Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1987 Apr 24;15(8):3369–3383. doi: 10.1093/nar/15.8.3369

Sequence and S1 nuclease mapping of the 5' region of the dihydrofolate reductase-thymidylate synthase gene of Leishmania major.

G M Kapler, K Zhang, S M Beverley
PMCID: PMC340735  PMID: 3554143

Abstract

The 5' structure of mRNA transcribed from the dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene of the protozoan parasite Leishmania major has been characterized. S1 nuclease mapping identifies a heterogenous 5' structure which is not affected by growth phase or developmental stage. The DNA sequence of the 5' region of the DHFR-TS gene does not reveal homology with other trypanosomatid genes, eukaryotic consensus genetic elements, or the mammalian DHFR promoter element. This latter finding is especially significant as we show that the 5' region of the E. coli DHFR gene exhibits homology to the mammalian DHFR promoter element, despite their greater evolutionary distance.

Full text

PDF
3369

Images in this article

Selected References

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

  1. 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]
  2. Banerji J., Rusconi S., Schaffner W. Expression of a beta-globin gene is enhanced by remote SV40 DNA sequences. Cell. 1981 Dec;27(2 Pt 1):299–308. doi: 10.1016/0092-8674(81)90413-x. [DOI] [PubMed] [Google Scholar]
  3. Belfort M., Maley G., Pedersen-Lane J., Maley F. Primary structure of the Escherichia coli thyA gene and its thymidylate synthase product. Proc Natl Acad Sci U S A. 1983 Aug;80(16):4914–4918. doi: 10.1073/pnas.80.16.4914. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Berk A. J., Sharp P. A. Sizing and mapping of early adenovirus mRNAs by gel electrophoresis of S1 endonuclease-digested hybrids. Cell. 1977 Nov;12(3):721–732. doi: 10.1016/0092-8674(77)90272-0. [DOI] [PubMed] [Google Scholar]
  5. Bernards A., Kooter J. M., Borst P. Structure and transcription of a telomeric surface antigen gene of Trypanosoma brucei. Mol Cell Biol. 1985 Mar;5(3):545–553. doi: 10.1128/mcb.5.3.545. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Beverley S. M., Coderre J. A., Santi D. V., Schimke R. T. Unstable DNA amplifications in methotrexate-resistant Leishmania consist of extrachromosomal circles which relocalize during stabilization. Cell. 1984 Sep;38(2):431–439. doi: 10.1016/0092-8674(84)90498-7. [DOI] [PubMed] [Google Scholar]
  7. Beverley S. M., Ellenberger T. E., Cordingley J. S. Primary structure of the gene encoding the bifunctional dihydrofolate reductase-thymidylate synthase of Leishmania major. Proc Natl Acad Sci U S A. 1986 Apr;83(8):2584–2588. doi: 10.1073/pnas.83.8.2584. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Beverley S. M., Ismach R. B., Pratt D. M. Evolution of the genus Leishmania as revealed by comparisons of nuclear DNA restriction fragment patterns. Proc Natl Acad Sci U S A. 1987 Jan;84(2):484–488. doi: 10.1073/pnas.84.2.484. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Biggin M. D., Gibson T. J., Hong G. F. Buffer gradient gels and 35S label as an aid to rapid DNA sequence determination. Proc Natl Acad Sci U S A. 1983 Jul;80(13):3963–3965. doi: 10.1073/pnas.80.13.3963. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Bodemer W., Niller H. H., Nitsche N., Scholz B., Fleckenstein B. Organization of the thymidylate synthase gene of herpesvirus saimiri. J Virol. 1986 Oct;60(1):114–123. doi: 10.1128/jvi.60.1.114-123.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Borst P. Discontinuous transcription and antigenic variation in trypanosomes. Annu Rev Biochem. 1986;55:701–732. doi: 10.1146/annurev.bi.55.070186.003413. [DOI] [PubMed] [Google Scholar]
  12. Breathnach R., Chambon P. Organization and expression of eucaryotic split genes coding for proteins. Annu Rev Biochem. 1981;50:349–383. doi: 10.1146/annurev.bi.50.070181.002025. [DOI] [PubMed] [Google Scholar]
  13. Campbell D. A., Thornton D. A., Boothroyd J. C. Apparent discontinuous transcription of Trypanosoma brucei variant surface antigen genes. 1984 Sep 27-Oct 3Nature. 311(5984):350–355. doi: 10.1038/311350a0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Cathala G., Savouret J. F., Mendez B., West B. L., Karin M., Martial J. A., Baxter J. D. A method for isolation of intact, translationally active ribonucleic acid. DNA. 1983;2(4):329–335. doi: 10.1089/dna.1983.2.329. [DOI] [PubMed] [Google Scholar]
  15. Coderre J. A., Beverley S. M., Schimke R. T., Santi D. V. Overproduction of a bifunctional thymidylate synthetase-dihydrofolate reductase and DNA amplification in methotrexate-resistant Leishmania tropica. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2132–2136. doi: 10.1073/pnas.80.8.2132. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Cornelissen A. W., Verspieren M. P., Toulmé J. J., Swinkels B. W., Borst P. The common 5' terminal sequence on trypanosome mRNAs: a target for anti-messenger oligodeoxynucleotides. Nucleic Acids Res. 1986 Jul 25;14(14):5605–5614. doi: 10.1093/nar/14.14.5605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Crouse G. F., Simonsen C. C., McEwan R. N., Schimke R. T. Structure of amplified normal and variant dihydrofolate reductase genes in mouse sarcoma S180 cells. J Biol Chem. 1982 Jul 10;257(13):7887–7897. [PubMed] [Google Scholar]
  18. De Lange T., Michels P. A., Veerman H. J., Cornelissen A. W., Borst P. Many trypanosome messenger RNAs share a common 5' terminal sequence. Nucleic Acids Res. 1984 May 11;12(9):3777–3790. doi: 10.1093/nar/12.9.3777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Deng T. L., Li D. W., Jenh C. H., Johnson L. F. Structure of the gene for mouse thymidylate synthase. Locations of introns and multiple transcriptional start sites. J Biol Chem. 1986 Dec 5;261(34):16000–16005. [PubMed] [Google Scholar]
  20. Dunn T. M., Hahn S., Ogden S., Schleif R. F. An operator at -280 base pairs that is required for repression of araBAD operon promoter: addition of DNA helical turns between the operator and promoter cyclically hinders repression. Proc Natl Acad Sci U S A. 1984 Aug;81(16):5017–5020. doi: 10.1073/pnas.81.16.5017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Dynan W. S., Tjian R. The promoter-specific transcription factor Sp1 binds to upstream sequences in the SV40 early promoter. Cell. 1983 Nov;35(1):79–87. doi: 10.1016/0092-8674(83)90210-6. [DOI] [PubMed] [Google Scholar]
  22. Ebrahimzadeh A., Jones T. C. A comparative study of different Leishmania tropica isolates from Iran: correlation between infectivity and cytochemical properties. Am J Trop Med Hyg. 1983 Jul;32(4):694–702. doi: 10.4269/ajtmh.1983.32.694. [DOI] [PubMed] [Google Scholar]
  23. Efstratiadis A., Posakony J. W., Maniatis T., Lawn R. M., O'Connell C., Spritz R. A., DeRiel J. K., Forget B. G., Weissman S. M., Slightom J. L. The structure and evolution of the human beta-globin gene family. Cell. 1980 Oct;21(3):653–668. doi: 10.1016/0092-8674(80)90429-8. [DOI] [PubMed] [Google Scholar]
  24. Farnham P. J., Schimke R. T. In vitro transcription and delimitation of promoter elements of the murine dihydrofolate reductase gene. Mol Cell Biol. 1986 Jul;6(7):2392–2401. doi: 10.1128/mcb.6.7.2392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Farnham P. J., Schimke R. T. Murine dihydrofolate reductase transcripts through the cell cycle. Mol Cell Biol. 1986 Feb;6(2):365–371. doi: 10.1128/mcb.6.2.365. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Farnham P. J., Schimke R. T. Transcriptional regulation of mouse dihydrofolate reductase in the cell cycle. J Biol Chem. 1985 Jun 25;260(12):7675–7680. [PubMed] [Google Scholar]
  27. Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  28. Ferone R., Roland S. Dihydrofolate reductase: thymidylate synthase, a bifunctional polypeptide from Crithidia fasciculata. Proc Natl Acad Sci U S A. 1980 Oct;77(10):5802–5806. doi: 10.1073/pnas.77.10.5802. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Garrett C. E., Coderre J. A., Meek T. D., Garvey E. P., Claman D. M., Beverley S. M., Santi D. V. A bifunctional thymidylate synthetase-dihydrofolate reductase in protozoa. Mol Biochem Parasitol. 1984 Apr;11:257–265. doi: 10.1016/0166-6851(84)90070-7. [DOI] [PubMed] [Google Scholar]
  30. Gasser C. S., Schimke R. T. Cell cycle regulation of transfected murine dihydrofolate reductase genes. J Biol Chem. 1986 May 25;261(15):6938–6946. [PubMed] [Google Scholar]
  31. Gonzalez A., Lerner T. J., Huecas M., Sosa-Pineda B., Nogueira N., Lizardi P. M. Apparent generation of a segmented mRNA from two separate tandem gene families in Trypanosoma cruzi. Nucleic Acids Res. 1985 Aug 26;13(16):5789–5804. doi: 10.1093/nar/13.16.5789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Gray M. W. Unusual pattern of ribonucleic acid components in the ribosome of Crithidia fasciculata, a trypanosomatid protozoan. Mol Cell Biol. 1981 Apr;1(4):347–357. doi: 10.1128/mcb.1.4.347. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Grumont R., Washtien W. L., Caput D., Santi D. V. Bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica: sequence homology with the corresponding monofunctional proteins. Proc Natl Acad Sci U S A. 1986 Aug;83(15):5387–5391. doi: 10.1073/pnas.83.15.5387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Hamada H., Seidman M., Howard B. H., Gorman C. M. Enhanced gene expression by the poly(dT-dG).poly(dC-dA) sequence. Mol Cell Biol. 1984 Dec;4(12):2622–2630. doi: 10.1128/mcb.4.12.2622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Irani M. H., Orosz L., Adhya S. A control element within a structural gene: the gal operon of Escherichia coli. Cell. 1983 Mar;32(3):783–788. doi: 10.1016/0092-8674(83)90064-8. [DOI] [PubMed] [Google Scholar]
  36. Jenh C. H., Geyer P. K., Johnson L. F. Control of thymidylate synthase mRNA content and gene transcription in an overproducing mouse cell line. Mol Cell Biol. 1985 Oct;5(10):2527–2532. doi: 10.1128/mcb.5.10.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Jenh C. H., Rao L. G., Johnson L. F. Regulation of thymidylate synthase enzyme synthesis in 5-fluorodeoxyuridine-resistant mouse fibroblasts during the transition from the resting to growing state. J Cell Physiol. 1985 Jan;122(1):149–154. doi: 10.1002/jcp.1041220122. [DOI] [PubMed] [Google Scholar]
  38. Kellems R. E., Morhenn V. B., Pfendt E. A., Alt F. W., Schimke R. T. Polyoma virus and cyclic AMP-mediated control of dihydrofolate reductase mRNA abundance in methotrexate-resistant mouse fibroblasts. J Biol Chem. 1979 Jan 25;254(2):309–318. [PubMed] [Google Scholar]
  39. Leys E. J., Kellems R. E. Control of dihydrofolate reductase messenger ribonucleic acid production. Mol Cell Biol. 1981 Nov;1(11):961–971. doi: 10.1128/mcb.1.11.961. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Liu A. Y., Van der Ploeg L. H., Rijsewijk F. A., Borst P. The transposition unit of variant surface glycoprotein gene 118 of Trypanosoma brucei. Presence of repeated elements at its border and absence of promoter-associated sequences. J Mol Biol. 1983 Jun 15;167(1):57–75. doi: 10.1016/s0022-2836(83)80034-5. [DOI] [PubMed] [Google Scholar]
  41. Mariani B. D., Slate D. L., Schimke R. T. S phase-specific synthesis of dihydrofolate reductase in Chinese hamster ovary cells. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4985–4989. doi: 10.1073/pnas.78.8.4985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Masters J. N., Attardi G. Discrete human dihydrofolate reductase gene transcripts present in polysomal RNA map with their 5' ends several hundred nucleotides upstream of the main mRNA start site. Mol Cell Biol. 1985 Mar;5(3):493–500. doi: 10.1128/mcb.5.3.493. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Maxam A. M., Gilbert W. A new method for sequencing DNA. Proc Natl Acad Sci U S A. 1977 Feb;74(2):560–564. doi: 10.1073/pnas.74.2.560. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. McGrogan M., Simonsen C. C., Smouse D. T., Farnham P. J., Schimke R. T. Heterogeneity at the 5' termini of mouse dihydrofolate reductase mRNAs. Evidence for multiple promoter regions. J Biol Chem. 1985 Feb 25;260(4):2307–2314. [PubMed] [Google Scholar]
  45. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  46. Mitchell P. J., Carothers A. M., Han J. H., Harding J. D., Kas E., Venolia L., Chasin L. A. Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene. Mol Cell Biol. 1986 Feb;6(2):425–440. doi: 10.1128/mcb.6.2.425. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Mount S. M. A catalogue of splice junction sequences. Nucleic Acids Res. 1982 Jan 22;10(2):459–472. doi: 10.1093/nar/10.2.459. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Navalgund L. G., Rossana C., Muench A. J., Johnson L. F. Cell cycle regulation of thymidylate synthetase gene expression in cultured mouse fibroblasts. J Biol Chem. 1980 Aug 10;255(15):7386–7390. [PubMed] [Google Scholar]
  49. Nelson R. G., Parsons M., Barr P. J., Stuart K., Selkirk M., Agabian N. Sequences homologous to the variant antigen mRNA spliced leader are located in tandem repeats and variable orphons in trypanosoma brucei. Cell. 1983 Oct;34(3):901–909. doi: 10.1016/0092-8674(83)90547-0. [DOI] [PubMed] [Google Scholar]
  50. Nishioka Y., Leder P. Organization and complete sequence of identical embryonic and plasmacytoma kappa V-region genes. J Biol Chem. 1980 Apr 25;255(8):3691–3694. [PubMed] [Google Scholar]
  51. Parsons M., Nelson R. G., Watkins K. P., Agabian N. Trypanosome mRNAs share a common 5' spliced leader sequence. Cell. 1984 Aug;38(1):309–316. doi: 10.1016/0092-8674(84)90552-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Pribnow D. Nucleotide sequence of an RNA polymerase binding site at an early T7 promoter. Proc Natl Acad Sci U S A. 1975 Mar;72(3):784–788. doi: 10.1073/pnas.72.3.784. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Proudfoot N. J., Brownlee G. G. 3' non-coding region sequences in eukaryotic messenger RNA. Nature. 1976 Sep 16;263(5574):211–214. doi: 10.1038/263211a0. [DOI] [PubMed] [Google Scholar]
  54. Reed R., Maniatis T. Intron sequences involved in lariat formation during pre-mRNA splicing. Cell. 1985 May;41(1):95–105. doi: 10.1016/0092-8674(85)90064-9. [DOI] [PubMed] [Google Scholar]
  55. Salser W. Globin mRNA sequences: analysis of base pairing and evolutionary implications. Cold Spring Harb Symp Quant Biol. 1978;42(Pt 2):985–1002. doi: 10.1101/sqb.1978.042.01.099. [DOI] [PubMed] [Google Scholar]
  56. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Sather S., Agabian N. A 5' spliced leader is added in trans to both alpha- and beta-tubulin transcripts in Trypanosoma brucei. Proc Natl Acad Sci U S A. 1985 Sep;82(17):5695–5699. doi: 10.1073/pnas.82.17.5695. [DOI] [PMC free article] [PubMed] [Google Scholar]
  58. Sazer S., Schimke R. T. A re-examination of the 5' termini of mouse dihydrofolate reductase RNA. J Biol Chem. 1986 Apr 5;261(10):4685–4690. [PubMed] [Google Scholar]
  59. Setzer D. R., McGrogan M., Schimke R. T. Nucleotide sequence surrounding multiple polyadenylation sites in the mouse dihydrofolate reductase gene. J Biol Chem. 1982 May 10;257(9):5143–5147. [PubMed] [Google Scholar]
  60. Smith D. R., Calvo J. M. Nucleotide sequence of the E coli gene coding for dihydrofolate reductase. Nucleic Acids Res. 1980 May 24;8(10):2255–2274. doi: 10.1093/nar/8.10.2255. [DOI] [PMC free article] [PubMed] [Google Scholar]
  61. Swinkels B. W., Gibson W. C., Osinga K. A., Kramer R., Veeneman G. H., van Boom J. H., Borst P. Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei. EMBO J. 1986 Jun;5(6):1291–1298. doi: 10.1002/j.1460-2075.1986.tb04358.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  62. Tschudi C., Young A. S., Ruben L., Patton C. L., Richards F. F. Calmodulin genes in trypanosomes are tandemly repeated and produce multiple mRNAs with a common 5' leader sequence. Proc Natl Acad Sci U S A. 1985 Jun;82(12):3998–4002. doi: 10.1073/pnas.82.12.3998. [DOI] [PMC free article] [PubMed] [Google Scholar]
  63. Van der Ploeg L. H., Liu A. Y., Michels P. A., De Lange T., Borst P., Majumder H. K., Weber H., Veeneman G. H., Van Boom J. RNA splicing is required to make the messenger RNA for a variant surface antigen in trypanosomes. Nucleic Acids Res. 1982 Jun 25;10(12):3591–3604. doi: 10.1093/nar/10.12.3591. [DOI] [PMC free article] [PubMed] [Google Scholar]
  64. Walder J. A., Eder P. S., Engman D. M., Brentano S. T., Walder R. Y., Knutzon D. S., Dorfman D. M., Donelson J. E. The 35-nucleotide spliced leader sequence is common to all trypanosome messenger RNA's. Science. 1986 Aug 1;233(4763):569–571. doi: 10.1126/science.3523758. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES