• Asian-Australasian Journal of Animal Sciences
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• v.14 no.6
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• pp.880-884
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• 2001

Rumen microbiology research has undergone several evolutionary steps: the isolation and nutritional characterization of readily cultivated microbes; followed by the cloning and sequence analysis of individual genes relevant to key digestive processes; through to the use of small subunit ribosomal RNA (SSU rRNA) sequences for a cultivation-independent examination of microbial diversity. Our knowledge of rumen microbiology has expanded as a result, but the translation of this information into productive alterations of ruminal function has been rather limited. For instance, the cloning and characterization of cellulase genes in Escherichia coli has yielded some valuable information about this complex enzyme system in ruminal bacteria. SSU rRNA analyses have also confirmed that a considerable amount of the microbial diversity in the rumen is not represented in existing culture collections. However, we still have little idea of whether the key, and potentially rate-limiting, gene products and (or) microbial interactions have been identified. Technologies allowing high throughput nucleotide and protein sequence analysis have led to the emergence of two new fields of investigation, genomics and proteomics. Both disciplines can be further subdivided into functional and comparative lines of investigation. The massive accumulation of microbial DNA and protein sequence data, including complete genome sequences, is revolutionizing the way we examine microbial physiology and diversity. We describe here some examples of our use of genomics- and proteomics-based methods, to analyze the cellulase system of Ruminococcus flavefaciens FD-1 and explore the genome of Ruminococcus albus 8. At Illinois, we are using bacterial artificial chromosome (BAC) vectors to create libraries containing large (>75 kbases), contiguous segments of DNA from R. flavefaciens FD-1. Considering that every bacterium is not a candidate for whole genome sequencing, BAC libraries offer an attractive, alternative method to perform physical and functional analyses of a bacterium's genome. Our first plan is to use these BAC clones to determine whether or not cellulases and accessory genes in R. flavefaciens exist in clusters of orthologous genes (COGs). Proteomics is also being used to complement the BAC library/DNA sequencing approach. Proteins differentially expressed in response to carbon source are being identified by 2-D SDS-PAGE, followed by in-gel-digests and peptide mass mapping by MALDI-TOF Mass Spectrometry, as well as peptide sequencing by Edman degradation. At Ohio State, we have used a combination of functional proteomics, mutational analysis and differential display RT-PCR to obtain evidence suggesting that in addition to a cellulosome-like mechanism, R. albus 8 possesses other mechanisms for adhesion to plant surfaces. Genome walking on either side of these differentially expressed transcripts has also resulted in two interesting observations: i) a relatively large number of genes with no matches in the current databases and; ii) the identification of genes with a high level of sequence identity to those identified, until now, in the archaebacteria. Genomics and proteomics will also accelerate our understanding of microbial interactions, and allow a greater degree of in situ analyses in the future. The challenge is to utilize genomics and proteomics to improve our fundamental understanding of microbial physiology, diversity and ecology, and overcome constraints to ruminal function.

• Microbiology and Biotechnology Letters
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• v.17 no.5
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• pp.447-453
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• 1989

In order to study the transfer of antibiotics resistance genes of the genetically cloned bacteria in water environments, DK1 strain, which is resistant to kanamycin (Km), chloramphenicol (Cm), streptomycin (Sm), and sulfadiazine (Su), was selected from the Gram-negative bacterial isolates from wastewater. One of 4 plasmids harboured in the DK1 strain was found to possess Km$^{${\gamma}$}$Cm$^{${\gamma}$}$ gene and be about 68 kb in size, and it was designated as pDK101. The plasmid of pDK101 was also found to have 16, 32, and 6 restriction sites for EcoRI. .PstI, and SalI, respectively. From the digestion fragments of pDK101 plasmid and pKT230 used as a vector by EcoRI restriction endonuclease, pDT309 and pDT529 were constructed as chimeric plasmids which possess Km$^{${\gamma}$}$Cm$^{${\gamma}$}$ gene and are 30.9 and 52.9 kb in size, respectively. When the chimeric plasmids were trasformed into E. coli C600 or E. coli HB101, transformants of DKC601, DKC602, DKH102, and DKH103 were obtained as cloned bacterial cells. The Km$^{${\gamma}$}$Cm$^{${\gamma}$}$ genes were well expressed in those cloned cells and the chimeric plasmids were clearly detected in the cloned cells of DKC601 and DKH103.

• Proceedings of the Korean Society of Plant Pathology Conference
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• 1999.04a
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• pp.16.3-16
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• 1999

• KOREAN JOURNAL OF CROP SCIENCE
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• v.52 no.4
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• pp.458-468
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• 2007

To tolerate environmentally adverse conditions such as cold, drought, and salinity, plants often synthesize and accumulate proline in cells as compatible osmolytes. ${\Delta}^1$-Pyrroline-5-carboxylate synthetase(P5CS) catalyzes the rate-limiting step of proline biosynthesis from glutamate. Two complete genes, MtP5CS1 and MtP5CS2, were isolated from the model legume Medicago truncatula by cDNA cloning and bacterial artificial chromosome library screening. Nucleotide sequence analysis showed that both genes consisted of 20 exons and 19 introns. Alignment of the predicted amino acid sequences revealed high similarities with P5CS proteins from other plant species. The two MtP5CS genes were expressed in response to high salt and low temperature treatments. Semi-quantitative reverse transcription-polymerase chain reaction showed that MtP5CS1 was expressed earlier than MtP5CS2, indicating differential regulation of the two genes. To evaluate the reverse genetic effects of nucleotide changes on MtP5CS function, a Targeting Induced Local Lesions in Genomes approach was taken. Three mutants each were isolated for MtP5CS1 and MtP5CS2, of which a P5CS2 nonsense mutant carrying a codon change from arginine to stop was expected to bring translation to premature termination. These provide a valuable genetic resource with which to determine the function of the P5CS genes in environmental stress responses of legume crops.

• The Plant Pathology Journal
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• v.20 no.1
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• pp.7-12
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• 2004

An important recent advance in the field of plant-microbe interactions has been the cloning of genes that confer resistance to specific viruses, bacteria, fungi or insects. Disease resistance (R) genes encode proteins with predicted structural motifs consistent with them having roles in signal recognition and transduction. Plant disease resistance is the result of an innate host defense mechanism, which relies on the ability of plant to recognize pathogen invasion and efficiently mount defense responses. In tomato, resistance to the pathogen Pseudomonas syringae pv. tomato is mediated by the specific recognition between the tomato serine/threonine kinase Pto and bacterial protein AvrPto or AvrPtoB. This recognition event initiates signaling events that lead to defense responses including an oxidative burst, the hypersensitive response (HR), and expression of pathogenesis- related genes.

• Ocean and Polar Research
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• v.27 no.2
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• pp.205-214
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• 2005

From ancient Antarctic glacier ice, we extracted total genomic DNA that was suitable for prokaryotic 16S rDNA gene cloning and sequencing, and bacterial artificial chromosome (BAC) library and end-sequencing. The ice samples were from the Dry Valley region. Age dating by $^{40}Ar/^{39}Ar$ analysis on the volcanic ashes deposited in situ indicated the ice samples are minimum 100,000-300,000 yr (sample DLE) and 8 million years (sample EME) old. Further assay proved the ice survived freeze-thaw cycles or other re-working processes. EME, which was from a small lobe of the basal Taylor glacier, is the oldest known ice on Earth. Microorganisms, preserved frozen in glacier ice and isolated from the rest of the world over a geological time scale, can provide valuable data or insight for the diversity, distribution, survival strategy, and evolutionary relationships to the extant relatives. From the 16S gene cloning study, we detected no PCR amplicons with Archaea-specific primers, however we found many phylotypes belonging to Bacteria divisions, such as Actinobacteria, Acidobacteria, Proteobacteria $({\alpha},\;{\beta},\;and\;{\gamma})$, Firmicutes, and Cytophaga-Flavobacterium-Bacteroid$. BAC cloning and sequencing revealed protein codings highly identical to phenylacetic acid degradation protein paaA, chromosome segregation ATPases, or cold shock protein B of present day bacteria. Throughput sequencing of the BAC clones is underway. Viable and culturable cells were recovered from the DLE sample, and characterized by their 16S rDNA sequences. Further investigation on the survivorship and functional genes from the past should help unveil the evolution of life on Earth, or elsewhere, if any.

• Journal of Microbiology and Biotechnology
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• v.17 no.8
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• pp.1344-1352
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• 2007

An opportunistic human pathogen, Pseudomonas aeruginosa, contains the major catalase KatA, which is required to cope with oxidative and osmotic stresses. As an attempt to uncover the $H_2O_2$-dependent regulatory mechanism delineating katA gene expression, four prototrophic $H_2O_2$-sensitive mutants were isolated from about 1,500 TnphoA mutant clones of P. aeruginosa strain PA14. Arbitrary PCR and direct cloning of the transposon insertion sites revealed that one insertion is located within the katA coding region and two are within the coding region of oxyR, which is responsible for transcriptional activation of several antioxidant enzyme genes in response to oxidative challenges. The fourth insertion was within PA3815 (IscR), which encodes a homolog of the Escherichia coli iron-sulfur assembly regulator, IscR. The levels of catalase and SOD activities were significantly reduced in the iscR mutant, but not in the oxyR mutant, during the normal planktonic culture conditions. These results suggest that both IscR and OxyR are required for the optimal resistance to $H_2O_2$, which involves the expression of multiple antioxidant enzymes including KatA.

• Proceedings of the Botanical Society of Korea Conference
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• 1987.07a
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• pp.323-347
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• 1987

The R locus of maize in one of several genes that regulate the anthocyanin pigments throughout the body of the plant and seed. The R gene product may regulate pigment deposition by controlling the expression of the flavonoid biosynthetic gene pathway in a tissue-specific manner. To understand the basis for tissue specific regulation and allelic variation at R, the molecular study has been done by cloning a portion of the R complex by transposon tagging with Ac. R specific probe were cloned from the R-nj mutant induced by Ac insertion mutagenesis. From southern analysis of R-r complex using the R-nj probe, the structure of R-r was proposed that R-r containes the three elements, (P)(Q)(S). These elements may organize as the inversion triplication model which (S) sequence was inverted in relation to (P) and (Q). The R-sc derivated from R-mb or R-nj was cloned with R-nj probe, and molecular genetical data showed that R-sc containes tissue specific and tissue nonspecific area, and the sequencing of R-sc are progressed now.

• Journal of Aquaculture
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• v.18 no.2
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• pp.81-85
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• 2005

We isolated two parvalbumin cDNAS by expressed sequence tag analysis (1,577 ESTs in total) from the self-fertilizing fish Rivulus marmoratus (Cyprinodontiformes, Rivulidae). Two isoforms of parvalbumin genes showed high similarity to those of carp at 88% and 91% amino acid residues identity, respectively, and showed 79.8% similarity between two parvalbumin isoforms. Of 1,577 ESTs from R. marmroatus sequenced, parvalbumin 1 gene was most abundant. This gene was strongly expressed in the order of muscle, eye, and brain, while it was expressed slightly in other tissues. In this paper, we discussed on the R. marmoratus parvalbumin genes on its sequence and basic characteristics.

• Korean Journal of Agricultural Science
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• v.39 no.1
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• pp.23-28
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• 2012

In order to cloning of PPO gene as a melanin formation related genes involved in hardening and pigmentation of insect integument or wing, we cloned cDNA and analyzed the sequence of PPO gene of H. axyridis. PPO2 primer were designed based on the sequences of PPO genes of Tribolium castaneum and Drosophila melanogaster, and then plasmid DNA were cloned from PCR products obtained from different two color patterns. When the plasmid DNA band pattern were digested by restriction enzymes, BamH1, Xba1, and EcoR1, we found same size band pattern. However, this sequence was not homologous to sequence of T. castaneum PPO gene. Using the primer designed based on the sequence of D. melanogaster, 209 bp PCR product was observed.