Ac induces homologous recombination at the maize P locus.

The maize P gene conditions red phlobaphene pigmentation to the pericarp and cob. Starting from two unstable P alleles which carry insertions of the transposable element Ac, we have derived 51 P null alleles; 47 of the 51 null alleles have a 17-kb deletion which removes the 4.5-kb Ac element and 12.5 kb of P sequences flanking both sides of Ac. The deletion endpoints lie within two 5.2-kb homologous direct repeats which flank the P gene. A P allele which contains the direct repeats, but does not have an Ac insertion between the direct repeats, shows very little sporophytic or gametophytic instability. The apparent frequency of sporophytic mutations was not increased when Ac was introduced in trans. Southern analysis of DNA prepared from the pericarp tissue demonstrates that the deletions can occur premeiotically, in the somatic cells during development of the pericarp. Evidence is presented that the deletions occurred by homologous recombination between the two direct repeats, and that the presence of an Ac element at the P locus is associated with the recombination/deletion. These results add another aspect to the spectrum of activities of Ac: the destabilization of flanking direct repeat sequences.

Insertional mutagenesis of the maize P gene by intragenic transposition of Ac.

The P-rr allele of the maize P gene regulates the synthesis of pigments derived from flavan-4-ol in the pericarp, cob glumes and other floral organs. We characterized 21 P alleles derived by intragenic transposition of Ac from three known positions. Ac transpositions can occur in either direction in the P gene, and with no apparent minimum distance: in one case Ac transposed just 6 bp from its original insertion site. However, the distribution of transposed Ac elements was markedly nonrandom: of 19 transposed Ac elements derived from a single Ac donor, 15 were inserted in a 1.1-kb region at the 5' end of P, while none had inserted in an adjacent 3.2-kb intronic region. All of the Ac insertions affect both pericarp and cob glume pigmentation, providing further evidence that the P-rr allele contains a single gene required for both pericarp and cob glume pigmentation. The distribution of the inserted Ac elements and the phenotype conditioned by each allele suggests a structure of P-rr which is similar to that previously determined molecularly. Possible explanations for the nonrandom distribution of transposed Ac elements are discussed.

Molecular genotyping shows that ataxia-telangiectasia heterozygotes are predisposed to breast cancer.

About 1.4% of the general population are heterozygous carriers of the gene for ataxiatelangiectasia (A-T), an autosomal recessive progressive neurologic syndrome in which cancer incidence of homozygotes is approximately 100-fold greater than the general population's rates. The hypothesis that A-T heterozygotes are predisposed to breast cancer was tested by the unbiased statistically powerful index-test method based on molecular genotyping. The A-T gene carrier status of 775 blood relatives in 99 A-T families was determined by tracing the A-T gene in each family through tightly linked flanking DNA markers. There were 33 women with breast cancer who could be genotyped; 25 of these were A-T heterozygotes, compared to an expected 14.9 (odds ratio 3.8, 95% confidence limits 1.7-8.4, one-sided p = .0001). This demonstrates that the A-T gene predisposes heterozygotes to breast cancer. For the 21 breast cancers with onset before age 60, the odds ratio was 2.9 (1.1-7.6, p = .009) and for the 12 cases with onset at age 60 or older, the odds ratio was 6.4 (1.4-28.8, p = .002). Thus the breast cancer risk for A-T heterozygous women is not limited to young women but appears even higher at older ages. Of all breast cancers in the United States, 6.6% may occur in women who are A-T heterozygotes. This proportion is several fold greater than the estimated proportion of carriers of BRCA1 mutations in breast cancer cases with onset at any age.

DNA marker D11S384 shows zero recombination with the ataxia-telangiectasia locus in North American families.

At the genetic locus D11S384, one probe detects a Taql RFLP and another detects a RFMP. In 52 pedigrees of North American A-T patients, parental haplotypes based on these two bi-allelic systems have a heterozygosity of 0.69 and a PIC of 0.64. No recombinant events between D11S384 and the A-T locus were detected in the 43 pedigrees in which this marker locus was informative.

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

We 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.

Alternatively spliced products of the maize P gene encode proteins with homology to the DNA-binding domain of myb-like transcription factors.

The Zea mays P gene has been postulated to regulate the biosynthetic pathway of a flavonoid-derived pigment in certain floral tissues [Styles, E. D. & Ceska, O. (1977) Can. J. Genet. Cytol. 19, 289-302]. We have characterized two P transcripts that are alternatively spliced at their 3' ends. One message of 1802 nucleotides encodes a 43.7-kDa protein with an N-terminal region showing approximately 40% homology to the DNA-binding domain of several members of the myb family of protooncogene proteins. A second message of 945 nucleotides encodes a 17.3-kDa protein that contains most of the myb-homologous domain but differs from the first protein at the C terminus. The deduced P-encoded proteins show an even higher homology (70%) in the myb-homologous domain to the maize regulatory gene C1. Additionally, the P and C1 genes are structurally similar in the sizes and positions of the first and second exons and first intron. We show that P is required for accumulation in the pericarp of transcripts of two genes (A1 and C2) encoding enzymes for flavonoid biosynthesis--genes also regulated by C1 in the aleurone.

Single base polymorphism linked to the ataxia-telangiectasia locus is detected by mismatch PCR.

A polymorphic site at D11S384, which shows zero recombination with the ataxia-telangiectasia (A-T) locus, was originally detected by Mutation Detection Enhancement Gel and Denaturing Gradient Gel Electrophoresis. In order to increase the throughput and decrease the complexity of the assays, we developed a site directed mutagenesis using primers with mismatched 3' ends, followed by restriction digestion, as a rapid, nonradioactive method for detecting polymorphisms/mutations.

Alleles of the maize P gene with distinct tissue specificities encode Myb-homologous proteins with C-terminal replacements.

The maize P gene is a transcriptional regulator of genes encoding enzymes for flavonoid biosynthesis in the pathway leading to the production of a red phlobaphene pigment. Multiple alleles of the P gene confer distinct patterns of pigmentation to specific floral organs, such as the kernel pericarp and cob tissues. To determine the basis of allele-specific pigmentation, we have characterized the gene products and transcript accumulation patterns of the P-wr allele, which specifies colorless pericarps and red cob tissues. RNA transcripts of P-wr are present in colorless pericarps as well as in the colored cob tissues; however, the expression of P-wr in pericarp does not induce the accumulation of transcripts from the C2 and A1 genes, which encode enzymes for flavonoid pigment biosynthesis. The coding sequences of P-wr were compared with the P-rr allele, which specifies red pericarp and red cob. The P-wr and P-rr cDNA sequences are very similar in their 5' regions. There are only two nucleotide changes that result in amino acid differences; both are outside of the Myb-homologous DNA binding domain. In contrast, the 3' coding region of P-rr is replaced by a unique 210-bp sequence in P-wr. The predicted P-wr protein has a C-terminal sequence resembling a cysteine-containing metal binding domain that is not present in the P-rr protein. These results indicate that the differential pericarp pigmentation specified by the P-rr and P-wr alleles does not result from an absence of P-wr transcripts in pericarps. Rather, the allele-specific patterns of P-rr and P-wr pigmentation may be associated with structural differences in the proteins encoded by each allele.

A maize Myb homolog is encoded by a multicopy gene complex.

The maize P gene encodes a Myb-homologous transcriptional regulator of flavonoid pigmentation in floral organs, and different P gene alleles condition precise tissue- and organ-specific pigmentation patterns. To determine the molecular basis for allele-specific expression patterns, we have isolated and compared two natural alleles of the P gene which differ in expression, structure and copy number. The P-rr allele is associated with pigmentation of most floral tissues and contains a single copy of the P gene. In contrast, the P-wr allele restricts pigmentation to a subset of floral tissues, and is composed of six gene copies arranged in a tandem head-to-tail array. Each of the six repeats contains a single P gene, including regulatory and coding sequences. Despite the six-fold tandem repetition of P-wr gene copies, P-wr mRNA levels in kernel pericarp are much reduced compared to mRNA levels from the single-copy P-rr gene. Moreover, the P-wr multicopy complex is hypermethylated relative to P-rr. Thus, maize P gene alleles may represent a natural system for studying the effects of methylation and gene copy number on tissue-specific gene expression. We discuss the possibility that somatic pairing of repeated gene copies may be involved in regulating gene expression.