Intron requirement for AFP gene expression in Trichoderma viride.

The 430 bp ORF of the Aspergillus giganteus antifungal protein (AFP) gene, containing two small introns, was fused between the promoter and the terminator of the Aspergillus nidulans trpC gene. The AFP gene in this vector produced detectable levels of spliced mRNA in Trichoderma viride. In contrast, in the same vector configuration, its 285 bp intronless derivative showed no accumulation of mRNA when transformed into T. viride. Such expression results were confirmed at the protein level. This fact demonstrated that the introns were required for AFP gene expression in T. viride. This is thought to be a novel phenomenon found in filamentous fungi. Although the mechanism of splicing in filamentous fungi might be similar to that in other eukaryotes, little is known of how it affects expression. This study suggests that the small introns in filamentous fungal genes may not only act as intervening elements, but may also play crucial roles in gene expression by affecting mRNA accumulation. Furthermore, it may provide new evidence for intron-dependent evolution.

[Secretory expression of an antifungal protein in the mold Trichoderma viride].

AFP (antifungal protein) secreted by the filamentous fungi Aspergillus giganteus MDH18894 is a strong basic protein (pI>10.65). Its mRNA has an 430 bp open reading frame encoding the 94 amino acids' precursor of AFP. The precursor of AFP is processed to form the mature AFP of 51 amino acids by a putative two-step processing. A 285 bp AFP-like gene (i.e. AFP cDNA) lacking introns was reported to exist in the genome of Trichoderma viride, another species of filamentous fungi, but the gene was silent. In order to investigate whether the genomic AFP gene from A. giganteus could be expressed in T. viride, an expression plasmid with the open reading frame of AFP gene fused between the promoter and terminator of trpC gene was constructed and was transformed into T. viride. SDS-PAGE and Western blot analysis showed that AFP was expressed and secreted into the culture supernatant of the transformant of T. viride. Our study provides a basis for further secretory expression of heterologous eukaryotic genes of important value in the system of T. viride.

Studies of three genes encoding Cinnamomin (a type II RIP) isolated from the seeds of camphor tree and their expression patterns.

Cinnamomin, which has three isoforms, is a type II ribosome-inactivating protein (RIP) purified from the mature seeds of camphor tree (Cinnamomum camphora). In a previous study, an incomplete cDNA that encoded the A- and B-chain of Cinnamomin but lacked signal peptide sequence was cloned. In the present paper, its full-length cDNA was obtained by 5' rapid amplification of cDNA ends (5'RACE). Subsequently, polymerase chain reaction (PCR) amplification of its genomic DNA was performed. Unexpectedly, sequence analysis of the PCR products revealed three cinnamomin genes with >98.0% sequence identity. One of them corresponded to the published cDNA and was designated as cinnamomin I, whereas the other two genes were named as cinnamomin II and cinnamomin III, respectively. RT-PCR amplification of the cDNAs of cinnamomin II and III manifested that these two genes were functional. The three genes have no intron. Three Cinnamomin precursors that were inferred from the cDNA sequence of three cinnamomin genes exhibited relatively high sequence homology with other type II RIPs. Northern blot analysis demonstrated that the cinnamomin genes only expressed in cotyledons of C. camphora seeds and the acmes of expression emerged at 75-90 DAF when seeds were close to maturity. It is proposed that the three cinnamomin genes may encode three isoforms of Cinnamomin. The physiological function of Cinnamomin in C. camphora seeds is briefly discussed.

Purification and characterization of an ethylene-induced antifungal protein from leaves of guilder rose (Hydrangea macrophylla).

An ethylene-induced, chitin-binding protein (designated as HM30) from leaves of Hydrangea macrophylla was identified and purified to apparent homogeneity by chitin affinity chromatography followed by FPLC on a Superose 12 column. The molecular mass of HM30 was 30,010.0 Da determined by mass spectrometry and its isoelectric point of 8.4 was estimated by isoelectric focusing. The amino acid composition of HM30 was also determined. The initial 15 amino acid residues of the N-terminal were found to be N-S-M-E-R-V-E-E-L-R-K-K-L-Q-D by automatic Edman degradation. This chitin-binding protein showed antifungal activity toward several crop fungal pathogens. Knowledge of properties of HM30 should be useful for its potential application as a plant fungicidal agent.

Effect of High Hydrostatic Pressure on Activity of Restriction Endonucleases.

The effect of high hydrostatic pressure on the activities of type II restriction enzymes HindIII and XbaI in digesting plasmid pSPORT1 was studied. The endonuclease activity of HindIII and XbaI at 37 degrees were gradually inhibited by increasing pressure and completely inhibited at 200 and 180 MPa, respectively. No obvious irreversible effect was observed for HindIII after suffering high pressure, while a considerable irreversible inactivation was observed for XbaI. The standard molar volume changes for HindIII and XbaI estimated from the inhibition of endonuclease activity at different pressures were 213 and 103 ml/mol, respectively. It was also concluded that pressurization did not change the substrate sequence specificity of both HindIII and XbaI.