Insertion of a nuclear factor kappa B DNA nuclear-targeting sequence potentiates suicide gene therapy efficacy in lung cancer cell lines.

Lung cancer currently causes the majority of cancer-related deaths worldwide and new treatments are in high demand. Gene therapy could be a promising treatment but currently lacks sufficient efficiency for clinical use, primarily due to limited cellular and nuclear DNA delivery. In the present study, we investigated whether it was possible to exploit the endogenous nuclear-shuttling activity by the nuclear factor kappa B (NFκB) system, which is highly prominent in many cancers as well as lung cancer. We observed that insertion of a DNA nuclear-targeting sequence (DTS) recognized by NFκB could improve plasmid nuclear delivery and enhance the therapeutic effect of a validated transcriptionally cancer-targeted suicide gene therapy system. A clear correlation between the number of inserted NFκB-binding sites and the therapeutic effect of the suicide system was observed in both small cell lung cancer (SCLC) and non-SCLC cell lines. The effect was observed to be due to elevated nuclear translocation of the suicide gene-encoding plasmids. The results show that a significant improvement of gene therapeutic efficiency can be obtained by increasing the intracellular trafficking of therapeutic DNA. This is to our knowledge the first time a DTS strategy has been implemented for suicide gene therapy.

New efficient synthesis of thymidine cyclic 3',5'-phosphorofluoridate and its sulfur analogue via the phosphoroamidite route.

We describe the convenient synthesis of thymidine cyclic 3', 5'-phosphorofluoridate 6, which is superior to that previously reported. Our procedure is based on a sequence of reactions utilizing 3 as the key substrate. Similar sequence of reaction leads to the sulfur analogues of 6 the thymidine cyclic 3',5'-phosphorofluoridothioate 7.

Accuracy of protein biosynthesis: quasi-species nature of proteins and possibility of error catastrophes.

Yeast aminoacyl-tRNA synthetases act in a multi-step process when recognizing their cognate amino acids; this identification event includes "physical" binding and "chemical" proof-reading steps. However, the various enzymes use these single steps at different degrees, and their specificities with regard to the 20 naturally occurring amino acids deviate considerably from each other. The characteristic discrimination factors D were determined for seven synthetases in vitro: the highest specificity with D values between 28,000 and > 500,000 were observed with tyrosyl-tRNA synthetase, the lowest values between 130 and 1700 for lysyl-tRNA synthetase. The tested class I enzymes are more specific than the investigated class II enzymes, and it may be put into discussion whether this observation can be generalized. Error rates in amino acid recognition differ not only between the individual aminoacyl-tRNA synthetases but also considerably for different amino acids sorted by the same enzyme. Strikingly, all investigated enzymes exhibit a poor specificity in discrimination of cysteine and tryptophan from their cognate substrates, and these cases may be regarded as "specificity holes". In view of the observed specificities a protein consisting of 700 amino acids would contain maximally up to five "incorrect" residues, if the in vitro error rates are also valid under in vivo conditions. Therefore the terminus "quasi-species", an expression which was originally created for nucleic acids, is justified. The "quasi-species" nature of proteins may become important when genes are translated in different organisms with different accuracies of the translation apparatus. In such cases different "quasi-species" will be obtained. Using our data in mathematical models which predict the stability of protein synthesizing systems, we find that they are consistent with a stable yeast organism which is not prone to die by an "error catastrophe". However, this appears only if average values from our experiments are used for calculations. If a single compound, e.g. the arginine analog canavanine, is discriminated very poorly from the cognate substrate, or if the "specificity holes" get larger, an "error catastrophe" must be envisaged.

Isolation and analysis of mutated histidyl-tRNA synthetases from Escherichia coli.

Amino terminally deleted and point-mutated histidyl-tRNA synthetases were purified from E. coli via betaGal fusion proteins. A hinge region proximal and distal to the factor Xa cleavage region was necessary to cut the betaGal-fusion proteins efficiently under very mild nondenaturing conditions. N-terminal addition of either methionine or valine to this enzyme (its starting N-formyl-methionine is in vivo post-translationally removed) or the deletion of 6 amino terminal amino acids decreased the specific aminoacylation activity 2- to 7-fold. Further N-terminal deletions of 10 or 17 amino acids caused significantly reduced aminoacylation (100-fold) and ATP/PPi exchange (10-fold) activities, and a reduced binding affinity for histidine. Removal of 18 or more amino acids from the N-terminus thereby removing residues from MOTIF 1 resulted in inactive histidyl-tRNA synthetase mutants. Two point mutations within the histidyl-adenylate binding pocket, R259Q and R259K, also blocked histidyl-tRNA synthetase activity without affecting histidine or ATP binding. The experiments shown identify a highly conserved N-terminal R/KG-patch in front of MOTIF 1 as well as R259 as vital for full enzymatic activity.

The modified wobble base inosine in yeast tRNAIle is a positive determinant for aminoacylation by isoleucyl-tRNA synthetase.

Earlier work by two independent groups has established the fact that anticodons GAU and LAU of Escherichia coli tRNAIle isoacceptors play a critical role in the tRNA identity. Yeast possesses two isoleucine transfer RNAs, a major one with anticodon IAU and a minor one with anticodon PsiAPsi which are derived from the post-transcriptional modification of AAU and UAU gene sequences, respectively. We present direct evidence which reveals that inosine is a positive determinant for yeast isoleucyl-tRNA synthetase. We also show that yeast tRNAMet with guanosine at the wobble position becomes aminoacylated with isoleucine while methionine acceptance is lost. As inosine and guanosine share the 6-keto and the N-1 hydrogen groups, this suggests that these hydrogen donor and acceptor groups are determinants for isoleucine specificity. The role of the minor tRNAIle anticodon pseudouridines in tRNA isoleucylation could not be tested directly but was deduced from a 40-fold decrease in the activity of the unmodified transcript. The presence of the NHCO structure in guanosine, inosine, pseudouridine, and lysidine suggests a unifying model of wobble base recognition by the yeast and E. coli isoleucyl-tRNA synthetase. In contrast to lysidine which switches the identity of the tRNA from methionine to isoleucine [Muramatsu, T., Nishikawa, K., Nemoto, F., Kuchino, Y., Nishimura, S., Miyazawa, T., & Yokoyama, S. (1988) Nature 336, 179-181], pseudouridine-34 does not modify the specificity of the yeast minor tRNAIle since U-34 is a strong negative determinant for yeast MetRS. Therefore, the major role of Psi-34 (in combination with Psi-36 or not) is likely in isoleucine AUA codon specificity and translational fidelity.

Phenylalanyl-tRNA synthetase from yeast and its discrimination of 19 amino acids in aminoacylation of tRNA(Phe)-C-C-A and tRNA(Phe)-C-C-A(3'NH2).

For discrimination between phenylalanine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for phenylalanine, discrimination factors, D, of 190-6300 have been determined from kcal and K(m) values. Generally, phenylalanyl-tRNA synthetase is more specific than the class II enzymes specific for Lys and Thr, but works with lower accuracy than the class I enzymes specific for IIe, Tyr, and Arg. In aminoacylation of tRNA(Phe)-C-C-A(3'NH2) discrimination factors D1 vary between 80-1610. Pre-transfer proof-reading factors II1 are in the range 2.3-74, post-transfer proof-reading factors II2 in the range 1.0-4.6, showing that pre-transfer proof-reading is the main correction step, post-transfer proofreading is less effective or negligible. Initial discrimination factors (I1 and I2) caused by differences in Gibbs free energies of binding between phenylalanine and non-cognate amino acids have been calculated assuming a two-step binding process. Factors I1 can be related to hydrophobic-interaction forces depending on accessible surface areas of the amino acids, factors I2 scatter about a low mean value and do not show any relation to amino acid structures or surfaces, indicating less checking of amino acid side chains in the putative second binding step.

Threonyl-tRNA synthetase from yeast. Discrimination of 19 amino acids in aminoacylation of tRNA(Thr)-C-C-A and tRNA(Thr)-C-C-A(2'NH2).

For discrimination between threonine and 18 other naturally occurring non-cognate amino acids by the class II aminoacyl-tRNA synthetase specific for threonine, discrimination factors (D) have been determined from Kca and Km values. The lowest values were found for Cys, Met, Val (D = 70-280), indicating that threonine is only 70-280-times more often esterified to tRNA(Thr)-C-C-A than are these non-cognate compounds at the same amino acid concentrations. The highest D values have been observed for Gly, Pro, Gln, Leu, Phe, and Lys (D = 1000-2000), for the other non-cognate amino acids D values are in the medium range 300-1000. Generally, threonyl-tRNA synthetase is less specific than the class I enzymes specific for Ile, Val, Tyr, Arg, but more specific than the only investigated class II enzyme specific for Lys. In aminoacylation of tRNA(Thr)-C-C-A(2'NH2) discrimination factors D1 are in the range 2-170. From D1 values and AMP-formation stoichiometry, pre-transfer proof-reading factors II1, were determined; post-transfer proof-reading factors II2 were determined from D values and AMP-formation stoichiometry in acylation of tRNA(Thr)-C-C-A. II1 values are in the range 1.8-33, II2 values in the range 1.4-22, thus threonyl-tRNA synthetase shows the highest post-transfer proof-reading activity of six investigated synthetases (specific for Ile, Val, Tyr, Arg, Lys). Initial discrimination factors caused by differences in Gibbs free energies of binding between threonine and non-cognate amino acids have been calculated from discrimination and proof-reading factors. Assuming a two-step binding process, two factors (I1 and I2) have been determined which can be related to hydrophobic interaction forces depending on accessible surface areas of the amino acids. The threonine side chain must be bound by hydrophobic forces and two hydrogen bonds. In contrast to proof-reading factors obtained with the synthetases specific for Ile, Val, Tyr, Arg, and Lys, proof-reading factors II1 and II2 obtained with threonyl-tRNA synthetase are also related to hydrophobic interaction of the amino acid side chains and the enzyme. Threonyl-tRNA synthetase examines side chain structures of amino acids in the four postulated recognition steps, for each step the enzyme uses special distinct structures or conformations of the binding cleft.

Lysyl-tRNA synthetase from yeast. Discrimination of amino acids by native and phosphorylated species.

Discrimination factors (D) which are characteristic for discrimination between lysine and 19 naturally occurring non-cognate amino acids have been determined from kcat and Km values for native and phosphorylated lysyl-tRNA synthetases from yeast. Generally, both species of this class II aminoacyl-tRNA synthetase are considerably less specific than the class I synthetases specific for isoleucine, valine, tyrosine, and arginine. D values of the native enzyme are in the range 90-1700, D values of the phosphorylated species in the range 40-770. The phosphorylated enzyme acts faster and less accurately. In aminoacylation of tRNALys-C-C-A(2'NH2) discrimination factors D1 vary over 30-980 for the native and over 8-300 for the phosphorylated enzyme. From AMP formation stoichiometry and D1 values pretransfer proof-reading factors (II1) of 1.1-56 were calculated for for the native enzyme, factors of 1.0-44 for the phosphorylated species. Post-transfer proof-reading factors (II2) were calculated from D values and AMP formation stoichiometry in acylation of tRNALys-C-C-A. Pretransfer proof-reading is the main correction step, posttransfer proof-reading is less effective or negligible (II2 approximately 1-8). Initial discrimination factors (I), which are due to differences in Gibbs free energies of binding between lysine and noncognate substrates (delta delta GI), were calculated from discrimination and proof-reading factors. In contrast to class I synthetases, for lysyl-tRNA synthetase only one initial discrimination step can be assumed and amino acid recognition is reduced to a three-step process instead of the four-step recognition observed for the class I synthetases. Plots of delta delta GI values against accessible surface areas of amino acids show clearly that phosphorylation of the enzyme changes the structures of the amino acid binding sites. This is illustrated by a hypothetical 'stopper model' of these sites.

Oxygen and acceleration.

Significant numbers of high performance fighter aircraft continue to be lost due to acceleration (Gz)-induced loss of consciousness. This is due to the rapid onset of high sustained Gz which results in the sudden loss of blood flow to the brain. Present research efforts to extend Gz tolerance are directed toward the maintenance of cerebral blood flow, i.e. straining maneuvers, anti-G suits, tilt seats. The purpose of this paper is to review the present situation and discuss the potential benefit of breathing 100% oxygen at high pressure. Basic science evidence and experience with hyperbaric oxygen in the clinical setting suggest that if the oxygen concentration in the brain tissue is increased, prior to the onset of Gz, additional time of useful consciousness may be realized. The advanced tactical fighter, now in the design stage, will have a sustained Gz capability of 12-14 Gz. This is above human tolerance at the present time and provides an impetus for future acceleration research. Continued aircraft loss due to Gz loss of consciousness will remain an operational problem in aerospace pathology in the 1990s and beyond.

Aminoacylation of tRNAs as critical step of protein biosynthesis.

Isoleucyl-tRNA synthetases isolated from commercial baker's yeast and E coli were investigated for their sequences of substrate additions and product releases. The results show that aminoacylation of tRNA is catalyzed by these enzymes in different pathways, eg isoleucyl-tRNA synthetase from yeast can act with four different catalytic cycles. Amino acid specificities are gained by a four-step recognition process consisting of two initial binding and two proofreading steps. Isoleucyl-tRNA synthetase from yeast rejects noncognate amino acids with discrimination factors of D = 300-38000, isoleucyl-tRNA synthetase from E coli with factors of D = 600-68000. Differences in Gibbs free energies of binding between cognate and noncognate amino acids are related to different hydrophobic interaction energies and assumed conformational changes of the enzyme. A simple hypothetical model of the isoleucine binding site is postulated. Comparison of gene sequences of isoleucyl-tRNA synthetase from yeast and E coli exhibits only 27% homology. Both genes show the 'HIGH'- and 'KMSKS'-regions assigned to binding of ATP and tRNA. Deletion of 250 carboxyterminal amino acids from the yeast enzyme results in a fragment which is still active in the pyrophosphate exchange reaction but does not catalyze the aminoacylation reaction. The enzyme is unable to catalyze the latter reaction if more than 10 carboxyterminal residues are deleted.

The pAX plasmids: new gene-fusion vectors for sequencing, mutagenesis and expression of proteins in Escherichia coli.

A family of plasmid cloning vectors have been constructed, allowing both the sequencing and mutagenesis of foreign genes and the easy isolation of their expression products via fusion proteins in Escherichia coli. Fusion proteins can be inducibly expressed and isolated by affinity chromatography on APTG-Sepharose. The fusion protein consists of beta-galactosidase at the N-terminus, linked by a collagen 'hinge' region containing blood coagulation factor Xa cleavage site to the foreign protein at the C terminus. The factor Xa cleavage site at the N-terminal side of the foreign protein allows the release of the desired amino acid sequence under mild conditions. A multiple cloning site in all three reading frames and stop codons followed by the strong lambda t0 terminator facilitate simple gene insertions and manipulations. The intergenic region of the phage f1 inserted in both orientations allows the isolation of single-stranded DNA from either plasmid-strand for sequencing and mutagenesis. This vector family has been successfully used for the expression and purification of the isoleucyl-tRNA synthetase from Saccharomyces cerevisiae and the histidyl-tRNA synthetase from E. coli.

Fidelity in the aminoacylation of tRNA(Val) with hydroxy analogues of valine, leucine, and isoleucine by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli.

Several analogues of valine, leucine, and isoleucine carrying hydroxyl groups in the gamma- or delta-position have been tested in the aminoacylation of tRNA by valyl-tRNA synthetases from Saccharomyces cerevisiae and Escherichia coli. Results of the ATP/PPi exchange and of the aminoacylation reactions indicate that the amino acid analogues not only can form the aminoacyl adenylate intermediate but are also transferred to tRNA. However, the fact that the reaction consumes an excess of ATP indicates that the misactivated amino acid analogue is hydrolytically removed. Thus, valyl-tRNA synthetase from S. cerevisiae shows a high fidelity in forming valyl-tRNA. Although the much bulkier amino acid analogues allo- and iso-gamma-hydroxyvaline and allo- and iso-gamma-hydroxyisoleucine are initially charged to tRNA, the misaminoacylated tRNA(Val) is enzymatically deacylated. This cleavage reaction is mediated by the hydroxyl groups of the amino acid analogues which are converted into the corresponding lactones.

Valyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Val)-C-C-A and tRNA(Val)-C-C-A(3'NH2).

For discrimination between valine and the 19 naturally occurring noncognate amino acids, as well as between valine and 2-amino-isobutyric acid by valyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values in aminoacylation of tRNA(Val)-C-C-A. The lowest values were found for Trp, Ser, Cys, Lys, Met and Thr (D = 90-870), showing that valine is 90-870 times more frequently attached to tRNA(Val)-C-C-A than the noncognate amino acids at the same amino acid concentrations. The other amino acids exhibit D values between 1,100 and 6200. Generally, valyl-tRNA synthetase is considerably less specific than isoleucyl-tRNA synthetase, but this may be partly compensated in the cell by valine concentrations higher than those of noncognate acids. In aminoacylation of tRNA(Val)-C-C-A(3'NH2) discrimination factors D1 are in the range of 40-1260. From D1 values and AMP formation stoichiometry, pretransfer proof-reading factors pi 1 were determined: post-transfer proof-reading factors II 2 were determined from D values and AMP formation stoichiometry in acylation of tRNA(Val)-C-C-A. II 1 values (7-168) show that pretransfer proof-reading is the main correction step, post-transfer proof-reading (II 2 approximately 1-7) is less effective and in some cases negligible. Initial discrimination factors were calculated from discrimination and proof-reading factors according to a two-step binding process. These factors, due to different Gibbs free energies of binding can be related to hydrophobic interaction forces, and a hypothetical 'stopper' model of the amino-acid-binding site is discussed.

Arginyl-tRNA synthetase from yeast. Discrimination between 20 amino acids in aminoacylation of tRNA(Arg)-C-C-A and tRNA(Arg)-C-C-A(3'NH2).

For discrimination between arginine and 19 other amino acids in aminoacylation of tRNA(Arg)-C-C-A by arginyl-tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values. The lowest values were found for Trp, Cys, Lys (D = 800-8500), showing that arginine is 800-8500 times more often incorporated into tRNA(Arg)-C-C-A than noncognate acids at the same amino acid concentrations. The other noncognate amino acids exhibit D values between 10,000 and 60,000. In aminoacylation of tRNA(Arg)-C-C-A(3'NH2) discrimination factors D1 are in the range 10-600. From these values and AMP formation stoichiometry, pretransfer proof-reading factors II1 were determined; from D values and AMP stoichiometry in aminoacylation of tRNA(Arg)-C-C-A, posttransfer proof-reading factors II2 could be calculated, II1 values between 2 and 120 show that pretransfer proof-reading is the main correction step, posttransfer proof-reading (II2 approximately 1-10) plays a marginal role. Initial discrimination factors due to different Gibbs free energies of binding between arginine and the noncognate amino acids were calculated from discrimination and proof-reading factors. According to a two-step binding process, two factors (I1 and I2) were determined. They can be related to hydrophobic interaction forces and hydrogen bonds that are especially formed by the arginine side chain. A hypothetical 'stopper' model of the amino acid recognition site is discussed.

Preparation of a novel psoralen containing deoxyadenosine building block for the facile solid phase synthesis of psoralen-modified oligonucleotides for a sequence specific crosslink to a given target sequence.

4,5',8-Trimethylpsoralen was attached to the C8-position of deoxyadenosine via a sulfur atom and a five carbon atom linker. The modified deoxyadenosine was then converted to a protected phosphoramidite and used as unusual as a building block for solid phase oligodeoxyribonucleotide synthesis. The efficiency of the photoreaction of a psoralen-modified oligonucleotide to a complementary matrix strand reached more than 90% within a 1 hour irradiation time at a wavelength of 345 nm.

Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-A.

For discrimination between isoleucine and 19 other amino acids by isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600, discrimination factors D have been determined from kcat and Km values in amino-acylation of cognate tRNA(Ile)-C-C-A. Factors D are also products of initial discrimination factors I' and proof-reading factors II'; D = I' II'. Factors II' were calculated from AMP formation stoichiometries and factors I' as quotients of D and II'; I' = D/II'. II' is considered as a product of a pre- and post-transfer proof-reading factor (II' = II1II2), I' as a product of initial discrimination factors I1 and I2 which are due to two steps of initial discrimination. With the yeast enzyme the highest accuracy is achieved in discrimination between isoleucine and valine (D = 38,000); other D values in a high range (10,000-20,000) are observed for Gly, Ser, Thr, Leu and Met; the lowest factors D belong to Cys, Asp, Asn and Trp (300-700); the remaining amino acids are discriminated with medium D values (1000-10,000). Discrimination factors D observed for isoleucyl-tRNA synthetase from E. coli are on average 2-3 times higher than for the yeast enzyme. Highest values were found in discrimination against Gly, Ala and Val (60,000-72,000), the lowest values for Cys, Arg and Trp (600-3000); the other amino acids exhibit D values between 20,000 and 50,000. Initial discrimination factors can be related to hydrophobic interaction forces between the substrates and the enzyme; a hypothetical model of the amino acid binding site is discussed.

Isoleucyl-tRNA synthetase from baker's yeast and from Escherichia coli MRE 600. Discrimination of 20 amino acids in aminoacylation of tRNA(Ile)-C-C-A(3'NH2).

For discrimination between isoleucine and the other 19 naturally occurring amino acids by isoleucyl-tRNA synthetases from baker's yeast and from Escherichia coli MRE 600 discrimination factors have been determined from kcat and Km values in aminoacylation of the modified tRNA(Ile)-C-C-A(3'NH2). Discrimination factors D1 are products of an initial discrimination factor and a proof-reading factor: D1 = I1.II1. From discrimination factors and AMP formation stoichiometry factors I1 and II1 were calculated. D1 values obtained with the enzyme from E. coli are generally higher than those observed with the yeast enzyme, in some cases up to ten times. With both enzymes low D1 values are found for cysteine, valine, and tryptophan (20-200), the highest values for glycine, alanine, and serine (600-4000). I1 values calculated for the E. coli enzyme are slightly higher (4-145) than the factors observed with the yeast enzyme (1-85), proof-reading factors II1 of the E. coli enzyme are scattering about a mean value about 70, those of the yeast enzyme about a mean value about 50. Initial discrimination factors I1 are directly related to hydrophobic interaction forces between the substrates and the enzymes. Plots of Gibbs free energy differences calculated from these factors are linearly related to the accessible surface areas of the amino acids. A hypothetical model of the binding site can be given in which selection of amino acids is achieved by hydrophobic forces and removal of steric hindrance.