An interactive clustering model based on positional weight matrices is described and results obtained using the model to analyze gene regulation patterns in archaea are presented. The 5' flanking sequences of ORFs identified in four archaea, Sulfolobus solfataricus, Pyrobaculum aerophilum, Halobacterium sp. NRC-1, and Pyrococcus abyssi, were clustered using the model. Three regular patterns of clusters were identified for most ORFs. One showed genes with only a ribosome-binding site; another showed genes with a transcriptional regulatory region located at a constant location with respect to the start codon. A third pattern combined the previous two. Both P. aerophilum and Halobacterium sp. NRC-1 exhibited clusters of genes that lacked any regular pattern. Halobacterium sp. NRC-1 also presented regular features not seen in the other organisms. This group of archaea seems to use a combination of eubacterial and eukaryotic regulatory features as well as some unique to individual species. Our results suggest that interactive clustering may be used to examine the divergence of the gene regulatory machinery in archaea and to identify the presence of archaea-specific gene regulation patterns.
Liu D, Lawrence ML, Austin FW (2004) Specific PCR identification of Pasteurella multocida based on putative transcriptional regulator genes. Journal of Microbiological Methods 58(2): 263-267
Pasteurella multocida is an important animal pathogen that may also infect humans through animal bites and scratches. After comparison of transcriptional regulator gene sequences from the P. multocida genome with other DNA sequences at GenBank, we identified two genes (i.e., Pm0762 and Pm1231) uniquely present in P. multocida. By using oligonucleotide primers (Pm0762F/R and Pm1231F/R) designed from these genes in PCR, it was found that specific DNA products of expected sizes were obtained with genomic DNA from P. multocida only, but not from other bacteria. These results indicated that the putative transcriptional regulator genes Pm0762 and Pm1231 are species-specific, and that the PCR methods targeting these genes provide a useful means of rapidly and precisely identifying P. multocida from other bacteria. Further elucidation of the roles and functions of these putative transcriptional regulator genes (Pm0762 and Pm1231) and their protein products may help provide valuable insight into the molecular mechanism of P. multocida virulence and pathogenicity.
Corzo A, Kidd MT, Burgess SC (2004) Whole-plasma MALDI-TOF proteomics for identification of biomarkers of nutritional status in the chicken. Journal of Animal & Veterinary Advances 3: 522-526
Little research has focused on biotechnology for animal nutrition. We wanted to investigate the possibility of simplifying a modern biotechnology for practical application to broiler research. A study was conducted to evaluate plasma protein status as affected by dietary lysine using Matrix-assisted-laser-desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS). Broiler chicks were fed either a diet deficient (1.00%) or adequate (1.22%) in lysine from 0 to 17 d of age. Body weight was determined at 17 d of age. Upon validation of different growth conditions, blood sample representative from each experimental unit was taken and pooled by treatment. Blood plasma was then submitted to MALDI-TOF MS analyses. Resultant spectra, representative of the smallest-mass subset (<25 kDa) of the proteins in each sample was collected. Despite the limited mass range, the data sets were extremely complex; each set of data had almost a quarter of a million datapoints per sample. Plasma from the chicks fed the lysine adequate diet appeared to have a higher number of highly stringent and differentially expressed protein peaks when compared to the underfed lysine chicks. There appears to be a tendency for increased variability as proteins of higher nominal mass are expressed in MALDI-TOF MS, based on calculation of coefficient of variation (CV). However, such CV is not surprising to any equipment or laboratory assay, and appears to be of low impact to this procedure (<0.1% CV). Results from this study exemplify the feasibility of MALDI-TOF MS for biomarker identification of nutritional status.
Liu D, Ainsworth AJ, Austin FW, Lawrence ML (2004) PCR detection of a putative N-acetylmuramidase gene from Listeria ivanovii facilitates its rapid identification. Veterinary Microbiology 101(2): 83-89
Listeria ivanovii is a Gram-positive bacterial pathogen that is capable of causing abortions and stillbirths in farm animals, particularly sheep and cattle. In terms of morphological, biochemical and molecular characteristics, L. ivanovii resembles other Listeria species such as L. monocytogenes, a pathogen of both man and animals. In this study, through comparative analysis of genomic DNA from the six Listeria species, a L. ivanovii specific clone (liv22-228) containing a 946 bp insert was isolated. This clone contained the 5' ends of two divergently transcribed L. ivanovii genes and an intergenic spacer region, similar in organization to homologous regions from the L. innocua and L. monocytogenes genomes. Regions of low homology in the clone were identified by comparing to the L. innocua and L. monocytogenes genomes, and oligonucleotide primers (liv22-228F and liv22-228R) were designed. These primers amplified a 463 bp band from genomic DNA of L. ivanovii strains only, but not from other Listeria species or common bacteria. Thus, PCR employing L. ivanovii specific primers (liv22-228F and liv22-228R) provides a useful and straightforward method for rapid and precise determination of L. ivanovii.
Paterson AH, Bowers JE, Chapman BA, Peterson DG, Rong J, Wicker TM (2004) Comparative genome analysis of monocots and dicots, toward characterization of angiosperm diversity. Current Opinion in Biotechnology 15(2): 120-125
The importance of angiosperms to sustaining humanity by providing a wide range of 'ecosystem services' warrants increased exploration of their genomic diversity. The nearly completed sequences for two species representing the major angiosperm subclasses, specifically the dicot Arabidopsis thaliana and the monocot Oryza sativa, provide a foundation for comparative analysis across the angiosperms. The angiosperms also exemplify some challenges to be faced as genomics makes new inroads into describing biotic diversity, in particular polyploidy (genome-wide chromatin duplication), and much larger genome sizes than have been studied to date.
Burgess SC (2004) Proteomics in the chicken: tools for understanding immune responses to avian diseases. Poultry Science 83(4): 552-573
The entire chicken genome sequence will be available by the time this review is in press. Chickens will be the first production animal species to enter the "postgenomic era." This fundamental structural genomics achievement allows, for the first time, complete functional genomics approaches for understanding the molecular basis of chicken normo- and pathophysiology. The functional genomics paradigm, which contrasts with classical functional genetic investigations of one gene (or few) in isolation, is the systematic holistic genetic analyses of biological systems in defined contexts. Context-dependent gene interactions are the fundamental mechanics of all life. Functional genomics uses high-throughput large-scale experimental methods combined with statistical and computational analyses. Projects with expressed sequence tags in chickens have already allowed the creation of cDNA microarrays for large-scale context-dependant mRNA analysis (transcriptomics). However, proteins are the functional units of almost all biological processes, and protein expression very often bears no correlation to mRNA expression. Proteomics, a discipline within functional genomics, is the context-defined analysis of complete complements of proteins. Proteomics bridges the "sequence-to-phenotype gap;" it complements structural and other functional genomics approaches. Proteomics requires high capital investment but has ubiquitous biological applications. Although currently the fastest-growing human biomedical discipline, new paradigms may need to be established for production animal proteomics research. The prospective promise and potential pitfalls of using proteomics approaches to improve poultry pathogen control will be specifically highlighted. The first stage of our recently established proteomics program is global protein profiling to identify differentially expressed proteins in the context of the commercially important pathogens. Our trials and tribulations in establishing our proteomics program, as well some of our initial data to understand chicken immune system function, will be discussed.
Liu D, Ainsworth AJ, Austin FW, Lawrence ML (2004) Use of PCR primers derived from a putative transcriptional regulator gene for species-specific determination of Listeria monocytogenes. International Journal of Food Microbiology 91(3): 297-304
Listeria monocytogenes is an opportunistic bacterial pathogen that has accounted for an important portion of human foodborne diseases worldwide. In this study, through comparative analysis of L. innocua and L. monocytogenes genomic sequences, we selected a L. monocytogenes specific gene (lmo0733) that has the potential for specific detection of L. monocytogenes. Using PCR primers (lmo0733F and lmo0733R) derived from this gene, a specific fragment of 453 bp was amplified only from genomic DNA of L. monocytogenes strains. PCR products from other Listeria species as well as other Gram-positive and -negative species were not detectable, confirming the specificity of this assay. Thus, the PCR test employing primers lmo0733F and lmo0733R represents an additional tool in the diagnostic arsenal for rapid, sensitive and specific detection and identification of human infections due to L. monocytogenes.
Pechan T, Ma PW, Luthe DS (2004) Heterologous expression of maize (Zea mays L.) Mir1 cysteine proteinase in eukaryotic and prokaryotic expression systems. Protein Expression & Purification 34(1): 134-141
Several heterologous expression systems were tested for their ability to express a unique maize cysteine proteinase Mir1. A baculovirus-based expression system using Trichoplusia ni larvae as host resulted in the expression of Mir1 that was correctly processed and exhibited proteinase activity. Expression in Escherichia coli resulted in accumulation of Mir1, but it had limited solubility and enzymatic activity. Large quantities of Mir1 were produced when Pichia pastoris was used as the host, but the enzyme was insoluble and inactive.
Corzo A, Kidd MT, Pharr GT, Burgess SC (2004) Initial mapping of the chicken blood plasma proteome. International Journal of Poultry Science 3(3): 157-162
The cereal species, of central importance to our diet, began to diverge 50-70 million years ago. For the past few thousand years, these species have undergone largely parallel selection regimes associated with domestication and improvement. The rice genome sequence provides a platform for organizing information about diverse cereals, and together with genetic maps and sequence samples from other cereals is yielding new insights into both the shared and the independent dimensions of cereal evolution. New data and population-based approaches are identifying genes that have been involved in cereal improvement. Reduced-representation sequencing promises to accelerate gene discovery in many large-genome cereals, and to better link the under-explored genomes of `orphan' cereals with state-of-the-art knowledge.
Peng Z, Shen Y, Feng S, Wang X, Chitteti BR, Vierstra RD,Deng XW (2003) Evidence for a physical association of the COP9 signalosome, the proteasome, and specific SCF E3 ligases in vivo. Current Biology 13(1): R504-R505
Cot-based cloning and sequencing (CBCS), a synthesis of Cot analysis, DNA cloning and high-throughput sequencing, promises to accelerate the study of eukaryotic genomes. In particular, CBCS will (1) permit efficient gene discovery in species with substantial quantities of repetitive DNA, (2) allow the sequence complexity (i.e. all the unique sequence information) of large genomes to be elucidated at a fraction of the cost of shotgun sequencing, and (3) enhance genome sequencing efforts by facilitating capture of low-copy sequences not secured by EST sequencing. CBCS should accelerate comparative genomics research, especially in large genomes such as those of many crops.
Pechan T, Cohen A, Williams WP, Luthe DS (2002) Insect feeding mobilizes a unique plant defense protease that disrupts the peritrophic matrix of caterpillars. Proceedings of the National Academy of Sciences of the United States of America 99(20): 13319-13323
Plants frequently respond to herbivorous insect attack by synthesizing defense proteins that deter insect feeding and prevent additional herbivory. Maize (Zea mays L.) lines, resistant to feeding by a number of lepidopteran species, rapidly mobilize a unique 33-kDa cysteine protease in response to caterpillar feeding. The accumulation of the 33-kDa cysteine protease in the maize mid-whorl was correlated with a significant reduction in caterpillar growth that resulted from impaired nutrient utilization. Black Mexican Sweetcorn callus transformed with mir1, the gene encoding the 33-kDa cysteine protease, expressed the protease and growth of caterpillars reared on the transgenic callus was reduced 60-80%. Scanning electron microscopy was used to examine the effect of plant material expressing the 33-kDa cysteine protease on the structure of the caterpillar peritrophic matrix. Because the peritrophic matrix surrounds the food bolus, assists in digestive processes, and protects the caterpillar midgut from physical and chemical damage, disruption of peritrophic matrix may reduce caterpillar growth. The results indicated that the peritrophic matrix was severely damaged when caterpillars fed on resistant maize plants or transgenic Black Mexican Sweetcorn. The accumulation of the 33-kDa cysteine protease in response to caterpillar feeding, and its ability to damage the insect peritrophic matrix, represents an unusual host-plant resistance mechanism that may have applications in agricultural biotechnology.