Molecular characterization of a novel beta-1,3-exoglucanase related to mycoparasitism of Trichoderma harzianum.

Trichoderma harzianum, a soil-borne filamentous fungus, is capable of parasitizing several plant pathogenic fungi. Secretion of lytic enzymes, mainly glucanases and chitinases, is considered the most crucial step of the mycoparasitic process. The lytic enzymes degrade the cell walls of the pathogenic fungi, enabling Trichoderma to utilize both their cell walls and cellular contents for nutrition. We have purified a 110kDa novel extracellular beta-1,3-exoglucanase from T. harzianum, grown with laminarin or in dual cultures with host fungi. The corresponding gene, lam1.3, and its cDNA were isolated and their nucleotide sequences determined. The deduced amino-acid sequence predicted a molecular mass of 110.7kDa of a mature protein excluding a signal peptide. LAM1.3 showed high homology to EXG1, a beta-1,3-exoglucanase of the phytopathogenic fungus Cochliobolus carbonum, and a lower homology to BGN13.1, a beta-1,3-endoglucanase isolated from T. harzianum. However, it contains a unique C-terminal embodying cysteine motifs. The expression of lam1.3 in growth with laminarin, but not with glucose, was found to be a result of differential accumulation of the corresponding mRNA.

Functional analysis of DNA sequences responsible for ethylene regulation of a bean chitinase gene in transgenic tobacco.

Expression of at least two genes from bean encoding the defense-related protein chitinase has been shown previously to be transcriptionally regulated by the phytohormone ethylene. We have determined the complete nucleotide sequence of one of these genes, the CH5B gene, which resides on a 4.7-kilobase fragment of bean genomic DNA. The structural gene consists of a single open reading frame and encodes the 301 amino acids of the mature protein and a 26-amino acid signal peptide. The CH5B gene has been introduced into tobacco plants using Agrobacterium Ti-plasmid vectors. Little or no expression of the bean gene was observed when transgenic tobacco plants were grown in air; however, exposure of these plants to an atmosphere containing 50 parts per million ethylene resulted in an approximately 20-fold to 50-fold increase in the level of the bean chitinase mRNA. Ethylene-dependent expression of a chimeric gene consisting of 1.6 kilobases of 5'-flanking DNA derived from the CH5B gene fused to the coding sequence of beta-glucuronidase indicates that this region of the CH5B gene is sufficient for ethylene-regulated expression. Deletion analysis of the CH5B promoter region has allowed us to localize these DNA sequences to within a 228-base pair region situated between -422 and -195 upstream of the transcriptional start site. This region is characterized by two short DNA sequences that are exactly conserved in a second ethylene-regulated bean chitinase gene.

Ethylene-regulated gene expression: molecular cloning of the genes encoding an endochitinase from Phaseolus vulgaris.

A full-length copy of bean leaf chitinase mRNA has been cloned. The 1146-base-pair insert of pCH18 encodes the 27-residue amino-terminal signal peptide of the precursor and 301 residues of the mature protein. Utilizing pCH18 as a hybridization probe, we have shown that the increase in translatable chitinase mRNA seen upon ethylene treatment of bean seedlings is due to a 75- to 100-fold increase in steady-state mRNA levels. Southern blot analysis of bean genomic DNA revealed that chitinase is encoded by a small, multigene family consisting of approximately four members. From our nucleotide sequence analysis of five additional chitinase cDNA clones, it appears that at least two of these genes are expressed. Three of the bean chitinase genes have been isolated from a Sau3A genomic library and partially characterized.

Characterization and derivation of the gene coding for mitochondrial carbamyl phosphate synthetase I of rat.

The nucleotide sequence of rat carbamyl phosphate synthetase I mRNA has been determined from the complementary DNA. The mRNA comprises minimally 5,645 nucleotides and codes for a polypeptide of 164,564 Da corresponding to the precursor form of the rat liver enzyme. The primary sequence of mature rat carbamyl phosphate synthetase I indicates that the precursor is cleaved at one of two leucines at residues 38 or 39. The derived amino acid sequence of carbamyl phosphate synthetase I is homologous to the sequences of carbamyl phosphate synthetase of Escherichia coli and yeast. The sequence homology extends along the entire length of the rat polypeptide and encompasses the entire sequences of both the small and large subunits of the E. coli and yeast enzymes. The protein sequence data provide strong evidence that the carbamyl phosphate synthetase I gene of rat, the carAB gene of E. coli, and the CPA1 and CPA2 genes of yeast were derived from common ancestral genes. Part of the rat carbamyl phosphate synthetase I gene has been characterized with two nonoverlapping phage clones spanning 28.7 kilobases of rat chromosomal DNA. This region contains 13 exons ranging in size from 68 to 195 base pairs and encodes the 453 carboxyl-terminal amino acids of the rat protein. Southern hybridization analysis of rat genomic DNA indicates the carbamyl phosphate synthetase I gene to be present in single copy.

The gene coding for carbamoyl-phosphate synthetase I was formed by fusion of an ancestral glutaminase gene and a synthetase gene.

A near full-length cDNA copy of rat carbamoyl-phosphate synthetase I (EC 6.3.4.16) mRNA has been cloned. The cDNA insert in the recombinant plasmid pHN234 is 5.3 kilobases long. Analysis of the sequence coding for carbamoyl-phosphate synthetase I indicates that the gene has arisen from a fusion of two ancestral genes: one homologous to Escherichia coli carA, coding for a glutaminase subunit, and the second homologous to the carB gene that codes for the synthetase subunit. A short amino acid sequence previously proposed to be part of the active site involved in glutamine amide nitrogen transfer in the E. coli and yeast carbamoyl-phosphate synthetases (EC 6.3.5.5) is also present in the rat enzyme. In the mammalian enzyme, however, the glutaminase domain lacks a cysteine residue previously shown to interact with glutamine. The cysteine is replaced by a serine residue. This substitution could, in part, account for the inability of mammalian carbamoyl-phosphate synthetase I to catalyze the hydrolysis of glutamine to glutamic acid and ammonia.

Regulation and expression of carbamyl phosphate synthetase I mRNA in developing rat liver and Morris hepatoma 5123D.

A cDNA clone complementary to mRNA encoding the precursor (Mr = 165,000) to the rat liver mitochondrial matrix enzyme carbamyl phosphate synthetase I (Mr = 160,000) was employed to compare relative amounts of the messenger in adult and fetal liver and in Morris hepatoma 5123D and 3924A cells. Northern blot analysis gave a size estimate for the messenger of 6,500-6,700 nucleotides. Carbamyl phosphate synthetase mRNA levels in 15-day-old fetal liver were less than 10% of adult levels; 5123D cells expressed the messenger at levels about 2-fold higher than normal adult liver, but the messenger was undetectable in 3924A cells. Albumin mRNA was also expressed in the former but not in the latter. Maintaining rats for 5 days on a diet containing 60% casein augmented the relative amount of carbamyl phosphate synthetase mRNA by about 2-fold, while a protein-free diet resulted in reduced levels of the mRNA (about 50% compared to animals on a normal diet). Finally, the pattern of hybridization of carbamyl phosphate synthetase cDNA to HindIII-digested genomic DNA showed no differences between normal liver and its corresponding hepatoma; however, a HindIII site polymorphism was observed between Buffalo and ACI rats.

Yeast carbamyl phosphate synthetase. Structure of the yeast gene and homology to Escherichia coli carbamyl phosphate synthetase.

A cloned fragment of yeast chromosomal DNA carrying the gene CPA2 coding for the large subunit of arginine-specific carbamyl phosphate synthetase has been sequenced. The cloned DNA has a 3,354-nucleotide long continuous reading frame coding for a polypeptide of 1,117 amino acids. The calculated molecular weight of the encoded polypeptide is 123,787, in good agreement with the reported molecular weight of the yeast carbamyl phosphate synthetase large subunit. The amino acid sequence of yeast carbamyl phosphate synthetase is homologous to the recently determined sequence of Escherichia coli carbamyl phosphate synthetase (Nyunoya, H., and Lusty, C. J. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 4629-4633) over almost the entire length of the protein. Like the E. coli large subunit, the yeast enzyme exhibits an extensive internal homology between its NH2- and carboxyl-terminal halves. The internal homology in both the yeast and E. coli proteins indicates that the gene coding for the large subunit of carbamyl phosphate synthetase was derived from a tandem duplication which occurred prior to the divergence of eukaryotes and prokaryotes.

Fluorescence studies of threonine-promoted conformational transitions in aspartokinase I using the substrate analogue 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate.

The trinitrophenyl derivative of ATP, 2'(3')-O-(2,4,6-trinitrophenyl) adenosine 5'-triphosphate, has been used as a spectroscopic probe to investigate threonine-promoted conformational changes in the aspartokinase region of aspartokinase-homoserine dehydrogenase I in an attempt to relate the structural effects of threonine binding to inhibition of enzymatic activity. Binding of this analogue substrate to the enzyme is characterized by a 9-fold enhancement in probe fluorescence. Saturating levels of the feedback inhibitor, threonine, produce a 77% increase in fluorescence enhancement, indicating an increase in the rigidity or hydrophobicity of the nucleotide-binding site in the inhibited form of the enzyme. Threonine titration studies indicate that the two inhibitor-binding sites found on each subunit do not contribute equally to the fluorescence-detected conformational change. Comparison of the spectral change with the inhibition of dehydrogenase activity has revealed the exclusive involvement of the non-kinase threonine sites. No transition can be detected as a consequence of inhibitor binding at the kinase subsites. The results of the 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate study have provided further evidence for a concerted kinase-dehydrogenase conformational change which is induced by threonine interaction with the high affinity binding sites and which provides maximal inhibition of homoserine dehydrogenase and the majority of aspartokinase inhibition. The failure to observe a distinct enzyme form produced by threonine occupation of the low affinity kinase sites suggests that no large structural reorganization of the kinase active site is produced as a result of this binding event. The conformational change, suggested by the cooperativity of threonine binding, must instead involve only a subtle or highly localized alteration which does not perturb the environment of the ATP-binding cleft.