To prepare complimentary overhangs for subsequent ligation, the PCR product and vector were digested with BamHI-HF and XbaI; the vector DNA was then CIP-treated. For an optimal digest, the vector DNA and PCR product concentrations were determined using a nanodrop spectrophotometer; the vector DNA and PCR product concentrations were found to be 404.2ng/ÂµL and 137ng/ÂµL, respectively. The success of the digest was verified via preparative AGE (see Figure 2).
A no-digest (negative) vector control was performed (Figure 2, lane 4); this shows the supercoiled circular vector DNA, which travels through the gel faster than linearized DNA of the same size. There are also several slow-travelling bands representing the CIP-nicked circular DNA, which unfolded and yet could not easily travel through the gel. The doubly-digested vector DNA band (Figure 2, lane 1) does not match the travel distance of either supercoiled or nicked circular vector DNA, and thus the doubly-digest vector DNA must have been linearized.
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Single-cut XbaI and single-cut BamHI-HF vector controls were run (Figure 2, lanes 2 and 3 respectively), and were both shown to have linearized the vector DNA. The bands are between the supercoiled and nicked circular DNA of the no-digest vector control (Figure 2, lane 4), indicating they have indeed been linearized. Since each enzyme worked individually, it is assumed that the enzymes both performed well for the double-digest of the vector and the PCR product (Figure 2, lane 5). Additionally, the doubly-digested vector DNA band (Figure 2, lane 1) matches the travel distance of the single-cut vector DNA controls (Figure 2, lanes 2 and 3), further indicating that the doubly-digested vector DNA was linearized.
The indicated doubly-digested PCR product and linearized vector DNA bands (Figure 2, lanes 1 and 5 respectively) were excised and purified. Careful isolation prevented the collection of any improperly digested DNA, such as supercoiled vector DNA or non-specific amplification products.
Ligation and Amplification of Vector and Insert
Upon isolation, the vector and genomic DNA were ligated together. To prevent the re-ligation of any singly-cut DNA, the vector DNA was CIP-treated (as aforementioned); this fostered the removal of any 5' phosphates from the cut DNA. Without 5' phosphates, the singly-cut DNA was not about to re-ligate. The doubly-digested vector DNA was unable re-ligate with itself because it contained non-complementary overhangs. However, it was able to ligate to the insert despite the CIP treatment. This was possible due to the remaining 5' overhangs on the insert.
The ligation reactions were amplified via transformation in E. coli. This allowed for the selection of only circular DNA that should only have been derived from the successful ligation of vector and insert DNA; therefore, any singly-cut vector DNA was not amplified. However supercoiled, unsuccessfully dephosphorylated, or gel extraction carryover DNA may have been present despite the isolation procedure. As such, cells transformed with empty vector could still survive on selective media. To account for this, cells were transformed with vector but no insert (Lig 1); the number of colonies on the Lig 1 plate should show the approximate prevalence of colonies with empty vectors. The Lig 1 plate has no colonies (see Table 1), and thus there are assumed to be no empty vectors on the Lig 2 (1:3 vector:insert) and Lig 3 (1:7 vector:insert) plates.
A negative water control was performed for the transformations. There is no colony growth on the water control (see Table 1), thus the transformations are free from contamination. A positive pUC19 control transformation was also performed. Since colony growth is observed on this plate (~500 colonies, see Table 1), the efficacy of the transformation protocol was confirmed.
The Lig 2 and Lig 3 reactions were transformed in E. coli. Since there are no Lig 1 colonies, all Lig 2 and Lig 3 colonies (~150 and ~200 colonies respectively; see Table 1) are assumed to be free of empty vectors. Four putative clones were picked (putative clones 1 & 2 from Lig 3; putative clones 3 & 4 from Lig 2) for later analysis.
Analysis of Putative Clones
An analytical AGE of the four putative clones was performed (see Figure 3) to determine whether or not they contained the insert. All four putative clones contain the desired 1.8kb genomic DNA insert. Putative clones 1 & 3 were selected for sequencing since they were originally from separate ligation reaction transformation plates (Lig 3 and Lig 2 respectively). Putative clones 1 & 3 were designated pMR1 and pMR2, and were prepared for subsequent sequencing.
Sequence Analysis and Gene Identification
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The pMR1 and pMR2 sequences were analyzed with FinchTV and (NCBI) ORFfinder; using (NCBI) protein BLAST, each sequence was found to align with the Saccharomyces cerevisiae Hog1 protein (see Table 2). Both alignments generated low E-values ( for pMR1; for pMR2), and so are highly unlikely to have been matched to the Hog1 gene by chance. The SGD website was used to generate a Hog1 chromosome map. The Hog1 gene is located on chromosome XII (371620-372927bp; see Figure 4).
The unknown gene was successfully amplified and sequenced, and can now be used for later experimentation. The sequenced gene, Hog1, is an important osmolarity-regulating protein in S. cerevisiae (Westfall etc al 2004). Ensuring the cell is in an appropriate osmotic equilibrium with its environment is a universal cellular challenge, and thus proteins involved in osmotic regulation, such as members of the mitogen-activated protein kinases (MAPK) superfamily, are highly conserved (Hohmann 2002).
The high osmolarity glycerol (HOG) pathway is activated by a hypertonic extracellular fluid. Hog1p is activated via threonine and tyrosine phosphorylation (Brewster et al 1993). Activated Hog1p can then enter the nucleus, where it acts to phosphorylate a vast array of transcription factors (Hohmann 2002). One of the end results is the production of glycerol. Glycerol serves to increase the cytosolic solute concentration, thus countering the osmotic gradient of the extracellular milieu relative to the intracellular fluid (Westfall et al 2004).
Hog1p serves as an interesting target for future studies, partly due to the ecumenical abundance of MAPK homologs. For instance, p38 is the mammalian counterpart to Hog1p, and is activated by stress (Han et al 1994). Similar to the osmotic-stress response with Hog1p in yeast, p38 is phosphorylated on tyrosine and threonine residues in response to cellular cytokine stimulation (Raingeaud et al 1995). Therefore further studying the effects of Hog1p activation could have an impact on the control of inflammation in mammalian systems. One study, for instance, could investigate possible activation of Hog1p in yeast using mammalian cytokines (and other stress signals); if a stress response occurs, the analogous stress-response pathway could be deduced. Additionally, the consequences of defective Hog1p activation could be assessed by creating mutations in the phosphorylated tyrosine and threonine residues (such as changing the residues to alanine); this would allow investigators to explore the non-nuclear effects of Hog1p (as opposed to entirely knocking Hog1p out).