Gene expression levels of the three hemoglobins of the clam Lucina pectinata differ between different tissues

Abstract

The clam Lucina pectinata has three different hemoglobins (Hb): hemoglobin I (HbI), a sulfide‐reactive protein and two oxygen‐reactive proteins, hemoglobin II (HbII) and hemoglobin III (HbIII). In order to better understand the physiological role of these hemoproteins, we evaluated their gene expression at the mRNA level by real time PCR. The Hb mRNAs expression levels were compared to that of the histone H3 mRNA and the 18S rRNA. Total RNA was isolated by the TRIzol method in order to obtain higher RNA yields than that obtained using column‐based methods. RNAs were further purified by n‐butanol and diethyl ether extractions in order to achieve better A260/A230 ratios and higher amplification efficiencies during one step real time RT‐PCR. The histone H3 gene was expressed in similar levels in most tissues except in visceral mass, while the 18S rRNA gene levels were more uniform throughout all tissues. Thus, we compared the three hemoglobin mRNA levels to that of the 18SrRNA using the clam's foot tissue as calibrator. The Hb mRNAs were most highly expressed in ctenidia, whereas foot and muscle had lower levels of expression. In ctenidia, oxygen‐reactive Hb mRNAs were expressed at higher levels than HbI mRNA, as reflected at the protein level in previous studies. In contrast, the visceral mass mRNA expression of HbI was higher than for HbII and HbIII. This research was supported by NIH grants 8G12‐MD007600 and 5R25GM088023.