Patrick Carnegie

Understanding the genesis of the block haplotype structure of the genome is a major challenge. With the completion of the sequencing of the Human Genome and the initiation of the HapMap project the concept that the chromosomes of the mammalian genome are a mosaic, or patchwork, of conserved extended block haplotype sequences is now accepted by the mainstream genomics research community. Ancestral Haplotypes (AHs) can be viewed as a recombined string of smaller Polymorphic Frozen Blocks (PFBs). How have such variant extended DNA sequence tracts emerged in evolution? Here the relevant literature on the problem is reviewed from various fields of molecular and cell biology particularly molecular immunology and comparative and functional genomics. Based on our synthesis we then advance a testable molecular and cellular model. A critical part of the analysis concerns the origin of the strand biased mutation signatures in the transcribed regions of the human and higher primate genome, A-to-G versus T-to-C (ratio ~1.5 fold) and C-to-T versus G-to-A (≥1.5 fold). A comparison and evaluation of the current state of the fields of immunoglobulin Somatic Hypermutation (SHM) and Transcription-Coupled DNA Repair focused on how mutations in newly synthesized RNA might be copied back to DNA thus accounting for some of the genome-wide strand biases (e.g., the A-to-G vs T-to-C component of the strand biased spectrum). We hypothesize that the genesis of PFBs and extended AHs occurs during mutagenic episodes in evolution (e.g., retroviral infections) and that many of the critical DNA sequence diversifying events occur first at the RNA level, e.g., recombination between RNA strings resulting in tandem and dispersed RNA duplications (retroduplications), RNA mutations via adenosine-to-inosine pre-mRNA editing events as well as error prone RNA synthesis. These are then copied back into DNA by a cellular reverse transcription process (also likely to be error-prone) that we have called "reverse transcription-mediated long DNA conversion." Finally we suggest that all these activities and others can be envisaged as being brought physically under the umbrella of special sites in the nucleus involved in transcription known as "transcription factories.".

A partial amino acid sequence of a serine protease from Dermatophilus congolensis allowed the design of oligonucleotide primers that were complemented with additional ones from previously published partial sequences of the gene encoding the enzyme. The polymerase chain reaction (PCR), using combinations of specific and degenerate oligonucleotide primers, allowed the amplification of a 1738-bp internal fragment of the gene, which was finally characterised by inverse PCR as the first full-length sequenced serine protease gene (nasp) from Dermatophilus congolensis. The deduced amino acid sequence of this enzyme, probably involved in the pathogenesis of dermatophilosis, links it to the subtilisin family of proteases. © 2004 Federation of European Microbiological Societies.

Random peptide libraries (RPL) displayed on the surface of filamentous bacteriophages have been extensively used as a tool to map epitopes or to identify antigenic mimics (mimotpoes) of disease-specific monoclonal antibodies or polyclonal sera. These RPL are engineered by the insertion of degenerate oligonucleotides, encoding a specific number of random amino acids, in frame with a bacteriophage gene specifying a virion surface protein. The RPL are constructed by inserting 21 to 36 random nucleotides into the gene for an appropriate coat protein in a phage that is propagated in E. coli. Particularly, clear mimotopes have been obtained with monoclonal antibodies and with a few polyclonal antibodies against viruses. The choice and processing of sera for the selection of the disease related mimotopes and sera to remove mimotopes reacting with ubiquitous antibodies are important in studies of RPL. We have used RPL to select mimotopes of antigens to be used for immunization against dermatophilosis. IgG from sheep protected from dermatophilosis with an enzyme preparation from Dermatophilus congolensis, was used to select peptides displayed on phage in the Ph.D.-7 random peptide library which contains 109 peptides. Phage displaying peptides that were unrelated to the mimotopes associated with protection to dermatophilosis was removed with IgG from sheep that were vaccinated with the enzyme preparation but were not protected. The IgG from the protected sheep, before vaccination, was also used for the negative selection. The selected mimotopes, those with clearly repeating motifs, were chosen and used to immunize sheep. A mixture of four phage mimotopes induced antibody to a recombinant protease from D. congolensis. The immunized sheep recovered more rapidly from the lesions caused by the strains when challenged with two strains of D. congolensis.

The major histocompatibility complex (MHC) region contains several hundred genes in addition to the well-known HLA Class I and II loci. Some of these will explain MHC-disease associations. We have used paralogous and syntenic mapping to short list candidates and conclude that non-HLA genes present on human 6,1 and 19 and murine 17, 4 and 7 regulate immune responses and autoimmune disease. These genes are likely to be components of the early pre-HLA MHC and include tenascin and possibly notch inter alia.

Random peptide libraries (RFL) displayed on phage are being increasingly used to identify mimics of epitopes (mimotopes) in antigens (1). These RPL's typically provide over 109 peptide at a fraction of the cost of chemically synthesized RPL's. The RFL are constructed by inserting 21 to 36 random nudeotides into the gene for an appropriate coat protein in a phage which is propagated in Ecoll. Particularly dear mimotopes have been obtained with monoclonal antibodies and with a few polyclonal antibodies against viruses. Now that good quality RPL are available commercially they will be used in attempts to identify novel autoantigens which react with antibodies present in patients with autoimmune diseases. Once the peptide mimic of the autoantigen is obtained, it can be used to affinity to purify the autoantibody, which can then be used to locate the real autoantigen in a cDNA library from the target organ. It is particularly difficult with human sera to obtain mimotopes which are specific for a disease. Strategies are necessary to increase the chance of selecting a specific peptide where the level of autoantibody in serum could be low and it is likely to be only one of thousands of antibodies with no relevance to the disease. Despite these difficulties interesting results are emerging in rheumatoid arthritis (2), immune thrombocy topenic purpura (3) and multiole sclerosis (4).

Serine proteases are thought to he involved in the initial attack on sheep skin by Dermatophilus congolensis and are obvious antigens for inclusion in a vaccine to prevent lumpy wool disease (dermato-philosis). Degenerate primers were designed after alignment of seven bacterial serine proteases. Inosine was incorporated into the primers at positions of three- and four-base redundancy, and this reduced the complexity of the primer mixtures from several thousand to sixteen different sequences for each primer. The primers were validated by production and sequencing of amplicons from serine protease genes in Bacillus suhtilis and Serratia marcescens. The primers were used with heat-soaked polymerase chain reaction (PCR) to produce amplicons from two D. congolensis strains, AG and MB. In the amplicon codons for arginine, rather than the expected serine, were found where inosine was used for both the first and third positions for a codon in the primer. A search with the deduced amino acid sequences of the amplicons showed significant similarity to a keratinase and other serine proteases from various organisms. Similarity was most apparent around the active site residues and other essential secondary structural elements.

Serum and synovial antibody reactivities of caprine arthritis encephalitis virus (CAEV) infected goats were assessed by Western blotting against purified CAEV antigen and the greatest intensity of reactivity in the serum of arthritic goats was to the gp45 transmembrane protein (TM). The extracytoplasmic domain of the TM gene was cloned into a pGEX vector and expressed in Escherichia coil as a glutathione S transferase fusion protein (GST-TM). This clone was found to be 90.5 and 89.2% homologous to published sequences of CAEV TM gene. Serum of 16 goats naturally infected with CAEV were examined by Western blotting for reactivity to the fusion protein. Antibody reactivity to the GST-TM correlated with clinically detectable arthritis (R = 0.642, P ≤ 0.007). The hypothesis that the immune response to the envelope proteins of the CAEV contributes to the severity of arthritis in goats naturally infected with CAEV via epitope mimicry was tested. Antibodies from 5 CAEV infected goats were affinity purified against the GST-TM fusion protein and tested for cross-reactivity with a series of goat synovial extracts and proteogylcans. No serum antibody response or cross-reactivity of affinity purified antibodies could be detected. Peptides of the CAEV SU that were predicted to be linear epitopes and a similar heat shock protein 83 (HSP) peptide identified by database searching, were synthesized and tested for reactivity in CAEV goats using ELISA, in vitro lymphocyte proliferation and delayed type hypersensitivity (DTH) assays. Peripheral blood lymphocytes from 10 of 17 goats with long term natural CAEV infections proliferated in vitro in response to CAEV and in vivo 3 of 7 CAEV infected goats had a DTH reaction to CAEV antigen. However, none of the peptides elicited significant cell mediated immune responses from CAEV infected goats. No antibody reactivity to the SU peptides or HSP peptide was found. We observed that the antibody reactivity to the CAEV TM protein associated with severity of arthritis however epitope mimicry by the envelope proteins of CAEV is unlikely to be involved.

Epitope mimicry is the theory that an infectious agent such as a virus causes pathological effects via mimicry of host proteins and thus elicits a cross-reactive immune response to host tissues. Weise and Carnegie (1988) found a region of sequence similarity between the pol gene of the Maedi Visna virus (MVV), which induces demyelinating encephalitis in sheep, and myelin basic protein (MBP), which is known to induce experimental allergic encephalitis (EAE) in laboratory animals. In this study, cross-reactions between sera raised in sheep against synthetic peptides of MVV (TGKIPWILLPGR) and 21.5 kDa MBP (SGKVPWLKPGR) were demonstrated using enzyme-linked immunosorbant assay (ELISA) and thin layer chromatography (TLC) immunoprobing. The antibody responses of MVV-infected sheep were investigated using ELISA against the peptides, and MBP protein, immunoprobing of the peptides on TLC plates and Western blotting against MBP. Slight significant reactions to the 21.5 kDa MBP peptide (P < 0.001) and to a lesser extent sheep MBP (P < 0.004) were detected in ELISA. The MBP peptide evoked stronger responses from more sera than the MVV peptide on immunoprobed TLC plates. On the Western blots, eight of the 23 sheep with Visna had serum reactivity to MBP. This slight reaction to MBP in MVV-infected sheep is of interest because of the immune responses to MBP evident in multiple sclerosis and EAE, but its relevance in Visna is limited since no correlation with disease severity was observed. The cell-mediated immune responses of MVV-infected sheep against similar peptides was assessed. The peptides did not stimulate proliferation of peripheral blood lymphocytes of MVV-infected sheep. Since the MVV peptide was not recognised by antibodies or T lymphocytes from MVV-infected and encephalic sheep, it was concluded that epitope mimicry of this 21.5 kDa MBP peptide by the similar MVV pol peptide was not contributing to the immunopathogensis of Visna. The slight antibody response to MBP and the MBP peptide can be attributed to by-stander effects of the immunopathology of MVV-induced encephalitis.

Gamma inulin and Algammulin, two new adjuvants, were examined and compared with alum and Freund's Complete/Incomplete Adjuvant (FCA/FIA), for potentiation of cell-mediated immunity (CMI) and humoral immunity in sheep to a recombinant Taenia ovis antigen. The ability to protect sheep when challenged with live T. ovis eggs was also assessed The results showed that gamma inulin and Algammulin induced a CMI response which was comparable to the FCA/FIA and alum groups and significantly higher than the control saline group. While gamma inulin, Algammulin and alum performed similarly and induced a significantly higher humoral immune response than the saline group, FCA/FIA elicited a much higher humoral immune response. Algammulin did not show the synergistic effect seen in mice and performed similarly to gamma inulin and alum alone. All the adjuvant groups induced significantly higher IgG1 and IgG2 levels than the saline group and they all favoured IgG1 production. When the sheep were challenged with live T. ovis eggs, at 25 weeks after primary immunization, the only group to show significant protection was the one which received FCA/FIA.