天花粉蛋白通过抗原加工提呈调节T细胞免疫应答

用三种抗原加工阻断剂预处理抗原提呈细胞（ＡＰＨ）１ｈ后再用天花粉蛋白（Ｔｋ）脉冲处理,观察经由ＡＰＣ递呈的Ｔｋ对ＰＭＡ和Ａ２３１８７诱发Ｔ细胞增殖的作用。结果表明,这些药物均能不同程度地阻断Ｔｋ的抑制效应,以氯喹和Ｌｅｕｐｅｐｔｉｎ的作用更为明显。用胶体金标记Ｔｋ（Ｔｋ－Ｇ）,电镜下观察Ｔｋ在Ｔ细胞和ＡＰＣ内的定位,发现Ｔｋ－Ｇ仅与ＡＰＣ发生细胞表面粘附,继而内化,先后出现在ＡＰＣ的内体和溶酶体中     

A method to distinguish between the de novo induction of thymidine kinase mutants and the selection of pre-existing thymidine kinase mutants in the mouse lymphoma assay

The mouse lymphoma assay (MLA) is the most widely used in vitro mammalian gene mutation assay. It detects various mutation events involving the thymidine kinase (Tk) gene in L5178Y/Tk+/- -3.7.2C mouse lymphoma cells. Mutants are detected using a thymidine analogue that arrests the growth of cells containing a functional Tk gene. However, there are a number of potential test chemicals that are thymidine analogues, and there is a problem when using the MLA to evaluate the mutagenicity of these chemicals. Thymidine analogues are activated by Tk before eliciting their toxicity. Therefore, any pre-existing Tk-/- mutants may avoid the toxicity of the test chemical and obtain a growth advantage over the Tk+/- cells, increasing the Tk mutant frequency (MF) in the culture via a selection mechanism. This potential mutant selection effect needs to be distinguished from de novo mutant induction in order to properly evaluate the mutagenicity of these chemicals. Here we describe a simple MLA study design that can differentiate between the selection of pre-existing mutants and de novo mutant induction. Trifluorothymidine (TFT), a thymidine analogue and the selection agent normally used in the MLA, and 4-nitroquinoline-1-oxide (4-NQO), a potent mutagen, were used to treat cells from two different Tk+/- mouse lymphoma cell cultures with different background MFs (approximately 112 and 305x10(-6)). Both agents significantly increased the Tk MFs in both the normal and high background cultures (p<0.01). In 4-NQO-treated cultures, the induced MFs (MF of treated culture-MF of control) for the cultures with different background MFs were about the same (p>0.1), while in TFT-treated cultures, they were significantly different (p<0.01). In TFT-treated cultures, the fold-increases of MF (MF of treated culture/MF of control) for the cultures with different background MFs were about the same (p>0.1), while in 4-NQO-treated cultures, they were significantly different (p<0.01). This study confirms that, when de novo mutations are induced, the induced MF is the same for cultures with normal and artificially high background MFs. In situations where the increase in MF is due solely to selection of pre-existing mutants, the "induced" MF will be a multiple of the background MF and the magnitude of the increase of the induced MF will depend upon the magnitude of the background MF. Our results demonstrate that it is possible, using this experimental design, to distinguish between chemicals acting primarily via the selection of pre-existing Tk mutants and those inducing de novo mutants in the MLA.     

A membrane-bound archaeal Lon protease displays ATP-independent proteolytic activity towards unfolded proteins and ATP-dependent activity for folded proteins

In contrast to the eucaryal 26S proteasome and the bacterial ATP-dependent proteases, little is known about the energy-dependent proteolysis in members of the third domain, Archae. We cloned a gene homologous to ATP-dependent Lon protease from a hyperthermophilic archaeon and observed the unique properties of the archaeal Lon. Lon from Thermococcus kodakaraensis KOD1 (Lon(Tk)) is a 70-kDa protein with an N-terminal ATPase domain belonging to the AAA(+) superfamily and a C-terminal protease domain including a putative catalytic triad. Interestingly, a secondary structure prediction suggested the presence of two transmembrane helices within the ATPase domain and Western blot analysis using specific antiserum against the recombinant protein clearly indicated that Lon(Tk) was actually a membrane-bound protein. The recombinant Lon(Tk) possessed thermostable ATPase activity and peptide cleavage activity toward fluorogenic peptides with optimum temperatures of 95 and 70 degrees C, respectively. Unlike the enzyme from Escherichia coli, we found that Lon(Tk) showed higher peptide cleavage activity in the absence of ATP than it did in the presence of ATP. When three kinds of proteins with different thermostabilities were examined as substrates, it was found that Lon(Tk) required ATP for degradation of folded proteins, probably due to a chaperone-like function of the ATPase domain, along with ATP hydrolysis. In contrast, Lon(Tk) degraded unfolded proteins in an ATP-independent manner, suggesting a mode of action in Lon(Tk) different from that of its bacterial counterpart.     

Characterization of a novel glucosamine-6-phosphate deaminase from a hyperthermophilic archaeon

A key step in amino sugar metabolism is the interconversion between fructose-6-phosphate (Fru6P) and glucosamine-6-phosphate (GlcN6P). This conversion is catalyzed in the catabolic and anabolic directions by GlcN6P deaminase and GlcN6P synthase, respectively, two enzymes that show no relationship with one another in terms of primary structure. In this study, we examined the catalytic properties and regulatory features of the glmD gene product (GlmD(Tk)) present within a chitin degradation gene cluster in the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Although the protein GlmD(Tk) was predicted as a probable sugar isomerase related to the C-terminal sugar isomerase domain of GlcN6P synthase, the recombinant GlmD(Tk) clearly exhibited GlcN6P deaminase activity, generating Fru6P and ammonia from GlcN6P. This enzyme also catalyzed the reverse reaction, the ammonia-dependent amination/isomerization of Fru6P to GlcN6P, whereas no GlcN6P synthase activity dependent on glutamine was observed. Kinetic analyses clarified the preference of this enzyme for the deaminase reaction rather than the reverse one, consistent with the catabolic function of GlmD(Tk). In T. kodakaraensis cells, glmD(Tk) was polycistronically transcribed together with upstream genes encoding an ABC transporter and a downstream exo-beta-glucosaminidase gene (glmA(Tk)) within the gene cluster, and their expression was induced by the chitin degradation intermediate, diacetylchitobiose. The results presented here indicate that GlmD(Tk) is actually a GlcN6P deaminase functioning in the entry of chitin-derived monosaccharides to glycolysis in this hyperthermophile. This enzyme is the first example of an archaeal GlcN6P deaminase and is a structurally novel type distinct from any previously known GlcN6P deaminase.     

Expression of cancer-associated simple mucin-type O-glycosylated antigens in parasites

Simple mucin-type O-glycan structures, such as Tn, TF, sialyl-Tn and Tk antigens, are among of the most specific human cancer-associated structures. These antigens are involved in several types of receptor-ligand interactions, and they are potential targets for immunotherapy. In the last few years several simple mucin-type O-glycan antigens were identified in different species belonging to the main two helminth parasite phyla, and sialyl-Tn bearing glycoproteins were detected in Trypanosoma cruzi. These results are of interest to understand new aspects in parasite glycoimmunology and may help identify new biological characteristics of parasites as well of the host-parasite relationship. Considering that different groups reported a negative correlation between certain parasite infections and cancer development, we could hypothesize that simple mucin-type O-glycosylated antigens obtained from parasites could be good potential targets for cancer immunotherapy.     

Tk+/- mouse model for detecting in vivo mutation in an endogenous, autosomal gene

Tk+/- transgenic mice were created using an embryonic stem cell line in which one allele of the endogenous thymidine kinase (Tk) gene was inactivated by targeted homologous recombination. Breeding Tk+/- parents produced viable Tk-/- knockout (KO) mice. Splenic lymphocytes from KO mice were used in reconstruction experiments for determining the conditions necessary for recovering Tk somatic cell mutants from Tk+/- mice. The cloning efficiency of KO lymphocytes was not affected by the toxic thymidine analogues 5-bromo-2'-deoxyuridine (BrdUrd) or trifluorothymidine (TFT), or by BrdUrd in the presence of lymphocytes from Tk+/- animals; however, it was easier to identify clones resistant to BrdUrd than to TFT when Tk+/- cells were present. Tk+/- mice were treated with vehicle or 100 mg/kg of N-ethyl-N-nitrosourea (ENU), and after 4 months, the frequency of Tk mutant lymphocytes was measured by resistance to BrdUrd. The frequency of Tk mutants was 22+/-5.9x10-6 in control animals and 80+/-31x10-6 in treated mice. In comparison, the frequency of Hprt mutant lymphocytes, as measured by resistance to 6-thioguanine, was 2.0+/-1.2x10-6 in control animals and 84+/-28x10-6 in the ENU-treated mice. Analysis of BrdUrd-resistant lymphocyte clones derived from the ENU-treated animals revealed point mutations in the non-targeted Tk allele. These results indicate that the selection of BrdUrd-resistant lymphocytes from Tk+/- mice may be used for assessing in vivo mutation in an endogenous, autosomal gene.     

Engineering of a type III rubisco from a hyperthermophilic archaeon in order to enhance catalytic performance in mesophilic host cells

The hyperthermophilic archaeon Thermococcus kodakaraensis harbors a type III ribulose 1,5-bisphosphate carboxylase/oxygenase (Rbc(Tk)). It has previously been shown that Rbc(Tk) is capable of supporting photoautotrophic and photoheterotrophic growth in a mesophilic host cell, Rhodopseudomonas palustris Delta3, whose three native Rubisco genes had been disrupted. Here, we have examined the enzymatic properties of Rbc(Tk) at 25 degrees C and have constructed mutant proteins in order to enhance its performance in mesophilic host cells. Initial sites for mutagenesis were selected by focusing on sequence differences in the loop 6 and alpha-helix 6 regions among Rbc(Tk) and the enzymes from spinach (mutant proteins SP1 to SP7), Galdieria partita (GP1 and GP2), and Rhodospirillum rubrum (RR1). Loop 6 of Rbc(Tk) is one residue longer than those found in the spinach and G. partita enzymes, and replacing Rbc(Tk) loop 6 with these regions led to dramatic decreases in activity. Six mutant enzymes retaining significant levels of Rubisco activity were selected, and their genes were introduced into R. palustris Delta3. Cells harboring mutant protein SP6 displayed a 31% increase in the specific growth rate under photoheterotrophic conditions compared to cells harboring wild-type Rbc(Tk). SP6 corresponds to a complete substitution of the original alpha-helix 6 of Rbc(Tk) with that of the spinach enzyme. Compared to wild-type Rbc(Tk), the purified SP6 mutant protein exhibited a 30% increase in turnover number (k(cat)) of the carboxylase activity and a 17% increase in the k(cat)/K(m) value. Based on these results, seven further mutant proteins were designed and examined. The results confirmed the importance of the length of loop 6 in Rbc(Tk) and also led to the identification of specific residue changes that resulted in an increase in the turnover number of Rbc(Tk) at ambient temperatures.     

Polyethylenimine-mediated suicide gene transfer induces a therapeutic effect for hepatocellular carcinoma in vivo by using an Epstein-Barr virus-based plasmid vector

The present study aimed to establish a novel efficient nonviral strategy for suicide gene transfer in hepatocellular carcinoma (HCC) in vivo. We employed branched polyethylenimine (PEI) and combined it with Epstein-Barr virus (EBV)-based plasmid vectors. The HCC cells transfected with an EBV-based plasmid carrying the herpes simplex virus-1 thymidine kinase (HSV-1 Tk) gene (pSES.Tk) showed up to 30-fold higher susceptibilities to ganciclovir (GCV) than those transfected with a conventional plasmid vector carrying the HSV-1 Tk gene (pS.Tk). The therapeutic effect in vivo was tested by intratumoral injection of the plasmids into HuH-7 hepatomas transplanted into C.B-17 scid/scid mutant (SCID) mice and subsequent GCV administrations. Treatment with pSES.Tk, but not pS.Tk, markedly suppressed growth of hepatomas in vivo, resulting in a significantly prolonged survival period of the mice. These findings suggest that PEI-mediated gene transfer system can confer efficient expression of the suicide gene in HCC cells in vivo by using EBV-based plasmid vectors.     

Disruption of a sugar transporter gene cluster in a hyperthermophilic archaeon using a host-marker system based on antibiotic resistance

We have developed a gene disruption system in the hyperthermophilic archaeon Thermococcus kodakaraensis using the antibiotic simvastatin and a fusion gene designed to overexpress the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase gene (hmg(Tk)) with the glutamate dehydrogenase promoter. With this system, we disrupted the T. kodakaraensis amylopullulanase gene (apu(Tk)) or a gene cluster which includes apu(Tk) and genes encoding components of a putative sugar transporter. Disruption plasmids were introduced into wild-type T. kodakaraensis KOD1 cells, and transformants exhibiting resistance to 4 microM simvastatin were isolated. The transformants exhibited growth in the presence of 20 microM simvastatin, and we observed a 30-fold increase in intracellular HMG-CoA reductase activity. The expected gene disruption via double-crossover recombination occurred at the target locus, but we also observed recombination events at the hmg(Tk) locus when the endogenous hmg(Tk) gene was used. This could be avoided by using the corresponding gene from Pyrococcus furiosus (hmg(Pf)) or by linearizing the plasmid prior to transformation. While both gene disruption strains displayed normal growth on amino acids or pyruvate, cells without the sugar transporter genes could not grow on maltooligosaccharides or polysaccharides, indicating that the gene cluster encodes the only sugar transporter involved in the uptake of these compounds. The Deltaapu(Tk) strain could not grow on pullulan and displayed only low levels of growth on amylose, suggesting that Apu(Tk) is a major polysaccharide-degrading enzyme in T. kodakaraensis.     

Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT(Tk)) was identified and characterized. The gene (cgt(Tk)) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the alpha-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80 degrees C and 5.5 to 6.0, respectively. The presence of Ca(2+) enhanced the enzyme activity and elevated the optimum temperature to 85 to 90 degrees C. With the addition of Ca(2+), the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85 degrees C, and a half-life of 20 min at 100 degrees C. CGT(Tk) could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT(Tk) harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with beta-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT(Tk)DeltaC) with 23 C-terminal amino acid residues deleted. CGT(Tk)DeltaC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT(Tk). In contrast, the cyclization activity of CGT(Tk)DeltaC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT(Tk) to properly catalyze the cyclization reaction.     

Bromate induces loss of heterozygosity in the thymidine kinase gene of L5178Y/Tk(+/-)-3.7.2C mouse lymphoma cells

Potassium bromate (KBrO(3)) induces DNA damage and tumors in mice and rats, but is a relatively weak mutagen in microbial assays and the in vitro mammalian Hprt assay. Concern that there may be a human health risk associated with bromate, a disinfectant by-product of ozonation, has accompanied the increasing use of ozonation as an alternative to chlorination for treatment of drinking water. In this study, we have evaluated the mutagenicity of KBrO(3) and sodium bromate (NaBrO(3)) in the Tk gene of mouse lymphoma cells. In contrast to the weak mutagenic activity seen in the previous studies, bromate induced a mutant frequency of over 100 x 10(-6) at 0.6mM with minimal cytotoxicity (70-80% survival) and over 1300 x 10(-6) at 3mM ( approximately 10% survival). The increase in the Tk mutant frequency was primarily due to the induction of small colony of Tk mutants. Loss of heterozygosity (LOH) analysis of 384 mutants from control and 2.7 mM KBrO(3)-treated cells showed that almost all (99%) bromate-induced mutants resulted from LOH, whereas in the control cultures 77% of the Tk mutants were LOH. Our results suggest that bromate is a potent mutagen in the Tk gene of mouse lymphoma cells, and the mechanism of action primarily involves LOH. The ability of the mouse lymphoma assay to detect a wider array of mutational events than the microbial or V79 Hprt assays may account for the potent mutagenic response.     

Stimulation of DNA-mediated transformation by UV irradiation of recipient (mouse FM3A) cells

DNA-mediated transformation for thymidine autotrophs (Tk+) was stimulated severalfold when the recipient (mouse FM3A) (Tk-) cells were preirradiated by UV light. The effect was most prominently seen with the cells irradiated 12-16 hours prior to DNA addition. A similar stimulatory effect was observed when the cells were treated with novobiocin or mitomycin C. The effect, however, was blocked when cycloheximide was present during postirradiation period, suggesting that de novo protein synthesis is required for producing the effect. Most of the Tk+ transformants arised from the UV-irradiated cells carried the donor DNA sequences in their chromosomes.     

Methionine sulfoxide reductase from the hyperthermophilic archaeon Thermococcus kodakaraensis, an enzyme designed to function at suboptimal growth temperatures

Methionine sulfoxide reductase (Msr) catalyzes the thioredoxin-dependent reduction and repair of methionine sulfoxide (MetO). Although Msr genes are not present in most hyperthermophile genomes, an Msr homolog encoding an MsrA-MsrB fusion protein (MsrAB(Tk)) was present on the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. Recombinant proteins corresponding to MsrAB(Tk) and the individual domains (MsrA(Tk) and MsrB(Tk)) were produced, purified, and biochemically examined. MsrA(Tk) and MsrB(Tk) displayed strict substrate selectivity for Met-S-O and Met-R-O, respectively. MsrAB(Tk), and in particular the MsrB domain of this protein, displayed an intriguing behavior for an enzyme from a hyperthermophile. While MsrAB(Tk) was relatively stable at temperatures up to 80 degrees C (with a half-life of approximately 30 min at 80 degrees C), a 75% decrease in activity was observed after 2.5 min at 85 degrees C, the optimal growth temperature of this archaeon. Moreover, maximal levels of MsrB activity of MsrAB(Tk) were observed at the strikingly low temperature of 30 degrees C, which also was observed for MsrB(Tk). Consistent with the low-temperature-specific biochemical properties of MsrAB(Tk), the presence of the protein was greater in T. kodakaraensis cells grown at suboptimal temperatures (60 to 70 degrees C) and could not be detected at 80 to 90 degrees C. We found that the amount of intracellular MsrAB(Tk) protein increased with exposure to higher dissolved oxygen levels, but only at suboptimal growth temperatures. While measuring background rates of the Msr enzyme reactions, we observed significant levels of MetO reduction at high temperatures without enzyme. The occurrence of nonenzymatic MetO reduction at high temperatures may explain the specific absence of Msr homologs in most hyperthermophiles. Together with the fact that the presence of Msr in T. kodakaraensis is exceptional among the hyperthermophiles, the enzyme may represent a novel strategy for this organism to deal with low-temperature environments in which the dissolved oxygen concentrations increase.     

Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone lambda HaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA.     

Analysis of mutations in the Tk gene of Tk(+/-) mice treated as neonates with 3'-azido-3'-deoxythymidine (AZT)

Mother-to-child transmission of the human immunodeficiency virus (HIV) is reduced by perinatal treatment with the antiretroviral nucleoside analogue 3'-azido-3'-deoxythymidine (AZT, zidovudine). AZT, however, is genotoxic and carcinogenic in mice when administered either transplacentally or neonatally, suggesting a possible cancer risk for children later in life. In a previous study we found that treating B6C3F1/Tk(+/-) mice on postnatal days 1 through 8 with intraperitoneal injections of 200 mg AZT per kg body weight per day significantly increased spleen lymphocyte mutant frequencies in the autosomal Tk gene. Allele-specific PCR of Tk mutants from treated mice indicated that 61% had lost the Tk(+) allele (loss of heterozygosity; LOH), compared with 35% of Tk mutants from control mice, a difference that was significant. In the present study, Tk mutant lymphocyte clones were analyzed further using polymorphic microsatellite markers that flank the Tk gene along the length of mouse chromosome 11. The analysis indicated that allele-loss mutations in control mice were due to either total chromosome loss, mitotic recombination, or both. The pattern of marker loss in mutants from AZT-treated mice differed significantly from the control mice and was consistent with chromosome loss, mitotic recombination, interstitial deletion, gene conversion, and an unusual discontinuous LOH. The results indicate that AZT induced a unique pattern of mutations in the Tk gene of mice and that the major mechanisms of mutation by AZT involved deletion and recombination.     

Increase in activation rate of Pro‐Tk‐subtilisin by a single nonpolar‐to‐polar amino acid substitution at the hydrophobic core of the propeptide domain

Tk‐subtilisin (Gly70‐Gly398) is a subtilisin homolog from Thermococcus kodakarensis. Active Tk‐subtilisin is produced from its inactive precursor, Pro‐Tk‐subtilisin (Gly1‐Gly398), by autoprocessing and degradation of the propeptide (Tk‐propeptide, Gly1‐Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk‐propeptide. Tk‐propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar‐to‐polar amino acid substitution at this core affects the activation rate of Pro‐Tk‐subtilisin, the Pro‐Tk‐subtilisin derivative with the Phe17→His mutation (Pro‐F17H), Tk‐propeptide derivative with the same mutation (F17H‐propeptide), and two active‐site mutants of Pro‐F17H (Pro‐F17H/S324A and Pro‐F17H/S324C) were constructed. The crystal structure of Pro‐F17H/S324A was nearly identical to that of Pro‐S324A, indicating that the mutation does not affect the structure of Pro‐Tk‐subtilisin. The refolding rate of Pro‐F17H/S324A and autoprocessing rate of Pro‐F17H/S324C were also nearly identical to those of their parent proteins (Pro‐S324A and Pro‐S324C). However, the activation rate of Pro‐F17H greatly increased when compared with that of Pro‐Tk‐subtilisin, such that Pro‐F17H is efficiently activated even at 40°C. The far‐UV circular dichroism spectrum of F17H‐propeptide did not exhibit a broad trough at 205–230 nm, which is observed in the spectrum of Tk‐propeptide. F17H‐propeptide is more susceptible to chymotryptic degradation than Tk‐propeptide. These results suggest that F17H‐propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk‐subtilisin. Thus, destabilization of the hydrophobic core of Tk‐propeptide by a nonpolar‐to‐polar amino acid substitution is an effective way to increase the activation rate of Pro‐Tk‐subtilisin.     

Acquired B antigen: further studies using synthetic oligosaccharides

The reactivity of acquired-B red cells with various antisera has been investigated by agglutination inhibition assays using four trisaccharides obtained by chemical synthesis. Two of these had the structure GalNAc alpha 1-3 (LFuc alpha 1-2). Gal and Gal alpha 1-3 (LFuc alpha 1-2) Gal which are characteristic of the A and B determinants respectively and were indeed strong inhibitors of human anti-A and -B antibodies. The other two sugars denoted B-OAc and B-NH2 are derivatives of the B-trisaccharide by substitution of the hydroxyl group on carbon-2 of the alpha-galactose residue with the O-acetyl group (B-OAc) or the amino group (B-NH2) respectively. We have shown that both B and B-NH2 trisaccharides inhibited strongly the agglutination of acquired B red cells by the anti-B reagents (crude or affinity purified) whereas sera containing "anti-acquired B" agglutinins were specifically inhibited by B-NH2 but not by the A, B or B-OAc structures. We have also shown that the agglutination of Tk-activated erythrocytes by the BS-II lectin is specifically inhibited by N-acetylglucosamine but not by B, B-OAc or B-NH2 structures. These results and the observation that anti-acquired B agglutinins cannot be adsorbed on Tk red cells suggest that (i) the "B-like" and the "acquired-B" determinants share a common structure best represented by B-NH2 and (ii) Tk and "acquired-B" antigens are not identical.     

Homologous recombination between overlapping thymidine kinase gene fragments stably inserted into a mouse cell genome.

We have constructed a substrate to study homologous recombination between adjacent segments of chromosomal DNA. This substrate, designated lambda tk2 , consists of one completely defective and one partially defective herpes simplex virus thymidine kinase (tk) gene cloned in bacteriophage lambda DNA. The two genes have homologous 984-base-pair sequences and are separated by 3 kilobases of largely vector DNA. When lambda tk2 DNA was transferred into mouse LMtk- cells by the calcium phosphate method, rare TK+ transformants were obtained that contained many (greater than 40) copies of the unrecombined DNA. Tk- revertants, which had lost most of the copies of unrecombined DNA, were isolated from these TK+-transformed lines. Two of these Tk- lines were further studied by analysis of their reversion back to the Tk+ phenotype. They generated ca. 200 Tk+ revertants per 10(8) cells after growth in nonselecting medium for 5 days. All of these Tk+ revertants have an intact tk gene reconstructed by homologous recombination; they also retain various amounts of unrecombined lambda tk2 DNA. Southern blot analysis suggested that at least some of the recombination events involve unequal sister chromatid exchanges. We also tested three agents, mitomycin C, 12-O-tetradecanoyl-phorbol-13-acetate, and mezerein, that are thought to stimulate recombination to determine whether they affect the reversion from Tk- to Tk+. Only mitomycin C increased the number of Tk+ revertants.     

A novel ADP-forming succinyl-CoA synthetase in Thermococcus kodakaraensis structurally related to the archaeal nucleoside diphosphate-forming acetyl-CoA synthetases

We have identified and characterized a structurally novel succinyl-CoA synthetase (SCS) from the hyperthermophilic Archaea Thermococcus kodakaraensis. The presence of an SCS completes the metabolic pathway from glutamate to succinate in Thermococcales, which had not been clarified because of the absence of classical SCS homologs on their genomes. The SCS from T. kodakaraensis (SCS(Tk)) is a heteromeric enzyme (alpha(2)beta(2)) encoded by TK1880 (alpha-subunit) and TK0943 (beta-subunit). Although both SCS(Tk) and classical SCSs harbor the five domains present in enzymes of the acyl-CoA synthetase (nucleoside diphosphate-forming) superfamily, the domain order and distribution among subunits in SCS(Tk) (alpha-subunit, domains 1-2-5; beta-subunit, domains 3-4) are distinct from those of classical SCSs (alpha-subunit, domains 1-2; beta-subunit, domains 3-4-5) and instead resemble the acetyl-CoA synthetases from Pyrococcus furiosus (ACSs I(Pf) and II(Pf)). Comparison of the four Thermococcales genomes revealed that each strain harbors five alpha- and two beta-subunit homologs. Sequence similarity suggests that the beta-subunit of SCS(Tk) is also a component of the presumed ACS II from T. kodakaraensis (ACS II(Tk)). We coexpressed the alpha/beta-genes of SCS(Tk) (TK1880/TK0943) and of ACS II(Tk) (TK0139/TK0943). ACS II(Tk) recognizes a broad range of hydrophobic/aromatic acid compounds, as is the case with ACS II(Pf), whereas SCS(Tk) displays a distinct and relatively strict substrate specificity for several acids, including succinate. This indicates that the alpha-subunits are responsible for the distinct substrate specificities of SCS(Tk) and ACS II(Tk).     

A DNA ligase from a hyperthermophilic archaeon with unique cofactor specificity

A gene encoding DNA ligase (lig(Tk)) from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, has been cloned and sequenced, and its protein product has been characterized. lig(Tk) consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 Da. Sequence comparison with previously reported DNA ligases and the presence of conserved motifs suggested that Lig(Tk) was an ATP-dependent DNA ligase. Phylogenetic analysis indicated that Lig(Tk) was closely related to the ATP-dependent DNA ligase from Methanobacterium thermoautotrophicum DeltaH, a moderate thermophilic archaeon, along with putative DNA ligases from Euryarchaeota and Crenarchaeota. We expressed lig(Tk) in Escherichia coli and purified the recombinant protein. Recombinant Lig(Tk) was monomeric, as is the case for other DNA ligases. The protein displayed DNA ligase activity in the presence of ATP and Mg(2+). The optimum pH of Lig(Tk) was 8.0, the optimum concentration of Mg(2+), which was indispensable for the enzyme activity, was 14 to 18 mM, and the optimum concentration of K(+) was 10 to 30 mM. Lig(Tk) did not display single-stranded DNA ligase activity. At enzyme concentrations of 200 nM, we observed significant DNA ligase activity even at 100 degrees C. Unexpectedly, Lig(Tk) displayed a relatively small, but significant, DNA ligase activity when NAD(+) was added as the cofactor. Treatment of NAD(+) with hexokinase did not affect this activity, excluding the possibility of contaminant ATP in the NAD(+) solution. This unique cofactor specificity was also supported by the observation of adenylation of Lig(Tk) with NAD(+). This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.