Isolation and molecular analysis of the maize P locus

Abstract

The maize P locus is involved in the synthesis of a red flavonoid pigment in the pericarp, cob and other floral tissues. The tissue-specific pattern of expression of certain P alleles suggests that P may be a complex locus, with more than one functional unit. The P-VV allele, which specifies variegated pericarp and variegated cob, however, shows that insertion and excision of the transposable element Ac affects both pericarp and cob expression as though cob and pericarp pigmentation are controlled by a single gene. Using Ac as a transposon tag, we have isolated 34 kb of genomic DNA from the P-VV and P-RR allele. The cloned DNA contains two 5.8 kb cross-hybridizing regions, in direct orientation relative to each other, separated by 6.6 kb of intervening DNA. A sequence motif of 250 bp is repeated at three locations within the cloned region: once within each of the 5.8 kb repeats, and once outside the 5.8 kb repeats. DNA fragments flanking the Ac element detect five transcripts in RNA from wild type (P-RR) that are absent from mutant (P-VV) tissues. To localize the transcribed sequences, DNA probes spanning the 34 kb of cloned DNA were used in Northern analysis of RNA from mutant and wild-type kernels. The results suggest the presence of a single transcriptional unit located primarily within the DNA between the 5.8 kb repeats. The five RNAs transcribed from this region may be formed by alternative splicing. The size of the P gene derived from the length of the transcribed region seems much smaller than the gene size estimated from Ac-induced P-VV mutations.

Progressive Loss of DNA Methylation Releases Epigenetic Gene Silencing From a Tandemly Repeated Maize Myb Gene

“…Total RNA was fractionated on denaturing gels, blotted onto nylon membranes, and hybridized with a 32 P-dCTP probe fragments as described previously (Boddu et al 2004). DNA fragments F15 and F8B belonging to an upstream regulatory region and second intron, respectively, were used as probes in Southern blot analysis (Lechelt et al 1989;Grotewold et al 1991;Sekhon et al 2007). Probe fragments used for Northern analysis were obtained from the plasmids pC2 containing a maize C2 cDNA (Wienand et al 1986), pChi1 containing a maize Chi1 cDNA (Grotewold and Peterson 1994), and pZmA1 with a maize DFR cDNA (Schwarz-Sommer et al 1987).…”

Ac-Immobilized, a Stable Source of Activator Transposase That Mediates Sporophytic and Gametophytic Excision of Dissociation Elements in Maize

“…A brief description of each allele used in this study is given below: P1-vvTAc: an unstable Ac insertion at the p1 locus required for the accumulation of a red phlobaphene-like pigment (Styles and Ceska 1977) resulting in variegated pericarp and cob tissues (Lechelt et al 1989). p1-wr: a complex p1 allele resulting in colorless pericarp and red cob tissues (Chopra et al 1998).…”

Molecular characterization of a mutable pigmentation phenotype and isolation of the first active transposable element from Sorghum bicolor

“…A BamHI size-selected (7.0-10.0 kb) subgenomic library from leaf DNA of candystripe sorghum line CS8110419 was constructed in -EMBL3͞BamHI vector by following standard methods (28). A total of 160,000 clones were screened with maize P1-rr genomic fragment 8A (29) as probe, and two positive clones were isolated. The clones appeared to be identical based on their restriction digest patterns.…”

“…Genomic fragments were partially filled in to generate ends compatible with the partially filled-in XhoI ends of the vector. From this library, a total of 8 ϫ 10 5 clones were screened by using the maize P1-rr gene fragment 12 (29). In this screen, three positive clones were identified and the largest clone (CS-3) was characterized further.…”