The transport of silicon is an integral part of the synthesis of the silicified cell wall of diatoms, yet knowledge of the number, features, and regulation of silicon transporters is lacking. We report the isolation and sequence determination of five silicon transporter (SIT) genes from Cylindrotheca fusiformis, and examine their expression patterns during cell wall synthesis. The encoded SIT amino acid sequences are highly conserved in their putative transmembrane domains. Nine conserved cysteines in this domain may account for the sensitivity of silicon uptake to sulfhydryl blocking agents. A less conserved C-terminal domain is predicted to form coiled-coil structures, suggesting that the SITs interact with other proteins. We show that SIT gene expression is induced just prior to, and during, cell wall synthesis. The genes are expressed at very different levels, and SIT1 is expressed in a different pattern from SIT 2-5. Hybridization experiments show that multiple SIT gene copies are present in all diatom species tested. From the data we infer that individual transporters play specific roles in silicon uptake, and propose that the cell regulates uptake by controlling the amount or location of each. The identification of all SIT genes in C. fusiformis will enhance our understanding of the mechanism and control of silicon transport in diatoms.

Although approximately 98% of the internal surface area of the lung is lined by alveolar type I cells, little is known about the functions of this cell type. Using freshly isolated rat type I and type II cells, we created a subtraction library by suppression subtractive hybridization to identify genes differentially expressed by type I cells. We identified twelve genes of known function that are differentially expressed by type I cells. Differential expression of all 12 genes was confirmed by Northern blotting; we confirmed differential expression by immunocytochemistry for 3 genes for which suitable antibodies were available. Most of the genes code for proteins that are multifunctional. From the known functions of these genes, we infer that type I cells may play a role in the maintenance of normal alveolar homeostasis and protection from injury, lung development and remodeling, host defense, tumor/growth suppression, and surfactant metabolism, among other functions.

The molecular characterization of components involved in nitrate uptake and assimilation in phytoplankton is likely to provide new insights into these processes, their regulation, and their effect on primary production. We report the cloning and initial characterization of the first nitrate transporter genes in a marine organism, from the diatom Cylindrotheca fusiformis Reimann et Lewin. A clone isolated from a silicon-responsive cDNA library was shown by sequence comparison to encode a homolog of high-affinity nitrate transporters. The C. fusiformis nitrate transporter cDNA was named NAT (NitrAte Transporter). The NAT cDNA was used to isolate a genomic clone that contained two additional nitrate transporter genes, NAT1 and NAT2, arranged in tandem. The cDNA and two genomic sequences were highly conserved, and only 18 of 1446 nucleotides in the coding region differed. At least four copies of NAT genes were present in C. fusiformis and as shown by hybridization, multiple copies were present in other diatom species. The transcript abundance of NAT genes in cultures with different nitrogen sources was monitored by RNase protection assays. NAT mRNA levels were high in the presence of nitrate, at nearly the same level during nitrogen starvation, and also high in urea-containing cultures. Lower mRNA levels occurred in nitrite-grown cultures. NAT transcript levels were highly repressed with NH Cl or NH NO as the nitrogen source, although very low amounts were detected. These results suggested that monitoring NAT mRNA levels could serve as a marker for (1) nitrate uptake in nitrate medium, (2) nitrogen starvation, and (3) ammonium use by virtue of absence of expression. NAT mRNA levels were not directly regulated by light or dark, but were apparently related to cellular growth and protein synthesis. Using light/dark synchronized cultures to monitor cell cycle responses, NAT mRNA levels were high in early G phase, decreased through the remainder of G , then increased during DNA synthesis in S phase and into G , and finally decreased after M phase. In silicon-starvation synchronized cultures, levels were high at the G /S phase boundary, high throughout S and G , and finally decreased after M phase. It was clear that NAT expression, and by inference nitrate uptake, did not occur at continuous levels throughout the cell cycle. The results of the RNase protection experiments suggested that transcriptional regulation is a major contributing factor in the control of diatom nitrate uptake. The cloning of the C. fusiformis nitrate transporter genes provides a new tool for investigating diatom nitrogen uptake and metabolism. In addition, the regulation of NAT expression by nitrogen source is likely to be useful in developing techniques to specifically control the expression of genes fused to NAT regulatory sequences in transgenic diatoms.