Mol Cell Biol, 1991 Aug, 11(8), 4111 - 20 The highly conserved N-terminal domains of histones H3 and H4 are required for normal cell cycle progression; Morgan BA et al.; The N-terminal domains of the histones H3 and H4 are highly conserved throughout evolution . Mutant alleles deleted for these N-terminal domains were constructed in vitro and examined for function in vivo in Saccharomyces cerevisiae . Cells containing a single deletion allele of either histone H3 or histone H4 were viable . Deletion of the N-terminal domain of histone H4 caused cells to become sterile and temperature sensitive for growth . The normal cell cycle progression of these cells was also altered, as revealed by a major delay in progression through the G2 + M periods . Deletion of the N-terminal domain of histone H3 had only minor effects on mating and the temperature-sensitive growth of mutant cells . However, like the H4 mutant, the H3 mutants had a significant delay in completing the G2 + M periods of the division cycle . Double mutants containing N-terminal domain deletions of both histone H3 and histone H4 were inviable . The phenotypes of cells subject to this synthetic lethality suggest that the N-terminal domains are required for functions essential throughout the cell division cycle and provide genetic evidence that histones are randomly distributed during chromosome replication. J Virol, 1991 Aug, 65(8), 4486 - 9 Bacterially expressed nucleoprotein of infectious hematopoietic necrosis virus augments protective immunity induced by the glycoprotein vaccine in fish; Oberg LA et al.; The ribonucleoprotein gene of infectious hematopoietic necrosis virus (IHNV) has been expressed in Escherichia coli as a trpE fusion protein . This viral protein does not induce protective immunity to lethal IHNV infection in fish, and virus-neutralizing antibodies do not react with this viral protein . However, when it was administered with a bacterial lysate containing a region of the IHNV glycoprotein, there was enhanced resistance in immunized fish to lethal virus infection. Biol Chem Hoppe Seyler, 1991 Aug, 372(8), 613 - 24 Mitochondrial 3-2trans-Enoyl-CoA isomerase . Purification, cloning, expression, and mitochondrial import of the key enzyme of unsaturated fatty acid beta-oxidation; Muller-Newen G et al.; 3-2trans-Enoyl-CoA isomerase (EC 5.3.3.8) is a key enzyme in mitochondrial beta-oxidation of unsaturated fatty acids in bacteria, plant and animal cells . The enzyme was isolated from rat liver mitochondria and purified to homogeneity by two chromatographic steps . Partial polypeptide sequences of the 29 kDa protein were derived from cyanogen bromide, tryptic, Lys-C, and protease V8 fragments by Edman degradation . Peptide-derived synthetic oligonucleotides were used for the isolation of a 990 bp long isomerase-specific cDNA from rat liver cDNA libraries . 867 bp encode the 289 amino-acid residues of the preisomerase with a molecular mass of 32,254 Da . The 1.3-kb mRNA is most strongly expressed in skeletal muscle followed by liver, heart, kidney, and weakly expressed in spleen and brain . In vitro transcription and translation yielded a 32 kDa polypeptide which was immunoprecipitated by anti rat isomerase antibodies . In the presence of mitochondria the 32 kDa precursor isomerase was processed during mitochondrial import to the 29 kDa mature form of the 3-2trans-enoyl-CoA isomerase with 264 amino-acid residues (Mr 29,706) . A N-terminal signal sequence of 25 amino-acid residues directs the import into the mitochondrial matrix and is cleaved in two successive steps passing through an intermediate form of Mr 30,475 . The two cysteine residues in positions 142 and 148 of the preisomerase are present as free thiol groups as shown by derivatization of the mature, native protein with the fluorescent label N-(iodoacetaminoethyl)-1-naphthylamine-5-sulfonic acid . The mitochondrial 3-2trans enoyl-CoA isomerase shows significant homology and conserved amino-acid exchanges with the mitochondrial enoyl-CoA hydratase, the N-terminal domain of the bifunctional peroxisomal enoyl-CoA-hydratase:3-hydroxyacyl-CoA dehydrogenase and to extended domains of the alpha-subunit of the procaryotic beta-oxidation complex sharing enoyl-CoA isomerase, D(-)3-hydroxyacyl-CoA epimerase, enoyl-CoA hydratase and L(+)3-hydroxyacyl-CoA dehydrogenase activity, encoded by the fad B operon of E . coli. J Gen Microbiol, 1991 Aug, 137 ( Pt 8), 1963 - 70 Positive regulation of colonization factor antigen I (CFA/I) production by enterotoxigenic Escherichia coli producing the colonization factors CS5, CS6, CS7, CS17, PCFO9, PCFO159:H4 and PCFO166; Hibberd ML et al.; Entertoxigenic Escherichia coli (ETEC) strains of nineteen serogroups which produced colonization factors (coli-surface-associated antigens CS5, CS6, CS7 and CS17, colonization factor antigen CFA/III and putative colonization factors PCFO159:H4, PCFO166 and PCFO9) were tested for hybridization with a DNA probe containing the cfaD sequence that regulates expression of CFA/I . Strong colony hybridization, similar to that with the CFA/I-positive control strain H10407, occurred with ETEC strains of serogroups O27, O159 and O169 which produced CS6 antigen, and with all the strains which produced PCFO166 fimbriae . Weak colony hybridization, compared to the control strain, was found with ETEC producing CS5 fimbriae with CS6 antigen, CFA/III fimbriae with CS6 antigen, CS7 fimbriae or PCFO159:H4 fimbriae . CS6-antigen-positive strains of serogroups O79, O89 and O148 and all the CS17-antigen-positive and PCFO9-fimbriae-positive strains were negative in colony hybridization tests with the cfaD probe . Plasmid DNA of nine ETEC strains and their colonization-factor-negative derivatives was tested for hybridization with the cfaD probe and with ST and LT oligonucleotide probes . The sequences that hybridized with the cfaD probe were on the plasmids which coded for enterotoxin production . Fifteen strains were transformed with NTP513, a recombinant plasmid which contains the CFA/I region 1 fimbrial subunit operon but lacks a functional cfaD sequence, in order to determine whether DNA in any of these strains could substitute for the cfaD sequence in the regulation of production of CFA/I fimbriae.(ABSTRACT TRUNCATED AT 250 WORDS) Can J Microbiol, 1991 Aug, 37(8), 650 - 3 Detection of Escherichia coli cytotoxins by enzyme-linked immunosorbent assays; Speirs JI et al.; Sandwich enzyme-linked immunosorbent assays (ELISAs) were developed to detect Escherichia coli cytotoxins . Wells were coated with monoclonal antibodies from hybridomas 13C4 and (or) 11E10, and biotin conjugates of these antibodies were used for detecting verotoxin 1 and Shiga-like toxin II, respectively . Sensitivities were about 100 and 200 cytotoxic doses, respectively . Verotoxin 2 was detected by ELISA with monoclonal antibody 11E10, but at a sensitivity of only about 4000 cytotoxic doses . ELISA results of polymyxin-treated cell extracts from cultures of 67 E . coli strains were in agreement with Vero cell assay as regards the presence and type of toxin. Zentralbl Veterinarmed B, 1991 Aug, 38(6), 445 - 52 Specific DNA fragments coding for ST1 and LT1 toxins, and K88 (F4) adhesin in enterotoxigenic Escherichia coli; Wasteson Y et al.; Ten porcine strains of enterotoxigenic Escherichia coli possessing the K88 (F4) adhesion fimbriae, were selected for study of enterotoxin- and fimbriae-encoding plasmids . Plasmid DNA, separated according to size by gel electrophoresis was transferred to nylon membranes by Southern blotting, and hybridized with enzyme-labelled oligonucleotide probes for ST1 and LT1 enterotoxins, and a 32P-labelled probe for the F4 fimbriae . Plasmids possessing the enterotoxin genes ranged from 50 MDa to 78 MDa in size . The ST1 genes were located on a common 8-MDa EcoR1 restriction endonuclease fragment, while the LT1 genes were located on a 4.5-MDa EcoR1 fragment from the different plasmids . Plasmids with the F4 genes ranged from 50 MDa to 118 MDa in size, but the F4 encoding genes were located on a common 3-MDa HindIII restriction endonuclease fragment . ST1 and LT1 genes were found on the same plasmid in only one strain, LT1 and F4 genes on the same plasmids in 5 strains, while no plasmid contained genes for both ST1 and F4. Radiat Res, 1991 Aug, 127(2), 202 - 10 Mutations induced by ionizing radiation in a plasmid replicated in human cells . II . Sequence analysis of alpha-particle-induced point mutations; Jaberaboansari A et al.; The human shuttle plasmid pZ189, containing the Escherichia coli supF gene as the mutational target, was irradiated in vitro with 210Po alpha particles and transfected into human lymphoblastoid cells . Plasmids which were replicated in human cells were recovered and those containing mutant supF genes were isolated by phenotypic screening in E . coli . The mutations were characterized by sequencing the tRNA gene . The mutant frequency increased linearly with the alpha-particle dose and, at 259 Gy, it was 16 times (0.29%) that observed in unirradiated controls (0.018%) . The distribution of alpha-particle-induced point mutations was highly nonrandom and similar to that observed in the unirradiated or X-irradiated plasmid DNAs . The majority of the mutations were G.C----A.T transitions and occurred selectively at most 5'-TC (3'-AG) and 5'-CC (3'-GG) sequences . For the unirradiated control DNA, these mutations at C's (G's) were preferentially located in the nontranscribed strand, similar to the observation previously made for mutations in X-irradiated DNA . Such a strand bias was not observed for mutations in the alpha-particle-irradiated DNA . The data suggest that, although similar types of point mutations are induced in unirradiated, X-irradiated, and alpha-particle-irradiated DNAs, the mechanisms of their induction and the exact nature of the lesions involved may be quite different. Radiat Res, 1991 Aug, 127(2), 190 - 201 Mutations induced by ionizing radiation in a plasmid replicated in human cells . I . Similar, nonrandom distribution of mutations in unirradiated and X-irradiated DNA; Waters LC et al.; The Escherichia coli supF gene encoding the suppressor tyrosine tRNA in a human shuttle plasmid, pZ189, was used as a target for molecular analysis of X-ray-induced mutations in human lymphoblastoid cells . Following replication of the in vitro-irradiated plasmid in human cells, the mutant supF-containing molecules were cloned by phenotypic screening in E . coli and the nature of the mutations was determined by direct sequencing of the tRNA gene . At 160 Gy the mutant frequency was 13 times (0.39%) that observed in unirradiated controls (0.031%) . When control plasmid was replicated directly in E . coli, the mutant frequency was 16 times less than that of the plasmid passaged through the human cells . The distribution of mutations was highly nonrandom and remarkably similar in both irradiated and control DNAs . The majority of the mutations were transitions involving G.C pairs and occurred selectively at most 5'-TC (3'-AG) sequences . These mutations at C's were preferentially distributed in the nontranscribed strand . We propose that mutations in the control plasmid result from oxidative damages that occur during and/or after its incorporation into human cells and that these damages are similar to those induced by ionizing radiation . The hot spots for mutations suggest that the proximate nucleotide sequence and the overall conformation of the target DNA are important in the production and/or processing of these damages during repair and replication. Radiat Res, 1991 Aug, 127(2), 156 - 63 Damage to the plasma membrane in Escherichia coli K-12 induced by far-ultraviolet radiation and its repair; Mody R et al.; Escherichia coli cells treated with low fluences of far-uv radiation (up to 90 J/m2) showed repairable damage to the plasma membrane . The loss of the ability of the cells to exclude citrate was evident from the respiratory stimulation of irradiated cells when citrate was provided exogenously . This loss of a barrier was a result of a structural disorganization of the plasma membrane as seen by freeze-etching electron microscopy . Analysis of the plasma membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a characteristic loss of certain membrane proteins . When irradiated cells were incubated in glucose minimal medium at 37 degrees C for various times, a gradual recovery of membrane structure and function was observed . The recovery process was inhibited in the absence of an energy source as well as protein synthesis . The majority of the recovery occurred in the initial 1 h of the postirradiation holding . These results demonstrated that far-uv radiation at a fluence less than the D10 value had a direct or indirect effect on plasma membrane proteins, causing their release from the membrane bilayer . The lost proteins were subsequently regained by de novo protein synthesis. FEMS Microbiol Lett, 1991 Aug 1, 66(2), 189 - 93 Transformation of Aspergillus terreus with the hygromycin B resistance marker from Escherichia coli; Ventura L et al.; Aspergillus terreus was transformed to hygromycin B resistance using a bacterial resistance gene under the control of Aspergillus nidulans regulatory sequences . Southern hybridization of transformants indicated that in most of the cases the vector DNA was integrated into the recipient chromosome in the form of tandem arrays . Transformants were mitotically stable in both selective and non-selective medium and retained their capacity to produce xylanase or glucoamylase activities. FEMS Microbiol Lett, 1991 Aug 1, 66(2), 137 - 41 Two different Escherichia coli capsular polysaccharide depolymerases each associated with one of the coliphage phi K5 and phi K20; Nimmich W et al.; The Escherichia coli capsular polysaccharides (K antigens) K5 and K20 are known as primary receptors for the coliphage phi K5 and phi K20, respectively . A host range study of the phage revealed that E . coli K5 strains were not only lysed by phi K5 but also by phi K20, and furthermore that the E . coli K95 test strain was attacked by phi K5 in addition to K5 strains . In order to find out whether the phage can degrade the K antigens, the interaction of the phage with isolated polysaccharides was studied . It could be demonstrated that phi K5 was able to depolymerize the K5 and K95 polysaccharides and that phi K20 showed degrading activity towards the antigens K20 and K5 . Obviously, each of the phages was associated with two different enzyme systems which enabled them to recognize and depolymerize chemically unrelated polysaccharides. Eur J Cell Biol, 1991 Aug, 55(2), 191 - 9 In vitro reconstitution of recombinant lamin A and a lamin A mutant lacking the carboxy-terminal tail; Gieffers C et al.; Xenopus lamin A and a lamin A mutant lacking the complete 280 amino acid long carboxy-terminal tail were expressed in bacteria and purified from inclusion bodies . Electron microscopic analysis of lamin A dimers revealed that the carboxy-terminal 280 amino acids correspond to the globular domain seen in rotary-shadowed wild-type lamin and that the rodlike domain consists of the short non-helical amino terminus and the alpha-helical region . During reconstitution lamin A dimers first formed polar head to tail aggregates which then associated laterally resulting in paracrystals with periodic repeats of 25 nm . In the mutant, the longitudinal and lateral association of dimers had not been influenced, however, periodic repeats were absent in the filament bundles formed . Thus our data clearly demonstrate that carboxy-terminal tails are localized in light-stained regions of negatively stained paracrystals and that they are responsible for the alternating light dark staining of paracrystals . Fibrils, 2 to 3 nm thick, were a common structural element of paracrystals and filament bundles. New Biol, 1991 Aug, 3(8), 729 - 33 Is the occurrence of some spontaneous mutations directed by environmental challenges? Hall BG. Cairnsian mutations have been defined as nonrandom mutations that occur as specific and direct responses to environmental challenges . This article reviews the evidence for the occurrence of such mutations in Escherichia coli, and concludes that under conditions of prolonged, intense selection Cairnsian mutations occur at several loci, and include base substitution mutations, frameshift mutations, and mutations mediated by excision of mobile genetic elements . Cairnsian mutations occur in nondividing cells . They are thus time-dependent, rather than replication-dependent . The process that produces Cairnsian mutations is so powerful that it can generate double mutations at rates (mutations per cell per day) that approach the rates of the component single mutations under identical conditions . Several mechanisms, including slow repair of mis-matched bases, mutagenic transcription, and a hypothetical "hypermutable" physiological state, have been proposed to explain the occurrence of Cairnsian mutations by an underlying random process, rather than by the instructional, or "directed" process originally proposed by Cairns . Recent evidence, however, argues strongly against all of those proposed mechanisms and leaves us without a viable model to explain this powerful, and potentially very important, process. Genet Anal Tech Appl, 1991 Aug, 8(5), 148 - 50 Agarose entrapment method for the production of SfiI linking library for Theileria parva; Young JR et al.; We have developed a simple method for isolation of SfiI linking clones from a eukaryotic genomic DNA . The method involves the physical separation of the small proportion of plasmids in a plasmid genomic library that are linearized by SfiI digestion, from the bulk of molecules that remain circular, by ordinary electrophoresis through high-percentage gels of SeaPlaque agarose . Following the isolation of linearized molecules, their recircularization, and introduction into Escherichia coli, 55% of recovered plasmids contained inserts of the expected size, and 73% of these had SfiI sites . This represented a 25-fold enrichment of linking clones expected to be present at a frequency of 1/60 in the original library of 4- 6-kb fragments of genomic DNA of the protozoan parasite Theileria parva . This approach is rapid and obviates the need for introduction of a selectable marker . It is uniquely appropriate for linking clones spanning SfiI sites as this enzyme leaves degenerate 3' overhanging ends that preclude the direct ligation into vector sites required by most alternative strategies, but that favor the recircularization reactions used here. Poult Sci, 1991 Aug, 70(8), 1709 - 15 Vitamin E as adjuvant in emulsified vaccine for chicks; Franchini A et al.; Mineral oil was partially replaced with D, L-alpha-tocopheryl acetate (vitamin E) in bacterial and viral inactivated emulsified vaccines . Vitamin E increased the immune response to the viral antigen (Newcastle disease virus) used but not to the bacterial antigen (Escherichia coli) when its presence in the oil phase did not exceed 30% . Inoculated vitamin E may have enhanced the immune response by interacting with the immune-competent cells involved in the inflammatory reaction that followed inoculation of emulsified vaccines. Genetics, 1991 Aug, 128(4), 695 - 701 Adaptive reversion of a frameshift mutation in Escherichia coli; Cairns J et al.; Mutation rates are generally thought not to be influenced by selective forces . This doctrine rests on the results of certain classical studies of the mutations that make bacteria resistant to phages and antibiotics . We have studied a strain of Escherichia coli which constitutively expresses a lacI-lacZ fusion containing a frameshift mutation that renders it Lac- . Reversion to Lac+ is a rare event during exponential growth but occurs in stationary cultures when lactose is the only source of energy . No revertants accumulate in the absence of lactose, or in the presence of lactose if there is another, unfulfilled requirement for growth . The mechanism for such mutation in stationary phase is not known, but it requires some function of RecA which is apparently not required for mutation during exponential growth. Chin Med J (Engl), 1991 Aug, 104(8), 669 - 72 Comparative evaluation of nine different methods for detecting enterotoxin of Escherichia coli; Zhu QY et al.; Nine different methods for detecting enterotoxin of Escherichia coli were studied and compared . We found rabbit ileal-loop test and suckling mouse assay were both quite accurate and reliable for detecting heat labile toxin (LT) and heat stable toxin (ST) of enterotoxigenic Escherichia coli (ETEC) . Mouse ileal-loop test was simple, but its sensitivity and specificity were comparatively low . CHO cell-culture assay might be more sensitive and specific . LT-DNA probe was the most sensitive and specific method . In practical application, PIHT (plate immunohemolytic test), Biken's, SPA-CoA and ELISA methods are recognized as simple, rapid, sensitive and specific methods for detecting ETEC-LT . These methods can be selected for use in clinical laboratory. Mol Gen Genet, 1991 Aug, 228(1-2), 249 - 57 Inactivation of lacZ gene expression by UV light and bound DNA photolyase implies formation of extended complexes in the genomes of specific Escherichia coli strains; Li BH et al.; In Escherichia coli strains WU and CS101, UV inactivation of lacZ gene expression is more effective when the cells contain amplified DNA photolyase, and flash photoreactivation (fPR) after 15 min of metabolism does not reverse inactivation by the photolyase-dimer complexes . In other strains, also studied with or without amplified DNA photolyase, there is no differential UV inactivation and fPR reverses inactivation by the complexes regardless of continued metabolism . The irreparable condition in strain WU is not due to dysfunction of photolyase: during post-UV metabolism, fPR still restores viability and dimers are removed from the region of the lac operon . When the wild-type lac promoter is replaced by the UV5 promoter, making expression insensitive to relaxed supercoiling and catabolite repression, inactivation by dimers alone becomes more resistant, i.e . requires higher fluences, but inactivation in WU and CS101 is still exceptionally sensitive to photolyase-dimer complexes . This indicates that dimers external to the wild-type lac operon may inhibit expression by altering supercoiling but that complexes must involve some other mechanism for their special effect in WU and CS101 . The exceptionally efficient inactivation and irreparable condition are consistent with the idea that, in two specific laboratory strains, photolyase bound to dimers at a considerable distance from the lac operon may initiate an aggregation of DNA with other cellular molecules that extends to, and inactivates expression from, the operon. Mutat Res, 1991 Aug, 253(1), 103 - 8 In vitro and in vivo analysis of somatic and germline mutability of 2-amino-N6-hydroxyadenine in Drosophila melanogaster; Smith PD et al.; Two complementary assays were employed to examine the mutagenicity of 2-amino-N6-hydroxyadenine (AHA) in Drosophila melanogaster . A lambda phage-based shuttle vector system, utilizing the supF transfer RNA gene of Escherichia coli, questioned the mutagenicity of AHA in established cell cultures derived from somatic tissue while the standard sex-linked recessive lethal assay measured mutational events in vivo . Consistent with studies in other systems, AHA appears strongly mutagenic when cells are exposed directly . Conversely, in vivo studies suggest that AHA is not a strong mutagen . Further studies will determine if AHA is weakly or not mutagenic in vivo and, using the supF system, what the nature of the mutational events at the molecular level is. J Cell Biol, 1991 Aug, 114(4), 671 - 9 Reconstitution of GTP-binding Sar1 protein function in ER to Golgi transport; Oka T et al.; In the yeast secretory pathway, two genes SEC12 and SAR1, which encode a 70-kD integral membrane protein and a 21-kD GTP-binding protein, respectively, cooperate in protein transport from the ER to the Golgi apparatus . In vivo, the elevation of the SAR1 dosage suppresses temperature sensitivity of the sec12 mutant . In this paper, we show cell-free reconstitution of the ER-to-Golgi transport that depends on both of these gene products . First, the membranes from the sec12 mutant cells reproduce temperature sensitivity in the in vitro ER-to-Golgi transport reaction . Furthermore, the addition of the Sar1 protein completely suppresses this temperature-sensitive defect of the sec12 membranes . The analysis of Sar1p partially purified by E . coli expression suggests that GTP hydrolysis is essential for Sar1p to execute its function. Proc Natl Acad Sci U S A, 1991 Aug 1, 88(15), 6603 - 7 Mutations in 16S rRNA that affect UGA (stop codon)-directed translation termination; Goringer HU et al.; Site-directed mutagenesis was performed on a sequence motif within the 3' major domain of Escherichia coli 16S rRNA shown previously to be important for peptide chain termination . Analysis of stop codon suppression by the various mutants showed an exclusive response to UGA stop signals, which was correlated directly with the continuity of one or the other of two tandem complementary UCA sequences (bases 1199-1204) . Since no other structural features of the mutated ribosomes were hampered and the translation initiation and elongation events functioned properly, we propose that a direct interaction occurs between the UGA stop codon on the mRNA and the 16S rRNA UCA motif as one of the initial events of UGA-dependent peptide chain termination . These results provide evidence that base pairing between rRNA and mRNA plays a direct role in termination, as it has already been shown to do for initiation and elongation. J Bacteriol, 1991 Aug, 173(16), 5244 - 6 Cysteine, even in low concentrations, induces transient amino acid starvation in Escherichia coli; Sorensen MA et al.; Cysteine, in concentrations down to 0.04 micrograms/ml, induces transient amino acid starvation in Escherichia coli growing in minimal medium . The duration depends on the concentration and is 5 min at 2 micrograms of cysteine per ml . At low cysteine concentrations, threonine and isoleucine almost completely abolish the starvation. J Bacteriol, 1991 Aug, 173(16), 5030 - 5 Cofactor requirements of BamHI mutant endonuclease E77K and its suppressor mutants; Xu SY et al.; A mutant BamHI endonuclease, E77K, belongs to a class of catalytic mutants that bind DNA efficiently but cleave DNA at a rate more than 10(3)-fold lower than that of the wild-type enzyme (S . Y . Xu and I . Schildkraut, J . Biol . Chem . 266:4425-4429, 1991) . The preferred cofactor for the wild-type BamHI is Mg2+ . BamHI is 10-fold less active with Mn2+ as the cofactor . In contrast, the E77K variant displays an increased activity when Mn2+ is substituted for Mg2+ in the reaction buffer . Mutations that partially suppress the E77K mutation were isolated by using an Escherichia coli indicator strain containing the dinD::lacZ fusion . These pseudorevertant endonucleases induce E . coli SOS response (as evidenced by blue colony formation) and thus presumably nick or cleave chromosomal DNA in vivo . Consistent with the in vivo result, the pseudorevertant endonucleases in the crude cell extract display site-specific partial DNA cleavage activity . DNA sequencing revealed two unique suppressing mutations that were located within two amino acid residues of the original mutation . Both pseudorevertant proteins were purified and shown to increase specific activity at least 50-fold . Like the wild-type enzyme, both pseudorevertant endonucleases prefer Mg2+ as the cofactor . Thus, the second-site mutation not only restores partial cleavage activity but also suppresses the metal preference as well . These results suggest that the Glu-77 residue may play a role in metal ion binding or in enzyme activation (allosteric transition) following sequence-specific recognition. Surgery, 1991 Aug, 110(2), 154 - 60; discussion 160-1 Maintenance of superior mesenteric arterial perfusion prevents increased intestinal mucosal permeability in endotoxic pigs; Fink MP et al.; Lipopolysaccharide increases intestinal mucosal permeability to hydrophilic compounds such as chromium 51-labeled edetate (51Cr-EDTA) . We sought to determine whether this phenomenon is partly mediated by lipopolysaccharide-induced mesenteric hypoperfusion . We assessed permeability in an isolated segment of ileum by measuring plasma-to-lumen clearances (C) for two probes, 51Cr-EDTA and urea, and expressing the results as a ratio (CEDTA/CUREA) . In control pigs (n = 6) resuscitated with Ringer's lactate (RL), mucosal permeability was unchanged during the 210-minute period of observation . In pigs (n = 7) infused with lipopolysaccharide (50 micrograms/kg) and similarly resuscitated with RL, mesenteric perfusion (Qsma) decreased significantly and permeability increased progressively and significantly . When endotoxic pigs (n = 6) were resuscitated with a regimen (RL plus hetastarch plus dobutamine) that preserved normal Qsma, lipopolysaccharide-induced mucosal hyperpermeability was prevented . Resuscitation of endotoxic pigs (n = 6) with RL plus hetastarch provided intermediate protection against both mesenteric hypoperfusion and increased permeability . These data suggest that diminished Qsma contributes to impaired ileal mucosal barrier function in experimental endotoxicosis. Arch Biochem Biophys, 1991 Aug 1, 288(2), 495 - 9 Increase in fidelity of rat liver Ile-tRNA formation by both spermine and the aminoacyl-tRNA synthetase complex; Kusama-Eguchi K et al.; To examine the polyamine effects on the fidelity at the aminoacylation level and the physiological significance of the existence of the aminoacyl-tRNA synthetase complex (ARSC) in animal cells, a single-chain Ile-tRNA synthetase (IRSS) was isolated from the complex by treatment with trypsin . Ile-tRNA formation by IRSS was strongly stimulated by spermine, similar to the results with ARSC . Two misacylations (Val-tRNAIle and Ile-tRNAiMet formation) by IRSS were measured . The error frequency was higher in Ile-tRNAiMet formation (tRNA misacylation) than in Val-tRNAIle formation (amino acid misacylation) . Spermine did not influence significantly Ile-tRNAiMet formation, but it stimulated Val-tRNAIle formation by IRSS . Accordingly, spermine decreased the error frequency of tRNA misacylation, but not amino acid misacylation . These results suggest that the conformational changes of individual tRNA by spermine differ from each other, meaning that spermine influences the interaction between individual tRNA and aminoacyl-tRNA synthetase variously . When the aminoacylations of tRNAIle from rat liver, yeast, and Escherichia coli were compared with ARSC and IRSS, the relative speed of Ile-tRNA formation with tRNAIle from other species was faster with IRSS than with ARSC . This indicates that ARSC can recognize tRNAIle from the same species more specifically than IRSS . These results show that both spermine and ARSC are involved in the increase of fidelity of rat liver Ile-tRNA formation. Arch Biochem Biophys, 1991 Aug 1, 288(2), 350 - 7 Pyrroline-5-carboxylate reductase in soybean nodules: isolation/partial primary structure/evidence for isozymes; Chilson OP et al.; Electrophoretic evidence was obtained for two forms of pyrroline-5-carboxylate reductase (P5CR) in soybean nodules . One form was purified over 2300-fold . The apparent sizes of the polypeptides comprising the pyrroline-5-carboxylate reductases from soybean cytosol (29,700) and Escherichia coli (28,000) were consistent with those predicted from the sequences of the genes encoding them (Deutch et al., 1982 Nucleic Acid Res . 10, 7701-7714; Delauney and Verma, 1990 Mol . Gen . Genet . 221, 299-305) . Primary structural analysis of the intact soybean P5CR subunit indicated that the amino-terminal residue is blocked . Analyses of a 12-mer and a 21-mer isolated from a cyanogen bromide digest were consistent with the proposition that the soybean P5CR isolated in these studies is very similar, although perhaps not identical, to the polypeptide predicted for the recently cloned soybean reductase (Delauney and Verma, 1990 Mol . Gen . Genet . 221, 299-305). Mol Gen Genet, 1991 Aug, 228(1-2), 62 - 4 An essential gene of Escherichia coli that has sequence similarity to a chloroplast gene of unknown function; Nagano Y et al.; The dedB gene of Escherichia coli has sequence similarity to the zfpA gene of the chloroplast chromosome . The functions of dedB and zfpA are unknown . We constructed derivatives of temperature-sensitive polA strains into whose chromosomes a plasmid containing the disrupted dedB gene was integrated by homologous recombination . These strains contained normal and disrupted dedB genes in their chromosomes . We then selected plasmid-segregated strains and found no cells containing the disrupted dedB gene, indicating that disruption of the dedB gene was lethal in polA strains of E . coli. Mol Gen Genet, 1991 Aug, 228(1-2), 307 - 11 cysB and cysE mutants of Escherichia coli K12 show increased resistance to novobiocin; Rakonjac J et al.; Mutations in the cysB and cysE genes of Escherichia coli K12 cause an increase in resistance to the gyrase inhibitor novobiocin but not to coumermycin, acriflavine and rifampicin . This unusual relationship was also observed among spontaneous novobiocin resistant (Novr) mutants: 10% of Novr mutants isolated on rich (LA) plates with novobiocin could not grow on minimal plates, and among those approximately half were cysB or cysE mutants . Further analyses demonstrated that cysB and cysE negative alleles neither interfere with transport of novobiocin nor affect DNA supercoiling. Mol Gen Genet, 1991 Aug, 228(1-2), 287 - 93 Direct genetic selection of a maize cDNA for dihydrodipicolinate synthase in an Escherichia coli dapA- auxotroph; Frisch DA et al.; Dihydrodipicolinate synthase (DHPS; EC 4.2.1.52) is the first committed enzyme in the lysine branch of the aspartate-derived amino acid biosynthesis pathway and is common to bacteria and plants . Due to feedback inhibition by lysine, DHPS serves in a regulatory role for this pathway in plant metabolism . To elucidate the molecular genetic characteristics of DHPS, we isolated a putative full-length cDNA clone for maize DHPS by direct genetic selection in an Escherichia coli dapA- auxotroph . The maize DHPS activity expressed in the complemented E . coli auxotroph showed the lysine inhibition characteristics of purified maize DHPS, indicating that the cDNA encoded sequences for both the catalytic function and regulatory properties of the enzyme . The N-terminal amino acid sequence of purified maize DHPS was determined by direct sequencing and showed homology to a sequence within the cDNA, indicating that the clone contained the entire coding region for a mature polypeptide of 326 amino acids plus a 54 amino acid transit peptide sequence . The molecular weight of 35,854, predicted from the deduced amino acid sequence, was similar to the 38,000 Mr determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) for the purified enzyme from maize . DHPS mRNAs complementary to the cDNA were detected in RNA isolated from developing maize endosperm and embryo tissues . Southern blots indicated the presence of more than one genomic sequence homologous to DHPS per haploid maize genome. Mol Gen Genet, 1991 Aug, 228(1-2), 258 - 64 Post-transcriptional regulation in higher eukaryotes: the role of the reporter gene in controlling expression; Gallie DR et al.; We have investigated whether reporter genes influence cytoplasmic regulation of gene expression in tobacco and Chinese hamster ovary (CHO) cells . Two genes, uidA encoding beta-glucuronidase (GUS) from Escherichia coli and Luc, encoding firefly luciferase (LUC), were used to analyze the ability of a cap, polyadenylated tail, and the 5'- and 3'-untranslated regions (UTR) from tobacco mosaic virus (TMV) to regulate expression . The regulation associated with the 5' cap structure and the TMV 5'-UTR, both of which enhance translational efficiency, was reporter gene-independent . The poly(A) tail and the TMV 3'-UTR, which is functionally equivalent to a poly(A) tail, increase translational efficiency as well as mRNA stability . The regulation associated with these 3' ends was highly reporter gene-dependent; their effect on GUS expression was almost an order of magnitude greater than that on LUC expression . In tobacco, the tenfold reporter gene effect on poly(A) tail or TMV 3'-UTR function could not be explained by a differential impact on mRNA stability; GUS and LUC mRNA half-life increased only twofold when either the poly(A) tail or TMV 3'-UTR was present . In CHO cells, however, GUS mRNA was stabilized to a greater extent by a poly(A) tail or the TMV 3'-UTR than was LUC mRNA. Mol Gen Genet, 1991 Aug, 228(1-2), 240 - 8 Sequence of the structural gene for granule-bound starch synthase of potato (Solanum tuberosum L.) and evidence for a single point deletion in the amf allele; van der Leij FR et al.; The genomic sequence of the potato gene for starch granule-bound starch synthase (GBSS; "waxy protein") has been determined for the wild-type allele of a monoploid genotype from which an amylose-free (amf) mutant was derived, and for the mutant part of the amf allele . Comparison of the wild-type sequence with a cDNA sequence from the literature and a newly isolated cDNA revealed the presence of 13 introns, the first of which is located in the untranslated leader . The promoter contains a G-box-like sequence . The deduced amino acid sequence of the precursor of GBSS shows a high degree of identity with monocot waxy protein sequences in the region corresponding to the mature form of the enzyme . The transit peptide of 77 amino acids, required for routing of the precursor to the plastids, shows much less identity with the transit peptides of the other waxy preproteins, but resembles the hydropathic distributions of these peptides . Alignment of the amino acid sequences of the four mature starch synthases with the Escherichia coli glgA gene product revealed the presence of at least three conserved boxes; there is no homology with previously proposed starch-binding domains of other enzymes involved in starch metabolism . We report the use of chimeric constructs with wild-type and amf sequences to localize, via complementation experiments, the region of the amf allele in which the mutation resides . Direct sequencing of polymerase chain reaction products confirmed that the amf mutation is a deletion of a single AT basepair in the region coding for the transit peptide.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1991 Aug, 228(1-2), 125 - 8 Physical linkage and transcriptional orientation of the tdc operon on the Escherichia coli chromosome; Schweizer HP et al.; The physical and genetic structure of 37 kilobases of DNA encompassing the tdc region at 68.3 min of the Escherichia coli chromosome was determined by DNA sequence analysis and restriction mapping . Re-examination of new data concerning the direction of transcription of the tdc operon revealed that in strain W3110 the tdc region is located on a transposable segment of DNA. J Trop Med Hyg, 1991 Aug, 94(4), 234 - 40 Guinea-pig ileal loop assay: a better replacement of the suckling mouse assay for detection of heat-stable enterotoxins of Escherichia coli; Choudhry MA et al.; The suitability of guinea-pig ileal loop assay (GILA) for the assay of heat-stable (ST) enterotoxin was confirmed . Secretory response against Escherichia coli heat-stable enterotoxin in this model was determined in terms of dilatation index (DI) . DI equal to 0.50 or more was considered as a positive secretory response . Kinetics of fluid accumulation and the titration of toxin in guinea-pig ileal loop suggest uniform secretory response throughout the small intestine and 31.5 microgram of crude ST toxin as the minimum effective dose to induce a DI of 0.5 . Guinea-pig intestine was found sensitive to both methanol soluble (STa) and methanol insoluble (STb) toxins of E . coli and so was considered superior to the existing suckling mouse assay (SMA), which is known to be sensitive only to STa toxin . In addition, GILA was also found to be more suitable and economical as at least 10 strains together with the positive and negative controls can be tested in one animal, whereas in SMA, four suckling mice were needed to test a single strain . Hence, in SMA individual susceptibility among mice cannot be ruled out . GILA was considered to be an alternative to the presently available test, SMA, in the determination of ST toxin of E . coli. J Trauma, 1991 Aug, 31(8), 1063 - 7 Experimental hemorrhage and blunt trauma do not increase circulating tumor necrosis factor; Stylianos S et al.; Tumor necrosis factor (TNF) is a potent cytokine mediator of the shock states associated with sepsis and burn injury . This experimental study was done to determine whether circulating TNF plays a major role in the vasomotor collapse seen following experimental hemorrhage and blunt injury . Twenty anesthetized pigs were divided into two groups . Ten animals were bled 60% of their calculated blood volume in 15 minutes . Animals in Group IA (n = 5) had no treatment, and Group IB animals (n = 5) were given twice the shed volume as crystalloid 30 minutes after hemorrhage . The other animals, groups IIa and IIb (n = 5 each), were first subjected to a blunt injury to the thigh sufficient to cause a midshaft femur fracture, then bled and similarly treated . In both groups, mean arterial pressure (MAP), cardiac output (CO), and serum TNF activity by L929 bioassay were measured at 15-minute intervals for 120 minutes after hemorrhage or hemorrhage and blunt injury . An additional three animals were infused with 4 x 10(8)/kg heat-killed E . coli to validate the TNF assay . All bled animals sustained a fall in MAP and CO to a mean of 33% of baseline values, with or without fracture . Group IB and IIB animals responded to fluid resuscitation by restoration of MAP and CO to 85%-97% of the baseline values . Tumor necrosis factor was not detectable before injury and remained undetectable in all these animals during the 120 minutes of the experiment despite hemorrhage alone or combined hemorrhage and blunt trauma, with or without fluid resuscitation . The test animals receiving the E . coli responded with markedly elevated TNF levels, which peaked at 90 minutes after injection.(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Virol, 1991 Aug, 72 ( Pt 8), 1801 - 9 Transgenic plants and insect cells expressing the coat protein of arabis mosaic virus produce empty virus-like particles; Bertioli DJ et al.; The 3' end of the RNA-2 of arabis mosaic virus (ArMV) was cloned and sequenced . The N-terminal amino acid sequence of the virion coat protein was determined by Edman degradation and the corresponding coding region identified . This gene was modified at the 5' and 3' ends by use of mismatched primers in the polymerase chain reaction (PCR), in order to facilitate the cloning of the gene, and to provide it with a methionine initiation codon . The modified cloned gene was expressed in transgenic plants, recombinant baculovirus-infected insect cells and bacteria . Both the insect cells and the plants expressing the modified coat protein gene contained empty virus-like particles (VLPs) similar to the empty virus shells found in plants infected with ArMV . These VLPs were not detected in the Escherichia coli expressing the coat protein . Analysis of the primary amino acid sequence in the ArMV coat protein revealed extensive regions of identity with that of grapevine fanleaf virus . Patterns in these identities may reflect a three-domain organization of the proteins. Biochem J, 1991 Aug 1, 277 ( Pt 3), 659 - 64 Structural and functional studies on the human hepatic interleukin-6 receptor . Molecular cloning and overexpression in HepG2 cells; Schooltink H et al.; cDNAs coding for the human hepatic interleukin-6 receptor (IL-6-R) have been isolated from a library made from poly(A) RNA of dexamethasone-treated human hepatoma cells (HepG2) . We found the hepatic IL-6-R to be identical to the one expressed by leucocytes . A polyclonal antiserum was raised in rabbits against the IL-6-R protein expressed in Escherichia coli . Although the entire IL-6-R protein was used for immunization, only antibodies to the cytoplasmic domain of the IL-6-R were obtained . It is demonstrated by affinity cross-linking and subsequent immunoprecipitation with antibodies against the ligand as well as against the receptor that the cloned cDNA codes for the functional IL-6-R on HepG2 cells . When the hepatic IL-6-R cDNA was overexpressed in HepG2 cells, these cells became more sensitive to low concentrations of IL-6 with respect to the induction of gamma-fibrinogen mRNA. Biochem J, 1991 Aug 1, 277 ( Pt 3), 643 - 5 Effect of magnesium ions on the inhibition of S-adenosylmethionine decarboxylase from Escherichia coli by {2-(amino-oxy)ethyl}(5'-deoxyadenosin-5'-yl)(methyl)sulphonium ; Weitkamp EL et al.; {2-(Amino-oxy)ethyl}(5'-deoxyadenosin-5'-yl)(methyl)sulphonium+ ++, the amino-oxy analogue of decarboxylated S-adenosylmethionine, is a potent irreversible inhibitor of Escherichia coli S-adenosylmethionine decarboxylase {Khomutov, Zavalova, Syrku, Artamonova & Khomutov (1983) Bioorg . Khim . 9, 130-131; Artamonova, Zavalova, Khomutov & Khomutov (1986) Bioorg . Khim . 12, 206-212} . We have shown that Mg2+ ions are required for the irreversible inhibition of the decarboxylase, and that S-adenosylmethionine protects against this inhibition. Am J Physiol, 1991 Aug, 261(2 Pt 1), L148 - 55 Effects of intratracheal endotoxin administration on hamster lung glycosaminoglycans; Karlinsky JB et al.; Incorporation of {3H}glucosamine and 35S into glycosaminoglycan (GAG) was measured in hamster lung explant cultures at 0, 1, 4, and 24 h after a single endotracheal instillation of Escherichia coli endotoxin . Lung content of GAG was measured in a second group of treated animals over an 8-day period . Albumin was detected after endotoxin treatment in bronchoalveolar lavage fluid at 24 h but was not found in lavage fluid 7 days later or in lavage fluid of saline-treated animals . Over the initial 24 h, increasing amounts of radiolabeled precursor molecules were incorporated into all classes of GAG . Proportionally more radiolabel was incorporated into hyaluronic acid and chondroitin sulfate, and less was incorporated into heparan sulfate . The proportion of radiolabel incorporated into dermatan sulfate did not change . Total lung content of hyaluronate and chondroitin sulfate was elevated at 24 h but was returning to baseline by 8 days . The lung content of dermatan sulfate was increased at 8 days; lung content of heparan sulfate did not change over the 8-day study period . Elevations in the amount of explant heparan sulfate that bound to antithrombin III (AT III) were found at 1 h after both saline and endotoxin treatment . Radiosulfated heparan sulfates were found in blood from hamsters treated with endotoxin 1 h previously; these heparan sulfates did not bind to AT III . However, blood contained heparin-like activity . We conclude that endotoxin differentially alters the metabolism of each class of hamster lung glycosaminoglycans and that metabolic changes begin very rapidly after endotoxin exposure . The relation of pulmonary endothelial injury to the presence of heparin-like activity in blood is not yet clear. Genes Dev, 1991 Aug, 5(8), 1453 - 63 Cooperativity at a distance promoted by the combined action of two replication initiator proteins and a DNA bending protein at the replication origin of pSC101; Stenzel TT et al.; We have investigated the interaction of the host-encoded DNA bending protein IHF, the host-encoded initiator DnaA, and the plasmid-encoded initiator RepA with the replication origin of pSC101 . We have discovered that DNA bending induced by IHF in vitro promoted the interaction of DnaA protein with two physically separated binding sites called dnaAs and dnaAw . This cooperative interaction at a distance, most probably, caused looping out of the ihf site . We have also discovered that RepA protein binding to its cognate sites promoted enhanced binding of DnaA protein to the physically distant dnaAs site, probably also by DNA looping . The addition of RepA to a binding reaction containing IHF and DnaA further enhanced the binding of DnaA protein to the dnaAs site . Thus, the three DNA-binding proteins interacted with the origin, generating a higher order structure in vitro . On the basis of the results of the known requirement of all three proteins for replication initiation, we have proposed a model for the structure of a preinitiation complex at the replication origin. FASEB J, 1991 Aug, 5(11), 2606 - 10 A rational approach in the search for potent inhibitors against HIV proteinase; Hui KY et al.; Synthetic peptides described as dog renin inhibitors were found to effectively inhibit the aspartyl protease of human immunodeficiency virus (HIV) . The selection of oligopeptides for the HIV protease inhibition study was based on 1) the current strategy of inhibiting aspartyl proteases with transition state analogs, and 2) our previous observations regarding optimal structural differentiation at the P2 position among human, dog, and rat renin inhibitors . In an in vitro assay system consisting of recombinant HIV protease and a synthetic decapeptide substrate (at pH 5.5), results show that HIV protease was unaffected by statine-containing analogs carrying histidine at the P2 position whereas analogs containing valine at the same position yielded anti-protease IC50 values ranging from 50 to 500 nM . As anticipated, some analogs were also shown to inhibit processing of recombinant polyprotein substrate by HIV protease in vitro . The anti-viral activity of three inhibitors was studied in HIV-infected CEM and MT-2 cells . Results showed that one compound, Ac-Naphthylalanyl-Pro-Phe-Val-Statine-Leu-Phe-NH2 (antiprotease IC50 value = 0.4 microM), protected the infected cells effectively with IC50 values (0.73 microM for CEM cells and 0.88 microM for MT-2 cells) . This antiviral effect is comparable to those obtained with AZT and ddC in parallel studies of MT-2 cells. Dev Biol, 1991 Aug, 146(2), 519 - 30 cut-1 a Caenorhabditis elegans gene coding for a dauer-specific noncollagenous component of the cuticle; Sebastiano M et al.; We have molecularly identified a new gene of Caenorhabditis elegans that codes for a component of the cuticle . The gene has been physically mapped on LGII near the locus sqt-1 . The structure and the sequence of the gene have been determined and antisera have been raised against parts of the protein produced as fusions in Escherichia coli . By transcription analysis, and by the use of specific antisera, we have determined that this gene is expressed specifically during dauer larva formation . In extracts of worms completing the dauer transformation the product of this gene migrates in sodium dodecyl sulfate acrylamide gels with an apparent molecular mass of 40 kDa . By immunofluorescence we have determined that it is a component of the cuticles of dauer larvae . It forms a ribbon approximately 2 microns wide running along the lateral lines underneath the alae . Once it is assembled in the cuticle the protein becomes insoluble even in the presence of strong detergents and reducing agents in a manner that is similar to that described for the noncollagenous, insoluble residue of nematode cuticles called cuticlins; therefore, we have named the gene cut-1 for cuticlin 1 . cut-1 represents the first gene for a noncollagenous component of C . elegans cuticle that has been characterized molecularly. Proc Natl Acad Sci U S A, 1991 Aug 1, 88(15), 6824 - 8 The C-terminal half of UvrC protein is sufficient to reconstitute (A)BC excinuclease; Lin JJ et al.; The UvrC protein is one of three subunits of the Escherichia coli repair enzyme (A)BC excinuclease . This subunit is thought to have at least one of the active sites for nucleophilic attack on the phosphodiester bonds of damaged DNA . To localize the active site, mutant UvrC proteins were constructed by linker-scanning and deletion mutagenesis . In vivo studies revealed that the C-terminal 314 amino acids of the 610-amino acid UvrC protein were sufficient to confer UV resistance to cells lacking the uvrC gene . The portion of the uvrC gene encoding the C-terminal half of the protein was fused to the 3' end of the E . coli malE gene (which encodes maltose binding protein), and the fusion protein MBP-C314C was purified and characterized . The fusion protein, in combination with UvrA and UvrB subunits, reconstituted the excinuclease activity that incised the eighth phosphodiester bond 5' and the fourth phosphodiester bond 3' to a psoralen-thymine adduct . These results suggest that the C-terminal 314 amino acids of UvrC constitute a functional domain capable of interacting with the UvrB-damaged DNA complex and of inducing the two phosphodiester bond incisions characteristic of (A)BC excinuclease. Proc Natl Acad Sci U S A, 1991 Aug 1, 88(15), 6519 - 22 Adaptation of bird hemoglobins to high altitudes: demonstration of molecular mechanism by protein engineering; Jessen TH et al.; Of two closely related species of geese, one, the greylag goose, lives in the Indian plains all year round, while the other, the bar-headed goose, lives at the Tibetan lakes and migrates across the Himalayas to winter in India . Another species, the Andean goose, lives in the High Andes all year round . Possession of a Hb with high oxygen affinity helps to adapt bar-headed and Andean geese to high altitudes . The Hb amino acid sequences of the bar-headed and the greylag geese differ by four substitutions, of which only one is unique among bird sequences: Pro-119 alpha (H2)----Ala . Perutz proposed that the two-carbon gap left by this substitution at the alpha 1 beta 1 contact raises the oxygen affinity, because it relaxes the tension in the deoxy or T structure {Perutz, M . F . (1983) Mol . Biol . Evol . 1, 1-28} . It was later found that the Hb of the Andean goose has a gap in the same position, due to the complementary substitution Leu-55 beta (D6)----Ser . We have tested Perutz's hypothesis by introducing each of these substitutions into human globin synthesized in Escherichia coli . The reconstituted Hbs combine cooperatively with oxygen . Their oxygen affinities exceed that of normal human Hb by an even larger factor than that found between the high-flying geese and the greylag goose . The mutant Hb Met-55 beta (D6)----Ser was crystallized . Its structure is the same as that of HbA, except in the immediate environment of the gap left by the substitution of the serine for the methionine side chain, which evidently causes the increased oxygen affinity of this Hb. Proc Natl Acad Sci U S A, 1991 Aug 1, 88(15), 6394 - 7 Allosteric and catalytic binding of S-adenosylmethionine to Escherichia coli DNA adenine methyltransferase monitored by 3H NMR; Bergerat A et al.; Adenine methylation of GATC sequences in DNA is carried out by the DNA adenine methyltransferase with the methyl group source being the cofactor S-adenosylmethionine . We report 3H NMR studies on the interaction of DNA adenine methyltransferase with S-adenosylmethionine and the reaction when the ternary complex is formed with an oligonucleotide containing a GATC site . The methylation reaction was also studied in the presence of a competitive inhibitor and this showed two successive stages involved in the methylation and two sites of binding for S-adenosylmethionine. Nature, 1991 Aug 1, 352(6334), 444 - 8 Transcription by single molecules of RNA polymerase observed by light microscopy; Schafer DA et al.; The kinetics of transcription by Escherichia coli RNA polymerase relate directly to the regulation of transcription and to the properties of processive enzymes in general, but analysis of RNA polymerase movement along the DNA template has so far been limited to the study of populations of enzyme molecules . The ability to view nanometre-sized particles with the light microscope suggested a method of monitoring transcription by individual RNA polymerase molecules . We describe here the behaviour of 40-nm-diameter particles of colloidal gold attached to the ends of DNA molecules being transcribed by RNA polymerase immobilized on a glass surface . The tethered gold particles are released from the surface at times after addition of nucleoside triphosphates that are consistent with the kinetics of transcription by RNA polymerase in solution . Analysis of the brownian motion of the gold particles enabled us to measure the movement along the template DNA of individual polymerase molecules. Mutat Res, 1991 Aug, 263(4), 217 - 22 Damage distribution and mutation spectrum: the case of 8-methoxypsoralen plus UVA in mammalian cells; Sage E et al.; We determined the distribution of monoadducts and biadducts induced in the supF tRNA gene carried by the shuttle vector pZ189, after exposure to 8-methoxypsoralen (8-MOP) plus a double UVA (365 nm) irradiation . These data were compared to our previously published 8-MOP-photoinduced mutation spectrum obtained after propagation of the damaged shuttle vector in mammalian cells . One mutational hot spot in an ATAT/TATA sequence is targeted at a hot spot of biaddition . A second hot spot is not related to the presence of photoadducts either at or near the site . Moreover, it is located in a sequence which can be defined as 'mutation-prone' . Mutations occurring at GC base pairs are not targeted at sites of photoaddition, and may result from a decrease in fidelity of DNA polymerase when copying the damaged vector. J Bacteriol, 1991 Aug, 173(16), 5200 - 6 A missense mutation in the rpoC gene affects chromosomal replication control in Escherichia coli; Petersen SK et al.; An RNA polymerase mutant with a single-base-pair change in the rpoC gene affects chromosome initiation control . The mutation, which is recessive, is a G to A transition leading to the substitution of aspartate for glycine at amino acid residue 1033 in the RNA polymerase beta' subunit . The chromosome copy number is increased twofold in the mutant at semipermissive growth temperatures (39 degrees C) . In a delta oriC strain, in which chromosome initiation is governed by an F replicon, chromosome copy number is not affected . Plasmid pBR322 copy number is also increased in the mutant at 39 degrees C . The mutation causes a more than fivefold increased expression of the dnaA gene at 39 degrees C . It is conceivable that it is this high DnaA concentration which causes the high chromosome copy number and that the mutant RNA polymerase beta' subunit exerts its effect by altering the expression of the dnaA gene . However, other factors must be affected as well to explain why the RNA polymerase mutant can grow in a balanced fashion with a high chromosome concentration . This is in contrast to wild-type cells, which exhibit higher origin concentrations when DnaA protein is overproduced, but in which the overall DNA concentration is only moderately affected. J Bacteriol, 1991 Aug, 173(16), 5181 - 7 Identification of the promoter region of the ribosome-releasing factor cistron (frr); Shimizu I et al.; Previous studies of the structure and expression of the ribosome-releasing factor (RRF) cistron (frr) have suggested that an efficient promoter region is located in the RRF cistron . We report here on the nucleotide sequence and in vivo function of the RRF promoter . The transcriptional start site was determined by primer extension to be 58 bp upstream of the translational initiation codon of frr . The location of the RRF promoter region was confirmed by means of (i) deletion analysis of the 5' proximal sequences of frr fused to the chloramphenicol acetyltransferase reporter gene, (ii) analysis of RRF produced in vivo from the deletion derivatives of frr cloned into pUC19, and (iii) gel retardation analysis with Escherichia coli RNA polymerase . The -35 and -10 regions were TTacCc and TATAcT, respectively . The strength of the RRF promoter was similar to that of the lac promoter, as determined by in vivo expression of chloramphenicol acetyltransferase activity . However, the RRF promoter was not affected by the intracellular cyclic AMP level despite the presence of a cyclic AMP receptor protein binding site downstream of the RRF promoter. J Bacteriol, 1991 Aug, 173(16), 5079 - 85 Importance of the position of TYR R boxes for repression and activation of the tyrP and aroF genes in Escherichia coli; Andrews AE et al.; Tyrosine-mediated repression of aroF and tyrP was studied by inserting DNA sequences between the two adjacent TYR R boxes which, in each case, overlap the respective RNA polymerase binding sites of these genes . In both cases, repression was greatest when homologous regions of these two TYR R boxes were on the same face of the DNA helix and the boxes were directly adjacent . An insertion of 3 bases was sufficient to abolish repression, which was reestablished as the boxes became separated by one full turn of the helix . These observations, coupled with the results of in vitro DNase I protection studies, supported the hypothesis that the binding of TyrR protein to the downstream boxes required cooperative interaction with TyrR protein already bound to the upstream boxes . In the case of tyrP, moving the upstream box also affected activation . Maximal activation was observed when the box was moved 3 or 12 to 14 residues upstream . Practically no activation was seen at intermediate positions, such as +7 and -4 . It is hypothesized that these results indicate positions allowing maximal interaction between TyrR protein bound to the upstream box and RNA polymerase bound to the RNA polymerase binding site. J Bacteriol, 1991 Aug, 173(16), 5068 - 78 Mutational analysis of repression and activation of the tyrP gene in Escherichia coli; Andrews AE et al.; In a previous report it had been suggested that the tyrP gene of Escherichia coli may be expressed from two separate promoters . We have endeavored to confirm this suggestion by primer extension studies and the separate subcloning of each of these promoters . In these studies, we found a single promoter whose expression was repressed by TyrR protein in the presence of tyrosine and activated by TyrR protein in the presence of phenylalanine . Two adjacent TYR R boxes, with the downstream one overlapping the tyrP promoter, are the likely targets for the action of TyrR protein . Mutational analysis showed that both TYR R boxes were required for tyrosine-mediated repression but that only the upstream box was required for phenylalanine-mediated activation . In vitro DNase protection studies established that whereas in the absence of tyrosine TyrR protein protected the region of DNA represented by the upstream box, at low TyrR protein concentrations both tyrosine and ATP were required to protect the region of DNA involving the downstream box and overlapping the RNA polymerase binding site. J Bacteriol, 1991 Aug, 173(16), 5017 - 23 Tryptophan gene cluster of Methanobacterium thermoautotrophicum Marburg: molecular cloning and nucleotide sequence of a putative trpEGCFBAD operon; Meile L et al.; A recombinant cosmid carrying the Methanobacterium thermoautotrophicum Marburg trp genes was selected by complementation of Escherichia coli trp mutations . A 7.3-kb fragment of the cloned archaeal DNA was sequenced . It contained the seven trp genes, arranged adjacent to each other in the order trpEGCFBAD . No gene fusions were observed . The trp genes were organized in an operonlike structure, with four short (5- to 56-bp) intergenic regions and two overlapping genes . There was no indication for an open reading frame encoding a leader peptide in the upstream region of trpE . The gene order observed in the M . thermoautotrophicum trp operon was different from all known arrangements of the trp genes in archaea, bacteria, and eucarya . The encoded sequences of the Methanobacterium Trp proteins were similar in size to their bacterial and eucaryal counterparts, and all of them contained the segments of highly similar or invariant amino acid residues recognized in the Trp enzymes from bacteria and eucarya . The TrpE, TrpG, TrpC, TrpA, and TrpD proteins were 30 to 50% identical to those from representatives of other species . Significantly less sequence conservation (18 to 30%) was observed for TrpF, and TrpB exhibited a high degree of identity (50 to 62%) to the sequences of representatives of the three domains . With the exception of TrpB, the beta subunit of tryptophan synthase, tryptophan was absent from all Trp polypeptides. J Bacteriol, 1991 Aug, 173(16), 4941 - 51 Genetic evidence for interaction between the CheW and Tsr proteins during chemoreceptor signaling by Escherichia coli; Liu JD et al.; This study presents two lines of genetic evidence consistent with the premise that CheW, a cytoplasmic component of the chemotactic signaling system of Escherichia coli, interacts directly with Tsr, the membrane-bound serine chemoreceptor . (i) We demonstrated phenotypic suppression between 10 missense mutant CheW proteins and six missense mutant Tsr proteins . Most of these mutant proteins had leaky chemotaxis defects and were partially dominant, implying relatively minor functional alterations . Their suppression pattern was allele specific, suggesting that the mutant proteins have compensatory conformational changes at sites of interactive contact . (ii) We isolated five partially dominant CheW mutations and found that four of them were similar or identical to the suppressible CheW mutant proteins . This implies that there are only a few ways in which CheW function can be altered to produce dominant defects and that dominance is mediated through interactions of CheW with Tsr . The amino acid replacements in these mutant proteins were inferred from their DNA sequence changes . The CheW mutations were located in five regularly spaced clusters in the first two-thirds of the protein . The Tsr mutations were located in a highly conserved region in the middle of the cytoplasmic signaling domain . The hydrophobic moments, overall hydrophobicities, and predicted secondary structures of the mutant segments were consistent with the possibility that they are located at the surface of the CheW and Tsr molecules and represent the contact sites between these two proteins. J Lab Clin Med, 1991 Aug, 118(2), 186 - 93 Detection of endotoxin in triglyceride-rich lipoproteins in vitro; Harris HW et al.; Numerous investigations have been performed in which volunteers have received infusions of triglyceride-rich lipoproteins without apparent screening of the infusates for bacterial endotoxin . This study was designed to examine the capacity of triglyceride-rich lipoproteins to mask their endotoxin content in vitro as measured by a chromogenic modification of the standard Limulus assay . Lipoproteins and lipoprotein-deficient plasma were isolated from normal human plasma by sequential ultracentrifugation under apyrogenic conditions . Individual lipoproteins and a synthetic lipid emulsion were suspended in 10% lipoprotein-deficient plasma . Samples were then incubated at 37 degrees C for 4 hours with increasing concentrations of E . coli (055:B5) endotoxin and assayed for detectable endotoxin activity . The capacity to inhibit detection of endotoxin in 10% lipoprotein-deficient plasma was significantly increased (10 to 100 times) by the addition of VLDL (1.0 mg triglyceride/ml), chylomicrons (1.0 mg triglyceride/ml), or the synthetic lipid emulsion (2.5 mg triglycerides/ml) . These data demonstrate that triglyceride-rich lipoproteins, and the synthetic lipid emulsion, can markedly inhibit the detection of endotoxin by the Limulus assay in vitro . In addition to the potential of harm to experimental subjects, infusion of endotoxin could vitiate kinetic studies by direct alteration of lipoprotein metabolism and by inducing changes in hepatic blood flow . Thus experimental protocols that involve the infusion of humans with triglyceride-rich lipoproteins should include detailed testing for the presence of endotoxin. J Bacteriol, 1991 Aug, 173(15), 4904 - 7 Role of translation of the pheA leader peptide coding region in attenuation regulation of the Escherichia coli pheA gene; Gavini N et al.; In Escherichia coli, the expression of the pheA gene is regulated by attenuation of transcription . To study the molecular details of pheA attenuation, we introduced mutations in the pheA leader peptide coding region and analyzed their effects by using pheA promoter-lacZ gene transcription fusions (pheAp-lacZ) . Mutations in the ribosome-binding site (pheAe1213) or in the translation initiation codon (pheAe24) of the pheA leader peptide coding region resulted in superattenuation of pheA expression . However, the presence of a stop codon upstream to the tandem phenylalanine codons (pheAe3334) led to an increase in the basal-level expression of pheA . This increase was further enhanced in the presence of prfA release factor mutant . The level of pheA expression in all three mutants was the same when cells were starved for phenylalanine . These results demonstrate that efficient translation of the pheA leader peptide coding region and the position of the ribosome on the leader transcript play decisive roles in the attenuation regulation of pheA. J Bacteriol, 1991 Aug, 173(15), 4862 - 76 The malX malY operon of Escherichia coli encodes a novel enzyme II of the phosphotransferase system recognizing glucose and maltose and an enzyme abolishing the endogenous induction of the maltose system; Reidl J et al.; Mutants lacking MalK, a subunit of the binding protein-dependent maltose-maltodextrin transport system, constitutively express the maltose genes . A second site mutation in malI abolishes the constitutive expression . The malI gene (at 36 min on the linkage map) codes for a typical repressor protein that is homologous to the Escherichia coli LacI, GalR, or CytR repressor (J . Reidl, K . Romisch, M . Ehrmann, and W . Boos, J . Bacteriol . 171:4888-4899, 1989) . We now report that MalI regulates an adjacent and divergently oriented operon containing malX and malY . MalX encodes a protein with a molecular weight of 56,654, and the deduced amino acid sequence of MalX exhibits 34.9% identity to the enzyme II of the phosphototransferase system for glucose (ptsG) and 32.1% identity to the enzyme II for N-acetylglucosamine (nagE) . When constitutively expressed, malX can complement a ptsG ptsM double mutant for growth on glucose . Also, a delta malE malT(Con) strain that is unable to grow on maltose due to its maltose transport defect becomes Mal+ after introduction of malI::Tn10 and the plasmid carrying malX . MalX-mediated transport of glucose and maltose is likely to occur by facilitated diffusion . We conclude that malX encodes a phosphotransferase system enzyme II that can recognize glucose and maltose as substrates even though these sugars may not represent the natural substrates of the system . The second gene in the operon, malY, encodes a protein of 43,500 daltons . Its deduced amino acid sequence exhibits weak homology to aminotransferase sequences . The presence of plasmid-encoded MalX alone was sufficient for complementing growth on glucose in a ptsM ptsG glk mutant, and the plasmid-encoded MalY alone was sufficient to abolish the constitutivity of the mal genes in a malK mutant . The overexpression of malY in a strain that is wild type with respect to the maltose genes strongly interferes with growth on maltose . This is not the case in a malT(Con) strain that expresses the mal genes constitutively . We conclude that malY encodes an enzyme that degrades the inducer of the maltose system or prevents its synthesis. J Bacteriol, 1991 Aug, 173(15), 4851 - 61 Mutational analysis and characterization of the Escherichia coli hya operon, which encodes {NiFe} hydrogenase 1; Menon NK et al.; Deletion mutants of Escherichia coli specific for hydrogenase isoenzyme 1 (HYD1) have been constructed and characterized . The hya operon, which contains genes for the two HYD1 structural subunits and four additional genes, was mapped at 22 min on the E . coli chromosome . The total hydrogenase activities of the HYD1-negative mutant and wild-type strains were similar . However, the formate dehydrogenase activity associated with the formate hydrogen lyase pathway was lower in the mutant . The hya mutant (strain AP1), complemented with only the hydrogenase structural genes (hyaAB), produced antigenically identifiable but inactive HYD1 protein . The first five genes of hya (hyaA to hyaE) were required for the synthesis of active HYD1, but wild-type levels of HYD1 activity were restored only when mutant cells were transformed with all six genes of the operon . When AP1 was complemented with hya carried on a high-copy-number plasmid, the HYD1 structural subunits were overexpressed, but the excess protein was unprocessed and localized in the soluble fraction of the cell . The products of hyaDEF are postulated to be involved in the processing of nascent structural subunits (HYAA and HYAB) . This processing takes place only after the subunits are inserted into the cell membrane . It is concluded that the biosynthesis of active HYD1 is a complex biochemical process involving the cellular localization and processing of nascent structural subunits, which are in turn dependent on the insertion of nickel into the nascent HYD1 large subunit. J Bacteriol, 1991 Aug, 173(15), 4751 - 6 Autoradiographic analysis of diaminopimelic acid incorporation in filamentous cells of Escherichia coli: repression of peptidoglycan synthesis around the nucleoid; Mulder E et al.; Peptidoglycan synthesis rate in nonconstricting filaments of Escherichia coli dnaX(Ts) has been studied by autoradiography of incorporated {3H}diaminopimelic acid . Analysis of autoradiograms of whole cells and sacculi showed that peptidoglycan is synthesized at a reduced rate in the nucleoid-containing parts of these filaments . The lower rate of peptidoglycan synthesis in the cell center coincides with a higher local rate of protein synthesis . DNA-less cell formation in dnaX(Ts), dnaX(Ts) sfiA, and the minB minicell-forming mutant is accompanied by a local increase in peptidoglycan synthesis at the constriction site. J Bacteriol, 1991 Aug, 173(15), 4736 - 41 Rifampin-resistant replication of pBR322 derivatives in Escherichia coli cells induced for the SOS response; Magee TR et al.; Replication of plasmid pBR322 in Escherichia coli cells normally requires RNA synthesis and thus is sensitive to rifampin, an inhibitor of RNA polymerase . In cells induced for the SOS response, however, derivatives of pBR322 were found to replicate in the presence of rifampin . This rifampin-resistant replication of pBR322 requires the insertion of certain sequences of DNA . The replication depends on recF+ and DNA polymerase I. J Bacteriol, 1991 Aug, 173(15), 4653 - 9 RNase I*, a form of RNase I, and mRNA degradation in Escherichia coli; Cannistraro VJ et al.; A previously unreported endoRNase present in the spheroplast fraction of Escherichia coli degraded homoribopolymers and small RNA oligonucleotides but not polymer RNA . Like the periplasmic endoRNase, RNase I, the enzyme cleaved the phosphodiester bond between any nucleotides; however, RNase I degraded polymer RNA as fast as homopolymers or oligomers . Both enzymes migrated as 27-kDa polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and could not be separated by various chromatographic procedures . In rna insertion mutants, both enzymes were completely missing; the spheroplast enzyme is called RNase I*, since it must be a form of RNase I . The two forms could be distinguished by physical treatments . RNase I could be activated by Zn2+, while RNase I* was inactive in the presence of Zn2+ . RNase I was inactivated very slowly at 100 degrees C over a wide pH range, while RNase I* was inactivated slowly by heat at pH 4.0 but much more rapidly as the pH was increased to 8.0 . In the presence of a thiol-binding agent, the inactivation at the higher pH values was much slower . These results suggest that RNase I*, but not RNase I, has free sulfhydryl groups . RNase I* activity in the cell against a common substrate was estimated to be several times that of RNase I . All four 2',3'-phosphomonoribonucleotides were identified in the soluble pools of growing cells . Such degradative products must arise from RNase I* activity . The activity would be suited for the terminal step in mRNA degradation, the elimination of the final oligonucleotide fragments, without jeopardizing the cell RNA . An enzyme with very similar specificity was found in Saccharomyces cerevisiae, suggesting that the activity may be widespread in nature. Infect Immun, 1991 Aug, 59(8), 2836 - 8 Antibody response of humans to the circumsporozoite protein of Plasmodium vivax; Franke ED et al.; We studied the interaction of sera from residents of an area in northern Peru where vivax malaria is endemic with four recombinant DNA-derived circumsporozoite (CS) proteins of Plasmodium vivax . The antigens used in the enzyme-linked immunosorbent assay included one Escherichia coli-produced and three Saccharomyces cerevisiae-produced recombinant proteins . Three of the proteins (NS1(81)V20, Vivax-1, and Vivax-2) contain the entire central repeat region of the P . vivax CS protein, and one protein (Vivax-3) contains only two repeat sequences . Vivax-1, Vivax-2, and Vivax-3 contain different lengths of sequences flanking the repeats . A higher percentage of the sera had antibodies to Vivax-2 and Vivax-3, the two proteins containing the longest nonrepeat sequences, than to NS1(81)V20 or Vivax-1 . Children less than 5 years of age did not have immunoglobulin G antibodies to NS1(81)V20; however, they had antibodies to Vivax-1, Vivax-2, and Vivax-3 . The finding that individuals living in a malaria-endemic area produce antibodies to peptides containing nonrepeat regions of the CS protein emphasizes the need to characterize the immune response to these regions in naturally exposed and experimentally immunized humans. Cancer Res, 1991 Aug 1, 51(15), 3930 - 7 DNA sequence specificity of doxorubicin-induced mutational damage in uvrB- Escherichia coli; Anderson RD et al.; In the absence of excision repair, doxorubicin caused a striking (41-fold) increase in the frequency of large deletion mutations extending from the lac operator (lacO) into the lac repressor gene (lacI) of Escherichia coli . In contrast, there was only a 2-fold increase in the frequency of small deletions despite a 3-fold increase in overall mutation frequency . The 5'-endpoints of doxorubicin-induced lacO and lacI/lacO deletions occurred at the DNA sequence 5'-pyTAA or 5'-AATpy (where py is pyrmidine) (16%), at runs of purines or pyrimidines (41%) and adjacent to 5'-dGdC or 5'-dCdG doublets (34%) . Ninety % (27 of 30) of the doxorubicin-induced deletions involving the region of the lacO palindrome had 3'-endpoints within the palindrome sequence as compared with 40% (4 of 10) spontaneous deletions in an untreated set . Doxorubicin-induced single base substitutions were highly focused at one site (4 of 6) in the i-d region of lacI, in contrast to the spontaneous distribution of point mutations, where 16 mutants were recovered at 12 different sites . An increased frequency (3-fold) of highly focused base substitutions was also observed at 2 sites in the lac operator region (at lacO +6, which is a transition "hotspot" in the spontaneous spectra of both wild type and uvrB- organisms and at the adjacent +5 site) . Notably, the frequency of 1- and 2-base frameshifts did not increase in the doxorubicin-induced spectrum, relative to the spontaneous mutation spectrum . These in vivo observations in E . coli suggest that in the absence of excision repair, doxorubicin causes highly focused deletions and base substitutions . These mutations occur adjacent to DNA sequences identified in previous in vitro studies as preferential sites of doxorubicin binding. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1991 Aug, 13(4), 309 - 12 {Plasmid pBR322 drug-resistance gene changes induced by glycidyl methacrylate}; Fang F; A mutagen, glycidyl methacrylate (GMA), discovered by us a few years ago has been used to investigate the mutation mechanism of drug-resistance genes of plasmid pBR322 . The results indicated that GMA binds strongly to pBR322 DNA, and this binding decreased the relative transformation efficiency using E . coli HB1O1 strain on LB plates containing ampicillin (Ap) or tetracycline (Tc) . The mutants, ARpTSc, AspTRc and ASpTSc, have been isolated and their drug-resistance proved to be heritable. Zhongguo Yi Xue Ke Xue Yuan Xue Bao, 1991 Aug, 13(4), 241 - 5 {Cloning and sequencing of a highly repeated DNA fragment of rabbit}; Ding J; We obtained highly repeated DNA fragments from a rabbit by using restriction endonuclease HindIII digestion of chromosomal DNA; the smallest highly repeated DNA fragment was cloned into vector plasmid pUC12 . We used the recombinant plasmid to transform E . coli JM101, selected and identified the positive clone by DNA probe in situ hybridization as well as Southern blotting . We obtained a recombinant clone {termed pRAb (Hind III)-1} which contains the highly repeated DNA fragment {termed RAb (Hind III)-1} . The complete nucleotide sequence of the 345bp RAb (Hind III)-1 fragment was determined by the dideoxynucleotide termination sequencing method . We analyzed the characteristics of the structure of the sequence with a microcomputer, and the results suggested that the RAb (Hind III)-1 sequence is quite different from alphoid DNA of primates. J Biochem (Tokyo), 1991 Aug, 110(2), 196 - 201 Molecular cloning and expression in Escherichia coli of cDNA encoding the subunit of sweet potato beta-amylase; Yoshida N et al.; Tuberous roots of the sweet potato are unusually rich in beta-amylase, and the beta-amylase polypeptides account for about 5% of the total soluble protein of the organ . Unlike beta-amylases from other origins, the sweet potato beta-amylase is a tetramer of identical subunits, and it also bears starch phosphorylase-inhibitor activity . A cDNA for the subunit of sweet potato beta-amylase was obtained by immunological screening of an expression cDNA library constructed by the vector-primer and linker method using a plasmid vector containing tac-SP6 promoters . The SP6 transcript of a 2,000 base-pair-long cDNA insert directed the synthesis in vitro of a precursor to the subunit of beta-amylase which was identical in size with the mature subunit, and the beta-amylase mRNA detected by Northern blot hybridization was identical in size with the SP6 transcript of the cDNA insert . The cDNA insert contained 1,494 base pairs of an open reading frame which codes for the 499-amino-acid-long precursor to the subunit of beta-amylase . An amino acid sequence identical to the N-terminal amino acid sequence of the mature subunit appeared immediately after the initiator methionine of the precursor, indicating that the subunit of beta-amylase is synthesized as a mature form . Comparison of the amino acid sequences of subunits of sweet potato beta-amylase and seed beta-amylases from barley and soybean indicated that these enzymes share about 68% amino acid identities among each other . Escherichia coli cells harboring the cDNA clone produced the mature-sized subunit of the beta-amylase, and the soluble extract exhibited amylolytic activity which migrated to the same position as the beta-amylase purified from the sweet potato in non-denaturing polyacrylamide gel containing soluble starch indicating that oligomerization of the subunit occurred properly in E . coli cells. Immunology, 1991 Aug, 73(4), 450 - 6 Activation of human natural killer cells by lipopolysaccharide and generation of interleukin-1 alpha, beta, tumour necrosis factor and interleukin-6 . Effect of IL-1 receptor antagonist; Conti P et al.; The presence in the body of an antigen species or a bacterial lipopolysaccharide (LPS) has a pleiotropic effect on the immune system activating macrophages, lymphocytes and natural killer (NK) cells . Recently it has been reported that human macrophages not only secrete interleukin-1 (IL-1) but also its inhibitor, called IL-1 receptor antagonist (IL-1ra), structurally similar to IL-1 beta, but with no IL-1-like activity and which binds to the IL-1 receptor . In this study we show that LPS stimulates NK cell activity and IL-1ra potentiates the stimulatory effect of human recombinant interleukin-2 (hrIL-2) on NK cell activity . In addition, we found that hrIL-1ra inhibits DNA synthesis in lymphocyte culture stimulated with phytohaemagglutinin (PHA) (20 micrograms/ml), presumably via IL-1 inhibition . We also found that LPS is a potent stimulator of monokines: IL-6, tumour necrosis factor-alpha (TNF-alpha), and IL-1 beta, as determined by radioimmunoassay method, and interferon-gamma (IFN-gamma), IL-2, TNF-alpha and IL-1 alpha, as determined by ELISA method, in peripheral blood mononuclear cells (PBMC) . We used PBMC as effector cells since LPS requires the presence of accessory cells to activate lymphocytes and bind to the HLA-DR molecule on accessory cells . The effect of LPS on PBMC cytotoxicity has been compared with an endotoxin-free extract of Escherichia coli, OM-8990, which did not provoke cytokine production nor did it cause enhancement of NK cell activity . We found that human recombinant IL-1ra potentiates the stimulatory effect of IL-2 on NK cell activity, similar to hrIL-1 beta . The potentiation of IL-2 in stimulating NK cell activity by IL-1ra is not yet understood . Since IL-1ra is a part of the IL-1 family, it may work in a similar fashion to IL-1, which also potentiates IL-2 to enhance NK cell activity but has been shown not to be directly important in tumour cell killing . In addition, hrIL-1ra can amplify the effect of IL-2 on NK activity, possibly by inhibiting the cyclo-oxygenase products, which are immunosuppressive and are generated in antigen-stimulated PBMC cultures . The generation of IFN-gamma by PBMC after treatment with LPS strongly suggests that the enhancement of NK cell activity may be indirectly due to IFN production. Am J Physiol, 1991 Aug, 261(2 Pt 2), R442 - 52 Comparison between effects of interleukin-1 alpha administration and sublethal endotoxemia in primates; Fischer E et al.; Interleukin (IL)-1 is an early mediator of host response to inflammation, although its contribution to individual components of the acute phase reaction is still unclear . To evaluate how the hemodynamic, metabolic, and hormonal responses to sublethal endotoxemia compare with IL-1 administration, baboons received intravenously either lipopolysaccharide (LPS) or 0.1, 10, or 100 micrograms/kg IL-1 alpha . LPS induced an early tachycardia and a fall in mean arterial pressure, as well as lacticacidemia and hypoaminoacidemia . Similar hemodynamic and metabolic changes were seen with 10 or 100 micrograms/kg of IL-1 alpha . An increase in adrenocorticotropic hormone and fall in serum iron were induced by IL-1 alpha but not by LPS . Plasma tumor necrosis factor-alpha (TNF-alpha) was not measurable after IL-1 alpha administration, whereas LPS induced a monophasic TNF-alpha response . IL-6 levels were significantly greater after LPS than IL-1 alpha administration . Histopathological lesions, similar in LPS- and 100 micrograms/kg IL-1 alpha-treated groups, were present only in the adrenal cortex . We conclude that many, but not all, of the effects of sublethal endotoxemia can be replicated by IL-1 alpha administration, and these responses are dose dependent. Mutat Res, 1991 Aug, 260(4), 349 - 67 Performance of 133 compounds in the lambda prophage induction endpoint of the Microscreen assay and a comparison with S . typhimurium mutagenicity and rodent carcinogenicity assays; Rossman TG et al.; The Microscreen assay was developed as a means of testing very small samples, as in complex mixture fractionation . It is a multi-endpoint assay which utilizes E . coli WP2s(lambda) . Exposure takes place to serial dilutions of the test compound in microtitre wells (250 microliters) followed by sampling from wells in which growth has occurred ('non-toxic wells') . Although a number of different endpoints can be measured, only the prophage induction endpoint (the first one developed) has been extensively tested . Results with 133 compounds are presented . These include 111 compounds which have been tested in the S . typhimurium assay and 66 compounds for which both rodent bioassay and S . typhimurium assay data exists . The concordance for the Microscreen assay and the S . typhimurium assay was 71% . For this group of compounds, the sensitivity of the Microscreen assay in detecting carcinogens was 76% compared with 58% for the S . typhimurium assay . However, the S . typhimurium assay was somewhat more specific (69%) compared with the Microscreen (56%) . The overall association between carcinogenicity and Microscreen results was statistically significant (p = 0.029), whereas for the S . typhimurium assay the association with carcinogenicity was non-significant (p = 0.086) . The Microscreen assay was able to detect halogenated compounds better than the S . typhimurium assay . The Microscreen assay should prove useful in complex mixture fractionation, or in other situations where sample size is limiting. Scand J Immunol, 1991 Aug, 34(2), 215 - 20 Expression and secretion of T-cell receptor V alpha and V beta domains using Escherichia coli as a host; Ward ES; An expression system for the production of recombinant T-cell receptor (TCR) variable domains would, inter alia, allow structural studies to be carried out and provide protein for the generation of anti-clonotypic antibodies . In this report the V alpha and V beta domain genes have been isolated from a T-cell hybridoma which is associated with the pathogenesis of experimental allergic encephalomyelitis (EAE) in the H-2u mouse . These have been expressed as secreted domains in Escherichia coli, using secretion vectors previously used for the production of immunoglobulin fragments . Both V alpha and V beta domains are secreted in milligram quantities into the culture supernatant, although the levels of the V alpha domain are about 10-20 fold higher than those of the V beta domain . This expression system offers a rapid route for the production of recombinant TCRs in soluble form. Proc Natl Acad Sci U S A, 1991 Aug 1, 88(15), 6545 - 9 Reconstitution of a protein translocation system containing purified SecY, SecE, and SecA from Escherichia coli; Akimaru J et al.; Reconstitution of the translocation machinery for secretory proteins from purified constituents was performed . SecY was solubilized from SecY/SecE-overproducing Escherichia coli cells and purified by chromatography on ion-exchange and size-exclusion columns . Proteoliposomes active in protein translocation were reconstituted from the purified preparations of SecY and SecE . The reconstituted translocation activity was SecA- and ATP-dependent . Although the purified preparations of SecY and SecE were still contaminated with minute amounts of other proteins, the elution profiles of SecY and SecE on column chromatographies coincided with the elution profiles of reconstituted translocation activity, indicating that SecY and SecE are the indispensable components in these preparations . We conclude that SecY, SecE, and SecA are essential components of the protein secretion machinery and that translocation activity can be reconstituted from only these three proteins and phospholipids. J Bacteriol, 1991 Aug, 173(16), 5220 - 3 A novel activity in Escherichia coli K-12 that directs restriction of DNA modified at CG dinucleotides; Kelleher JE et al.; The restriction systems McrA and McrB of Escherichia coli K-12 are known to attack DNA containing modified cytosine . In strains lacking both activities, however, we observed that DNA methylated at CG dinucleotides (as is mammalian DNA) was still significantly restricted . We show that this substantial barrier to the acceptance of 5-methylcytosine-containing DNA is attributable to a hitherto unknown activity of the Mrr restriction system . Strikingly, the multiple systems used by this gut inhabitant to determine the fate of invading DNA will all limit genetic exchange with its mammalian host(s), reinforcing the idea that one role of DNA methylation is to serve as a "molecular passport" (E . A . Raleigh, R . Trimarchi, and H . Revel, Genetics 122:279-296, 1989). Protein Expr Purif, 1991 Aug, 2(4), 296 - 303 Expression of recombinant human apolipoprotein A-I in Chinese hamster ovary cells and Escherichia coli; Brissette L et al.; Human recombinant apolipoprotein (apo) A-I was produced by Chinese hamster ovary (CHO) cells and Escherichia coli with expression vectors containing cDNAs encoding preproapoA-I or apoA-I, respectively . The apoA-I from CHO cells was purified from the culture medium by ammonium sulfate precipitation, phenyl-Sepharose chromatography, and affinity purification on anti-apoA-I immunoabsorber . Human apoA-I was produced in E . coli as a fusion protein with glutathione S-transferase . A four amino acid linker, which separated the two proteins, was specifically recognized and cut by Factor Xa . The purification was accomplished by chromatography of E . coli extracts on glutathione-Sepharose and digestion with Factor Xa . The highest production level was found to be 0.5 micrograms/ml of culture medium per 48 h for a clone of stable transformant of CHO cells, whereas E . coli could produce as much as 20 micrograms/ml of bacterial culture . These apoA-I forms were compared in terms of molecular weight, isoelectric point, and expression of several epitopes . Recombinant apoA-I obtained from CHO cells appears intact and its isoelectric point is compatible with that of the mature form and the proform of apoA-I, whereas a part of the apoA-I produced by E . coli does not contain the COOH-terminus . Also, two of six epitopes are expressed to a greater extent in apoA-I obtained from E . coli than in apoA-I obtained from human plasma. Protein Expr Purif, 1991 Aug, 2(4), 287 - 95 Characterization of homogeneous recombinant glutaredoxin from Escherichia coli: purification from an inducible lambda PL expression system and properties of a novel elongated form; Bjornberg O et al.; We have constructed a plasmid, pAHOB1, with a 482-b AluI fragment containing the Escherichia coli glutaredoxin gene (grx) cloned under lambda PL promoter control . Growth of E . coli N4830/pAHOB1 cells at 30 degrees C followed by heat induction at 40 degrees C for 5 h resulted in expression of glutaredoxin as 20% of the soluble E . coli protein . Methods for the preparation of gram amounts of glutaredoxin and 5 mM solutions suitable for NMR studies were developed . About 10% of the glutaredoxin activity showed an unexpected higher isoelectric point and was isolated by DEAE-cellulose chromatography at pH 6.0 . Sequence analysis demonstrated that this novel form (grx-90) contained five additional N-terminal residues (Met-Arg-Arg-Glu-Ile) added to the glutaredoxin molecule with 85 residues (grx-85) . Grx-90 originates from an alternative ATG initiation codon present 5' of the previously identified translation start site on the grx gene in E . coli . Despite the highly charged N-terminal extension, grx-90 showed full activity as a GSH-disulfide oxidoreductase and the same apparent Km value (0.14 microM) as glutaredoxin in GSH-dependent reduction of CDP by ribonucleotide reductase . Both grx-90 and grx-85 showed identical competition curves in radioimmunoassays . The presence of grx-90 was also demonstrated in log-phase E . coli C600 cells as 5 to 10% of total glutaredoxin by immunological techniques . The molar extinction coefficient of native glutaredoxin (12,500 M-1 cm-1 at 280 nm) was 15% higher than expected from its content of one Trp and four Tyr residues. Protein Expr Purif, 1991 Aug, 2(4), 270 - 7 Rapid isolation of homogeneous cloned T7 gene 5 protein and T7 DNA polymerase by affinity chromatography on immobilized thioredoxin; Slaby I et al.; Phage T7 DNA polymerase consists of a strong 1:1 complex of T7 gene 5 protein (80 kDa) and the reduced form of Escherichia coli thioredoxin (12 kDa) . Immobilization of E . coli thioredoxin on the agarose matrix Affi-Gel retained both its redox activity and its ability to bind T7 gene 5 protein . This was used to develop a simple and fast high-yield purification method . Cloned T7 gene 5 protein, expressed in a thioredoxin-negative host cell, was isolated in pure and highly active form after elution from Affi-Gel--thioredoxin with a pH gradient from 10 to 12 . This purification step separated gene 5 protein from variable amounts of two sets of reconstituting large polypeptide fragments without catalytic activity . Proteolytic cleavage in vivo probably gave rise to the fragments, the generation of which was mimicked by trypsin cleavage of pure gene 5 protein . The gene 5 protein preparation had an inherent low DNA polymerase and double-stranded 3'-exonuclease activity, which was stimulated at least 30-fold by the presence of reduced thioredoxin . Highly active and pure T7 DNA polymerase was obtained by reconstitution of gene 5 protein with thioredoxin and was isolated by phosphocellulose or FPLC Mono Q chromatography . The gene 5 protein and T7 DNA polymerase preparations are suitable for further physicochemical characterization and as reagents in DNA sequencing. Antonie Van Leeuwenhoek, 1991 Aug, 60(2), 95 - 9 Cloning of the HIS3 gene of Yarrowia lipolytica; Prodromou C et al.; The HIS3 gene of the yeast Yarrowia lipolytica has been cloned from a genomic library by complementation of the his3 mutation of Saccharomyces cerevisiae . The gene was subsequently subcloned in Escherichia coli and characterized by restriction enzyme mapping. Mol Cell Probes, 1991 Aug, 5(4), 271 - 5 Evaluation of three new techniques for the detection of STb-positive Escherichia coli strains; Lortie LA et al.; A study was conducted to compare different techniques for the detection of heat-stable enterotoxin b (STb)-positive E . coli strains . Antisera against purified STb was used to develop an enzyme-linked immunosorbent assay (ELISA) . STb-positive strains identified by ELISA were tested for bioactivity in rat jejunal loops . Our ELISA was as sensitive as, but less specific than, the bioassay for detection of STb-positive strains . A non-radioactive DNA probe to detect the gene coding for STb was also developed by incorporating digoxigenin-11-dUTP into DNA by the random primed labelling technique . The non-radioactive digoxigenin-labelled DNA probe demonstrated a similar detectability to the radioactive probe and was more convenient to manipulate but was less sensitive and specific than the bioassay and the radioactive probe . In addition, the polymerase chain reaction (PCR) was used to amplify a specific portion of the gene coding for STb . The PCR was a highly specific and practical technique for the detection of STb-positive strains . All E . coli strains tested containing the STb gene produced the STb toxin. Mol Gen Mikrobiol Virusol, 1991 Aug, (8), 8 - 11 {Analysis of the nucleotide sequence of a small colicinogenic plasmid Cold gene coding for lysis}; Negorev DG et al.; The nucleotide sequence of a DNA fragment of a small colicigonenic plasmid Cold-CA23 containing the lysis gene cdl has been defined . An open reading frame has been found within the fragment for 48 amino acid polypeptide with the molecular mass 4920 Da . The polypeptide is homologous to the lysis proteins encoded by the small colicinogenic plasmids ColE1 and CloDF13 . The homology was also found for the structural and functional arrangement of the cdl gene and the plasmid CloDF13 lysis gene . The analysis of the possible secondary structures of the lysis protein encoded by cdl gene has revealed the amino acid sequences able to form the alternative structures . The described feature is supposed to be required for lysis protein translocation from cytoplasmatic to outer membrane of Escherichia coli cells. Biochem Soc Trans, 1991 Aug, 19(3), 719 - 23 ATP-promoted interaction between Clp A and Clp P in activation of Clp protease from Escherichia coli; Maurizi MR; Clp protease is a high relative molecular mass, ATP-dependent protease found in the cytoplasm of Escherichia coli . Clp protease is composed of two protein components, Clp A, which has ATPase activity, and Clp P, which has the proteolytic active site and is activated by Clp A in the presence of ATP . Clp P subunits (Mr = 21,500) are arranged in two hexagonal rings directly superimposed on each other, and under low salt conditions two dodecamers associate to form a particle with Mr approximately 440,000 . Clp A (subunit Mr = 83,000) and Clp P do not associate in the absence of nucleotide, but Clp A with ATP bound associates with Clp P to form an active proteolytic complex with Mr approximately 700,000 . Although adenosine 5'-{beta gamma-imido}triphosphate (AMPPNP) weakly promotes association between Clp A and Clp P, non-hydrolysable analogues of ATP do not activate proteolysis, indicating that association between the components is not sufficient to allow proteolysis . Association between Clp A and Clp P does not alter the basal ATPase activity of Clp A, but addition of protein substrates is accompanied by an increase in ATP hydrolysis by Clp A . Chemically-inactivated Clp P or inactive mutants of Clp P also associate with Clp A, but no increase in the ATPase activity of Clp A is observed, either in the presence or absence of protein substrates, when Clp P is inactive . Thus the increased ATP hydrolysis is dependent on active proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS) J Biomol Struct Dyn, 1991 Aug, 9(1), 169 - 86 The mechanism of recognition of templates by DNA polymerases from pro- and eukaryotes as revealed by affinity modification data; Kolocheva TI et al.; Pt(2+)-containing derivatives of oligodeoxyribonucleotides were used to evaluate the ligand affinity to the template sites of Klenow fragment of DNA polymerase I from E . coli and DNA polymerase alpha from human placenta . The values of Kd and Gibb's energy (delta G degree) for the complexes of oligodeoxyribonucleotides and their derivatives with the template sites of these enzymes were determined from the effects protecting the enzyme from inactivation by Pt(2+)-containing oligonucleotides . Kd and delta G degree values of the complexes made by DNA polymerases and orthophosphate, triethylphosphate, d(pC)n, d(pT)n, d(pG)n, d(pA)n (where n = 1-25), heterooligonucleotides of various length and structure, and oligothymidylates with partially and completely ethylated internucleotide phosphates were evaluated . The obtained data enabled us to suggest 19-20 mononucleotide units of the template to interact with the protein . Only one template internucleotide phosphate forms a Me(2+)-dependent electrostatic contact (delta G = -1.1...-1.7 kcal/mol) and a hydrogen bond (delta G = -4.4...-4.9 kcal/mol) with the enzyme . It is likely that the mononucleoside units of the template form hydrophobic contacts with the enzymes . The efficiency of such interaction changes with the hydrophobicity of the bases: C less than T less than G approximately A . For both homo- and heterooligonucleotides the contributions of nucleoside units to the affinity of the templates to the enzymes is due to the complementary interactions with the primers . A hypothetical model for the template-primer interaction with DNA polymerases is suggested. J Biomol Struct Dyn, 1991 Aug, 9(1), 143 - 57 Stereochemical analysis of interaction of signal peptide with phospholipids at the initiation of protein translocation across the membrane; Kajava AV et al.; Stereochemical analysis of signal peptide interaction with E . coli membrane phospholipids revealed the structural complementarity of N-terminus of signal peptide alpha-helix and acid phospholipids . The formation of their complex leads to neutralization of charges and decrease in hydrophilicity of both components, and promotes insertion of peptide and phospholipid into the membrane, not separately but as a complex . Interaction of acid phospholipids with the E . coli alkaline phosphatase (AP) signal peptide was thoroughly analyzed, and it was shown that in this case a complex of signal peptide alpha-helix with phosphatidylglycerol is inserted into the membrane with the lowest energy expense . On the basis of the results of stereochemical analysis and the available experimental data, a molecular mechanism of protein translocation initiation across the membrane has been proposed, in which the key events are the formation of the complex "signal peptide alpha-helix-acid phospholipid", the coupled insertion of hydrophobic peptide-lipid complex into a nonpolar membrane interior and translocation across the membranes. Biochem Int, 1991 Aug, 24(6), 1033 - 42 Chemical modification of PABA synthase; McLeish MJ et al.; p-Aminobenzoic acid (PABA) synthase catalyses the first step in folic acid biosynthesis, the conversion of chorismate to p-aminobenzoate . In general, difficulties in purification have permitted only limited investigation of this enzyme . However, in an attempt to identify possible active site residues, the E . coli enzyme has been incubated with a range of protein modifying agents . Results indicate that cysteine, histidine, arginine and tyrosine residues are important for enzyme activity . Attempts were made to determine the subunits upon which these residues were located. Circ Shock, 1991 Aug, 34(4), 364 - 70 Relationship between the lung and systemic response to endotoxin: comparison of physiologic change and the degree of lipid peroxidation; Demling RH et al.; The lung and systemic response to Escherichia coli endotoxin either 2 or 5 micrograms/kg was measured in 16 sheep with chronic lung and soft tissue lymph fistulae . Oxidant-induced lung and liver lipid peroxidation was measured as tissue malondialdehyde (MDA) . Conjugated dienes were also monitored . Both doses produced a comparable pulmonary hypertension and hypoxia as well as a comparable increase in protein-rich lymph flow, QL . However, lung MDA was significantly greater with the 5 micrograms/kg than with the 2 micrograms/kg dose, both being more than twofold greater than controls . The systemic physiologic responses between the two doses were quite different . The 5 microgram/kg dose resulted in a significant increase in oxygen delivery (DO2), oxygen consumption (VO2), and decrease in arterial O2 extraction in the 3-5 hr postendotoxin period compared with the 2 microgram/kg dose . A twofold increase in protein-rich soft tissue QL was also seen after the 5 micrograms/kg dose, whereas QL was not changed after 2 micrograms/kg . Liver MDA was only increased by 30% over controls with both doses . We conclude that the relationship between oxidant change and physiologic response varies considerably between lung and systemic tissues after endotoxemia with the degree of lung lipid peroxidation corresponding with the degree of impairment in systemic tissue O2 extraction and the onset of delivery-dependent O2 consumption. Curr Opin Cell Biol, 1991 Aug, 3(4), 580 - 4 The protein translocation apparatus of the rough endoplasmic reticulum, its associated proteins, and the mechanism of translocation; Gilmore R; The demonstration that the yeast Sec61, Sec62, and Sec63 proteins are assembled into a multisubunit complex in the yeast endoplasmic reticulum was of particular significance in a year when protein, and nucleic-sequence analyses revealed interesting homologies between pathways of protein transport in mammals and yeast, and possibly in Escherichia coli. J Cell Sci, 1991 Aug, 99 ( Pt 4), 823 - 36 Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation; Atkinson SJ et al.; We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM . The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule . Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M . Constructs in which the 'skip' residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly . Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region . The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin. Appl Environ Microbiol, 1991 Aug, 57(8), 2255 - 9 Transcription of the Escherichia coli fliC gene is regulated by metal ions; Guzzo A et al.; luxAB gene fusions in the Escherichia coli genome were used to screen for clones displaying transcriptional changes in the presence of aluminum . One clone was found that contained a luciferase gene fusion in which transcription was increased in the presence of aluminum and which was subsequently shown to be induced by copper, iron, and nickel . Cloning of the metal-regulated gene, hybridization to the ordered phage lambda bank of the E . coli chromosome, and sequencing of DNA adjacent to the luxAB fusion revealed that the insertion occurred within the fliC (hag) gene of E . coli . This gene encodes flagellin, the filament subunit of the bacterial motility organ, and is under the control of several regulatory cascades . These results suggest that environmental metals may play a role in the regulation of the motility potential of E . coli and that this bioluminescent gene fusion clone (or derivatives thereof) may be used to prepare a biosensor for the rapid detection of metal contamination in water samples. Mol Microbiol, 1991 Aug, 5(8), 1983 - 91 Regulation of nitrogen fixation in Azorhizobium caulinodans: identification of a fixK-like gene, a positive regulator of nifA; Kaminski PA et al.; The nucleotide sequence of a 1 kb fragment upstream of Azorhizobium caulinodans fixL was established . An open reading frame of 744 bp was identified as a fixK homologue . A kanamycin cartridge was inserted into the cloned fixK-like gene and recombined into the host genome . The resulting mutant was Nif-Fix-, suggesting that FixK was required for nitrogen fixation both in symbiotic conditions and in the free-living state . Using a pfixK-lacZ fusion, the FixLJ products were shown to control the expression of fixK . Using a pnifA-lacZ fusion, the FixK product was shown to regulate positively the transcription of nifA in bacteria grown in the free-living state . In addition, a double ntrC-fixL mutant was constructed and was shown to be completely devoid of nitrogenase activity . A model of regulation, based on these data, is presented and might explain the unusual ability of A . caulinodans to fix nitrogen both under symbiotic conditions and in the free-living state. M