Phenylalanine ammonia-lyase (PAL) catalyses the first step in the phenylpropanoid pathway and is induced during differentiation and by various stimuli. PAL activity is slowly induced during anthocyanin synthesis which occurs in a medium lacking 2,4-D and is also induced rapidly and transiently by transfer of cells to fresh medium and lowering the cell density. From the carrot genomic library, four clones of PAL genes, g&PAL1,2,3 and 4, were obtained. Analyses of nucleotide sequences revealed that only the gLkPAL3 gene is responsible for the induction of anthocyanin synthesis by 2,4-D. Several cis-elements, boxes M, P, A, L, and G, exist in the proximal promoter region of gDcPAL3. Transient expression experiments in carrot protoplasts using deletion mutants of the proximal promoter region of gLkPAL3 gene showed that boxes M and L, both of which contain core sequences of the Myb binding sites, might play an important role in gkPAL3 promoter activity. Four myb cDNAs, Dcmyb8, K), 12 and 14 were obtained from a carrot subtracted cDNA library and their structure and expression patterns were analyzed. In addition to the analysis of the proximal region of gDcpAL3 promoter, the possibility of the regulation of gene expression by genomic DNA structure and chromatin modification in metabolic differentiation is discussed.

The phenylalanine ammonia-lyase (PAL) gene, DcPAL3, was expressed during the synthesis of anthocyanin in suspension-cultured cells of carrot (Daucus carota). There were two putative cis-elements in the DcPAL3 promoter region: the box-L and GCC-box homologs. Both of these are committed to the upregulation of promoter activity. Although box-L is known as the conserved cis-element present in the promoter region of most PAL genes of many plant species targeted by the R2R3-MYB protein, among PAL genes, the GCC-box homolog is unique to the promoter region of the DcPAL3 gene. We have isolated two proteins belonging to the ethylene-responsive element-binding factor (ERF) family, DcERF1 and DcERF2, from two different cDNA libraries prepared from anthocyanin-synthesizing cells of different cultured cell lines of carrot. The methodology employed was yeast one-hybrid screening with the GCC-box homolog as a bait. Both DcERF1 and DcERF2 bound to the GCC-box homolog sequence in vitro. Transient expression analysis showed that, in carrot protoplasts, DcERF1 was able bind to the GCC-box homolog and act as an activator of the DcPAL3 promoter. In contrast, DcERF2 itself had no ability to activate DcPAL3 promoter activity, possibly because transiently expressed DcERF2 may not be exported into the nucleus. These results suggest that DcERF1 and DcERF2 may function in different ways in committing to the upregulation of the DcPAL3 promoter activity in anthocyanin-synthesizing cells of carrot.

Genetically modified (GM) sugar beets have been bred for use as food and animal feed. To evaluate the applicability of GMO analyses to beet sugar products, we investigated residual DNA in eight sorts of in-process beet sugar samples and commercial beet sugar products. Polymerase chain reaction (PCR) analyses with taxon-specific primers indicated that sugar beet DNA was degraded at an early stage of sugar processing, and no PCR amplification was detected from the investigated sugar products because of low DNA recovery and/or PCR inhibition.

In suspension-cultured carrot cells, a phenylalanine ammonia-lyase gene, DcPAL3, plays an important role in regulation of anthocyanin synthesis. In the DcPAL3 promoter region, a putative cis-element, box-L, which is committed to the upregulation of promoter activity, has been identified. Here, we isolated DcMYB5 cDNA using yeast one-hybrid screening with box-L as a bait from a cDNA library prepared from cells of a variant cultured cell line constitutively synthesizing anthocyanin. Although expression of DcMYB2, 3, and 4 was observed, all of which were previously isolated by plaque hybridization from a subtracted cDNA library of anthocyanin-synthesizing cells of a normal cultured cell line cultured in medium lacking 2,4-dichlorophenoxyacetic acid, DcMYB5 expression was not observed in cells of the normal cultured cell line; high expression of DcMYB5 was observed in cells of the variant cultured cell line. Although not only DcMYB5 but also DcMYB2, 3, and 4 could bind to the box-L sequence in yeast, DcMYB3 and 5 showed strong transcriptional activation activity for DcPAL3 promoter in carrot protoplasts. These results suggest that DcMYB3 and 5 might play an important role in the upregulation of DcPAL3 promoter activity in the different regulatory paths between the normal and variant cultured cell lines.

Polymerase chain reaction (PCR) primers were prepared to amplify the DNA fragment between the genomic DNA sequence adjacent to the 5′-integration site of Roundup Ready ® (RR) soybeans neighboring the transgene and the parts of the coding region of the transgene, together with the primer set for the internal host gene, the ␣' subunit of ␤-conglycinin storage protein gene (Cong gene). Using the primers for the transgene and Cong gene, the DNA fragments were amplified from the individual genomic DNAs prepared from 72 samples of RR soybean isolated from imported soybean seeds labeled "not segregated." Although the frequency of alterations of the nucleotide sequences in both the transgene and Cong gene were almost the same, the mutations that caused alterations to the amino acid sequence were more highly repressed in the transgene than in the Cong gene. In the nucleotide sequence upstream of the coding region of the transgene, the number of alterations of the nucleotide in the proximal promoter region was smaller than that in the further upstream region, suggesting that the mutants missing or being weak glyphosate-tolerance by an alteration of the critical nucleotide sequences in the promoter or coding region might be discarded artificially. It is supposed that the selective bias on the transgene might be extremely high, which indicates that the nucleotide sequence of the transgene might be stable and maintained in inbred RR soybean lines.