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 and 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.
Proteomics is the study of the entire protein compliment of an organism. The blood plasma is the only tissue in which an organism's entire proteome may be potentially represented. First results toward mapping the broiler plasma proteome are presented here. Blood was taken from eight 18 day-old representative commercial broiler chickens. Plasma was isolated from each sample and pooled. For initial sample fractioning a 0.4 µl aliquot of the pooled plasma was run on one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. Based on relative amounts of protein, the gel was divided into three fractions. The proteins were in-gel digested with trypsin. Two-dimensional liquid chromatography in-line with electrospray ionization tandem mass spectrometry was then used for "shot-gun" qualitative plasma proteomics. The resulting tandem mass spectra were then searched against the non-redundant chicken protein database. Generally accepted high stringency statistical criteria for protein identification were used. Eighty-four chicken proteins were identified. Our work demonstrates the future potential for plasma proteomics for identifying biomarkers of disease and production in chickens.
Paterson AH, Bowers JE, Peterson DG, Estill JC, Chapman BA (2003) Structure and evolution of cereal genomes. Current Opinion in Genetics and Development 13: 644-650.
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.
Liu D, Ainsworth AJ, Austin FW, Lawrence ML (2003) Characterization of virulent and avirulent Listeria monocytogenes strains by PCR amplification of putative transcriptional regulator and internalin genes. Journal of Medical Microbiology 52(12): 1065-1070.
Listeria monocytogenes is an opportunistic bacterial pathogen that is an important cause of human food-borne illness worldwide. However, L. monocytogenes strains demonstrate considerable variation in pathogenic potential. In this report, virulent and avirulent L. monocytogenes isolates were compared by using a comparative screening strategy. Two clones were identified that contained DNA that was only present in virulent L. monocytogenes strains. PCR primers were designed for three genes from these clones and for five other selected L. monocytogenes genes. All eight primer sets predominantly detected virulent L. monocytogenes isolates, as determined by a mouse virulence assay; one of the putative internalin genes, lmo2821, was detected in all strains that were considered to be virulent. Primers from these eight genes were then tested by PCR against a larger panel of bacterial strains; each of the genes was detected predominantly in clinical or food L. monocytogenes isolates, rather than environmental isolates. The findings from this study suggest that virulent L. monocytogenes strains may possess genes that are not present in avirulent isolates, which could serve as markers for PCR assessment of L. monocytogenes virulence.
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.
Serino G, Su H, Peng Z, Tsuge T, Wei N, Deng XW (2003) Characterization of the last subunit of the arabidopsis COP9 signalosome: Implications for the overall structure and origin of the complex. Plant Cell 15(3): 719-731.
The COP9 signalosome (CSN) is an evolutionarily conserved protein complex that resembles the lid subcomplex of proteasomes. Through its ability to regulate specific proteasome-mediated protein degradation events, CSN controls multiple aspects of development. Here, we report the cloning and characterization of AtCSN2, the last uncharacterized CSN subunit from Arabidopsis. We show that the AtCSN2 gene corresponds to the previously identified FUS12 locus and that AtCSN2 copurifies with CSN, confirming that AtCSN2 is an integral component of CSN. AtCSN2 is not only able to interact with the SCF(TIR1) subunit AtCUL1, which is partially responsible for the regulatory interaction between CSN and SCF(TIR1), but also interacts with AtCUL3, suggesting that CSN is able to regulate the activity of other cullin-based E3 ligases through conserved interactions. Phylogenetic analysis indicated that the duplication and subsequent divergence events that led to the genes that encode CSN and lid subunits occurred before the divergence of unicellular and multicellular eukaryotic organisms and that the CSN subunits were more conserved than the lid subunits during evolution. Comparative analyses of the subunit interaction of CSN revealed a set of conserved subunit contacts and resulted in a model of CSN subunit topology, some aspects of which were substantiated by in vivo cross-link tests.
Peterson DG, Wessler SR, Paterson AH (2002) Efficient capture of unique sequences from eukaryotic genomes. Trends in Genetics 18(11): 547-550.
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.
Burnham MR, Branton SL, Peebles ED, Lott BD, Gerard PD (2002) Effects of F-strain Mycoplasma gallisepticum inoculation at twelve weeks of age on performance and egg characteristics of commercial egg-laying hens. Poultry Science 81(10): 1478-1485.
The effects of F-strain Mycoplasma gallisepticum (FMG) inoculation during the pullet period on the subsequent performance and egg characteristics of commercial Single Combed White Leghorn hens were evaluated. In two trials, BW, feed consumption, egg production (EP), egg weight, egg size class, relative eggshell water vapor conductance, and relative percentages of eggshell, yolk and albumen weights were determined through approximately 60 wk of age. In each trial, pullets at 12 wk of age were randomly assigned to negative pressure biological isolation units. Birds in one-half of the total units were inoculated with FMG, and the other half were sham-inoculated with sterile media. In both trials, onset of lay was delayed approximately 1 wk in layers inoculated with FMG. Control birds that had not been previously inoculated with FMG laid their first egg at 18 wk of age, while birds that had been previously inoculated with FMG laid their first egg at 19 wk of age. In Trial 1, FMG-inoculated hens laid significantly fewer total eggs, which became apparent at each week after Week 42. In Trial 2, a numerical decrease in total EP occurred, and the percentage of undersized eggs laid by FMG-inoculated birds was significantly lower at 19 wk of age but was higher at 20 and 21 wk when compared to controls. Mortality was not significantly different between the treatments in either trial. These data demonstrate that when birds are housed in isolation facilities and inoculated with FMG at 12 wk of age, onset of lay is delayed. These data also suggest that FMG may lead to delays in undersize EP and decreases in total EP. However, because significant FMG effects on these parameters were observed in only one trial, additional studies may be necessary to verify these effects.