Abstract

     All cells within a eukaryotic organism contain the same DNA. What makes these cells different is the regulated expression of the genes in the DNA. When a cell differentiates, selected genes are up or down regulated to provide the required proteins for the transformation the cell needs to achieve. Without gene regulation all cells would become unable to differentiate and function properly.

     These patterns of gene regulation can be observed by conducting a microarray analysis comparing two different cell types or cells in different situations. In the lab project wild type, zms1D, zms2D and zms1Dzms2D genes were analyzed to discover which genes were up and down regulated. Just as in many other cells, I hypothesize that there will be a distinct pattern of genes that are up and down regulated for the zms1D, zms2D and zms1Dzms2D. This information will provide data supporting the idea that each mutation has a specific and constant set of up ad down regulated genes associated with the mutation. It is just like a fingerprint of the mutation itself. This is called cluster analysis.

    Cluster analysis is a popular method currently used in gene expression analysis. Cluster analysis can be used to simplify complicated data and provide for identification of genes that could possess similar functions. If two or three genes are up regulated similarly in the microarray data then they might be involved in a similar function.

    This experiment investigates zms1D and zms2D mutants against wild type expression. I will be analyzing eight grids from four slides to attempt to find correlations and patterns amongst up and down regulated genes of the zms1D and zms2D genes.

Methods

    Methods are the same as those used for the primary experiment conducted. For a list of the methods CLICK HERE. Additional methods involved analyzing four more grids (grids 13 and 14 were used in all data analysis) and scanning all genes for a three fold up or down regulation consistent in at least four of the six grids used.

Results

    Results concerning the yeast cultures, yeast RNA extraction, quantification, RNA degradation, cDNA dye labeling, and microarray hybridization and analysis are presented within the corresponding groups web results. For those results click accordingly Group 1, Group 4, Group 7 and Group8.

    Prior to standardization box plots displayed values that were not a 1:1 ratio as would be desired for accurate analysis as seen in Figure 1. Standardizing the data provides correction for the disproportional fluorescence of either the red or green dye by setting the mean ratio of the arrays to zero. Doing so will help avoid false findings of up or down regulation.

    Upon standardization gene expression was analyzed using Magic Tool. Any gene displaying an up or down regulation of at least three fold or higher in four or more of the columns was seen as significant. On the Log2 scale that is approximately equal to ± 1.59. Five genes were found to meet this criteria (Table 1). The YDR218C gene was down regulated three fold or higher in five of the six columns. Genes YHR053C, YHR094C and YOR153W were all found to be up regulated in four of the six columns. The YKL096WA gene is the only exception having only 3 of the six columns showing three fold or higher expression. This gene was accepted anyway due to the average of the six columns equating to 2.026 which exceeds the 1.59 cutoff. In addition, the YKL096WA gene is one of the only genes displaying approximately two fold up regulation in all columns.

        Gene functions were then determined using the Magic Tool gene function data base. The function of YDR218C, a down regulated gene, was identified as a structural constituent of the cytoskeleton (Table 2). Genes found up regulated had the following functions: YHR053C - copper ion binding activity, YHR094 - glucose transporter activity, YKL096WA - structural constituent of cell wall and YOR153W - xenobiotic-transporting ATPase activity (Table 3).

Discussion

        This experiment investigated possible up and down regulated genes found in both zms1Δ and zms2Δ  when compared to the wild type. Cluster analysis was done to attempt to develop a gene expression pattern of constantly up or down regulated genes. Up or down regulation was determined to be anything over or under expressed by three fold when compared to the wild type. A total of five genes were up or down regulated according to the aforementioned criteria in the zms1Δ and zms2Δ mutants.

        The only gene found to be down regulated was YDR218C (alias SPR28). The protein produced by the YDR218C gene functions in cell wall organization and biogenesis (Longtine 1996). Down regulation of a gene with this function was not expected and cannot be explained at this time. Genes found to be up regulated were YHR053C (alias CUP1-1), YHR094C (alias HXT1), YKL096WA (alias CWP2) and YOR153W (alias PDR5). Each up regulated gene had a very different function than the next. The genes YHR053C and YHR094C function in copper binding activity (Winge 1985) and glucose transporter activity (Wieczorke 1999) respectively. The YKL096WA gene was found to function as a structural constituent of the cell wall (Van der Vaart 1995). The final gene found to be up regulated was YOR153W, which functions in xenobiotic-transporting ATPase activity (Wolfger 2001).

        The only similarity between genes was YDR218C, a down regulated gene, and YKL096WA, an up regulated gene, both having functions as a structural constituent of the cell wall. Upon further research the YDR218C gene was found to be expressed at high levels during meiotic divisions (Longtine 1996). The reasoning as to why this gene would be down regulated in both samples of zms1Δ and zms2Δ when compared to the wild type is unclear. More of the wild type strains could have been undergoing  meiotic divisions than the mutant strains. This would account for the difference but is only hypothetical. Looking further into the function of YKL096WA revealed it also is involved in low pH resistance (Van der Vaart 1995). Reasons the YKL096WA gene would be up regulated in zms1Δ and zms2Δ is also not clear. Oxidative stress could be causing the yeast cells to have low pH then the up regulation of YKL096WA would be expected. Further research could be done to determine if this hypothesis is correct.

        There was not a strong correlation found amongst genes up or down regulated in zms1Δ and zms2Δ. However, all five genes found to be up or down regulated did show a strong pattern between each set of microarray data. Every gene showed at least a three fold up or down regulation in a minimum of four of the six samples. Even samples not exceeding a three fold increase or decrease usually where very close to doing so or are considered to be errors due to strong deviation from the other samples. Further analysis can be done on more grids to gain more data for interpretation. In addition to obtaining more data, further analysis can be done on the genes that were found to be up or down regulated in this experiment to investigate reasons for the changes in regulation.

literature Cited

Longtine MS, DeMarini DJ, Valencik ML, Al-Awar OS, Fares H, De Virgilio C, Pringle JR. The septins: roles in cytokinesis and other processes. Curr Opin Cell Biol. 1996 Feb

Van der Vaart JM, Caro LH, Chapman JW, Klis FM, Verrips CT. Identification of three mannoproteins in the cell wall of Saccharomyces cerevisiae. J Bacteriol. 1995 Jun 177(11):3104-10.

Wieczorke R, Krampe S, Weierstall T, Freidel K, Hollenberg CP, Boles E. Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett. 1999 Dec 31;464(3):123-8.

Winge DR, Nielson KB, Gray WR, Hamer DH. Yeast metallothionein. Sequence and metal-binding properties. J Biol Chem. 1985 Nov 25;260(27):14464-70.106-19.

Wolfger H, Mamnun YM, Kuchler K. Fungal ABC proteins: pleiotropic drug resistance, stress response and cellular detoxification. Res Microbiol. 2001 Apr-May;152(3-4):375-89.