To validate 2DE results, we performed relative quantification real-time PCR and semi quantitative RT-PCR on 4 genes representative of housekeeping metabolism, cell cycle control and pathological states.
For instance, differences may occur in the levels of cellular components needed for protein expression (e.g. variations in the global pool of housekeeping genes, cell cycle position or fluctuations of environmental conditions discussed later).
housekeeping metabolism, (GAPDH), cell cycle control (MAP kinases), ROS management and pathological links (PARK7) and DNA synthesis (PHB).
It is clear that HSP90 plays an important role in parasite housekeeping, metabolism, and cell cycle, particularly considering the huge number of client proteins that are under HSP90 control.
Motif cc_6 is the cognate binding motif for the RpoD sigma factor, which drives the expression of biosynthetic and housekeeping genes throughout the cell cycle [26] (Figure 2B); as expected, the great majority of the genes with cc_6 motif (27 out of 34) were down-regulated upon carbon starvation in our experiments.
This could at least in part be explained by the fact that the CS genes are highly enriched with genes involved in the regulation of basic, housekeeping processes such as cell cycle, cell growth, programmed cell death, DNA repair, and cellular response to stress (Suppl. Table 2).
Two housekeeping genes (18S RNA and glyceraldehyde 3-phosphate dehydrogenase (GAPDH)) were included in cell cycle analyses, and three housekeeping genes (18S RNA, RORα, and GAPDH) were included for normalization (primer sequences available upon request).
In the eukaryote/opisthokont core, other housekeeping processes such as cellular division, cell cycle and cellular organization processes constitute a larger fraction of the total protein set.
On the other hand, confluent monolayers of UFL-Ag-286 cells (1 × 106 cells/ml) were infected with AgMNPV-SF (MOIs 0.1, 1 and 10) under the same conditions previously mentioned, and harvested at 1, 12 and 24 h post infection to analyze the transcription level of ie1, gp64 and polh AgMNPV genes into the viral cycle respect a housekeeping cell gene (actin).
Over-expression of the human housekeeping porphobilinogen deaminase in the glioma cells, using the housekeeping-porphobilinogen deaminase plasmid, induced a G1 cell cycle attenuation accompanied by increases in enzyme activity and c6 differentiation toward astrocytes.
The results of this analysis highlighted that the skin transcriptome presented the most distant fingerprint from the global set, since the categories related to housekeeping processes (i.e. protein fate, cell cycle and DNA processing) were under-represented while those related to secondary, amino acid and lipid metabolism were highly expressed, if compared to pulp and seeds [ 17].
