Kathryn
THIS IS KATHRYN'S PAGE
Notes 2/4/16 What are do we want from our research?
How do we get there?
What are we going to do with each of our 12 data sets? What do we need to do to evaluate these?
Notes 1/28/16 - 2/2/16 What are we doing?
Need to match each script to a gene and figure out how many times that is expressed.
How do we normalize that gene expression given that we don't get the same number of reads from each sample?
1) use DESeq which is a program that is able to normalize for the length of the gene per million reads so that we are able to compare across samples, good to normalize for the length as well as the #,
this is what we did using R, were able to compare across organs and then individually for intestine with fed vs unfed
2)benchmark to a housekeeping gene?
How do we know we have the right tissue?
1)look at the over represented sequences and blast this to see if we get a match?
- Did this and mostly was rRNA which means that cleaning up didn't go as well as we thought it did
Notes 1/12/16
Only .1 g of organ taken: possibility that connective tissue was taken, not representative of entire organ possibly total RNA > mRNA using beads that attach to polyA tails of mRNA - randomly fragment mRNA (since you can only read from an end to 75 base pairs) now get a lot more accurate reads, now know more about entire sequence mRNA > CDNA: using reverse transcriptase, dNTP, use primers that has every possibly combination of 6 nucleotides so all mRNA is transcribed CDNA: has been transcribed as mRNA and then changed into DNA to make more stable form
Research
BoAT1:
The molecular correlate of system B0, the major apical neutral amino acid transporter in kidney and intestine, is B0AT1 (SLC6A19) (46), a protein of 634 amino acids. Currently, no splice variants of the transporter have been reported. The human SLC6A19 shows very little activity in heterologous expression systems; hence, the mouse transporter has been characterized in more detail. In agreement with functional studies, B0AT1 transports all neutral amino acids, albeit to a varying extent. Vmax values appear to be fairly similar, but affinities are different for each amino acid. The order of preference is Met = Leu = Ile = Val > Gln = Asn = Phe = Cys = Ala > Ser = Gly = Tyr = Thr = His = Pro > Trp > Lys. This order is in partial agreement with studies in the intestine (280). The transporter shows some affinity for lysine; In situ hybridization and immunocytochemical analysis showed that the transporter is expressed in the kidney proximal convoluted tubule (46, 297) and in all parts of the small intestine but not in the colon (Fig. 1). Expression increases from the duodenum to the ileum (297, 373). The transporter is confined to the apical membrane. The signal was more intense towards the tip of the villi it has been reported that expression of the B0AT1 protein in the brush-border membrane requires coexpression of collectrin Find Collection gene?
Collectrin-deficient mice had low levels of B0AT1 and other members of the SLC6 family in the brush-border membrane. Transcript levels, in contrast, were unaltered, suggesting a posttranscriptional mechanism
Three different Na+-dependent components were identified, namely, a system A-like activity, a system ASC-like activity, and a novel activity. All Na+-dependent transporters have affinities in the micromolar range (356). They most likely serve to recruit nutritional amino acids when the intestine is inactive or starved. System ASC activity was also detected in the brush-border membrane in both kidney and intestine In summary, there is strong evidence for the presence of system B0 in the intestine; the role of system ASC in amino acid absorption remains to be determined
ASCT2:
The ASCT2 transporter is Na+ dependent but not electrogenic. This apparent discrepancy is explained by the mechanism of ASCT2, which involves an obligatory exchange of substrate amino acids against each other and a nonproductive Na+/Na+ exchange (48). It appears that there is no fixed ratio between the number of Na+ exchanged and the number of amino acids exchanged (176). Because of its antiport mechanism, ASCT2 cannot contribute to net transport of neutral amino acids across the apical membrane. ASCT2 transports small neutral amino acids with Km values of ∼20 μM; glycine, leucine and methionine are transported with Km values of 300–500 μM (387). Immunohistochemical analysis and reconstitution experiments suggest its presence in the apical membrane in the kidney and intestine (13, 251). In the kidney, expression is confined to the proximal tubule; in the intestine, expression is high in the jejunum and colon but lower in duodenum and ileum
Other Possible Genes:
Cationic amino acids : rBAT 4F2/LAT2, 4F2/y+LAT1
Anionic amino acids: ASCT2, EAAT3, EAAT2
Proline and Glycine: PAT1 (rat), IMINO (rabbit) BOAT1
Beta-amino acids: TauT, PAT1
