ALDH2

1: To find general information about the promoter-

General search:
http://www.aldh.org/superfamily.php Description: ALDH 2 - promoter Search: http://www.aldh.org/superfamily.php Description: ALDH 2 Family The ALDH2 gene product is a mitochondrial matrix homotetramer (~56.4 kDa subunits). Based on the current nomenclature system, ALDH2 should be considered a member of the ALDH1B subfamily (>70% sequence homology) however, it was granted inclusion in the ALDH2 family due to the volume of literature regarding this enzyme as ALDH2.ALDH2 has a broad expression pattern and is most notably involved in the second step of ethanol metabolism, the oxidation of acetaldehyde. A large portion of individuals of Far East asian descent (~40%) have a functional polymorphism in the gene encoding ALDH2 (ALDH2*2) leading to a partially inactive form of the enzyme. This results in acetaldehyde accumulation and an alcohol-induced flushing reaction (10606490) , an increased level of response to alcohol and as a consequence, lower rates of alcoholism in this population (7625477) , (11925062) . In the mutant ALDH2 enzyme Glu487 has been replaced by a lysine and does not bind to AMP-sepharose chromatographic medium due to a decreased affinity for NAD+ (7910607). More recently, ALDH2 has been identified as a major enzyme responsible for bioactivation of nitroglycerin, used to treat angina and heart failure (17055381). Important Note: ...is most notably involved in the second step of ethanol metabolism... Search: http://switchgeargenomics.com/genecards.php?id=704324 Description: Gene symbol Product ID ALDH2 S704324

Type of product Human promoter reporter construct: transfection-ready

Suggested sequence (903bp) - commercial: CAGAGAAGGAAGGGAGTCTTGGTTATCAGATTATCT CTGCAGCTGATCTCCAAACCTATT TCCCTTCTTCCTGGCACAGGGGCCACTCATTTCCCAGACTCTTTTGCATGGAGGCATGGT TGTGTGATTGAAATCCAGCCAGGCGAGCAGGCTCAGCCCACAGAAACCTCCCATGGGCCA CCCCCATGCCTTTTCTTCCTCTGGTTTGTGTGGAATGGTGCTGAGCCCCCAGGGCGACTT TGGTGGAATCTGGTGTTGAAATCAGAGTCCCACCGCCGGGCGCGGTGGCTCACGCCTGTA ATCCCAGCACTTTGGGAGGCCGAGGCAGGCGGATCATGAGGTGAGGAGATCGAGACCATC CTGGCTAACACGGTGAAACCCCGTCTCTACTGAAAATACAAAAAATTAGCCGGGCGTGGT G G C A G G C G C C T G T G G TC C C A G C T A C T C C G G A G G C G G A G G C A G G A G A C T G G T G T G A A C C C G GGAGGCAGAGCTTGCAGTGAGCCGAGATCGTGCCACTGCACTCCAGCCTAGGCGACACAG TGAGACTCCGTCTCAAAAAAAAAAAAAAAAAAAAGAATAAGCAAATCATGCAAATCATCC TGTTCTAAACTCTGAACCGCTGCTTCCAAGACCGTCCCTGCCCTTGCCCTGGCCGCCTCT CTCCTTGTTAACTGGGCTCCATTCATTCTTTCCATTTCTCTAACACGTGCCAGGTGGTCT CATCTCCTGGCCTTTGCCCTTGCTGTTCCCTGTCATCATTCAGGTCTCACTTGTCATTTC CTGACCATGGTACTTATAAAAGCAGTGCCGTCTGCCCCATCCATGTCACCTCGTTCATCT CCTTCACCTCCGAAATGATCTCGCTTTTGGGTTTACGGCCGGTCTCTTCACCTGGAGCAT CAG The site highlighted in red is the PstI restriction site. Where is this referenced in the literature? Search: http://www.ncbi.nlm.nih.gov/gene/217 Suggested region: 112203518-112204421 =>903bp

Result of search (as link): http://tinyurl.com/bww3gmd

Annotated screenshot Locating biobrick site: Search: http://tools.neb.com/NEBcutter2/

FOUND PstI site.

In order to produce the primers with biobrick prefix / suffix we may need to do a point mutation. Also: Primer test: http://ginkgobioworks.com/cgi/primer.cgi

Warning: PstI site(s) found in sequence Forward primer: gtttcttcgaattcgcggccgcttctagagcagagaaggaagggagtcttggttatc Reverse primer: gtttcttcctgcagcggccgctactagtactgatgctccaggtgaagagacc
Suggestion; http://openwetware.org/wiki/Site-directed_mutagenesis There was also a suggestion by Mark that instead of going for site directed mutagenesis, we omit the sequence with the restriction site. The restriction site is found at the beginning of the promoter sequence, therefore this should be okay-(needs to be looked at). Mark also suggested pulling out some additional flanking sequence either side of the promoter region so that primers can be designed to these regions. I am not sure if this any better because we have an EcoRI site just before the beginning of the primer, so we cant take a flanking sequence upstream- because this will mean another site directed mutagenesis. Otherwise we can manually design the primers to amplify just the 903 bp region. But the suggested primer above is designed to include all the 903 bp.