Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1985 Feb;5(2):330–341. doi: 10.1128/mcb.5.2.330

Structure and expression of the human gene encoding major heat shock protein HSP70.

B Wu, C Hunt, R Morimoto
PMCID: PMC366716  PMID: 2858050

Abstract

We have cloned a human gene encoding the 70,000-dalton heat shock protein (HSP70) from a human genomic library, using the Drosophila HSP70 gene as a heterologous hybridization probe. The human recombinant clone hybridized to a 2.6-kilobase polyadenylated mRNA from HeLa cells exposed to 43 degrees C for 2 h. The 2.6-kilobase mRNA was shown to direct the translation in vitro of a 70,000-dalton protein similar in electrophoretic mobility to the HSP70 synthesized in vivo. From the analysis of S1 nuclease-resistant mRNA-DNA hybrids, the HSP70 gene appears to be transcribed as an uninterrupted mRNA of 2.3 kilobases. We show that the cloned HSP70 gene contains the sequences necessary for heat shock-induced expression by two criteria. First, hamster cells transfected with a subclone containing the HSP70 gene and flanking sequences synthesized a HSP70-like protein upon heat shock. Second, human cells transfected with a chimeric gene containing the 5' flanking sequences of the HSP70 gene and the coding sequences of the bacterial chloramphenicol acetyltransferase gene transcribed the chimeric gene upon heat shock. We show that the HSP70 mRNA transcribed in an adenovirus 5 transformed human cell line (293 cells) is identical to the HSP70 mRNA induced by heat shock.

Full text

PDF
330

Images in this article

333Image
on p.333
333Image
on p.333
334Image
on p.334
335Image
on p.335
335Image
on p.335
336Image
on p.336
337Image
on p.337
338Image
on p.338
338Image
on p.338
338Image
on p.338

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ashburner M., Bonner J. J. The induction of gene activity in drosophilia by heat shock. Cell. 1979 Jun;17(2):241–254. doi: 10.1016/0092-8674(79)90150-8. [DOI] [PubMed] [Google Scholar]
  2. Aviv H., Leder P. Purification of biologically active globin messenger RNA by chromatography on oligothymidylic acid-cellulose. Proc Natl Acad Sci U S A. 1972 Jun;69(6):1408–1412. doi: 10.1073/pnas.69.6.1408. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Babich A., Feldman L. T., Nevins J. R., Darnell J. E., Jr, Weinberger C. Effect of adenovirus on metabolism of specific host mRNAs: transport control and specific translational discrimination. Mol Cell Biol. 1983 Jul;3(7):1212–1221. doi: 10.1128/mcb.3.7.1212. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Banerji S. S., Theodorakis N. G., Morimoto R. I. Heat shock-induced translational control of HSP70 and globin synthesis in chicken reticulocytes. Mol Cell Biol. 1984 Nov;4(11):2437–2448. doi: 10.1128/mcb.4.11.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bell G. I., Pictet R., Rutter W. J. Analysis of the regions flanking the human insulin gene and sequence of an Alu family member. Nucleic Acids Res. 1980 Sep 25;8(18):4091–4109. doi: 10.1093/nar/8.18.4091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beltz G. A., Flint S. J. Inhibition of HeLa cell protein synthesis during adenovirus infection. Restriction of cellular messenger RNA sequences to the nucleus. J Mol Biol. 1979 Jun 25;131(2):353–373. doi: 10.1016/0022-2836(79)90081-0. [DOI] [PubMed] [Google Scholar]
  7. Berk A. J., Sharp P. A. Spliced early mRNAs of simian virus 40. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1274–1278. doi: 10.1073/pnas.75.3.1274. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Blin N., Stafford D. W. A general method for isolation of high molecular weight DNA from eukaryotes. Nucleic Acids Res. 1976 Sep;3(9):2303–2308. doi: 10.1093/nar/3.9.2303. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Cato A. C., Sillar G. M., Kioussis J., Burdon R. H. Molecular cloning of cDNA sequences coding for the major (beta-, gamma-, delta- and epsilon-) heat-shock polypeptides of HeLa cells. Gene. 1981 Dec;16(1-3):27–34. doi: 10.1016/0378-1119(81)90058-5. [DOI] [PubMed] [Google Scholar]
  10. Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
  11. Cleveland D. W., Lopata M. A., MacDonald R. J., Cowan N. J., Rutter W. J., Kirschner M. W. Number and evolutionary conservation of alpha- and beta-tubulin and cytoplasmic beta- and gamma-actin genes using specific cloned cDNA probes. Cell. 1980 May;20(1):95–105. doi: 10.1016/0092-8674(80)90238-x. [DOI] [PubMed] [Google Scholar]
  12. Collins P. L., Hightower L. E., Ball L. A. Transcription and translation of Newcastle disease virus mRNA's in vitro. J Virol. 1978 Oct;28(1):324–336. doi: 10.1128/jvi.28.1.324-336.1978. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Darnell J. E., Jr Implications of RNA-RNA splicing in evolution of eukaryotic cells. Science. 1978 Dec 22;202(4374):1257–1260. doi: 10.1126/science.364651. [DOI] [PubMed] [Google Scholar]
  14. Deininger P. L., Jolly D. J., Rubin C. M., Friedmann T., Schmid C. W. Base sequence studies of 300 nucleotide renatured repeated human DNA clones. J Mol Biol. 1981 Sep 5;151(1):17–33. doi: 10.1016/0022-2836(81)90219-9. [DOI] [PubMed] [Google Scholar]
  15. Drouin J. Cloning of human mitochondrial DNA in Escherichia coli. J Mol Biol. 1980 Jun 15;140(1):15–34. doi: 10.1016/0022-2836(80)90354-x. [DOI] [PubMed] [Google Scholar]
  16. Feramisco J. R., Smart J. E., Burridge K., Helfman D. M., Thomas G. P. Co-existence of vinculin and a vinculin-like protein of higher molecular weight in smooth muscle. J Biol Chem. 1982 Sep 25;257(18):11024–11031. [PubMed] [Google Scholar]
  17. Fritsch E. F., Lawn R. M., Maniatis T. Molecular cloning and characterization of the human beta-like globin gene cluster. Cell. 1980 Apr;19(4):959–972. doi: 10.1016/0092-8674(80)90087-2. [DOI] [PubMed] [Google Scholar]
  18. Gilbert W. Why genes in pieces? Nature. 1978 Feb 9;271(5645):501–501. doi: 10.1038/271501a0. [DOI] [PubMed] [Google Scholar]
  19. Gorman C. M., Moffat L. F., Howard B. H. Recombinant genomes which express chloramphenicol acetyltransferase in mammalian cells. Mol Cell Biol. 1982 Sep;2(9):1044–1051. doi: 10.1128/mcb.2.9.1044. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Graham F. L., Smiley J., Russell W. C., Nairn R. Characteristics of a human cell line transformed by DNA from human adenovirus type 5. J Gen Virol. 1977 Jul;36(1):59–74. doi: 10.1099/0022-1317-36-1-59. [DOI] [PubMed] [Google Scholar]
  21. Grunstein M., Hogness D. S. Colony hybridization: a method for the isolation of cloned DNAs that contain a specific gene. Proc Natl Acad Sci U S A. 1975 Oct;72(10):3961–3965. doi: 10.1073/pnas.72.10.3961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hamer D. H., Leder P. Splicing and the formation of stable RNA. Cell. 1979 Dec;18(4):1299–1302. doi: 10.1016/0092-8674(79)90240-x. [DOI] [PubMed] [Google Scholar]
  23. Hentschel C. C., Birnstiel M. L. The organization and expression of histone gene families. Cell. 1981 Aug;25(2):301–313. doi: 10.1016/0092-8674(81)90048-9. [DOI] [PubMed] [Google Scholar]
  24. Holmgren R., Livak K., Morimoto R., Freund R., Meselson M. Studies of cloned sequences from four Drosophila heat shock loci. Cell. 1979 Dec;18(4):1359–1370. doi: 10.1016/0092-8674(79)90246-0. [DOI] [PubMed] [Google Scholar]
  25. Houck C. M., Rinehart F. P., Schmid C. W. A ubiquitous family of repeated DNA sequences in the human genome. J Mol Biol. 1979 Aug 15;132(3):289–306. doi: 10.1016/0022-2836(79)90261-4. [DOI] [PubMed] [Google Scholar]
  26. Ingolia T. D., Craig E. A. Drosophila gene related to the major heat shock-induced gene is transcribed at normal temperatures and not induced by heat shock. Proc Natl Acad Sci U S A. 1982 Jan;79(2):525–529. doi: 10.1073/pnas.79.2.525. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Ingolia T. D., Slater M. R., Craig E. A. Saccharomyces cerevisiae contains a complex multigene family related to the major heat shock-inducible gene of Drosophila. Mol Cell Biol. 1982 Nov;2(11):1388–1398. doi: 10.1128/mcb.2.11.1388. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Jagadeeswaran P., Forget B. G., Weissman S. M. Short interspersed repetitive DNA elements in eucaryotes: transposable DNA elements generated by reverse transcription of RNA pol III transcripts? Cell. 1981 Oct;26(2 Pt 2):141–142. doi: 10.1016/0092-8674(81)90296-8. [DOI] [PubMed] [Google Scholar]
  29. Jelinek W. R., Toomey T. P., Leinwand L., Duncan C. H., Biro P. A., Choudary P. V., Weissman S. M., Rubin C. M., Houck C. M., Deininger P. L. Ubiquitous, interspersed repeated sequences in mammalian genomes. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1398–1402. doi: 10.1073/pnas.77.3.1398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Kao H. T., Nevins J. R. Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection. Mol Cell Biol. 1983 Nov;3(11):2058–2065. doi: 10.1128/mcb.3.11.2058. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Kelley P. M., Schlesinger M. J. Antibodies to two major chicken heat shock proteins cross-react with similar proteins in widely divergent species. Mol Cell Biol. 1982 Mar;2(3):267–274. doi: 10.1128/mcb.2.3.267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Kingston R. E., Baldwin A. S., Jr, Sharp P. A. Regulation of heat shock protein 70 gene expression by c-myc. Nature. 1984 Nov 15;312(5991):280–282. doi: 10.1038/312280a0. [DOI] [PubMed] [Google Scholar]
  33. Kioussis D., Eiferman F., van de Rijn P., Gorin M. B., Ingram R. S., Tilghman S. M. The evolution of alpha-fetoprotein and albumin. II. The structures of the alpha-fetoprotein and albumin genes in the mouse. J Biol Chem. 1981 Feb 25;256(4):1960–1967. [PubMed] [Google Scholar]
  34. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  35. Lamb R. A., Lai C. J. Sequence of interrupted and uninterrupted mRNAs and cloned DNA coding for the two overlapping nonstructural proteins of influenza virus. Cell. 1980 Sep;21(2):475–485. doi: 10.1016/0092-8674(80)90484-5. [DOI] [PubMed] [Google Scholar]
  36. Laskey R. A., Mills A. D. Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur J Biochem. 1975 Aug 15;56(2):335–341. doi: 10.1111/j.1432-1033.1975.tb02238.x. [DOI] [PubMed] [Google Scholar]
  37. Lawn R. M., Fritsch E. F., Parker R. C., Blake G., Maniatis T. The isolation and characterization of linked delta- and beta-globin genes from a cloned library of human DNA. Cell. 1978 Dec;15(4):1157–1174. doi: 10.1016/0092-8674(78)90043-0. [DOI] [PubMed] [Google Scholar]
  38. Lis J. T., Ish-Horowicz D., Pinchin S. M. Genomic organization and transcription of the alpha beta heat shock DNA in Drosophila melanogaster. Nucleic Acids Res. 1981 Oct 24;9(20):5297–5310. doi: 10.1093/nar/9.20.5297. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Livak K. J., Freund R., Schweber M., Wensink P. C., Meselson M. Sequence organization and transcription at two heat shock loci in Drosophila. Proc Natl Acad Sci U S A. 1978 Nov;75(11):5613–5617. doi: 10.1073/pnas.75.11.5613. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Maniatis T., Hardison R. C., Lacy E., Lauer J., O'Connell C., Quon D., Sim G. K., Efstratiadis A. The isolation of structural genes from libraries of eucaryotic DNA. Cell. 1978 Oct;15(2):687–701. doi: 10.1016/0092-8674(78)90036-3. [DOI] [PubMed] [Google Scholar]
  41. Maniatis T., Jeffrey A., van deSande H. Chain length determination of small double- and single-stranded DNA molecules by polyacrylamide gel electrophoresis. Biochemistry. 1975 Aug 26;14(17):3787–3794. doi: 10.1021/bi00688a010. [DOI] [PubMed] [Google Scholar]
  42. Maxam A. M., Gilbert W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980;65(1):499–560. doi: 10.1016/s0076-6879(80)65059-9. [DOI] [PubMed] [Google Scholar]
  43. McDonell M. W., Simon M. N., Studier F. W. Analysis of restriction fragments of T7 DNA and determination of molecular weights by electrophoresis in neutral and alkaline gels. J Mol Biol. 1977 Feb 15;110(1):119–146. doi: 10.1016/s0022-2836(77)80102-2. [DOI] [PubMed] [Google Scholar]
  44. McDongall J. K., Dunn A. R., Gallimore P. H. Recent studies on the characteristics of adenovirus-infected and -transformed cells. Cold Spring Harb Symp Quant Biol. 1975;39(Pt 1):591–600. doi: 10.1101/sqb.1974.039.01.073. [DOI] [PubMed] [Google Scholar]
  45. Nagata S., Mantei N., Weissmann C. The structure of one of the eight or more distinct chromosomal genes for human interferon-alpha. Nature. 1980 Oct 2;287(5781):401–408. doi: 10.1038/287401a0. [DOI] [PubMed] [Google Scholar]
  46. Nevins J. R. Induction of the synthesis of a 70,000 dalton mammalian heat shock protein by the adenovirus E1A gene product. Cell. 1982 Jul;29(3):913–919. doi: 10.1016/0092-8674(82)90453-6. [DOI] [PubMed] [Google Scholar]
  47. Pelham H. R., Jackson R. J. An efficient mRNA-dependent translation system from reticulocyte lysates. Eur J Biochem. 1976 Aug 1;67(1):247–256. doi: 10.1111/j.1432-1033.1976.tb10656.x. [DOI] [PubMed] [Google Scholar]
  48. Ricciardi R. P., Miller J. S., Roberts B. E. Purification and mapping of specific mRNAs by hybridization-selection and cell-free translation. Proc Natl Acad Sci U S A. 1979 Oct;76(10):4927–4931. doi: 10.1073/pnas.76.10.4927. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Rigby P. W., Dieckmann M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J Mol Biol. 1977 Jun 15;113(1):237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  50. Ryffel G. U., Muellener D. B., Wyler T., Wahli W., Weber R. Transcription of single-copy vitellogenin gene of Xenopus involves expression of middle repetitive DNA. Nature. 1981 Jun 4;291(5814):429–431. doi: 10.1038/291429a0. [DOI] [PubMed] [Google Scholar]
  51. Schmid C. W., Jelinek W. R. The Alu family of dispersed repetitive sequences. Science. 1982 Jun 4;216(4550):1065–1070. doi: 10.1126/science.6281889. [DOI] [PubMed] [Google Scholar]
  52. Shen C. K., Maniatis T. The organization of repetitive sequences in a cluster of rabbit beta-like globin genes. Cell. 1980 Feb;19(2):379–391. doi: 10.1016/0092-8674(80)90512-7. [DOI] [PubMed] [Google Scholar]
  53. Singer M. F. SINEs and LINEs: highly repeated short and long interspersed sequences in mammalian genomes. Cell. 1982 Mar;28(3):433–434. doi: 10.1016/0092-8674(82)90194-5. [DOI] [PubMed] [Google Scholar]
  54. Slater A., Cato A. C., Sillar G. M., Kioussis J., Burdon R. H. The pattern of protein synthesis induced by heat shock of HeLa cells. Eur J Biochem. 1981 Jul;117(2):341–346. doi: 10.1111/j.1432-1033.1981.tb06343.x. [DOI] [PubMed] [Google Scholar]
  55. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  56. Sugden B., De Troy B., Roberts R. J., Sambrook J. Agarose slab-gel electrophoresis equipment. Anal Biochem. 1975 Sep;68(1):36–46. doi: 10.1016/0003-2697(75)90676-4. [DOI] [PubMed] [Google Scholar]
  57. Thomas P. S. Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5201–5205. doi: 10.1073/pnas.77.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Velazquez J. M., Sonoda S., Bugaisky G., Lindquist S. Is the major Drosophila heat shock protein present in cells that have not been heat shocked? J Cell Biol. 1983 Jan;96(1):286–290. doi: 10.1083/jcb.96.1.286. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. Wang C., Gomer R. H., Lazarides E. Heat shock proteins are methylated in avian and mammalian cells. Proc Natl Acad Sci U S A. 1981 Jun;78(6):3531–3535. doi: 10.1073/pnas.78.6.3531. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Wigler M., Pellicer A., Silverstein S., Axel R., Urlaub G., Chasin L. DNA-mediated transfer of the adenine phosphoribosyltransferase locus into mammalian cells. Proc Natl Acad Sci U S A. 1979 Mar;76(3):1373–1376. doi: 10.1073/pnas.76.3.1373. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Yu-Lee L. Y., Rosen J. M. The rat casein multigene family. I. Fine structure of the gamma-casein gene. J Biol Chem. 1983 Sep 10;258(17):10794–10804. [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES