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You are given a cloned gene from Homo sapiens and are asked to identify structurally similar genes (potential orthologues) in a. Arabidopsis. b. Bombyx mori (silk moth).  How will you determine the expression pattern of the gene?

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Introduction

You are given a cloned gene from Homo sapiens and are asked to identify structurally similar genes (potential orthologues) in a. Arabidopsis. b. Bombyx mori (silk moth). How will you determine the expression pattern of the gene? Before analysis of related genes can begin it is important to extract as much information about or cloned gene is possible, as this will aid us in our search for potential orthologues. Firstly I am assuming that the cloned gene is derived from a DNA library and is contained within a vector as part of recombinant DNA. Knowledge about the type of library from which it originated is helpful (either cDNA or Chromosomal) for analysis of the sequence but I will examine that later. The first step is to determine the DNA sequence of this cloned gene. Assuming that I have been given only one colony of the bacteria containing the cloned gene for good experimental technique it is necessary to increase the amount of bacteria and so copies of the gene, before starting analysis. I would do this by setting up a liquid phase culture (LB for example) and incubating the colony I have been given until I have an appropriately sized quantity. In order to start analysis I must first extract the cloned gene from the bacteria and the plasmid vector to which it is attached. I would start by separating the components of the cell by size, in order to get rid of cellular debris. Initially I will have to break apart the cell to gain access to the plasmids, done by the addition of Lysozyme and EDTA. Because the plasmids used are of a lower weight than the rest of the cell contents after centrifuging they will stay in the supernatant whilst the rest will form a pellet at the bottom. See diagram. There is now a solution containing many types of damaged plasmids and small strands of DNA, which will affect the quality of our sequencing unless properly removed. ...read more.

Middle

All you have to do to search for an orthologue is to go to a website, e.g. http://www.arabidopsis.org/blast/ and use the BLAST program. Then simply type in your sequence hit search and any matches within the database are displayed graphically and then with a more informative text output telling you the percentage accuracy of the match and the gene it is matched to. Here is an example of a match I performed earlier just using some randomly typed in DNA sequence. Bombyx mori (silk moth) The search for potential orthologues becomes increasingly more difficult in the silk moth because unlike the Aribidopsis it is not yet fully sequenced. However you can still use the partially completed database to search at http://210.145.41.132/cgi-bin/lib_proc_MX . However when I tried using the same random sequence above it returned no matches at all and only 18 thousand sequences had been determined so far. Because I cannot simply BLAST the sequence I have to search for any orthologues manually. This can be done in two methods, depending on whether the gene is expressed frequently enough to detect in the silk moth or not at all. To determine whether the gene is expressed I would take a number of samples of tissue from the moth (muscle, neuronal) and then using the protein derived from the human gene as a template to would check for that protein by ding a biochemical analysis of the gene products. This would only be a quick search as the chances are that the gene in question is not expressed or is so rare that I will not be able to detect it. If however I do manage to find that the gene in question does have an orthologue that is expressed I can then move onto trying to ascertain the DNA sequence from the mRNA transcript and studying the similarities. Firstly I would have to isolate all the mRNA from the cell. ...read more.

Conclusion

Studying the translation product of the cloned gene can give us information as to how large the product is and how to look for it using other techniques. Hybrid-release translation (HRT) is used to create gene products using cell free translation systems. The gene products from the mRNA are created in vitro and are usually labelled through a particular amino acid. Which can then be run on a gel with another protein ladder and the size of the gene product can be determined. With this method it would therefore be possible to find out the absorbance of the product at a particular wavelength. The cells could be lysed and proteins purified and run it through an HPLC column attached to a UV detector (or mass spec if exact weight could be calculated) to see how much of the particular protein was being expressed. You could also use a GC but I think the protein would be too big for the coils and clog them up. The only other way I can think of to determine the gene expression pattern of the different organisms is to use antibodies. These would be created by challenging a rabbit with the protein in question. The rabbit would be left for a couple of days to create antibodies in an immunological response to the foreign protein. Then the antibodies in the blood could be extracted and have a luminescent marker attached, i.e. horseradish peroxidase. Now when the cells are plated out and exposed to the antibody if the protein in question if being expressed then they will bind to the protein. If the cells are then exposed to luminol the cells expressing the gene light up due to the chemiluminescence with the modified antibody. All the above methods are ways of detecting the gene products and hence expression patterns of the organisms in question. I have not mentioned the use of deletion sequences to determine the promoters and activators of the genes because in eukaryotes there is usually fatal damage done to the embryo if these sequences are disrupted, much more than prokaryotes. ...read more.

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