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The following projects have been offered for this year:

Laboratory Projects

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

Identification of differentially expressed genes in a model of metastatic breast cancer.

P9Ka (also known as S100A4) is a protein which co-operates with the oncogene c-erbB-2 to yield metastatic mammary cancer cells. Four subtracted cDNA libraries have been created consisting of genes that are differentially expressed during the progression of metastatic breast cancer. The aim of the project is to screen these libraries to identify those cloned cDNAs which show a pattern of expression associated with the metastatic cells. The techniques will include preparing filters containing cDNAs, and their hybridisation to cDNA populations (Reverse Northern blot) to screen in a semi high-throughput manner, clones from the subtracted libraries. The project will involve using ³²P-labelled PCR-generated - cDNA probes, and will therefore suit a technically able student who is willing to work with radioactivity and to comply with current procedures of safe practice. If time permits, selected cloned DNAs may then be subcloned into suitable vectors for further study providing additional experience of molecular biology techniques.

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

The interaction of E.coli topoisomerase IV with supercoiled DNA

Topoisomerase IV is a homologue of DNA gyrase, and is primarily responsible for the ATP-dependent decatenation (unlinking) of the daughter chromosomes at the termination of replication in bacteria. The enzyme will also relax negatively supercoiled DNA. The enzyme operates by making a transient double-strand break in DNA, then passing a second double strand through the break. As such, it must interact with two regions of DNA double helix. In supercoiled DNA, there is evidence that enzymes related to topo IV interact with crossed helices in the DNA structure. This project aims to identify such interactions using E. coli topo IV. The primary experiment will involve relaxing supercoiled DNA with a second topoisomerase in the presence of topo IV. Binding to crossovers will result in the trapping of supercoils which will not be removed, and can be detected by agarose gel electrophoresis.

3. Dr B.F. Flanagan (Department of Immunology, Duncan Building)

Chemokine and chemokine receptor polymorphisms in health and disease

The chemokines are a family of low molecular weight cytokines known to be important in recruiting inflammatory cells to sites of inflammation. To date around thirty different chemokines, which fall into two main subfamilies, the cc and cxc chemokines have been identified. These molecules act through distinct cell surface receptors differentially expressed on inflammatory cell types such as macrophages or eosinophils. A number of polymorphisms associated with chemokines or their receptors have been defined including one in receptor CCR5 associated with resistance to HIV infection. The object of this project will be to look at two recently defined polymorphisms one in macrophage chemotactic protein 1 and the second in Receptor CCR3. The student will firstly design and test a pcr based strategy to detect these alternative forms and determine the gene frequencies using genomic DNA from control and Patient groups, including asthmatics. Such polymorphisms are of most interest if they can be shown to influence gene expression or protein function. This aspect will form the second section of the project if satisfactory progress is made.

4. Dr. M.X. Caddick (School of Biological Sciences, Donnan Labs)

Molecular genetic analysis of gene regulation mechanisms

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 nitrogen 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 translated when ammonium or glutamine are present.

Up to four 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 for 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 asses the available literature and after discussion determine which area of the project they wish to explore. More than one students can take on a specific aspect, if they so wish. The work will provide experience in a variety of molecular and genetic techniques.

5. Prof. P. Strike & Dr. L.A. Iwanejko (School of Biological Sciences, Donnan Labs)

Isolation and Modification of DNA repair genes from E.coli and Saccharomyces cerevisiae: Development of tools for the detection of damage in DNA.

Eight related projects are offered, in two groups of four. One group will work with E.coli, the other with Saccharomyces. There will be group meetings to decide on target genes, each group identifying four genes with which to work. Each individual student will therefore have a unique gene to clone, characterise and manipulate. Projects will be conducted and written up individually, but common techniques and approaches will be used so that there will be constant group support during the progress of the work. Likely techniques involved will be DNA isolation, primer design, PCR, sequencing, cloning, gene tagging and over-expression. If time permits, there may be an opportunity to undertake protein purification.

Living organisms constantly have to deal with damage to their DNA. There are numerous causes, types and consequences of DNA damage. For instance oxidative damage to DNA can occur simply as a result of normal metabolic processes and oxidative phosphorylation in the mitochondria. In humans oxidative damage to macromolecules such as DNA and proteins is implicated in carcinogenesis and the ageing process. Evidence for this comes from the observation that one of the most frequently studied products of oxidative DNA damage, 8-OHdG, is mutagenic and accumulates with age. The majority of these studies have measured total amount of 8-OhdG in cells or excreted in the urine.

It may not however be appropriate to specifically address the biological relevance of any type DNA damage by studying total DNA damage or by measuring excretion of modified bases. Low levels of damage in specific target genes for instance may be biologically more important than high levels of damage in so called ‘junk’ DNA.

The aims of these projects are to design and develop strategies for identifying damage ‘hotspots’. Researchers in our lab are currently developing a novel, PCR based, technique that will be used to identify the target sequences of oxidative DNA damage in cells of various human tissues. The technique exploits the affinity that the yeast repair enzyme, OGG1 has for 8-OhdG in DNA. At the outset of these projects, we will make a presentation in which the details of this approach are explained and the results to date will be presented. The aims of these projects will be to develop a similar approach, using appropriate enzymes, to identify a wide range of adducts in DNA.

You will work in two groups of 4 students. One group will use E. coli repair enzymes and the second group will work with yeast repair enzymes. A major part of the project will be deciding which adduct(s) your group wishes to study, and designing the approach for developing and testing the technique. Although you will be working as part of a team, the aim will be that the group divides the work appropriately so that each member of the group has their own project to conduct.

Both Professor Strike and Dr Iwanejko will jointly supervise all 8 students. Suitable background reading on DNA repair mechanisms can be found in ‘DNA Repair and Mutagenesis’ Freidberg (1995). More specific recent literature will be identified in the group discussions.

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

Cloning of cDNAs encoding the ultraspiracle gene product (USP)

Moulting hormones (ecdysteroids) regulate many physiological processes in insects. They act by binding of the hormone to the ecdysone receptor (EcR) in the target tissues, which then forms a dimer with a retinoid X receptor (RXR) and this complex then binds to the upstream regulatory elements of the target genes in the nucleus and regulates their expression. The ultraspiracle gene product (USP) is a member of the RXR family of nuclear receptors and it would be useful to clone this gene from our model organisms to allow us to study in more detail how regulation is achieved. The nuclear receptors for the different steroid hormones have common structural features and some domains are highly conserved. This should allow the design of degenerate primers to amplify cDNA fragments of USP by PCR. This will be done using RNA from both Spodoptera littoralis (an insect) and from C. pagurus (a crustacean) and any resulting PCR clones will be sequenced to establish whether they encode USP. Genomic libraries of both these organisms are available in the lab for screening with USP cDNAs to permit isolation of the complete genes. The project will provide experience in modern molecular biology techniques.

7. Dr. M.R.H. White (School of Biological Sciences, Life Sciences Building)

Cloning and characterisation of mammalian promoters

One of the major research activities in this laboratory is to identify and isolate the promoters of mammalian genes involved in the cell cycle, apoptosis and in response to important signalling pathways. A choice of possible promoters is available. These include the promoters for proliferating cell nuclear antigen which is (involved in DNA repair and the cell cycle); GADD45 (which is involved in DNA repair); PIG 3 (which is regulated by p53 and may be involved in apoptosis); and cyclins D1-3 (which are involved in regulation of the cell cycle). The student will be able to choose one or two of these promoters and to design primers to allow the promoters to be amplified from human genomic DNA by PCR. The PCR promoter fragments will be cloned into a plasmid and sequenced in order to ensure that the correct promoter sequence has been amplified without any polymerase errors. The promoter will then be cloned in front of the firefly luciferase gene. If progress is good it may then be possible for the student to study the expression of the plasmid in mammalian cells by luminescence imaging of firefly luciferase expression. The project could provide training in PCR (including primer design), gene cloning, restriction mapping, small and large scale plasmid preparation, DNA sequence analysis, mammalian cell transfection and luciferase assays.

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

The construction of chimeric human estrogen receptors

There are now known to be two different estrogen receptors (ERa & ERb) which show strong conservation of amino acid sequence in the regions of the two proteins responsible for hormone binding and DNA-binding. However, they show little if any homology in the N-terminal domain. This region of the ERa is known to be involved in two important aspects of receptor function: (i) it contains one of the two transcriptional activation functions (ii) it is essential for the "ligand-independent" activation of ERa by growth factors such as EGF. Expression vectors will be constructed that code for ERs in which the N-terminal domains have been interchanged between ERa & ERb, so that the consequences for the overall behaviour of the receptors may be determined.

Dry Projects

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

Design and construction of a DNA Topology Web site

ADB is the co-author of a textbook on DNA Topology, which includes all aspects of DNA supercoiling, topoisomerase enzymes and the effects of these processes on biological systems. The original book was published in 1993, and a second edition is currently in preparation. It is hoped to complement the physical text with a Web site, which will incorporate more complex versions of some of the figures in the book, most notably crystal structures of topoisomerase enzymes and cartoons or animations of the mechanisms of some of the enzymes involved. The site will also contain links to other relevant material on the Web. The intention of this project is to begin the design and construction of this Web site, in collaboration with ADB and his co-author, Professor Tony Maxwell from the University of Leicester. The project will require the development of some feel for the subject matter, as well as the methods for Web site construction, notably the use of the HTML language and the Chime utility for displaying 3-D structures.

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

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 (temporarily at http://www.liv.ac.uk/~dgfernig/proteoglycans.html), and, if time allows, overhaul the look and feel of the site to make it more user-friendly. Knowledge of HTML is not needed, though a very basic familiarity with the internet would be useful. This project would be ideal for a student interested in moving into bioinformatics or some other aspect of information technology. Training will be provided in producing and mounting web pages on the internet. For "inside" information, contact last year’s student, Monica Barclay (Mbarclay@btinternet.com).

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

The serine/threonine protein kinases of Caenorhabditis elegans

The free-living nematode C. elegans, is an important model multicellular organism. Sequencing of the C. elegans genome has now been completed and we have recently been able to make use of this information in the characterization of the cyclic AMP-dependent protein kinase(s) expressed in this organism. The purpose of this project is to survey the occurrence of a wide range of serine/threonine protein kinases in C. elegans. The experimental approach will be based upon retrieval of sequence information from protein databases followed by utilization of a range of Internet resources for structural and evolutionary analysis. This project will suit somebody who is familiar with the University PC Network for Internet access.

12. Dr. J.A. Smith (School of Biological Sciences, Life Sciences Building)

Use of sequence data to identify growth factor receptors in Caenorhabditis elegans

The complete genomic sequence of C. elegans has now become available. Although a relatively simple organism with a defind number of cells, C. elegans has most of the organ systems found in mammals. It therefore offers a minimalistic system in which to identify inter-organ signalling pathways which are complicated to study in higher organisms. This project will involve using sequences common to all receptor tyrosine kinases (RTKs) in mammals to identify the complete set of receptor tyrosine kinases in C. elegans. These will then be related to known RTKs in mammals to allow an impression to be gained of the likely number of RTKs still to be discovered in higher organisms.

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