? ? In our initial studies we have performed a series of cDNA microarrays in a Δspe3 Δfms1 strain to investigate the responsiveness of yeast genes to supplementation with spermidine and spermine. Microarrays were used to identify those genes responsive to the addition of either excess spermidine or spermine (10-5 M) compared to a control culture containing 10-8 M spermidine. 360 genes were up-regulated >2-fold, and 21 genes were up-regulated more than 10-fold after spermidine addition. Functional categorization of the genes showed induction of transport related genes, genes involved in methionine, arginine, lysine, NAD and biotin biosynthesis. 296 genes, were down-regulated more than 2-fold, and 9 genes were down-regulated more than 10-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 (38) were significantly responsive to spermine. Thus, global gene expression analyses show that spermidine plays a more major role in modulating gene expression in yeast than spermine. ? ? Since, the above microarray studies showed major effects of spermidine addition on methionine and arginine and ornithine metabolism, in our current studies we have compared the early and late responses of spermidine additions using a yeast double mutants deleted in both spe1and spe2 genes. This polyamine auxotroph 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 spermidine. Yeast cultures were harvested at 30, 60, 120 and 180 min intervals after addition of 10-5 M spermidine and RNA was isolated and microarray signals were compared at each time point with control cultures. To avoid any complexity due to modified hypusinated eIF5A effect on these microarrays , we have studied the early points of gene expression, as we found that the modified eIF5A is unchanged up to 180 min after spermidine addition. 176 genes were up or down regulated with in 30-60 min after spermidine (10-5 M) addition to a control (10-8 M spermidine supplemented) culture, while 313 genes changed the expression with in 120-180 min. Some of the induced genes at earlier time points are GAD1 (glutamate decarboxylas), CTR1 (copper transporter), DDR2 (multiple stress response proteins, GDH3 (glutamate dehydrogenase), several HSPs, GSP2 (a GTP binding protein). Some of the repressed genes are MUP1 (methionine permease), MBF1 (transcriptional co-activator), NPL3 (RNA binding protein). The most interesting sets of up-regulated genes are TYB, TyA Gag-proteins, OPT1-an oligo peptide transporter, HAS1 (ATP dependent RNA helicase), HSPs. The repressed genes at later time points include URP1 (actin associated protein), CDC50 (involved in cell polarity), MED7 (subunit of RNA polymerase II), PSK1 (serine/threonine protein kinase). ? ? In a related study, we have compared the whole proteome of yeast polyamine auxotrophs (spe1Δ spe2Δ) 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, duplicate 2D gels from each sample were scanned with a laser densitometer and differentially expressed peptides were detected by computerized comparison of the two gels. The peptides that are affected by different concentration of spermidine will be identified by for mass spectrometric analysis and presumably will give further insight into specific physiologic functions of polyamine in yeast.
| Chattopadhyay, Manas K; Tabor, Celia White; Tabor, Herbert (2006) Polyamine deficiency leads to accumulation of reactive oxygen species in a spe2Delta mutant of Saccharomyces cerevisiae. Yeast 23:751-61 |
