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The following projects have been offered for this year:

Dr B.R. Barraclough (School of Biological Sciences, Life Sciences Building)

1. Production in bacterial cells of a novel eukaryotic protein encoded by an estrogen-responsive expressed sequence tag.

A screen of differentially-expressed cDNAs between an estrogen receptor a-positive, and an estrogen receptor a negative cell line has yielded an number of previously unidentified expressed sequence tags that are products of potentially novel estrogen responsive genes. For two of these, full-length cDNAs are available with potential open reading frames. In order to obtain the protein products and to provide material for raising specific antisera, recombinant proteins must be produced. The aim of this project is to sub-clone one of the cDNAs into an inducible bacterial expression vector and to establish conditions for the production of the protein in bacterial cells. The project will initially provide experience of PCR, gene cloning, of the expression of proteins in bacteria and of polyacrylamide gel electrophoresis. If the project goes well, there might be the opportunity to purify the protein and to begin a preliminary characterisation of its properties. The project will suit a student who took BIOL234 last year.

Dr A.D. Bates, (School of Biological Sciences, Life Sciences Building)

2. Properties of a functional domain of E. coli topoisomerase IV

Topoisomerase IV is a bacterial enzyme related to the more well-known DNA gyrase. Like gyrase, topo IV operates on DNA using the energy of ATP hydrolysis to pass one double-helical segment of DNA through a transient break in another, but is specialised in operating on DNA segments from different molecules, leading to the unlinking of daughter chromosomes after replication. The enzyme consists of two subunits, ParE and ParC, which form an active E2C2 tetramer. We are investigating the mechanism of this enzyme, in part by expressing and purifying fragments of the subunits corresponding to functional domains of the protein. We have expressed a fragment of the C protein, which is responsible for the breaking and rejoining of the DNA fragment, and wish to further characterise its activity in combination with the E subunit (which carries out the ATPase reaction). This will involve assays of the cleavage, relaxation, and decatenation and possibly ATPase activity of the enzyme in comparison with that of the wild-type enzyme. Additionally, some experience of protein purification may be involved.

3. DNA topoisomerase II from Deinococcus radiodurans (one of two projects)

DNA topoisomerase enzymes manipulate DNA Topology (supercoiling and linking of DNA in all cells). In bacteria, two related type II topoisomerases occur. They operate by passing one double-stranded DNA segment through a transient break in another using the energy of ATP hydrolysis, and have specific distinct roles. DNA gyrase introduces negative supercoiling and topoisomerase IV is responsible for unlinking (decatenating) daughter chromosomes after replication, so they can be partitioned into the dividing cells.

An increasing number of complete sequences of bacterial genomes are now available, and it has become apparent that a number of species manage with only one type II enzyme, which looks most similar to a DNA gyrase by comparison of the sequence. We are in the process of cloning and expressing the genes for the two subunits of the single enzyme from the highly stress-sensitive bacterium, Deinococcus radiodurans. We hope to investigate the properties of this enzyme in vitro to determine whether it is capable of carrying out both type II reactions. Depending on the current state of the project, the work may involve DNA cloning, protein purification, and assays of topoisomerase activity. One possibility would be to have two project students, one working on each subunit.

Prof S.W. Edwards (School of Biological Sciences, Life Sciences Building)

4. Expression of fluorescent death and survival proteins in immune cells

All mammalian cells possess proteins that determine whether they die or survive in response to external signals. These proteins are termed the Bcl-2 family and they comprise proteins that either control death (pro-apoptotic) or promote survival (anti-apoptotic). Cells of the immune system must have carefully regulated death pathways to ensure elimination of the cells when the immune response terminates. Neutrophils (phagocytic immune cells) express a number of death proteins (e.g. Bax), but also two, highly labile survival proteins, called A1 and Mcl-1. Death proteins and survival proteins

can interact with each other and this interaction is often important in determining cell fate. The expression of the two survival proteins is highly regulated by cytokines and their levels determine whether a neutrophil dies or survives.

We have already cloned cDNAs for Bax, Mcl-1 and A1 and in this project they will be sub-cloned into vectors that will allow them to be expressed as fluorescent fusion proteins. If these fusion proteins fluoresce at different wavelengths, then it will be possible to determine (a) where these proteins are located in living cells; (b) if the cellular location of these proteins changes during cell death and (c) which of the survival proteins physically interacts with the death protein, Bax.

This project will provide training in the following techniques: cDNA cloning, transfection of mammalian cells, tissue culture, fluorescence imaging

Dr. D.G. Fernig (School of Biological Sciences, Life Sciences Building)

5. A glycosyltransferase bibliography

Internet databases represent a key resource for biologists. The glycobiology community has produced a number of such databases, which include the CCRC (http://www.ccrc.uga.edu/) and the WWW Guide to Cloned Glycosyltransferases (http://www.vei.co.uk/tgn/gt_guide.htm). With the accretion of new knowledge, these resources require continual updating and occasionally a complete overhaul to the"look and feel" of the site and/or its data structure. This project will aim to update the proteoglycan section of the Glycosyltransferase Guide, and, if time allows, overhaul the look and feel of the site to make it more user-friendly. Knowledge of HTML not needed, though familiarity with the internet is essential. This project would be ideal for a student interested in moving into bioinformatics or some other aspect of information technology. Training provided in producing and mounting web pages on the internet.

An alternative project on the phylogeny of growth factors is also available. This will require a student who already has excellent IT skills, including programming (language irrelevant), and whose ambitions are to become a bioinformatician.

Dr M. J. Fisher (School of Biological Sciences, Life Sciences Building)

6. Structural analysis of protein splice-junctions

Protein splicing is a recently discovered post-translational modification that involves proteolytic cleavage and subsequent ligation events whereby distinct, functional proteins can be produced from a single polypeptide. This is analogous to the splicing events commonly observed in the processing of mRNA and represents a hitherto unexplored mechanism for the generation of multiple products from a single structural gene. The aim of this project is to investigate the structural features that predict the occurrence of splicing in proteins and thereby obtain a better understanding of the prevalence and mechanistic features underlying this novel form of covalent modification. The experimental approach will be based upon retrieval of sequence information from protein databases followed by utilisation of a range of Internet resources for structural analysis. This project will suit somebody who is familiar with using the University PC Network for Internet access.

Dr C.D. Green (School of Biological Sciences, Life Sciences Building)

7.Comparison of the activities of different isoforms of estrogen receptor beta

Until recently it was believed that there was only a single gene in mammalian species that encoded the estrogen receptor (ER alpha). However, in 1996 a second gene coding for an estrogen receptor (ER beta) was discovered. The two receptors show similarities in their sequences and in their abilities to stimulate gene expression but they also show sequence and functional differences. Two isoforms of ER beta have been reported that differ in their N-terminal amino acid sequences. This project will use transient transfection of ER expression vectors & estrogen-responsive "reporter" genes into mammalian cells in culture to compare the relative activities of the ER beta isoforms with each other and with ER alpha.

8. Writing a Grant Proposal for your breast cancer research project.

An important present day aspect of conducting research is, of course, obtaining the funding to support that research. Writing a Grant Proposal is an activity that involves a variety of skills - not only does the project have to be scientifically "sound" it also needs to convince the reviewers that it is likely to produce useful results. This project will involve the production of a grant proposal for the investigation of some molecular aspect of breast cancer. It will be necessary to identify an appropriate subject area, to produce an account of what is already known in the area and what needs to be understood next, to describe a logical & detailed programme of research and to calculate the cost of this programme. This will be written up in a form based upon a real grant application form. It is anticipated that computing skills (use of Web search engines, databases and word processing) will be central to this project.

Dr. P.C. Turner (School of Biological Sciences, Life Sciences Building)

9. Design and construction of a ribozyme to cleave specific target RNA molecules.

Ribozymes (catalytic RNA molecules) have the potential to be useful therapeutic reagents for disorders which could be treated by destroying specific RNA molecules, such as overexpressed oncogenes or viral transcripts. Although in some situations ribozymes can be delivered as drugs, expressing ribozymes in vivo from DNA constructs has many advantages and will be useful in gene therapy protocols. We have been developing DNA constructs to express hammerhead ribozymes in vivo which are based on the U7 snRNA gene. Although these do work, their efficiency could be improved. A project will be offered in which a new ribozyme construct will be made. Synthetic oligonucleotides will be cloned into a suitable vector and the resulting plasmid constructs will be sequenced to verify correct construction. Further rounds of modification may be necessary to create the desired ribozyme construct. The project will use a variety of molecular biology techniques.

Birikh, K. R., Heaton, P. A. and Eckstein, F. The structure, function and application of the hammerhead ribozyme. Eur J Biochem 1997; 245:1-16.

James, H. A. and Gibson, I. The therapeutic potential of ribozymes. Blood 1998; 91:371-382.

Dr John Jenkins, (Department of Medicine. phone: 1254067, e-mail : John.Jenkins@liv.ac.uk)

10. Is The Interaction Between Topoisomerase II And PKC Part Of The Cellular Stress Response Mechanism?

Eukaryotic topoisomerase II enzymes are essential for efficient chromosome DNA segregation in both mitosis and meiosis (1, 2). This makes them attractive targets for cytotoxic agents. To extend our understanding of the way topoisomerase II functions within its intercellular environment we carried out a protein-protein interactor screen, using the N-terminal domain of the yeast topoisomerase II. Yeast is a far more tractable organism and is the classical model system for studying the function of topoisomerases. Using the yeast two-hybrid screen, we identified the yeast protein kinase C 1 enzyme (Pkc1), the yeast homologue of the mammalian calcium dependent PKC (3). Pkc1 belongs to the group of serine/threonine kinases that are characterised by a high level of homology in their catalytic domains and cystine-rich region. A paper published recently establishes a functional link between the Pkc1 and cell cycle control in yeast proposing that there is a conserved mechanism that links signal transduction pathways and cell cycle machinery. Yeast Pkc 1 is also essential for cell growth and a role in bud emergence has also been suggested.

There is conflicting data over the role and effect of topoisomerase II and PKC modulators, with respect to their corresponding affect on the other enzyme. However they point to a role for PKC phosphorylation of topoisomerase II during drug treatment of cells.

Hypothesis: In yeast Pkc1 has been shown to be an essential component of the cellular survival mechanism in response to both heat osmotic and shock. What other proteins interact with this important domain of PKC?

This is a proven system, which represents a powerful tool in the study of the functional components of the Pkc1 pathway. We have converted the Pkc1 clone that we previously identified to a bait and propose to use it to screen a yeast two hybrid library.

1. Holm, C., T. Goto, J. C. Wang, and D. Botstein. 1985. DNA topoisomerase II is required at the time of mitosis in yeast. Cell 41:553-563.

2. Rose, D., W. Thomas, and C. Holm. 1990. Segregation of recombined chromosomes in meiosis I requires DNA topoisomerase II. Cell 60:1009-1017.

3. Watanabe, M., Chen, C.-Y., and Levin, D. E. (1994) Saccharomyces cerevisiae PKC1 Encodes a Protein Kinase C (PKC) Homolog with a Substrate Specificity Similar to That of Mammalian PKC*. J. Biol. Chem., 269, 16829-16836.

4.Tsai-Pflugelder, M., L. F. Liu, A. A. Liu, K. M. Tewey, J. Whang-Peng, T. Knutsen, K. Huebner, C. M. Croce, and J. C. Wang. 1988. Cloning and sequencing of cDNA encoding human DNA topoisomerase II and localization of the gene to chromosome region 17q21-22. Proc. Natl. Acad. Sci. USA

5. Jenkins, J. R., P. Ayton, T. Jones, S. L. Davies, D. L. Simmons, A. L. Harris, D. Sheer, and I. D. Hickson. 1992. Isolation of cDNA clones encoding the  isozyme of human DNA topoisomerase II and localisation of the gene to chromosome 3p24. Nucleic Acids Res. 20:5587-5592.

6. Hammonds TR, Maxwell A, Jenkins JR 1998. Use of a rapid throughput in vivo screen to investigate inhibitors of eukaryotic topoisomerase II enzymes. Antimicrob Agents Chemother 42(4):889-94

Professor S.J. Kemp (School of Biological Sciences, Life Sciences Building)

11. Identifying and characterising damselfly Microsatellites.

The Southern damselfly is an endangered species and exists in a number of more-or-less isolated sub populations. A panel of microsatellite markers is under development to characterise these populations.

A damselfly library has already been made and evaluated. This project will screen the library for microsatellite-containing clones, sequence positive clones, design primers to amplify the microsatellites and evaluate their usefulness as genetic markers.

Refs.

http://www.pcweb.liv.ac.uk/pmiller/groups/sjkemp/frames1.htm

Arnaud Estoup & Julie Turgeon. Microsatellite markers: isolation with non-radioactive probes and amplification. Version of 12/1996. Laboratoire de Genetique des Poissons, INRA 78352 Jouy-en-Josas, France. (paper copy available from Dr Kemp)

Kemp, S.J., Hishida , O., Wambugu, J., Rink, A., Longeri, M.L., Ma, R.Z. and Da, Y. A panel of polymorphic bovine, ovine and caprine microsatellites. Animal Genetics 26: 299-306, 1995.

Kumari, P. and Kemp, S.J. Polymorphic microsatellite markers in the ostrich (struthio camelus). Mol.Ecol. 7: 133-134, 1998.

Professor A.B. Tomsett and Ms. Helene Charrel (School of Biological Sciences, Life Sciences Building)

12. A molecular analysis of defined DNA sequences of weedy rice from South East Asia.

'Weedy rice' are a new type of rice, or rice mimic, which have appeared spontaneously in and around rice fields in various parts of the world. They exhibit unwanted wild characters, which lower yield and value of the crop.

The long term aim of this project is to understand the genesis of weedy rice. Three theoretical mechanisms have been proposed to explain their emergence: invasion and persistence of annual wild rice plants; introgression of genes from wild rice into cultivars; segregation and selection of weedy characters within the genome of exiting cultivars.

We are investigating these weedy rice using a range of genetic markers, including microsatellites, ITS sequences within rDNA genes and the catalase gene.

The student will use a range of molecular techniques with one or more of these to determine whether weedy rice have different DNA sequences to those in cultivars from the same fields.

Dr Mark Caddick (School of Biological Sciences, Life Sciences Building)

13. Molecular genetic analysis of gene regulation mechanisms (one of three projects)

Much of our research is concerned with the regulation of gene expression in Eukaryotes. As a model system we are currently working on the regulation of genes involved in nitrogen metabolism in Aspergillus nidulans. This research has lead to a detailed understanding and many of the components in the regulatory system are known. The main regulatory gene, areA, is required for the expression of most genes involved in the utilisation of nitrogen sources other than glutamine or ammonium. Its product, AREA, monitors the level of available nitrogen within the cell such that when nitogen levels are low AREA facilitates gene expression, allowing the organism to utilise a wide variety of nitrogen sources. Much of our work is currently focused on how AREA activity is modulated by the levels of available nitrogen. We know that at least two mechanisms are involved; one acts at the protein level where a negative regulatory protein. NmrA, interacts with AREA and in some way inactivates the transcription factor. The second mechanism involves destabilising the areA transcript so that very little AREA protein is translate when ammonium or glutamine are present.

Up to three projects will be available to investigate this system. The areas of interest include:

 
1. Devising ways of monitoring AREA and NmrA protein by introducing an epitope tag into the functional protein. This will allow us to characterise protein levels, their localisation within the cell, determine if they are phosphorylated under different conditions and determine if they interact with any specific proteins.

    

2. Devise selection techniques, including novel reporter constructs, to monitor the transcript degradation mechanisms which are important for areA and at least three other genes involved in nitrogen metabolism. This will allow us to select mutations which disrupt this signalling mechanism.

    

3. Mutation analysis of the regions of the transcripts known to be responsible f or regulated transcript degradation. The aim being to define the specific structures and sequences involved.

    

4. Mutagenesis of nmrA. This gene has been cloned, as has its homologue from Neurospora crassa. However, little is known about the functional organisation of either protein. Therefore deletion analysis, directed by sequence comparisons, will be undertaken.

    

5. There is evidence that a key element involved in modulating AREA activity has not been identified. This would be consistent with a co-activator being present in A. nidulans, which interacts with AREA and thus increases its activity. In yeast one such protein (Ada1p) has been identified which interacts with the two AREA homologues. It will be interesting to devise appropriate selection strategies to isolate mutants disrupted in any such activity or use molecular techniques to isolate homologues to the yeast ADA1 gene.

The students allocated to this project will initially be required to assess the available literature and after discussion determines which area of the project they wish to explore. More than one student can take on a specific aspect, if they so wish. The work will provide experience in a variety of molecular and genetic techniques.

Prof J.A. Gallagher and Dr D. McCreavy (Dept. of Human Anatomy & Cell Biology)

14. A study of the differential expression of a panel of prohormone convertases within human tissue using the Roche Lightcycle

A number of hormone or hormone-like molecules including parathyroid hormone-related protein (PTHrP) contain multiple dibasic sequences which potentially represent substrates on which pro-hormone convertases (PC) may act. A family pf PC's have been identified and it is believed that individual members act during post-translational processing in a tissue specific manner. Consequently a molecule such as PTHrP may be processed to liberate different mature molecules dependent upon tissue type or whether stage of differentiation.

The aim of this project is to determine and optimise Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) conditions for the amplification of members of the subtilisin-like family of PC's using a range of tissues believed to express them. Secondly to apply these conditions to study the differential expression of PC's within a variety of tissues using Quantitative PCR (Q-PCR).

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