Previous studies have suggested that the R locus of maize is responsible for determining the temporal and spatial pattern ofanthocyanin 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 helixloop-helix DNA-binding/dimerization motif found in the Lmyc 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 anthocyanin biosynthetic pathway of maize has proven to be an ideal system for understanding genetic interactions between regulatory and structural genes (for review see ref.

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.

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The genome of Arabidopsis thaliana (Linnaeus) Heynhold was shown to contain an c-tubulin gene family consisting of at least four genes and/or pseudogenes. The primary structure of a transcribed a-tubulin gene was determined. A comparison of the predicted amino acid sequence of the A. thaliana a-tubulin with the predicted amino acid sequences of a-tubulins of Chlamydomonas reinhardtii, Stylonychia lemnae, and Homo sapiens reveals a high degree of homology; 90%, 87%, and 83% identity, respectively. Thus, a plant a-tubulin exhibits a high degree of homology to the a-tubulins of protists and animals. The coding sequence of the A. thaliana a-tubulin gene is interrupted by four introns, which occur at positions different from those of the less numerous introns of C. reinhardtii and rat cv-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 a-tubulin gene in leaves, roots, and flowers by hybridization to total RNA isolated from these tissues. The results showed that the a-tubulin gene was transcribed in all three tissues.Microtubules play central roles in several of the most basic processes of eukaryotic cells: cell division, cell motility, intracellular transport, and the control of cell shape. In plant cells, rigid cell walls obviate the need for direct cytoskeletal maintenance of cell shape. However, wall formation and the division of walled cells require the action of several microtubule arrays unique to plant systems. These arrays include the cortical microtubules involved in orientation of cellulose microfibrils, the preprophase band (which delineates the plane of the ensuing cell division), and the phragmoplast (which forms at the equatorial plane of the spindle to participate in formation of the new cell wall) (see refs. 1-3 for reviews).Approaches to understanding the diversity of microtubule function in animal and fungal systems have included the molecular and genetic analysis of genes coding for

Microtubules are important components of the cytoskeleton of plant cells and play key roles in plant growth and morphogenesis. Recent molecular studies have begun to elucidate the structure and expression of plant genes coding for the major components of microtubules, a-and 0-tubulin. Tubulin amino acid sequences deduced from the DNA sequences of eight higher plant tubulin genes are 79-87 % homologous with constitutively expressed mammalian tubulins. The genome of the model plant system Arabidopsis thulium contains four dispersed a-tubulin sequences and at least seven 0-tubulin sequences, only two of which appear to be linked. Of the five A. thaliuna genes whose expression has been analyzed, the transcripts of one a-tubulin and one 0-tubulin gene are constitutively expressed in roots, leaves, and flowers. A second a-tubulin gene is expressed predominately in flowers; the transcripts of the second and third 0-tubulin genes are found predominately in leaves or in roots, respectively.

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.

Defects of insulin receptor binding and tyrosine kinase activity have been described in genetically diabetic (db/db) and obese (ob/ob) mice. To determine if these changes were related to an abnormality in insulin receptor mRNA expression or structure of the receptor gene, we quantitated receptor mRNA from db/db and ob/ob homozygous, heterozygous (db/x, ob/x) and unaffected [db(x/x), ob(x/x)] mice and also analyzed restriction fragment length patterns of genomic DNA. Northern blot analysis of insulin receptor mRNA in livers from each of the genotypes revealed two major species of 7.5 and 9.5 kilobases. In contrast to known decreased receptor number in various tissues of ob/ob and db/db mice, quantitation of liver insulin receptor mRNA revealed that both homozygous affected strains had 2-fold or more increased levels of both major mRNA species compared to unaffected control groups. (P less than 0.05). Restriction fragment length analysis revealed no major insertion or deletion mutations in either the db/db or ob/ob insulin receptor gene. From the number and size of the fragments generated by this analysis, the minimal size of the mouse insulin receptor gene was calculated to be 97 kilobases, and the minimal number of exons was 16. These data indicate that the insulin receptor gene in ob/ob and db/db mice exhibits no major structural abnormality. Decreases in insulin receptor binding and/or kinase activity in affected mice appear to be due to a defect at the posttranscriptional level and occur despite increased levels of receptor mRNA.

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