# Chris Rhodes Week 14

## Materials we need

• Table of significant genes at p< 0.05,0.01,0.001
• Top ten significant genes
• Top ten increased/decreased GO terms
• Make a list of the functions of the top ten genes.
• Take all of this data and hypothesize why under the conditions of the experiment they would be up or down regulated.
• How do your top ten significant genes relate to the GO terms? How do they relate to the experimental conditions?
• How do your results and hypothesis align with the paper you read?

## Methods

All important files used in this weeks experiment can be found in the Files section above.

Using the updated data spread sheet from the Week 12 Journal we used excell to calculate the average log2 value, the p-values, and the tstat values for each experiment, fast/slow,2.5%/50%, and 0.6%/50%.

• Average= AVERAGE(A2:E2)
• TStat= AVERAGE(N2:P2)/(STDEV(N2:P2)/SQRT(number of replicates)) where number of replicates=5
• P-value=TDIST(ABS(R2),degrees of freedom,2) where degrees of freedom=(5-1)=4

Using the filter function in excel, the experiment with the largest amount of significant genes (p<0.05) was found. This was the 2.5%/50% experiment with a total of 215 significant genes. This was a relatively small number considering the total number of genes studied was 6947. Using another filter function the top ten most significant genes were found in the 2.5%/50% experiment data. These genes, their p values, and their functions are listed below. The data from the 2.5/50 experiment was compiled and run through MAPPFinder in order to find increased and decreased ontologies in the experiment. MAPPFinder produced 2 txt files 1 for increased ontologies and another for decreased ontologies. By uploading these txt files into excel and using a filter function, the top ten most significantly increased and decreased ontologies were found. These ontologies and a description of their functions can be found below.

## Decreased GO Terms

1. fructuronate reductase activity: Catalysis of the reaction: D-mannonate + NAD(+) = D-fructuronate + H(+) + NADH AmiGO Link
2. precorrin-3B C17-methyltransferase activity: Catalysis of the reaction: S-adenosyl-L-methionine + precorrin-3B = S-adenosyl-L-homocysteine + precorrin AmiGO Link
3. precorrin-2 C20-methyltransferase activity:Catalysis of the reaction: S-adenosyl-L-methionine + precorrin-2 = S-adenosyl-L-homocysteine + H(+) + precorrin-3A AmiGO Link
4. coniferyl-aldehyde dehydrogenase activity: Catalysis of the reaction: coniferyl aldehyde + H2O + NAD(P)+ = ferulate + NAD(P)H + H+ AmiGO Link
5. inositol-1(or 4)-monophosphatase activity: Catalysis of the reaction: myo-inositol phosphate + H2O = myo-inositol + phosphate AmiGO Link
7. aldehyde dehydrogenase [NAD(P)+] activity: Catalysis of the reaction: an aldehyde + NAD(P)+ + H2O = an acid + NAD(P)H + H+ AmiGO Link
8. oxidoreductase activity:The catalysis of redox reactions specifically relating to energy functions such as NAD+/NADH AmiGO Link
9. inositol phosphatase activity: Catalysis of the reaction: inositol phosphate(n) + H2O = inositol phosphate(n-1) + phosphate. This reaction is the removal of a phosphate group from an inositol phosphate AmiGO Link
10. cellular aldehyde metabolic process: The chemical reactions and pathways involving aldehydes, any organic compound with the formula R-CH=O, as carried out by individual cells AmiGO Link

## Increased GO Terms

1. RNA metabolic process: The cellular chemical reactions and pathways involving RNA, ribonucleic acid, one of the two main type of nucleic acid, consisting of a long, unbranched macromolecule formed from ribonucleotides joined in 3',5'-phosphodiester linkage AmiGO Link
2. regulation of transcription: Any process that modulates the frequency, rate or extent of cellular DNA-dependent transcription AmiGO Link
3. regulation of nitrogen compound metabolic process: Any process that modulates the frequency, rate or extent of the chemical reactions and pathways involving nitrogen or nitrogenous compounds AmiGO Link
4. regulation of nucleobase, nucleoside, nucleotide and nucleic acid metabolic process: Any cellular process that modulates the frequency, rate or extent of the chemical reactions and pathways involving nucleobases, nucleosides, nucleotides and nucleic acids AmiGO Link
5. regulation of macromolecule biosynthetic process: Any process that modulates the rate, frequency or extent of the chemical reactions and pathways resulting in the formation of a macromolecule. AmiGO Link
6. regulation of gene expression: Any process that modulates the frequency, rate or extent of gene expression. This includes the production of an RNA transcript as well as any processing to produce a mature RNA product or an mRNA (for protein-coding genes) and the translation of that mRNA into protein AmiGO Link
7. regulation of cellular macromolecule biosynthetic process: Any process that modulates the frequency, rate or extent of cellular macromolecule biosynthetic process AmiGO Link
8. regulation of cellular biosynthetic process: Any process that modulates the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of substances, carried out by individual cells AmiGO Link
9. regulation of biosynthetic process: Any process that modulates the frequency, rate or extent of the chemical reactions and pathways resulting in the formation of substances AmiGO Link
10. regulation of macromolecule metabolic process: Any process that modulates the frequency, rate or extent of the chemical reactions and pathways involving macromolecules.AmiGO Link

## Top Ten Regulated Genes

ID Number: Protein Name, p-value, basic function

1. MSMEG_0410: MmpL Protein, 0.001619392, Lipid Transport Protein
2. MSMEG_0471: Transcriptional Regulator LysR Family Protein, 0.000589115, Transcriptional Regulator
3. MSMEG_1127: Probable Conserved Transmembrane Protein, 0.001345606, Transmembrane Protein
4. MSMEG_1161: Taurine Transport System Permease Protein TauC, 0.002122214, Taurine Transport
5. MSMEG_2951: [2Fe-2S] Binding Domain Protein, 0.002674215, Ferredoxin Enzyme
6. MSMEG_3446: Hypothetical Protein, 0.002569072, NA
7. MSMEG_4511: Linear Gramicidin Synthetase mbtf, 0.00059067, Antibiotic Synthesis
8. MSMEG_4525: Putative Oxygen-Independent Coproporhyrinogen III Oxidase, 0.003370748, Oxidase Activity
9. MSMEG_6415: Conserved Hypothetical Protein, 0.003256267, NA
10. MSMEG_6852: Putative Carboxylesterase/Lipase, 0.000659307, Lipid Hydrolysis

## Table of Significant Genes

Showing the number of genes with a p value below certain thresholds in the 50%/2.5% Oxygen Limited Experiment

p<     Number of Genes
0.05         215
0.01         42
0.001        3


## Discussion

Our results showed both similarities and differences between the source paper data. The source paper indicated that in conditions of oxygen limitation the cell would up-regulate the use of cytochromes, up-regulate the use of NAD+/NADH independent enzymes such as ferredoxin enzymes, and up-regulate the use of hydrogenases. All of these changes were a means of conserving energy and oxygen in the cell and more efficiently using the resources available to the cell. Out decreased expression ontologies are almost all related to pathways that involve the utilization of NAD+/NADH for cellular energy. This is consistent with the sources papers conclusion of the up-regulation of NAD+/NADH independent enzymes. If NAD+/NADH independent enzymes are being up-regulated then it stands to reason that NAD+/NADH dependent enzymes would be down-regulated to avoid excessive interference with the independent enzymes. Our increased ontologies show the up regulation of numerous regulatory pathways. This is consistent with the cellular behavior of resource conservation. By regulating systems such as macromolecule production, gene expression, and RNA metabolism the cell is attempting to limit the amount of energy and carbon spent on creating any substance unnecessary for immediate survival. One of the top ten differential expressed genes was a ferredoxin enzyme which corresponds to the source papers results of up-regulated ferredoxin enzymes. However we were unable to determine if the ferredoxin in our analysis was down-regulated or up-regulated so we cannot make any definitive statements about its agreement with the source paper. The other top 9 genes represented a wide range of functions that seemed to have no discernible connect to one another or to the results of the source paper. However Linear Gramicidin Synthetase mbtf which is involved in natural antibiotic synthesis could be a cellular survival mechanism in low oxygen conditions to try and kill off any other cells that would otherwise use up the remaining oxygen supply and energy resources. Additionally though our data didn't implicitly disagree with the source paper's findings our analysis didn't show any up-regulation of hydrogenases or cytochromes. This is probably do to the significance thresholds used when sorting the data as we really only looked at 10 genes and 20 ontologies and not a broad scope of all significantly expressed genes.