cnwokemodoih's blog

Which of the pathways is hampered by the ABSENCE of oxaloacetate in the mitochondrion of the cell?    A.) malonyl-CoA formation        B.) acyl-CoA transportation       C.) glycolysis

The correct answer is malonyl-CoA formation (A). Oxaloacetate needs to react with acetyl-CoA to form citrate, which can move out of the mitochondrion and re-form acetyl-CoA. Without extramitochondrial acetyl-CoA, malonyl-CoA cannot be formed, and the subsequent steps of fatty acid synthesis will not occur. Acyl-CoA transportation (B) is wrong because it happens even before acetyl-CoA formation and carnitine, not oxaloacetate, is required for that process. Glycolysis (C) is completely wrong because it has nothing to do with lipid metabolism.

We cut thin cross-sections of wild type and mutant stem internodes and stained them with phloroglucinol-HCl and toluidine blue (Figure 4). Phloroglucinol-HCl stains mostly lignin while toluidine blue stains polysaccharides and lignin. Comparing, the wild type and mutant internode cross-sections, we noticed that the wild type wall is thicker than the mutant wall, shown by the presence of more staining in the wild type cross-section (Figure 4a, 4c) than in the mutant cross-section (Figure 4b, 4d) for both phloroglucinol-HCl and toluidine blue stains. This indicates that there are possibly higher levels of lignin and polysaccharide in the wild type cell wall. Since the mutant shows less staining, we can predict that our gene is involved in the assembly of cell wall components.

Figure 4. Brachypodium distachyon internal stem internode anatomy. (a.)The stem internode cross-section from a wild type plant, stained with phloroglucinol-HCl. (b.)The stem internode cross-section from a mutant plant, stained with phloroglucinol-HCl. (c.)The stem internode cross-section from a wild type plant stained with toluidine blue. (d.)The stem internode cross-section from a mutant plant stained with toluidine blue. The positions of the epidermis (Ep), cortex (Co), vascular bundles (Vb) and pith (Pi) are indicated.

In an effort to extend the expression data in Phytozome, we designed an experiment to study Bradi3g27407 gene expression under 5% glucose growth conditions. To explore this, we conducted an experiment to study expression levels in root samples in the presence and absence of 5% glucose. We chose root samples because that is where our gene is most expressed, as indicated by our results from the e-FP browser. We used 8 root samples (5cm, young) from Brachypodium distachyon plants, growing 4 experimental samples in MS medium plates containing 5% glucose and another 4 control samples in MS medium plates without 5% glucose. We grew the plants at optimum temperature and light conditions: 24℃ day, 18 ℃ night. We created primers for the reverse transcription reaction, using primer3 software, so that they flanked introns but bound to exon sequences. The forward primer was 5’-tacaaggggaagatcagggc-3’ and the reverse primer was 5’-ccgcttgatctccttctcca-3’. These were so that the length of the sequence between the primers (not including the intron sequence) was 321bp (Figure S3). In Figure S3, the texts highlighted in yellow are the exon sequences, that highlighted in green is the intron sequence and the texts with red font color are the primers.

In an effort to extend the expression data in Phytozome, we designed an experiment to study Bradi3g27407 gene expression under 5% glucose growth conditions. To explore this, we conducted an experiment to study expression levels in root samples in the presence and absence of 5% glucose. We chose root samples because that is where our gene is most expressed, as indicated by our results from the e-FP browser. We used 8 root samples (5cm, young) from Brachypodium distachyon plants, growing 4 experimental samples in MS medium plates containing 5% glucose and another 4 control samples in MS medium plates without 5% glucose. We grew the plants at optimum temperature and light conditions: 24℃ day, 18 ℃ night. We created primers for the reverse transcription reaction, using primer3 software, so that they flanked introns but bound to exon sequences. The forward primer was 5’-tacaaggggaagatcagggc-3’ and the reverse primer was 5’-ccgcttgatctccttctcca-3’. These were so that the length of the sequence between the primers (not including the intron sequence) was 321bp (Figure S3). In Figure S3, the texts highlighted in yellow are the exon sequences, that highlighted in green is the intron sequence and the texts with red font color are the primers.

This semester, I learned how to genotype zebrafish. I had never genotyped before, so performing the steps regularly helped me to understand the process better. I learned how to analyze and interpret fragment analysis data. Importantly, I became more comfortable with running gels. Its importance lies in the fact that we run gels so often in the lab and it is an essential skill to have.  I also performed some TOPO-cloning, chemical transformation, inoculation and plasmid extraction, alone for the first time. This gave me more confidence to execute these processes in the future. Presenting before other members of the lab allowed me to get a feel of what it is like to make a scientific presentation. I learned not just from my presentation but from everyone else’s presentation. The feedback I received played an enormous role in my learning process here.

The DNA is the foundation of variability. The different arrangements of four nitrogenous bases, thymine, adenine, guanine and cytosine, determine how cells function and what characteristics an orgamism possesses. An organism's genome contains millions of genes which code for proteins with a variety of functions. With a million possible bases comes a million possible errors. Each base in the genome has a probability of being switched out for the wrong base due to mutations from UV radiation, errors during DNA replication and even mutagenic substances. Mutations that arise from changing single bases are called point mutations and these account for numerous genetic disorders like muscular dystrophy, sickle-cell anemia, phenylketonuria etc. With the advent of CRISPR-Cas9 system, it is posisble to create double-stranded breaks that allow the integration of random sequences. This is useful in research settings where knockout mutations are the aim but for therapy, the traditional CRISPR/Cas9 machinery may only aggravate the disease. The need to change single nucleotides spurred the discovery of base editors. Base editors consist of inactive Cas9 scissors paired with a protein that catalyzes the desired base change. In order to circumvent potential revertion to the incorrect base by the cell's proof-reading machinery, the base editor creates a nick in the other strand of DNA. This marks that strand for correction, allowing the proof-reading machinery to integrate the correct complementary base without hindering the correction. 

The DNA is the foundation of variability. The different arrangements of four nitrogenous bases, thymine, adenine, guanine and cytosine, determine how cells function and what characteristics an orgamism possesses. An organism's genome contains millions of bases which code for proteins with a variety of functions. With a possible bases comes a million possible errors. Each base in the genome has a probability of being switched out for the wrong base due to mutations from UV radiation, errors during DNA replication and even mutagenic substances. Mutations that arise from changing single bases are called point mutations and these account for numerous genetic disorders muscular dystrophy, sickle-cell anemia, phenylketonuria etc. With the advent of CRISPR-Cas9 system, it is posisble to create double-stranded breaks that allow the integration of random sequences. This is useful in research settings where knockout mutations are the aim but for therapy, the traditional CRISPR/Cas9 machinery may only aggravate the disease. The need to change single nucleotides spurred the discovery of base editors. Base editors consist of inactive Cas9 scissors paired with a protein that catalyzes the desired base change. In order to circumvent potential revertion to the incorrect base by the cell's proof-reading machinery, the base editor creates a nick in the other strand of DNA. This marks that strand for correction, allowing the proof-reading machinery to integrate the correct complementary base. 

Glucose growth condition is typically a form of osmotic stress. When a gene, SFAR4, was knocked out in Arabidopsis, the mutant plant was susceptible to glucose osmotic conditions and had lower germination rates than overexpression transgenic lines and wild-type. Under mannitol osmotic stress conditions, germination rates in mutant plants were not significantly lower than those in overexpression transgenic lines and wild-type. This indicates that the susceptibility to glucose is not due to osmotic stress but due to the mutated gene.

To get a basic idea of the Bradi3g27407.2 gene expression pattern, we used the e-FP browser. This web-based tool gave us a graphical summary of our gene expression data. We analyzed the pictogram, chart and table outputs and noted the tissues and organs where our gene is expressed. We explored more about our gene expression pattern using the PlaNet gene expression clustering program. In addition, we retrieved gene expression data from Phytozome for different Brachypodium distachyon plant growth conditions. We made figures for select conditions.

A novel GDSL-type esterase, SFAR4 was found in Arabidopsis thaliana. In this study, knockout mutants and overexpression lines were established. The absence and overexpression of the SFAR4 gene had no effect on germination rate when compared with the wild-types. However, in the presence of glucose, the lines with overexpressed SFAR4 had significantly higher germination rates than wild-type and knockout mutants. In contrast, the presence of mannitol did not affect germination rates. This finding suggests that perhaps SFAR4, a GDSL-type esterase plays a role in glucose susceptibility and glucose metabolic pathway regulation in germinating seeds. In this same study, the expression of fatty acid metabolic genes are shown to be differential in SFAR4 overexpression lines and knockout mutants. SFAR4 overexpression lines show increases levels of metabolism pathway components while knockout mutant lines are accompanied by lower levels of fatty acid metabolism gene expression. This indicates that SFAR4 plays a role in fatty acid metabolism.