Regeneration of injured CNS tracts poses a significant clinical challenge due to the multifactorial nature of the degenerative processes that ensues and technically complex delivery of therapeutic molecules. To circumvent these problems, highly disabled Herpes Simplex 1 (HSV1) based vectors were used to deliver NT3, BDNF and CNTF to the injured rat CNS. The ability of these vectors to promote the regeneration of corticospinal (CST), rubrospinal (RST) and optic nerve tracts was evaluated. Transgene expressing constructs were based on the backbone HSVl.pR19CMV that carries a combination of essential and non-essential gene deletions. These render it replication incompetent in vivo whilst allowing it to maintain transgene expression in the long term via expression cassettes that employ HSV1 latency associated transcript promoters. Via retrograde transport, a single spinal cord inoculation results in widespread, stable transgene expression in neurons throughout the CNS, from spinal cord to hypothalamus. In spinal cord regeneration experiments, NT3-expressing vector alone failed to promote regeneration in the chronically injured CST. In combination with transplanted embryonic day-14 spinal cord segments however, it led to enhanced CST sprouting and axon elongation up to 600ujn within the lesion site. In the chronically injured RST, BDNF and CNTF-expressing vectors produced no significant regeneration. In the optic nerve crush model however, both robust transduction and substantial axonal regeneration resulted from inoculation of retinal ganglion cell (RGC) targets. A combination of CNTF, BDNF, bFGF and Neurturin-expressing vectors induced regeneration of injured RGC axons in optic nerve, up to 1000 pjn distal to the crush site. Further vector development led to less-disabled vectors that supported strong transgene expression and targeted spinal cord, RGCs and even CST neurons, with high efficiency. By counteracting the host's immune response, minimally disabled ILlO-expressing vectors supported long-term expression of transgenes in DRG neurons following footpad inoculation. HSV1-based vectors are therefore powerful and versatile gene therapy tools for CNS regeneration.

The fission yeast centromere is packaged as transcriptionally silent heterochromatin which serves as a platform for kinetochore assembly. The centromere consists of two distinct domains; the outer repeats and the central core. It has been shown previously that these regions associate with distinct sets of proteins, for example, the fission yeast homologue of CENP-A, Cnp1p, is present at the central core, together with Mis6 and Mis12, whilst the heterochromatin protein Swi6 associates with the outer repeats. Marker genes placed in the centromere are transcriptionally silenced. This feature of the fission yeast centromere was utilised to screen for potential kinetochore components or regulators. Mutants with alleviated silencing at the central core were isolated and seven complementation groups identified; sim1 to 7, for silencing in the middle of the centromere. All the sim mutants display chromosome segregation defects and elevated rates of loss of a non-essential minichromosome. This study describes the ongoing characterisation of <i>sims</i> <i>1,6</i> and <i>7</i>. GFP tagged Sim1 associates with the central core of the centromere suggesting that Sim1 is also a novel kinetochore protein. Our working model is that Sim1 may be required for the assembly of Cnp1p chromatin. The <i>sim6</i> mutant is unusual as it alleviates silencing at both the central core and outer repeat regions. In the <i>sim7</i> mutant at the restrictive temperature, Cnp1, a crucial component of the centromere, shows greatly reduced localisation.

The spindle checkpoint monitors the bipolar attachment of sister chromatids to the mitotic spindle. A screen for Saccharomyces cerevisiae mutants sensitive to the microtubule depolymerising drug, benomyl, isolated 6 components of the spindle checkpoint; <i>MAD1</i>, <i>MAD2, MAD3, BUB1, BUB2, BUB3. </i>None of these components are essential for growth under normal conditions. <i>MPS1</i>, which encodes an essential kinase involved in spindle pole duplication, was also found to have a dual role in the spindle checkpoint. This work identifies the Saccharomyces cerevisiae open reading frame, YJL013c, as encoding for the <i>MAD3</i> gene. Mutations in this ORF are benomyl sensitive and divide faster on benomyl plates in a manner similar to that of previously characterised spindle checkpoint components. Mad3p localises to the nucleus when overexpressed and its expression levels are not cell cycle regulated. Mad3p shares two regions of homology with Bub1p; region I and region II. This work shows that region I of Mad3p is required for association with the target of the spindle checkpoint, Cdc20p. Region II is required for binding to Bub3p. A two hybrid assay using Mad3p as bait identified 8 possible interactors one of which is a regulatory subunit of the yeast protein phosphatase, Glc7p, a protein recently implicated in chromosome segregation. The loss or gain of all or part of a chromosome could lead to or propagate a cancerous cell. The mitotic checkpoint prevents the unequal segregation of sister chromatids and so protects against the loss or gain of chromosomes. Using an assay developed by Hieter <i>et al </i>the chromosome loss rates of spindle checkpoint mutants were calculated. Surprisingly, mutants in the different components had significantly different loss rats suggesting additional roles for Bub1p and Bub3p in the efficient segregation of sister chromatids. Point mutants in <i>mad3 </i>and <i>bub1 </i>were analysed in further detail and the effects of overexpression of checkpoint components was also analysed.

No description available.

Fission yeast has three centromeres and their structure resembles that of metazoans. They are composed of a central domain that constitutes the kinetochore surrounded by outer repeats packaged in silent chromatin. Silent chromatin is characterized by underacetylated histone H3 and H4 and methylated histone H3 on lysine 9 by the SET-domain methyltransferase Clr4. These specific marks allow the binding of chromodomain proteins such as Swi6 and subsequent recruitment of cohesin. Non-coding transcripts have been detected from both strands of the outer repeats. The RNAi pathway processes these transcripts and the resulting siRNAs direct silent chromatin formation over these repeats causing transcriptional repression of an inserted marker gene. In order to find new components involved in the assembly of a functional kinetochore, the csp temperature sensitive mutants, previously shown to alleviate silencing of marker genes inserted at the outer repeats of centromere, were analyzed. Specifically components involved in RNA transcription and processing such as a subunit of RNA pol II (Csp3/Rpb7) and splicing factors (Csp4/Cwf10 and Csp5/Prp39) were required for the formation of silent chromatin by affecting the production of centromeric transcripts. Fission yeast silent chromatin formation is dependent on methylation of histone H3 on lysine 9. In order to identify additional factors that might directly affect modification of histones tails, four genes encoding for SET domain protein which may represent putative histone methyltransferase were deleted (<i>set3, set6, set7</i> and <i>set9</i>). No affect on silent chromatin formation was detected. In a collaborative study Set9 was found to specifically methylate lysine 20 on histone H4 and to be involved in the general genome integrity. Methylation on lysine 20 of histone H4 together with phosphorylation of serine 129 of histone 1-12A is necessary to maintain the DNA damage checkpoint activity through the recruitment of Cbr2 at the sites of DNA damage.

The I factor is a Long Interspersed Nuclear Element of <i>Drosophila melanogaster. </i>Transposition of the I factor occurs via an RNA intermediate and is restricted to the germline of hybrid females that have received one half of their chromosome complement from a parent devoid of active I factors. If the I factor devoid chromosomes are of maternal origin, I factor transposition is associated with lethality of the F2 embryos and high frequency of lethal mutations in the F3 generation. In contrast to its restricted pattern of transposition, I factor expression is detectable in somatic issues of all flies that carry active I factors. Accumulation of I factor copies over the generations is associated with cessation of transposition and a decrease in expression in the female germline. As previously shown, the I factor 5' Untranslated Transcribed Region (UTR) can impose on a reporter gene a pattern of expression similar to that observed for complete I factors, i.e. high levels of expression in the germline of permissive females and lower yet significant levels in other tissues and in males. Furthermore, the I factor 5'UTR is sensitive to the presence of complete I factors and multiple copies of this sequence significantly reduce of the F2 lethality associated with I factor transposition. This work investigates the role of the 5' UTR in determining parental effects and in mediating processes of Polycomb-dependent I factor silencing. In addition, the possibility that the high Adenine-Thymine content of the 5'UTR might impose on this sequence an unusual conformation that could be responsible for some of the biological properties of the I factor is also investigated.

ERCC1-XPF is a structure specific endonuclease that acts in nucleotide excision repair (NER), and is also involved in repair of DNA interstrand crosslinks and removal of non-homologous 3’ tails during recombination. The ERCC1 null mouse is runted and dies of liver failure before weaning, at about 3 weeks old. By three weeks after birth, some ERCC1 null hepatocytes have developed polyploidy. The liver phenotype of the ERCC1 null mouse is not observed in other NER deficiency models, which indicates that the loss of non-NER roles of ERCC1 are responsible for the liver phenotype. Development of liver polyploidy in the ERCC1 null hepatocytes has been tracked from before birth to death using FACS analysis and nuclear area distribution measurements. This has been compared to hepatocyte polyploidy development in wild type mice. The ERCC1 null mice undergo an accelerated development of polyploidy compared to age matched wild type mice. The cell cycle status of ERCC1 null hepatocytes has been investigated using a fluorescence immunostaining assay for centromeres. This reveals that the enlarged ERCC1 null hepatocytes are primarily in G2/M. Enlarged old wild type hepatocytes are also primarily in G2/M. However, normal ERCC1 null, old wild type and young wild type hepatocytes are primarily in G1/G0. The premature polyploidy liver phenotype of the ERCC1 null mouse is very similar to that seen in a transgenic mouse that overexpresses p21 in liver. Immunohistochemistry shows that p21 protein levels are increased in ERCC1 null liver. The increase in p21 protein levels was seen in old wild type livers when measured by immunohistochemistry and Northern blot analysis. A p53-ERCC1 double null mouse was made to test if the ERCC1 phenotype was dependent on p53. ERCC1-p53 null hepatocytes have the same accelerated polyploidy and increased p21 levels as ERCC1 null hepatocytes.

The phenotypic effects of a novel X-chromosome linked mutation were studied in the offspring of male mice treated with the mutagen ethyl nitrosourea (ENU). Studies of X-chromosome inactivation patterns in females heterozygous for the mutation (ENU/+) has delineated four distinct cell lineages affected by the mutation, namely, B, pre-B and T lymphoctyes, erythrocytes and skeletal myocytes. The penetrance of the mutation depended on the age of mice, the cell lineage affected by the mutation and the stage of maturation of the cell lineage. Studies of B cells in females heterozygous for the mutation and the X-linked mutation, xid that affects B but not pre-B cells, indicated that the two mutations were not allelic. Factors influencing differences in X-chromosome inactivation between cells and hybrids and their relationship to alleles of the X-chromosome controlling element (Xce) in the ENU-mutant and parental strains were studied. It was not possible to identify the effects of the mutation on the immune system in functional terms either by flow cytometric analysis of leukocytes or after sensitisation to oxazolone. Results imply the mutation renders the affected cell lineages susceptible to competition with normal cells in the heterozygote, rather than there being any fundamental defect in cell function and that the mutation may be in a gene encoding a component of the cell cycle or controlling a maturation step.

In this project, a fast accurate global pairwise alignment of noncoding DNA sequences, MCALIGN2, is developed based on explicit models of indel evolution. A pair-hidden-Markov-Model (pair-HMM) of seven states and a golden-Section-Search algorithm are employed in this method to search for the most probable alignment between two homologous sequences. This method is then used to align and analyze noncoding DNA sequences in <i>Drosophila</i>. Comparative genomic analysis in this project shows that INE-1 elements, one of the most abundant TEs in <i>Drosophila</i>, along with sites within short introns and fourfold degenerate sites are the fastest evolving nucleotides in the genomes of <i>Drosophila</i> <i>melanogaster</i>, <i>D. simulans</i> and <i>D. sechellia</i>. Fourfold sites tend to be evolving (relatively) slightly more slowly than the other two classes of nucleotides, probably due to selection acting on protein translation efficiency. The observed substitution rate in these fastest evolving sites appears to be strongly influenced by the recombinational environment in which they are located. This rate may be influenced by several factors including ancestral polymorphisms, variation in mutation rate, natural selection and random genetic drift. The relative importance of these factors varies depending on the time since speciation. This project also fully investigates the distribution and rate of evolution of three major TE classes (LTR, non-LTR retrotransposons and DNA transposons) in the <i>Drosophila</i> euchromatic genomeusing a gene-centric approach. The study demonstrates that LTR elements outnumber non-LTR and DNA elements in all intergenic, intronic and exonic regions, and LTR elements also show relatively lower mean divergences than the other two classes between <i>D. melanogaster</i> and <i>D. yakuba</i>. The findings suggest that some TEs, rather than being “junk” and “selfish”, may be conserved between species, and therefore, play vital roles in gene regulation and host genome evolution.

Research predominantly on malaria parasites has been directed towards characterising, from a vaccine development viewpoint, surface antigens and their genes. Attempts at understanding the molecular biology of these amazing intracellular parasites has been much neglected. This thesis has focussed on understanding the molecular biology of two novel biological features in <i>Plasmodium falciparum</i>. Plasmodia rely on scavenging preformed host purines by the salvage pathway for nucleic acid synthesis. A pivotal enzyme in this pathway is hypoxanthine-guanine phosphoribosyltransferase (HGPRTase). The gene is present in a single copy in the <i>P.falciparum</i> genome and is located on a 1600 kb chromosome size molecule. The chromosomal copy of the HGPRTase gene has been cloned from the isolate K1 and also the cloned line HB3. DNA sequence analysis of the genes has been greatly hampered by their profound instability in conventional host-vector cloning and sequencing systems. However, some of these problems have now been surmounted and most of the gene sequenced. The picture that has emerged is fascinating. In all mammalian species examined, including man, the HGPRTase protein is encoded by less than 1kb of DNA. However, the genes span over a massive distance of 30-40 kb, the coding sequence is interrupted by eight intervening sequences which are present at precisely identical positions in all mammalian species. The organisation of the gene in <i>P.falciparum</i>, in contrast, is quite remarkable. It is contained within 2 kb of DNA and the coding sequence is interrupted by probably just a single intron at a position different from the introns in the mammalian gene. The coding sequence between different mammalian HGPRTase genes display over 90% sequence identity to each other, but only 70% identity to <i>P.falciparum</i>, most significantly the putative twenty residue catalytic domain of the protein is conserved. The monoclonal antibody, McAb 7.7, recognises a 34 kD parasite encoded protein exp-2 which has been characterised at the molecular level. The protein is synthesised as a 35 kD primary translation product which is presumably processed and exported outwith the parasite to reside in the parasitophorous vacuolar membrane and also vesicle-like structures in the erythrocyte cytoplasm. There is indirect evidence to suggest that vesicle-like structures are involved in trans-erythrocyte transport. The protein exp-2 has been affinity purified from differential detergent extracts. A number of proteins have been observed to reproducibly copurify with exp-2 of which one has been identified to be the erythrocyte anion-transporter band III. exp-2 has been convincingly demonstrated to be immunogenic in man. With a view to cloning the gene in expression libraries polyclonal antisera directed against exp-2 have been successfully raised. In conclusion the novel results that have emerged from this thesis are not only of great medical importance, but have widespread biological and evolutionary implications.