Borthakur D, Basche M, Buikema W J, Borthakur P B, Haselkorn R
Department of Molecular Genetics and Cell Biology, University of Chicago, Illinois 60637.
Mol Gen Genet. 1990 Apr;221(2):227-34. doi: 10.1007/BF00261725.
A 1.8 kb transcript corresponding to a region of the Anabaena 7120 chromosome 4 kb downstream of the nifHDK operon appears 12-18 h after heterocyst induction. The DNA corresponding to this transcript was sequenced and found to contain two open reading frames, designated ORF 1 and ORF 2. Two polypeptides, of 30 kDa and 13 kDa, encoded by these ORFs were expressed in Escherichia coli. An apparent start site for the transcript, detected by S1 nuclease protection, was located 42 bp upstream of the ATG start codon of ORF 1. ORF 2 shows strong sequence similarity to ORF 6 in the nif gene region of Azotobacter vinelandii. ORF 1 was interrupted using a 1.4 kb neomycin resistance cassette and the resulting mutant grew very slowly on medium lacking combined nitrogen. The mutant had 45% of wild-type acetylene reduction activity, which could be complemented by a 2.8 kb EcoRI fragment of wild-type Anabaena DNA containing only ORF 1 and ORF 2. Thus, one or both of these ORFs is required for efficient nitrogen fixation in Anabaena.
与鱼腥藻7120染色体上固氮酶操纵子下游4 kb区域相对应的一个1.8 kb转录本在异形胞诱导后12 - 18小时出现。对与该转录本相对应的DNA进行测序，发现其包含两个开放阅读框，分别命名为ORF 1和ORF 2。由这些开放阅读框编码的两种多肽，分子量分别为30 kDa和13 kDa，在大肠杆菌中表达。通过S1核酸酶保护检测到的该转录本的一个明显起始位点位于ORF 1的ATG起始密码子上游42 bp处。ORF 2与棕色固氮菌固氮基因区域中的ORF 6具有很强的序列相似性。使用一个1.4 kb的新霉素抗性盒中断ORF 1，得到的突变体在缺乏化合态氮的培养基上生长非常缓慢。该突变体具有野生型乙炔还原活性的45%，这可以被仅包含ORF 1和ORF 2的野生型鱼腥藻DNA的一个2.8 kb EcoRI片段所互补。因此，这些开放阅读框中的一个或两个对于鱼腥藻中高效的固氮作用是必需的。
