CONTENTS | NEXT PAGE

The following projects have been offered for this year.

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

Analysis of cloned cDNAs from a subtracted library corresponding to mRNAs of potential tumour suppressor genes.

We have used a technique to produce subtracted cDNA libraries which contain specifically cDNAs to mRNAs which are differentially-expressed between a benign human breast tumour-derived cell line and a malignant breast cancer cell line. We have already examined clones expressed at a higher level in the malignant cells relative to the benign cells and have identified a number of known mRNAs, unknown mRNAs and expressed sequence tags. The aim of this project is to identify and study some of the clones for mRNAs which are expressed at a higher level in the benign cells than in the malignant cells. Some of these might be clones for the products of potential tumour suppressor genes. The project will involve a range of molecular biology techniques including PCR, Southern blotting, DNA sequencing and the use of computers for database searching. This project will also provide an opportunity to become familiar with local rules for the safe handling of radioactivity.

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

Engineering mutants of the E.coli topoisomerase IV subunit, ParC, with altered drug sensitivity

Topoisomerase IV is a homologue of DNA gyrase, and is primarily responsible for the decatenation (unlinking) of the daughter chromosomes at the termination of replication in bacteria. The enzyme is the primary target of the clinically important quinolone antibiotics (e.g. ciprofloxacin) in many pathogenic Gram-positive bacterial species. In E. coli, however, the enzyme is less sensitive to these drugs than is DNA gyrase. The enzyme is heterotetrameric, consisting of two subunits, ParC and ParE, which are homologous to the A and B subunits of DNA gyrase, respectively. We plan to produce a ParC subunit which contains identical residues to GyrA within the so-called quinolone resistance determining region (QRDR) using site-directed mutagenesis. The mutant protein subunit will then be overproduced and purified, and its enzymatic properties and interaction with drugs analysed and compared with those of wild-type topoisomerase IV and gyrase. These experiments will test whether the QRDR is specifically responsible for the differing sensitivities of gyrase and topo IV.

3. Dr. M.X. Caddick (School of Biological Sciences, Donnan Laboratories)

Cloning and characterisation of the Aspergillus nidulans tRNA_CUG

In Sacharomcyces cerevisiae the tRNA_CUG has been shown to have an important role in signalling the levels of available nitrogen within the cell. The CUG anticodon decodes the CAG codon, introducing glutamine into the polypeptide being synthesised. Glutamine has for a long time been regarded as one of the principal molecules which signal nitrogen availability within the cell. The aim of this project will be to clone and sequence the equivalent tRNA coding gene from Aspergillus with the aim of determining if it has a similar role in nitrogen signalling. The project will employ PCR, sequencing, directed mutagenesis and transformation.

4. Dr. D.H. Edgar (Department of Human Anatomy and Cell Biology, Ashton Street)

Investigating the expression profiles of HNF4a 1 and HNF4a 7 in wild-type and basement membrane-deficient embryoid bodies

HNF4 is a transcription factor which plays an essential role in early development, knockout mice displaying defective gastrulation and dying at around embryonic day 8. HNF4 has recently been shown to up-regulate E-cadherin in hepatocytes, leading to the suggestion that it may induce some of the epithelial characteristics of these cells. Previous work in this laboratory using embryonic stem cell-derived embryoid bodies (a tissue culture model system for early embryogenesis) has shown that whereas the mRNA level of HNF4 increases twenty-five fold by day 12 in wild-type embryoid bodies, there is no such increase in their basement membrane-deficient counterparts. The HNF4 gene has at least 7 different splice variants, HNF4a 1 being the most ubiquitously expressed, and HNF4a 7 being the only variant expressed in undifferentiated embryonic stem cells. We intend to identify which splice variant is up-regulated during embryoid body maturation, and to determine the location of HNF4 expression. This project will involve the following techniques; mammalian cell culture, semi-quantitative reverse transcription PCR and whole mount in situ hybridisation.

5. Dr B.F. Flanagan & Dr S.E. Christmas (Dept. of Immunology, Duncan Building)

Expression of killer inhibitory receptor isoforms by human peripheral and decidual natural killer cells.

Natural killer (NK) cells make up around 10% of human peripheral blood lymphocytes but are the major cell type in decidualised endometrium during early pregnancy. These related cell types differ in cell surface phenotype and function. NK cells recognise target cells that lack cell surface expression of major histocompatibility complex (MHC) molecules. This is mediated via killer inhibitory receptors (KIRs) on the NK cell that interact with target cell MHC molecules, leading to the delivery of a negative, or inhibitory signal to the NK cell.

Several groups of families of human KIRs are known, each of which interacts with groups of related alleles of class I HLA molecules. One KIR family is part of the immunoglobulin supergene family (IgSF) and includes p50, p58 and p70 molecules. These closely related molecules have either two (p50, p58) or three (p70) extracellular IgSF domains. The p50 and p58 families differ in their cytoplasmic tails and have activatory or inhibitory functions, respectively.

Recently, cDNA clones of both p58 and p70 molecules have been obtained that completely lack either the first or second IgSF domain. It has been suggested that these result from alternative splicing. In this project, reverse transcriptase PCR using primers spanning the putative spliced domain will be carried out on cDNA from peripheral and decidual NK cells. This will show whether there are any differences in the proportions of transcripts containing different numbers of IgSF domains between the two cell populations. Techniques to be used include lymphocyte purification, RNA extraction, reverse transcription and PCR.

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

Screening a 2-hybrid library to isolate cDNAs coding for proteins that bind to the AF-1 region of the human oestrogen receptor.

The yeast two-hybrid system is a new and powerful method for isolating cDNA coding for the binding partner of a known protein. We are interested in identifying "coactivator" proteins that interact with the AF-1, transcriptional activation region of the human oestrogen receptor (ERa). We have already identified one such protein but there is reason to think that there may be others. The objective of this project will be to screen a pre-constructed cDNA library, from MCF-7 breast cancer cells, for cDNAs coding for proteins that bind to the AF-1 region of ERa. Clones isolated in this way will be characterised by sequencing, followed by database searches for homologous sequences.

7. Prof. B.E.H. Maden and Dr. L. Paraoan (School of Biological Sciences, Life Sciences Building)

Analysis of clones in a cDNA library of the human retinal pigment epithelium (RPE)

The RPE is the layer of cells behind the retina. It carries out essential supportive functions for the retina. Analysis of sequences within a cDNA library ("expressed sequence tags", or ESTs) is a key molecular approach to detailed functional characterization of a given tissue. Our laboratory possesses a sizeable aliquot of a cDNA library of human RPE, originally produced by Dr Dean Bok, University of California, Los Angeles. Dr L Paraoan analyzed some 200 ESTs from the library for her recently completed PhD, and has classified the clones into various functional categories or as "unidentified". There is a need to characterize many more of these clones. The relevant techniques of phagemid excision, DNA sequencing and computer analysis of the sequences are central techniques in modern molecular biology. The succesful student will work alongside two Biochemistry Honours students on this topic. The initial methods will be common to the three projects, but the data obtained will be unique for each student as the projects develop in parallel. Literature references on the RPE will be supplied to intending students.

8. Dr D.J. Moss (Department of Human Anatomy and Cell Biology, Ashton Street)

Identification of alternatively spliced forms of GP55a

GP55a is one member of a family of glycoproteins which is important during axon guidance and cell recognition in the developing nervous system. All three members of the family are attached to the plasma membrane by a lipid anchor - glycosyl phosphatidyl inositol (GPI). CEPU-1 (a second member of the family) has been shown to exist in two alternatively spliced forms, one of which is not membrane linked but secreted. The secreted form is produced as a result of an alternative exon at the 3´-end which removes the hydrophobic tail required for processing of the GPI linkage. The aim of this project is to test whether GP55a has any alternatively spliced forms using PCR and particularly whether there is a secreted form using 3´ RACE. The presence of secreted forms of these glycoproteins will shed light on the possible mechanisms by which this family of proteins may act in vivo.

9. Prof. H.H. Rees (School of Biological Sciences, Life Sciences Building)

Cloning and characterisation of ecdysteroid-metabolising enzymes from an insect species.

Moulting and metamorphosis in insects is regulated by the insect moulting hormones (ecdysteroids). Several processes are involved in regulating the changing hormone concentrations during development, including inactivation reactions. One such process involves ecdysone oxidase - catalyzed formation of 3-dehydroecdysone (3DE), followed by irreversible reduction to the hormonally inactive 3-epiecdysone (3a-hydroxyl) catalysed by 3DE 3a-reductase. A related enzyme, 3DE 3b-reductase also exists that reduces 3DE back to ecdysone (3b -hydroxyl). We are currently cloning and characterising these enzymes from the cotton leafworm, Spodoptera littoralis and exploiting their biotechnological application. This project is concerned with probing for analogous sequences in another important pest species, the tobacco hornworm, Manduca sexta, and undertaking initial stages of cloning the oxidase gene. Initially, cross-hybridization of sequences from S. littoralis in northern blots of mRNA from M. sexta tissues at different developmental times will be undertaken, to establish the similarity of the sequences and provide information on their expression. Further work still concentrate on cloning the ecdysone oxidase gene using a PCR approach and known S. littoralis sequence as a basis. The project will provide training in a range of modern molecular biology techniques.

10. Prof. P. Strike (School of Biological Sciences, Donnan Laboratories)

Characterisation of a putative RAD2 homologue from Aspergillus nidulans.

Using degenerate primers, a chromosomal fragment of Aspergillus nidulans homologous to the S. cerevisiae RAD2 gene has been cloned and sequenced. The sequenced fragment is 2.4kb, and contains most of the coding sequence. However, both 5´ and 3´ sequences are incomplete. In order to complete the sequencing, and to map the cloned gene to a specific location within the genome, a cosmid gene bank of the Aspergillus genome will be screened with the cloned sequence, and cosmids carrying the sequence will be identified. By reference to the Fungal Genome WEB site, the position of the cosmid will be determined on the Aspergillus genome. The cosmid will be digested with restriction enzymes, and fragments hybridising to the cloned RAD2 sequence will be identified. These will subsequently be cloned into appropriate cloning vectors, and their sequence will be determined. In this way, we should arrive at the complete sequence of the gene. If time permits, attempts will be made to clone the corresponding cDNA, and to determine whether expression of the gene is induced by DNA damage.

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

Analaysis of alcohol regulated gene expression in mammalian cells

Ethanol-utilization in Aspergillus nidulans is mediated in part by alcohol dehydrogenase I encoded by the alcA gene. AlcA is under the transcriptional control of the specific activator AlcR. The groups of Professor A.B. Tomsett and Dr M.X. Caddick in this Department have shown that when an expression vector encoding AlcR is introduced into plants, the alcA promoter becomes inducible by ethanol. This system constitutes an exciting new switch for use in plant biotechnology applications. In this project, the student will investigate the use of this system in mammalian cells. A series of plasmids have already been constructed which express– (1) the easily measurable firefly luciferase reporter gene under the control of the alcA promoter; (2) the alcR gene under the control of different mammalian promoters, and (3) a fusion gene of the alcR gene fused to a strong transcriptional activation domain which is placed under the control of a mammalian promoter. The student will introduce these vectors into mammalian cells by transfection and analyse the levels of luciferase reporter gene expression at different times in the presence and absence of ethanol. There will be potential for the student to gain experience of the preparation of new constructs depending on the initial results.

CONTENTS | NEXT PAGE