Gomi K, Kitamoto K, Kumagai C
National Research Institute of Brewing, Tokyo, Japan.
Gene. 1991 Dec 1;108(1):91-8. doi: 10.1016/0378-1119(91)90491-s.
We have isolated an acetamidase-encoding gene (amdS) from Aspergillus oryzae by heterologous hybridization using the corresponding Aspergillus nidulans gene as a probe. The gene is located on a 3.5-kb SacI fragment and its nucleotide (nt) sequence was determined. Compared with the A. nidulans amdS gene, the coding region of A. oryzae gene consists of seven exons interrupted by six introns and encodes 545 amino acid (aa) residues. The deduced aa sequence has a high degree of homology with that of the A. nidulans acetamidase protein. Three introns (IVS-1, IVS-2, and IVS-4) exist at the same positions as those of A. nidulans amdS, whilst three additional introns (IVS-3, IVS-5, and IVS-6) are also present. There is no preference in its codon usage (G + C content in the third position of codons is 51%). Gene disruption experiments demonstrate that the resulting mutants show significantly reduced growth on acetamide-containing medium, indicating that the A. oryzae amdS gene encodes a functional acetamidase that is required for acetamide utilization. Transcriptional analysis by Northern blot reveals a 1.8-kb transcript in RNA extracted from mycelium grown in medium containing acetamide or acetate plus beta-alanine as the sole carbon and nitrogen sources.
我们以构巢曲霉的相应基因作为探针，通过异源杂交从米曲霉中分离出了一个编码乙酰胺酶的基因（amdS）。该基因位于一个3.5kb的SacI片段上，并测定了其核苷酸（nt）序列。与构巢曲霉的amdS基因相比，米曲霉基因的编码区由7个外显子和6个内含子组成，编码545个氨基酸（aa）残基。推导的氨基酸序列与构巢曲霉乙酰胺酶蛋白的序列具有高度同源性。三个内含子（IVS-1、IVS-2和IVS-4）与构巢曲霉amdS的内含子位于相同位置，同时还存在另外三个内含子（IVS-3、IVS-5和IVS-6）。其密码子使用没有偏好（密码子第三位的G + C含量为51%）。基因破坏实验表明，产生的突变体在含乙酰胺的培养基上生长显著减少，这表明米曲霉amdS基因编码一种功能性乙酰胺酶，该酶是利用乙酰胺所必需的。通过Northern印迹进行的转录分析显示，从在含有乙酰胺或乙酸盐加β-丙氨酸作为唯一碳源和氮源的培养基中生长的菌丝体提取的RNA中，有一个1.8kb的转录本。
