![]() Invited Symposium: Molecular Mechanisms of Ageing |
Results Results To begin to identify mRNAs that are selectively translated at synapses we isolated synaptoneurosomes from the cortex of 12 day-old Long-Evans rats.
Total RNA isolated from these synaptoneurosomes was compared to total cortex RNA using differential display (DDRT-PCR) with the following set of primers: Table 1. 3' Primers Sequence _____________________________________________ dT 51 TTT TTT TTT TTT T 31 dT1 51 GCG CAA GCT12 GC 31 dT2 51 GCG CAA GCT12 GG 31 dT3 51 GCG CAA GCT12 GA 31 5' Primers Sequence _______________________________________________ A1 51 CGG ATC GAC TCC AAG 31 A2 51 CGG TAT CGA TAC AGG 31 A3 51 CGG CTG ATC CAT GC 31 A5 51 CGG GTC TGC TAG GTA 31 A6 51 CGG AGA CGC TAG TGT 31 A7 51 CGG ATC TTT CTA CCC 31 _______________________________________________A selection of differentially displayed is shown in Figure 3.
A total of 60 such bands were isolated using this procedure, 20 of which were cloned and sequenced. A summary of the different clones that were isolated is given in Table 2. Table 2. Primer Pair1Size Identity _________________________________________________ Actin 246 Ax-actin 1-1 246 Tip60a 1-1 138 Osteonectin 1-2 395 EST193443 1-2 231 Ceramide gluc. transferase 2-2 388 ZBP-89 3-1 155 rpL18a 1-5 95 calmodulin binding protein 2-5 143 EST UI-R-E0-cj-d-08-0-UI.S1 2-5 140 EST-UI-R-E0-bw-f-06-UI.S1 2-5 118 Mitochondrial cytochrome B 2-6 140 Unique 2-7 220 Unique 2-7 197 EST HUM404H10B 2-7 116 rpS29 _________________________________________________ 1. Primer Pair= 31dTx/51Ay where x and y are the different primers shown in Table 1.Touchdown RT-PCR was then used to quantify the message levels in synaptoneurosome RNA and cortex RNA; this allowed us to determine whether the cloned cDNAs were, in fact, derived from mRNAs enriched in synpatoneurosomes. The strategy used is outlined in Figure 4.
Three independent synaptoneurosome/cortex RNA pairs were used in this analysis. The results are summarized in Figure 5.
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Sung, Y-J.; Weiler, I. J.; Greenough, W. T.; Denman, R. B.; (1998). Selectively Enriched Messenger RNAs in Rat Synaptoneurosomes. Presented at INABIS '98 - 5th Internet World Congress on Biomedical Sciences at McMaster University, Canada, Dec 7-16th. Invited Symposium. Available at URL http://www.mcmaster.ca/inabis98/higuchi/sung0691/index.html | |||||||||||
© 1998 Author(s) Hold Copyright |