Gomes S L, Gober J W, Shapiro L
Departamento de Bioquimica, Universidade de Sao Paulo, Brazil.
J Bacteriol. 1990 Jun;172(6):3051-9. doi: 10.1128/jb.172.6.3051-3059.1990.
Caulobacter crescentus has a single dnaK gene that is highly homologous to the hsp70 family of heat shock genes. Analysis of the cloned and sequenced dnaK gene has shown that the deduced amino acid sequence could encode a protein of 67.6 kilodaltons that is 68% identical to the DnaK protein of Escherichia coli and 49% identical to the Drosophila and human hsp70 protein family. A partial open reading frame 165 base pairs 3' to the end of dnaK encodes a peptide of 190 amino acids that is 59% identical to DnaJ of E. coli. Northern blot analysis revealed a single 4.0-kilobase mRNA homologous to the cloned fragment. Since the dnaK coding region is 1.89 kilobases, dnaK and dnaJ may be transcribed as a polycistronic message. S1 mapping and primer extension experiments showed that transcription initiated at two sites 5' to the dnaK coding sequence. A single start site of transcription was identified during heat shock at 42 degrees C, and the predicted promoter sequence conformed to the consensus heat shock promoters of E. coli. At normal growth temperature (30 degrees C), a different start site was identified 3' to the heat shock start site that conformed to the E. coli sigma 70 promoter consensus sequence. S1 protection assays and analysis of expression of the dnaK gene fused to the lux transcription reporter gene showed that expression of dnaK is temporally controlled under normal physiological conditions and that transcription occurs just before the initiation of DNA replication. Thus, in both human cells (I. K. L. Milarski and R. I. Morimoto, Proc. Natl. Acad. Sci. USA 83:9517-9521, 1986) and in a simple bacterium, the transcription of a hsp70 gene is temporally controlled as a function of the cell cycle under normal growth conditions.
新月柄杆菌有一个单一的dnaK基因，它与热休克基因的hsp70家族高度同源。对克隆并测序的dnaK基因的分析表明，推导的氨基酸序列可编码一个67.6千道尔顿的蛋白质，该蛋白质与大肠杆菌的DnaK蛋白有68%的同一性，与果蝇和人类hsp70蛋白家族有49%的同一性。在dnaK末端3'方向165个碱基对处的一个部分开放阅读框编码一个190个氨基酸的肽，该肽与大肠杆菌的DnaJ有59%的同一性。Northern印迹分析揭示了一个与克隆片段同源的单一4.0千碱基mRNA。由于dnaK编码区为1.89千碱基，dnaK和dnaJ可能作为一个多顺反子信息进行转录。S1图谱分析和引物延伸实验表明，转录起始于dnaK编码序列5'方向的两个位点。在42℃热休克期间鉴定出一个单一的转录起始位点，预测的启动子序列符合大肠杆菌的共有热休克启动子。在正常生长温度（30℃）下，在热休克起始位点3'方向鉴定出一个不同的起始位点，该位点符合大肠杆菌σ70启动子共有序列。S1保护分析以及与lux转录报告基因融合的dnaK基因的表达分析表明，dnaK的表达在正常生理条件下受到时间控制，转录发生在DNA复制起始之前。因此，在人类细胞（I.K.L.Milarski和R.I.Morimoto，《美国国家科学院院刊》83:9517 - 9521，1986）和一种简单细菌中，hsp70基因的转录在正常生长条件下作为细胞周期的函数受到时间控制。
