Molecular characterisation of behavioural functions in Agrobacterium tumefaciens

Tn5 insertion behavioural mutants of A. tumefaciens C58C(^1) were available. Cloning of the kanamycin resistance gene allowed isolation of Tn5 flanking sequences from a number of the mutants. Flanking sequences from five mutants were used to isolate cosmids, overlapping the Tn5 insertion sites of the mutantsfrom a C58C(^1) library. Two cosmids, pDUB1900 and 1905 have been characterised. pDUB1900 contains the insertion sites of eight motility mutants, with another immediately adjacent. The pDUB1905 insert overlaps sequences interrupted in another three mutants. Behavioural genes in Agrobacterium are clustered together on the chromosome, as in other motile bacteria. Restriction maps of the isolated cosmids show that none of motility mutants analysed was the result of mactivation of pscA or chvB which would lead to an altered behavioural phenotype. Flanking sequences from three of the mutants hybridised to R. meliloti chromosomal DNA, but not to DNA from other motile genera. DNA adjacent to the insertion site of fla-11 hybridised to the insert of pRZ-2, a cosmid containing behavioural genes from R. meliloti. Experiments were undertaken to investigate the occurrence of proteins homologous to the MCP's of enteric bacteria in C58C(^1) DNA hybridisation to oligonucleotide probes showed DNA that could potentially code for MCP-like proteins exists m both Agrobacterium and Rhizobium spp. In addition, an antibody raised against the E. coli MCP Tar cross-reacted with a protein of approximately 60kDa. in C58C(^1). In vivo protein methylation experiments using C58C(^1) resulted in the labelling of a 45kDa protein, whose methylation pattern did not change upon addition of chemostimuli. Possible reasons for the difference in size between the labelled protein and that revealed by the antibodv are discussed

Identification of potential gene coding sequences within large cloned DNA arrays : analysis of zinc finger motif

Imperial Users onl

Global expression mapping of mammalian genomes

he aim of genome projects is to decipher all the information contained within the DNA of an organism and to study the way this information is processed in physiological processes. It is believed that more than 95% of the information content of the mammalian genome is represented in the protein coding sequences that make up only approximately 2% of the DNA sequence. Consequently much effort is being invested in the study of coding sequences in the form of cDNA analysis. This thesis is concerned with the development of a new strategy for a highly parallel approach to analyse entire cDNA libraries. The strategy is based upon generating sufficient sequence information to identify uniquely more than 100,000 cDNA clones by hybridisation with short oligonucleotides, typically 7 - 10 mers. Each oligonucleotide is hybridised to all cDNA clones in parallel and under stringent conditions positively identifies a subset (3 - 10%) of clones. Oligonucleotides are designed in such a way that each will positively identify a different subset of clones and statistical simulations estimate that approximately 200 such hybridisation events are required to identify uniquely upto 100,000 cDNA sequences. Such a fingerprint can be generated from many cDNA libraries constructed from different tissue mRNAs and will not only lead to the identification of most sequecnes expressed from the genome but also indicate the level of expression by determining the number of times any given sequence is represented across different cDNA libraries. A human foetal brain cDNA library has been constructed and 100,000 clones arrayed into microtitre plates and on nylon membranes. All the required technological developments have been carried out successfully and are presented. In excess of 200 oligonucleotide hybridisations have been performed on a subset of 32,000 cDNA clones and 1,000 sequenced control clones. A detailed analysis of the data on the control clones is presented and the implications for cDNA fingerprinting discussed

Optimisation of interphase fluorescence in situ hybridisation for detection of common aneuploidies

The optimisation of a simple, reliable and practical method of interphase FISH which allows prenatal diagnosis of major chromosome aneuploidies using a minimum volume of amniotic fluid sample was the overall objective of this study. When all the probes required were available, the study continued by developing the technique of ratio-mixing FISH for simultaneous detection of the five major chromosome aneuploidies. The technique of five-colour ratio mixing FISH which has been presented here is simple and straightforward, since only two haptenisation and detection systems have been employed to visualise simultaneously five different targets in five distinguishable colours. The steps of denaturation, hybridisation and detection are the same as those used in a uni-colour FISH experiment. The results obtained from hybridisation of an unselected series of 20 uncultured lymphocytes and 27 uncultured amniocytes indicate that the technique is reliable and can be used for simultaneous detection of major chromosome aneuploidies. In order to provide a practical strategy for clinical diagnostic purposes, the use of a three colour ratio-mixing FISH and a dual colour was investigated to visualise the five probe sets on two slides from the same sample. A total unselected series of 45 uncultured lymphocytes and 60 uncultured aminocytes were hybridised with different probe combinations using three colour ratio-mixing FISH. The results indicate that the major chromosome aneuploidies can be simply and reliably identified on two slides from the same sample, using a three colour ratio-mixing FISH to detect the chromosomes X, Y and 21 and a dual colour to detect chromosomes 13 and 18. The failure rate was reduced to 4 per cent using this approach

Physical, Transcriptional and Comparative Mapping on the Human X Chromosome

Progress in the study of the human genome has resulted in the production of a complete clone map and associated 'working draft' sequence. This will underpin the completion of the sequence itself, the annotation of genes and other features, and application of this new found knowledge. This thesis focuses on the evolving methods to determine the map, and to use the emerging sequence for the study of genes, incorporating new studies of other genomes to enhance progress in understanding and interpretation to biology and medicine. The success of the endeavours is necessarily accompanied by the development and evolution of new technologies and by critical assessment of the progress in acquiring knowledge of the genomic information. Evolution of mapping technologies included the development of the larger insert bacterial cloning systems (PACs) and (BACs), and an increase in available landmarks both from Y AC maps and RH maps. The work described in chapter 3, followed this evolution and was applied to construct a 6 Mb sequence-ready bacterial clone contig map in Xq22. A minimum set of clones was chosen for genomic sequencing. The resulting sequence map was compared to previously published maps and analysed both for common repeats, and previously unidentified low copy repeats. The availability of the emerging sequence of the human genome provided a resource for identification of the features encoded within. In chapter 4, the sequence of a 7 Mb region in Xq23-24 was analysed for the presence of genes using a combination of sequence similarity searches against both protein and DNA databases, and ab initio gene prediction. Predicted genes were confirmed where possible, by generating novel cDNA sequence. The region contained 33 confirmed genes (of which 14 were confirmed during this study), 11 predicted genes and 20 pseudogenes. Comparative genome sequence analysis is a powerful method both for aiding human gene identification and identifying other features encoded within the human genome such as regulatory elements. Comparing the genomes of two or more species also provides insights into the evolution of the species since the divergence from a common ancestor. Sequence from a 1 Mb region in human Xq24 was compared in two other species, mouse and zebrafish. In chapter 5, bacterial clone contigs for sequencing were constructed in the mouse by designing mouse-specific STSs orthologous to human sequence for clone isolation. In chapter 6, bacterial clone contigs for sequencing were constructed in zebrafish using STS from exons of human genes to identify zebrafish BAC clones by reduced stringency hybridisation. Comparative analysis of the region showed that humans and mice are more highly conserved than humans and zebrafish, in terms of gene content and organisation. A combination of comparative sequence analysis tools identified 14 novel potential conserved sequences between human and mouse, one of which was also conserved in zebrafish

Genes of the ovine major histocompatibility complex class II region

The major histocompatibility complex (MHC) is a multi-gene family encoding proteins which play important roles in the immune response to antigenic challenge. Three distinct regions designated class 1, 11, and 111 have been defined in the MHC of mouse and man. This thesis focuses on the genes of the class 11 region of the sheep. The products of the class 11 alpha (A) and beta (B) genes are heterodimeric glycoproteins whose physiological function is to present exogenous peptides to helper T cells. The recognition of MHC class 11/peptide complexes by the T cell receptor signals the release of a cytokine cascade resulting in T and B cell proliferation, macrophage activation and B cell differentiation with the production of increased amounts of pathogen-specific antibody.Much is known about the detailed structure and function of the MHC of man and mouse. However, when this project began little was known about the detailed structure of the MHC of the ungulates, the economically important group of animals which contains cattle, pigs, horses and sheep. As part of a study investigating fundamental cellular immunology in the sheep, this thesis describes the characterisation and expression of the genes of the sheep MHC class 11 region.Cosmid libraries prepared from DNA from three unrelated sheep were screened with probes from the DP, DQ and DR sub-regions of the human and mouse MHC class 11 regions. Cosmids were used because they facilitate the cloning of relatively large genomic inserts. Restriction maps of the cosmids have been produced showing that some of the clones overlapped. The MHC A and B genes within the clones have been sequenced and assigned to a specific sub-type. Functional genes have been identified by the reaction of their products with anti-sheep class 11 monoclonal antibodies following DNA-mediated transfection into the mouse L cell, a fibroblast cell line which does not express endogenous mouse class 11 genes. Transcription of some of the genes has been demonstrated by Northern blots and reverse transcription polymerase chain reaction.A restriction map of the sheep class 11 DQ sub-region has been constructed and shown to contain two distinct DQA loci with associated DQB genes. The DQ1 A/B gene pair was expressed in the mouse L cell. The sheep class 11 DQ1 product at the cell surface reacted with a sub-set of the available anti-sheep class 11 monoclonal antibodies. The DQ2 genes were transcribed and some evidence for their cell surface expression was obtained, although this was not formally proved.A previous study demonstrated the expression of a putative sheep DRA gene when co-transfected with a sequenced DRB gene. The sequence of the sheep DRA gene is described here together with sequence data from a number of DRB genes or pseudogenes which show that, depending on haplotype, the sheep DR sub-region may contain up to five DRB genes.The cloning and sequencing of ruminant orthologues of the HLA-DNA and -DOB genes is described for the first time. Although evidence was obtained for the transcription of the sheep DNA gene, its DOB gene partner is transcriptionally silent.A class 11 locus designated DY which is not found in mouse or man is described from the sheep MHC. Cloning and sequencing has shown that it contains a class 11 A/B gene pair like that of the expressed DQ1 locus, however, it appears to be transcriptionally silent

A molecular analysis of chromosome 11 band q23 in haematopoietic malignancy

Rearrangements of chromosome 11 at band q23 have been observed in haematopoietic malignancies. These rearrangements are mainly translocations and involve other chromosomes, the most common being t(4;11)(q21;q23), t(9;11)(p22;q23) and t(11;19)(q23;p13). The molecular basis of these translocations is as yet unknown. The aim of this thesis is to characterise the 11q23 region at the molecular level and to study leukaemic patients with 11q23 involvement. Three main approaches have been used, namely mapping, rearrangement studies and isolation of novel probes. Mapping of various genes localised to 11q23 in relation to the leukaemic translocation breakpoints have been carried out using somatic cell hybrids. By studying different translocation breakpoints, relative positions of the breakpoints and genes in this region were determined. A novel method of gene mapping by the enzymatic amplification of flow-sorted chromosomes is described. This was used to map the c-ets-1, Thy-1 and CD3 genes distal to the breakpoint of the constitutional t(11;22)(q23;q11) translocation. Long range rearrangement studies of the Hq23 region by pulsed field gel electrophoresis are also described. Emphasis was on the c-ets-1, Thy-1 and CD3 genes. CD3G, a member of the CD3 gene complex, was found to be rearranged in the t(4;11) translocation and positioned within 200kb of the translocation breakpoint. In an attempt to clone the t(4;11) translocation breakpoint, yeast artificial chromosome clones positive for CD3 were isolated. They did not encompass the breakpoint and were localized approximately 100kb proximal to it. End-cloning of one of the clones were performed in order to isolate an overlapping telomeric yeast artificial chromosome clone. The pulsed field gel and yeast artificial chromosome studies lead to a more precise localisation of the t(4;11) translocation breakpoint. A novel technique of Alu-PCR was investigated and used on flow-sorted chromosomes for the generation of probes specific for the Hq23-q24 region. Eight novel probes to this region were generated and mapped in relation to the t(4;11) and the Ewing's sarcoma t(11;22) translocation breakpoints. The studies described have contributed to the knowledge of the molecular organization of the 11q23 region. This is important for the understanding of the molecular basis of 11q23 abnormalities, and their association with haematopoietic malignancy