Purification of transketolase from baker's yeast by an immunoadsorbent

Baker's yeast transketolase has been purified by immunoaffinity chromatography on specific TK antibodies covalently linked to CNBr-agarose. Affinity chromatography allows a 480-fold purification of TK from yeast extracts and about 80% recovery of the original activity. The isolated enzyme has a specific activity of 12-60 U/mg and during polyacrylamide gel electrophoresis performed at pH 8.9 migrates as two protein bands possessing a transketolase activity which corresponds to two isoforms of the enzyme.     

Gene expansion and retention leads to a diverse tyrosine kinase superfamily in amphioxus

Tyrosine kinase (TK) proteins play a central role in cellular behavior and development of animals. The expansion of this superfamily is regarded as a key event in the evolution of the complex signaling pathways and gene networks of metazoans and is a prominent example of how shuffling of protein modules may generate molecular novelties. Using the intron/exon structure within the TK domain (TK intron code) as a complementary tool for the assignment of orthology and paralogy, we identified and studied the 118 TK proteins of the amphioxus Branchiostoma floridae genome to elucidate TK gene family evolution in metazoans and chordates in particular. Unlike all characterized metazoans to date, amphioxus has members of all known widespread TK families, with not a single loss. Putting amphioxus TKs in an evolutionary context, including new data from the cnidarian Nematostella vectensis, the echinoderm Strongylocentrotus purpuratus, and the ascidian Ciona intestinalis, we suggest new evolutionary histories for different TK families and draw a new global picture of gene loss/gain in the different phyla. Surprisingly, our survey also detected an unprecedented expansion of a group of closely related TK families, including TIE, FGFR, PDGFR, and RET, due most probably to massive gene duplication and exon shuffling. Based on their highly similar intron/exon structure at the TK domain, we suggest that this group of TK families constitute a superfamily of TK proteins, which we termed EXpanding TK, after their seemingly unique propensity to gene duplication and exon shuffling, not only in amphioxus but also across all metazoan groups. Due to this extreme tendency to both retention and expansion of TK genes, amphioxus harbors the richest and most diverse TK repertoire among all metazoans studied so far, retaining most of the gene complement of its ancestors, but having evolved its own repertoire of genetic novelties.     

Expression and characterization of the thymidine kinase gene of African swine fever virus.

The thymidine kinase (TK) gene of African swine fever virus (ASFV) was located within the viral genome by using two degenerate oligonucleotide probes derived from sequences of the vaccinia virus and cellular TK genes. The TK gene was mapped within a 0.72-kbp BglII-XhoI fragment (0.242 to 0.246 map units) derived from a 23.9-kbp SalI-B fragment of the ASFV genome. Identification of this region as the ASFV TK gene was confirmed by expression of TK in Escherichia coli and by the synthesis of active TK in a cell-free system programmed with RNA synthesized in vitro. The sequenced gene for TK includes an open reading frame of 588 nucleotides encoding a protein of 196 amino acids. The deduced amino acid sequence shows 32.4% identity with the TK of vaccinia virus.     

人体胸苷激酶(TK)基因在共转染过程中结构改变的研究

用磷酸钙沉淀法,我们把带有人体TK基因片段的重组噬菌体DNA共转染小鼠Ltk~-细胞,得到TK~+转化细胞克隆。同时用HeLa细胞DNA转染Ltk~-细胞,得到第一代TK~+转化细胞,再进行第二轮、第三轮转染,得到第二代、第三代TK~+转化细胞。比较其转化效率,结果基因组DNA转化率大于基因两个片段的共转化率,更大于不加携带者DNA的共转化率。限制性内切酶消化各种TK~+转化细胞的DNA,与TK基因探针作Southern印迹杂交,结果表明两个TK基因片段共转染Ltk~-细胞时,它们可以在受体细胞里重建成一个具有完整功能的遗传单位,但在连接过程中结构可以发生改变。当用HeLa纽胞DNA转染Ltk~-细胞时,虽然连续三代转染,每一代TK~+转化细胞中人TK基因的结构未发现变化。但也不能排除基因结构改变的频率很低未能有效检出的可能性。     

Structure and properties of an engineered transketolase from maize

The gene specifying plastid transketolase (TK) of maize (Zea mays) was cloned from a cDNA library by southern blotting using a heterologous probe from sorghum (Sorghum bicolor). A recombinant fusion protein comprising thioredoxin of Escherichia coli and mature TK of maize was expressed at a high level in E. coli and cleaved with thrombin, affording plastid TK. The protein in complex with thiamine pyrophoshate was crystallized, and its structure was solved by molecular replacement. The enzyme is a C2 symmetric homodimer closely similar to the enzyme from yeast (Saccharomyces cerevisiae). Each subunit is folded into three domains. The two topologically equivalent active sites are located in the subunit interface region and resemble those of the yeast enzyme.     

Identification of novel small-molecule inhibitors for human transketolase by high-throughput screening with fluorescent intensity (FLINT) assay

The metabolic enzyme transketolase (TK) plays a crucial role in tumor cell nucleic acid synthesis, using glucose through the elevated nonoxidative pentose phosphate pathway (PPP). Identification of inhibitors specifically targeting TK and preventing the nonoxidative PPP from generating the RNA ribose precursor, ribose-5-phosphate, provides a novel approach for developing effective anticancer therapeutic agents. The full-length human transketolase gene was cloned and expressed in Escherichia coli and the recombinant human transketolase protein purified to homogeneity. A fluorescent intensity (FLINT) assay was developed and optimized. Library compounds were screened in a high-throughput screening (HTS) campaign using the FLINT assay. Fifty-four initial hits were identified. Among them, 2 scaffolds with high selectivity, ideal physiochemical properties, and low molecular weight were selected for lead optimization studies. These compounds specifically inhibited in vitro TK enzyme activity and suppressed tumor cell proliferation in at least 3 cancer cell lines: SW620, LS174T, and MIA PaCa-2. Identification of these active scaffolds represents a good starting point for development of drugs specifically targeting TK and the nonoxidative PPP for cancer therapy.     

Transfer of the herpes simplex thymidine kinase gene from human cells to mouse cells by means of metaphase chromosomes.

Thymidine kinase (TK)-deficient human cells were infected with ultraviolet light-inactivated Herpes simplex virus type 1, and "transformed" cells that expressed Herpes TK activity were isolated. Purified metaphase chromosomes were isolated from the transformed human line and incubated with TK-deficient mouse cells. TK+ cells were selected, and it was shown that these cells were gene transferents which expressed Herpes TK activity, identical to that found in the transformed human cells. The gene transferents contained no intact human chromosomes. When removed from selective pressure, the gene transferents rapidly lost the TK+ phenotype. However, upon continued growth in nonselective medium, a subpopulation in which the TK+ phenotype had become more stabilized appeared. These results suggest that the Herpes gene for thymidine kinase has integrated into the genome of the HSV-transformed human cells and that it can be transferred to other cells by means of purified metaphase chromosomes.     

Immobilised enzyme microreactor for screening of multi-step bioconversions: characterisation of a de novo transketolase-ω-transaminase pathway to synthesise chiral amino alcohols

Complex molecules are synthesised via a number of multi-step reactions in living cells. In this work, we describe the development of a continuous flow immobilized enzyme microreactor platform for use in evaluation of multi-step bioconversion pathways demonstrating a de novo transketolase/ω-transaminase-linked asymmetric amino alcohol synthesis. The prototype dual microreactor is based on the reversible attachment of His₆-tagged enzymes via Ni-NTA linkage to two surface derivatised capillaries connected in series. Kinetic parameters established for the model transketolase (TK)-catalysed conversion of lithium-hydroxypyruvate (Li-HPA) and glycolaldehyde (GA) to l-erythrulose using a continuous flow system with online monitoring of reaction output was in good agreement with kinetic parameters determined for TK in stop-flow mode. By coupling the transketolase catalysed chiral ketone forming reaction with the biocatalytic addition of an amine to the TK product using a transaminase (ω-TAm) it is possible to generate chiral amino alcohols from achiral starting compounds. We demonstrated this in a two-step configuration, where the TK reaction was followed by the ω-TAm-catalysed amination of l-erythrulose to synthesise 2-amino-1,3,4-butanetriol (ABT). Synthesis of the ABT product via the dual reaction and the on-line monitoring of each component provided a full profile of the de novo two-step bioconversion and demonstrated the utility of this microreactor system to provide in vitro multi-step pathway evaluation.     

Regulation of chimeric phosphoenolpyruvate carboxykinase genes by the trans-dominant locus TSE1.

Extinction of phosphoenolpyruvate carboxykinase (PCK) gene expression in hepatoma x fibroblast hybrids is mediated by a trans-acting genetic locus designated tissue-specific extinguisher 1 (TSE1). To identify PCK gene sequences required for extinction, hepatoma transfectants expressing PCK-thymidine kinase (TK) chimeric genes were fused with TK- fibroblasts and PCK-TK expression in the resulting hybrids was monitored. Expression of a PCK-TK chimera containing PCK sequences between base pairs -548 and +73 was extinguished in four of five hepatoma transfectants tested, although hybrids derived from one transfectant clone failed to extinguish PCK-TK expression. In contrast, crosses between hepatoma transfectants expressing the herpesvirus TK gene from its own promoter and TK- fibroblasts produced TK+ hybrids; extinction of the transfected TK gene was not observed. Thus, rat PCK gene sequences between base pairs -548 and +73 are sufficient for tissue-specific extinction in hybrid cells. Extinction of PCK-TK gene expression in transfectant microcell hybrids mapped specifically to human chromosome 17, the site of human TSE1.     

Construction and isolation of a transmissible retrovirus containing the src gene of Harvey murine sarcoma virus and the thymidine kinase gene of herpes simplex virus type 1

We constructed lambda recombinants containing the Harvey murine sarcoma virus genome and the thymidine kinase (tk) gene of herpes simplex virus type 1 linked to each other. The tk gene was located in a position downstream from both the long terminal repeat and the src gene of Harvey murine sarcoma virus. The DNAs of the lambda recombinants were used to transfect NIH3T3 mouse fibroblasts in order to obtain Harvey murine sarcoma virus DNA-induced foci of transformed cells. The transformed foci were superinfected with a helper-independent retrovirus, and new individual retrovirus were isolated from the superinfected foci. The new viruses could induce focus formation on NIH3T3 cells and could convert NIH3T3(TK-) cells into TK+ cells by carrying the herpes simplex virus type 1 tk gene into the TK- cells. From virus-infected cells, we isolated nonproducer foci on NIH3T3 cells and TK+ transformants on NIH3T3(TK-) cells containing one such new viral genome coding for the dual properties. The new retroviral sequence in the nonproducer cells could be rescued into virus particles at high titers by superinfection with a helper-independent retrovirus. A hybridization analysis indicated that the recombinant virus contained both the Harvey murine sarcoma virus src sequence and the tk gene sequence in a single RNA species approximately 4.9 kilobases long. We concluded that retroviruses can be used as true vectors for genes other than genes that lead to oncogenesis.     

The SR splicing factors ASF/SF2 and SC35 have antagonistic effects on intronic enhancer-dependent splicing of the beta-tropomyosin alternative exon 6A.

Exons 6A and 6B of the chicken beta-tropomyosin gene are mutually exclusive and selected in a tissue-specific manner. Exon 6A is present in non-muscle and smooth muscle cells, while exon 6B is present in skeletal muscle cells. In this study we have investigated the mechanism underlying exon 6A recognition in non-muscle cells. Previous reports have identified a pyrimidine-rich intronic enhancer sequence (S4) downstream of exon 6A as essential for exon 6A 5'-splice site recognition. We show here that preincubation of HeLa cell extracts with an excess of RNA containing this sequence specifically inhibits exon 6A recognition by the splicing machinery. Splicing inhibition by an excess of this RNA can be rescued by addition of the SR protein ASF/SF2, but not by the SR proteins SC35 or 9G8. ASF/SF2 stimulates exon 6A splicing through specific interaction with the enhancer sequence. Surprisingly, SC35 behaves as an inhibitor of exon 6A splicing, since addition to HeLa nuclear extracts of increasing amounts of the SC35 protein completely abolish the stimulatory effect of ASF/SF2 on exon 6A splicing. We conclude that exon 6A recognition in vitro depends on the ratio of the ASF/SF2 to SC35 SR proteins. Taken together our results suggest that variations in the level or activity of these proteins could contribute to the tissue-specific choice of beta-tropomyosin exon 6A. In support of this we show that SR proteins isolated from skeletal muscle tissues are less efficient for exon 6A stimulation than SR proteins isolated from HeLa cells.     

De novo methylation as major event in the inactivation of transfected herpesvirus thymidine kinase genes in human cells

Spontaneous inactivation of integrated thymidine kinase genes was studied in three human cell lines, one with multiple copies and two with a single copy of a transfected shuttle plasmid containing two selectable genes: the HSV tk gene and the Eco gpt gene. Selection for gpt expression prevented the isolation of TK- mutants which are the result of plasmid loss. Under these conditions TK- clones were isolated with a frequency of 5.10(-6) both with the cell line containing 5 or 6 copies of the tk gene and with one of the two cell lines containing one copy of this gene. This inactivity of the tk gene was associated with de novo methylation as the number of HAT-resistant (TK+) clones strongly increased after growth of the TK- derivatives in the presence of the demethylating agent, 5-azacytidine. Digestion with methylation-sensitive restriction enzymes revealed two different patterns of DNA methylation in the genomic DNA of TK- variants. In the TK- derivatives of the cell line containing multiple copies of the tk gene many HpaII restriction sites in the gene copies were insensitive to digestion. These HpaII sites were, however, not methylated in TK- variants of the cell line containing one copy of the plasmid, and methylated CpGs could be detected only with EcoRI which recognizes the cGAATTCg sequence in the tk promoter region. With the other of the two single-copy TK+ cell lines no TK- mutants were obtained, suggesting that the position of a gene in the genome is an important factor in determining the frequency and the extent of de novo methylation. Additionally, we observed that remethylation is an even more efficient process of gene inactivation as TK+ clones reactivated with 5-azacytidine can become TK- again at a 100-fold higher rate than the original TK+ cell line.     

Influence of donor substrate on kinetic parameters of thiamine diphosphate binding to transketolase

The two-step mechanism of interaction of thiamine diphosphate (ThDP) with transketolase (TK) has been studied: TK + ThDP <--> TK...ThDP <--> TK*-ThDP. The scheme involves the formation of inactive intermediate complex TK...ThDP followed by its transformation into catalytically active holoenzyme, TK*-ThDP. The dissociation and kinetic constants for individual stages of this process have been determined. The values of forward and backward rate constants change in the presence of the donor substrate hydroxypyruvate. This finally leads to an increase in the overall affinity of the coenzyme to TK.     

Identification and nucleotide sequence of the thymidine kinase gene of Shope fibroma virus.

The thymidine kinase (TK) gene of Shope fibroma virus (SFV), a tumorigenic leporipoxvirus, was localized within the viral genome with degenerate oligonucleotide probes. These probes were constructed to two regions of high sequence conservation between the vaccinia virus TK gene and those of several known eucaryotic cellular TK genes, including human, mouse, hamster, and chicken TK genes. The oligonucleotide probes initially localized the SFV TK gene 50 kilobases (kb) from the right terminus of the 160-kb SFV genome within the 9.5-kb BamHI-HindIII fragment E. Fine-mapping analysis indicated that the TK gene was within a 1.2-kb AvaI-HaeIII fragment, and DNA sequencing of this region revealed an open reading frame capable of encoding a polypeptide of 176 amino acids possessing considerable homology to the TK genes of the vaccinia, variola, and monkeypox orthopoxviruses and also to a variety of cellular TK genes. Homology matrix analysis and homology scores suggest that the SFV TK gene has diverged significantly from its counterpart members in the orthopoxvirus genus. Nevertheless, the presence of conserved upstream open reading frames on the 5' side of all of the poxvirus TK genes indicates a similarity of functional organization between the orthopoxviruses and leporipoxviruses. These data suggest a common ancestral origin for at least some of the unique internal regions of the leporipoxviruses and orthopoxviruses as exemplified by SFV and vaccinia virus, respectively.     

Single and Coincident Intragenic Mutations Attributable to Gene Conversion in a Human Cell Line

Two polymorphic sites are located within the heterozygous TK1 locus in the human lymphoblastoid cell line TK6: an inactivating frameshift in exon 4 of the nonfunctional allele and a phenotypically silent frameshift in exon 7 of the functional allele. Through the use of these intragenic polymorphisms and microsatellite markers that flank TK1, we demonstrate that partial gene conversion accounts for 3/75 (0.04) spontaneous and 9/163 (0.06) X-ray-induced TK1(-) mutants, thus comprising a significant component of forward mutations at this locus. In all cases, the conversion tract is <1 cM, rendering double exchange a remote alternate explanation for these results. Sequence analysis of full length TK1 cDNA provides rigorous exclusion of deletion events as a mechanism for generation of these allelotypes. Detailed examination of allelotypes in TK1(-) mutants identified two mechanisms for the generation of coincident sequence alterations that sometimes accompanied gene conversions. Mutations within the conversion tract were attributed to either error-prone gap filling synthesis during recombinational repair or mismatch repair within a heteroduplex region following branch migration. These findings suggest that a proportion of point mutations may not be targeted to sites of DNA base damage, but rather may arise as secondary consequences from the repair of DNA strand breaks.