• Korean Journal of Microbiology
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• v.53 no.4
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• pp.286-291
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• 2017

In eukaryotic cells, histone modification is an important mechanism to regulate the chromatin structure. The methylation of the fourth lysine on histone H3 (H3K4) by Set1 complex is one of the various well-known histone modifications. Set1 complex has seven subunits including Swd2, which is known to be important for H2B ubiquitination dependent on H3K4 methylation. Swd2 was reported to regulate Set1's methyltransferase activity by binding to near RNA recognition motif (RRM) domain of Set1 and to act as a component of CPF (Cleavage and Polyadenylation Factors) complex involved in RNA 3' end processing. According to the recent reports, two functions of Swd2 work independently of each other and the lethality of Swd2 knockout strain was known to be caused by its function as a component of CPF complex. In this study, we found that Swd2 could influence the Set1's stability as well as histone methyltransferase activity through the association with RRM domain of Set1. Also, we found that ${\Delta}swd2$ mutant bearing truncated-Set1, which cannot interact with Swd2, lost its lethality and grew normally. These results suggest that the dual functions of Swd2 in H3K4 methylation and RNA 3' end processing are not independent in Saccharomyces cerevisiae.

• Journal of Microbiology
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• v.39 no.1
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• pp.37-41
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• 2001

The fission yeast Schizosaccharomyces pombe contains two distinct superoxide dismutase (SOD) activities, one in the cytosol encoded by the $sod2^{+}$ gene and the other in mitochondria. The $sod2^{+}$ gene encoding putative mitochondrial manganese superoxide dismutase (MnSOD) was isolated from the S. pombe genomic library using a PCR fragment as the probe. The nucleotide sequence of the $sod2^{+}$ gene and its flanking region (4051 bp HindIII fragment) was determined. An intron of 123 nt in size was predicted and confirmed by sequencing the cDNA following reverse transcription PCR. The predicted Sod2p consists of 218 amino acid residues with a molecular mass of 24,346 Da. The deduced amino acid sequence showed a high degree of homology with other MnSODs, especially in the metal binding residues at the active site and their relative positions. The transcriptional start site was mapped by primer extension at 231 at upstream from the ATG codon. A putative TATA box(TATAAAA) was located 58 nt upstream from the transcriptional start site and putative polyadenylation sites were located at 1000, 1062, and 1074 nt downstream from the ATG start codon.

• Asian-Australasian Journal of Animal Sciences
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• v.26 no.8
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• pp.1057-1064
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• 2013

p70 ribosomal S6 kinase (p70S6K) can integrate nutrient and growth factor signals to promote cell growth and survival. We report our molecular characterization of the complementary DNA (cDNA) that encodes the goat p70S6K gene 40S ribosomal S6 kinase 1 (S6K1) (GenBank accession GU144017) and its 3' noncoding sequence in Inner Mongolia Cashmere goats (Capra hircus). Goat S6K1 cDNA was 2,272 bp and include an open reading frame (ORF) of 1,578 bp, corresponding to a polypeptide of 525 amino acids, and a 694-residue 3' noncoding sequence with a polyadenylation signal at nucleotides 2,218 to 2,223. The relative abundance of S6K1 mRNA was measured by real-time PCR in 6 tissues, and p70S6K expression was examined by immunohistochemistry in heart and testis. The phosphorylation of p70S6K is regulated by mitogen-activated protein kinase (MAPK) signaling in fetal fibroblasts.

• Journal of Sericultural and Entomological Science
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• v.39 no.2
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• pp.180-185
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• 1997

To develope the novel baculovirus transfer vector, the p10 gene was cloned from the Bombyx mori nuclear polygedrosis virus (BmNPV) vB2 strain isolated from the B. mori larvae of sericultural farms. The novel transfer vector was constructed by using the p10 gene of BmNPV vB2 strain was 210 bp. The TAAG sequence at the -71 bp of upstream from translation initiator ATG and two polyadenylation signal site at the downstream from terminator TAA were also detected in the p10 gene. The 5' and 3' flanking region of the p10 gene amplified by PCR was cloned into pBluescriptII SK(+) and then transfer vector pBm10 was construceted. The 7.9 kb pBm10 was analysed by restriction enzymes and the map was confirmed. In order to determine the expression of foreign gene of pBm10, $\beta$-galactosidase gene was inserted in the SmaI site of foreign gene cloning site of pBm10. The pBm10 containing $\beta$-galactosidase gene was cotranfected wth genomic DNA of BmNPV vB2 into BmN-4 cells. The recombinant baculovirus expressing $\beta$-galactosidase was also produced polygedra in the infected cells. The results indicated that pBm10 is functional, suggesting that in the baculovirus expression vector system, the recombinant virus produced by pBm10 was effective by oral infection for the producing recombinant proteins in in vivo expression.

• YAKHAK HOEJI
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• v.31 no.2
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• pp.116-125
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• 1987

It is still difficult and time consuming to obtain cDNA sequences that contain the entire nucleotide sequence of the corresponding mRNA. A rapid and high efficient cloning method to obtain full-length cDNA segments is thus developed. The cloning procedure described here consists of the construction of oligo(dT)-tailed vector primer using pWR34 plasmid, polyadenylation of mRNA-cDNA heteroduplex using terminal deoxytransferase, and replacement of MRNA strand with DNA by RNase H and DNA polymerase I. The restriction endonuclease analysis shows that the size of inserted-cDNA is in the range of 1.5~4.0 kb long suggesting that most of cloned cDNA are full-length or nearly full-length cDNA. The plasmid-DNA recombinants obtained were 4$\times$$10^5$~$10^{6}$ per $\mu\textrm{g}$ of rat liver poly (A$^+$)mRNA, which is 4 to 10 fold higher cloning efficiency in comparison to the presently used methods for full-length cDNA cloning. The results indicate that the described cloning system is much simpler, less time consuming, and very efficient cloning method to construct a cDNA library.

• Journal of Microbiology and Biotechnology
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• v.2 no.3
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• pp.174-182
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• 1992

The hemagglutinin-esterase glycoprotein (HE) gene of bovine coronavirus, coupled with a simian virus 40 early promoter and polyadenylation signal, was inserted into a human adenovirus transfer vector. The transfer vector was used to co-transfect 293 cells along with adenovirus genomic DNA. The hemagglutinin-esterase transcription unit was rescued into the adenovirus genome by homologous in vivo DNA recombination between the vector plasmid DNA and the adenovirus genomic DNA, and a recombinant adenovirus was isolated by several rounds of plaque assays. Thus the recombinant adenovirus carries the hemagglutinin-esterase gene in the early transcription region 3 (E3) of the adenovirus genome in the parallel orientation to the E3 transcription. The recombinant adenovirus synthesized the HE polypeptide in HeLa cells as demonstrated by immunoprecipitation with anti-coronavirus rabbit antisera. The recombinant HE polypeptide could be labelled by $[^3H]$glucosamine, demonstrating that the recombinant HE was glycosylated. Cells expressing the HE polypeptide exhibited hemadsorption activity when incubated with mouse erythrocytes. The HE was transported to the plasma membrane as shown by the cell surface immunofluorescence, indicating that the recombinant HE polypeptide retained its biological activities. Potential for the use of infectious recombinant adenovirus as a live virus-vectored vaccine candidate for bovine coronavirus disease is discussed.

• Archives of Pharmacal Research
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• v.19 no.6
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• pp.497-501
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• 1996

A gene coding for triosephosphate isomerase (TPI) from a rat skeletal muscle cDNA library was cloned and its nucleotide sequence was determined. The 1, 348-bp cDNA clone contains 24 bp $5^I$ noncoding region, the entire 750 bp coding region corresponding to a protein of 249 amino acids, $547bp 3^I$ noncoding region and part of a poly(A) tail. It also contains a polyadenylation signal, AATAAA, starting from 17 bp upstream of the poly(A) tail. The calculated molecular weight of rat TPI is 27.8 kDa and the net charge is +4. The deduced amino acid sequence from rat TPI CDNA sequence has 93% and 94% homology with that of mouse and human clones, respectively. The amino acids at the residue of Asn12, Lys14, His96, Glu 166, His96, His101, Ala177, Tyr165, Glu13O, Tyr2O9, and Ser212 in catalytic site are completely identical, confirming that the functional residues in TPI proteins are highly conserved throughout evolution. The most profound characteristic of rat TPI enzyme, compared with other TPIs, is that there are five cysteine substitutions at the residue of 21, 27, 159, 195 and 204. A Glu123 instead of Gly was found in rabbit, rhesus, mouse and human sequences. Through the method of RT-PCR, the mRNA transcription level of TPI gene was found to be different among various tissues and was highest in muscle.

• Journal of Sericultural and Entomological Science
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• v.41 no.3
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• pp.147-153
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• 1999

Chitinolytic enzymes such as ${\beta}$-N-acetylglucosaminidase are major hydrolases involved in insect molting. We have isolated, sequenced a CDNA encoding ${\beta}$-N-acetylglucosaminidase from the silkworm, Bombyx mandarina, and compared its sequence with genes encoding chitinolytic enyzmes from other sources. The insert DNA in the clone is 3,284 nucleotides long with an open reading frame of 1,788 nucleotides that encodes a protein of 596 amino acids with a molecular weight of 68.2 kDa. There is a 3’-untranslated region composed with 1.479 nucleotides and are several potential polyadenylation signals. The predicted amino acid sequence apparently contains a leader peptide of 23 amino acids. A search of the amino acids sequence databases for sequences similarities to other ${\beta}$-N-acetylglucosaminidases or ${\beta}$-N-acetylhexosaminidases. The highest similarity matched with the enzyme from B. mori, which has a sequence identity of 95%. On the other hand, the identity between the B. mandarina enzyme and those from M. sexta and human are 70% and 24%, respectively.

• Journal of Marine Life Science
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• v.3 no.1
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• pp.1-8
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• 2018

Myosin is considered as the vital motor protein in vertebrates and invertebrates. Our present study was conducted to decipher the occurrence of myosin in dog fish (Squalus mitsukurii). We isolated one clone containing 979 bp cDNA sequence, which consisted of a complete coding sequence of 453 bp and a deduced amino acid sequence of 150 amino acids from the open reading frame with molecular weight, isoelectric point and aliphatic index are 16.72 Kda, 4.49 and 78.00, respectively. It contained 428 bp long 3' UTR with single potential polyadenylation signals (AATAAA). The predicted EF CA²⁺ binding domains were identified in residue 6-41, 83-118 and 133-150. A BLAST search indicates this protein exhibits a strong similarity to whale shark (Rhincodon typus) MLC3 (91% identical) and also house mouse (Mus musculus) MLC isoform 3f (81% identical). Phylogenetic analysis revealed that this protein is a MLC 3 isoform like protein. This protein also demonstrates highly conserved region with other myosin proteins. Homology modeling of S. mitsukuri was performed using crystal structure of Gallus gallus skeletal muscle myosin II based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR results exhibits dogfish myosin protein is highly expressed in muscle tissue.

• BMB Reports
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• v.31 no.4
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• pp.413-417
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• 1998

Transgenic mice containing a bovine ${\beta}-Casein/Human$ lysozyme fusion gene (pBZ) were generated in order to produce human lysozyme in their milk. The expression vector was a quadripartite fusion consisting of a 2 kb upstream DNA of the bovine ${\beta}-casein$ gene, human lysozyme gene, intron II of the rabbit ${\beta}-globin$ gene, and the polyadenylation/termination signals of SV40 DNA. Fertilized mouse zygotes were microinjected with pBZ, then transferred into the oviduct of foster mothers. Out of 20 mice born, 11 survived until postweaning and three were identified as positivetransgenic by Southern blot analysis (one male and two females). The founder mice were mated to BCFl mice to produce transgenic progeny. It was confirmed by RT-PCR and Northern blot analyses that the transgene was specifically expressed in the mammary gland of the founder mice. Furthermore, the artificial introns within the transgenic RNA was proven to be correctly spliced out as judged by RT-PCR analysis. These results indicated that transgenic mice generated in this study properly expressed the human lysozyme RNA in their mammary gland.