56 PRODUCTION OF TRANSGENIC RECLONED MINIATURE PIGS EXPRESSING HUMAN DECAY ACCELERATING FACTOR

This study was performed to produce transgenic recloned miniature pigs expressing human decay-accelerating factor (hDAF) to overcome the hyperacute rejection of pig-to-human xenotransplantation. The expression vector, named hDAF-Neo, was constructed by subcloning the amplified 2.2 kb hDAF cDNA fragment into the NheI and NotI site of the pEGFP-N1 (Clontech, Palo Alto, CA, USA) vector containing the chicken beta-actin promoter and rabbit globin poly A without a EGFP fragment. Day 30 fetal fibroblast cells (GN0) were used for producing the newborn cloned pigs (GN1). GN1 fibroblasts cultured from the newborn cloned pig derived from GN0 were used for transfection of the hDAF-Neo plasmid using FuGENE-6� (Roche Diagnostics, Indianapolis, IN, USA). Transfected fibroblast cell colonies were selected with neomycin. All data were analyzed by one-way ANOVA and the protected least significant difference (LSD) test using general linear models in a statistical analysis system (SAS Institute, Inc., Cary, NC, USA) program to determine differences among experimental groups. Significant difference among the treatment groups was determined when the P value was less than 0.05. There was no significantly different development rate between GN0 and GN1 (12.8 vs. 13.0%) and between GN1-hDAF and GN2 (9.3 vs. 8.2%). Transfected recloned embryos (GN1-hDAF) had no significantly different blastocyst formation compared to normal (GN1). After embryo transfer, we obtained two transgenic recloned pigs. hDAF gene integration in transgenic recloned piglets was confirmed by polymerase chain reaction. Cultured fibroblasts of transgenic recloned pigs showed 1.0-2.3 times higher levels of hDAF protein than did human umbilical vein endothelial cells. The levels of C3 deposition on cultured fibroblast cells after incubation with 10% human serum were decreased in transgenic recloned pigs compared to normal miniature pigs. In conclusion, the recloning procedure can be used to produce multiple genetically modified cloned pigs without any severe epigenetic abnormality. This study was supported by grants from the Korean MOST (Top Scientist Fellowship) and MAF (Biogreen 21 #20050301-034-443-026-01-00).

Molecular and enzymic properties of recombinant 1,2-α-mannosidase from Aspergillus saitoi overexpressed in Aspergillus oryzae cells

For the construction of an overexpression system of the intracellular 1,2-α-mannosidase (EC 3.2.1.113) gene (msdS) from Aspergillus saitoi (now designated Aspergillus phoenicis), the N-terminal signal sequence of the gene was replaced with that of the aspergillopepsin I (EC 3.4.23.18) gene (apnS) signal, one of the same strains as described previously. Then the fused 1,2-α-mannosidase gene (f-msdS) was inserted into the NotI site between P-No8142 and T-agdA in the plasmid pNAN 8142 (9.5 kbp) and thus the Aspergillus oryzae expression plasmid pNAN-AM1 (11.2 kbp) was constructed. The fused f-msdS gene has been overexpressed in a transformant A. oryzae niaD AM1 cell. The recombinant enzyme expressed in A. oryzae cells was purified to homogeneity in two steps. The system is capable of making as much as about 320 mg of the enzyme/litre of culture. The recombinant enzyme has activity with methyl-2-O-α-ᴅ-mannopyranosyl α-ᴅ-mannopyranoside at pH 5.0, while no activity was determined with methyl-3-O-α-ᴅ-mannopyranosyl α-ᴅ-mannopyranoside or methyl-6-O-α-ᴅ-mannopyranosyl α-ᴅ-mannopyranoside. The substrate specificity of the enzyme was analysed by using pyridylaminated (PA)-oligomannose-type sugar chains, Man9-6(GlcNAc)2-PA (Man is mannose; GlcNAc is N-acetylglucosamine). The enzyme hydrolysed Man8GlcNAc2-PA (type ‘M8A’) fastest, and ‘M6C’ {Manα1-3[Manα1-2Manα1-3(Manα1-6)Manα1-6]Manβ1-4GlcNAcβ1-4GlcNAc-PA} slowest, among the PA-sugar chains. Molecular-mass values of the enzyme were determined to be 63 kDa by SDS/PAGE and 65 kDa by gel filtration on Superose 12 respectively. The pI value of the enzyme was 4.6. The N-terminal amino acid sequence of the enzyme was GSTQSRADAIKAAFSHAWDGYLQY, and sequence analysis indicated that the signal peptide from apnS gene was removed. The molar absorption coefficient, ϵ, at 280 nm was determined as 91539 M-1·cm-1. Contents of the secondary structure (α-helix, β-structure and the remainder of the enzyme) by far-UV CD determination were about 55, 38 and 7% respectively. The melting temperature, Tm, of the enzyme was 71 °C by differential scanning calorimetry. The calorimetric enthalpy, ∆Hcal, of the enzyme was calculated as 13.3 kJ·kg of protein-1. Determination of 1 g-atom of Ca2+/mol of enzyme was performed by atomic-absorption spectrophotometry.

The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail

The RPC31 gene encoding the C31 subunit of Saccharomyces cerevisiae RNA polymerase C (III) has been isolated, starting from a C-terminal fragment cloned on a lambda gt11 library. It is unique on the yeast genome and lies on the left arm of chromosome XIV, very close to a NotI site. Its coding sequence perfectly matches the amino acid sequence of two oligopeptides prepared from purified C31. It is also identical to the ACP2 gene previously described as encoding an HMG1-like protein (W. Haggren and D. Kolodrubetz, Mol. Cell. Biol. 8:1282-1289, 1988). Thus, ACP2 and RPC31 are allelic and encode a subunit of RNA polymerase C. The c31 protein has a highly acidic C-terminal tail also found in several other chromatin-interacting proteins, including animal HMG1. Outside this domain, however, there is no appreciable homology to any known protein. The growth phenotypes of a gene deletion, of insertions, and of nonsense mutations indicate that the C31 protein is strictly required for cell growth and that most of the acidic domain is essential for its function. Random mutagenesis failed to yield temperature-sensitive mutants, but a slowly growing mutant was constructed by partial suppression of a UAA nonsense allele of RPC31. Its reduced rate of tRNA synthesis in vivo relative to 5.8S rRNA supports the hypothesis that the C31 protein is a functional subunit of RNA polymerase C.

The RPC31 gene of Saccharomyces cerevisiae encodes a subunit of RNA polymerase C (III) with an acidic tail.

The RPC31 gene encoding the C31 subunit of Saccharomyces cerevisiae RNA polymerase C (III) has been isolated, starting from a C-terminal fragment cloned on a lambda gt11 library. It is unique on the yeast genome and lies on the left arm of chromosome XIV, very close to a NotI site. Its coding sequence perfectly matches the amino acid sequence of two oligopeptides prepared from purified C31. It is also identical to the ACP2 gene previously described as encoding an HMG1-like protein (W. Haggren and D. Kolodrubetz, Mol. Cell. Biol. 8:1282-1289, 1988). Thus, ACP2 and RPC31 are allelic and encode a subunit of RNA polymerase C. The c31 protein has a highly acidic C-terminal tail also found in several other chromatin-interacting proteins, including animal HMG1. Outside this domain, however, there is no appreciable homology to any known protein. The growth phenotypes of a gene deletion, of insertions, and of nonsense mutations indicate that the C31 protein is strictly required for cell growth and that most of the acidic domain is essential for its function. Random mutagenesis failed to yield temperature-sensitive mutants, but a slowly growing mutant was constructed by partial suppression of a UAA nonsense allele of RPC31. Its reduced rate of tRNA synthesis in vivo relative to 5.8S rRNA supports the hypothesis that the C31 protein is a functional subunit of RNA polymerase C.