Lakhotia Subhash C, Srivastava Priya, Prasanth K V
Cytogenetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi 221 005, Uttar Pradesh, India.
Cell Stress Chaperones. 2002 Oct;7(4):347-56. doi: 10.1379/1466-1268(2002)007<0347:rohsph>2.0.co;2.
It is known from earlier studies that the heat shock (HS) response in Malpighian tubules (MTs) of Drosophila larvae is different from that in other tissues because instead of the Hsp70 and other common heat shock proteins, Hsp64 and certain other new proteins are induced immediately after HS. In the present study, we examined the kinetics of the synthesis of Hsp70 and Hsp64 immediately after HS and during recovery from HS by 35S-methionine labeling and Western blotting. In addition, we also examined the transcriptional activity of hsp70 genes in larval MT cells at different times after HS by in situ hybridization and Northern blotting. The HS-induced synthesis of Hsp64 ceased by 1 hour of recovery from the HS when synthesis of the Hsp70 commenced. Our results revealed that the induced synthesis of Hsp64 immediately after HS was dependent on new transcription. Although the levels of Hsp70 in MT cells rapidly increased after its synthesis began during recovery, the levels of Hsp64 remained unaltered irrespective of its new synthesis occurring during or after HS. Inhibition of new Hsp64 synthesis by transcriptional or translational inhibitors also did not affect the total amount of this protein in MTs. The Hsp64 polypeptides synthesized in response to HS are degraded rapidly. Apparently, the cells in MTs maintain a balance between new synthesis of Hsp64 and its turnover so that under all conditions a more or less constant level of this protein is maintained. Although the Hsp70 synthesis started only after 1 hour of recovery, the hsp70 genes were transcriptionally activated immediately after HS and they continued to transcribe till at least 4 hours after the HS. The hsp70 transcripts in MT cells that recovered for 2 hours or longer did not contain the 3' untranslated regions (UTRs), which may allow their longer stability and translatability at normal temperature. Synthesis of Hsp70 during recovery period was dependent on continuing transcription. Assessment of the beta-galactosidase activity in 2 transgenic lines carrying the LacZ reporter gene under hsp70 promoter and different lengths of the 5'UTR suggested that the delayed translation of hsp70 transcripts in MTs is probably regulated by some elements in the 5'UTR.
早期研究表明，果蝇幼虫马氏管（MTs）中的热休克（HS）反应与其他组织不同，因为热休克后，诱导产生的不是Hsp70和其他常见的热休克蛋白，而是Hsp64和某些其他新蛋白。在本研究中，我们通过35S-甲硫氨酸标记和蛋白质免疫印迹法，检测了热休克后立即以及热休克恢复过程中Hsp70和Hsp64的合成动力学。此外，我们还通过原位杂交和Northern印迹法，检测了热休克后不同时间幼虫MT细胞中hsp70基因的转录活性。从热休克恢复1小时后，热休克诱导的Hsp64合成停止，此时Hsp70的合成开始。我们的结果显示，热休克后立即诱导合成的Hsp64依赖于新的转录。尽管恢复过程中Hsp70合成开始后，MT细胞中Hsp70的水平迅速增加，但无论热休克期间还是热休克后Hsp64发生新的合成，其水平都保持不变。转录或翻译抑制剂抑制Hsp64的新合成，也不影响MTs中该蛋白的总量。热休克诱导合成的Hsp64多肽迅速降解。显然，MTs中的细胞在Hsp64的新合成与其周转之间保持平衡，以便在所有条件下维持该蛋白或多或少恒定的水平。尽管Hsp70的合成仅在恢复1小时后开始，但hsp70基因在热休克后立即被转录激活，并且至少在热休克后4小时仍继续转录。恢复2小时或更长时间的MT细胞中的hsp70转录本不包含3'非翻译区（UTR），这可能使其在常温下具有更长的稳定性和可翻译性。恢复期间Hsp70的合成依赖于持续的转录。对携带hsp70启动子和不同长度5'UTR的LacZ报告基因的2个转基因品系中的β-半乳糖苷酶活性进行评估，结果表明MTs中hsp70转录本的延迟翻译可能受5'UTR中某些元件的调控。
