Tamara E. English, Ph.D. Biology 2000

Differential gene expression in response to freezing and anoxia in the intertidal marine gastropod, Littorina littorea.



The intertidal zone is a highly variable environment where temperature, salinity and oxygen availability all fluctuate on a daily and seasonal basis. As a result, animals that inhabit this zone possess a high degree of metabolic plasticity. Littorina littorea, an intertidal marine gastropod, is tolerant of both freezing and anoxia. This study searched for changes in gene expression that may underlie the animal’s ability to endure these stresses. Screening of cDNA libraries was employed to identify freezing- and anoxia-induced genes in foot muscle of L.littorea; two libraries were synthesized from mRNA isolated from foot muscle of animals exposed to either 1, 12 and 24 hours of freezing or 1, 12 and 24 hours of anoxia. Differential screening of the frozen or anoxic libraries with mRNA isolated from stressed versus control (5°C)snails, followed by northern blot analysis, resulted in six transcripts that were confirmed as stress-upregulated. DNA sequence analysis identified 3 clones as myosin heavy chain, beta actin and cytochrome oxidase subunit 2. These clones were isolated from the freezing library but showed greatest transcript accumulation during recovery after stress. The translated amino acid sequence of a fourth clone, LLMET, elicited a putative identification of metallothionein, a heavy metal binding protein with a possible antioxidant role. The remaining two clones, LLGRP and LLAFW, were novel, showing little or no homology to DNA or amino acid sequences in various databases. Analysis of their translated amino acid sequences indicated that both proteins possessed a secretory signal at the N terminus, suggesting that they had either a membrane location or are excreted from the cell. Structural predictions based on previously analyzed proteins, suggested that LLGRP is a membrane channel protein of the porin class whereas LLAFW fits the criteria of an anti-parallel bundle (apb) protein of the alpha-class. None of the six genes/proteins found by this study has a cellular function that integrates well with any of the previously-identified biochemical adaptations that support anoxia or freezing tolerance but each suggests that there is still much more to learn about the types of molecular adjustments that are needed to support natural stress tolerance.