Cloning of a Conserved Surface Glycoprotein
of the Human Parasite, Giardia lamblia.
By Trisha Foster
Sponsor Dr. Aley
Bridges for the Future Program
The purpose of this research is to study Giardia lamblia. More specifically, to study a surface protein called GP49. This glycoprotein of ~ 49kD is the only known invariant protein described on the surface of this protozoan. This protein has been identified on the surface of every isolate of Giardia tested, indicating that it is essential to the well being of the parasite, however nothing is known of its function. The goal of the research is to clone the gene that expresses this protein. From the clone of the gene, then try to obtain an unambiguous sequence of the DNA for future biochemical studies. Retrieving the double stranded sequence will be done through PCR techniques, using restriction enzyme digest and Agarose gel electrophoresis, Southern blots, Sub-cloning, ligations, and sequencing of the DNA.
Giardia lamblia is a parasitic protozoan that is the major cause of waterborne enteric disease (1). Its life cycle consists of two stages, the cyst and the trophozoite. The cyst is a dormant stage of the parasite that can survive in cold water, outside of the host, for long periods and is responsible for infection of any animal drinking contaminated water. The trophozoite is the growing form of the parasite that lives in the human small intestine and is responsible for disease (2). In the small intestine the trophozoite is subjected to both natural and immune defenses including bile salts, digestive enzymes and mucosal immune defenses. Because of the constant attack on the surface of the trophozoite, the most common proteins found on the surface of the trophozoite are variable. This means that as soon as, or even before, the host defenses find a way to attack the surface proteins, they change to new proteins and the body’s defenses have to start over again (1). GP49, first identified by Das, et al (3) is different in that it does not seem to change its structure. Even when different strains of G. lamblia are isolated from people, sheep, or other animals, the GP49 protein seems to be the same (3). Because this protein remains the same despite the attack of the host defenses, it is likely that the function of this protein is essential to the well being of the parasite (2). However, studies as to what the function is of GP49 have been hampered by the lack of information as to the sequence of the protein and to the lack of significant amounts of purified protein. Using microanalysis techniques, Aley and Das have determined the amino-terminal amino acid sequence (Aley and Das, personal communication), but subsequent attempts to use this information for cloning of the gene have proven to be difficult.
A breakthrough in the identification of this gene came when a routine search of preliminary data from a whole genome sequencing project identified a small stretch of uncharacterized DNA which matches the amino terminal sequence (Reiner, personal communication). Using this small piece of DNA as a tag, the putative GP49 gene is being isolated and cloned (Kalemegham, personal communication). Determining unambiguous sequence of this gene would help us to understand the structure and function of this membrane protein. This is turn can help in the understanding of the outer surface of Giardia and possibly help us find ways for eliminating Giardia infection from the small intestine.
I propose to clone and sequence the putative GP49 gene, analyze the predicted protein structure for clues to the function of this protein to aid future biochemical studies.
A large sized piece of DNA containing the putative GP49 clone has already been identified. My first effort will be to subclone (clone out smaller pieces of DNA) those that still contain the desired gene. This subcloning is essential for sequencing the gene because our DNA procedure can only read about 800 bases of DNA in one experiment. The total size of this gene is predicted to be about 1500 bases of DNA.
As sufficiently small pieces of DNA are made, they will be sequenced using the non-radioactive LICOR sequencer in the Biology Department. To unambiguously sequence the gene, each strand of DNA must be sequenced in both directions and overlapping sequences resolved. The subcloning and multiple sequencing of this gene are expected to take the greatest portion of my time on this project.
Sequence will then be analyzed both by the DNASIS package and by BLAST searches of the GENBANK national sequence database in Washington, DC.
Materials and Method
DNA Isolation: After identifying the large piece of DNA that reacted with the probe, restriction enzyme digests and electrophoresis is used to isolate smaller pieces of DNA. Cocktails of DNA, restriction enzyme, buffer, and water are put into a microcentrifuge tube. The cocktail is then heated at 37C for one hour. This allows the enzyme to cut the DNA strand and therefore create smaller fragments of the original DNA. Electrophoresis gels are also helpful in telling the size and concentration of the fragment of DNA.
A Southern blot was also used to help identify correctly what fragments of DNA from the gel contain the gene desired. The original gel is put on a filter, which transfers the DNA. The original probe made earlier as told in the introduction then probes that filter. The probe then reacts with the sites containing the gene desired. From there we then use the restriction enzyme digests and the electrophoresis to retrieve that fragment.
Ligations and Subcloning: From the electrophoresis gel and the Southern, we then know what fragments to ligate and subclone. The fragment is cut out of the original DNA, either by gel extraction or by a deletion. Gel extraction is done by using a kit that extracts the gel around the fragment and leaves the DNA. A deletion is done when an entire portion of the DNA sequence is cut out and the remaining plasmid religates back upon itself. When either of these processes are done then a ligation can be done. With the gel extracted fragment it needs to be put back into a vector. A vector called pBluscript was used. A cocktail of the fragment, the vector, ligase, and buffer are put into a microcentrifuge tube and then put into a cold water bath overnight for the ligation to occur. After the ligation, a transformation of the cells is done. In a 15ml tube, 100ul of competent cells and 10ul of the ligation are put on ice for one hour. Then 0.9ml of TSS-glucose is added to the tube and put on a rocker in the incubator at 37C. The transformation is then plated onto X-gal + IPTG + Pen plates. The X-gal and IPTG act as marker to which colonies contain our insert and which are just the vector without insert. When the insert is in the vector, a B-gal site is not intact because the insert is there. When the B-gal site is expressed, the X-gal and IPTG breakdown and the colony turns blue. Thus we know that the colonies desired are the white ones and the ones not containing our insert are blue. This is called a blue/white colony selection.
From the selection of colonies, subclones are then created. The colonies are put in 5ml of media called TB and 5ul of PenG in 50ml tubes. Then the tubes are placed overnight in the incubator to grow. A prep of the DNA is then done to extract only the DNA desired and not the bacteria. An alkaline mini-prep is most commonly used. From this process, the fragment has successfully been inserted into the vector and has been created into a sample that can be used for sequencing.
Sequencing: From the sub-clones, sequencing reactions are performed. Sequencing starts at either a T3 or T7 site that are founded on the pBluscript plasmid connected to the insert sites. A cocktail of the DNA sample, the primer for the T3 or T7, polymerase, buffer and water are made for the reaction. These are mixed thoroughly. Then in separate tubes reagents of ddATP, ddCTP, ddGTP, ddTTP are added because they have specific chain terminators. An equal portion of the cocktail is then added to each reagent and put into the PCR. First the sample is melted at 92C. This breaks apart the double stranded sequence. Then the sample is annealed at 50C that allows the primers to attach to the sequence. From there the sample is extended at 70C until the reagent stops the chain by added either a ddATP, ddCTP, ddGTP, ddTTP which attach to the OH group and stop the chain from continuing. This reaction steps are repeated 29 more times. After the reaction is done stop buffer is added to stop the enzyme reaction. The samples are then denatured before being loaded onto the sequencing gel to pull apart the strands. From the gel, the program automatically reads the gel lanes, usually determining between 500 to 1,000 base pairs. The samples are then edited manually for greater accuracy.
Results and Discussion
The results are that I have retrieved almost all of the double stranded sequence of a band that reacted with the probe.
The test of the sequence is that it should show particular features. Since GP49 is a surface protein, it is expected to see a signal sequence that starts the protein. Also it is expected to see a transmembrane sequence that runs the protein between the membrane layers. It is also expected that a GPI anchor sequence to be shown for the anchor to form. Finally, the protein should be expected to be around 40kDaltons.
After creating a consensus sequence of this band, it does not show these features shown above. Through further studying of the initial gel though, it might be possible that this particular band was right above another band. Meaning that the band that reacted with the Southern might actually be hidden under this band.
From the point in my research, I am continuing my efforts to locate and isolate GP49. I am going to try and isolate GP49 from a cosmid and from some ligations that I have done of that cosmid during this program. From that point I hope to retrieve the sequence, then I am going to try and express the protein. The expression could show to be of importance to find a way to penetrate the surface.
With the help of the Bridges program, I have transferred to UTEP. I submitted a proposal for a grant and scholarship to continue my research and have found out that I am going to receive it. I am very excited about this opportunity to continue my research and continue my education at UTEP.
I would like to thank everyone in the Bridges program, especially Nic Lannutti and Dr. Johnson. If Dr. Johnson had not told me about this program, I would have never known. I also would like to thank Dr. Aley. His patience and support has been wonderful. Also, I want to thank Priya Kalamegham, whom I worked with and taught me a great deal and Dr. Aley’s entire lab in which everyone helped me whenever I had a question (which was a lot!).
- Adam RD. The Biology of Giardia spp. Microbial Rev 1991; 55:706-32.
- Aley S, Gillin, F. Specialized Surface Adaptations of Giardia lamblia. Infections Agents and Disease 1995; 4:161-6.
- Das S, Traynor-Kaplan A, Reiner D, Meng T, Gillin F. A surface Antigen of Giardia lamblia with a Glycosylphosphatidylinositol Anchor. Journal of Biological Chemistry 1991; 26:21318-23.