Kevin F. Larade, Ph.D. Biology 2002

Response to anoxia exposure by the marine snail, Littorina littorea: transcription and translation patterns of differentially expressed genes and proteins



Situated in the intertidal zone, the marine periwinkle Littorina littoreahas modified a number of biochemical mechanisms in order to endure extended periods of oxygen deprivation that accompany aerial exposure at low tide. To cope with decreased ATP production during anaerobiosis, organisms often suppress energy-consuming processes such as those involved in macromolecular synthesis. Analysis of hepatopancreas samples from snails exposed to 24-96 h anoxia showed a gradual disaggregation of polysomes into monosomes, signifying a decrease in protein synthesis, with a subsequent re-aggregation of polysomes observed after 3 h of aerobic recovery. Upon examination of protein synthesis, the rate of [3H] leucine incorporation into newly translated protein of hepatopancreas isolated from 48 h anoxic snails was determined to be 49% relative to normoxic controls. Western blots examining the phosphorylation state of eIF-2alpha, a factor involved with initiation of protein translation, also supported the proposal that metabolic suppression during anoxia in L. littorea involves a decrease in protein translation. The rate of overall mRNA synthesis in anoxia was also examined; [32P] UTP incorporation into RNA transcripts of nuclei obtained from 48 h anoxic snails was determined to be 31.7% relative to normoxic controls. Although these data show that transcription and translation are suppressed overall during anoxic exposure, specific RNA transcripts were up-regulated during anoxia as identified via differential screening of a hepatopancreas cDNA library. Several anoxia-induced clones were identified as homologues of known genes (e.g. ribosomal protein L26, ferritin heavy chain) and others were deemed novel (e.g.kvn). Northern blots showed gene-specific patterns of transcript elevation over a time course of anoxia. Nuclear run-off assays confirmed transcriptional up-regulation during anoxia, whereas organ culture experiments implicated selected second messengers and protein kinases in signal transduction pathways regulating gene expression. Examination of the protein products of anoxia-induced genes was accomplished with western blotting when antibodies to specific proteins were available. These results suggest that metabolic suppression in L. littoreainvolves a general decrease in macromolecular synthesis, whereas specific transcripts that may help cope with oxygen lack are up-regulated during anoxia exposure.