No abstract

Four mutants of Arabidopsis thaliana that are deficient in adenine phosphoribosyl transferase (APRT) activity have been isolated by selecting for germination of seeds and growth of the plantlets on a medium containing 2,6-diaminopurine (DAP), a toxic analog of adenine. In all mutants, DAP resistance is due to a recessive nuclear mutation at a locus designated apt. The mutants are male sterile due to pollen abortion after meiosis. Furthermore, it has been shown that metabolism of cytokinins is impaired in the mutant BM3, which has the lowest level of APRT activity among the mutants tested. However, three different cDNAs encoding APRT have been isolated in A. thaliana and this raised the question of the nature of the mutation which results in low APRT activity. The mutation was genetically mapped to chromosome I and lies within 6 cM of the phenotypic marker dis2, indicating that the mutation affects the APT1 gene, a result confirmed by sequencing of mutant alleles. The mutation in the allele apt1-3 is located at the 5' splicing site of the third intron, and eliminates a BstNI restriction site, as verified by Southern blotting and PCR fragment length analysis.

Adenine phosphoribosyltransferase (APRTase) is an important enzyme for its ability to convert adenine, a byproduct of many biochemical reactions, into AMP. By functional complementation of an Escherichia coli mutant, cDNAs encoding two APRTases have been cloned from Arabidopsis thaliana. One of the cDNAs (ATapt1) has been previously identified while the second (ATapt2) is of a previously unknown type. Kinetic analysis of the two enzymes purified from E. coli expressing the two cDNAs indicates that ATapt2 has a higher affinity for cytokinin than the ATapt1. RNase protection studies indicate that the ATapt2, is not expressed in leaves. Analysis of the gene structure indicates that ATapt2 has identical intron positions to ATapt1, but neither the intron sequence nor intron size are conserved between the two genes. The implications of a second, differentially expressed APRTase with affinity for both adenine and cytokinin are discussed.

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Tropomyosins (TMs) constitute a group of contractile proteins encoded by a multigene family showing distinct cell-type-specific and developmental expression patterns. In mammals and birds, the alpha-TM gene is the most complex and can produce several muscle and non-muscle isoforms. We report here the characterization of the 5' region of the Xenopus laevis alpha-TM gene and its developmental expression. The 5' region of the gene is structurally related to the avian and mammalian cognates and presents two promoters flanking a pair of alternatively spliced exons, 2a/2b, where exon 2a is a smooth-muscle-specific exon. The internal promoter is used to generate a non-muscle low molecular weight TM whilst muscle TM isoforms originate from the distal promoter. RNase protection analysis shows that the two promoters have distinct temporal programs of activation. The internal promoter is activated early in oogenesis and non-muscle transcripts are found throughout oogenesis, embryogenesis and in adult tissues. Only low molecular weight non-muscle TM-encoding mRNAs are expressed in oogenesis. The distal promoter is silent during oogenesis, and the skeletal muscle alpha-TM transcripts accumulate from stage 15 in the embryo and are expressed in adult striated muscle tissues. In situ hybridization indicates that these transcripts are expressed in both the somites and heart of the embryo. Ectopic expression of myogenic factors, but not the MEF2 myocyte-specific enhancer factor 2 factors SL1 and SL2, can induce the expression of the alpha-TM gene suggesting that the gene is a direct target for myogenic but not for MEF2 factors. The amphibian alpha-TM gene constitutes a gene marker for studying the developmental control expression of muscle genes in the different myogenic lineages.