Exploring DNA replication in the third domain of life

I am currently a student working towards an integrated master’s degree in biology at the University of Nottingham. The first three years of the degree were designed to provide a comprehensive overview of scientific knowledge, and to teach the skills required to efficiently access and interpret scientific literature. The 4th and final year of my master’s degree has been dominated by an 80 credit (that’s about 66% of the year’s grades) laboratory project. Now that I have a good background in scientific knowledge I have been unleashed into a laboratory environment, to learn the methods and skills needed to undertake practical scientific work. This project has been immensely beneficial. Actually learning how to carry out the techniques I had previously only read about has been an eye-opening experience into the life of a scientist. I have learnt many new skills. To list a few: I can now run a PCR, run an agarose gel, carry out miniprep and maxiprep, carry out transformations in both E. coli and Haloferax volcanii and I am learning how to carry out a number of assays. Valuable skills that would serve as an advantage when applying for lab based jobs.

Specifically, I have been studying archaea in a lab run by Thorsten Allers. Archaea are microorganisms, much like bacteria in appearance, but in cellular and genetic terms they widely differ. Archaea make up the third domain of life. The other two being bacteria and eukaryotes. A tree showing the evolutionary relationship can be seen in Figure 1. Humans fall into the domain eukarya. The tree shows that genetically we are more closely related to archaea than bacteria. This is one of the reasons that they are such interesting organisms to study. I am looking at a gene cluster found in H. volcanii involved with DNA replication and repair processes. Many of the genes found in archaea related to replication and repair have homologues in humans (Delmas et al., 2009). This means that increasing the understanding of how replication works in archaea could have important implications for the study of replication in humans. Deficiencies in replication can contribute towards the onset of cancer. Research into this area could therefore highlight cellular targets for cancer therapies (Chernikova et al., 2012) .

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Figure 1: A simplified family tree showing the three domains of life. Archaea and eukarya are more closely related to one another than to bacteria. Taken from: https://www.learner.org/courses/envsci/unit/text.php?unit=9&secNum=3#common_ancestor

 

The gene cluster I am looking at is shown in Figure 2. I am creating several different deletion constructs and observing what effect these gene deletions have on H. volcanii compared to the wild type. From the data gained, the lab can start piecing together the puzzle of how all these genes function and interact with one another. Much of my lab work so far has involved me trying in vain to amplify a genomic clone of the gene dnaK via PCR. dnaK is not directly in the gene cluster of interest but it is suspected to have a functional link as a chaperone protein, helping to disassemble proteins at a damaged replication fork so that the fork can be fixed (Thorsten Allers, personal communication). Creating a deletion plasmid will therefore be useful for the labs future research, and I learn the process of creating a genomic clone. The H. volcanii genome is notoriously difficult to work with. This is because it contains a main chromosome and several megaplasmids (Hartman et al., 2010). This can make amplifying specific sections of the genome via PCR pretty difficult. To get around this I am carrying out an older technique called a genomic assay (who said the new ways were better?), which involves using E. coli to amplify the dnaK gene instead of a PCR machine. While it has been disheartening to see my efforts fail for so long, at least I will be taught a new technique I might not otherwise have learnt, and hopefully this new method will bear fruit. Once I have a copy of the gene amplified I can create a plasmid containing the regions flaking the gene but lacking the actual dnaK gene itself.

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I’m using different gene deletion combinations at each stage of this project simply because I do not have enough time in the lab to carry out the whole project sequentially with a single deletion strain. Work on each section is being undertaken concurrently. The next stage of my work involves inserting plasmids that have already been created into H. volcanii. This is done through a process called transformation, in which H. volcanii cells are made to take up the deletion plasmid and insert the DNA into their genomes using random recombination, this is called ‘pop-in’. The plasmid is then removed from the genome but hopefully the deletion remains, this part is called the ‘pop-out’. It is achieved via a series of overnight dilutions in which the plasmid is hopefully removed via recombination. A more indepth discription is shown in Figure 3 for those curious. The pop out then needs to be verified using both colony hybridisation and southern blot. Finally, I will carry out a number of different assays, to study the effects of gene deletions on Haloferax. These assays will screen a strain with a deletion of the entire 5 gene region of interest, and compare its traits to a wildtype strain.  For example, defects that prevent DNA repair shall be screened for using a UV survival assay and defects in replication will be highlighted using a MMC (mitomycin C) survival assay (Lestini et al., 2010). My work contributes towards a much larger project in which the genetic and protein interactions carried out by this gene cluster will hopefully become a lot clearer.

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Figure 3: An example of the pop-in/pop-out method for gene knockout. A) The H. volcanii strain is originally ∆pyrE2 which means that it is unable to produce its own uracil. The process called crossover inserts the plasmid into the genome (pop-in). The plasmid is then lost through intrachromosomal crossing over. This can occur to the left of the deletion, which restores the gene to wild type, or to the right which deletes the desired gene. In both instances the cell is ∆pyrE2 and unable to produce uracil, making it resistant to 5-FOA (5-fluoroorotic acid), because it cannot convert it to the toxic 5-fluorouracil. B) A trpA marker is added which allows the organism to produce its own tryptophan. The strain is originally ∆pyrE2 ∆trpA but is transformed to produce its own uracil and tryptophan. The gene deletion wanted can be selected for in on step as it will be able to grow on agar plates without tryptophan (as it can produce its own) and in the presence of 5-FOA (as it cannot break it down into its toxic form). Taken from: Allers et al., 2004.

 

My time spent in Thorsten Allers’ lab has been the most enlightening and informative part of my degree. If someone is looking to enter a lab based career and gets the opportunity to gain some practical experience as part of their degree course, I would recommend it immensely.

References:

Allers, T., Ngo, H.P., Mevarech, M., Lloyd, R.G. (2004) Development of additional selectable markers for the halophilic archaeon Haloferax volcanii based on the leuB and trpA genes. Applied and Environmental Microbiology 70, 943-953

Chernikova, S.B., Game, J.C., Brown, J.M. (2012) Inhibiting homologous recombination for cancer therapy. Cancer Biology & Therapy 13, 61-68

Delmas, S., Shunburne, L., Ngo, H.P., Allers, T. (2009) Mre11-Rad50 promotes rapid repair of DNA damage in the polyploid archaeon Haloferax volcanii by restraining homologous recombination. Public Library of Science Genetics 5, 1-12

Hartman, A.L., Norais, C., Badger, J.H., Delmas, S., Haldenby, S., Madup, R., Robinson, J., Khouri, H., Ren, Q., Lowe, T.M., Mauplin-Furlow, J., Pohlschroder, M., Daniels, C., Pfeiffer, F., Allers, T., Eisen, J.A. (2010) The complete genome sequence of Haloferax volcanii DS2, a model Archaeon. Public Library of Science ONE 5, 1-20

Lestini, R., Duan, Z., Allers, T. (2010) The archaeal Xpf/Mus81/FANCM homolog Hef and the Holliday junction resolvase Hjc define alternative pathways that are essential for cell viability in Haloferax volcanii. DNA repair 9, 994-1002

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