Liu X, Gorovsky M A
Department of Biology, University of Rochester, NY 14627, USA.
Nucleic Acids Res. 1996 Aug 1;24(15):3023-30. doi: 10.1093/nar/24.15.3023.
A truncated cDNA clone encoding Tetrahymena thermophila histone H2A2 was isolated using synthetic degenerate oligonucleotide probes derived from H2A protein sequences of Tetrahymena pyriformis. The cDNA clone was used as a homologous probe to isolate a truncated genomic clone encoding H2A1. The remaining regions of the genes for H2A1 (HTA1) and H2A2 (HTA2) were then isolated using inverse PCR on circularized genomic DNA fragments. These partial clones were assembled into intact HTA1 and HTA2 clones. Nucleotide sequences of the two genes were highly homologous within the coding region but not in the noncoding regions. Comparison of the deduced amino acid sequences with protein sequences of T. pyriformis H2As showed only two and three differences respectively, in a total of 137 amino acids for H2A1, and 132 amino acids for H2A2, indicating the two genes arose before the divergence of these two species. The HTA2 gene contains a TAA triplet within the coding region, encoding a glutamine residue. In contrast with the T. thermophila HHO and HTA3 genes, no introns were identified within the two genes. The 5'- and 3'-ends of the histone H2A mRNAs; were determined by RNase protection and by PCR mapping using RACE and RLM-RACE methods. Both genes encode polyadenylated mRNAs and are highly expressed in vegetatively growing cells but only weakly expressed in starved cultures. With the inclusion of these two genes, T. thermophila is the first organism whose entire complement of known core and linker histones, including replication-dependent and basal variants, has been cloned and sequenced.
利用来源于梨形四膜虫H2A蛋白序列的合成简并寡核苷酸探针，分离出了编码嗜热四膜虫组蛋白H2A2的截短cDNA克隆。该cDNA克隆用作同源探针，分离出了编码H2A1的截短基因组克隆。然后，通过对环化基因组DNA片段进行反向PCR，分离出H2A1（HTA1）和H2A2（HTA2）基因的其余区域。这些部分克隆被组装成完整的HTA1和HTA2克隆。两个基因的核苷酸序列在编码区内高度同源，但在非编码区则不然。将推导的氨基酸序列与梨形四膜虫H2A的蛋白质序列进行比较，结果显示，对于H2A1的总共137个氨基酸和H2A2的132个氨基酸，分别仅有两个和三个差异，这表明这两个基因在这两个物种分化之前就已出现。HTA2基因在编码区内含有一个TAA三联体，编码一个谷氨酰胺残基。与嗜热四膜虫HHO和HTA3基因不同，在这两个基因中未鉴定出内含子。通过RNase保护以及使用RACE和RLM-RACE方法进行PCR定位，确定了组蛋白H2A mRNA的5'端和3'端。这两个基因均编码聚腺苷酸化的mRNA，在营养生长的细胞中高表达，但在饥饿培养物中仅微弱表达。随着这两个基因的加入，嗜热四膜虫成为首个其全部已知核心组蛋白和连接组蛋白（包括复制依赖性和基础变体）均已被克隆和测序的生物体。
