Thomas Peterson

The myb-homologous P gene controls phlobaphene pigmentation in maize floral organs by directly activating a flavonoid biosynthetic gene subset

The maize P gene, which specifies red pigmentation of the kernel pericarp, cob, and other floral organs, has been an important model since the early days of modern genetics. Here we show that P encodes a Myb homolog that recognizes the sequence CCT/AACC, in sharp contrast with the C/TAACGG bound by vertebrate Myb proteins. P binds to and activates transcription of the A1 gene required for 3-deoxy flavonoid and phlobaphene biosynthesis, but not the Bz1 gene required for anthocyanin biosynthesis. The maize C1 gene, which also encodes a Myb homolog, activates both the A1 and Bz1 genes, but only in the presence of a basic-helix-loop-helix coactivator encoded by the maize genes R or B. These results indicate that Myb homologs can differentially regulate gene expression by binding different DNA sequences, through combinatorial interactions with other factors, or both.

Isolation and molecular analysis of the maize P locus

SummaryThe 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 by 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.

Isolation and characterization of a maize gene encoding chalcone flavonone isomerase.

We report here the first cloning of a chalcone flavonone isomerase gene (CHI) from maize. Northern blot experiments indicate that the maize CHI gene (ZmCHI1) is regulated in the pericarp by the P gene, a myb homologue. The ZmCHI1 gene encodes a 24.3 kDa product 55% and 58% identical to CHI-A and CHI-B from Petunia, respectively. This maize CHI gene has four exons and an intron-exon structure identical to the CHI-B gene of Petunia hybrida. RFLP mapping data indicate that some inbred lines contain two additional CHI-homologous sequences, suggesting an organization more complex than that found in Petunia or bean. The possibility that the additional CHI-homologous sequences are responsible for the lack of CHI mutants in maize will be discussed.

A possible hot spot for Ac insertion in the maize P gene

SummaryWe have analyzed the footprints left by a single Ac transposable element during its intragenic transposition to different positions in the maize P gene. One site appears to have been visited twice by transposons, indicating that it may be an insertion hot spot. Implications of this finding for the origin of the P-vv allele are discussed. Analysis of transposon footprints may prove generally useful for establishing pedigree relationships among gene alleles.

Isolation of Sequences Flanking Ac Insertion Sites by Ac Casting

Localizing Ac insertions is a fundamental task in studying Ac-induced mutation and chromosomal rearrangements involving Ac elements. Researchers may sometimes be faced with the situation in which the sequence flanking one side of an Ac/Ds element is known, but the other flank is unknown. Or, a researcher may have a small sequence surrounding the Ac/Ds insertion site and needs to obtain additional flanking genomic sequences. One way to rapidly clone unknown Ac/Ds flanking sequences is via a PCR-based method termed Ac casting. This approach utilizes the somatic transposition activity of Ac during plant development, and provides an efficient means for short-range genome walking. Here we describe the principle of Ac casting, and show how it can be applied to isolate Ac macrotransposon insertion sites.

Plant Transposable Elements

Research on transposable elements began nearly 100 years ago with classical genetic experiments. Remarkably, many of the activities of transposable elements, such as the ability to transpose, to induce chromosome rearrangements, to undergo cycles of activity and inactivity, and to affect expression of neighboring genes, were described by geneticists long before transposons were molecularly isolated. This chapter traces the historical roots of transposable element research, describing the scientists, their observations, and interpretations as they sought to understand the enigma of transposable elements.

Aspects of the Ac/Ds Transposable Element System in Maize

SUMMARY Studies of the Ac (Activator) transposable element provided the data which led Barbara McClintock to postulate that certain segments of chromosomes could transpose to different locations in the genome. McClintock also recognized the existence of Ds (Dissociation) elements which could transpose, but only in the presence of a trans-acting Ac element elsewhere in the genome. DNA sequences corresponding to Ds and Ac have now been identified, and an understanding of many of the properties of these transposable elements in the maize genome has been acquired in recent years. It is known that cryptic Ac elements and members of at least two families of Ds elements occur in the genome of all maize lines examined. Ds elements also occur in Teosinte and the more distantly related Tripsacum. We discuss the possible origin of these elements and consider the mechanism of activation of cryptic Ac elements. A recent molecular analysis of a transition of an Ac-derived Ds-element back to an active Ac element suggests one molecular mechanism by which changes in the activity state of Ac may occur. Distinctive phenotypes created by controlling elements within a target gene have been shown to be governed by the properties of the insertion element and the position of the insertion within the gene. Genetic effects include modulation of gene expression, alteration of gene products, instability of mutant phenotypes, deletion and duplication of chromosome segments and the production of chromosome rearrangements. We describe an example where a Ds insertion generates an additional intron in the Adh1 gene which reduces gene expression through mRNA instability. We also discuss an Ac-dependent modulation of P gene activity in glume and pericarp tissues of maize which may be attributed to an alteration either in patterns of gene expression or the developmental biology of the flower. The molecular consequences of Ac and Ds insertions and excisions are known at the DNA sequence level but little is known of the mechanism of transposition. An initial approach has been to analyse Ac transcription. Preliminary results showing transcription of a limited region of Ac are discussed. The corresponding upstream regions have been linked to the coding region of chloramphenicol acetyltransferase (CAT) and show promoter activity following electroporation into tobacco protoplasts.