Porcine TCR CD3zeta-chain and eta-chain.

Complete porcine CD3zeta-chain cDNA sequence was obtained for the first time, and its genomic nucleotide sequence was investigated from exon 2 down to CD3eta-chain exon 8. The sequence of porcine CD3zeta-chain showed homologous amino acid sequence with human and murine counterparts, in contrast to CD3eta-chain exon 8 with diversity among animals previously investigated. CD3eta-chain peptide is an alternative splice form of CD3zeta-chain exon 7 splicing to CD3eta-chain exon 8 instead of CD3zeta-chain exon 8. The genomic sequences revealed that the splice acceptor sequences of CD3eta-chain exon 8 of all animals investigated to be completely uniform. Further, CD3eta-chain exon 8 amino acid sequences retained the unique characters of having high proline (Pro) and positively charged amino acid content except for rats and mice. Although the biological role of CD3eta-chain remains to be enigmatic, these evidences suggests the evolutional pressure to maintain its sequence.

Development of novel monoclonal antibody 4G8 against swine leukocyte antigen class I alpha chain.

A mouse monoclonal antibody (MAb) was generated against swine leukocyte antigen (SLA) class I alpha chain. A newly developed series of MAb clones that react with pan leukocytes were selected and tested by immuno-histochemistry using SLA class I alpha chain expressing Cos-7 cells. Among them, MAb 4G8 was characterized by the following features: (1) 4G8 reacted with Cos-7 cells transfected with SLA class I alpha chain from the d haplotype, (2) 4G8 recognized epitopes that were different from those of commercially available anti-SLA class I MAbs 74-11-10 and PT85A, and (3) 4G8 could be used to immunostain frozen sections of thymus, spleen, lymph node, kidney, and liver tissues with good results.

Analysis of a full-length cDNA library constructed from swine olfactory bulb for elucidation of expressed genes and their transcription initiation sites.

The olfactory system is indispensable to the survival of animals in finding foods and for the reproductive process. Odorant signals are conveyed through olfactory sensory neurons to the olfactory bulb, which modifies the signals and relays them to the neocortex. In the present study, a "full-length" cDNA library was constructed from the main and accessory olfactory bulbs of 5-week-old male pigs, in order to elucidate the expressed genes. The average insert size of the library was estimated to be 1.7 kb based on 54 randomly-selected clones. One thousand randomly selected clones were subjected to sequencing, and the resulting 883 sequences were then clustered into 753 sequences based on similarity. Since 723 of the 753 sequences had sufficient sequence information for homology analysis, the 723 sequences were subjected to BLAST analysis against GenBank/EMBL/DDBJ; 655 out of the 723 sequences showed similarities with known genes, and the remaining 68 were indicated to be novel sequences. The full-length rate of the library was estimated to be ca. 80%, using 70 sequences corresponding to human full-length cDNAs. The full-length cDNA sequences of a single gene appearing more than 6 times in the analysis were aligned to determine major transcription initiation sites for SLC25A, CKB, TUBB4, TUBB, YWHAH, TUBB2, and CNP genes.

PEDE (Pig EST Data Explorer): construction of a database for ESTs derived from porcine full-length cDNA libraries.

We generated the PEDE (Pig EST Data Explorer; http://pede.dna.affrc.go.jp/) database using sequences assembled from porcine 5' ESTs from oligo-capped full-length cDNA libraries. Thus far we have performed EST analysis of various organs (thymus, spleen, uterus, lung, liver, ovary and peripheral blood mononuclear cells) and assembled 68,076 high-quality sequences into 5546 contigs and 28,461 singlets. PEDE provides a search interface for getting results of homology searches and enables users to obtain information on sequence data and cDNA clones of interest. Single-nucleotide polymorphisms detected through comparison of the EST sequences are classified by origin (western and oriental breeds) and are searchable in the database. This database system can accelerate analyses of livestock traits and yields information that can lead to new applications in pigs as model systems for medical research.

Amino acid sequence of the alpha- and beta-globin chains of the Erabu sea snake (Laticaudia semifasciata).

We determined the complete amino acid sequences of the Erabu sea snake (Laticaudia semifasciata) hemoglobin by analyzing the intact globin chains, enzymatically digested fragments, and chemical cleavage fragments to clarify the molecular evolution and phylogenetic classification of the sea snake. The Erabu sea snake has two types of hemoglobin components, Hb-I and Hb-II, which contain different alpha- and beta-chains. This is the second report of the complete primary structure for hemoglobin of snakes. The sequences were compared with those of other reptilian hemoglobins. Amino acids at positions critical for the structure and physiological functions of hemoglobin were loosely conserved. The requirements for binding of ATP and of diphosphoglycerate as allosteric effectors of beta-globins seemed to be fulfilled.

Biochemical and genetic characterization of mundticin KS, an antilisterial peptide produced by Enterococcus mundtii NFRI 7393.

Mundticin KS, a bacteriocin produced by Enterococcus mundtii NFRI 7393 isolated from grass silage in Thailand, is active against closely related lactic acid bacteria and the food-borne pathogen Listeria monocytogenes. In this study, biochemical and genetic characterization of mundticin KS was done. Mundticin KS was purified to homogeneity by ammonium sulfate precipitation, sequential ion-exchange chromatography, and solid-phase extraction. The gene cluster (mun locus) for mundticin KS production was cloned, and DNA sequencing revealed that the mun locus consists of three genes, designated munA, munB, and munC. The munA gene encodes a 58-amino-acid mundticin KS precursor, munB encodes a protein of 674 amino acids involved in translocation and processing of the bacteriocin, and munC encodes a mundticin KS immunity protein of 98 amino acids. Amino acid and nucleotide sequencing revealed the complete, unambiguous primary structure of mundticin KS; mundticin KS comprises a 43-amino-acid peptide with an amino acid sequence similar to that of mundticin ATO6 produced by E. mundtii ATO6. Mundticin KS and mundticin ATO6 are distinguished by the inversion of the last two amino acids at their respective C termini. These two mundticins were expressed in Escherichia coli as recombinant peptides and found to be different in activity against certain Lactobacillus strains, such as Lactobacillus plantarum and Lactobacillus curvatus. Mundticin KS was successfully expressed by transformation with the recombinant plasmid containing the mun locus in heterogeneous hosts such as E. faecium, L. curvatus, and Lactococcus lactis. Based on our results, the mun locus is located on a 50-kb plasmid, pML1, of E. mundtii NFRI 7393.

Amino acid sequence of the alpha- and beta-globin chains of the Hiroo sea snake (Laticauda laticaudata).

We determined the hemoglobin complete amino acid sequences of the Hiroo sea snake (Laticaudia laticuada) from the intact globin chain, enzymatically digested fragments, and chemical cleavage fragments to analyze molecular evolution for classification of the sea snake. The Hiroo sea snake has two hemoglobin components, Hb-I and Hb-II, which contain different alpha- and beta-chains, respectively. This is the first report of the complete primary structure of a snake hemoglobin. The sequences were compared with those of other reptilian hemoglobins. Amino acid replacements at positions critical for structure and physiological role of hemoglobin were loosely conserved. The requirements for binding of ATP and of diphosphoglycerate as allosteric effectors at beta-globins seemed to be fullfilled.

Isolation of enterocin SE-K4-encoding plasmid and a high enterocin SE-K4 producing strain of Enterococcus faecalis K-4.

Enterococcus faecalis K-4, which produces a class IIa bacteriocin, enterocin SE-K4, carries two plasmids, pEK4S (approximately 60 kb) and pEK4L (approximately 75 kb). Plasmid-curing experiments showed that pEK4S was involved in the production of and immunity to enterocin SE-K4 in strain K-4. A derivative strain, M6, with pEK4S produced a higher amount of enterocin SE-K4 than the parental strain K-4, although its growth rate was lower than that of parental strain K-4. Phenotypic changes in strain M6 are attributed to an increase in plasmid copy number.