• Archives of Pharmacal Research
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• v.20 no.6
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• pp.550-554
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• 1997

Chlorambucil is known to alkylate primarily N7 of guanine and N3 of adenine to induce DNA monofunctional adducts and interstrand cross-links (ISC). We have investigated the sequence specificity for DNA ISC induced by chlorambucil using duplex oligomers containing a defined cross-linkable sequences $ 5^{I}-A*TT, 5^{I}-G*TTor5^{I}-G*CC$ under bar which asterisk indicates the potential cross-linking site and underlined base indicates the potential cross-linking site on the opposite strand. An analysis of 20% denaturing polyacrylamide gel electrophoresis showed that chlorambucil was albe to induce DNA ISC in the duplex oligomers containing a sequence $5^I-GCC$. The formation of DNA ISC was not observed in the duplex oligomers containing sequences $5^I-ATT$. or $5^I-GTT$. These results indicate that chlorambucil induces guanine-guanine DNA ISC but not guanine-adenine or adenine-adenine DNA ISC. In addition, we have tested the ability of chlorambucil to induce DNA ISC within $5^I-GNNC$ or $5^I-GNNC$sequences using duplex oligomers containing the sequence$5^I-G^4G^3G^2^C$. The result of DNA strand cleavage assay showed that DNA ISC was formed at the $5^I-GGC$ sequence (an 1,3 cross-link, $G^1-G^3$) but not at $5^I-GGGC$ (an 1,4 cross-link, $G^1-G^4$) or $5^I-GC$ sequence (an 1,2 cross-link, $G^1-G^2$).

• KSBB Journal
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• v.9 no.1
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• pp.63-71
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• 1994

During the last decade enzyme reaction in organic solvent has been studied to show that specificity in buffer is different from that in organic solvent. The specificity of restriction enzyme was effected by various factors such as ionic strength, salt organic solvent and temperature. In this study, restriction enzyme PvuII which is used most frequently in genetic engineering and the substrate was vector pGEM3 whose sequence was already known were used. As a result the recognition sequence site was changed in the presence of organic solvents whose Log P are -1.5∼0. Their specificities were contrast with activities were contrasted. Specificities were not changed in organic solvent easily in inactivating enzyme. We think that the enzyme recognition site was not changed randomly but by preferential order. A recombinant vector which does not contain typical cleavage site CAG↓CTG was cleaved in 20% ethanol solution. This result might show that restriction enzyme could be used to cleave at unusual sites by changing the reaction conditions.

• BMB Reports
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• v.53 no.12
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• pp.611-621
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• 2020

Bacterial endoribonuclease toxins belong to a protein family that inhibits bacterial growth by degrading mRNA or rRNA sequences. The toxin genes are organized in pairs with its cognate antitoxins in the chromosome and thus the activities of the toxins are antagonized by antitoxin proteins or RNAs during active translation. In response to a variety of cellular stresses, the endoribonuclease toxins appear to be released from antitoxin molecules via proteolytic cleavage of antitoxin proteins or preferential degradation of antitoxin RNAs and cleave a diverse range of mRNA or rRNA sequences in a sequence-specific or codon-specific manner, resulting in various biological phenomena such as antibiotic tolerance and persister cell formation. Given that substrate specificity of each endoribonuclease toxin is determined by its structure and the composition of active site residues, we summarize the biology, structure, and substrate specificity of the updated bacterial endoribonuclease toxins.

• Journal of Microbiology and Biotechnology
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• v.12 no.1
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• pp.25-30
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• 2002

A lipase of Staphylococcus aureus B56 was purified from a culture supernatant and its molecular weight was estimated to be 45 kDa by SDS-PAGE. The optimum temperature and pH for the hydrolysis of olive oil was $42^{\circ}C$ and pH 8-8.5, respectively. The enzyme was stable up to $55^{\circ}C$ in the presence of $Ca^++$ at pHs 5-11. The lipase gene was cloned using the PCR amplification method. The sequence analysis showed an open reading frame of 2,076 bp, which encoded a preproenzyme of 691 amino acids. The preproenzyme was composed of a signal sequence (37 aa), propeptide (255 aa), and mature enzyme (399 aa). Based on a sequence comparison, lipase B56 constituted of a separate subgroup among the staphylococcal lipase groups, such as S. aureus PS54 and S. haemolyticus L62 lipases, and was distinct from other lipases in their optimum pH and substrate specificity.

• BMB Reports
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• v.44 no.4
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• pp.250-255
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• 2011

In multicellular organisms, including humans, understanding expression specificity at the tissue level is essential for interpreting protein function, such as tissue differentiation. We developed a prediction approach via generated sequence features from overrepresented patterns in housekeeping (HK) and tissue-specific (TS) genes to classify TS expression in humans. Using TS domains and transcriptional factor binding sites (TFBSs), sequence characteristics were used as indices of expressed tissues in a Random Forest algorithm by scoring exclusive patterns considering the biological intuition; TFBSs regulate gene expression, and the domains reflect the functional specificity of a TS gene. Our proposed approach displayed better performance than previous attempts and was validated using computational and experimental methods.

• YAKHAK HOEJI
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• v.36 no.5
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• pp.469-473
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• 1992

Yeast alcohol dehydrogenase (YADH) has an acidic residue that interacts with the 2'- and 3'-hydroxyl groups of the adenosine ribose of the $NAD^+$ coenzyme. The acidic residue of Asp-223 (according to horse liver alcohol dehydrogenase amino acid sequence) is supposed to determine the coenzyme specificity for $NAD^+$ rather than $NADP^+$. We mutated Asp-223 to leucine and the mutant YADH was expressed in yeast and characterized for the coenzyme specificity. The turnover numbers of mutant enzyme for $NAD^+$ and ethanol were decreased 3.5- and 4.8-fold compared to wild-type enzyme, respectively. Contrastively, catalytic specificity for $NADP^+$ was increased 13-fold. As a result, the mutant YADH also employed $NADP^+$ as a coenzyme.

• YAKHAK HOEJI
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• v.52 no.2
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• pp.125-130
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• 2008

The long terminal repeat (LTR) of retroviral DNA genome plays an important role in the integration process by providing substrate recognition site for viral integrase (IN). The dinucleotide CA near the 3'-end of the LTR termini is completely conserved among retoviruses. In order to study specificity of interaction between prototype foamy virus (PFV) IN and its U5 LTR DNA, the effect of mutagenesis of the CA sequence was investigated by studying reactivity of PFV IN to the mutant LTR substrates. Replacement of only the C or the A allowed 60 to 100% of the reactivity of the wild type LTR substrate. In addition, replacement of the C and the A showed 50 to 80% of the reactivity of the wild type LTR substrate, indicating that PFV IN has less specificity on the conserved CA sequence when it is compared to the other retroviral INs. Therefore it is suggested that PFV IN is less dependent on the conserved sequence of LTR termini for its enzymatic reaction.

• BMB Reports
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• v.31 no.6
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• pp.607-610
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• 1998

Transcription of mitochondrial DNA in the yeast S. cerevisiae depends on recognition of a consensus nonanucleotide promoter sequence by mitochondrial RNA polymerase specificity factor, which is a 43 kDa polypeptide encoded by the nuclear MTF1 gene. Mtf1p has only limited amino acid sequence homology to bacterial sigma factors, but functions in many ways like sigma in that it is required for promoter recognition and initiation of transcription. To analyze the corebinding region of Mtf1p, monoclonal antibodies to this protein were prepared. Recombinant Mtf1p overproduced in E. coli was purified to near homogeneity and used to raise monoclonal antibodies (mAbs). From fused cells screened for Mtf1p mAbs by immunodot blot analysis, 19 positive clones were initially isolated. Further analysis of positive clones by Western blotting resulted in 4 mAbs of Mtf1p.

• Journal of Photoscience
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• v.9 no.2
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• pp.278-280
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• 2002

We have cloned a hydromedusan opsin cDNA and showed that the deduced amino acid sequence of the cytoplasmic loop between helices 5 and 6 (loop 5-6) was clearly different from that reported so far. The amino acid sequence of the loop 5-6 is important on determination of the specificity for the coupled G- protein. To clarify which class of G-protein mediates the phototransduction system in the ocellus of the hydromedusan, we investigated G-proteins expressed in the ocellus. By PCR against the cDNA of the ocellus with primers designed according to the conserved amino acid sequence in G-protein a subunit, we obtained three kinds of cDNA fragments. Based on the sequence similarities, ttwo of them (JGI and JG3) were classified as $G_{i}$ and $G_{q}$, respectively. The other one (JG2) was a new subtype within $G_{*}$ class. Electron microscopic immunocytochemistry with the antiserum against the C-terminal sequence of $G_{q}$ or $G_{t}$ revealed the presence. of the both classes in the ocellus. The similarity of the C-terminal sequence of the JG2 with that of bovine $G_{t}$ suggests that the anti- $G_{t}$ antiserum would bind to JG2. These results suggest the possibility that the hydromedusan rhodopsin decides the specificity for the coupled G-protein by the other domain than the loop 5-6.oop 5-6.5-6.

• Bulletin of the Korean Chemical Society
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• v.20 no.10
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• pp.1126-1128
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• 1999