1. I.INTRODUCTION Huntington’s disease (HD) is an autosomal dominant neurological disorder, leading to progressive degeneration of the nervous system. Usually becoming apparent in mid-life and progressing for 15-20 years, with increased motor and cognitive impairment. Huntington’s disease is a type I polyglutamine(CAG) expansion disease. The disease causes severe changes in the striatum Huntington’s disease is associated with a mutant huntingtin gene, responsible for producing the huntingtin protein, since HD is a dominant allele disorder it exhibits mendelian inheritance. The focus of the research conducted in the lab was on the HAP1 promoter gene. HAP1 was one of the first proteins identified to interact with mutant huntingtin protein (htt). HAP1, or htt-associated protein-1 is ubiquitous in the brain and colocalizes with htt in the cytoplasm of neurons. HAP1 and htt are believed to be associated with intracellular trafficking and endocytosis due to their involvement with other trafficking proteins such as dynactin p150 and kinesin light chain. HAP1 is primarily found to be involved with axonal trafficking and endocytosis possibly playing the role as an adaptor protein between cargos and intracellular transporters. It has been found that in some mutations of the disease mutant huntingtin protein binds more tightly to HAP1 and can cause variation in the onset of the disease. The promoter region governs where initial transcription will begin for a gene. Through the use of PCR, restriction digests, ligation and transformation of bacteria we can learn much more about the promoter of the HAP1 gene. To find the promoter region of the HAP1 gene, we isolate bands of DNA from an agarose gel using electrophoresis. The isolated DNA is then placed into constructs, transformed into bacteria and transfected into cells for viewing fluorescence. The focus of research for this gene is whether removing its promoter region will still allow this gene to transcribe the protein. To test this idea we will use several different sizes of Rat DNA that contains the HAP-1 gene and insert them into our plasmid to identify whether or not the GFP(Green Fluorescent Protein)gene will be expressed. Following the transformation, DNA was extracted and a restriction digest performed. By comparing these results to the initial plasmid, we are in the process of determining which region of the HAP1 gene was successfully transformed into bacterial cells. We will then determine which of the constructs retained functional protein production from the HAP1 gene. II. METHOD AND PROCEDURES Finding the Primers  Upstream from the start codon we find the reverse primer, which is conserved in all the PCR runs.  Upstream from the start codon, the forward primer can be located at its respective base pair length. The Primer lengths used:  6kb, 4kb, 2kb, 1kb,0.5kb, 0.4kb, 0.3kb, 0.2kb  Primers were diluted to 100 uM PCR Preparation PCR solution was prepared consisting of. 10 µL Redox solution (Mg+, dnTPs, Taq Polymerase)  For a total of a 20 µL PCR sample  Denaturation: 95 C, Annealing: 59 C, Elongation 70 C  The solution was run for 35 cycles The DNA was then run through an agarose gel, The bands cut out the gel using the UV light box to measure the size. Using GeneJET TM Gel Extraction Kit (K0691) I purified the DNA sample that was extracted from the gel. To quantify the amounts within, we ran the samples in the spectrophotometer at 260 and 280 wavelengths (Table I). III. RESULTS V.DISCUSSION The main goal of this project has been to isolate specific regions of the promoter for the HAP1 gene, insert it into a vector and successfully transform it into bacteria. To achieve this we performed PCR amplification, Restriction Digest, Ligation, and Transformation techniques in the lab. The next step in the project would be to test the transformed colonies and determine if the plasmid had the inserted promoter of HAP1.  However, when the plasmids were isolated using a Plasmid Extraction Kit, there were no readings for the presence of our plasmid using UV spectroscopy or Ethidium bromide agarose gel.  One possibility, the plasmids are somehow lost in the plasmid extraction process, leading to no observable DNA when running the gel, and spec.  Additionally the growth in our broth cultures was low and there may not have been enough growth in the broth culture. Thank you to Fran Norflus Assoc. Prof. Clayton State University and Dr. Claire-Anne Gutekunst Asst. Prof. Emory University. This work was funded by the department of Natural Sciences at Clayton State University & Minorities Affairs Committee of the American Society of Cell Biology Restriction Digest and Transformation of the pEGFP-C1 vector involving the Huntington Associated Protein 1 1 Clayton State University Department of Natural Sciences and 2 Emory University Department of Neurology Addias Mervin 1, Natasha Dixon 1,Claire-Anne Gutekunst 2 and Fran Norflus 1 Restriction Digest We performed a double digest on our plasmid and PCR fragments so the ends would be complimentary.  Two enzymes were used for restriction digest  ASEI  AGEI  The samples were placed in water bath for 15 min at 37˚C for 5 minutes and deactivate enzymes in 65 ˚- 85 ˚C water bath for 5 minutes.  After the plasmid was cut with the enzyme, we used Alkaline Phosphatase to cleave the phosphates from the end of the plasmid and reduce background during the transformation. Both Plasmid and PCR fragments were purified using the GeneJET TM Gel Extraction Kit (K0691) Ligation Ligation setup was performed according to Fermentas Rapid DNA Ligation Kit(#K1422). The appropriate amount of DNA insert to add was calculated using Fermentas Ligation Calculator (http://www.fermentas.com/ using a 1:3 vector to insert ratio. The set up for each insert is listed in Table II. Transformation Obtain E.coli (pUC) & streak plates gather one single colony for the inoculation of C media overnight at 37 o C in shaker. Add 5 mL of DNA to 50mL of cells and transfer to Kanamycin plates for growth. Incubate overnight at 37 o C and observe whether growth occurred. If transformation was successful we should see growth on antibiotic plates. A positive control was prepared with the original plasmid to compare the plates Plasmid Extraction after transformation The colonies grown on the antibiotic (Kanamycin) plates, were picked and allowed to incubate in broth culture overnight at 37 o C in a shaker. Once growth was confirmed in the broth, I used GeneJET TM Plasmid Miniprep Kit (K0502) to extract the plasmid. 4 kbp 2 kbp1 kbp300 bp200 bp Positive & Negative control 5'-ATCTGACT + GATGCGTATGCT-3' 3'-TAGACTGACTACG CATACGA-5' 5'-ATCTGACTGATGCGTATGCT-3‘ 3'-TAGACTGACTACGCATACGA-5' These two “sticky “ ends are compatible: The plasmid and the DNA should ligate and make complementary base pairs. 6kb 4kb1kb500b 400b 300b 200b DNA Marker Original Plasmid Cut w/ both enzymes, 2 fragments Cut w/ Age I Cut w/ Ase I Fig 1. Restriction Digest Fig 2. PCR results Experimental Samples Fig. 3 Transformation results Plasmid Rat DNA InsertConcentration (ng/uL) 200 bp490 300 bp610 1 kbp460 2 kbp310 4 kbp310 4x kbp1580 Table I