The N-terminus and several internal tryptic peptides of endopolygalacturonase purified from Fusarium moniliforme were sequenced. The amino acid sequences of the N-terminus and of one of the tryptic peptides were used to design redundant oligonucleotides. A DNA fragment of 288 bp, encoding part of the polygalacturonase gene, was amplified by the polymerase chain reaction (PCR) using the oligonucleotides as primers and, as a template, cDNA synthesized from poly(A)+ RNA extracted from mycelium grown on pectin as a sole carbon source. The PCR-generated fragment was utilized as a probe to screen a genomic and a cDNA library of F. moniliforme. Genomic and cDNA copies of the endopolygalacturonase gene were isolated and sequenced. A cloned 1272 bp cDNA consisted of a single open reading frame encoding 359 amino acids (including the entire 349 aa mature protein and part of the N-terminal signal peptide), the 3' transcribed untranslated region (173 nt), and the poly(A) tail. The nucleotide sequence of the genomic endopolygalacturonase gene was interrupted by four short introns (50-54 bp) and revealed the presence of a 24 amino-acid signal peptide. By RNAse protection experiments, multiple sites of initiation for the transcription of the endopolygalacturonase gene were demonstrated, from 33 to 45 nt downstream from the putative TATA box.
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/S0953-7562(09)80138-X|
|Codice identificativo ISI:||WOS:A1993KZ53100013|
|Codice identificativo Scopus:||2-s2.0-0000101957|
|Titolo:||Cloning and characterization of a gene encoding the endopolygalacturonase of Fusarium moniliforme|
|Appare nelle tipologie:||1.1 Articolo in rivista|