Progress 09/01/00 to 08/31/04
Outputs
Certain plant and animal introns increase expression of protein-coding sequences when placed in the 5' region of the transcription unit. The mechanisms of intron-mediated enhancement have not been defined, but are generally accepted to be post- or cotranscriptional in character. One of the most effective plant introns in stimulating gene expression is the 1,028-bp first intron of the Sh1 gene that encodes maize (Zea mays) sucrose synthase. To address the mechanisms of intron-mediated enhancement, we used reporter gene fusions to identify features of the Sh1 first intron required for enhancement in cultured maize cells. A 145-bp derivative conferred approximately the same 20- to 50-fold stimulation typical for the full-length intron in this transient expression system. A 35-bp motif contained within the intron is required for maximum levels of enhancement but not for efficient transcript splicing. The important feature of this redundant 35-bp motif is T-richness rather
than the specific sequence. When transcript splicing was abolished by mutations at the intron borders, enhancement was reduced to about 2-fold. The requirement of splicing for enhancement was not because of upstream translation initiation codons contained in unspliced transcripts. On the basis of our current findings, we conclude that splicing of the Sh1 intron is integral to enhancement, and we hypothesize that transcript modifications triggered by the T-rich motif and splicing may link the mRNA with the trafficking system of the cell.
Impacts
The observations reported above, coupled with the fact that enhancing introns vary widely in their ability to stimulate expression, strongly suggest that different mechanisms of intron-enhanced expression occur in plants. Individual introns likely facilitate one or more of the processes of transcript transport, stabilization, or translation to differing degrees. A complete understanding of intron-mediated enhancement of gene expression must account for these specific findings and for the intriguing observation that the magnitude of enhancement is generally greater for weaker promoters. The evolution of different mechanisms for intron-enhanced gene expression in plants points to the critical importance of this phenomenon in plant gene expression.
Publications
- Spielbauer G, Margl L, Hannah LC, Romisch W, Ettenhuber C, Bacher A, Gierl A, Eisenreich W, Genschel U. 2006 Robustness of central carbohydrate metabolism in developing maize kernels. Phytochemistry 67: 1460 - 1475.
- Lal, S.K., L. C. Hannah 2005 Helitrons contribute to the lack of gene colinearity observed in modern maize. Proc. Natl Acad. Sci 102: 9993-9994.
- Lal, S. K., L. C. Hannah. 2005 Massive changes of the maize genome are caused by Helitrons. Nature: Heredity 95, 421-422. -
- McCarty, D. R., Settles, A. M., Suzuki, M., Tan, B. C., Latshaw, S., Porch, T., Robin, K., Baier, J., Avigne, W., Lai, J., Messing, J., Koch, K., and L. Curtis Hannah 2005 Steady-state transposon mutagenesis in inbred maize. Plant J. 44: 52-61.
- Hannah, L. C. 2005. Starch synthesis in the maize endosperm. Maydica, 50: 497-506.
- Boehlein, Susan K., Aileen K. Sewell, Joanna Cross, Jon D. Stewart and L. Curtis Hannah 2005. Purification and characterization of adenosine diphosphate glucose pyrophosphorylase from maize/potato hybrids. Plant Physiol 138:1552-1562.
- Linebarger, C., Boehlein, S.K., Aileen K. Sewell, A. K., Shaw, J. R. and Hannah, L. C. 2005 Heat stability of maize endosperm ADP-glucose pyrophosphorylase is conveyed by insertion of a cysteine in the N-terminus of the small subunit. Plant Physiol, 139: 1625-1634.
- Ettenhuber, C., Gerti Spielbauer, G., Hannah, L. C., Genschel, U., Bacher, A., Gierl. A., and Wolfgang Eisenreich, W., 2005 Changes in flux pattern of the central carbohydrate metabolism during kernel development in maize. Phytochemistry 66: 2632-2642.
- Cross, J. M., Clancy, M., Shaw, J., Boehlein, S., Greene, T. Schmidt, R. Okita, T. and L. C. Hannah. 2005. A polymorphic motif in the small subunit of ADP-glucose pyrophosphorylase modulates interactions between the small and large subunits. Plant Journal 41: 501-511.
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Progress 10/01/02 to 10/01/03
Outputs
A new transposable element, termed a Helitron, was found in maize. Evidence for recent transposition was gleaned and published. Transgenics with and without the Sh1 first intron were produced and materials for analysis are in hand. A method to completely purify the reporter gene product, adenosine diphosphate glucose pyrophosphorylase (AGP), was developed using monoclonal column affinity with antibodies synthesized against the small subunit. A bank of monoclonal antibodies synthesized against the AGP small subunit was screened for epitope binding. Some antibodies recognized the maize endosperm AGP small subunit but would not recognize the small subunit of the highly related wheat endosperm AGP small subunit, some recognized the amino terminal portion while others recognized only the carboxyl terminus. One antibody seems to react only with intact small subunit. Partly degraded small subunit was not recognized. Through these transformations, several cases of gene
silencing of the reporter gene, Sh2, were found. Because these are dominant, they likely have a role in sweet corn improvement since one can plant a starch-rich seed yet harvest an ear for consumption that is high in sugar. This was tested by analysis of sugar data as a function of development. The silenced Sh2 allele conditioned sugar levels as high as the recessive sh2 allele now found in commerce.
Impacts
The impact of alterations in gene expression have been described previously. The potential of dominant, loss-of-function mutants of shrunken-2 for the sweet corn industry is significant.
Publications
- Hannah, L. Curtis and Cliff Weil. Transposons and Allelic Diversity. in Encyclopedia of Plant and Crop Science, Update 1, Ed. Robert Goodman Marcel Dekker, Inc.: New York, 2004; www.dekker.com (in press).
- Smidansky, E.D., J.M. Martin, L.C. Hannah, A. M. Fischer, and M.J. Giroux. (2003) Seed yield and plant biomass increases in rice are conferred by deregulation of endosperm ADP-glucose pyrophosphorylase. Planta 216:656-664.
- Lal, S. K, Giroux, M. J. Brendel, V. Vallejos, C. E. and Hannah, L. C. (2003) The Maize Genome Contains a Helitron Insertion. Plant Cell 15: 381-391.
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Progress 10/01/01 to 10/01/02
Outputs
Introns play an important role in controlling levels of gene expression. Experiments focus on the 1028 nt shrunken 1 (Sh1) first intron. A sequence needed for enhancement but not for splicing has been identified. This sequence must be within an intron to enhance expression. Splice site mutations abolish splicing and intron-mediated enhancement of gene expression. Splicing is required for intron-mediated enhancement of gene expression. Second generation transgenic plants have been synthesized to test whether the intron functions in plants as it does in tissue culture cells are in the process of analysis.
Impacts
These studies should yield fundamentally important insight into the mechanism(s) by which this intron in particular and perhaps introns in general modulate gene expression. Because this intron is now being used in agriculturally-relevant transgenics, advances in our understanding of the mechanism by which it modulates gene expression is directly relevant to extant and future plant improvement programs.
Publications
- Smidansky E. D, Maureen Clancy, Fletcher D. Meyer, Susan P. Lanning, Nancy K. Blake, Luther E. Talbert, and Michael J. Giroux 2002 ADP-glucose Pyrophosphorylase Activity in Wheat Endosperm Increases Seed Yield Proc. Natl. Acad. Sciences, 99: 1724-1729
- Eric D. Smidansky, L. Curtis Hannah, and Michael J. Giroux. 2002 Seed yield and plant biomass increases in rice are conferred by deregulation of endosperm ADP glucose pyrophosphorylase Planta, in press
- Lal, S. K, Giroux, M. J. Brendel, V. Vallejos, C. E. and Hannah, L. C. 2003 The maize genome contains an active Helitron - type transposable element. Plant Cell, in press.
- Cross, J. M. Clancy, M., Shaw, J., Greene, T. W., Schmidt, R. R. Okita, T. W. and Hannah, L. C. 2003 Both subunits of ADP-glucose pyrophosphorylase are regulatory. Plant Cell, submitted.
- Brian T. Burger, Joanna M. Cross, Janine R. Shaw, Joel R. Caren, Thomas W. Greene, Thomas W. Okita and L. Curtis Hannah. 2003 Relative turnover numbers of maize endosperm and potato tuber ADP-glucose pyrophosphorylases in the absence and presence of 3-PGA. Planta in press.
- Clancy, M. and L. Curtis Hannah 2002. Splicing of the maize Sh1 first intron is essential for enhancement of gene expresion and a T-rich motif increases expression without affecting splicing. Plant Physiol 130: 918-929.
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Progress 10/01/00 to 10/01/01
Outputs
Introns play an important role in controlling levels of gene expression. Expression-enhancing introns do not share obvious sequence domains, do not function as classical enhancers, must lie within the 5' transcribed region and, in at least one case, operate post-transcriptionally. Experiments focus on the 1028 nt shrunken 1 (Sh1) first intron. Our recent unpublished results show the following: Up to 86% of the Sh1 first intron can be deleted without affecting the enhancement of gene expression. A sequence needed for enhancement but not for splicing has been identified. Composition rather than specific sequence is the important characteristic of this element. This sequence must be within an intron to enhance CAT activity Splice site mutations abolish splicing and intron-mediated enhancement of gene expression. Splicing is required for intron-mediated enhancement of gene expression. Translation start codons contained within Sh1 intron1 derivatives do not affect reporter
enzyme activity or transcript splicing efficiency. Reporter enzyme activity is elevated more than transcript levels in Sh1 intron 1 mediated enhancement of gene expression. Transgenic plants, produced through the Maize Transformation Core, Iowa State University have been synthesized to test whether the intron functions in plants as it does in tissue culture cells.
Impacts
These studies should yield fundamentally important insight into the mechanism(s) by which this intron in particular and perhaps introns in general modulate gene expression. Because this intron is now being used in agriculturally-relevant transgenics, advances in our understanding of the mechanism by which it modulates gene expression is directly relevant to extant and future plant improvement programs.
Publications
- Hannah, L. C., Janine R. Shaw, Michael Giroux, Agnes Reyss, Jean-Louis Prioul, Jung-Myung Bae and Jung-Youn Lee. 2001. Maize genes encoding the small subunit of ADP-glucose pyrophosphorylase. Plant Physiol, 127 173-183.
- Smidansky E. D, Maureen Clancy, Fletcher D. Meyer, Susan P. Lanning, Nancy K. Blake, Luther E. Talbert, and Michael J. Giroux 2002 Enhanced ADP-glucose Pyrophosphorylase Activity in Wheat Endosperm Increases Seed Yield Proc. Natl. Acad. Sciences, accepted
- Smidansky E. D., L. C. Hannah and M. J. Giroux. 2002. Seed yield and plant biomass increases in rice are conferred by deregulation of endosperm ADP-glucose pyrophosphorylase. Planta submitted
- Burger B.T, J. Cross, T. W. Okita and L. C. Hannah. 2002. Relative turnover numbers of maize endosperm and potato tuber ADP-glucose pyrophosphorylases in the absence and presence of 3-PGA. Plant Physiol, submitted
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Progress 10/01/99 to 09/30/00
Outputs
The first intron of the shrunken1 (Sh1) gene of maize increases gene expression some 70 fold. Using a transient expression system, we performed a deletion analysis within the Sh1 first intron. We identified a 35 bp portion of the Sh1 first intron, that is essential for maximum enhanced gene expression but not efficient splicing. Subsequent work showed that the base composition (AT richness) rather than the specific sequence of the 35 bp intron element is important for enhanced gene expression. Furthermore it was shown that the sequence must be within the intron for stimulation of gene expression. Splicing of the intron is essential for stimulation of gene activity. Constructs containing point mutations that alter the terminal dinucleotides of both the 5' and the 3' intron splice sites were synthesized and expressed. RNA analysis revealed that only unspliced transcripts were detectable. Thus, the mutations abolished splicing at the authentic sites, and no cryptic sites
were used. Subsequent enzymic assay of the reporter gene showed no enhancement of gene expression.. Intron deletion constructs with intact splice junctions are spliced at varying efficiencies, and unspliced transcripts accumulate. These unspliced transcripts contain one or more AUG codons, depending on the length of the Sh1 intron derivative. These intron AUGs are out of frame with the reporter gene and/or are followed by multiple stop codons. To investigate whether initiation of translation at any of these upstream sites affected reporter gene activity, the ATGs were altered by single base substitutions. These mutations have no effect on intron-mediated enhancement of gene expression or transcript splicing efficiency Subsequently, the intron ATGs were mutated in a construct in which splicing was abolished. No enhancement of gene expression occurred, the same result seen when the ATGs were present in this construct Thus, upstream AUGs have no effect and CAT activity is reduced to
about the level of the intron less control constructs when splicing is blocked. We conclude that splicing of the intron is essential for stimulation of gene activity.
Impacts
The ability of the shrunken1 first intron to enhance gene expression provides other molecular biologist with a tool to enhance expression of their transgenes.
Publications
- Peterson, D. M., L. Curtis Hannah, W. D. Hitz, A. J. Kinney, W. R. Meredith, M. A. L. Smith and P. J. White. 2000. Genetic Potential. In: "Designing Crops for Added Value" pp. 147-150. Ed. by C. F. Murphy and D. M. Peterson, American Society of Agronomy, Inc. Madison Wisconsin, USA.
- Boyer, C. D and L. C. Hannah. 2000. Kernel mutants of corn. In: Speciality Corn, Second edition. pp. 1-32. Ed. by A. R. Hallaner. CRC Press, Boca Raton, Fl.
- Vasil, V., M. A. Clancy, R. J. Ferl, I. K. Vasil and L. C. Hannah. 1999. Means for enhancing gene expression. United States Patent 5,955,330.
- Maitz, M, G Santandrea, Z. Zhang, S. Lal, L. C. Hannah, F. Salamini, and R. Thompson 2000. Rgf, a mutation reducing grain filling in maize through effects on basal endosperm and pedicel development. Plant Journal 23:29-42.
- Hannah, L. C. 2000. Starch Biosynthesis and Genetic Potential. In: "Designing Crops for Added Value" pp. 181-200. Ed. by C. F. Murphy and D. M. Peterson, American Society of Agronomy, Inc. Madison Wisconsin, USA.
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