Identification of altered gene expression in skeletal muscle during sepsis using differential display.
Different aspects of muscle metabolism are altered during sepsis and there is evidence that some of these changes may be regulated at the gene level. Differential display is a recently described technique to identify genes whose expression has changed during a biological process. This technique utilizes reverse transcriptase-polymerase chain reaction (RT-PCR) to compare mRNA signals in tissues during two different conditions. We used differential display to test the hypothesis that gene expression is altered in skeletal muscle during sepsis. Sepsis was induced in rats by cecal ligation and puncture (CLP). Control rats were sham-operated. Sixteen hours after CLP or sham operation, extensor digitorum longus muscles were harvested and RNA was extracted. Following differential display, 30 fragments (F1-F30) were identified that appeared to be uniquely expressed in muscles from sham-operated or septic rats. These fragments were reamplified by PCR and used as probes in Northern blot analysis. Messenger RNA levels corresponding to 2 of the 30 fragments (F5 and F24) were confirmed to be increased by Northern blot analysis in septic muscle. Following cloning and sequencing, F5 was found to display significant homology to the gene sequence of the guanine nucleotide releasing protein MSS4. The sequence of F24 did not match any reported gene sequence and may therefore represent a previously unidentified gene. The results support the hypothesis that gene expression is altered in skeletal muscle during sepsis.
Tiao
G M
GM
Department of Surgery, University of Cincinnati, Ohio, USA.
Hudson
K
K
Lieberman
M A
MA
Fischer
J E
JE
Hasselgren
P O
PO
D000818:Animals; D001483:Base Sequence; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression; D020662:Guanine Nucleotide Exchange Factors; D007239:Infections / Q000235:genetics*; D008297:Male; D015335:Molecular Probes / Q000235:genetics; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000502:physiology*; D016133:Polymerase Chain Reaction; D011506:Proteins / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D018076:DNA, Complementary; D020662:Guanine Nucleotide Exchange Factors; D015335:Molecular Probes; D011506:Proteins