Collett M A, Bradshaw R E, Scott D B
Department of Microbiology and Genetics, Massey University, Palmerston North, New Zealand.
Gene. 1995 May 26;158(1):31-9. doi: 10.1016/0378-1119(95)00143-t.
A fragment of the Claviceps purpurea pyr4 gene, encoding orotidine-5'-monophosphate decarboxylase (OMP decarboxylase), was used to screen a genomic library from an isolate of a fungus, Acremonium sp. (designated Lp1), which grows as an endophyte in perennial ryegrass (Lolium perenne). Three positive clones, lambda MC11, lambda MC12 and lambda MC14, were isolated. Two of these clones, lambda MC12 and lambda MC14, were overlapping clones from the same locus, while lambda MC11 was from a different locus. Fragments of these clones which hybridised with C. purpurea pyr4 were sequenced and found to have similarity with pyr4 from other Pyrenomycete fungi. The pyr4 gene from lambda MC12 and lambda MC14 was designated pyr4-1 and that from lambda MC11 was designated pyr4-2. The predicted ORFs of the two genes were highly conserved, with 97.5% identity at the nucleotide level, the 5' non-coding sequences were the least conserved with 88.5% identity and the 3' non-coding sequences had 93.0% identity. RT-PCR analysis of total RNA from Lp1 demonstrated that transcripts from the two genes were present at similar levels, and hybridisation of pyr4-1 to Northern blots of total RNA from Lp1 showed that full-length transcripts were being produced. Genomic fragments containing pyr4 were transformed into a strain of Aspergillus nidulans which has a mutation in pyrG (encoding OMP decarboxylase). Both pyr4-1 and pyr4-2 complemented the pyrG mutation in A. nidulans, indicating that both encode functional OMP decarboxylases. It has been proposed [Schardl et al., Genetics 136 (1994) 1307-1317] that the two pyr4 in Lp1 arose by interspecific hybridisation, most likely between the ryegrass choke pathogen, Epichloë typhina, and another endophyte from perennial ryegrass, Acremonium lolii. Analysis by PCR amplification and direct sequencing of the variable 5' non-coding regions of pyr4, from possible ancestors to Lp1 supports this hypothesis. Comparisons of these sequences to the 5' non-coding sequences from pyr4-1 and pyr4-2 demonstrated that E. typhina and A. lolii were the most likely ancestors of the two pyr4 found in Lp1.
用编码乳清苷 - 5'-单磷酸脱羧酶（OMP脱羧酶）的麦角菌（Claviceps purpurea）pyr4基因片段，从一种真菌——顶孢霉属（Acremonium sp.）（命名为Lp1）的分离株中筛选基因组文库，该真菌作为内生菌生长在多年生黑麦草（Lolium perenne）中。分离出了三个阳性克隆，λMC11、λMC12和λMC14。其中两个克隆，λMC12和λMC14，是来自同一基因座的重叠克隆，而λMC11来自不同基因座。对这些与麦角菌pyr4杂交的克隆片段进行测序，发现它们与其他核菌纲真菌的pyr4具有相似性。来自λMC12和λMC14的pyr4基因被命名为pyr4 - 1，来自λMC11的被命名为pyr4 - 2。这两个基因的预测开放阅读框高度保守，核苷酸水平上的同一性为97.5%，5'非编码序列保守性最低，同一性为88.5%，3'非编码序列的同一性为93.0%。对Lp1总RNA的RT - PCR分析表明，这两个基因的转录本水平相似，并且pyr4 - 1与Lp1总RNA的Northern杂交结果显示产生了全长转录本。将含有pyr4的基因组片段转化到在pyrG（编码OMP脱羧酶）中存在突变的构巢曲霉（Aspergillus nidulans）菌株中。pyr4 - 1和pyr4 - 2都能互补构巢曲霉中的pyrG突变，表明它们都编码有功能的OMP脱羧酶。有人提出[Schardl等人，遗传学136（1994）1307 - 1317]，Lp1中的两个pyr4是通过种间杂交产生的，最有可能是在黑麦草内生真菌——黑麦草内生菌（Epichloë typhina）和多年生黑麦草的另一种内生菌——黑麦草顶孢霉（Acremonium lolii）之间。通过对pyr4可变5'非编码区从Lp1可能的祖先进行PCR扩增和直接测序分析，支持了这一假设。将这些序列与pyr4 - 1和pyr4 - 2的5'非编码序列进行比较，表明黑麦草内生菌和黑麦草顶孢霉最有可能是Lp1中发现的两个pyr4的祖先。
