCdc42p was detected in ectomycorrhiza-forming hyphae if growth and differentiation of the symbiotic hyphae took place. Cdc42p and actin were localized at the tips of S. bovinus vegetative hyphae. Similar to yeast, in filamentous fungi Cdc42p may be necessary to maintain the actin cytoskeleton at hyphal tips, making the polarized growth of the hyphae possible. In developing ectomycorrhiza, Cdc42p and actin were visualized in association with plasma membrane in swollen cells typical to the symbiotic hyphae. The role of Cdc42p and actin in regulation of the growth pattern and morphogenesis of ectomycorrhizal hyphae is discussed.

Cellulose and xylan degrading enzymes in Thecotheus pelletieri

The ability to produce cellulose and xylan degrading enzymes by different strains of Thecotheus pelletieri, in liquid synthetic media with cellulose and xylan as inducers, was compared. All the strains tested were able to grow and produce cellulases and xylanases, being the strain BAFC 2077 the best producer. Several cultural conditions were analysed in order to optimise enzyme production by strain 2077. Shaking cultures gave higher yields of cellulases and xylanases compared with stationary ones. Asparagine at 0.75 g N/L was the best nitrogen source in promoting enzyme production. The influence of different surfactants on enzyme production was studied. Tween 80 exhibited no effect on growth and enzyme production, whereas Tween 20 and Triton X-100 were inhibitory. By means of studies of variation of cellulose/xylan ratio in the culture medium we determined that cellulose and xylan induced cellulase and xylanase synthesis, being the specific substrates the most effective. The inducible behavior of cellulases and xylanases in T. pelletieri was determined by means of inhibition of protein synthesis by cycloheximide and ethidium bromide. Moreover, we found that glucose as well as xylose repressed cellulase and xylanase synthesis in T. pelletieri.(AU)

Cellulose and xylan degrading enzymes in Thecotheus pelletieri

The ability to produce cellulose and xylan degrading enzymes by different strains of Thecotheus pelletieri, in liquid synthetic media with cellulose and xylan as inducers, was compared. All the strains tested were able to grow and produce cellulases and xylanases, being the strain BAFC 2077 the best producer. Several cultural conditions were analysed in order to optimise enzyme production by strain 2077. Shaking cultures gave higher yields of cellulases and xylanases compared with stationary ones. Asparagine at 0.75 g N/L was the best nitrogen source in promoting enzyme production. The influence of different surfactants on enzyme production was studied. Tween 80 exhibited no effect on growth and enzyme production, whereas Tween 20 and Triton X-100 were inhibitory. By means of studies of variation of cellulose/xylan ratio in the culture medium we determined that cellulose and xylan induced cellulase and xylanase synthesis, being the specific substrates the most effective. The inducible behavior of cellulases and xylanases in T. pelletieri was determined by means of inhibition of protein synthesis by cycloheximide and ethidium bromide. Moreover, we found that glucose as well as xylose repressed cellulase and xylanase synthesis in T. pelletieri.

Cellulose and xylan degrading enzymes in Thecotheus pelletieri.

The ability to produce cellulose and xylan degrading enzymes by different strains of Thecotheus pelletieri, in liquid synthetic media with cellulose and xylan as inducers, was compared. All the strains tested were able to grow and produce cellulases and xylanases, being the strain BAFC 2077 the best producer. Several cultural conditions were analysed in order to optimise enzyme production by strain 2077. Shaking cultures gave higher yields of cellulases and xylanases compared with stationary ones. Asparagine at 0.75 g N/L was the best nitrogen source in promoting enzyme production. The influence of different surfactants on enzyme production was studied. Tween 80 exhibited no effect on growth and enzyme production, whereas Tween 20 and Triton X-100 were inhibitory. By means of studies of variation of cellulose/xylan ratio in the culture medium we determined that cellulose and xylan induced cellulase and xylanase synthesis, being the specific substrates the most effective. The inducible behavior of cellulases and xylanases in T. pelletieri was determined by means of inhibition of protein synthesis by cycloheximide and ethidium bromide. Moreover, we found that glucose as well as xylose repressed cellulase and xylanase synthesis in T. pelletieri.

Cellulose and xylan degrading enzymes in Thecotheus pelletieri.

The ability to produce cellulose and xylan degrading enzymes by different strains of Thecotheus pelletieri, in liquid synthetic media with cellulose and xylan as inducers, was compared. All the strains tested were able to grow and produce cellulases and xylanases, being the strain BAFC 2077 the best producer. Several cultural conditions were analysed in order to optimise enzyme production by strain 2077. Shaking cultures gave higher yields of cellulases and xylanases compared with stationary ones. Asparagine at 0.75 g N/L was the best nitrogen source in promoting enzyme production. The influence of different surfactants on enzyme production was studied. Tween 80 exhibited no effect on growth and enzyme production, whereas Tween 20 and Triton X-100 were inhibitory. By means of studies of variation of cellulose/xylan ratio in the culture medium we determined that cellulose and xylan induced cellulase and xylanase synthesis, being the specific substrates the most effective. The inducible behavior of cellulases and xylanases in T. pelletieri was determined by means of inhibition of protein synthesis by cycloheximide and ethidium bromide. Moreover, we found that glucose as well as xylose repressed cellulase and xylanase synthesis in T. pelletieri.

Influence of temperature and pH on cellulase activity and stability in Nectria catalinensis.

The influence of temperature and pH on the activity and stability of the cellulase system (endoglucanase, exoglucanase and cellobiase) was investigated in Nectria catalinensis. Optimal temperature for the activity of the cellulase system ranged from 50 to 55 degrees C, with an optimum for stability between 23 degrees C and 37 degrees C after a 72 h incubation period. For the different enzymes, maximal activity was registered between pH 4.2-5.8, with pH 4.8 being close to optimal for all stability studies. The activation energy was 4.97 Kcal mol-1 for endoglucanase, 4.37 Kcal mol-1 for exoglucanase and 13.73 Kcal mol-1 for cellobiase. The K(m) and Vmax values were 1.73 mg CMC ml-1 and 0.45 mumol glucose min-1 mg protein-1 for endoglucanase, 0.22 mg microcrystalline cellulose ml-1 and 57.1 nmol glucose min-1 mg protein-1 for exoglucanase, and 2.95 mM cellobiose and 0.17 mumol glucose min-1 mg protein-1 for cellobiase.

Influence of temperature and pH on cellulase activity and stability in Nectria catalinensis

The influence of temperature and pH on the activity and stability of the cellulase system (endoglucanase, exoglucanase and cellobiase) was investigated in Nectria catalinensis. Optimal temperature for the activity of the cellulase system ranged from 50 to 55 degrees C, with an optimum for stability between 23 degrees C and 37 degrees C after a 72 h incubation period. For the different enzymes, maximal activity was registered between pH 4.2-5.8, with pH 4.8 being close to optimal for all stability studies. The activation energy was 4.97 Kcal mol-1 for endoglucanase, 4.37 Kcal mol-1 for exoglucanase and 13.73 Kcal mol-1 for cellobiase. The K(m) and Vmax values were 1.73 mg CMC ml-1 and 0.45 mumol glucose min-1 mg protein-1 for endoglucanase, 0.22 mg microcrystalline cellulose ml-1 and 57.1 nmol glucose min-1 mg protein-1 for exoglucanase, and 2.95 mM cellobiose and 0.17 mumol glucose min-1 mg protein-1 for cellobiase.

Influence of temperature and pH on cellulase activity and stability in Nectria catalinensis.

The influence of temperature and pH on the activity and stability of the cellulase system (endoglucanase, exoglucanase and cellobiase) was investigated in Nectria catalinensis. Optimal temperature for the activity of the cellulase system ranged from 50 to 55 degrees C, with an optimum for stability between 23 degrees C and 37 degrees C after a 72 h incubation period. For the different enzymes, maximal activity was registered between pH 4.2-5.8, with pH 4.8 being close to optimal for all stability studies. The activation energy was 4.97 Kcal mol-1 for endoglucanase, 4.37 Kcal mol-1 for exoglucanase and 13.73 Kcal mol-1 for cellobiase. The K(m) and Vmax values were 1.73 mg CMC ml-1 and 0.45 mumol glucose min-1 mg protein-1 for endoglucanase, 0.22 mg microcrystalline cellulose ml-1 and 57.1 nmol glucose min-1 mg protein-1 for exoglucanase, and 2.95 mM cellobiose and 0.17 mumol glucose min-1 mg protein-1 for cellobiase.

Influence of different cultural conditions on cellulase production by Nectria catalinensis.

The production of the extracellular cellulolytic enzyme system (endoglucanase, exoglucanase and cellobiase) of N. catalinensis was tested with different nitrogen sources, inorganic and organic ones, in liquid culture medium with microcrystalline cellulose. The nitrogen compounds used were: potassium nitrate, sodium nitrate, ammonium nitrate, ammonium phosphate, ammonium sulphate, ammonium chloride, ammonium carbonate, ammonium acetate, ammonium tartrate, urea, casamino acids, glycine, L-alanine, L-leucine, L-proline, L-lysine, L-aspartic acid, L-glutamic acid, L-asparagine, L-glutamine, L-phenylalanine, L-tyrosine, L-tryptophan, L-methionine and L-cysteine. Among these, ammonium nitrate and ammonium tartrate gave the highest yields of cellulases in 20-day-old cultures at a concentration equivalent to 0.75 g N/l in both cases. Optimal temperature for cellulase production, growth and cellulose degradation was 23 degrees C. On the other hand, an initial pH of 6.5 gave the highest yields of endoglucanase and cellobiase. In the same way, at pH 6.5, maximal growth and cellulose degradation were achieved. However, maximal exoglucanase production and glycogen content were reached at pH 7.5.

[Endoglucanase induction in Nectria catalinenesis with various carbon sources]. / Inducción de endoglucanasa en Nectria catalinensis con distintas fuentes de carbono.

The effect of different carbon sources on endoglucanase synthesis in Nectria catalinensis was investigated. Many different kinds of sugars (mono, di and polysaccharides) were added to the culture in order to investigate their effect on endoglucanase induction. The highest yield of cellulase was achieved with microcrystalline cellulose as the carbon source but the best inducer was CMC-Na because a very poor biomass amount was capable of producing the second yield of endoglucanase. On the other hand, glucose and cellobiose gave the highest yields of biomass.

[Cellulolytic activity of coprophilic fungi]. / Actividad celulolítica de hongos coprófilos.

Fourteen isolates, members of the Ascobolaceae family, representing two widespread genera, Ascobolus and Saccobolus, were obtained from dung of herbivorous animals. All the isolates were capable of growing and producing clearing zones in CMC agar media. The species of Ascobolus gave larger clearing zones than the species of Saccobolus. S. verrucisporus and S. longevisporus were the most cellulolytic species in this genera while A. bistisii was the most cellulolytic among all the species tested. The results obtained showed that more than one method of screening must be employed to analyse the cellulolytic ability of different species.

Amino acid sequence of the heavy-chain variable region of the crystallizable human myeloma protein Dob.

The amino acid sequence of the heavy-chain variable region of the crystallizable human myeloma protein Dob has been determined. This protein has previously been shown to have a deletion in the hinge region [Lopes, A. D., & Steiner, L. A. (1973) Fed. Proc., Fed. Am. Soc. Exp. Biol. 32, 1003; Steiner, L. A., & Lopes, A. D. (1979) Biochemistry (preceding paper in this issue)]. The complete sequence was established by analysis, in the automated sequenator, of the intact Fd' piece and of three large overlapping fragments prepared from Fd' by digestion with cyanogen bromide, by tryptic digestion of the citraconylated Fd', and by cleavage with hydroxylamine. Portions of the sequence were confirmed by examination of the amino acid composition and the partial sequence of a variety of small peptides obtained by enzymatic degradation. The Dob heavy-chain variable region appears to belong to the VHIII subgroup, but there are several unusual substitutions. Residue 45 in the Dob sequence is proline, although all other known heavy-chain sequences in man, mouse, rabbit, and guinea pig have leucine at this position. Positions 10 (aspartic acid), 68 (alanine), and 82 (leucine) in the Dob sequence are also atypical. There is no deleted segment in the variable region of the Dob heavy chain nor any abnormality in the variable-constant joining region. The hinge-region deletion appears to be the only gross structural anomaly in the Dob heavy chain.

The primary structure of a human immunoglobulin G2 (IgG2) pFc' fragment.

Amino acid sequence analysis of the pFc' fragment obtained by pepsin digestion of a human IgG2 myeloma protein PIG Gm (n or 23) negative shows it to consist of 112 residues. It starts at position 334 (gamma1 numbering) and contains eight residues from the Cgamma2 region, and the whole Cgamma3 domain. Comparison with the sequence of gamma1 shows two differences at positions 339 and 397. Each of them can be explained by a single base substitution. This high degree of homology among gamma-chain subclasses suggests a recent diversification.

[Idiopathic intestinal lymphangiectasis. Evolution with M.C.T. (author's transl)]. / Linfangiectasia intestinal idiopática. Evolución con triglicéridos de cadena media

A case of idiopathic intestinal lymphangiectasis is reported in a three month old child. Clinical course and laboratory findings are given in relation to administration of three diets containing different concentrations and types of fat. Short term improvement was only noticed with diets containing low concentrations of long chain triglycerides supplement with medium chain triglycerides. Clinical manifestations related to fat malabsortion improved greatly but there was no relationship with serum protein level. No effect on low level of gamma-globulins and lymphatic displasia was found as sawn in an intestinal biopsy performed after three months of treatment. Nevertheless, long-term results were poor and only were evident in a diminution of steatorrhea and normalization of stools.