Little is known about the regulation of gene expression in diatoms, partly because genetic transformation was not possible in this group of algae before NREL’s transformation system was developed. The availability of this transformation system now allows the study of the roles of regulatory DNA sequences in gene expression. As a first step toward a better understanding of gene transcription in diatoms, NREL researchers Paul Roessler and Steve Milstrey used the firefly luciferase reporter gene (discussed earlier), to study the level of gene expression as controlled by various DNA regulatory sequences from the diatom C. cryptica and other organisms. They also used this system to try to define the regions of the ACCase gene promoter involved in the Si-depletion response.

Various plasmids were constructed in which different combinations of 5’ regulatory DNA sequences (promoters) and 3 ’ regulatory DNA sequences (terminators) were linked to the firefly luciferase gene (luc). The regulatory sequences used in this study included both the ACCase promoter and the UDP-glucose pyrophosphorylase/phosphoglucomutase promoters from C. cryptica. Also tested were the simian virus 40 (SV40) promoter, which drives high levels of gene expression in mammalian cells, and the cauliflower mosaic virus 35 S RNA promoter (CaMV35S), which is a strong constitutive promoter in plants. These plasmids were introduced into C. cryptica via cotransformation with the selectable marker plasmid pACCNPT5.1 as described earlier. Approximately half of the transformed strains produced in this manner contained the luc gene, as determined by PCR analysis. Based on past results, it is expected that the plasmid DNA was integrated into the genome of the cells. Luciferase activities in randomly

chosen transformants (eight from each plasmid type) were determined by the use of a luminometer.

As expected, the promoter regions of both C. cryptica genes drove luciferase expression in the transformed C. cryptica cell at high levels. Less predictable was the finding that the SV40 mammalian promoter also drove luciferase expression in C. cryptica at relatively high levels (although lower than seen using the homologous promoters), but the CaMV35S promoter was much less effective. In most of the constructs used in this study, the 3′ terminator regulatory region was from the C. cryptica aac1 gene. Replacement of this sequence with the SV40 terminator did not affect the levels of luciferase expression driven by the accl promoter, indicating that the source of the terminator sequence used may not be a critical determinant of gene transcription efficiency.

Previous results at NREL indicated that Si deficiency may affect the expression of the acc1 gene in C. cryptica (see Section II. B.2.d.). To try to identify regions of the acc1 promoter that might be responsive to Si levels, three plasmids that contained varying lengths of the acc1 promoter region (900, 445, and 396 bp, respectively) fused to the luc gene were used to transform C. cryptica. Under Si replete conditions, the average luciferase activities of transformants containing these plasmids were very similar. Furthermore, the luciferase activity increased to the same extent (approximately twofold) 6 hours after transfer into Si-free medium. This suggests that the Si-responsive elements are either within the shortest (396-bp) promoter region tested or in a separate area of the genome.

These results indicate that the firefly luciferase gene can be expressed in recombinant C. cryptica cells, to provide a sensitive reporter system for analyzing gene expression and promoter function in diatoms. This and similar systems will likely be extremely useful for gaining a better understanding of the molecular biology of this important group of organisms.