Protein synthesis is one of the greatest energy consuming activities in all cells (e.g., using about 36% of total ATP turnover in normoxic turtle hepatocytes)  for it requires over 4 ATP equivalents per peptide bond formed . Cell systems under stress typically suppress protein synthesis as an early response to energy limitation and our studies of freezing and/or anoxia tolerant organisms have shown that global suppression of transcription and translation is a critical part of the metabolic rate depression that supports anoxia/freezing survival ,  and . However, against this background of general suppression, our studies in recent years have shown that both freezing and anoxia exposure trigger the up-regulation of a selected small number genes whose protein products appear to address specific functional needs for stress endurance. In particular, we have analyzed freeze-responsive gene expression in wood frogs (R. sylvatica) and anoxia-responsive gene expression in both vertebrates (adult red-eared slider turtles T. s. elegans) and invertebrates (marine snails Littorina littorea) using techniques of cDNA library screening and DNA array screening (reviewed in ,  and ). Interestingly, our work on these systems, as well as on gene expression in mammalian hibernation  and , is highlighting several consistent themes in gene expression that recur across phylogenetic lines and across multiple forms of stress-induced metabolic arrest. As discussed below, our analysis of freeze-responsive gene expression in hatchling C. p. marginata concurs with and extends our understanding of both freeze and anoxia responsive gene expression in other systems.
Survival of anoxia or freezing undoubtedly requires a broad suite of protein adjustments addressing multiple cellular needs. For example, one of the known protein responses to anoxia in adult turtles is enhanced expression of selected heat shock proteins in an organ-specific manner  and ; these chaperones may aid the long term stability of other proteins over extended periods of hypometabolism. Our initial expectation when we began to search for freeze-responsive genes was that we would find a considerable number of genes whose protein products would be associated with some of the known physiological and biochemical mechanisms that had previously been identified as important to freeze tolerance (e.g., genes for proteins involved in cryoprotectant metabolism and distribution, or in cell volume regulation, etc.). In some cases we were correct (e.g. some genes associated with glucose metabolism are up-regulated by freezing exposure in wood frogs ) but in other cases our screening results surprised us by revealing genes/proteins that had never before been associated with freeze tolerance . These suggest that many different and previously unsuspected areas of metabolism require adjustment to create coordinated cell, organ, and organismal survival of freezing. For example, in wood frogs the genes identified to date as being freeze responsive suggest that freezing survival requires alterations to blood clotting capacity, membrane transporters, ribosomal function, adenosine receptor signaling, hypoxia tolerance, natriuretic peptide regulation of fluid dynamics, defenses against the accumulation of advanced glycation end products, and improved antioxidant defenses .
Our initial analysis of stress-induced gene expression in turtles used differential screening of cDNA libraries to identify anoxia-responsive genes in organs of adult T. s. elegans  and . Interestingly, the results included multiple examples of the up-regulation of genes encoded on the mitochondrial genome during anoxia exposure. Screening of a heart library made from anoxic turtles (20 h submergence in N2-bubbled water at 7 °C) showed up-regulation of Cox1 that encodes cytochrome c oxidase subunit 1 (COX1) and Nad5 that encodes subunit 5 of NADH-ubiquinone oxidoreductase (ND5) in anoxia, as compared with aerobic controls . Cox1 and Nad5 transcript levels in heart rose within 1 h under anoxic conditions to 4.5- and 3-fold higher than control values, respectively, and remained high over 20 h of anoxia before declining during aerobic recovery. Both genes also responded to anoxia in red muscle, brain, and kidney  whereas other mitochondrially encoded genes (Nad4, Cytb that encodes cytochrome b) were anoxia responsive in liver . Furthermore, when Cox1 and Nad5 expression were analyzed in liver of control versus frozen hatchling C. p. maginata, we found that these two mitochondrially encoded genes were also freeze-responsive . This suggests that some gene expression responses to freezing by turtle hatchlings are responses to the anoxia/ischemia that develops due to plasma freezing (a concept that is further supported by the results of DNA array screening reported below). Indeed, this idea was first put forward from our studies of freeze tolerance in wood frogs . Two of the major consequences of freezing are cellular dehydration due to water exit into extracellular ice masses and ischemia due to the freezing of extracellular body fluids. We mimicked each of these stresses by separately exposing wood frogs to either dehydration or anoxia at 5 °C and found that some freeze responsive genes were also up-regulated under dehydration stress (fibrinogen subunits, inorganic phosphate carrier, FR10) whereas others responded to anoxia stress (ADP-ATP translocase, FR47, Li16, P0) ,  and . Note that FR10, FR47 and Li16 are freeze-responsive novel proteins not found in other vertebrates and whose functions have yet to be identified whereas P0 is a protein in the 60S ribosomal subunit that is essential for translation. P0 up-regulation also occurred in response to anoxia in freeze intolerant leopard frogs  and its levels peaked during periods of low oxygen consumption in diapausing insects  which confirms its link with ribosomal function under oxygen-limited conditions.
DNA array screening is an extremely powerful technique that is allowing major advances to be made in our understanding of the gene expression responses that underlie animal adaptation to environmental stresses. Array screening has key advantages including (a) thousands of genes, representing hundreds of different cell functions, can be screened simultaneously, (b) coordinated responses by families of genes or by functional groups of genes can be identified, and (c) the breadth of gene response to a particular stress can be viewed, often identifying genes and gene families that have never before been considered as contributing to stress tolerance . We have made extensive use of human 19,000 gene cDNA arrays produced by the Microarray Center at the University of Toronto to provide overviews of gene expression changes in multiple animal systems including hibernating mammals, freeze tolerant frogs and turtles, and anoxia tolerant turtles and snails , , , ,  and . The use of heterologous probing means that cross-reaction is never 100% but after optimizing binding/washing conditions, a high percentage hybridization can typically be achieved. Of the total 19,200 spots on the array, 2% showed an increase in signal intensity of 1.5-fold or more under experimental (freezing or anoxia) conditions compared with controls and 0.45% showed 2.0-fold or greater signal when C. p. marginata tissues were analyzed (for a description of the methodology see the legend of Table 2). Enhanced expression of a variety of genes was seen in response to freezing or anoxia with binding intensities of labeled probe from experimental samples that were 1.5- to 3.5-fold higher than in controls. In particular, our analysis sought commonalities between the two organs examined (heart, liver) and between the two stresses (freezing, anoxia) tested. Selected genes meeting these criteria fell into three categories: (1) iron binding proteins, (2) enzymes of antioxidant defense, and (3) serine protease inhibitors (serpins). These are listed in Table 2 and discussed below.
Heterologous array screening requires confirmatory methods to validate the data for the species under study. We have used two methods to do this and in numerous studies have shown that results from heterologous array screening hold up when examined by other methods ,  and . One of these methods is the screening of a species-specific cDNA library to find up-regulated genes followed by Northern blotting to assess mRNA expression levels in different experimental tissues/conditions. The other is a PCR-based approach involving the design of DNA primers based on a consensus sequence of the gene of interest (put together from available sequences in GenBank), use of the primers to retrieve the species-specific cDNA via PCR and then design and use a species-specific probe to evaluate organ-, time-, and stress-specific gene expression via Q-PCR. Western blotting can also be used to directly determine whether the protein products of putatively up-regulated genes are also elevated. For the data on freeze responsive genes we turned to a cDNA library that we had constructed from tissues of frozen hatchlings using the Strategene Lambda Zap kit, as described previously . A pooled cDNA library was constructed from a combination of five tissues (muscle, heart, liver, kidney, and brain) sampled from C. p. marginata hatchlings given 1, 4, 12 or 20 h freezing at −2.5 °C. The pooled method is highly effective for gaining a broad picture of prominently up-regulated genes in control versus stressed tissues, especially when tissue sample size is limiting, as it is with tiny hatchlings. Tissue-specific and time-specific expression of key stress-responsive genes are subsequently sorted out by Northern blotting. One result from the screening of this cDNA library (performed as in ) was the prominent freeze-responsive up-regulation of the gene encoding the hemoglobin α chain. Northern blots confirmed its up-regulation (prominently in liver) and validated the results from array screening.