J Pediatr Gastroenterol Nutr, 1999 Feb, 28(2), 147 - 51 Lymphocyte subset profile of young healthy children residing in a rural area: possible role of recurrent gastrointestinal infections; Granot E et al.; BACKGROUND: Lymphocyte subsets in healthy children are currently characterized by age-related standards . Because antigenic stimuli play a role in maturation of the immune system after birth, there is a question of whether cellular immune development differs in infants whose living conditions entail extensive antigenic exposure and infants growing up in a more protected environment . METHODS: Peripheral blood lymphocyte subsets were studied in two populations of children of similar age and nutritional status; children belonging to a rural population residing in proximity with farm animals and children from an economically privileged urban population . In each population, children studied included a group with an acute diarrheal episode and a healthy control group . RESULTS: Among rural population children, 65% had experienced at least one episode of gastroenteritis within the previous 3-month-period, compared with less than 10% of urban population children . In the rural population group 15% had experienced two or more episodes of gastroenteritis . The proportion of helper T cells was similar in rural population and urban population children . Among helper T cells, the proportion of CD29+ "memory" cells of the total CD4+ helper T cells was more than two times higher than those in rural population children . The proportion of CD8 cells was higher in rural population children than in urban population children, and the proportion of natural killer cells, CD56+ and CD57+, was two to three times higher in rural population children . Within each population, peripheral blood lymphocyte subsets did not differ between the healthy control group and those with acute diarrhea . CONCLUSIONS: In young children exposure to environmental pathogens and specifically to gastrointestinal antigenic stimuli is a major factor affecting development of the cellular immune response . Young children who have experienced enhanced infectious exposure have a peripheral blood lymphocyte profile similar to that of adults. Prog Nucleic Acid Res Mol Biol, 1999, 62, 55 - 108 Degradation of mRNA in Escherichia coli: an old problem with some new twists; Coburn GA et al.; Metabolic instability is a hallmark property of mRNAs in most if not all organisms and plays an essential role in facilitating rapid responses to regulatory cues . This article provides a critical examination of recent progress in the enzymology of mRNA decay in Escherichia coli, focusing on six major enzymes: RNase III, RNase E, polynucleotide phosphorylase, RNase II, poly(A) polymerase(s), and RNA helicase(s) . The first major advance in our thinking about mechanisms of RNA decay has been catalyzed by the possibility that mRNA decay is orchestrated by a multicomponent mRNA-protein complex (the "degradosome") . The ramifications of this discovery are discussed and developed into mRNA decay models that integrate the properties of the ribonucleases and their associated proteins, the role of RNA structure in determining the susceptibility of an RNA to decay, and some of the known kinetic features of mRNA decay . These models propose that mRNA decay is a vectorial process initiated primarily at or near the 5' terminus of susceptible mRNAs and propagated by successive endonucleolytic cleavages catalyzed by RNase E in the degradosome . It seems likely that the degradosome can be tethered to its substrate, either physically or kinetically through a preference for monphosphorylated RNAs, accounting for the usual "all or none" nature of mRNA decay . A second recent advance in our thinking about mRNA decay is the rediscovery of polyadenylated mRNA in bacteria . Models are provided to account for the role of polyadenylation in facilitating the 3' exonucleolytic degradation of structured RNAs . Finally, we have reviewed the documented properties of several well-studied paradigms for mRNA decay in E . coli . We interpret the published data in light of our models and the properties of the degradosome . It seems likely that the study of mRNA decay is about to enter a phase in which research will focus on the structural basis for recognition of cleavage sites, on catalytic mechanisms, and on regulation of mRNA decay. Gene, 1999 Jan 21, 226(2), 365 - 73 Isolation and expression of the genes encoding the acidic ribosomal phosphoproteins P1 and P2 of the medfly Ceratitis capitata; Gagou ME et al.; The genes of the acidic ribosomal proteins P1 and P2 (CcP1 and CcP2) of the medfly Ceratitis capitata were isolated from a genomic library using homologue DNA probes prepared by PCR . Sequencing and characterization of the two genes revealed strong similarities of the encoded amino acid sequence to the homologous proteins of Drosophila melanogaster and other eukaryotic species . The predicted amino acid sequences of the CcP1 and CcP2 proteins shared an almost identical carboxyl terminal sequence of 10 amino acids common to most known acidic ribosomal proteins . The CcP2 gene lacked intervening sequences in contrast to the CcP1 gene, which was interrupted by an intron of 188 nucleotides . Both genes were cloned in expression pT7 vectors and were expressed in Esherichia coli . The 17- and 15-kDa recombinant proteins reacted with a monoclonal antibody specific to the highly conserved carboxyl terminus of eukaryotic acidic ribosomal proteins, confirming their equivalence to these ribosomal components . Both recombinant proteins were electrophoretically identical to acidic proteins extracted from purified ribosomes of C . capitata. Gene, 1999 Jan 21, 226(2), 263 - 71 Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding; Subrahmanyam S et al.; We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein . This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis . By the use of a radioactively labeled DNA-binding protein and nonradioactive DNA coupled with PCR amplification from gel slices, we show that specific binding sites can be isolated from Escherichia coli genomic DNA . We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E . coli . We have also isolated a second binding site for BirA, the biotin operon repressor/biotin ligase, from the E . coli genome that has a very low binding efficiency compared with the bio operator region. Gene, 1999 Jan 21, 226(2), 233 - 42 Isolation and confirmation of function of the Coccidioides immitis URA5 (orotate phosphoribosyl transferase) gene; Yu JJ et al.; The OPRTase (URA5) gene of the human pathogenic fungus, Coccidioides immitis (Ci), was cloned, sequenced, chromosome-mapped and expressed both by transformation of Escherichia coli and by complementation of wdura5Delta, an auxotrophic strain of Wangiella dermatitidis (Wd) with a disrupted URA5 gene . A functional assay of the recombinant URA5 expressed by E . coli was conducted to ensure that the isolated Ci gene encodes the appropriate enzyme . In the absence of a transformation system for Ci, we also used a reported method of introduction of heterologous DNA into cells of the phylogenetically related fungus, Wangiella dermatitidis, to confirm the function of the Ci URA5 gene . Both the genomic and cDNA sequences of the Ci URA5 gene are presented . The transcription start point and two poly(A) addition sites were confirmed . The gene contains a 714-bp ORF that translates a 238-amino-acid (aa) protein of 25.5kDa and pI of 6.5 . No introns are present . The translated protein contains a single, putative N-glycosylation site . The deduced Ci protein showed 55-63% aa sequence similarity to reported fungal OPRTases . The URA5 gene was mapped to chromosome IV of Ci, and was shown to be a single copy gene by Southern and Northern hybridizations . Transformation of the wdura5Delta mutant to prototrophy was accomplished by electroporation of Wd yeast cells with the Ci URA5 gene . Cellular uptake of the heterologous DNA was confirmed by Southern hybridization . The stable transformants were unable to grow on a medium containing 5-FOA . Expression of the Ci URA5 gene can be used as a selectable marker for a transformation system, and the latter is essential for molecular studies of this pathogenic fungus. Gene, 1998 Dec 11, 224(1-2), 117 - 22 Molecular cloning of Ras cDNA from Penaeus japonicus (Crustacea, decapoda): geranylgeranylation and guanine nucleotide binding; Huang CF et al.; A cDNA was isolated from the shrimp Penaeus japonicus by homology cloning . The shrimp hepatopancreas cDNA encodes a 187-residue polypeptide whose predicted amino acid sequence shares 85% homology with mammalian K-Ras 4B protein and demonstrates identity in the guanine nucleotide binding domains . Expression of the shrimp cDNA in Escherichia coli yielded a 21-kDa polypeptide with a positive reactivity towards the monoclonal antibodies against mammalian Ras . The GTP binding of the shrimp ras-encoded fusion protein was approximated to be 30000units/mg of protein, whereas the binding for GDP was 5000units/mg of protein . Fluorography analysis demonstrated that the prenylation of both shrimp Ras GDP and shrimp Ras GTP by protein geranylgeranyltransferase I of shrimp Penaeus japonicus exceeded the shrimp Ras nucleotide-free form by 10-fold, and fourfold, respectively; that is, the shrimp protein geranylgeranyltransferase I prefers to react with the shrimp ras-encoded p25 fusion protein in the GDP-bound form. Gene, 1998 Dec 28, 225(1-2), 97 - 105 Selection of a cDNA clone for chicken high-mobility-group 1 (HMG1) protein through its unusually conserved 3'-untranslated region, and improved expression of recombinant HMG1 in Escherichia coli; Lee KB et al.; Screening of cDNA libraries for the homologous vertebrate proteins high mobility group (HMG) 1 and 2 using DNA probes based on the coding sequences is likely to result in isolation of both HMG1 and HMG2 clones, as well as pseudogenes, which may be transcribed at low levels . However, the 3'-untranslated regions (UTRs) of HMG1 and 2 are quite distinct, and unusually conserved across species . We have used this property to select the true chicken HMG1 cDNA clone from a chicken lymphocyte cDNA library in lambdagt11, using a probe based on the 3'-UTR of rat HMG1 cDNA . The chicken HMG1 cDNA clone is very similar to all the complete HMG1 cDNA clones isolated so far . We suggest that the sequence designated chicken HMG1 in the GenBank Data Library (Accession number D14314) is, in fact, that of HMG2a {and moreover that the recently reported mouse clone (Accession number AF022465), proposed to encode a new HMG protein, HMG4, is also likely to encode an HMG2a, based on the translated amino-acid sequence and 3'-UTR} . We also report much improved expression of intact recombinant HMG1 in Escherichia coli by the use of chloramphenicol rather than ampicillin selection and conditions that limit cell growth . This should be general for all members of the HMG1 (and 2) family which may be toxic to cells (possibly because of the long acidic tail), and may also prove useful in the production of other such proteins. Gene, 1998 Dec 28, 225(1-2), 31 - 8 Establishment of framework P1 clones for map-based cloning and genome sequencing: direct RFLP mapping of large clones; Shibata D et al.; Large insert capacity, clone stability and convenient propagation in Escherichia coli have made bacterial artificial chromosome and phage P1 vector-based libraries the first choice for large-scale sequencing projects, and these libraries have also proven useful for chromosome walking . The application of these libraries for either purpose is greatly facilitated by the establishment of a set of framework clones distributed across the genome . Using a P1-based library of Arabidopsis thaliana with genomic inserts of 70-90kb (Liu, Y.-G., Mitsukawa, N., Vazquez-Tello, A., Whittier, R.F., 1995 . Generation of a high-quality P1 library of Arabidopsis suitable for chromosome walking . Plant J . 7, 351-358), we have now established such a set of framework clones . To date, such clones have usually been identified by hybridization to smaller, previously mapped clones that detect restriction fragment length polymorphisms (RFLPs) . In order to establish framework clones more efficiently, we refined protocols for P1 clone DNA isolation and RFLP detection in order to employ whole P1 clones directly as probes . This strategy enabled a very high rate of RFLP detection, and obviated the need to screen the P1 library with smaller RFLP probes . Altogether 95 clones were mapped providing a framework into which further clones can be integrated by physical overlap. Biochem J, 1999 Feb 15, 338 ( Pt 1), 41 - 8 Alternatively spliced mRNA variants of chloroplast ascorbate peroxidase isoenzymes in spinach leaves; Yoshimura K et al.; We have previously shown that stromal and thylakoid-bound ascorbate peroxidase (APX) isoenzymes of spinach chloroplasts arise from a common pre-mRNA by alternative splicing in the C-terminus of the isoenzymes {Ishikawa, Yoshimura, Tamoi, Takeda and Shigeoka (1997) Biochem . J . 328, 795-800} . To explore the production of mature, functional mRNA encoding chloroplast APX isoenzymes, reverse transcriptase-mediated PCR and S1 nuclease protection analysis were performed with poly(A)+ RNA or polysomal RNA from spinach leaves . As a result, four mRNA variants, one form of thylakoid-bound APX (tAPX-I) and three forms of stromal APX (sAPX-I, sAPX-II and sAPX-III), were identified . The sAPX-I and sAPX-III mRNA species were generated through the excision of intron 11; they encoded the previously identified sAPX protein . Interestingly, the sAPX-II mRNA was generated by the insertion of intron 11 between exons 11 and 12 . The use of this insertional sequence was in frame with the coding sequence and would lead to the production of a novel isoenzyme containing a C-terminus in which a seven-residue sequence replaced the last residue of the previously identified sAPX . The recombinant novel enzyme expressed in Escherichia coli showed the same enzymic properties (except for molecular mass) as the recombinant sAPX from the previously identified sAPX-I mRNA, suggesting that the protein translated from the sAPX-II mRNA is functional as a soluble APX in vivo . The S1 nuclease protection analysis showed that the expression levels of mRNA variants for sAPX and tAPX isoenzymes are in nearly equal quantities throughout the spinach leaves grown under normal conditions . The present results demonstrate that the expression of chloroplast APX isoenzymes is regulated by a differential splicing efficiency that is dependent on the 3'-terminal processing of ApxII, the gene encoding the chloroplast APX isoenzymes. J Mol Biol, 1999 Feb 12, 286(1), 257 - 65 Using loop length variants to dissect the folding pathway of a four-helix-bundle protein; Nagi AD et al.; Rop is a four-helix-bundle protein formed by the association of two helix-loop-helix monomers . The short helix-connecting loop was replaced with a series of polyglycine linkers of increasing length . These mutant proteins all appear to fold via the same general mechanism as that of the wild-type protein, even at the longest loop lengths . Replacement of the wild-type two-residue loop (Asp-Ala) with a (Gly-Gly) linker accelerates both unfolding and refolding rates . These changes in folding and unfolding kinetics likely indicate an alteration in the energy of the transition state . As the length of the glycine linker is further increased, the unfolding rate increases while the refolding rates decrease . The influence of loop length is not limited to these rates, but also impacts upon the stability of the folding intermediate . These dependences underscore the importance of loop closure and help refine the model for Rop's folding, implicating a dimeric intermediate involving hairpin formation . These observations show that loop alteration may be useful as a general technique for dissecting protein folding pathways . J Mol Biol, 1999 Feb 12, 286(1), 219 - 32 Nuclear magnetic resonance and molecular dynamics studies on the interactions of the Ras-binding domain of Raf-1 with wild-type and mutant Ras proteins; Terada T et al.; The Ras protein and its homolog, Rap1A, have an identical "effector region" (residues 32-40) preceded by Asp30-Glu31 and Glu30-Lys31, respectively . In the complex of the "Ras-like" E30D/K31E mutant Rap1A with the Ras-binding domain (RBD), residues 51-131 of Raf-1, Glu31 in Rap1A forms a tight salt bridge with Lys84 in Raf-1 . However, we have recently found that Raf-1 RBD binding of Ras is indeed reduced by the E31K mutation, but is not affected by the E31A mutation . Here, the "Rap1A-like" D30E/E31K mutant of Ras was prepared and shown to bind the Raf-1 RBD less strongly than wild-type Ras, but slightly more tightly than the E31K mutant . The backbone 1H, 13C, and 15N magnetic resonances of the Raf-1 RBD were assigned in complexes with the wild-type and D30E/E31K mutant Ras proteins in the guanosine 5'-O-(beta,gamma-imidotriphosphate)-bound form . The Lys84 residue in the Raf-1 RBD exhibited a large change in chemical shift upon binding wild-type Ras, suggesting that Lys84 interacts with wild-type Ras . The D30E/E31K mutant of Ras caused nearly the same perturbations in Raf-1 chemical shifts, including that of Lys84 . We hypothesized that Glu31 in Ras may not be the major salt bridge partner of Lys84 in Raf-1 . A molecular dynamics simulation of a model structure of the Raf-1 RBD.Ras.GTP complex suggested that Lys84 in Raf-1 might instead form a tight salt bridge with Asp33 in Ras . Consistent with this, the D33A mutation in Ras greatly reduced its Raf-I RBD binding activity . We conclude that the major salt bridge partner of Lys84 in Raf-1 may be Asp33 in Ras . J Mol Biol, 1999 Feb 12, 286(1), 95 - 104 Cross-functional analysis of the Microviridae internal scaffolding protein; Burch AD et al.; The assembly of the viral structural proteins into infectious virions is often mediated by scaffolding proteins . These proteins are transiently associated with morphogenetic intermediates but not found in the mature particle . The genes encoding three Microviridae (phiX174, G4 and alpha3) internal scaffolding proteins (B proteins) have been cloned, expressed in vivo and assayed for the ability to complement null mutations of different Microviridae species . Despite divergence as great as 70% in amino acid sequence over the aligned length, cross-complementation was observed, indicating that these proteins are capable of directing the assembly of foreign structural proteins into infectious particles . These results suggest that the Microviridae internal scaffolding proteins may be inherently flexible . There was one condition in which a B protein could not cross-function . The phiX174 B protein cannot productively direct the assembly of the G4 capsid at temperatures above 21 degreesC . Under these conditions, assembly is arrested early in the morphogenetic pathway, before the first B protein mediated reaction . Two G4 mutants, which can productively utilize the phiX174 B protein at elevated temperatures, were isolated . Both mutations confer amino acid substitutions in the viral coat protein but differ in their relative abilities to utilize the foreign scaffolding protein . The more efficient substitution is located in a region where coat-scaffolding interactions have been observed in the atomic structure and may emphasize the importance of interactions in this region . J Mol Biol, 1999 Feb 12, 286(1), 71 - 81 The effect of a hydrophobic N-terminal probe on translational pausing of chloramphenicol acetyl transferase and rhodanese; Tsalkova T et al.; The effect on translational pausing of a hydrophobic probe, coumarin, at the N terminus of nascent peptides was investigated . Two different proteins, bacterial chloramphenicol acetyltransferase and bovine rhodanese, were synthesized by coupled transcription/translation in a cell-free system derived from Escherichia coli . Protein synthesis was initiated with N-formyl-Met-tRNAf or N-acetyl-S-coumarin-Met-tRNAf . Cotranslational incorporation of the coumarin derivative generated nascent polypeptides with a hydrophobic residue at their N termini . The effect of the two N-terminal groups on the size distribution and quantity of the peptides formed by translational pausing was investigated . The N-terminal coumarin caused an accumulation of nascent chloramphenicol acetyltransferase peptides in the mass range of 3.5-4.0 kDa that reflects a delay in translation at this point . No similar effect on rhodanese pause-site peptides was observed . This effect on translational pausing cannot be explained by either mRNA secondary structure or rare codons and tRNA abundance . It is suggested that the effect of N-terminal coumarin on translational pausing is the result of the interaction of the nascent peptide with components of the large ribosomal subunit along the path it follows between the peptidyl transferase center and the exit site on the distal surface . Nature, 1999 Jan 21, 397(6716), 271 - 4 Protein translation and folding are coupled by an endoplasmic-reticulum-resident kinase; Harding HP et al.; Protein synthesis and the folding of the newly synthesized proteins into the correct three-dimensional structure are coupled in cellular compartments of the exocytosis pathway by a process that modulates the phosphorylation level of eukaryotic initiation factor-2alpha (eIF2alpha) in response to a stress signal from the endoplasmic reticulum (ER) . Activation of this process leads to reduced rates of initiation of protein translation during ER stress . Here we describe the cloning of perk, a gene encoding a type I transmembrane ER-resident protein . PERK has a lumenal domain that is similar to the ER-stress-sensing lumenal domain of the ER-resident kinase Ire1, and a cytoplasmic portion that contains a protein-kinase domain most similar to that of the known eIF2alpha kinases, PKR and HRI . ER stress increases PERK's protein-kinase activity and PERK phosphorylates eIF2alpha on serine residue 51, inhibiting translation of messenger RNA into protein . These properties implicate PERK in a signalling pathway that attenuates protein translation in response to ER stress. Nature, 1999 Jan 21, 397(6716), 267 - 70 Direct interaction of microtubule- and actin-based transport motors; Huang JD et al.; The microtubule network is thought to be used for long-range transport of cellular components in animal cells whereas the actin network is proposed to be used for short-range transport, although the mechanism(s) by which this transport is coordinated is poorly understood . For example, in sea urchins long-range Ca2+-regulated transport of exocytotic vesicles requires a microtubule-based motor, whereas an actin-based motor is used for short-range transport . In neurons, microtubule-based kinesin motor proteins are used for long-range vesicular transport but microtubules do not extend into the neuronal termini, where actin filaments form the cytoskeletal framework, and kinesins are rapidly degraded upon their arrival in neuronal termini, indicating that vesicles may have to be transferred from microtubules to actin tracks to reach their final destination . Here we show that an actin-based vesicle-transport motor, MyoVA, can interact directly with a microtubule-based transport motor, KhcU . As would be expected if these complexes were functional, they also contain kinesin light chains and the localization of MyoVA and KhcU overlaps in the cell . These results indicate that cellular transport is, in part, coordinated through the direct interaction of different motor molecules. Biochemistry, 1999 Feb 2, 38(5), 1652 - 8 Site-directed mutagenesis of charged and potentially proton-carrying residues in the beta subunit of the proton-translocating nicotinamide nucleotide transhydrogenase from Escherichia coli . Characterization of the beta H91, beta D392, and beta K424 mutants; Hu X et al.; Conserved and semiconserved acidic and basic residues of the beta subunit of the proton-pumping nicotinamide nucleotide transhydrogenase from Escherichia coli potentially involved in proton pumping were investigated . Out of 16 charged residues studied, 6 have not been previously investigated . The most dramatic effects of mutation were observed with beta H91, beta D392, and beta K424 . beta H91E showed a pronounced shift of the pH optimum for both reduction of thio-NADP+ by NADH (forward reaction) and reduction of 3-acetylpyridine-NAD+ by NADPH (reverse reaction) to lower pH . This mutant catalyzed a cyclic reduction of 3-acetylpyridine-NAD+ by NADH in the presence of NADP(H) with a pH profile also shifted toward a lower pH . These results are consistent with a mechanism where the normal forward and reverse reactions are indeed limited by protonation/deprotonation of beta H91 . The cyclic reaction was affected by mutations of beta H91, probably through conformational changes involving the active NADP(H) site . The beta D392A mutant was inactive with regard to forward and reverse reactions, but showed a wild-type-like pH dependence for the partly active cyclic reaction . However, Km,app for NADP(H) in this reaction was elevated 50-100-fold, suggesting that beta D392 is located in or near the NADP(H)-binding site . Transhydrogenases contain a conserved beta K424-beta R425-beta S426 sequence that has been proposed to be important for NADP(H) binding . beta K424R was strongly inhibited and showed an 18-fold increased Km,app for NADPH in the reverse reaction as compared to wild type . Consequently, this mutation affected all NADP(H)-linked activities and essentially abolished the unspecific interaction of NAD(H) with this site . The pH dependences of the forward and reverse reactions, as well as the cyclic reaction, were shifted to a lower pH as compared to the wild-type enzyme, and the salt dependence was also altered. Biochemistry, 1999 Feb 2, 38(5), 1643 - 51 Dissecting the role of acyltransferase domains of modular polyketide synthases in the choice and stereochemical fate of extender units; Lau J et al.; Modular polyketide synthases (PKSs), such as the 6-deoxyerythronolide B synthase (DEBS), are large multifunctional enzyme complexes that are organized into modules, where each module carries the domains needed to catalyze the condensation of an extender unit onto a growing polyketide chain . Each module also dictates the stereochemistry of the chiral centers introduced into the backbone during the chain elongation process . Here we used domain mutagenesis to investigate the role of the acyl transferase (AT) domains of individual modules in the choice and stereochemical fate of extender units . Our results indicate that the AT domains of DEBS do not influence epimerization of the (2S)-methylmalonyl-CoA extender units . Hence, stereochemical control of the methyl-branched centers generated by DEBS most likely resides in the ketosynthase (KS) domains of the individual modules . In contrast, several recent studies have demonstrated that extender unit specificity can be altered by AT domain substitution . In some of these examples, the resulting polyketide was produced at considerably lower titers than the corresponding natural product . We analyzed one such attenuated mutant of DEBS, in which the methylmalonyl transferase domain of module 2 was replaced with a malonyl transferase domain . As reported earlier, the resulting PKS produced only small quantities of the expected desmethyl analogue of 6-deoxyerythronolide B . However, when the same hybrid module was placed as the terminal module in a truncated 2-module PKS, it produced nearly normal quantities of the expected desmethyl triketide lactone . These results illustrate the limits to modularity of these multifunctional enzymes . To dissect the role of specific amino acids in controlling AT substrate specificity, we exchanged several segments of amino acids between selected malonyl and methylmalonyl transferases, and found that a short (23-35 amino acid) C-terminal segment present in all AT domains is the principal determinant of their substrate specificity . Interestingly, its length and amino acid sequence vary considerably among the known AT domains . We therefore suggest that the choice of extender units by the PKS modules is influenced by a "hypervariable region", which could be manipulated via combinatorial mutagenesis to generate novel AT domains possessing relaxed or altered substrate specificity. Biochemistry, 1999 Feb 2, 38(5), 1394 - 401 Allosteric dominance in carbamoyl phosphate synthetase; Braxton BL et al.; A linked-function analysis of the allosteric responsiveness of carbamoyl phosphate synthetase (CPS) from E . coli was performed by following the ATP synthesis reaction at low carbamoyl phosphate concentration . All three allosteric ligands, ornithine, UMP, and IMP, act by modifying the affinity of CPS for the substrate MgADP . Individually ornithine strongly promotes, and UMP strongly antagonizes, the binding of MgADP . IMP causes only a slight inhibition at 25 degreesC . When both ornithine and UMP were varied, models which presume a mutually exclusive binding relationship between these ligands do not fit the data as well as does one which allows both ligands (and substrate) to bind simultaneously . The same result was obtained with ornithine and IMP . By contrast, the actions of UMP and IMP together must be explained with a competitive model, consistent with previous reports that UMP and IMP bind to the same site . When ornithine is bound to the enzyme, its activation dominates the effects when either UMP or IMP is also bound . The relationship of this observation to the structure of CPS is discussed. Biochemistry, 1999 Jan 26, 38(4), 1377 - 85 Effects of core mutations on the folding of a beta-sheet protein: implications for backbone organization in the I-state; Lorch M et al.; A series of core mutations were introduced into beta-strand segments of an immunoglobulin fold (the isolated first domain of CD2, CD2.d1) to examine their influence on the rapidly formed intermediate state (I-state) which transiently accumulates in the folding reaction {Parker, M . J., and Clarke, A . R . (1997) Biochemistry 36, 5786-5794} . The residue changes were chemically conservative, each representing the removal of one or two methylene groups from aliphatic side chains . Predictably, the mutations destabilize the folded state with respect to the unfolded state by about 1.1 +/- 0.7 kcal mol-1 per methylene group removed . However, when the folding reaction is dissected by transient kinetic analysis into its component steps, six out of the nine mutations lead to a stabilization of the I-state . The direction and magnitude of these effects on the global stability of the transient intermediate are well correlated with changes in secondary structure propensity occasioned by the substitutions . The results show that, although side chain interactions are extremely weak in this early phase of folding, the beta-strand conformation of the polypeptide chain is established . In the next phase of the reaction, the rate-limiting transition state is attained by the formation of a tightly localized hydrophobic nucleus which includes residues V30, I18, and V78 . Interestingly, in almost all immunoglobulin domains of extracellular proteins, the latter pair are cysteine residues which form a disulfide bridge. Biochemistry, 1999 Jan 26, 38(4), 1371 - 6 Modification of ribonuclease T1 specificity by random mutagenesis of the substrate binding segment; Hubner B et al.; Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far . Therefore, we applied a semirational approach by randomizing the guanine binding site . A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations of 6 amino acid positions within the recognition loop was screened on RNase indicator plates . The specificity profiles of 180 individual clones showing RNase activity revealed that variant K41S/N43W/N44H/Y45A/E46D (RNaseT1-8/3) exhibits an altered preference toward purine nucleotides . The ApC/GpC preference in the cleavage reaction of this variant was increased 4000-fold compared to wild-type . Synthesis experiments of dinucleoside monophosphates from cytidine and the corresponding 2'3'-cyclic diesters using the reverse reaction of the transesterification step showed a 7-fold higher ApC synthesis rate of RNase 8/3 than wild-type, whereas the GpC synthesis rates for both enzymes were comparable . This study shows that site-directed random mutagenesis is a powerful additional tool in protein design in order to achieve new enzymatic specificities. Biochemistry, 1999 Jan 26, 38(4), 1346 - 55 Mapping of subunit-subunit contact surfaces on the beta subunit of Escherichia coli RNA polymerase; Nomura T et al.; The RNA polymerase core enzyme of Escherichia coli is composed of 2alpha, 1beta, and 1beta' subunits . Previously we mapped the alpha-alpha, alpha-beta, and alpha-beta' contact sites on the alpha subunit . Here we analyzed the alpha subunit contact sites on the beta subunit by using various experimental approaches: (i) comparison of the proteolytic cleavage map between the unassembled free beta subunit and the alpha2 beta complex; (ii) analysis of the binary complex formation between His6-tagged intact alpha subunit and various truncated beta fragments; and (iii) analysis of the complex formation between the alpha subunit and various His6-tagged beta fragments . The results altogether indicate that two regions of the beta subunit are involved in the full activity of alpha binding, that is, the primary contact site between residues 737 and 904 and the secondary region with assembly control activity downstream from residue 1138 . All of the alpha subunit-beta fragment binary complexes identified in this study were found to bind beta' subunit and form pseudo-core complexes, indicating that the regions of beta involved in alpha subunit contact also participate in interaction with the beta' subunit. Biochemistry, 1999 Jan 26, 38(4), 1316 - 22 Spontaneous propeptide processing of mini-stromelysin-1 mutants blocked by APMA ((4-Aminophenyl)mercuric acetate); Galazka G et al.; Human stromelysin-1 (SL-1) is a member of the stromelysin subfamily of matrix metalloproteinases (MMPs) . The MMPs play a major role in the degradation of the extracellular matrix (ECM) during normal and pathological conditions . SL-1 like the other MMPs can be activated in vitro by the stepwise removal of the propeptide that contains a single unpaired cysteine which coordinates the active site zinc . Other residues in the propeptide also play a role in maintaining the latency of the enzymes . Deletion mutants and single-site amino acid replacements within the propeptide of a carboxyl-terminally truncated stromelysin-1 (mini-SL-1) were constructed and expressed in Escherichia coli to further examine what amino acids within the propeptide of SL-1 are important for maintaining latency . While the natural enzyme displayed some limited tendency to spontaneously (autolytically) convert to lower Mr in a stepwise manner and finally to the fully processed form, all of the truncation mutants of more than 19 amino acids generated in E . coli showed greatly accelerated self-cleavage indicative of diminished stability and/or resistance to proteolysis of the residual propeptide . Mutant Delta63 as well as other mutants in which most of the propeptide had been deleted no longer responded to exposure to the organomercurial APMA by accelerated autolytic processing . Rather, APMA inhibited the autolytic processing in these mutants, further confirming the complexity of the action of this organomercurial in the activation of pro-MMPs. Biochemistry, 1999 Jan 26, 38(4), 1310 - 5 Role of carboxyl-terminal charges on S-modulin membrane affinity and inhibition of rhodopsin phosphorylation; Matsuda S et al.; S-Modulin shows a higher affinity for urea-stripped frog rod outer segment membranes than s26 (a cone homologue of S-modulin) . NaCl at a concentration of several hundred millimolar reduced the membrane affinity of S-modulin to the s26 level . Chimeric S-modulin and s26 whose respective 23 and 29 amino acids at the carboxyl terminus were swapped showed membrane affinites similar to those of s26 and S-modulin, respectively . The membrane affinity of an S-modulin mutant lacking C-terminal positive charges was reduced to the s26 level, while another S-modulin mutant lacking C-terminal negative charges has a higher membrane affinity than wild-type S-modulin . When the molar ratio of recombinant S-modulins to rhodopsin is 0.5, there was no large difference in the inhibition efficiency . However, S-modulin and mutants with high membrane affinities inhibit rhodopsin phosphorylation more efficiently than s26 and mutants with low membrane affinities at the molar ratio of 0.1 . These results indicate that the C-terminal positive charges of these Ca2+-binding proteins enhance the membrane affinity and the inhibitory effect on rhodopsin phosphorylation by increasing the concentration of S-modulin on the membrane. Biochemistry, 1999 Jan 26, 38(4), 1243 - 51 Quaternary structure sensitive tyrosine residues in human hemoglobin: UV resonance raman studies of mutants at alpha140, beta35, and beta145 tyrosine; Nagai M et al.; Recent studies noted the contribution of alpha42Tyr to the T-R-dependent UV resonance Raman (UVRR) spectral changes of HbA {Nagai, M., et al . (1996) J . Mol . Struct . 379, 65-75; Huang, S., et al . (1997) Biochemistry 36, 6197-6206}, but the observed UVRR changes of the Tyr residue cannot be fully interpreted with alpha42Tyr alone . To identify the remaining contributions, the 235 nm-excited UVRR spectra of Tyr mutant Hbs at alpha140, beta35, and beta145 were investigated here . The Fe-His stretching mode demonstrated that all of these mutant Hbs take the T structure in the deoxy form under these experimental conditions . The UVRR change of the Trp residue of these mutants upon the T-R transition was the same as that in HbA, indicating that the T-R-dependent UVRR change of beta37Trp is not due to stacking with Tyr residues but is due to the formation or destruction of a hydrogen bond . The recombinant Hbs beta35Tyr --> Phe and beta35Tyr --> Thr both exhibited UVRR spectra identical with that of HbA, meaning that beta35Tyr is not responsible . In the spectra of des(beta146His,beta145Tyr)Hb with inositol hexaphosphate, the frequency shift of the Tyr RR bands was the same as that in HbA but the intensity enhancement in the CO form was small, suggesting that beta145Tyr contributes to a part of the intensity change, but scarcely relates to the frequency shift . In the spectra of Hb Rouen (alpha140Tyr --> His), the frequency shifts of bands at 1617 (Y8a) and 1177 (Y9a) cm-1 following ligation were half of those in HbA, while the intensity enhancement was not detected . This result means that alpha140Tyr is responsible for both the frequency shift and the intensity changes . It is suggested that the frequency shift of the Tyr RR bands upon the T --> R transition is due to changes in the hydrogen bonding state of alpha42- and alpha140Tyr and that the intensity enhancement is due to changes in the environment of the penultimate Tyr in both alpha and beta subunits (alpha140 and beta145) . These alterations in the vibrational spectra clearly demonstrate which tyrosine residues are involved in the T-R transition as a result of modification of their local environments. Biochemistry, 1999 Jan 26, 38(4), 1193 - 202 Functions of His107 in the catalytic mechanism of human glutathione S-transferase hGSTM1a-1a; Patskovsky YV et al.; Domain interchange analyses and site-directed mutagenesis indicate that the His107 residue of the human subunit hGSTM1 has a pronounced influence on catalysis of nucleophilic aromatic substitution reactions, and a H107S substitution accounts for the marked differences in the properties of the homologous hGSTM1-1 (His107) and hGSTM4-4 (Ser107) glutathione S-transferases . Reciprocal replacement of His107 and Ser107 in chimeric enzymes results in reciprocal conversion of catalytic properties . With 1-chloro-2, 4-dinitrobenzene as a substrate, the His107 residue primarily influences the pH dependence of catalysis by lowering the apparent pKa of kcat/Km from 7.8 for the Ser107-containing enzymes to 6.3 for the His107-containing enzymes . There is a parallel shift in the pKa for thiolate anion formation of enzyme-bound GSH . Y6F mutations have no effect on the pKa for these enzymes . Crystal structures of hGSTM1a-1a indicate that the imidazole ring of His107 is oriented toward the substrate binding cleft approximately 6 A from the GSH thiol group . Thus, His107 has the potential to act as a general base in proton transfer mediated through an active site water molecule or directly following a modest conformational change, to promote thiolate anion formation . All wild-type enzymes and H107S chimera have nearly identical equilibrium constants for formation of enzyme-GSH complexes (Kd values of 1-2 x 10(-)6 M); however, KmGSH and Ki values for S-methylglutathione inhibition determined by steady-state kinetics are nearly 100-fold higher . The functions of His107 of hGSTM1a-1a are unexpected in view of a substantial body of previous evidence that excluded participation of histidine residues in the catalytic mechanisms of other glutathione S-transferases . Consequences of His107 involvement in catalysis are also substrate-dependent; in contrast to 1-chloro-2,4-dinitrobenzene, for the nucleophilic addition reaction of GSH to ethacrynic acid, the H107S substitution has no effect on catalysis presumably because product release is rate-limiting. J Neurochem, 1999 Feb, 72(2), 549 - 56 A 127-kDa protein (UV-DDB) binds to the cytoplasmic domain of the Alzheimer's amyloid precursor protein; Watanabe T et al.; Alzheimer amyloid precursor protein (APP) is an integral membrane protein with a short cytoplasmic domain of 47 amino acids . It is hoped that identification of proteins that interact with the cytoplasmic domain will provide new insights into the physiological function of APP and, in turn, into the pathogenesis of Alzheimer's disease . To identify proteins that interact with the cytoplasmic domain of APP, we employed affinity chromatography using an immobilized synthetic peptide corresponding to residues 645-694 of APP695 and identified a protein of approximately 130 kDa in rat brain cytosol . Amino acid sequencing of the protein revealed the protein to be a rat homologue of monkey UV-DDB (UV-damaged DNA-binding protein, calculated molecular mass of 127 kDa) . UV-DDB/p127 co-immunoprecipitated with APP using an anti-APP antibody from PC12 cell lysates . APP also co-immunoprecipitated with UV-DDB/p127 using an anti-UV-DDB/p127 antibody . These results indicate that UV-DDB/p127, which is present in the cytosolic fraction, forms a complex with APP through its cytoplasmic domain . In vitro binding experiments using a glutathione S-transferase-APP cytoplasmic domain fusion protein and several mutants indicated that the YENPTY motif within the APP cytoplasmic domain, which is important in the internalization of APP and amyloid beta protein secretion, may be involved in the interaction between UV-DDB/p127 and APP. Protein Eng, 1998 Dec, 11(12), 1285 - 92 Human pancreatic RNase1-human epidermal growth factor fusion: an entirely human 'immunotoxin analog' with cytotoxic properties against squamous cell carcinomas; Psarras K et al.; The gene encoding human pancreatic ribonuclease 1 (hpRNasel) was fused with a gene encoding human epidermal growth factor (hEGF) . The hybrid human protein was isolated from Escherichia coli inclusion bodies, refolded and purified to homogeneity . The fusion protein competed with 125I-hEGF for binding to hEGF receptors (EGFR) and had ribonucleolytic activities approaching those of hpRNase1 . Several conformations having different enzymatic activities could be detected after reversed-phase high-performance liquid chromatographic analysis, the less hydrophobic molecules being the most active . The hybrid protein was specifically cytotoxic to A431, an EGFR overexpressing squamous carcinoma cell line, with an IC50 of approximately 10(-7) M . In contrast, recombinant hpRNase1 had an IC50 higher than 10(-4) M . A mixture of free hEGF and free hpRNasel was not more cytotoxic than hpRNasel alone and no cytotoxicity was detected in EGFR-deficient control cells . Taken together, these data suggest that this construct might be useful for targeted therapy of esophageal, lung and other squamous cell carcinomas and also breast cancers overexpressing EGFR, which correlate with a poor prognosis and cannot be cured by surgery alone . Engineering hybrid molecules with endogenous human proteins for targeted therapy may alleviate the dose-limiting immunogenicity and toxicity of conventional immunotoxins. Protein Eng, 1998 Dec, 11(12), 1257 - 65 Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein; Dimasi N et al.; The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed into functional polypeptides by both host- and virus-encoded proteases . The HCV-encoded NS3 protease and its cofactor peptide NS4A form a non-covalent complex, which participates in processing the viral polyprotein . This proteolytic activity is believed to be essential for virus proliferation and thus the NS3 protease is a prime target for developing anti-HCV pharmacological agents . Recent X-ray crystallography structural studies have revealed the nature of this non-covalent complex between NS3 protease and the 'active' central segment of NS4A, providing the opportunity to design a single-chain polypeptide . To this end, the DNA sequence encoding for the NS4A peptide (residues 21-34) was genetically fused via a short linker, capable of making a beta-turn, to the N-terminus of the NS3 protease domain . This engineered single-chain NS3-protease (scNS3) is fully active with kinetic parameters virtually identical with those of the NS3/ NS4A non-covalent complex . Moreover, the scNS3 protease can be displayed on filamentous phage and affinity selected using an immobilized specific inhibitor . The scNS3 expressed as a soluble protein and in a phage-display format facilitates enzyme engineering for further structural studies and in vitro selection of potential drug-resistant mutants . These are important steps towards developing effective anti-protease compounds. Protein Eng, 1998 Dec, 11(12), 1249 - 56 Expression of apolipoprotein(a) kringle IV type 9 in Escherichia coli: demonstration of a specific interaction between kringle IV type 9 and apolipoproteinB-100; Rahman M et al.; A number of studies have provided evidence that lipoprotein(a) {Lp(a)} assembly is a two-step process in which initial non-covalent interactions between apolipoprotein(a) {apo(a)} and apolipoproteinB-100 (apoB-100) precede specific disulfide bond formation . We have designed a construct encoding apo(a) kringle IV type 9 (KIV9) in which the unpaired cysteine at position 67 in this kringle is replaced with a tyrosine . The single kringle was expressed in bacteria and purified to homogeneity from cell homogenates . The purified derivative (designated KIV9deltaCys) was assessed for its ability to bind to purified human LDL . This interaction was detected either by ELISA using immobilized LDL or by column chromatography in which LDL binding to KIV9deltaCys immobilized on Ni2+-Sepharose was determined . In both cases, the interaction of KIV9deltaCys and LDL was observed . Further, we demonstrated that the binding interaction was sensitive to the addition of amino acids including lysine, the lysine analogue epsilon-aminocaproic acid, arginine, phenylalanine and proline, with arginine and lysine having the greatest inhibitory effect . Binding of KIV9deltaCys to an immobilized apoB peptide spanning residues 3732-3745 of apoB was also demonstrated by ELISA . As was the case for LDL, this binding interaction was sensitive to the addition of arginine and lysine . Computer modeling of KIV9 demonstrated an excellent fit with residues 3732-3738 (PSCKLDF) of the apoB peptide . The modeling predicts the presence of overlapping lysine and phenylalanine-binding pockets in KIV9 which explains the inhibitory effects of lysine, arginine and phenylalanine which were observed in the binding assays . In summary, this study represents the first demonstration that KIV9 can interact directly with LDL through non-covalent interactions which may contribute to the first step of Lp(a) formation. Protein Eng, 1998 Dec, 11(12), 1243 - 7 Protein engineering of chimeric Serpins: an investigation into effects of the serpin scaffold and reactive centre loop length; Bottomley SP et al.; The exposed Serpin reactive centre loop controls the specificity of the serpin proteinase interaction . Mutations within this region have been used to generate novel potentially therapeutic inhibitors . In this study we examine the effect of the serpin scaffold and reactive centre loop length upon the generation of such inhibitors . The reactive centre loop regions, P7-P3', of alpha1-antitrypsin and alpha1-antichymotrypsin were replaced by the corresponding residues of the viral serpin, Serp1, to form AT/Serp1 and ACT/Serp1, respectively . AT/Serp1 formed SDS stable complexes with a range of proteinases with association rate constants for plasmin, tissue plasminogen activator, urokinase, thrombin and factor Xa of approximately 10(4) M(-1)s(-1) and a stoichiometry of inhibition of approximately 1 for all of them . ACT/Serp1, however, formed SDS-stable complexes with only plasmin and thrombin with association rate constant 100-fold slower than AT/Serp1 and an increased stoichiometry of inhibition . The reactive centre loop of ACT/Serp1 is four amino acid residues longer than AT/Serp1 . These four additional residues (VETR) were inserted into AT/Serp1 to form AT/Serp1(VETR) . AT/Serp1(VETR) formed SDS stable complexes with plasmin, thrombin and tissue plasminogen activator similar to AT/Serp1, however, the association rate constants were 10-fold slower than those observed with AT/Serp1, while the stoichiometry of inhibition remained around 1 . These results suggest that the additional reactive centre loop residues effect the rate of initial complex formation by placing the reactive centre loop in a non-ideal conformation . This study demonstrates that both reactive centre loop length and serpin scaffold are important in defining the inhibitory characteristics of a serpin. Protein Eng, 1998 Dec, 11(12), 1229 - 34 Engineered human carboxypeptidase B enzymes that hydrolyse hippuryl-L-glutamic acid: reversed-polarity mutants; Edge M et al.; Variants of human pancreatic carboxypeptidase B (HCPB), with specificity for hydrolysis of C-terminal glutamic acid and aspartic acid, were prepared by site-directed mutagenesis of the human gene and expressed in the periplasm of Escherichia coli . By changing residues in the lining of the S1' pocket of the enzyme, it was possible to reverse the substrate specificity to give variants able to hydrolyse prior to C-terminal acidic amino acid residues instead of the normal C-terminal basic residues . This was achieved by mutating Asp253 at the base of the S1' specificity pocket, which normally interacts with the basic side-chain of the substrate, to either Lys or Arg . The resulting enzymes had the desired reversed polarity and enzyme activity was improved significantly with further mutations at residue 251 . The {G251T,D253K}HCPB double mutant was 100 times more active against hippuryl-L-glutamic acid (hipp-Glu) as substrate than was the single mutant, {D253K}HCPB . Triple mutants, containing additional changes at Ala248, had improved activity against hipp-Glu substrate when position 251 was Asn . These reversed-polarity mutants of a human enzyme have the potential to be used in antibody-directed enzyme prodrug therapy of cancer. Protein Eng, 1998 Dec, 11(12), 1219 - 27 Thermostable glycerol kinase from a hyperthermophilic archaeon: gene cloning and characterization of the recombinant enzyme; Koga Y et al.; The Pk-glpK gene, which encodes glycerol kinase (GK) from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, was cloned and expressed in Escherichia coli . The amino acid sequence of this enzyme (Pk-GK) deduced from the nucleotide sequence showed 57% identity with that of E . coli GK and 47% identity with that of human GK . Pk-GK, which has a molecular weight of 55902 (497 amino acid residues), was purified from E . coli and characterized . Despite the high sequence similarity, Pk-GK and E . coli GK are greatly divergent in structure and function from each other . Unlike E . coli GK, which exists as a tetramer, Pk-GK exists as a dimer . The preferred divalent cation for Pk-GK is Co2+, instead of Mg2+ . The optimum pH and temperature for Pk-GK activity are 8.0 and 80 degrees C, respectively . Pk-GK can utilize other nucleoside triphosphates than ATP as a phosphoryl donor . It is fairly resistant to an allosteric inhibitor of E . coli GK, fructose-1,6-bisphosphate . Determination of the kinetic parameters indicates that the Km value of the enzyme is 15.4 microM for ATP and 111 microM for glycerol and its kcat value is 940 s(-1) . The enzyme was shown to be fairly resistant to irreversible heat inactivation and still retained 50% of its enzymatic activity even after heating at 100 degrees C for 30 min . Construction of a model for the three-dimensional structure of the enzyme suggests that the formation of extensive ion-pair networks is responsible for the high stability of this enzyme. Protein Eng, 1998 Dec, 11(12), 1211 - 7 Misfolding of chloramphenicol acetyltransferase due to carboxy-terminal truncation can be corrected by second-site mutations; Van der Schueren J et al.; Folding of chloramphenicol acetyltransferase (CAT) in Escherichia coli is hampered by deletion of the carboxy-terminal tail including the last residue of the carboxy-terminal alpha-helix . Such truncated CAT polypeptides quantitatively aggregate into cytoplasmic inclusion bodies, which results in absence of a chloramphenicol-resistant phenotype for the producing host . In this paper, a genetic approach is presented to examine this aggregation process in more detail . Random mutagenesis of inactive CAT followed by direct phenotypic selection for revertants with restored chloramphenicol resistance was used to isolate second-site suppressors of inactive truncation mutants of CAT . Two random mutagenesis procedures, independently of each other, yielded a unique substitution of Phe for Leu at amino acid position 145 . This second-site mutation does not drastically affect the proteins' stability under normal growth conditions of E . coli . Hence, the introduction of Phe at amino acid position 145 improves the ability of the protein to fold into a soluble, enzymatically active conformation . The conservative character of the Leu145Phe replacement indicates that limited changes at crucial positions can have important effects on protein folding in vivo. Scand J Gastroenterol, 1998 Dec, 33(12), 1256 - 61 Extracts of Helicobacter pylori reduce gastric mucosal blood flow through a VacA- and CagA-independent pathway in rats; Atuma C et al.; BACKGROUND: Helicobacter pylori may interfere with gastroduodenal protective mechanisms . Such effects could be due to a direct interaction with gastric epithelial cells but also to the action of a wide range of secreted and membrane-bound virulence factors . Our aim was to study the acute effects of water extracts produced from H . pylori on gastric mucosal blood flow and acid secretion and to relate them to VacA and CagA activity . METHOD: Extracts were produced from strains 88-23 and A5, both wild type; A5VacA, an isogenic mutant lacking expression of the vacuolating cytotoxin (VacA) and the immunodominant antigen (CagA); and Escherichia coli strain ATCC-25922 . Bacterial extracts were applied on the exteriorized gastric corporal mucosa in inactin-anaesthetized rats after removal of as much as possible of the mucus layer, during intravital microscopy . Blood flow was measured by means of laser-Doppler flowmetry . RESULTS: All H . pylori extracts, including the extract from 88-23 heated to 100 degrees C for 30 min, significantly reduced blood flow by 15%-19%, whereas E . coli had no significant effect on blood flow . CONCLUSION: A factor or a combination of factors, other than VacA and CagA released from H . pylori, might compromise the natural defence of the gastric corporal mucosa by reducing mucosal blood flow . The factor is heat-stable and lacking or less potent in E . coli. Gene Ther, 1998 Nov, 5(11), 1571 - 4 Provision of positive and negative selections in retroviral vectors containing the cytosine deaminase gene; Shiau AL et al.; The E . coli cytosine deaminase (CD) provides a negative selection system for suicide gene therapy as CD transfectants are eliminated following 5-fluorocytosine (5FC) treatment . Here we report a positive selection system for the CD gene using 5-fluorouracil (5FU) and cytosine in selection medium to screen for CD-positive transfectants . It is based on the relief of 5FU toxicity by uracil which is converted from cytosine via CD catalysis, as uracil competes with the toxic 5FU in subsequent pyrimidine metabolism . Hence, a retroviral vector containing the CD gene may provide both positive and negative selections after gene transfer . The CD transfectants selected with the positive selection system showed susceptibility to 5FC in subsequent negative selection in vitro and in vivo . Therefore, this dual selection system is useful not only for combination therapy with transgene and CD gene, but can also act to eliminate selectively transduced cells after the transgene has furnished its effects or upon undesired conditions if 5FC is applied for negative selection in vivo. Gene Ther, 1998 Nov, 5(11), 1481 - 7 Gene transfer vectors derived from equine infectious anemia virus; Olsen JC; Equine infectious anemia virus (EIAV) is a lentivirus in the retrovirus family of viruses . Replication-defective EIAV vectors have been constructed that encode bacterial puromycin-N-acetyl transferase and E . coli beta-galactosidase . These vectors could be prepared with titers greater than 10(5) infectious units/ml and were able to act as vehicles to carry genes into cultured human cells . In addition, stable helper cell lines were created by modifying human 293 cells to express EIAV proteins . Unlike retroviral vectors based on murine leukemia virus, EIAV lentiviral vectors transduce nondividing (aphidicolin-arrested) cells . These properties make EIAV vectors promising gene transfer vehicles. Arch Virol, 1998, 143(12), 2461 - 9 The nucleotide sequence of the 3'-terminal region of dasheen mosaic virus (Caladium isolate) and expression of its coat protein in Escherichia coli for antiserum production; Li RH et al.; A caladium isolate of dasheen mosaic virus (DsMV-Ch) was cloned as cDNA from genomic RNA . The sequence of the 3'-terminal 3158 nucleotides, which consisted of the 3'-terminus of the NIa gene, the NIb gene, the coat protein (CP) gene, and a 246-nucleotide non-coding region, was between 57-68% similar at the nucleotide level and 72-82% similar at the amino acid level when compared with other potyviruses . Phylogenetic analysis of aligned, selected potyviral CP sequences indicate that DsMV-Ch is similar to DsMV isolates infecting taro and closely related to the bean common mosaic virus subgroup in the genus Potyvirus . A recombinant DsMV-Ch CP (approximately 39 kDa) expressed in E . coli was used as an immunogen and the resulting antiserum reacted with DsMV and several other potyviruses in Western blots and indirect ELISA. Arch Virol, 1998, 143(12), 2443 - 51 Construction of full-length cDNA clones of lettuce mosaic virus (LMV) and the effects of intron-insertion on their viability in Escherichia coli and on their infectivity to plants; Yang SJ et al.; A full length cDNA copy of the genomic RNA of lettuce mosaic virus (LMV) was constructed under the control of an enhanced CaMV 35S promoter and of the NOS terminator . This construct was found infectious when inoculated to lettuce plants . The intron II of the bean nitrite reductase gene was engineered into the LMV FL cDNA in order to relieve possible deleterious effects of viral sequences to Escherichia coli cells and to evaluate the effects of the presence of the intron on the FL cDNA infectivity . The intron-less FL cDNA was found to be as stable as its intron-containing counterpart in E . coli . Sequence analysis of progeny RNA derived from plants inoculated with the intron-containing FL cDNA demonstrated that the inserted intron was perfectly spliced out . The symptoms induced in lettuce by either the intron-less or the intro-containing constructs were identical to those caused by the wild-type virus . However a slight delay in the establishment of infection in lettuce and a more obvious lag in Nicotiana benthamiana were observed with the intron-containing FL cDNA. Rocz Panstw Zakl Hig, 1998, 49(3), 293 - 8 The level of endotoxin contamination in biopreparations; Aleksandrowicz J et al.; Bacterial endotoxins as contamination of biopreparations have been estimated by chromogenic LAL test . Study on some compounds (aluminium hydroxide, formaldehyde and merthiolate) being components of vaccines showed no effect on the result of LAL test . The level of endotoxins in virus vaccines with the limits defined in producers certificate was adequate, the level of endotoxin was also low in virus vaccines of undefined requirements . The concentration of endotoxin in bacterial vaccines was differentiated . Considering the results of our experiments, as well as the fact, that the requirements for endotoxin contamination of bacterial vaccines are not available it seems necessary to establish the limits for these group of biopreparations. Genetika, 1998 Oct, 34(10), 1338 - 44 {Further genetic study of tandem duplication formation in the region of the deo operon in the process of Escherichia coli K-12 conjugational recombination}; Sukhodolets VV; The formation of heterozygous duplications in the region of the deo operon was studied in conjugational matings (male) HfrH deoC deoD thr::Tn9 thyA x HfrH deoA deoB::Tn5 thyA . When recombinants that inherited the donor marker thr::Tn9 (Cml r) were selected on a medium containing thymine and chloramphenicol, but not threonine, more than 80% of the offspring were heterozygous tandem duplications extending to the region of the deo operon . In matings with a thymidine-dependent HfrH deoA deoB::Tn5 thyA strain as a recipient, when recombinants were selected on a medium containing thymine, i.e., under conditions of thymine starvation of merozygotes, the recombinogenic effect was observed . However, this effect did not change the frequency of duplication formation . The integration of genetic markers via homologous recombination into the chromosomal regions adjacent to duplications occurred at a lower frequency . An analysis of the formation of haploid segregants by duplications showed that, in most cases of duplication formation, the proximal segment of the donor chromosome is integrated into the distal position, i.e., after the homologous segment of the recipient chromosome. Bioorg Khim, 1998 Oct, 24(10), 756 - 9 {Expression of mutant horse cytochrome c genes in Escherichia coli}; Dolgikh DA et al.; Here we describe genetically engineered constructs for the expression in Escherichia coli of genes for horse cytochrome c mutants . These constructs allow the expression of the cytochrome c genes together with hemeligase, an enzyme which covalently links heme to cytochrome . Careful selection of producer strains and the adjustment of the conditions of expression provided for expression levels of 10-15 mg of protein per liter of culture . This is by an order of magnitude greater than the expression previously achieved in yeast . A series of horse cytochrome c mutants were obtained in this way. Biophys J, 1999 Feb, 76(2), 709 - 15 Single-molecule imaging of RNA polymerase-DNA interactions in real time; Harada Y et al.; Using total internal reflection fluorescence microscopy, we have directly observed individual interactions of single RNA polymerase molecules with a single molecule of lambda-phage DNA suspended in solution by optical traps . The interactions of RNA polymerase molecules were not homogeneous along DNA . They dissociated slowly from the positions of the promoters and sequences common to promoters at a rate of approximately 0.66 s-1, which was more than severalfold smaller than the rate at other positions . The association rate constant for the slow dissociation sites was 9.2 x 10(2) bp-1 M-1 s-1 . The frequency of binding to the fast dissociation sites was dependent on the A-T composition; it was larger in the AT-rich regions than in the GC-rich regions . RNA polymerase molecules on the fast dissociation sites underwent linear diffusion (sliding) along DNA . The binding to the slow dissociation sites was greatly enhanced when DNA was released to a relaxed state, suggesting that the binding depended on the strain exerted on the DNA . The present method is potentially applicable to the examination of a wide variety of protein-nucleic acid interactions, especially those involved in the process of transcription. FEBS Lett, 1999 Jan 15, 442(2-3), 241 - 5 Conformational stabilities of the rat alpha- and beta-parvalbumins; Henzl MT et al.; It is widely believed that beta-parvalbumin (PV) isoforms are intrinsically less stable than alpha-parvalbumins, due to greater electrostatic repulsion and an abbreviated C-terminal helix . However, when examined by differential scanning calorimetry, the apo-form of the rat beta-PV (i.e . oncomodulin) actually displays greater thermal stability than the alpha-PV . Whereas the melting temperature of the a isoform is 45.8 degrees C at physiological pH and ionic strength, the Tm for the beta isoform is more than 7 degrees higher (53.6 degrees C) . This result suggests that factors besides net charge and C-terminal helix length strongly influence parvalbumin conformational stability . Extension of the F helix in the beta-PV, by insertion of Ser-109, has a modest stabilizing effect, raising the Tm, by 1.1 degrees . Truncation of the alpha-PV F helix, by removal of Glu-108, has a more profound impact, lowering the Tm by 4.0 degrees. FEBS Lett, 1999 Jan 15, 442(2-3), 231 - 4 Induction of cytokines in a human colon epithelial cell line by Shiga toxin 1 (Stx1) and Stx2 but not by non-toxic mutant Stx1 which lacks N-glycosidase activity; Yamasaki C et al.; Stx1 and Stx2 produced by Shiga toxin-producing Escherichia coli are cytotoxic due to their N-glycosidase activity on 28S rRNA . In this study, we have shown that proinflammatory cytokine mRNAs, especially IL-8, were induced by Stx1 and Stx2 in Caco-2 cells . A non-toxic mutant of Stxl which lacks N-glycosidase activity did not induce cytokine mRNAs . IL-8 production at the protein level was enhanced by Stx1 and Stx2, but not by the mutant Stx1 . These results demonstrate that Shiga toxins induce expression and synthesis of cytokines in Caco-2 cells and their N-glycosidase activity is essential for the induction. FEBS Lett, 1999 Jan 15, 442(2-3), 198 - 202 Purification of histidine tagged bacteriorhodopsin, pharaonis halorhodopsin and pharaonis sensory rhodopsin II functionally expressed in Escherichia coli; Hohenfeld IP et al.; Bacteriorhodopsin (BR) from Halobacterium salinarum as well as halorhodopsin (pHR) and sensory rhodopsin II (pSRII) from Natronobacterium pharaonis were functionally expressed in E . coli using the method of Shimono et al . IFEBS Lett . (1997) 420, 54-56} . The histidine tagged proteins were purified with yields up to 1.0 mg/l cell culture and characterized by ESI mass spectrometry and their photocycle . The pSRII and pHR photocycles were indistinguishable from the wild type proteins . The BR photocycle was considerably prolonged . pSOII is located in the cytoplasmic membrane and the C-terminus is oriented towards the cytoplasm as determined by immunogold labelling. FEBS Lett, 1999 Jan 15, 442(2-3), 183 - 8 A recombinant single-chain antibody fragment that neutralizes toxin II from the venom of the scorpion Androctonus australis hector; Mousli M et al.; Monoclonal antibody 4C1 specifically binds to and neutralizes the most potent neurotoxin (AahII) of the scorpion Androctonus australis . The cDNAs encoding the variable regions of this antibody were isolated by PCR-mediated cloning . A single-chain Fv gene was engineered and expressed in Escherichia coli . The recombinant protein had neutralizing activity similar to that of the intact antibody in vitro and in vivo . We have thus neutralized the pharmacological and biological properties of a scorpion neurotoxin with a single-chain Fv, which opens new perspectives for the treatment of envenomizations. FEBS Lett, 1999 Jan 15, 442(2-3), 143 - 6 Calcium-dependent interaction of annexin I with annexin II and mapping of the interaction sites; Lee KH et al.; Annexins are multifunctional intracellular proteins with Ca2+- and phospholipid-binding properties . Their structures consist of four conserved repeat domains that form the core and a diverse N-terminal tail, from which their functional differences may arise . We searched for cellular proteins that interact with the N-terminal tail plus domain I of annexin I (ANX1) by using the yeast two-hybrid method . Screening of a HeLa cell cDNA library yielded annexin II (ANX2) cDNA . The interaction between ANX1 and ANX2 also occurred in vitro in a Ca2+-dependent manner . Mapping of the interaction sites revealed that interaction between domain I of ANX1 and domain IV of ANX2 was stronger than the other combinations. Annu Rev Genet, 1998, 32, 437 - 59 Evolution and mechanism of translation in chloroplasts; Sugiura M et al.; The entire sequence (120-190 kb) of chloroplast genomes has been determined from a dozen plant species . The genome contains from 87 to 183 known genes, of which half encode components involved in translation . These include a complete set of rRNAs and about 30 tRNAs, which are likely to be sufficient to support translation in chloroplasts . RNA editing (mostly C to U base changes) occurs in some chloroplast transcripts, creating start and stop codons and changing codons to retain conserved amino acids . Many components that constitute the chloroplast translational machinery are similar to those of Escherichia coli, whereas only one third of the chloroplast mRNAs contain Shine-Dalgarno-like sequences at the correct positions . Analyses conducted in vivo and in vitro have revealed the existence of multiple mechanisms for translational initiation in chloroplasts. Annu Rev Genet, 1998, 32, 163 - 84 The genetics of disulfide bond metabolism; Rietsch A et al.; Disulfide bonds are required for the stability and function of a large number of proteins . Genetic analysis in combination with biochemical studies have elucidated the main catalysts involved in facilitating these processes in the cell . All enzymes involved in thiol-disulfide metabolism have a conserved active site that consists of two cysteine residues, separated by two intervening amino acids, the Cys-Xaa-Xaa-Cys motif . While these enzymes are capable of catalyzing both disulfide bond formation and reduction, they have evolved to perform one or the other reaction more efficiently . In the cytoplasm, multiple pathways are involved in the reduction of disulfide bonds that occur as part of the catalytic cycle of a variety of metabolic enzymes . In the bacterial periplasm, a system for the efficient introduction as well as isomerization of disulfide bonds is in place . In eukaryotes, disulfide bonds are introduced into proteins in the endoplasmic reticulum . Genetic studies have recently begun to reveal new features of this process . While the enzyme mechanisms of thiol-disulfide oxidoreductases have been the subject of much scrutiny, questions remain regarding where and when they act in vivo, their specificities, and the maintenance of the redox environment that determines their function. Annu Rev Genet, 1998, 32, 59 - 94 Targeting and assembly of periplasmic and outer-membrane proteins in Escherichia coli; Danese PN et al.; Escherichia coli must actively transport many of its proteins to extracytoplasmic compartments such as the periplasm and outer membrane . To perform this duty, E . coli employs a collection of Sec (secretion) proteins that catalyze the translocation of various polypeptides through the inner membrane . After translocation across the inner membrane, periplasmic and outer-membrane proteins are folded and targeted to their appropriate destinations . Here we review our knowledge of protein translocation across the inner membrane . We also discuss the various signal transduction systems that monitor extracytoplasmic protein folding and targeting, and we consider how these signal transduction systems may ultimately control these processes. Ann N Y Acad Sci, 1998 Dec 13, 864, 131 - 5 Properties of artificial proteins with random sequences; Yomo T et al.; A library of artificial proteins of 141 amino acid residues, of which 95 are random and which include 20 kinds of amino acids, was prepared . As the properties of the artificial random proteins are free from the evolutionary constraint, they can be used as a standard to discriminate the specialized properties of natural proteins . Out of the 25 identified random proteins, 5 are soluble in the cell lysate, indicating that about 20% of the random proteins expressed in Escherichia coli are expected to be soluble . Therefore, as natural soluble or insoluble proteins can arise from the line of soluble or insoluble ancestry, respectively, solubility does not seem a specialized property of natural proteins . The soluble random proteins RP3-42 and RP3-45 were purified and their properties were investigated. Nucleic Acids Res, 1999 Feb 15, 27(4), 1198 - 204 Double strand break rejoining by mammalian mitochondrial extracts; Lakshmipathy U et al.; DNA end-joining was measured by incubating linearized plasmid DNA with mitochondrial protein extracts . A spectrum of end-joined molecules ranging from re-circularized monomer to dimer and higher molecular weight forms was observed . The DNA end-joining reaction required ATP and Mg2+, and was inhibited by sodium chloride . Both cohesive- and blunt-ended DNA molecules were end-joined, although the former were more efficient substrates . Molecular analysis of rejoined molecules revealed that >95% of the linearized DNA were precisely end-joined . The few imprecisely end-joined molecules recovered, sustained deletions that spanned direct repeat sequences . The deletions observed are strikingly similar to those present in mitochondrial genomes of patients with Kearns-Sayre or Pearson syndromes, certain ophthalmic myopathies and the aged . These results suggest that mammalian mitochondria possess a DNA double strand break repair activity similar to that seen in the nucleus, and that this repair pathway may play a role in the generation of mitochondrial DNA deletions associated with a number of human pathologies. Nucleic Acids Res, 1999 Feb 15, 27(4), 1118 - 25 Thermodynamics of RNA hairpins containing single internal mismatches; Meroueh M et al.; Thermodynamic parameters and circular dichroism spectra are presented for RNA hairpins containing single internal mismatches in the stem regions . Three different sequence contexts for the G*U mismatch and two contexts for C*A, G*A, U*U, A*C and U*G mismatches were examined and compared with Watson-Crick base-pair stabilities . The RNA hairpins employed were a microhelix and tetraloop representing the Escherichia coli tRNAAlaacceptor stem and sequence variants that have been altered at the naturally occurring G*U mismatch site . UV melting studies were carried out under different conditions to evaluate the effects of sodium ion concentration and pH on the stability of mismatch-containing hairpins . Our main findings are that single internal mismatches exhibit a range of effects on hairpin stability . In these studies, the size and sequence of the loop and stem are shown to influence the overall stability of the RNA, and have a minor effect on the relative mismatch stabilities . The relationship of these results to RNA-ligand interactions involving mismatch base-pairs is discussed. Nucleic Acids Res, 1999 Feb 15, 27(4), 1094 - 103 Vaccine candidate MSP-1 from Plasmodium falciparum: a redesigned 4917 bp polynucleotide enables synthesis and isolation of full-length protein from Escherichia coli and mammalian cells; Pan W et al.; The Plasmodium falciparum malaria parasite is the causative agent of malaria tropica . Merozoites, one of the extracellular developmental stages of this parasite, expose at their surface the merozoite surface protein-1 complex (MSP-1), which results from the proteolytic processing of a 190-200 kDa precursor . MSP-1 is highly immunogenic in humans and numerous studies suggest that this protein is an effective target for a protective immune response . Although its function is unknown, there are indications that it may play a role during invasion of erythrocytes by merozoites . The parasite-derived msp-1 gene, which is approximately 5000 bp long, contains 74% AT . This high AT content has prevented stable cloning of the full-size gene in Escherichia coli and consequently its expression in heterologous systems . Here, we describe the synthesis of a 4917 bp gene encoding MSP-1 from the FCB-1 strain of P . falciparum adjusted for human codon preferences . The synthetic msp-1 gene (55% AT) was cloned, maintained and expressed in its entirety in E.coli as well as in CHO and HeLa cells . The purified protein is soluble and appears to possess native conformation because it reacts with a panel of mAbs specific for conformational epitopes . The strategy we used for synthesizing the full-length msp-1 gene was toassemble it from DNA fragments encoding all of the major proteolytic fragments normally generated at the parasite's surface . Thus, after subcloning we also obtained each of these MSP-1 processing products as hexahistidine fusion proteins in E.coli and isolated them by affinity chromatography on Ni2+agarose . The availability of defined preparations of MSP-1 and its major processing products open up new possibilities for in-depth studies at the structural and functional level of this important protein, including the exploration of MSP-1-based experimental vaccines. Nucleic Acids Res, 1999 Feb 15, 27(4), 1015 - 24 Synthesis and enzymatic processing of oligodeoxynucleotides containing tandem base damage; Bourdat AG et al.; Several studies have shown that ionizing radiation generates a wide spectrum of lesions to DNA including base modifications, abasic sites, strand breaks, crosslinks and tandem base damage . One example of tandem base damage induced by @OH radical inX-irradiated DNA oligomers is N -(2-deoxy-beta-d- erythro -pentofuranosyl)-formylamine/8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodGuo) . In order to investigate the biological significance of such a tandem lesion, both 8-oxo-7,8-dihydroguanine and formylamine were introduced into synthetic oligonucleotides at vicinal positions using the solid phase phosphoramidite method . For this purpose, a new convenient method of synthesis of 8-oxodGuo was developed . The purity and integrity of the modified synthetic DNA fragments were assessed using different complementary techniques including HPLC, polyacrylamide gel electrophoresis, electrospray and MALDI-TOF mass spectrometry . The piperidine test applied to the double modified base-containing oligonucleotides revealed the high alkaline lability of formylamine in DNA . In addition, various enzymatic experiments aimed at determining biochemical features of such multiply damaged sites were carried out using the synthetic substrates . The pro-cessing of the vicinal lesions by nuclease P1, snake venom phosphodiesterase, calf spleen phospho-diesterase and repair enzymes including Escherichia coli endonuclease (endo) III and Fapy-glycosylase was studied and is reported. Nucleic Acids Res, 1999 Feb 15, 27(4), 979 - 83 Nucleotide excision repair 3' endonuclease XPG stimulates the activity of base excision repairenzyme thymine glycol DNA glycosylase; Bessho T; An ionizing radiation-induced DNA lesion, thymine glycol, is removed from DNA by a thymine glycol DNA glycosylase with an apurinic/apyrimidinic (AP) lyase activity encoded by the Escherichia coli endonuclease III ( nth ) gene and its homolog in humans . Cells from Cockayne syndrome patients with mutations in the XPG gene show approximately 2-fold reduced global repair of thymine glycol . Hence, I decided to investigate the molecular mechanism of the effect of XPG protein observed in vivo on thymine glycol removal by studying the interactions of XPG protein and human endonuclease III (HsNTH) protein in vitro and the effect of XPG protein on the activity of HsNTH protein on a substrate containing thymine glycol . The XPG protein stimulates the binding of HsNTH protein to its substrate and increases its glycosylase/AP lyase activity by a factor of approximately 2 through direct interaction between the two proteins . These results provide in vitro evidence for a second function of XPG protein in DNA repair and a mechanistic basis for its stimulatory activity on HsNTH protein. Bioinformatics, 1998, 14(10), 839 - 45 SALSA: improved protein database searching by a new algorithm for assembly of sequence fragments into gapped alignments; Rognes T et al.; MOTIVATION: Optimal sequence alignment based on the Smith-Waterman algorithm is usually too computationally demanding to be practical for searching large sequence databases . Heuristic programs like FASTA and BLAST have been developed which run much faster, but at the expense of sensitivity . RESULTS: In an effort to approximate the sensitivity of an optimal alignment algorithm, a new algorithm has been devised for the computation of a gapped alignment of two sequences . After scanning for high-scoring words and extensions of these to form fragments of similarity, the algorithm uses dynamic programming to build an accurate alignment based on the fragments initially identified . The algorithm has been implemented in a program called SALSA and the performance has been evaluated on a set of test sequences . The sensitivity was found to be close to the Smith-Waterman algorithm, while the speed was similar to FASTA (ktup = 2) . AVAILABILITY: Searches can be performed from the SALSA homepage at using a wide range of databases . Source code and precompiled executables are also available . CONTACT: torbjorn.rognes@labmed.uio.no Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 939 - 44 Model of maltose-binding protein/chemoreceptor complex supports intrasubunit signaling mechanism; Zhang Y et al.; The Tar protein of Escherichia coli is unique among known bacterial chemoreceptors in that it generates additive responses to two very disparate ligands, aspartate and maltose . Aspartate binds directly to the periplasmic (extracytoplasmic) domain of Tar . Maltose first binds to maltose-binding protein (MBP) . MBP then assumes a closed conformation in which it can interact with the periplasmic domain of Tar . MBP residues critical for binding Tar were identified in a screen of mutations that cause specific defects in maltose chemotaxis . Mutations were introduced into a plasmid-borne malE gene that encodes a mutant form of MBP in which two engineered Cys residues spontaneously generate a disulfide bond in the oxidizing environment of the periplasmic space . This disulfide covalently crosslinks the NH3-terminal and COOH-terminal domains of MBP and locks the protein into a closed conformation . Double-Cys MBP confers a dominant-negative phenotype for maltose taxis, and we reasoned that third mutations that relieve this negative dominance probably alter residues that are important for the initial interaction of MBP with Tar . The published three-dimensional structures of MBP and the periplasmic domain of E . coli Tar were docked in a computer simulation that juxtaposed the residues in MBP identified in this way with residues in Tar that have been implicated in maltose taxis . The resulting model of the MBP-Tar complex exhibits good complementarity between the surfaces of the two proteins and supports the idea that aspartate and MBP may each initiate an attractant signal through Tar by inducing similar conformational changes in the chemoreceptor. Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 863 - 8 TOXCAT: a measure of transmembrane helix association in a biological membrane; Russ WP et al.; The noncovalent association of transmembrane alpha-helices is a fundamental event in the folding of helical membrane proteins . In this work, a system (TOXCAT) is developed for the study of transmembrane helix-helix oligomerization in a natural membrane environment . This assay uses a chimeric construct composed of the N-terminal DNA binding domain of ToxR (a dimerization-dependent transcriptional activator) fused to a transmembrane domain (tm) of interest and a monomeric periplasmic anchor (the maltose binding protein) . Association of the tms results in the ToxR-mediated activation of a reporter gene encoding chloramphenicol acetyltransferase (CAT) . The level of CAT expression indicates the strength of tm association . The assay distinguishes between a known dimerizing tm and a mutant in which dimerization is disrupted . In addition, modulation of the chimera concentration shows that the dimerization exhibits concentration dependence in membranes . TOXCAT also is used to select oligomeric tms from a library of randomized sequences, demonstrating the potential of this system to reveal novel oligomerization motifs . The TOXCAT system has been used to investigate glycophorin A tm-mediated dimerization . Although the overall sensitivity of glycophorin A tm dimerization to mutagenesis is found to be similar in membranes and in detergent micelles, several significant differences exist . Mutations to polar residues, which are generally disruptive in SDS, exhibit sequence specificity in membranes, demonstrating both the limitations of detergent micelles and the wider range of application of the TOXCAT system. Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 823 - 8 Three-dimensional structure of phosphoenolpyruvate carboxylase: a proposed mechanism for allosteric inhibition; Kai Y et al.; The crystal structure of phosphoenolpyruvate carboxylase (PEPC; EC 4 . 1.1.31) has been determined by x-ray diffraction methods at 2.8-A resolution by using Escherichia coli PEPC complexed with L-aspartate, an allosteric inhibitor of all known PEPCs . The four subunits are arranged in a "dimer-of-dimers" form with respect to subunit contact, resulting in an overall square arrangement . The contents of alpha-helices and beta-strands are 65% and 5%, respectively . All of the eight beta-strands, which are widely dispersed in the primary structure, participate in the formation of a single beta-barrel . Replacement of a conserved Arg residue (Arg-438) in this linkage with Cys increased the tendency of the enzyme to dissociate into dimers . The location of the catalytic site is likely to be near the C-terminal side of the beta-barrel . The binding site for L-aspartate is located about 20 A away from the catalytic site, and four residues (Lys-773, Arg-832, Arg-587, and Asn-881) are involved in effector binding . The participation of Arg-587 is unexpected, because it is known to be catalytically essential . Because this residue is in a highly conserved glycine-rich loop, which is characteristic of PEPC, L-aspartate seemingly causes inhibition by removing this glycine-rich loop from the catalytic site . There is another mobile loop from Lys-702 to Gly-708 that is missing in the crystal structure . The importance of this loop in catalytic activity was also shown . Thus, the crystal-structure determination of PEPC revealed two mobile loops bearing the enzymatic functions and accompanying allosteric inhibition by L-aspartate. Proc Natl Acad Sci U S A, 1999 Feb 2, 96(3), 811 - 7 Origins of DNA-binding specificity: role of protein contacts with the DNA backbone; Schildbach JF et al.; A central question in protein-DNA recognition is the origin of the specificity that permits binding to the correct site in the presence of excess, nonspecific DNA . In the P22 Arc repressor, the Phe-10 side chain is part of the hydrophobic core of the free protein but rotates out to pack against the sugar-phosphate backbone of the DNA in the repressor-operator complex . Characterization of a library of position 10 variants reveals that Phe is the only residue that results in fully active Arc . One class of mutants folds stably but binds operator with reduced affinity; another class is unstable . FV10, one member of the first class, binds operator DNA and nonoperator DNA almost equally well . The affinity differences between FV10 and wild type indicate that each Phe-10 side chain contributes 1.5-2.0 kcal to operator binding but less than 0.5 kcal/mol to nonoperator binding, demonstrating that contacts between Phe-10 and the operator DNA backbone contribute to binding specificity . This appears to be a direct contribution as the crystal structure of the FV10 dimer is similar to wild type and the Phe-10-DNA backbone interactions are the only contacts perturbed in the cocrystal structure of the FV10-operator complex. J Surg Res, 1999 Feb, 81(2), 156 - 63 Concomitant increase in neutrophil adhesion to inflammatory peritoneum and remote organs during peritonitis; Fukatsu K et al.; BACKGROUND: Neutrophils contribute to the host defense mechanism, but they can cause remote organ injury in peritonitis . The purpose of this study was to examine neutrophil adhesion to the peritoneum and remote organs simultaneously in peritonitis using a fluorescence microscopic method . STUDY DESIGN: Experiment 1: Sprague-Dawley rats (n = 16) were injected intraperitoneally (ip) with saline solution or 10(5), 10(7), or 10(9) Escherichia coli . Five hours after challenge, 1 x 10(6) fluorescein-labeled neutrophils were infused . Two minutes after neutrophil injection, five peritoneal samples (the greater omentum, mesentery, parietal peritoneum, colon, and ileum), both lungs, the liver, and the right kidney were harvested for counting of labeled neutrophils under epifluorescent microscopy . Lung myeloperoxidase (MPO) activity was also determined . Experiment 2: Rats (n = 23) were given 10(9) E . coli ip . Before challenge (0 h) or at 1, 5, or 10 h after challenge, labeled neutrophils were infused . Then, the labeled neutrophil numbers in organs and lung MPO activities were assessed as described for Experiment 1 . Hemodynamic and arterial blood gas data were also obtained in another set of rats before and at 1, 5, 8 and 10 h after 10(9) E . coli ip challenge . RESULTS: Experiment 1: The labeled neutrophil numbers in the peritoneum, lungs, and kidney showed significant positive correlations with the injected bacterial numbers . Lung MPO also positively correlated with E . coli number and labeled neutrophil number in the lungs . Experiment 2: Labeled neutrophil numbers in the peritoneum and kidney peaked at 5 h . The pulmonary labeled neutrophil number rose, reaching a plateau at 5 h . No remarkable change was observed in the hepatic labeled neutrophil number . There was a positive correlation between lung MPO activity and pulmonary labeled neutrophil number . Hemodynamic and blood gas data reflected a hyperdynamic state . CONCLUSIONS: Concomitant dose-dependent increases in neutrophil adhesion in the peritoneum, lungs, and kidney were observed in this peritonitis model . Increased neutrophil adhesion was transient in the peritoneum and kidney but persistent in the lungs . Strategies modulating neutrophil adhesion in organs are anticipated to be useful for the treatment of peritonitis . J Surg Res, 1999 Feb, 81(2), 129 - 38 Hepato-splanchnic blood flow and oxygen extraction capabilities during experimental tamponade: effects of endotoxin; Zhang H et al.; We studied the hepato-splanchnic vascular response and changes in O2 extraction capabilities to a reduction in blood flow following endotoxemia . Fourteen anesthetized and mechanically ventilated dogs were divided into two groups of seven each . Group 1 received 2 mg/kg of E . coli endotoxin, and group 2 served as a control . After initial fluid resuscitation following endotoxic shock, regional blood flow estimated by an ultrasonic technique increased similarly in the hepatic artery, portal vein, and mesenteric artery, but microvascular blood flow estimated by a laser Doppler technique was lower in the liver than in the intestinal mucosa . When blood flow was reduced by cardiac tamponade, endotoxin-treated animals had greater whole body and regional critical O2 delivery (DO2crit) and lower whole body, liver, and intestinal critical O2 extraction ratios (O2ERcrit) . DO2crit was higher in the liver than in intestine but O2ERcrit was similar in the two organs . Whole body DO2crit at the onset of organ O2 supply dependency was similar under control (9.4 +/- 1.9 mL/kg . min for whole body, 10.3 +/- 4.7 mL/kg . min for liver, and 10.0 +/- 2.6 mL/kg . min for intestine) and endotoxic conditions (13.6 +/- 3.2 mL/kg . min for whole body, 15.6 +/- 2.7 mL/kg . min for liver, and 15.4 +/- 8.7 mL/kg . min for intestine) . We conclude that fluid-resuscitated endotoxic shock in dogs is characterized by blood flow redistribution within the liver and intestine . Microvascular depression may be more severe in the liver than in the intestinal mucosa, although the whole body, the liver, and the intestine became O2 supply-dependent simultaneously . Genetics, 1999 Feb, 151(2), 439 - 46 Escherichia coli mutM suppresses illegitimate recombination induced by oxidative stress; Onda M et al.; DNA damage by oxidative stress is one of the causes of mutagenesis . However, whether or not DNA damage induces illegitimate recombination has not been determined . To study the effect of oxidative stress on illegitimate recombination, we examined the frequency of lambdabio transducing phage in the presence of hydrogen peroxide and found that this reagent enhances illegitimate recombination . To clarify the types of illegitimate recombination, we examined the effect of mutations in mutM and related genes on the process . The frequency of lambdabio transducing phage was 5- to 12-fold higher in the mutM mutant than in the wild type, while the frequency in the mutY and mutT mutants was comparable to that of the wild type . Because 7,8-dihydro-8-oxoguanine (8-oxoG) and formamido pyrimidine (Fapy) lesions can be removed from DNA by MutM protein, these lesions are thought to induce illegitimate recombination . Analysis of recombination junctions showed that the recombination at Hotspot I accounts for 22 or 4% of total lambdabio transducing phages in the wild type or in the mutM mutant, respectively . The preferential increase of recombination at nonhotspot sites with hydrogen peroxide in the mutM mutant was discussed on the basis of a new model, in which 8-oxoG and/or Fapy residues may introduce double-strand breaks into DNA. EMBO J, 1999 Feb 1, 18(3), 771 - 83 The internal workings of a DNA polymerase clamp-loading machine; Turner J et al.; Replicative DNA polymerases are multiprotein machines that are tethered to DNA during chain extension by sliding clamp proteins . The clamps are designed to encircle DNA completely, and they are manipulated rapidly onto DNA by the ATP-dependent activity of a clamp loader . We outline the detailed mechanism of gamma complex, a five-protein clamp loader that is part of the Escherichia coli replicase, DNA polymerase III holoenzyme . The gamma complex uses ATP to open the beta clamp and assemble it onto DNA . Surprisingly, ATP is not needed for gamma complex to crack open the beta clamp . The function of ATP is to regulate the activity of one subunit, delta, which opens the clamp simply by binding to it . The delta' subunit acts as a modulator of the interaction between delta and beta . On binding ATP, the gamma complex is activated such that the delta' subunit permits delta to bind beta and crack open the ring at one interface . The clamp loader-open clamp protein complex is now ready for an encounter with primed DNA to complete assembly of the clamp around DNA . Interaction with DNA stimulates ATP hydrolysis which ejects the gamma complex from DNA, leaving the ring to close around the duplex. EMBO J, 1999 Feb 1, 18(3), 595 - 604 Propeptide cleavage conditions sortilin/neurotensin receptor-3 for ligand binding; Munck Petersen C et al.; We recently reported the isolation and sequencing of sortilin, a new putative sorting receptor that binds receptor-associated protein (RAP) . The luminal N-terminus of sortilin comprises a consensus sequence for cleavage by furin, R41WRR44, which precedes a truncation originally found in sortilin isolated from human brain . We now show that the truncation results from cellular processing . Sortilin is synthesized as a proform which, in late Golgi compartments, is converted to the mature receptor by furin-mediated cleavage of a 44 residue N-terminal propeptide . We further demonstrate that the propeptide exhibits pH-dependent high affinity binding to fully processed sortilin, that the binding is competed for by RAP and the newly discovered sortilin ligand neurotensin, and that prevention of propeptide cleavage essentially prevents binding of RAP and neurotensin . The findings evidence that the propeptide sterically hinders ligands from gaining access to overlapping binding sites in prosortilin, and that cleavage and release of the propeptide preconditions sortilin for full functional activity . Although proteolytic processing is involved in the maturation of several receptors, the described exposure of previously concealed ligand-binding sites after furin-mediated cleavage of propeptide represents a novel mechanism in receptor activation. EMBO J, 1999 Feb 1, 18(3), 555 - 64 Retrograde transport from the yeast Golgi is mediated by two ARF GAP proteins with overlapping function; Poon PP et al.; ARF proteins, which mediate vesicular transport, have little or no intrinsic GTPase activity . They rely on the actions of GTPase-activating proteins (GAPs) for their function . The in vitro GTPase activity of the Saccharomyces cerevisiae ARF proteins Arf1 and Arf2 is stimulated by the yeast Gcs1 protein, and in vivo genetic interactions between arf and gcs1 mutations implicate Gcs1 in vesicular transport . However, the Gcs1 protein is dispensable, indicating that additional ARF GAP proteins exist . We show that the structurally related protein Glo3, which is also dispensable, also exhibits ARF GAP activity . Genetic and in vitro approaches reveal that Glo3 and Gcs1 have an overlapping essential function at the endoplasmic reticulum (ER)-Golgi stage of vesicular transport . Mutant cells deficient for both ARF GAPs cannot proliferate, undergo a dramatic accumulation of ER and are defective for protein transport between ER and Golgi . The glo3Delta and gcs1Delta single mutations each interact with a sec21 mutation that affects a component of COPI, which mediates vesicular transport within the ER-Golgi shuttle, while increased dosage of the BET1, BOS1 and SEC22 genes encoding members of a v-SNARE family that functions within the ER-Golgi alleviates the effects of a glo3Delta mutation . An in vitro assay indicates that efficient retrieval from the Golgi to the ER requires these two proteins . These findings suggest that Glo3 and Gcs1 ARF GAPs mediate retrograde vesicular transport from the Golgi to the ER. Am J Respir Crit Care Med, 1999 Feb, 159(2), 610 - 2 Contaminated aerosol recovery from pulmonary function testing equipment; Hiebert T et al.; Clinically, the spread of infectious agents between subjects undergoing spirometry is quite uncommon . There is almost no documentation in the medical literature on this subject . We studied the retrieval of nonpathogenic Escherichia coli after aerosolizing organisms into standard pulmonary function tubing of a type that is frequently used by volume-sensing spirometers . The arrival of the aerosol at the distal end of the tubing was documented by culture . After delays of 0, 1, 5, and 10 min, respectively, air was forcibly withdrawn from the proximal end of the tubing through a special petri plate assembly . The plates were cultured and the colonies were counted . Immediately after insufflation of organisms, air withdrawn from the proximal tubing had counts similar to the air sampled at the distal end . After a 1-min delay, the proximal samples contained only rare organisms . No organisms were recovered from proximal air samples after a delay of 5 or 10 min after insufflation of organisms . The absence of detectable aerosolized E . coli after delays of 5 and 10 min after insufflation of organisms into spirometry tubing supports the hypothesis that a significant transfer of aerosolized organisms does not occur during routine pulmonary function testing as long as an interval of 5 min or more is allowed between tests. Am J Respir Crit Care Med, 1999 Feb, 159(2), 563 - 70 Response to inhaled nitric oxide in acute lung injury depends on distribution of pulmonary blood flow prior to its administration; Gust R et al.; Responses to inhaled nitric oxide (iNO) in acute lung injury (ALI), as evidenced by improvements in oxygenation, are variable . We hypothesized that the effect of iNO may be related to the pre-iNO distribution of pulmonary blood flow (PBF) . In the present study we evaluated the effect of iNO on PBF in normal healthy dogs and in a canine model of ALI induced by oleic acid (OA) . In Group "OA only" (n = 5), ALI was induced by central venous injection of 0.08 ml/kg OA . In Group "E+OA" (n = 5), hypoxic pulmonary vasoconstriction after ALI was blocked with low-dose endotoxin (15 microg/kg of Escherichia coli endotoxin) administered 30 min before giving the same dose of OA . Measurements of regional PBF and lung water concentration (LWC) using positron emission tomography (PET) and H215O were performed before and after OA or placebo, and then again at concentrations of 10, 40, and 0 ppm iNO . One hundred twenty minutes after OA injury, PaO2/FIO2 fell significantly in Group OA only, from 567 +/- 32 to 437 +/- 67 mm Hg . In these animals, PBF redistributed from the dorsal edematous regions of the lungs to the nondependent zones, thus partially preserving normal ventilation/ perfusion relationships . As in the normal animals, in Group OA only, iNO did not significantly change either PBF or oxygenation . In Group E+OA, the administration of low-dose endotoxin eliminated perfusion redistribution from the dorsal edematous lung regions . As a result, PaO2/FIO2 fell from 558 +/- 70 to 119 +/- 53 mm Hg, a decrease that was significantly greater than that in Group OA only . In Group E+OA, administration of iNO restored perfusion redistribution to a similar level as in Group OA only, which was associated with a significant improvement in PaO2/FIO2, from 119 +/- 53 to 251 +/- 159 (10 ppm iNO), and 259 +/- 165 mm Hg (40 ppm iNO) . We conclude that the effect of iNO on oxygenation after ALI depends on the pre-iNO perfusion pattern, which may help explain the variable response to iNO often observed in patients with acute respiratory distress syndrome. Endocrinology, 1999 Feb, 140(2), 568 - 74 Characteristics of a highly labile human type 5 17beta-hydroxysteroid dehydrogenase; Dufort I et al.; 17Beta-hydroxysteroid dehydrogenases (17betaHSDs) play an essential role in the formation of active intracellular sex steroids . Six types of 17betaHSD have been described to date, which only share approximately 20% homology . Human type 5 17betaHSD complementary DNA is unique among the 17betaHSDs because it belongs to the aldo-keto reductase family, whereas the others are members of the short chain alcohol dehydrogenases . The characteristics of human type 5 17betaHSD were investigated in human embryonic (293) cells stably transfected with human and mouse type 5 17betaHSD, as well as human type 3 3alphaHSD . Using intact transfected cells, type 5 17betaHSD shows a substrate specificity pattern comparable to those of human type 3 17betaHSD and mouse type 5 17betaHSD . These enzymes catalyze more efficiently the transformation of androstenedione (4-dione) to testosterone, whereas the transformation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol is much lower . In contrast, type 3 3alphaHSD catalyzes more efficiently the transformation of dihydrotestosterone to 5alpha-androstane-3alpha,17beta-diol, whereas the transformation of 4-dione to testosterone represents only 7% of the 3alphaHSD activity . However, upon homogenization, human type 5 17betaHSD activity decreases to approximately 10% of the activity in intact cells and remains stable at this level together with the 3alphaHSD activity . Under the same conditions, however, the mouse enzyme is not altered by homogenization . Indeed, using purified human 17betaHSD overexpressed in Escherichia coli, we could confirm that a much greater amount of protein is required to produce activity similar to the enzymatic activity measured in intact transfected cells . The present data provide the answer to the question of why previous researchers could hardly detect type 5 17betaHSD activity . Indeed, all previous publications used cell or tissue homogenates or purified enzymes . Under such conditions, only the low level, but stable, 3alphaHSD and 17betaHSD activities could be measured, whereas the high level, but highly unstable, 17betaHSD activity could not be measured . As type 5 17betaHSD shares 84%, 86%, and 88% amino acid identity with types 1 and 3 3alphaHSD and 20alphaHSDs, respectively, Northern blot analysis used in previous studies could not provide unequivocal information . In this report, we used a more specific ribonuclease protection assay and could thus show that human type 5 17betaHSD is expressed in the liver, adrenal, and prostate; in prostatic cancer cell lines DU-145 and LNCaP; as well as in bone carcinoma (MG-63) cells . By analogy with type 3 17betaHSD, which is responsible for the formation of androgens in the testis, the expression of type 5 17betaHSD in the prostate and bone cells suggests that this enzyme is involved in the formation of active intracellular androgens in these tissues. Virus Genes, 1998, 17(3), 207 - 11 Sequence and expression in Escherichia coli of the coat protein gene of the dwarfing strain of soybean dwarf luteovirus; Smith OP et al.; The nucleotide sequence of the coat protein gene of the dwarfing (D) strain of soybean dwarf luteovirus (SbDV) was determined from cloned cDNA . The gene contains 600 nucleotides and encodes a protein of 200 amino acids with a calculated molecular mass of 22.2 kDa . A major portion of the coat protein open reading frame (ORF) was expressed in Escherichia coli as a pET fusion protein and the product was detected by western blot analysis using SbDV-D polyclonal antibodies . Comparison of the deduced coat protein amino acid sequence to that from the yellowing (Y) strain of SbDV demonstrated 88% identity. Acta Anaesthesiol Scand, 1999 Jan, 43(1), 56 - 63 Intestinal and hepatic perfusion and metabolism in hypodynamic endotoxic shock . Effects of nitric oxide synthase inhibition; Dahm PL et al.; BACKGROUND: Inhibition of nitric oxide synthase (NOS) has been claimed to be beneficial in septic shock . We investigated the overall and regional effects of a NOS-inhibitor on perfusion and metabolism during severe endotoxic shock . METHODS: Nineteen anaesthetised pigs were catheterised and ultrasonic flow-probes were placed around the portal vein, the hepatic artery, and the superior mesenteric artery . Thirteen animals were given a 3-h infusion of endotoxin; in 6 of these an infusion of NG-nitro-L-arginine-methyl-ester (L-NAME) was started an hour after the start of endotoxin while 7 animals served as controls and received endotoxin only . Six animals were sham operated with no further intervention . RESULTS: Endotoxin produced a hypodynamic shock with pulmonary hypertension . L-NAME did not increase arterial blood pressure, but deepened the fall in cardiac output and enhanced the increase in systemic and pulmonary vascular resistance . The infusion of endotoxin caused a decrease in flows in all regions . The addition of L-NAME induced a further decrease in the mesenteric artery flow only . L-NAME had no additional effect on hepatic artery flow ratio, while a transient decrease was seen in mesenteric flow ratio . Portal flow ratio decreased in the control group only . Global as well as regional oxygen extraction increased in both groups, more so in the L-NAME group . Lactate levels increased with no differences between the groups . CONCLUSION: In hypodynamic endotoxic shock, L-NAME infusion enhanced pulmonary vasoconstriction and increased left ventricular afterload . The resulting hypoperfusion caused an increase in mortality . The effects of L-NAME on global and mesenteric blood flow and metabolism were similar, while L-NAME had no additional effects on hepatic hypoperfusion or oxygen extraction . Thus, nitric oxide does not seem to be a major factor in the preservation of hepatic perfusion during unresuscitated endotoxic shock. J Mol Biol, 1999 Feb 5, 285(5), 1977 - 91 Classification of Arabidopsis thaliana gene sequences: clustering of coding sequences into two groups according to codon usage improves gene prediction; Mathe C et al.; While genomic sequences are accumulating, finding the location of the genes remains a major issue that can be solved only for about a half of them by homology searches . Prediction methods are thus required, but unfortunately are not fully satisfying . Most prediction methods implicitly assume a unique model for genes . This is an oversimplification as demonstrated by the possibility to group coding sequences into several classes in Escherichia coli and other genomes . As no classification existed for Arabidopsis thaliana, we classified genes according to the statistical features of their coding sequences . A clustering algorithm using a codon usage model was developed and applied to coding sequences from A . thaliana, E . coli, and a mixture of both . By using it, Arabidopsis sequences were clustered into two classes . The CU1 and CU2 classes differed essentially by the choice of pyrimidine bases at the codon silent sites: CU2 genes often use C whereas CU1 genes prefer T . This classification discriminated the Arabidopsis genes according to their expressiveness, highly expressed genes being clustered in CU2 and genes expected to have a lower expression, such as the regulatory genes, in CU1 . The algorithm separated the sequences of the Escherichia-Arabidopsis mixed data set into five classes according to the species, except for one class . This mixed class contained 89 % Arabidopsis genes from CU1 and 11 % E . coli genes, mostly horizontally transferred . Interestingly, most genes encoding organelle-targeted proteins, except the photosynthetic and photoassimilatory ones, were clustered in CU1 . By tailoring the GeneMark CDS prediction algorithm to the observed coding sequence classes, its quality of prediction was greatly improved . Similar improvement can be expected with other prediction systems . J Mol Biol, 1999 Feb 5, 285(5), 1935 - 50 Trans-splicing ribozymes for targeted gene delivery; Kohler U et al.; Ribozymes are potential tools for genetic manipulation, and various naturally occurring catalytic RNAs have been dissected and used as the basis for the design of new endoribonuclease activities . While such cleaving ribozymes may work well in vitro, they have not proved to be routinely effective in depleting living cells of the chosen target RNA . Recently, trans-splicing ribozymes have been employed to repair mutant mRNAs in vivo . We have designed modified trans-splicing ribozymes with improved biological activity . These allow accurate splicing of a new 3' exon sequence into a chosen site within a target RNA, and in frame fusion of the exon can result in expression of a new gene product . These trans-splicing ribozymes contain catalytic sequences derived from a self-splicing group I intron, which have been adapted to a chosen target mRNA by fusion of a region of extended complementarity to the target RNA and precise alteration of the guide sequences required for substrate recognition . Both modifications are required for improved biological activity of the ribozymes . Whereas cleaving ribozymes must efficiently deplete a chosen mRNA species to be effective in vivo, even inefficient trans-splicing can allow the useful expression of a new gene activity, dependent on the presence of a chosen RNA . We have targeted trans-splicing ribozymes against mRNAs of chloramphenicol acetyltransferase, human immunodeficiency virus, and cucumber mosaic virus, and demonstrated trans-splicing and delivery of a marker gene in Escherichia coli cells . The improved trans-splicing ribozymes may be tailored for virtually any target RNA, and provide a new tool for trigger