Skip to main content
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1982 May 25;10(10):3099–3116. doi: 10.1093/nar/10.10.3099

Construction and expression in vivo of an internally deleted mouse alpha-fetoprotein gene: presence of a transcribed Alu-like repeat within the first intervening sequence.

P R Young, R W Scott, D H Hamer, S M Tilghman
PMCID: PMC320693  PMID: 6179043

Abstract

An alpha-fetoprotein (AFP) 'minigene' was constructed by joining the first two exons along with 0.9 kilobases of 5' flanking sequence of the mouse AFP gene to its last exon and 0.4 kilobases of 3' flanking sequence. This 'minigene' was tested for activity by inserting it into an SV40 vector and infecting African green monkey kidney cells. Correct initiation, termination, polyadenylation and splicing were observed. Furthermore a 220 nucleotide transcript was detected and mapped to a mouse Alu-like or B1 repeat on the opposite strand to that encoding the AFP gene in the first intervening sequence.

Full text

PDF
3106

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. Benoist C., Chambon P. In vivo sequence requirements of the SV40 early promotor region. Nature. 1981 Mar 26;290(5804):304–310. doi: 10.1038/290304a0. [DOI] [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. Bogenhagen D. F., Sakonju S., Brown D. D. A control region in the center of the 5S RNA gene directs specific initiation of transcription: II. The 3' border of the region. Cell. 1980 Jan;19(1):27–35. doi: 10.1016/0092-8674(80)90385-2. [DOI] [PubMed] [Google Scholar]
  6. Breathnach R., Benoist C., O'Hare K., Gannon F., Chambon P. Ovalbumin gene: evidence for a leader sequence in mRNA and DNA sequences at the exon-intron boundaries. Proc Natl Acad Sci U S A. 1978 Oct;75(10):4853–4857. doi: 10.1073/pnas.75.10.4853. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cochet M., Gannon F., Hen R., Maroteaux L., Perrin F., Chambon P. Organization and sequence studies of the 17-piece chicken conalbumin gene. Nature. 1979 Dec 6;282(5739):567–574. doi: 10.1038/282567a0. [DOI] [PubMed] [Google Scholar]
  8. Di Segni G., Carrara G., Tocchini-Valentini G. R., Shoulders C. C., Baralle F. E. Selective in vitro transcription of one of the two Alu family repeats present in the 5' flanking region of the human epsilon-globin gene. Nucleic Acids Res. 1981 Dec 21;9(24):6709–6722. doi: 10.1093/nar/9.24.6709. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dziadek M., Adamson E. Localization and synthesis of alphafoetoprotein in post-implantation mouse embryos. J Embryol Exp Morphol. 1978 Feb;43:289–313. [PubMed] [Google Scholar]
  10. Eiferman F. A., Young P. R., Scott R. W., Tilghman S. M. Intragenic amplification and divergence in the mouse alpha-fetoprotein gene. Nature. 1981 Dec 24;294(5843):713–718. doi: 10.1038/294713a0. [DOI] [PubMed] [Google Scholar]
  11. Fitzgerald M., Shenk T. The sequence 5'-AAUAAA-3'forms parts of the recognition site for polyadenylation of late SV40 mRNAs. Cell. 1981 Apr;24(1):251–260. doi: 10.1016/0092-8674(81)90521-3. [DOI] [PubMed] [Google Scholar]
  12. Fowlkes D. M., Shenk T. Transcriptional control regions of the adenovirus VAI RNA gene. Cell. 1980 Nov;22(2 Pt 2):405–413. doi: 10.1016/0092-8674(80)90351-7. [DOI] [PubMed] [Google Scholar]
  13. Fuhrman S. A., Deininger P. L., LaPorte P., Friedmann T., Geiduschek E. P. Analysis of transcription of the human Alu family ubiquitous repeating element by eukaryotic RNA polymerase III. Nucleic Acids Res. 1981 Dec 11;9(23):6439–6456. doi: 10.1093/nar/9.23.6439. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Galli G., Hofstetter H., Birnstiel M. L. Two conserved sequence blocks within eukaryotic tRNA genes are major promoter elements. Nature. 1981 Dec 17;294(5842):626–631. doi: 10.1038/294626a0. [DOI] [PubMed] [Google Scholar]
  15. Ghosh P. K., Roy P., Barkan A., Mertz J. E., Weissman S. M., Lebowitz P. Unspliced functional late 19S mRNAs containing intervening sequences are produced by a late leader mutant of simian virus 40. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1386–1390. doi: 10.1073/pnas.78.3.1386. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gluzman Y., Sambrook J. F., Frisque R. J. Expression of early genes of origin-defective mutants of simian virus 40. Proc Natl Acad Sci U S A. 1980 Jul;77(7):3898–3902. doi: 10.1073/pnas.77.7.3898. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Grosschedl R., Birnstiel M. L. Spacer DNA sequences upstream of the T-A-T-A-A-A-T-A sequence are essential for promotion of H2A histone gene transcription in vivo. Proc Natl Acad Sci U S A. 1980 Dec;77(12):7102–7106. doi: 10.1073/pnas.77.12.7102. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. 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]
  19. Gruss P., Dhar R., Khoury G. Simian virus 40 tandem repeated sequences as an element of the early promoter. Proc Natl Acad Sci U S A. 1981 Feb;78(2):943–947. doi: 10.1073/pnas.78.2.943. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Gruss P., Efstratiadis A., Karathanasis S., König M., Khoury G. Synthesis of stable unspliced mRNA from an intronless simian virus 40--rat preproinsulin gene recombinant. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6091–6095. doi: 10.1073/pnas.78.10.6091. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Gruss P., Lai C. J., Dhar R., Khoury G. Splicing as a requirement for biogenesis of functional 16S mRNA of simian virus 40. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4317–4321. doi: 10.1073/pnas.76.9.4317. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hamer D. H., Kaehler M., Leder P. A mouse globin gene promoter is functional in SV40. Cell. 1980 Oct;21(3):697–708. doi: 10.1016/0092-8674(80)90433-x. [DOI] [PubMed] [Google Scholar]
  23. Hamer D. H., Smith K. D., Boyer S. H., Leder P. SV40 recombinants carrying rabbit beta-globin gene coding sequences. Cell. 1979 Jul;17(3):725–735. doi: 10.1016/0092-8674(79)90279-4. [DOI] [PubMed] [Google Scholar]
  24. Haynes S. R., Jelinek W. R. Low molecular weight RNAs transcribed in vitro by RNA polymerase III from Alu-type dispersed repeats in Chinese hamster DNA are also found in vivo. Proc Natl Acad Sci U S A. 1981 Oct;78(10):6130–6134. doi: 10.1073/pnas.78.10.6130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Haynes S. R., Toomey T. P., Leinwand L., Jelinek W. R. The Chinese hamster Alu-equivalent sequence: a conserved highly repetitious, interspersed deoxyribonucleic acid sequence in mammals has a structure suggestive of a transposable element. Mol Cell Biol. 1981 Jul;1(7):573–583. doi: 10.1128/mcb.1.7.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. 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]
  27. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  28. Hogan B. L., Taylor A., Adamson E. Cell interactions modulate embryonal carcinoma cell differentiation into parietal or visceral endoderm. Nature. 1981 May 21;291(5812):235–237. doi: 10.1038/291235a0. [DOI] [PubMed] [Google Scholar]
  29. Huang A. L., Ostrowski M. C., Berard D., Hager G. L. Glucocorticoid regulation of the Ha-MuSV p21 gene conferred by sequences from mouse mammary tumor virus. Cell. 1981 Dec;27(2 Pt 1):245–255. doi: 10.1016/0092-8674(81)90408-6. [DOI] [PubMed] [Google Scholar]
  30. 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]
  31. Jelinek W., Leinwand L. Low molecular weight RNAs hydrogen-bonded to nuclear and cytoplasmic poly(A)-terminated RNA from cultured Chinese hamster ovary cells. Cell. 1978 Sep;15(1):205–214. doi: 10.1016/0092-8674(78)90095-8. [DOI] [PubMed] [Google Scholar]
  32. 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]
  33. Krayev A. S., Kramerov D. A., Skryabin K. G., Ryskov A. P., Bayev A. A., Georgiev G. P. The nucleotide sequence of the ubiquitous repetitive DNA sequence B1 complementary to the most abundant class of mouse fold-back RNA. Nucleic Acids Res. 1980 Mar 25;8(6):1201–1215. doi: 10.1093/nar/8.6.1201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Leder A., Miller H. I., Hamer D. H., Seidman J. G., Norman B., Sullivan M., Leder P. Comparison of cloned mouse alpha- and beta-globin genes: conservation of intervening sequence locations and extragenic homology. Proc Natl Acad Sci U S A. 1978 Dec;75(12):6187–6191. doi: 10.1073/pnas.75.12.6187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Lee F., Mulligan R., Berg P., Ringold G. Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids. Nature. 1981 Nov 19;294(5838):228–232. doi: 10.1038/294228a0. [DOI] [PubMed] [Google Scholar]
  36. Maniatis T., Jeffrey A., Kleid D. G. Nucleotide sequence of the rightward operator of phage lambda. Proc Natl Acad Sci U S A. 1975 Mar;72(3):1184–1188. doi: 10.1073/pnas.72.3.1184. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. 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]
  38. McKnight S. L., Gavis E. R., Kingsbury R., Axel R. Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell. 1981 Aug;25(2):385–398. doi: 10.1016/0092-8674(81)90057-x. [DOI] [PubMed] [Google Scholar]
  39. Moreau P., Hen R., Wasylyk B., Everett R., Gaub M. P., Chambon P. The SV40 72 base repair repeat has a striking effect on gene expression both in SV40 and other chimeric recombinants. Nucleic Acids Res. 1981 Nov 25;9(22):6047–6068. doi: 10.1093/nar/9.22.6047. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. 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]
  41. Page G. S., Smith S., Goodman H. M. DNA sequence of the rat growth hormone gene: location of the 5' terminus of the growth hormone mRNA and identification of an internal transposon-like element. Nucleic Acids Res. 1981 May 11;9(9):2087–2104. doi: 10.1093/nar/9.9.2087. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Pavlakis G. N., Hizuka N., Gorden P., Seeburg P., Hamer D. H. Expression of two human growth hormone genes in monkey cells infected by simian virus 40 recombinants. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7398–7402. doi: 10.1073/pnas.78.12.7398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Sakonju S., Bogenhagen D. F., Brown D. D. A control region in the center of the 5S RNA gene directs specific initiation of transcription: I. The 5' border of the region. Cell. 1980 Jan;19(1):13–25. doi: 10.1016/0092-8674(80)90384-0. [DOI] [PubMed] [Google Scholar]
  44. Schibler U., Tosi M., Pittet A. C., Fabiani L., Wellauer P. K. Tissue-specific expression of mouse alpha-amylase genes. J Mol Biol. 1980 Sep 5;142(1):93–116. doi: 10.1016/0022-2836(80)90208-9. [DOI] [PubMed] [Google Scholar]
  45. Sharp P. A. Speculations on RNA splicing. Cell. 1981 Mar;23(3):643–646. doi: 10.1016/0092-8674(81)90425-6. [DOI] [PubMed] [Google Scholar]
  46. Shortle D., Koshland D., Weinstock G. M., Botstein D. Segment-directed mutagenesis: construction in vitro of point mutations limited to a small predetermined region of a circular DNA molecule. Proc Natl Acad Sci U S A. 1980 Sep;77(9):5375–5379. doi: 10.1073/pnas.77.9.5375. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Sleigh M. J., Topp W. C., Hanich R., Sambrook J. F. Mutants of SV40 with an altered small t protein are reduced in their ability to transform cells. Cell. 1978 May;14(1):79–88. doi: 10.1016/0092-8674(78)90303-3. [DOI] [PubMed] [Google Scholar]
  48. 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]
  49. Struhl K. Deletion mapping a eukaryotic promoter. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4461–4465. doi: 10.1073/pnas.78.7.4461. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Stumph W. E., Kristo P., Tsai M. J., O'Malley B. W. A chicken middle-repetitive DNA sequence which shares homology with mammalian ubiquitous repeats. Nucleic Acids Res. 1981 Oct 24;9(20):5383–5397. doi: 10.1093/nar/9.20.5383. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. 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]
  52. Trapnell B. C., Tolstoshev P., Crystal R. G. Secondary structures for splice junctions in eukaryotic and viral messenger RNA precursors. Nucleic Acids Res. 1980 Aug 25;8(16):3659–3672. doi: 10.1093/nar/8.16.3659. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Ullrich A., Shine J., Chirgwin J., Pictet R., Tischer E., Rutter W. J., Goodman H. M. Rat insulin genes: construction of plasmids containing the coding sequences. Science. 1977 Jun 17;196(4296):1313–1319. doi: 10.1126/science.325648. [DOI] [PubMed] [Google Scholar]
  54. Villa-Komaroff L., Efstratiadis A., Broome S., Lomedico P., Tizard R., Naber S. P., Chick W. L., Gilbert W. A bacterial clone synthesizing proinsulin. Proc Natl Acad Sci U S A. 1978 Aug;75(8):3727–3731. doi: 10.1073/pnas.75.8.3727. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wahl G. M., Stern M., Stark G. R. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl-paper and rapid hybridization by using dextran sulfate. Proc Natl Acad Sci U S A. 1979 Aug;76(8):3683–3687. doi: 10.1073/pnas.76.8.3683. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Wigler M., Silverstein S., Lee L. S., Pellicer A., Cheng Y. c., Axel R. Transfer of purified herpes virus thymidine kinase gene to cultured mouse cells. Cell. 1977 May;11(1):223–232. doi: 10.1016/0092-8674(77)90333-6. [DOI] [PubMed] [Google Scholar]
  57. Wuilmart C., Urbain J., Givol D. On the location of palindromes in immunoglobulin genes. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2526–2530. doi: 10.1073/pnas.74.6.2526. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES