ISOLATION AND CHARACTERIZATION OF 2 CDNAS ENCODING 4-COUMARATE-COA LIGASE IN LITHOSPERMUM CELL-CULTURES

Two near full-length cDNAs (LE4CL-1, LE4CL-2), which encode 4-coumarat e:CoA ligase (4CL), were cloned from a library of Lithospermum erythro rhizon cell suspension cultures by the use of heterologous probe of po tato 4CL. These cDNAs are 2.1 kb and 2.2 kb in length, respectively. L E4CL-1 encodes 636 amino acids, whose homologies to the 4CL protein se quences known to potato, parsley, pine and rice, were found to be 68%, 66%, 56% and 50% (identities on amino acid level), respectively, wher eas those of the predicted translation product of LE4CL-2 (594 amino a cids) to the above 4CL proteins were 49 similar to 54%. The similarity of the deduced amino acid sequences between the two 4CLs from Lithosp ermum cell cultures was 49% in identity. Northern analyses showed that the mRNA levels of both LE4CL-1 and LE4CL-2 were much higher under il lumination than in the dark, as reported for the 4CL genes of such pla nts as parsley. In comparison of mRNA levels of LE4CL-1 and LE4CL-2, t he former was demonstrated to be generally higher than the latter by m eans of an application of RT-PCR. The genomic southern blot experiment s suggested that there are probably three copies of LE4CL-1 in the Lit hospermum genome DNA, whereas only one copy was detected for LE4CL-2.