Blaiseau P L, Kunz C, Grison R, Bertheau Y, Brygoo Y
Institut de Génétique et Microbiologie, Université Paris Sud, Orsay, France.
Curr Genet. 1992 Jan;21(1):61-6. doi: 10.1007/BF00318656.
Recombinant clones from a cDNA library of an Aphanocladium album chitinase-overproducing mutant strain were isolated by screening with antiserum against a 39 kDa chitinase purified from this hyperparasitic fungus. Analysis of the isolated positive clones indicated that most of them carried the same cDNA. A cDNA from this group was used as a hybridization probe to isolate an 8 kb DNA fragment from a genomic library of the wild-type strain. The chitinase 1 gene was mapped to this fragment by two independent approaches. Its partial DNA sequence was in perfect agreement with an amino-terminal peptide sequence obtained by sequencing 23 amino acids of the 39 kDa chitinase. Its transfer in Fusarium oxysporum resulted in a transformant producing both a protein of about 39 kDa that cross-reacted with the chitinase antiserum and a chitinase activity that was inhibited by the same antiserum. Northern blot analysis indicates that the cloned chitinase gene was subject to catabolite repression and appeared inducible by chitin.
通过用针对从这种重寄生真菌中纯化的39 kDa几丁质酶的抗血清进行筛选，从产几丁质酶的水云状菌突变株的cDNA文库中分离出重组克隆。对分离出的阳性克隆的分析表明，它们中的大多数携带相同的cDNA。来自该组的一个cDNA被用作杂交探针，从野生型菌株的基因组文库中分离出一个8 kb的DNA片段。通过两种独立的方法将几丁质酶1基因定位到该片段上。其部分DNA序列与通过对39 kDa几丁质酶的23个氨基酸进行测序获得的氨基末端肽序列完全一致。它在尖孢镰刀菌中的转移导致一个转化体产生一种与几丁质酶抗血清发生交叉反应的约39 kDa的蛋白质，以及一种被相同抗血清抑制的几丁质酶活性。Northern印迹分析表明，克隆的几丁质酶基因受到分解代谢物阻遏，并且在几丁质诱导下出现。
