ADDITIONAL INFORMATION:

Note: This contains the information needed for Section 6. Section 8 "ADDITIONAL INFORMATION is not required, but helpful to describe the development and purpose of the RescueMu construct).

ADDITIONAL INFORMATION:

RescueMu is a modified Mutator1 (Mu1) transposon transformed into maize to permit mutagenesis and subsequent recovery of mutant alleles by plasmid rescue. The RescueMu transposing element consists of a bacterial pBluescript plasmid inserted into an intact Mu1 transposon which has characteristic terminal inverted repeat sequences. RescueMu is inserted in the leader sequence of Lc(R) cDNA (leaf color transcription factor of the R gene family in maize required for anthocyanin production). RescueMu::Lc is co-transformed with pAHC20 (a plasmid encoding the bar gene that confers resistance to the herbicide glufosinate) into maize by biolistic transformation. In the resulting RescueMu::Lc/pAHC20 transgene, pAHC20 is used as a selectable marker of stable insertion of RescueMu into maize, and Lc expression is used as a screenable marker of RescueMu excision. RescueMu transgenic lines must be crossed with lines with active MURA transposase (MUDR gene) in order for Mu transposition. Characterization of mutant alleles is made easy by 5-20 kb of Mu-flanking maize genomic DNA recovered in plasmid form after pBluescript rescue. Seed containing MuDR/RescueMu transposing elements can be hand-selected for plasmid rescue based on the appearance of frequent single-cell red spotting on the kernel (aleurone) surface when viewed under a microscope, indicating where tissue pigmentation has been restored after excision of RescueMu. To distinguish between different RescueMu elements, a short (approx 400 bp) unique tag from the nodulin gene of Sinorhizobium melioti was inserted into RescueMu during development to permit easier mutant allele-transposon cosegreation analysis. (Raizada, M.N. 2003. RescueMu protocols for maize functional genomics. In: Grotewold, E, editor. Methods in Molecular Biology, Vol. 236. Plant Functional Genomics. New Jersey, Humana Press. p 37-58; Raizada, M.N., G.L. Nan and V. Walbot. 2001. Somatic and germinal mobility of the RescueMu transposon in transgenic maize. The Plant Cell, Vol. 13, 1587-1608).

PHENOTYPE(S)

MG - Transposon inserted - red spots upon transposon excision

GENOTYPE(S)

Gene(s) of Interest:

Promoter: CaMV from Cauliflower mosaic caulimovirus - CaMV promoter for strong expression of the Lc::RescueMu transgene.

Trancription Factor: Lc (R) (Leaf color anthocyanin) from Zea mays - Lc (R) is a transcription factor of the R gene family in maize which is required for anthocyanin production. Serves as a marker of transposon excision where transposition of RescueMU from the Lc::RescueMu transgene leads to restoration of pigmentation.

Transposon: RescueMu2 from Zea mays - RescueMu is a modified Mutator1 transposon (Mu1) containing bacterial pBluescript plasmid and transformed into maize to permit mutagenesis and subsequent recovery of mutant alleles by plasmid rescue.

Vector Sequence: pBluescript from Escherichia coli - pBluescript (commercial cloning vector plasmid from Stratagene) contains a bacterial origin of replication and an antibiotic resistance gene (ampicillin) to permit plasmid rescue.

Marker Gene: 200 bp marker DNA from Sinorhizobium melioti - To distinguish between different RescueMu mobile elements, a unique 200 bp tag from the S. melioti nodulin gene has been inserted into the RescueMu2 transposon to permit easier mutant allele-transposon cosegreation analysis. No gene is expressed in transgenic maize as a result of this marker.

Terminator: RuBisCO small subunit (rbcS) from Pisum sativum - Terminator sequence of pea rbcS-E9 gene which in this case halts transcription of the maize Lc (R) anthocyanin transcription factor.

Gene(s) of Interest

Promoter: CaMV from Cauliflower mosaic caulimovirus - CaMV promoter for strong expression of the Lc::RescueMu transgene.

Trancription Factor: Lc (R) (Leaf color anthocyanin) from Zea mays - Lc (R) is a transcription factor of the R gene family in maize which is required for anthocyanin production. Serves as a marker of transposon excision where transposition of RescueMU from the Lc::RescueMu transgene leads to restoration of pigmentation.

Transposon: RescueMu3 from Zea mays - RescueMu is a modified Mutator1 transposon (Mu1) containing bacterial pBluescript plasmid and transformed into maize to permit mutagenesis and subsequent recovery of mutant alleles by plasmid rescue.

Marker Gene: 400 bp marker DNA from Sinorhizobium melioti - To distinguish between different RescueMu mobile elements, a unique 400 bp tag from the S. melioti nodulin gene has been inserted into the RescueMu3 transposon to permit easier mutant allele-transposon cosegreation analysis. No gene is expressed in transgenic maize as a result of this marker.

Selectable Marker

Promoter: Ubiquitin from Zea mays - Ubiquitin promoter for strong expression of bar gene

Enhancer: Ubiquitin I from Zea mays - Enhances trancription of bar gene

Gene: phosphinothricin acetyl transferase from Streptomyces hygroscopicus - Bialaphos resistance (bar) gene from pAHC20 plasmid encoding the phosphinothricin acetyl transferase (pat) enzyme which confers glufosinate tolerance, which in this case is used as a selectable marker to indicate the stable insertion of RescueMu.

Terminator: nopaline synthase (nos) from Agrobacterium tumefaciens - Terminator sequence of nopaline synthase which in this case halts transcription of the bar gene