The naturally occurring polyamines putrescine, spermidine or spermine are ubiquitous in all cells. Although polyamines have prominent regulatory roles in cell division and growth, precise molecular and cellular functions are not well established in vivo. In our first studies we performed microarray experiments with a spermidine synthase/ spermine oxidase mutant (deleted spe3 deleted fms1) strain to investigate the responsiveness of yeast genes to supplementation with spermidine or spermine. Expression analysis identified genes responsive to the addition of either excess spermidine (10-5 M) or spermine (10-5 M) compared to a control culture containing 10-8 M spermidine. 247 genes were up-regulated >2-fold, and 11 genes were up-regulated more than 10-fold after spermidine addition. Functional categorization of the genes showed induction of transport related genes, and genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 268 genes were down-regulated more than 2-fold, and 6 genes were down-regulated more than 8-fold after spermidine addition. A majority of the down-regulated genes are involved in nucleic acid metabolism and various stress responses. In contrast, only few genes (18) were significantly responsive to spermine. Thus, these results demonstrate a more major role for spermidine in modulating gene expression in yeast than spermine. In our current experiments we studied the effect of spermidine addition in a yeast double mutant deleted in both spe1 and spe2 genes. We have compared the early (30 min and 60 min) and late (120 min and 180 min) responses to spermidine additions to avoid any complications due to changes in hypusinated eIF5A since we found that the modified eIF5A is unchanged up to 180 min after spermidine addition. This polyamine auxotroph has the advantage over our earlier studies since it does not accumulate decarboxylated S-adenosylmethionine or putrescine and thus the effect of gene expression will be mainly due to change in spermidine concentration. As spermine didnt show much of an effect in earlier studies, we focused on the response to spermidine. Yeast cultures were harvested at different time intervals intervals after addition of 10-5 M spermidine and RNA was isolated and microarray signals were compared at each time point with control cultures (total 27 arrays). 176 genes were up or down regulated within 30-60 min after spermidine (10-5 M) addition while the expressin of 313 genes changed within 120-180 min. Expression of 40 genes were changed at all time points from 30 min to 180 min. Some of these genes were ADH5, BNA1, MDH2, OPI1, RPL22B, RPS22B, TDH1 HTB2, TOF2, QCR10, 7 and 9, RIP1, ROX1, GAD1, CTR1, DDR2, GDH3, several HSPs, GSP2, MBF1, NPL3 and OPT1. The most interesting sets of up-regulated genes are genes encoding the retrotransposons TyB, TyA Gag-proteins. We have also compared the whole proteome of the yeast polyamine auxotroph (spe1 spe2) grown in two different concentrations (low 10-8 M and high 10-5 M) of spermidine by 2D peptide arrays. Samples were run on 2D gels, and were scanned with a laser densitometer and differentially expressed peptides were detected by computerized comparison of the two gels. 195 differentially expressed peptides were noted from these study. The samples were reanalyzed by staining with coomassie stain and we are currently analyzing by mass spectrometry 20 differentially expressed peptides in collaboration with the NIDDK core facility. The multi-gene E. coli polyamine mutant has been used to feed C. elegans polyamine mutant (odc-1) in a different study in collaboration with Dr. Kevin OConnell of our laboratory. Initial studies have shown developmental defect during L1 to L4 larval development and embryonic lethality in C. elegans due to polyamine deficiency. E. coli grown in 10-5 to 10-6 M putrescine can rescue the C. elegans from this larval defect. Recently, Dr. OConnell has developed a mutant in combination with odc-1 and an oocyte developmental marker to study the developmental defect in detail.
| Chattopadhyay, Manas K; Chen, Weiping; Poy, George et al. (2009) Microarray studies on the genes responsive to the addition of spermidine or spermine to a Saccharomyces cerevisiae spermidine synthase mutant. Yeast 26:531-44 |
| Chattopadhyay, Manas K; Park, Myung Hee; Tabor, Herbert (2008) Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine. Proc Natl Acad Sci U S A 105:6554-9 |
