To determine the gene expression profile of aging in rockfish, total RNA was isolated from two pools of rockfish liver samples: twelve year olds and older rockfish in their thirties. Sufficient numbers of samples were available to isolate RNA from two groups in each age range to allow for biological replication of results. RNA was labeled using standard amino-allyl labeling protocols, and hybridized overnight to an array with oligonucleotides spotted in duplicate in separate quadrants across a glass slide. A long-oligonucleotide (65mers) zebrafish array set was obtained from Compugen, Inc. (http://www.labonweb.com/), which contains 16,399 oligonucleotides, representing 16,228 LEADSTM clusters (likely genes) plus 171 control oligonucleotides.

Because standard protocols generate lower signal intensities when applied to long oligonucleotide spotted arrays, several modifications to the standard labeling and hybridization protocols have been developed to improve signal-to-noise and overall signal intensity on the long oligonucleotide platform. Our expectation is that the high degree of sequence conservation among fish species will enable hybridization of rockfish RNA with the zebrafish sequences that are commercially available. The homology between zebrafish and rockfish is perhaps 60% (an estimate by Phylonix, a contract research organization providing in vivo zebrafish assays). Hybridization conditions will be relaxed should signal intensities be insufficient. With the large number of genes present on the oligonucleotide array, we expect to obtain a large amount of information regarding changes in the gene expression profile in rockfish liver during aging. These changes will be compared with expression data from shorter lived species to identify genes that may be responsible for extreme longevity in rockfish.

The first experiment with the rockfish RNA did not produce results. The controls worked, but there were no signals from the rockfish RNA. The experiment is being repeated under different conditions that may increase the signal, with new RNA being prepped for the next experiment.

Caption: Zebrafish Micro-array fluorescence output shown, following hybridization of fluorescently labeled zebrafish RNA to robotically spotted oligonucleotides affixed to a glass slide. This technique was used for the rockfish liver samples.

Recent literature has pointed out that at least some gene mutations that extend lifespan are in genes that involve increased resistance to oxidative stress, thermal stress and ultraviolet radiation. Increased lifespan could depend on the organisms ability to fend off insult by increased constitutive levels of protein products of some or all of these protective genes. Hsp70 (heat shock protein 70) and hsc70 (heat shock cognate 70) statement has been shown to play a role in the cascade of events dealing with protection from oxidative, thermal and ultraviolet insult. Prolonged exposure to innumerable stressors can result in cell death. The presence of these and other molecular chaperones may become extremely important in understanding how non-dividing cells survive a longer lifespan.

This study looked at the gene statement of the heat shock 70 family in rockfish. We compared constitutive levels in long-lived species of rockfish with those of other short- and long-lived organisms already examined to determine whether hsp70 levels change with age. We used western blot analysis, with antibodies available against a region that is vey homologus across species, and has been used in some of the published fish studies thus far.

Protein levels of MnSOD and CuZnSOD in liver samples obtained from rockfish were quantified by Western blot (first figure) and plotted versus age (second figure). A discordant relationship with age appears to exist for the two enzymes: MnSOD fell with age while CuZnSOD increased with the age of the rockfish. The increase with age in CuZnSOD is an intriguing finding given the powerful antioxidant status of SOD.

(4 - October 2001) Electron transport abnormalities and mDNA mutations. Judd Aiken, Department of Animal Health and Biomedical Sciences, University of Wisconsin-Madison, Madison, WI.

This study is investigating electron transport abnormalities and mitochondrial DNA mutations in rockfish heart tissue ranging in age from 12 to 83 years old. Judd has successfully amplified a portion of the cytochrome b gene. This was critical as his initial experimental design involved using the cytochrome b gene as the starting point for full genome PCR amplification.

With the recent publication of the complete mitochondrial genome for a closely related species, Sebastes schlegeli, he has revised his experimental protocol. New primer sets, based on the S. schlegeli and the limited sequence data for S. ruberrimus, have been designed that will allow him to amplify the major arc of the mitochondrial genome. Currently those primers are being used to investigate abnormalities and mutations.

(5 - September 2001) Turtle Biochemical profiling. Justin D. Congdon, Savannah River Ecology Laboratory, Aiken, SC.

In a joint collaboration, the project's turtle ecologist will use the Kronos Science Complete Assessment tests (shown in chart for Project 8 - Rockfish Biochemical Profiling). Turtles have been marked for many decades at several reserves in the United States (Congdon 1996). At the E.S. George Reserve in Michigan, some Blanding's turtles were marked as adults in the 1950's and remain in the population today; see picture below. Blood serum will be drawn from these and younger turtles. The measurements will identify a set of biochemical markers of age, which can then be compared with rockfish and other animals. Justin attempted to trap several turtles as part of an ecology assessment in 2001 but was unsuccessful in collecting samples. He has subsequently applied for general funding with which he can collect and analyze blood serum samples for this project.

(6 - July 2001) Mitochondria and nuclear DNA damage. Tory Hagen, Linus Pauling Institute, Oregon State University, Corvallis, OR.

Tory received 16 rockfish liver samples ranging in age from teen-age years to over 90 years old. He measured DNA damage in both mitochondrial and nuclear DNA (Helbock et al. for protocol). Since up to 95% of free-radical production occurs in the mitochondria, this information from a long-lived species could be very revealing. The preliminary evidence suggests there is no age-related increase in steady state 8-oxo-deoxyguanosine among the different aged samples (shown is table comparing 8-oxo-dG damage). The numbers, however, were several times higher than seen in rat liver tissue, so a larger sample size is necessary to verify if this is due to species differences or to artificial oxidation, which would still tend to increase relative differences uniformly.