Cloning, characterization and expression analysis of porcine microRNAs

Background: MicroRNAs (miRNAs) are small ~22-nt regulatory RNAs that can silence target genes, by blocking their protein production or degrading the mRNAs. Pig is an important animal in the agriculture industry because of its utility in the meat production. Besides, pig has tremendous biomedical importance as a model organism because of its closer proximity to humans than the mouse model. Several hundreds of miRNAs have been identified from mammals, humans, mice and rats, but little is known about the miRNA component in the pig genome. Here, we adopted an experimental approach to identify conserved and unique miRNAs and characterize their expression patterns in diverse tissues of pig.Results: By sequencing a small RNA library generated using pooled RNA from the pig heart, liver and thymus; we identified a total of 120 conserved miRNA homologs in pig. Expression analysis of conserved miRNAs in 14 different tissue types revealed heart-specific expression of miR-499 and miR-208 and liver-specific expression of miR-122. Additionally, miR-1 and miR-133 in the heart, miR-181a and miR-142-3p in the thymus, miR-194 in the liver, and miR-143 in the stomach showed the highest levels of expression. miR-22, miR-26b, miR-29c and miR-30c showed ubiquitous expression in diverse tissues. The expression patterns of pig-specific miRNAs also varied among the tissues examined.Conclusion: Identification of 120 miRNAs and determination of the spatial expression patterns of a sub-set of these in the pig is a valuable resource for molecular biologists, breeders, and biomedical investigators interested in post-transcriptional gene regulation in pig and in related mammals, including humans.Peer reviewedBiochemistry and Molecular BiologyAnimal Scienc

Diverse reaction behaviors of artificial ubiquinones in mitochondrial respiratory complex I

The ubiquinone (UQ) reduction step catalyzed by NADH-UQ oxidoreductase (mitochondrial respiratory complex I) is key to triggering proton translocation across the inner mitochondrial membrane. Structural studies have identified a long, narrow, UQ-accessing tunnel within the enzyme. We previously demonstrated that synthetic oversized UQs, which are unlikely to transit this narrow tunnel, are catalytically reduced by native complex I embedded in submitochondrial particles but not by the isolated enzyme. To explain this contradiction, we hypothesized that access of oversized UQs to the reaction site is obstructed in the isolated enzyme because their access route is altered following detergent solubilization from the inner mitochondrial membrane. In the present study, we investigated this using two pairs of photoreactive UQs (pUQ(m-1)/pUQ(p-1) and pUQ(m-2)/pUQ(p-2)), with each pair having the same chemical properties except for a similar to 1.0 angstrom difference in side-chain widths. Despite this subtle difference, reduction of the wider pUQs by the isolated complex was significantly slower than of the narrower pUQs, but both were similarly reduced by the native enzyme. In addition, photoaffinity-labeling experiments using the four [I-125]pUQs demonstrated that their side chains predominantly label the ND1 subunit with both enzymes but at different regions around the tunnel. Finally, we show that the suppressive effects of different types of inhibitors on the labeling significantly changed depending on [I-125]pUQs used, indicating that [I-125]pUQs and these inhibitors do not necessarily share a common binding cavity. Altogether, we conclude that the reaction behaviors of pUQs cannot be simply explained by the canonical UQ tunnel model.Peer reviewe

The diversity and biotechnological application of marine microbes producing omega-3 fatty acids

PhD ThesisOmega-3 polyunsaturated fatty acids (PUFAs), such as eicosapentaenoic acid (EPA, 20:5ω3) and docosahexaenoic acid (DHA, 22:6ω3) play a role in the modulation and prevention of human diseases, in particular cardiovascular diseases. The omega-3 family is found mainly in fish, of which wild stocks are becoming limited. Therefore production of omega-3 PUFAs by marine microbes may provide an alternative source of such componds. The diversity of marine microbes was studied using 16S/18S rRNA gene sequencing of different marine biota with 1500 bacterial strains and 50 microalgae were isolated. The diversity of culturalbe microorganisms inhabiting Mid-Atlantic Ridge (MAR) non-vent sediments was examined for the first time in this area with findings of high diversily of Gram-positive strains, good production of squalene by an unusual strain Bacillus sp. MAR089 and the highest yield of EPA ever recovered from strain Shewanella sp. MAR441. North Sea sponge associated Vibrio sp. strain NSP560 produced considerable levels of EPA, whereas no PUFAs producers were found from tropic Caribbean marine sponge associated bacteria. Photobacterium sp. strain MA665, isolated from the coast of North Sea, was described for the first time of this genus and could be cultured easily under atmospheric conditions with appreciable levels of EPA -1 with up to 25 % of total fatty acids (TFA) (or 10.6 mg g in dried cell). Strain MAR441 was identified as a new species, designated as Shewanella dovemarina sp. T nov. (Type strain MAR441 ). The level of EPA production of strain MAR441 has been optimized by varying fermentation conditions, and 15-25 % EPA of TFA (or 17-30 mg -1 g in dried cell) could be achieved with 40 % improvement. In order to understand the PUFAs biosynthesis pathways and better predict the maximum EPA production, EPA gene clusters (pfaA, pfaB, pfaC, pfaD and pfaE) were cloned and sequenced from the following three species Shewanella, Vibrio and Photobacterium. Great potential was found in marine algae Phaeodactylum tricornutum strain M7 with lipid content of 10 % in dry wt biomass and 22-30 % EPA of TFA when it was cultured outdoors under local weather conditions in UK. Under anaerobic conditions, strain MAR441 contained less -2 amount of EPA and produced electricity of ~100 mW m . Enhanced electricity production using artificial consortia of estuarine bacteria grown as biofilms was -2 observed with power generation of ~200 mW m . In conclusion, bacteria taxonomic resolution based on complete cell fatty acid composition is possible and marine microbes with considerable production of EPA could be potential candidates for industrial production of PUFAs

The molecular pathology of metastasis with reference to the SL 12 murine lymphoma model

The SL 12 murine T-lymphoma cell line established by MacLeod et. al., has two sister clones SL 12.3 and SL 12.4. These two cell lines with similar amount of DNA have been shown to have different surface antigens, tumourigenicity and pattern of metastasis. This was thought to be due to differential expression of genes, either repressed or activated, by the two cell lines. This study has re-established the SL 12.3 and SL 12.4 cell lines, hi tissue culture, both cell lines grow well in suspension in modified Dulbecco medium (DMEM). SL 12.4 cells grow in clumps whereas SL 12.3 cells grow diffusely in vitro. Growth curves of SL 12.3 and SL 12.4 cell lines over five days show similar characteristics but with wide variation in SL 12.4 cell counts resulting in large standard error of mean. Clumping of SL 12.4 cells may be responsible for this variation due to cell counting difficulties. Therefore, although growth curves between the two cell lines are shown to be similar', it is not possible to conclude that the growth rates and cell doubling times are identical. In macroscopic studies, mice injected with 106 SL 12.3 cells via the tail vein deteriorate over a median of 17 days (range 11 to 24 days). All mice (100%) have huge hepatosplenomegaly. On the other hand, only one mouse (9.1%) injected with similar number of SL 12.4 cells deteriorated with metastases at 22 days. When the inoculum of SL 12.4 cells was increased to 107 cells, 78.5% of mice developed metastasis over a median of 48 days (range 31-56 days). Metastases were seen in ovaries, kidneys and lymph nodes macroscopically in contrast to SL 12.3 cells. Microscopically, it was found that both SL 12.3 and SL 12.4 cells were similarly distributed in various organs that were not involved macroscopically indicating that the observed differing pattern of metastasis is more likely to be due to differential 'in-organ' growth rates rather than cells homing to a particular site after injection. This hypothesis was further investigated by in vivo cellular tracking studies using 125IUDR labelled cells. The mean number of SL 12.4 cells per gram of wet tissue was found to be higher in ovarian tissues at 60 minutes post injection but after 3 hours post injection, both cell lines showed no significant differences in tissue distribution. This again suggested no evidence of a 'homing' phenomenon. The percentage of incorporation was poorly reproducible and this markedly reduced the sensitivity of the study producing wide variation making the data interpretation difficult and therefore, conclusion based in the data non-valid. Molecular characterisation of the two cell lines was performed using the technique of Differential Display to further investigate the possibility of differential gene expression or repression that could be responsible for the differing in vivo growth rates. (Abstract shortened by ProQuest.)

An investigation into the role of autoimmunity in vitiligo

Vitiligo is an acquired depigmenting disorder characterised by the loss of functional melanocytes from the cutaneous epidermis. A role for autoimmunity is supported by the presence of circulating antibodies and T lymphocytes which react against melanocyte antigens in patients with vitiligo. The identification and characterisation of these autoantigens will improve understanding of the immune response in vitiligo, and may allow development of better therapies and diagnostic tools. Candidate immunoregulatory genes may predispose to vitiligo. However, this study failed to find an association between vitiligo and a polymorphism of the cytotoxic T lymphocyte antigen-4 (CTLA-4), although the polymorphism increases the likelihood of autoimmune endocrinopathy patients developing vitiligo. The epitopes on melanocyte-specific antigens tyrosinase and Pmel17 which are recognised by antibodies in vitiligo patient sera were identified by molecular mapping. Multiple regions of tyrosinase and at least two domains on PmeI17 were identified as B cell epitopes. Sequence analysis revealed that the tyrosinase epitopes are likely to be cross-reactive with tyrosinase-related proteins but that the antibody response to Pmel17 is distinct. Antibody reactivity to a melanocyte protein, MelanA, targeted by a cellular immune response in vitiligo and melanoma was investigated by immunoblotting and radioimmunoassay. No MelanA-specific antibodies were isolated suggesting that either it is not a target of the humoral immune response in vitiligo, or that antibody reactivity was not detectable by the methods used. To identify novel vitiligo autoantigens, a melanoma cDNA expression library was constructed in a phage-display cloning system and immunoscreened with vitiligo patient IgG. Several possible autoantigens were enriched by this technique, including proteins previously characterised as autoantigens in other disorders. Additionally, humoral reactivity was identified to a protein with a possible role in pigmentation, the melanin-concentrating hormone receptor 1 (MCHR1). MCHR1-specific antibodies were detected in 16.4% (9/55) of vitiligo patients but not in other diseases or healthy control subjects. The study demonstrates the usefulness of phage-display for further autoantigen identification in vitiligo

X-ray diffraction of a gram-negative bacterial membrane mimetic

This thesis applies x-ray diffraction to measure he membrane structure of lipopolysaccharides and to develop a better model of a LPS bacterial melilbrane that can be used for biophysical research on antibiotics that attack cell membranes. \iVe ha'e Inodified the Physics department x-ray machine for use 3.'3 a thin film diffractometer, and have lesigned a new temperature and relative humidity controlled sample cell.\Ve tested the sample eel: by measuring the one-dimensional electron density profiles of bilayers of pope with 0%, 1%, 1G :VcJ, and 100% by weight lipo-polysaccharide from Pse'udo'lTwna aeTuginosa. Background VVe now know that traditional p,ntibiotics ,I,re losing their effectiveness against ever-evolving bacteria. This is because traditional antibiotic: work against specific targets within the bacterial cell, and with genetic mutations over time, themtibiotic no longer works. One possible solution are antimicrobial peptides. These are short proteins that are part of the immune systems of many animals, and some of them attack bacteria directly at the membrane of the cell, causing the bacterium to rupture and die. Since the membranes of most bacteria share common structural features, and these featuret, are unlikely to evolve very much, these peptides should effectively kill many types of bacteria wi Lhout much evolved resistance. But why do these peptides kill bacterial cel: '3 , but not the cells of the host animal? For gramnegative bacteria, the most likely reason is that t Ileir outer membrane is made of lipopolysaccharides (LPS), which is very different from an animal :;ell membrane. Up to now, what we knovv about how these peptides work was likely done with r !10spholipid models of animal cell membranes, and not with the more complex lipopolysa,echaricies, If we want to make better pepticies, ones that we can use to fight all types of infection, we need a more accurate molecular picture of how they \vork. This will hopefully be one step forward to the ( esign of better treatments for bacterial infections

The accuracy of filtered basis functions for the first principles modelling of defects in semiconductors

PhD ThesisThis work presents the results of calculations using filtered basis functions performed with the ab initio modelling program (AIMPRO). The filtration method works by projecting out (filtering) components of a reference basis function that are not required for a description of the occupied states, thereby producing functions that are localised in energy. This leads to a significant reduction in the number of functions that are needed. It is demonstrated that when studying diamond, silicon and defects in these materials, the use of filtered basis sets using just four basis functions per atom can achieve a comparable accuracy to conventional calculations that use 30–40 basis functions. This enables a massive increase in computational efficiency that could have far reaching consequences for first principles modelling calculations. The accuracy of the filtration method is first examined for the bulk materials diamond and silicon, in which the energy, lattice constant, bulk modulus and band structure are studied. It is shown that the filtration approximation applied with an efficient spatial cut-off is able to reproduce current calculated values for these to a very high degree of accuracy. A study of the energies of various reconstructed surfaces in diamond and silicon is then presented. It is first demonstrated that the AIMPRO modelling software without filtration reproduces previous published values of surface energies to within about 100 meV per 1x1 surface cell, with this difference being related to different choices for the pseudo-potential and other details of the calculation. It is also demonstrated that iv changes of this degree also occur when changing the exchange-correlation functional used to model the surface. In contrast, the use of filtered basis sets changes these energies by only 1–2 meV, one hundred times smaller, indicating the excellence of this approach and showing that filtered basis calculation with efficient cut-off radii are of essentially equal quality to those of conventional localised basis functions. Finally a series of defect structures in diamond is considered, including both native defects and nitrogen containing defects. Properties studied include formation energies, binding energies, localised vibrational modes, and hyperfine coupling matrices. In all these cases it is shown that the filtration method produces results which closely match those with conventional basis sets and demonstrate that this method has excellent potential for modelling defecting semiconductor structures in the future. The asymptotic speed up of two to three orders of magnitude will then enable a new range of systems with significantly increased size and complexity to be modelled

The use of agroinfectious clones to investigate recombination between distinct maize streak virus strains

Bibliography: pages 199-213.The infectivity of the replicative form (RF) DNAs of MSV-Kom, MSV-Set and PanSV-Kar contained in the plasm ids pKom500, pSet100 and pPS100 was established by agroinoculating susceptible Jubilee sweetcorn with partial homodimeric Agrobacterium tumefaciens (C58C1) clones of RF-DNAs. Biological characteristics typical of Mastreviruses; such as, the appearance and leafhopper transmissibility of streak symptoms on infected plants, the presence of 18x30nm geminate particles in electron micrographs of leaf-dip preparations, and the presence of single-stranded and double-stranded DNA in Southern blot tests of infected plant DNA extracts, indicated that the RF-DNAs in pKom500, pSet100and PanSV-Kar represent the entire genomes of MSV-Kom, MSV-Set and PanSV-Karrespectively. The complete nucleotide (nt) sequence of the genome of MSV-Set was determined and characterised, and compared with those of MSV-Kom and PanSV-Kar. The genome sizes of MSV-Kom, MSV-Set and PanSV-Kar are 2701, 2690 and 2705 nt respectively, and all share Mastreviral genomic features. Phylogenetic analyses on the nt sequences and the putative amino acid sequences of the movement, coat and replication-associated proteins (MP, CP and Rep respectively) indicate that MSV-Set is grouped with, yet distinct from the MSV group of viruses isolated from maize. MSV-Set shares a 78% nt sequence identity with MSV-Kom which shares a >96% nt sequence identity with other MSVs. The PanSV-Kar genome shares a 60% nt sequence identity with the MSV group and89% with the Kenyan PanSV-Ken. PanSV-Kar causes mild non-persistent streak in Jubilee sweetcorn. MSV-Kom (previously isolated from maize in Komatipoort, Mpumalanga) and MSV-Set (previously isolated from a Setaria species in Mt. Edgecombe, Kwazulu/Natal) have different pathogenicities, and have overlapping, but non-identical, host ranges. Leafhopper transmission tests determined that MSV-Kom and MSV-Set generally cause severe and moderate streak in maize cultivars, or mild and severe streak in wheat cultivars respectively