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C Kang

Publications and source records attributed to C Kang.

At least 91 records · Page 5Linked to original sources
PubMed1994

Characterization of the putative GTP-binding site residues of Escherichia coli adenylosuccinate synthetase by site-directed mutagenesis.

Adenylosuccinate synthetase contains amino acid sequences in its GTP-binding domain that are homologous to other G-proteins. This homology includes a glycine-rich phosphate-binding loop, GXXXXGK, and a guanine-specific binding region, (N/T/Q)KXD; however, virtually no other sequence homology exists between other G-proteins and adenylosuccinate synthetase. On the basis of X-ray diffraction studies, the folding topologies of the synthetase and the p21 ras proteins are different. Yet, residues that interact with GTP in the p21 ras proteins are present in the synthetase in nearly identical positions. We chose therefore to study the G15V mutant, a phosphate-binding loop mutant, and K331L and K331R, two mutants of Lys331 that are involved in guanine ring binding. The Km values for GTP of adenylosuccinate synthetase mutants, K331L and K331R, when compared to those of the wild-type enzyme, were 27- and 20-fold increased, respectively, without any significant change in the Km values for IMP. Because both mutations affected the Km values for GTP similarly, whereas the kcat and secondary structure were essentially unchanged, it is suggested that Lys331 is located in the GTP-binding site of adenylosuccinate synthetase and the terminal N zeta of the Lys is not necessarily important in GTP-binding on the enzyme. Therefore, Lys331 may interact with GTP through hydrophobic interactions between its linear side chain and the aromatic ring of the guanine base of GTP. Also, structural characterization of the G15V mutant was carried out using circular dichroism (CD) spectrometry, NMR spectroscopy, and spectrofluorometry. The CD spectral data indicated that the secondary structure of the G15V mutant was significantly altered by GTP and IMP, whereas that of the wild-type enzyme is not changed; however, the two enzymes exhibited similar secondary structures in the absence of substrates. The NMR spectra of both enzymes were also similar in the absence of substrates. The dissociation constant (Kd) for IMP of the G15V mutant was 4.8-fold larger than its Km value which was 1.5-fold increased compared to the wild-type enzyme. From these findings, it was concluded that the phosphate-binding region of adenylosuccinate synthetase is involved in a conformational change induced by GTP and IMP binding, and that GTP and IMP binding depend on the presence of the other substrate at the active site of the enzyme. These results suggest that the Lys331 of adenylosuccinate synthetase may play similar roles in the function and structure to that of GTP-binding proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenylosuccinate Synthase↗
PubMed1994

Comparison of chromosome aberrations in leiomyoma and leiomyosarcoma using FISH on archival tissues.

Fluorescence in situ hybridization (FISH) with chromosome-specific probes was used to study cytogenetic changes in five cases of leiomyosarcoma (LMS) and nine cases of uterine leiomyoma (LM). Biotinylated DNA probes for the centromeric regions of chromosomes 1, 6, 8, 9, 17, and 18, painting probes for chromosomes 1 and 22, and the cosmid probe for chromosome region 21q22.3 were used on nuclei isolated from paraffin blocks. Four of five LMS cases revealed major chromosomal aberrations, while the only case with minor clonal aberrations was subsequently found not to be a typical LMS. The most common numerical aberrations found in the LMS cases were extra copies of chromosome 8 (three of five cases), loss of chromosome 1 (three of five cases), and loss of chromosome 6 (two of five cases). One of two LMS cases studied with a chromosome 1 painting probe demonstrated translocations of chromosome 1. In contrast to LMS, only five of nine uterine LM cases had abnormal clones, and these were smaller than those in LMS. Two LM cases showed 9% tetrasomy 8 with 17 or 20% monosomy 6, and three other cases had monosomy 6 clones in 18-34% of cells. These results indicate that typical LMS is characterized by multiple chromosomal aberrations affecting most of the cells, whereas borderline LMS and LM have fewer affected chromosomes and less clonal involvement.

Adult↗
PubMed1993

Two base pairs at -9 and -8 distinguish between the bacteriophage T7 and SP6 promoters.

Bacteriophage T7 and SP6 RNA polymerases and their promoters share a high degree of their primary structure homology, but each polymerase exclusively recognizes its own promoter sequence. To reveal the molecular basis of this specificity, 4 base pairs at positions -12, -10, -9, and -8 of the T7 promoter were substituted individually and multiply by SP6 promoter-specific base pairs, and 3 base pairs at -10, -9, and -8 of the SP6 promoter were replaced by T7 promoter-specific base pairs. Promoter activities of 28 sequences were measured in vitro with T7 and SP6 polymerases separately under optimal conditions at 6 mM MgCl2. Single and double substitutions at -12 and -10 do not significantly affect the T7 promoter activity, although they are almost exclusively conserved among T7 genomic promoters. Changes at -10 of SP6 promoter hardly affect the activity. However, any T7 variants that contain either or both changes at -9 and -8 show greatly reduced activity. Interestingly, the double substitution at -9 and -8 yields significant SP6 promoter activities and virtually no T7 promoter activity. Furthermore, the SP6 promoter variants with both T7-specific -9C and -8T show good T7 promoter activities, although they still show some SP6 promoter activities. However, under high salt conditions (either 20 mM MgCl2 or 100 mM NaCl plus 6 mM MgCl2), they show only slight SP6 promoter activity. No other SP6 variants show any T7 promoter activity. All these results indicate that the 2 base pairs at -9 and -8 of both the T7 and SP6 promoters are the primary (if not the only) determinants of specificity and that the hierarchy of importance of positions for promoter activity is -8, -9 > > -10 > -12. Also, a phylogenic relationship among the T3, T7, K11, and SP6 promoters is suggested based on dissimilarities in their sequences from -12 to -8.

Bacteriophage T7↗
PubMed1993

Iron(II)/hydroperoxide (Fenton reagent)-induced activation of dioxygen for (A) the direct ketonization of methylenic carbon and (B) the dioxygenation of cis-stilbene.

Several iron complexes [FeII(PA)2 (PA = picolinate), FeII(bpy)2(2+), FeII(OPPh3)4(2+), FeII(MeCN)4(2+), (Cl8TPP)FeII(py)2 (Cl8TPP = tetrakis(2,6-dichlorophenyl)porphyrin), and FeIIICl3] in combination with R'OOH (R' = H, t-Bu) catalytically activate O2 to oxygenate hydrocarbons [e.g., c-C6H12-->c-C6H10(O) [9 O2 turnovers per FeII(PA)2 or FeII(bpy)2(2+), and 13 per (Cl8TPP)-FeII(py)2]; PhCH2CH3-->PhC(O)CH3 (up to 25 O2 turnovers per FeIILx); c-C6H10-->c-C6H8(O) (up to 9 O2 turnovers per FeIILx); PhCH(Me)2-->PhC(O)Me, Ph(Me)2COH, and Ph(Me)C = CH2 (up to 5 O2 turnovers per FeIILx); and cis-PhCH = CHPh-->2PhCH(O) (up to 2 O2 turnovers per FeLx)]. With large R'OOH/FeLx ratios spontaneous decomposition occurs to give free O2 that is incorporated into the substrates. The product profiles for the various FeIILx/R'OOH, O2/RH systems and their electrochemical characterization during steady-state turnover confirm that the first-formed intermediate is a one-to-one R'OOH/FeIILx adduct (e.g., [(PA)2-FeIIOOR' + pyH+] (1)) (Fenton reagent), which reacts with (a) excess FeII(PA)2 to give (PA)2FeIIIOR', (b) excess c-C6H12 to give (c-C6H11)py (kinetic isotope effect, [KIE] = kc-C6H12/kc-C6D12, 4.6 with t-BuOOH and 1.7 with HOOH), (c) excess R'OOH to give [(PA)2FeIV(OH)(OOR')] (3), then [(PA)2FeIV(O2)] (7) and O2, and (d) O2 to form an adduct, [(PA)2-FeIII(O2)(OOR') + pyH+] (5), that reacts with c-C6H12 to form c-C6H10(O), [K] = 8.2 (t-BuOOH) and 2.1 (HOOH). When PhCH2CH3 or c-C6H10 are the substrates (RH), 5 reacts to form [(PA)2FeIV(OH)(OOR)] (6), which in turn reacts with RH and O2 in a catalytic cycle to give PhC(O)Me or c-C6H8(O) [up to 7 O2 turnovers per iron with FeII(OPPh3)4(2+)]. Species 7 reacts with cis-PhCH = CHPh to give PhCH(O).

Gas Chromatography-Mass Spectrometry↗
PubMed1993

A comparison of techniques in the assessment of chest wall thickness and composition.

The thickness and composition of defined regions of the anterior chest wall are important factors in the assessment of pulmonary plutonium by low-energy x ray counting. Estimates of these quantities are reported for seven male subjects investigated by three laboratories using ultrasonic methods and by a fourth laboratory using magnetic resonance imaging. No important bias was found in any one laboratory's estimates of chest wall thickness relative to those of the others, but differences of up to 6 mm were noted for individual subjects. The discrepancies are believed principally to reflect the different sampling regimes adopted to reach a representative mean chest wall thickness over the region of interest from measurements at selected points. The adipose-tissue component was consistently found to be lower when assessed by magnetic resonance imaging compared with estimates by ultrasound, but the differences were unimportant in the context of plutonium assessment.

Adipose Tissue↗
PubMed1993

All 4 bases of both strands at -9 and -8 in T7 promoter are needed to be substituted by SP6-specific bases to switch promoter specificity.

It has previously been shown that a substitution of two base pairs at -9 and -8 in T7 promoter by SP6 promoter-specific base pairs alone is sufficient to make the T7 variant become an SP6 promoter, abolishing T7 promoter activity [Lee, S. S., and Kang, C. (1992) Biochem. Int. 26, 1-5]. In order to determine which of the 4 bases on both strands at the two positions is involved in the discriminative specificity, each base or each pair of bases was substituted by SP6-specific base(s), allowing one or two mismatches. The results show that all-four-base substitution only is capable of switching promoter specificity, and that all the four bases are substantially involved in T7 promoter activity.

Bacteriophage T7↗
PubMed1993

One-step insertion of oligonucleotide linkers or adapters to DNA using unphosphorylated oligonucleotides.

A simple and efficient method was developed for insertion of oligonucleotide sequences into plasmids. In this method, an unphosphorylated oligonucleotide was ligated to the restriction-digested phagemid DNA. Only the single strand of the oligonucleotide was ligated at the 5' end of the phagemid, and this resulted in the creation of a long self-complementary single-strand overhang. These single-strand overhang-possessing phagemids were used to transform XL-1 cells. This simple ligation and transformation reaction rendered approximately 7.5 x 10(4) to 5 x 10(5) of white colonies per microgram DNA from the isopropyl-beta-D-thiogalactopyranoside and 5-bromo-4-chloro-3-indolyl-beta-D-galacto-pyranoside plate. This number is almost the same or even higher than the number of blue colonies from the control reaction in which ligase was used without the oligonucleotide. By this method we could mutate one enzyme site to another or create ribozyme and substrate phagemid very easily. Fidelity of this method was checked by restriction digestion, DNA sequencing and ribozyme reaction. By DNA sequencing, we observed that 100% of the white colonies contained a single oligonucleotide sequence.

Base Sequence↗
PubMed1993

Effects of multiple mutations at the conserved TATA sequence of bacteriophage SP6 promoter on transcription efficiency.

Mutations of A<==>T were introduced individually and multiple to TATA from -4 to -1 of the phage SP6 promoter and their effects on transcription initiation efficiency measured in vitro. All 15 mutants tested were less active than the wild type. Mutation at -4T nearly abolishes promoter activity independent of other changes, and alteration at -3A reduces promoter activity substantially. On the other hand, effects of mutations at -2T and -1A depend on other changes, suggesting their role should be associated with neighboring base pairs. These results suggest that -4T and -3A are involved in SP6 RNA polymerase binding and -2T and -1A are involved in DNA unwinding. This bipartite role of the SP6 promoter TATA contrasts with the single role of T7 promoter TATA on DNA unwinding. The polymerase binding region extends further downstream in the SP6 promoter than in the T7 promoter.

Bacteriophages↗
PubMed1992

Crystal structure of four-stranded Oxytricha telomeric DNA.

The sequence d(GGGGTTTTGGGG) from the 3' overhang of the Oxytricha telomere has been crystallized and its three-dimensional structure solved to 2.5 A resolution. The oligonucleotide forms hairpins, two of which join to make a four-stranded helical structure with the loops containing four thymine residues at either end. The guanine residues are held together by cyclic hydrogen bonding and an ion is located in the centre. The four guanine residues in each segment have a glycosyl conformation that alternates between anti and syn. There are two four-stranded molecules in the asymmetric unit showing that the structure has some intrinsic flexibility.

Animals↗
PubMed1992

A two-base-pair substitution in T7 promoter by SP6 promoter-specific base pairs alone abolishes T7 promoter activity but reveals SP6 promoter activity.

The phage T7 and SP6 RNA polymerase-promoter systems are very similar in many characteristics, but maintains stringent specificity for each. In order to identify the base pair element that distinguishes between T7 and SP6 promoters, the base pairs at -12, -10, -9, and -8 of the T7 promoter consensus sequence were changed singly and multiply to the SP6 promoter-specific base pairs, and assayed for T7 and SP6 promoter activities. The results indicate that the primary discrimination element is the base pairs at -8 and -9. The two-base-pair substitution alone in T7 promoter by SP6-specific base pairs is sufficient to make the T7 variant be a SP6 promoter, abolishing T7 promoter activity.

Base Composition↗
PubMed1992

Transcript RNA having trans-acting antitermination activity on the T7 transcription terminator.

The efficiency of the phage T7 intrinsic terminator was determined in pulse-labeling in vitro transcription reactions. While the factor-independent terminator subcloned in pET3a showed consistently high (approximately 80%) efficiency, the efficiency of the same terminator in pGEM3ZT was initially approximately 60% but exponentially decreased to approximately 20%, although the 39-bp terminator, and its 73-bp upstream and 32-bp downstream sequences are identical in the two plasmids. When transcription product mixture of pGEM3ZT was added to an on-going reaction of pET3a, the terminator efficiency from pET3a was immediately reduced to approximately 40%. Furthermore, when the pGEM3ZT product mixture was subjected to the promoter-cleaving HinfI digestion and then phenol/chloroform extraction, the mixture still maintained the trans-acting antitermination activity. The results suggest that the trans-acting component(s) are RNA synthesized from pGEM3ZT.

DNA-Directed RNA Polymerases↗
PubMed1991

Metabolism of injected barium in six healthy men.

Data are presented on the early metabolism and long-term retention of 133Ba (half-life 10.74 y) injected into six healthy male volunteers at ages 25-81 y. The tracer appeared to be mainly skeletal within several days, much earlier than predicted by the ICRP's model of alkaline earth metabolism. Excretion was mainly fecal, the relative fecal:urinary clearance up to 14 d ranging from 6 to 15 in the six subjects. The whole-body retention at 50 d (mean 8%, range 4.5 to 12%) was similar to that inferred from published data on the retention of injected Ra in man (mean 7%, range 3-13%). For about 1 y thereafter, the retention of Ba could be represented by simple power functions of time, with the rate of loss correlating with the excretory plasma clearance rate inferred over the first 4 d. In the subject aged 81 y, the pattern differed from that established following an earlier injection at age 60, but the differences were not necessarily related to advancing age.

Adult↗
PubMed1991

Activity of chloramphenicol acetyltransferase overproduced in E. coli with wild-type and mutant GroEL.

The homo-oligomeric protein chloramphenicol acetyltransferase (CAT) has previously been shown to interact with a chaperone GroEL in vitro, suggesting a possible involvement of GroEL in CAT assembly. CAT was overproduced to various levels in the presence and absence of GroEL overproduction, and in groEL mutants. CAT was accumulated to 9-45% of total cellular protein in a fully soluble form, without formation of inclusion bodies. In all cases, even with groEL mutants, CAT specific activity was shown proportional to the amount of protein produced, indicating the formation of active trimer CAT structure does not need GroEL in Escherichia coli.

Bacterial Proteins↗
PubMed1990

Metabolism of Ca and Sr in late adult life.

Data are presented on the metabolism of Ca and Sr in a healthy male volunteer who, in a series of investigations conducted between the ages of 53 and 82 y, received controlled intakes of 45Ca, 47Ca, or 85Sr. No age-related trends were established, either in factors affecting the skeletal deposition of the tracers or in their subsequent retention studied for up to 462 d after intake. The data thus lend support to an important working postulate in the ICRP's model of alkaline earth metabolism.

Aged↗
PubMed1990

[Physico-chemical analysis of Ostreas gigas Thumberg from Xinghua].

This paper presents a comparative physico-chemical analysis of the ancient Ostrea gigas with the medical Ostrea gigas concha, and shows that the protein and amino acid contents in the former are obviously lower than those in the latter, but for the contents of trace elements Mn, Fe, Ni and Pb (with the exception of As), the former appears higher than the latter.

Amino Acids↗
PubMed1987

Studies on SP6 promoter using a new plasmid vector that allows gene insertion at the transcription initiation site.

All the phage-promoter containing subcloning vectors available for in vitro transcription reactions contain a polylinker away from the transcription initiation site. A new SP6 transcription subcloning vector, pCKSP6, has been constructed, in which a gene can be inserted precisely at the transcription initiation site. This was achieved by bringing the BamHI cleavage site into the initiation site. When DNA ends of both insert gene and BamHI cleaved pCKSP6 are made blunt-ended using a single strand specific nuclease, the in vitro transcripts of the recombinant DNA by SP6 RNA polymerase will contain only the gene sequence immediately after the initiation base G. Mung bean nuclease was used to generate a series of mutants resulting from step-wise deletion of single base pairs around the initiation site. Transcription assays with these SP6 promoter mutants revealed that not only the sequence immediately upstream of the initiation site but also the six base pairs from position +1 to +6 are important elements for promoter binding and/or transcription initiation activity. Furthermore, there appears to be a hierarchy of importance of each base pair in the order of position +1 greater than +2 greater than +3 greater than +4, +5, +6, -1, -2.

Animals↗
PubMed1985

Enzymatic synthesis of uniformly 32P-labeled polyribonucleotides and high-specific-activity ribonucleoside 5'-[alpha-32P]diphosphates.

Uniformly 32P-labeled polyribonucleotides of high specific activity can be rapidly and easily synthesized from commercially available ribonucleoside 5'-[alpha-32P]triphosphates by using two enzymes in sequence. Myosin ATPase completely and irreversibly converted any triphosphates to diphosphates in 10 min. The product diphosphates, without purification, can be polymerized by polynucleotide phosphorylase (PNPase) in 1 h with an average yield of 60%. By choosing the desired molar ratio of radioactive and nonradioactive tri- or diphosphates, polymers of a wide range of specific activity can be obtained. Since myosin ATPase and PNPase both have little base specificity, the method can be used to synthesize a radiolabeled polymer of any desired base composition.

Adenosine Triphosphatases↗