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Amino Acid Sequencing with // Bats // University of New Hampshire Email: rei36@unh.edu WIKI created by: Robin E. Renzi

====** Project Objective: ** //By using several recent scientific research articles, a biochemistry driven technique will be thoroughly explained and elaborated as to how this technique was used in each research case//. ==== **Method or technique**: //Amino Acid Sequencing first begins by establishing how many ends which includes the C and N termini there are in a single protein. Disulfide bonds must then be destoyed through denaturization processes to eliminiate the cross-link within the protein chain creating the primary structure of the protein. The chains of protein, should there be more than one, are then seperated through gel filtration which segregates out chains (also known as subunits) by molecular weight. An amino acid sequencing machine is then used to find the actual number of different amino acids within each chain which does so by separating out the amino acids based on their ionic charge differences. Once the number of amino acids has been established, the protein is then cleaved by certain enzymes which fragments the amino acids chains that can then be analyzed by overlapping known amino acid compositions.//

WIKI Layout 1. Article 2. Background 3. Method 4. Results

Research Article 1  Citation: Shen et al, "Parallel and Convergent Evolution of the Dim-Light Vision Gene //RH1// in Bats (Order: Chiroptera)", PLoS One.2010 January 21. doi: 10.1371/journal.pone.0008838.

Used to discover differences in base pairs of the Dim-light Vision Gene RH1 in Bats(Order: Chiroptera), Amino Acid Sequencing directly reflects the differences of mechanisms used for sight (1). ====**__ Background __**: All bats are nocturnal mammals. This can cause a problem when the light conditions at night are of very dim quality. Under the enviromental pressure of a nocturnal niche, bats have developed two mechanisms of coping with such conditions. Microbats which eat insects, use the method of echolocation to detect food. This involves sending out an acoustic sound that bounces off objects and reverts back to the bat with a shape or image. Megabats have a diet that consists mostly of fruit such as the bat pictured at right (fig 1.). These bats have eyes that have evolved with elaborate complexity to allow for sight in variable light conditions. Initially studying phylogenic convergence of the order Chiroptera, with regard to vision differences, amino acid sequencing was performed and differences were found that reflect directly to the phylogenic outcome (1). ====



​**__Method__: ** Twenty three bats were aquired by unfortunately having to kill them, but was done so humanely. The eyes of these bats were removed and placed in liquid nitrogen that was frozen. RNA was obtained from the eyes by using RNAiso TM. Then using the isolated RNA, Polymerase Chain Reaction (PCR) was then performed which gave amino acid results that could be interpreted.  A further visual representation of PCR can be watched at this link: [|PCR Animation] Copyright 2002, John Wiley & Sons Publishers, Inc.

To the right in figure three, the same protein that provides for humans to see is the same as bats who use visual sight. The highlighted area in yellow is the action site of the rhodopsin protein. This particular area is where the protein changes from cis to trans configuration. This protein if damaged at the active site leads to blindness. The process begins when a light photon is absorbed by the eyes an impulse is sent down the optic nerve to the vertebrate photoreceptor protein reacting with the double bonds in the retinal portion of the rhodopsin protein located at carbon atoms 11 and 12 causing a //cis-trans// transition. At this covalent bond the 11-cis carbon isomerizes after proton absorption to form an all-trans geometry shape.
 * __Results__: ** What is important about figure 2. is the colored portions represent differences in amino acid sequences among the order Chiroptera discovered by the extraction of RNA that was then used to perform PCR.
 * **RNAiso** is a total RNA extraction reagent which can isolate RNA easily and rapidly from animal or plant tissues and cultured cells. After homogenizing the tissues or cells in the RNAiso solution, chloroform is then added to the homogenate solution. After mixing well the solution is then centrifuged to separate the solution into three layers. The top layer will be a clear liquid that contains the RNA, the middle layer will be a semi-solid containing DNA, and the bottom layer will be a red colored organic solvent containing proteins, polysaccharides, fatty acid, cell debris, and small amount of DNA. The top liquid layer is collected and pipetted into a new tube. Being careful not to collect any of the middle layer is important to prevent contamination of the sample. An isopropanol precipitation is then done to extract the total RNA. Using the RNAiso, the total RNA extraction process can be done in about one hour. The isolated total RNA is intact. [[image:Rhodopsin_protein.gif width="413" height="344" align="right" caption="Figure 3. The rhodopsin protein. Posted with permission from Bellarmine University" link="http://cas.bellarmine.edu/tietjen/HumanBioogy/sensory_systems.htm"]]

Research Article 2 <span style="font-family: "Trebuchet MS",Helvetica,sans-serif;">Citation: Somporn TANSKUL, Kazumi HIRAGA, Katsumi TAKADA, Suchart RUNGRATCHOTE, Prasert SUNTINANALERT and Kohei ODA, “An Alkaline Serine-Proteinase from a Bacterium Isolated from Bat Feces: Purification and Characterization”, //Biosci. Biotechnol. Biochem.//, Vol. **73**, 2393-2398 (2009).

<span style="font-family: "Comic Sans MS",cursive;">Using a combination of procedures of which included amino acid sequencing, reserchers were lead to conclude that Bat feces collected from Wat Swankuha cave in Thailand contains an alkaline serine-proteinase with resistance to detergents that is very stable (3).

<span style="font-family: "Comic Sans MS",cursive; font-size: 120%;">**__Background__:** <span style="font-family: "Comic Sans MS",cursive; font-size: 110%;">The i <span style="font-family: "Comic Sans MS",cursive; font-size: 105.6%;">ndustrial world market depends greatly on bacteria. Specifically, enzymes which bacteria produce. Alkaline serine-proteinase is used in commercial washing formulas and food processing. There has been only one known bacteria source of this enzyme until 2009 when this very enzyme was found in bat feces (3).

<span style="font-family: "Comic Sans MS",cursive; font-size: 120%;">**__Method__:** <span style="font-family: "Comic Sans MS",cursive; font-size: 108%;"> Although individual bats were not used for experimentation, fifty samples of feces were obtained from a limestone cave named Wat Suwankuha in Thailand. Identification of the bacteria was discovered through a process of elimination by Bergey's Manual of Systematic Bacteriology. [|Free Online Version of Bergey's Manual of Systematic Bacteriology] The rDNA was extracted from the bacteria grown from the bat feces using the 16th subunit. This rDNA was then amplified through PCR to compare the results to that of known bacteria results. The enzyme purified, was then sequenced for individual amino-acid analysis by running an SDS-PAGE. A protein-blotting technique was used to stain a membrane of polyvinylidene difluoride. This membrane once stained was then transferred into an animo acid sequencer.

**__<span style="font-family: "Comic Sans MS",cursive; font-size: 120%;">Results __:** <span style="font-family: "Comic Sans MS",cursive; font-size: 108%;">Based on collaborative results from quantifiable tests which included amino acid sequencing, the bacteria discovered in the bat feces was alkaline serine-proteinase. As pictured in figure five, each row is an amino-terminal sequence with row one corresponding to the proteinase found in bat feces. This (row one) was a 100% match to the known sequence of alkaline serine-proteinase. Respectively, rows two through four show differences in the sequence that reflect relatedness to other proteinases that were also found in bat feces.




 * <span style="font-family: "Comic Sans MS",cursive; font-size: 140%;">__References__: **


 * 1) <span style="font-family: "Comic Sans MS",cursive;"><span style="font-family: "Trebuchet MS",Helvetica,sans-serif;">Shen et al, "Parallel and Convergent Evolution of the Dim-Light Vision Gene //RH1// in Bats (Order: Chiroptera)", PLoS One.2010 January 21. doi: 10.1371/journal.pone.0008838. <span style="font-family: "Comic Sans MS",cursive;">[|PubMed Bat (Chiroptera) Article]  This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 * 2) <span style="font-family: "Comic Sans MS",cursive;"><span style="font-family: "Trebuchet MS",Helvetica,sans-serif;">Wibbelt G, Kurth A, Yasmum N, Bannert M, Nagel S, Nitsche A, Ehlers B."Discovery of herpesviruses in bats" J Gen Virol . 2007 Oct;88(Pt 10):2651-5. [|Pubmed Bats w/Herpesvirus Article] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 * 3) <span style="font-family: "Comic Sans MS",cursive;">Somporn TANSKUL, Kazumi HIRAGA, Katsumi TAKADA, Suchart RUNGRATCHOTE, Prasert SUNTINANALERT and Kohei ODA, “An Alkaline Serine-Proteinase from a Bacterium Isolated from Bat Feces: Purification and Characterization”, //Biosci. Biotechnol. Biochem.//, Vol. **73**, 2393-2398 (2009). <span style="font-family: "Comic Sans MS",cursive;">[|Pubmed Bat Poo Article] This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
 * 4) <span style="font-family: "Comic Sans MS",cursive;">[|PCR Animation]