Stark M J, Milner J S
Leicester Biocentre, University of Leicester, U.K.
Yeast. 1989 Jan-Feb;5(1):35-50. doi: 10.1002/yea.320050106.
The TRP1 gene of the yeast Kluyveromyces lactis has been cloned from a genomic library by complementation of the Saccharomyces cerevisiae trp1-289 mutation. The gene was located within the clone by transposon mutagenesis and the coding region identified by DNA sequencing. This has indicated that K. lactis TRP1 encodes a 210-amino acid polypeptide which shows 53% identity to the homologous S. cerevisiae protein. The K. lactis TRP1 gene has been disrupted by substituting the S. cerevisiae URA3 gene for a large part of the TRP1 coding sequence. Replacement of the chromosomal TRP1 locus with this construction has enabled the production of non-reverting trp1- strains of K. lactis, while a genetic analysis of the disrupted allele confirmed that the TRP1 gene had been cloned. DNA sequencing has also shown that the K. lactis TRP1 sequence is flanked by genes encoding inorganic pyrophosphatase and histone H3, which we have designated IPP and HHT1 respectively. Hybridization studies have shown that in common with S. cerevisiae, K. lactis has two copies of the histone H3 gene. Each H3 gene is closely linked to a gene encoding histone H4 and in both yeast species the IPP gene is tightly linked to one of the histone gene pairs.
乳酸克鲁维酵母的TRP1基因已通过对酿酒酵母trp1 - 289突变的互补作用从基因组文库中克隆出来。通过转座子诱变确定该基因位于克隆片段内，并通过DNA测序鉴定了编码区。这表明乳酸克鲁维酵母TRP1编码一种210个氨基酸的多肽，与同源的酿酒酵母蛋白具有53%的同一性。通过用酿酒酵母URA3基因取代TRP1编码序列的大部分，破坏了乳酸克鲁维酵母的TRP1基因。用这种构建体替换染色体TRP1位点能够产生不回复的乳酸克鲁维酵母trp1 - 菌株，而对破坏的等位基因的遗传分析证实已克隆到TRP1基因。DNA测序还表明，乳酸克鲁维酵母TRP1序列两侧分别是编码无机焦磷酸酶和组蛋白H3的基因，我们分别将其命名为IPP和HHT1。杂交研究表明，与酿酒酵母一样，乳酸克鲁维酵母有两个组蛋白H3基因拷贝。每个H3基因都与一个编码组蛋白H4的基因紧密相连，并且在这两种酵母中，IPP基因都与其中一对组蛋白基因紧密相连。
