A regulatory gene as a novel visible marker for maize transformation.

The temporal and spatial patterns of anthocyanin pigmentation in the maize plant are determined by the presence or absence of the R protein product, a presumed transcriptional activator. At least 50 unique patterns of pigmentation, conditioned by members of the R gene family, have been described. In this study, microprojectiles were used to introduce into maize cells a vector containing the transcription unit from one of these genes (Lc) fused to a constitutive promoter. This chimeric gene induces cell autonomous pigmentation in tissues that are not normally pigmented by the Lc gene. As a reporter for gene expression studies in maize, R is unique because it can be quantified in living tissue simply by counting the number of pigmented cells following bombardment. R may also be useful as a visible marker for selecting stably transformed cell lineages that can give rise to transgenic plants.

Lc, a member of the maize R gene family responsible for tissue-specific anthocyanin production, encodes a protein similar to transcriptional activators and contains the myc-homology region.

Previous studies have suggested that the R locus of maize is responsible for determining the temporal and spatial pattern of anthocyanin pigmentation in the plant. In this report we demonstrate that three members of the R gene family, P, S, and Lc, encode homologous transcripts 2.5 kilobases in length. The structure of one R gene, Lc, was determined by sequencing cDNA and genomic clones. The putative Lc protein, deduced from the cDNA sequence, is composed of 610 amino acids and has homology to the helix-loop-helix DNA-binding/dimerization motif found in the L-myc gene product and other regulatory proteins. It also contains a large acidic domain that may be involved in transcriptional activation. Consistent with its proposed role as a transcriptional activator is our finding that a functional R gene is required for the accumulation of transcripts of at least two genes in the anthocyanin biosynthetic pathway. We discuss the possibility that the diverse patterns of anthocyanin pigmentation conditioned by different R genes reflect differences in the R gene promoters rather than their gene products.

The α1-tubulin gene of Arabidopsis thaliana: primary structure and preferential expression in flowers.

The primary structure of the α1-tubulin gene of Arabidopsis thaliana was determined and the 5' and 3' ends of its transcript were identified by S1 nuclease mapping experiments. The information obtained was used to (i) predict the amino acid sequence of the α1-tubulin, (ii) deduce the positions of introns within the α1-tubulin gene, and (iii) construct 3' noncoding gene-specific hybridization probes with which to study the pattern of α1-tubulin transcript accumulation in different tissues and at different stages of development. The predicted amino acid sequence of the α1-tubulin has 92% identity with the predicted product of the previously characterized A. thaliana α3-tubulin gene. The coding sequence of the α1-tubulin gene is interrupted by four introns located at positions identical to those of the four introns in the α3 gene. RNA blot hybridization studies carried out with an α1-tubulin gene-specific probe showed that the α1 gene transcript accumulates primarily in flowers, with little transcript present in RNA isolated from roots or leaves. In order to investigate the pattern of α-tubulin gene expression in developing flowers, RNA was isolated from flowers at five different stages of development: flower buds, unopened flowers with pollen, open flowers, flowers with elongating carpels, and green seed pods. RNA blot hybridizations performed with 3' noncoding gene-specific probes showed that the α3 tubulin gene transcript is present in flowers at all stages of development, whereas the α1-tubulin gene transcript could only be detected in RNA from unopened flowers with pollen, open flowers, and flowers with elongating carpels.

Characterization of the alpha-tubulin gene family of Arabidopsis thaliana.

The genome of Arabidopsis thaliana (Linnaeus) Heynhold was shown to contain an alpha-tubulin gene family consisting of at least four genes and/or pseudogenes. The primary structure of a transcribed alpha-tubulin gene was determined. A comparison of the predicted amino acid sequence of the A. thaliana alpha-tubulin with the predicted amino acid sequences of alpha-tubulins of Chlamydomonas reinhardtii, Stylonychia lemnae, and Homo spaiens reveals a high degree of homology; 90%, 87%, and 83% identity, respectively. Thus, a plant alpha-tubulin exhibits a high degree of homology to the alpha-tubulins of protists and animals. The coding sequence of the A. thaliana alpha-tubulin gene is interrupted by four introns, which occur at positions different from those of the less numerous introns of C. reinhardtii and rat alpha-tubulin genes. S1 nuclease mapping data showed that transcription is initiated 99 +/- 1 base pairs upstream from the translation initiation codon. Both 5' and 3' noncoding gene-specific probes were used to examine the expression of the alpha-tubulin gene in leaves, roots, and flowers by hybridization to total RNA isolated from these tissues. The results showed that the alpha-tubulin gene was transcribed in all three tissues.

The nucleotide sequence of a mitochondrial replicon from maize.

The 1913-bp maize mitochondrial (mt) plasmid was isolated from a suspension culture of a Black Mexican Sweet maize strain, cloned into M13mp vectors, and sequenced by a unidirectional progressive deletion method. The 1.9-kb extrachromosomal double-stranded circular DNA plasmid was found to contain regions of sequence which in other systems are known to be part of origins of replication (ori). This plasmid could be used as a carrier for chimeric genes and a molecular probe for replication.

High frequency callus formation from maize protoplasts.

A solid feeder layer technique was developed to improve callus formation of Black Mexican Sweet maize (Zea mays L.) suspension culture protoplasts. Protoplasts were plated in 0.2 ml liquid media onto a cellulose nitrate filter on top of agarose-solidified media in which Black Mexican Sweet suspension feeder cells were embedded. Callus colony formation frequencies exceeding 10% of the plated protoplasts were obtained for densities of 10(3)-10(5) protoplasts/ 0.2 ml, which was 100- to 1,000-fold higher than colony formation frequencies obtained for conventional protoplast plating methods such as liquid culture or embedding in agarose media. Compared with conventional methods, the feeder layer method gave higher colony formation frequencies for three independently maintained Black Mexican Sweet suspension lines. Differences among the three lines indicated that colony formation frequencies might also be influenced by the suspension culture maintenance regime and length of time on different 2,4-dichlorophenoxyacetic acid concentrations. The callus colony formation frequency reported is an essential prerequesite for recovering rare mutants or genetically transformed maize protoplasts.