Sherson S, Gy I, Medd J, Schmidt R, Dean C, Kreis M, Lecharny A, Cobbett C
Department of Genetics, University of Melbourne, Parkville, Australia.
Plant Mol Biol. 1999 Mar;39(5):1003-12. doi: 10.1023/a:1006181908753.
The arabinose-sensitive ara1-1 mutant of Arabidopsis is deficient in arabinose kinase activity. A candidate for the ARA1 gene. ISA1, has been previously identified through the Arabidopsis genome sequencing initiative. Here we demonstrate that (1) the ARA1 gene coincides with ISA1 in a positional cloning strategy; (2) there are mutations in the ISA1 gene in both the ara1-1 mutant and an intragenic suppressor mutant; and (3) the ara1-1 and suppressor mutant phenotypes can be complemented by the expression of the ISA1 cDNA in transgenic plants. Together these observations confirm that ISA1 is the ARA1 gene. ARA1 is a member of the galactose kinase family of genes and represents a new substrate specificity among this and other families of sugar kinases. A second gene with similarities to members of the galactose kinase gene family has been identified in the EST database. A 1.8 kb cDNA contained an open reading-frame predicted to encode a 496 amino acid polypeptide. The GAL1 cDNA was expressed in a galK mutant of Escherichia coli and in vitro assays of extracts of the strain expressing GAL1 confirmed that the cDNA encodes a galactose kinase activity. Both GAL1 and ARA1 cross-hybridise at low stringency to other sequences suggesting the presence of additional members of the galactose kinase gene family.
拟南芥对阿拉伯糖敏感的ara1-1突变体缺乏阿拉伯糖激酶活性。通过拟南芥基因组测序计划先前已鉴定出ARA1基因的一个候选基因ISA1。在此我们证明:(1)在定位克隆策略中ARA1基因与ISA1基因一致;(2)ara1-1突变体和一个基因内抑制突变体的ISA1基因均存在突变;(3)ara1-1和抑制突变体的表型可通过在转基因植物中表达ISA1 cDNA来互补。这些观察结果共同证实ISA1就是ARA1基因。ARA1是半乳糖激酶基因家族的成员,代表了该家族及其他糖激酶家族中的一种新的底物特异性。在EST数据库中已鉴定出另一个与半乳糖激酶基因家族成员相似的基因。一个1.8 kb的cDNA包含一个预计编码496个氨基酸多肽的开放阅读框。GAL1 cDNA在大肠杆菌的一个galK突变体中表达,对表达GAL1的菌株提取物进行的体外测定证实该cDNA编码半乳糖激酶活性。GAL1和ARA1在低严谨度下均与其他序列交叉杂交,这表明半乳糖激酶基因家族存在其他成员。