Skip to main content
PMC home page
Genetics logoLink to Genetics
. 1988 Dec;120(4):1111–1124. doi: 10.1093/genetics/120.4.1111

Maize Glutamine Synthetase Cdnas: Isolation by Direct Genetic Selection in Escherichia Coli

D P Snustad 1, J P Hunsperger 1, B M Chereskin 1, J Messing 1
PMCID: PMC1203574  PMID: 2906306

Abstract

Maize glutamine synthetase cDNA clones were isolated by genetic selection for functional rescue of an Escherichia coli ΔglnA mutant growing on medium lacking glutamine. The Black Mexican Sweet cDNA library used in this study was constructed in pUC13 such that cDNA sense strands were transcribed under the control of the lac promoter. E. coli ΔglnA cells were transformed with cDNA library plasmid DNA, grown briefly in rich medium to allow phenotypic expression of the cDNAs and the pUC13 amp(r) gene, and challenged to grow on agar medium lacking glutamine. Large numbers of glutamine synthetase cDNA clones have been identified in individual 150-mm Petri dishes; all characterized cDNA clones carry complete coding sequences. Two cDNAs identical except for different 5' and 3' termini have been sequenced. The major open reading frame predicts a protein with an amino acid sequence that exhibits striking similarity to the amino acid sequences of the predicted products of previously sequenced eukaryotic glutamine synthetase cDNAs and genes. In addition, the maize glutamine synthetase cDNAs were shown to contain a 5' mini-ORF of 29 codons separated by 37 nucleotide pairs from the major ORF. This mini-ORF was shown not to be essential for the functional rescue of the E. coli ΔglnA mutant. Expression of the cDNAs in E. coli is presumed to be due to the function of a polycistronic hybrid lac messenger RNA or translational fusions encoded by the pUC plasmids. Proteins of the expected sizes encoded by two different pUC clones were shown to react with antibodies to tobacco glutamine synthetase.

Full Text

The Full Text of this article is available as a PDF (4.8 MB).

Selected References

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

  1. Benton W. D., Davis R. W. Screening lambdagt recombinant clones by hybridization to single plaques in situ. Science. 1977 Apr 8;196(4286):180–182. doi: 10.1126/science.322279. [DOI] [PubMed] [Google Scholar]
  2. Bondaryk R. P., Paulus H. Expression of the gene for Bacillus subtilis aspartokinase II in Escherichia coli. J Biol Chem. 1985 Jan 10;260(1):592–597. [PubMed] [Google Scholar]
  3. Colombo G., Villafranca J. J. Amino acid sequence of Escherichia coli glutamine synthetase deduced from the DNA nucleotide sequence. J Biol Chem. 1986 Aug 15;261(23):10587–10591. [PubMed] [Google Scholar]
  4. Cullimore J. V., Gebhardt C., Saarelainen R., Miflin B. J., Idler K. B., Barker R. F. Glutamine synthetase of Phaseolus vulgaris L.: organ-specific expression of a multigene family. J Mol Appl Genet. 1984;2(6):589–599. [PubMed] [Google Scholar]
  5. Dale R. M., McClure B. A., Houchins J. P. A rapid single-stranded cloning strategy for producing a sequential series of overlapping clones for use in DNA sequencing: application to sequencing the corn mitochondrial 18 S rDNA. Plasmid. 1985 Jan;13(1):31–40. doi: 10.1016/0147-619x(85)90053-8. [DOI] [PubMed] [Google Scholar]
  6. DasSarma S., Tischer E., Goodman H. M. Plant glutamine synthetase complements a glnA mutation in Escherichia coli. Science. 1986 Jun 6;232(4755):1242–1244. doi: 10.1126/science.2871626. [DOI] [PubMed] [Google Scholar]
  7. Devereux J., Haeberli P., Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. doi: 10.1093/nar/12.1part1.387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Gebhardt C., Oliver J. E., Forde B. G., Saarelainen R., Miflin B. J. Primary structure and differential expression of glutamine synthetase genes in nodules, roots and leaves of Phaseolus vulgaris. EMBO J. 1986 Jul;5(7):1429–1435. doi: 10.1002/j.1460-2075.1986.tb04379.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. 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]
  10. Hanahan D., Meselson M. Plasmid screening at high colony density. Gene. 1980 Jun;10(1):63–67. doi: 10.1016/0378-1119(80)90144-4. [DOI] [PubMed] [Google Scholar]
  11. Hanahan D. Studies on transformation of Escherichia coli with plasmids. J Mol Biol. 1983 Jun 5;166(4):557–580. doi: 10.1016/s0022-2836(83)80284-8. [DOI] [PubMed] [Google Scholar]
  12. Heidecker G., Messing J. Sequence analysis of zein cDNAs obtained by an efficient mRNA cloning method. Nucleic Acids Res. 1983 Jul 25;11(14):4891–4906. doi: 10.1093/nar/11.14.4891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Helfman D. M., Feramisco J. R., Fiddes J. C., Thomas G. P., Hughes S. H. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci U S A. 1983 Jan;80(1):31–35. doi: 10.1073/pnas.80.1.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Karlin-Neumann G. A., Tobin E. M. Transit peptides of nuclear-encoded chloroplast proteins share a common amino acid framework. EMBO J. 1986 Jan;5(1):9–13. doi: 10.1002/j.1460-2075.1986.tb04170.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kirihara J. A., Hunsperger J. P., Mahoney W. C., Messing J. W. Differential expression of a gene for a methionine-rich storage protein in maize. Mol Gen Genet. 1988 Mar;211(3):477–484. doi: 10.1007/BF00425704. [DOI] [PubMed] [Google Scholar]
  16. Kozak M. Bifunctional messenger RNAs in eukaryotes. Cell. 1986 Nov 21;47(4):481–483. doi: 10.1016/0092-8674(86)90609-4. [DOI] [PubMed] [Google Scholar]
  17. Kozak M. Compilation and analysis of sequences upstream from the translational start site in eukaryotic mRNAs. Nucleic Acids Res. 1984 Jan 25;12(2):857–872. doi: 10.1093/nar/12.2.857. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kozak M. How do eucaryotic ribosomes select initiation regions in messenger RNA? Cell. 1978 Dec;15(4):1109–1123. doi: 10.1016/0092-8674(78)90039-9. [DOI] [PubMed] [Google Scholar]
  19. Martin M. L., Kirkpatrick H. Nursing assessment of the aggressive elderly. Perspectives. 1987 Fall;11(3):8–10. [PubMed] [Google Scholar]
  20. McMullen M. D., Hunter B., Phillips R. L., Rubenstein I. The structure of the maize ribosomal DNA spacer region. Nucleic Acids Res. 1986 Jun 25;14(12):4953–4968. doi: 10.1093/nar/14.12.4953. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. McNally S. F., Hirel B., Gadal P., Mann A. F., Stewart G. R. Glutamine Synthetases of Higher Plants : Evidence for a Specific Isoform Content Related to Their Possible Physiological Role and Their Compartmentation within the Leaf. Plant Physiol. 1983 May;72(1):22–25. doi: 10.1104/pp.72.1.22. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Mozer T. J. Partial purification and characterization of the mRNA for alpha-amylase from barley aleurone layers. Plant Physiol. 1980 May;65(5):834–837. doi: 10.1104/pp.65.5.834. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Reed K. C., Mann D. A. Rapid transfer of DNA from agarose gels to nylon membranes. Nucleic Acids Res. 1985 Oct 25;13(20):7207–7221. doi: 10.1093/nar/13.20.7207. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Shure M., Wessler S., Fedoroff N. Molecular identification and isolation of the Waxy locus in maize. Cell. 1983 Nov;35(1):225–233. doi: 10.1016/0092-8674(83)90225-8. [DOI] [PubMed] [Google Scholar]
  27. Steege D. A. 5'-Terminal nucleotide sequence of Escherichia coli lactose repressor mRNA: features of translational initiation and reinitiation sites. Proc Natl Acad Sci U S A. 1977 Oct;74(10):4163–4167. doi: 10.1073/pnas.74.10.4163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Tabor S., Richardson C. C. DNA sequence analysis with a modified bacteriophage T7 DNA polymerase. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4767–4771. doi: 10.1073/pnas.84.14.4767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Tingey S. V., Coruzzi G. M. Glutamine Synthetase of Nicotiana plumbaginifolia: Cloning and in Vivo Expression. Plant Physiol. 1987 Jun;84(2):366–373. doi: 10.1104/pp.84.2.366. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Tingey S. V., Walker E. L., Coruzzi G. M. Glutamine synthetase genes of pea encode distinct polypeptides which are differentially expressed in leaves, roots and nodules. EMBO J. 1987 Jan;6(1):1–9. doi: 10.1002/j.1460-2075.1987.tb04710.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Vieira J., Messing J. Production of single-stranded plasmid DNA. Methods Enzymol. 1987;153:3–11. doi: 10.1016/0076-6879(87)53044-0. [DOI] [PubMed] [Google Scholar]
  32. Walker J. E., Gay N. J. Analysis of Escherichia coli ATP synthase subunits by DNA and protein sequencing. Methods Enzymol. 1983;97:195–218. doi: 10.1016/0076-6879(83)97133-1. [DOI] [PubMed] [Google Scholar]
  33. Weinstock R., Sweet R., Weiss M., Cedar H., Axel R. Intragenic DNA spacers interrupt the ovalbumin gene. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1299–1303. doi: 10.1073/pnas.75.3.1299. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. de Bruijn F. J., Ausubel F. M. The cloning and transposon Tn5 mutagenesis of the glnA region of Klebsiella pneumoniae: identification of glnR, a gene involved in the regulation of the nif and hut operons. Mol Gen Genet. 1981;183(2):289–297. doi: 10.1007/BF00270631. [DOI] [PubMed] [Google Scholar]

Articles from Genetics are provided here courtesy of Oxford University Press

RESOURCES