Our Molecular Biology/Biochemistry Research Laboratory focuses on understanding eukaryotic differential gene expression under various laboratory and environmental conditions. The general strategy of this research uses messenger ribonucleic acid (mRNA) differential display polymerase chain reaction (PCR), RT-PCR, to synthesize and amplify partial cDNA sequences from subsets of mRNAs of the adult rat soleus (slow-twitch) muscle exposed to the atrophy of microgravity.

Extended exposure of humans and other animals to spaceflight produces a progressive loss of skeletal muscle mass and strength. This atrophy has been well documented by NASA. This process must be understood to design effective countermeasures. In the complex of hindlimb muscles, the greatest atrophic effects have been seen in slow-twitch muscles, e.g., soleus muscle.

This research relates to NASA's interest in outer space exploration missions and investigations of musculoskeletal changes that occur during spaceflight as specified in NASA's publication "Research and Technology Objective and Plans (RTOP) Summary (RTOP, NASA TM-105441 and W92-70279: NASA Ames) and to NIH's interest in musculoskeletal protein structure, genetic, and connective tissue diseases. Although the "Tail-suspension Model of Microgravity" has long been available and has had a significant impact on our knowledge of spaceflight induced skeletal muscle atrophy; we still know little about adult muscle repair and maintenance in the presence of normal (ground-based) muscular tensions such as household and occupational injuries.

Initially we examined total lactate dehydrogenase (LDH) expression patterns in the left soleus muscle after seven days of hindlimb suspension followed by six hours of recovery using eight-week-old male rats and their parallel controls. All animals were used in accordance with NASA-ARC AHB 7180-1 guidelines and NIH regulations. LDH specific activity in the seven-day suspended/no recovery rats was 690 units/liter as compared to 160 units/liter in the seven-day suspended/six hour recovery rats. These results reflect a four-fold increase in total LDH activity in the soleus muscle of the seven-day suspended/no recovery rats. LDH specific activity in the parallel control rats ranged from 60 to 100 units/liter. Suspended rats did, however, show a loss of mass in their left soleus muscle when compared to their parallel controls. The exact nature of this expression pattern in suspended soleus muscle is yet to be determined. We are hopeful that these molecular biology studies will provide some insight into this NASA concern.

Higher organisms contain about 100,000 different genes, of which only a small fraction, 15% (15,000), are expressed in any individual cell. Experiments are now underway to determine if any of the 15,000 and other individual mRNA species are uniquely expressed in microgravity-exposed soleus muscle in the rat compared to their parallel controls.

The key element is to use a set of oligonucleotide primers, one (3"-oligo-dT, 5"-TMN) being anchored to the polyadenylate [poly (A)] tail of a subset of mRNAs, the other (5'-10mer) being short and arbitrary in sequence (arbitrary primer) so that it anneals at different positions relative to the first primer. A primer such as 5'-TCA will allow anchored annealing to mRNAs containing TG located just up-stream from their poly (A) tails. This primer set permits initiation of reverse transcription of only this mRNA sub-population. Unique PCR amplified cDNAs from the soleus muscles of rats exposed to and not exposed simulated microgravity will be characterized by dideoxy DNA sequence analysis after cloning them into a suitable vector and compare the sequence data to known DNA sequences using the GeneBank and European Molecular Biololgy Laboratory (EMBLE) DNA protein databases. These unique PCR amplified cDNAs will also be used probes to verify that these cDNAs are differentially expressed in microgravity exposed and non-microgravity exposed soleus muscles.

LaTanya Garvin and Rawlins Clark, IV, two first-year biology undergraduate students, completed their first 10-week summer research residency (summer 1998) learning reverse transcription-PCR protocols, DNA sequence gel electrophoresis, and DNA gel sequence analysis under the direct supervision of Dr. Larry L. Lowe. LaTanya and Rawlins also attended the 9^th Annual NASA JOVE Retreat in Cocoa Beach, Florida and Kennedy Space Center where they meet NASA research students and scientist from other colleges and universities. LaTanya is currently developing her DNA sequencing skills using the Stratagene Eagle Eye II DNA Imaging and Sequence Analysis System. Rawlins is currently learning to purify total RNA to be used in trial reverse transcription-PCR reactions. LaTanya and Rawlins will be presenting the results of their preliminary findings at the National Meeting of the Beta Kappa Chi Scientific Honor Society (Oklahoma City, OK) and at the Annual Meeting of the South Carolina Academy of Science (Lander University) in the spring of 1999.