Escherichia coli

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.

Mol Microbiol, 1991 Aug, 5(8), 1817 - 22
The pathogenic mechanisms of Shiga toxin and the Shiga-like toxins; Tesh VL et al.; It is now well documented that some enteric bacteria which cause diarrhoeal and/or dysenteric disease produce, at high levels, one or more of a family of protein toxins referred to as Shiga toxin and Shiga-like toxins (SLTs; alternatively called verocytotoxins or VTs) . Within the past few years, there have been considerable advancements made in our understanding of the biochemistry and molecular biology of Shiga toxin and SLTs . However, the precise role of the toxins in mediating colonic disease, as well as their contribution to the development of extra-intestinal sequelae (e.g . the haemolytic uraemic syndrome and neurological disorders), remain less clear . In this MicroReview, we will briefly summarize recent progress in Shiga toxin- and SLT-related research and present evidence supporting the concept that these toxins contribute to pathogenesis by directly damaging vascular endothelial cells, thereby disrupting the homeostatic properties of these cells . We will also discuss data which suggest that toxin-mediated damage in the kidney may not be limited to glomerular endothelial cells but may include tubular epithelial cells . Thus, the role of the toxins in renal disease may not be limited to the glomeruli, as was initially hypothesized when the association of infection with toxin-producing strains and the development of acute renal failure was established.

J Clin Microbiol, 1991 Aug, 29(8), 1652 - 8
Mycobacterium paratuberculosis antigen D: characterization and evidence that it is a bacterioferritin; Brooks BW et al.; By using a combination of agarose and polyacrylamide gel electrophoresis, Mycobacterium paratuberculosis antigen D was resolved from a crude sonicated preparation of the organism and characterized as a component with a molecular mass of approximately 400,000 Da . While this component was composed mainly of protein, with unusually high proportions of glutamic acid and leucine, it was resistant to digestion with a number of proteolytic enzymes . Structural detail revealed by electron microscopy, amino acid sequence data, and the demonstration of a Soret band in its absorption spectrum indicated that antigen D was similar to an Escherichia coli bacterioferritin.

J Biochem (Tokyo), 1991 Aug, 110(2), 284 - 91
Production and characterization of functional domains of human fibronectin expressed in Escherichia coli; Kimizuka F et al.; An efficient expression system was constructed in Escherichia coli that produced a 33-kDa fragment, C-274, of human fibronectin with a strong cell-adhesive activity . The entire sequence of the heparin-binding domain with 271 amino acids, H-271, was also expressed . Deletion analysis of the type III repeats showed that the heparin-binding site was at type III-13 . The cell-adhesive activity of a fusion protein, CH-271, containing the cell- and the heparin-binding domains was twice that of C-274 when BHK but not B16-F10 melanoma cells were tested; H-271 alone was inactive . Recombinant proteins containing the CS1 sequence of the IIICS region were more active than C-274 and CH-271 with B16-F10 . However, H-296, which contained both H-271 and CS1, was almost inactive with BHK . CH-296, which contained CS1 at the C-terminus of CH-271, was more active with B16-F10 than H-296 and C-CS1, which was produced by the deletion of H-271 from CH-296 . Thus, the cell-binding domain was active with both kinds of cells . The heparin-binding domain promoted the adhesion of both kinds of cells only when linked to the cell-binding domain or CS1 . CS1 was specific for the adhesion of B16-F10 but was not essential.

J Biochem (Tokyo), 1991 Aug, 110(2), 166 - 8
A continuous cell-free protein synthesis system for coupled transcription-translation; Kigawa T et al.; A continuous cell-free protein synthesis system {Spirin et al . (1988) Science 242, 1162-1164} was modified so as to be suitable for coupled transcription-translation, a process useful for obtaining products of cloned genes or cDNAs . A reaction chamber equipped with an ultrafiltration membrane was newly designed and an HPLC pump was used to supply a low molecular weight substrate solution at a constant rate to the viscous reaction mixture in the chamber . By using an Escherichia coli S30 extract in this modified flow system (1 ml), coupled transcription-translation could be continuously performed for 17 h, the synthesized chloramphenicol acetyltransferase (congruent to 0.1 mg) being subsequently eluted through the chamber's membrane and then purified.

J Cell Biochem, 1991 Aug, 46(4), 321 - 30
Secretion of mutant leucine-specific binding proteins with internal deletions in Escherichia coli; Adams MD et al.; The leucine-specific binding protein, encoded by the livK gene, is located in the periplasm of E . coli . The present study is an attempt to identify intragenic regions that determine the efficiency of its secretion into the periplasm . C-terminal deletions or fusions of the livK gene to trpA (encoding the alpha subunit of tryptophan synthetase) were secreted with little loss of efficiency {1} . A series of deletions was constructed at the unique Sphl site within livK, near the 5' end of the region coding for the mature protein . Between 16 and 113 amino acids were deleted in the amino-terminal one-third of the protein . A few of these deletions were located within a few amino acids of the signal sequence processing site . Deletions extending within thirteen residues of the processing site were processed and secreted more slowly than normal . Secondary structure predictions suggested that the alpha-helical core region of the signal sequence extends into the mature protein in the case of the slow processing mutants, perhaps interfering with the recognition site for leader peptidase or other secretory components . These results suggest that the conformation around the signal processing site may be a critical factor in determining the efficiency of secretion . During the course of this study, it was found that the difference in molecular weight between precursor and mature forms of some binding protein mutants, as judged by SDS-PAGE, was much greater than could be accounted for by processing of the signal sequence . This anomalous mobility on gels, however, could be eliminated by performing SDS-PAGE in the presence of 6 M urea.

Antibiot Khimioter, 1991 Aug, 36(8), 25 - 8
{Obtaining human recombinant (serine-17) beta-interferon by the method of oligonucleotide-directed mutagenesis and its expression in Escherichia coli}; Shekhter II et al.; The gene of human mutant (serine-17) fibroblast interferon was isolated with the use of highly efficient oligonucleotide-directed mutagenesis . On the basis of the constructed expression plasmid pPR-IFN Ser17 a strain producing human mutant beta-interferon (VKPM V-4678) was developed . It was shown that the specific activity of the human mutant (serine-17) fibroblast interferon was 1 order of magnitude higher than that of the recombinant interferon which reaches the specific activity of natural fibroblast interferon.

Physiol Behav, 1991 Aug, 50(2), 461 - 3
Lipopolysaccharide increases ambient temperature preference in C57BL/6J adult mice; Akins C et al.; The hypothesis was tested that animals exposed to a potentially dangerous endotoxin would attempt to behaviorally elevate their body temperature, perhaps in an effort to engage those immunological mechanisms which would counter the adverse effects of the endotoxin . Lipopolysaccharide (LPS) from Escherichia coli injected subcutaneously (100 micrograms) in adult C57BL/6J mice increased gradient temperature preference by 2.4 degrees C over saline controls . The increase in body temperature of 1.1 degrees C after LPS injection was due to the preference for higher ambient temperatures and was not the result of a systemic reaction to LPS (animals not exposed to the gradient did not differ in body temperature) . In summary, our data indicate that adult mice self-induce a febrile response, perhaps as an attempt to compensate for the physiological impact of the endotoxin.

Vaccine, 1991 Aug, 9(8), 595 - 601
Foreign epitopes in immunodominant regions of hepatitis B core particles are highly immunogenic and conformationally restricted; Brown AL et al.; The presentation of heterologous amino acid sequences on the surface of hepatitis B core antigen (HBcAg) particles has been studied using a defined linear neutralization site from human rhinovirus (HRV) . Previous work has shown that fusion particles, in which the HRV peptide sequence is linked to the amino terminus of the HBcAg protein, induce excellent immune responses in experimental animals . Using predictive models of HBcAg particulate structure and the approximate location of the major immunogenic regions we have designed and constructed bacterial expression vectors which direct synthesis of chimeric particles in which heterologous sequences are presented within an immunodominant area on the particle . Immunological responses to the heterologous peptide sequence are improved by at least tenfold when compared with amino terminal fusions of the same peptide sequence to HBcAg . Moreover, the restriction placed on the heterologous peptide by its linkage at both ends within the HBcAg protein results in a more constrained structure . In the case of the rhinovirus peptide sequence this results in an antigenic conformation more closely resembling that on the native virus particle . Such a system lends itself well as a general approach to the induction of high titre antibodies against defined epitopes.

Mol Microbiol, 1991 Aug, 5(8), 1961 - 73
The rifampicin-inducible genes srnB from F and pnd from R483 are regulated by antisense RNAs and mediate plasmid maintenance by killing of plasmid-free segregants; Nielsen AK et al.; The gene systems srnB of plasmid F and pnd of plasmid R483 were discovered because of their induction by rifampicin . Induction caused membrane damage, RNase I influx, degradation of stable RNA and, consequently, cell killing . We show here that the srnB and pnd systems mediate efficient stabilization of a mini-R1 test-plasmid . We also show that the killer genes srnB' and pndA are regulated by antisense RNAs, and that the srnC- and pndB-encoded antisense RNAs, denoted SrnC- and PndB-RNAs, are unstable molecules of approximately 60 nucleotides . The srnB and pndA mRNAs were found to be very stable . The differential decay rates of the inhibitory antisense RNAs and the killer-gene-encoding mRNAs explain the induction of these gene systems by rifampicin . Furthermore, the observed plasmid-stabilization phenotype associated with the srnB and pnd systems is a consequence of this differential RNA decay: the newborn plasmid-free cells inherit the stable mRNAs, which, after decay of the unstable antisense RNAs, are translated into killer proteins, thus leading to selective killing of the plasmid-free segregants . Thus our observations lead us to conclude that the F srnB and R483 pnd systems are phenotypically indistinguishable from the R1 hok/sok system, despite a 50% dissimilarity at the level of DNA sequence.

Mol Microbiol, 1991 Aug, 5(8), 1863 - 72
Structure of two retrons of Escherichia coli and their common chromosomal insertion site; Lim D; It has been shown that certain strains of myxobacteria and of Escherichia coli have a genetic element encoding a reverse transcriptase (RT) . This element, called a 'retron', produces a covalently linked RNA-DNA compound (msDNA-RNA) . Here, I report the complete nucleotide sequence of retron EC-86, the retron in E . coli B, together with its flanking regions . Retron EC-86 contains genes for msDNA-RNA (msd, and msr), a gene for RT (ret) and a gene for an open reading frame whose function is unknown . The upstream junction is composed of the sequence GCGCGCGC, but there are no direct or inverted repeats at the retron-host junctions . It is also shown that another retron of E . coli, EC-67, which was isolated originally from the clinical strain CL1 and was later found to be present also in a clinical E . coli isolate from Brazil, is inserted at the same chromosomal site as retron EC-86 . Retron EC-67 contains only msd, msr, and ret . I suggest that these two retrons were independently inserted into the same site of their host strains via a novel mechanism of integration.

Mol Biochem Parasitol, 1991 Aug, 47(2), 143 - 50
A Plasmodium falciparum malaria vaccine candidate which contains epitopes from the circumsporozoite protein and a blood stage antigen, 5.1; Caspers P et al.; The previously described Plasmodium falciparum blood stage antigen, 5.1 (also referred to as exp-1) was expressed at a high level in Escherichia coli . Saimiri monkeys immunised with purified recombinant antigen 5.1 were partially protected from P . falciparum blood stage parasite challenge . The gene coding for 5.1 was combined with DNA coding for an (Asn-Ala-Asn-Pro)19 sequence (abbreviated (NANP)19 in the one-letter amino acid code) . To facilitate purification of the recombinant protein, DNA coding for a hexahistidine (His6) sequence was introduced at the 5' end of the gene (proteins containing His6 have high affinity for Ni(2+)-chelate columns even in the presence of 6 M guanidine HCl) . The recombinant protein, His6-5.1-(NANP)19 with an apparent molecular size of 40 kDa could be highly purified by a combination of 4 steps: (1) release and solubilization of the recombinant fusion protein from E . coli in the presence of 6 M guanidine-HCl; (2) precipitation of over 60% of the bacterial proteins by the addition of ammonium sulphate to 50% saturation; (3) affinity chromatography on a Ni(2+)-chelate column in the presence of 6 M guanidine-HCl; (4) adsorption onto a cation exchange resin in the presence of 6 M urea, and elution with an increasing NaCl gradient . Compared with the previously tested tetanus toxoid-(NANP)3 malaria vaccine, this protein elicits an anti-(NANP)n response which more closely resembles that evoked by native sporozoites . The recombinant vaccine also induces the production of antibodies against the blood stages of the malaria parasite.

Curr Genet, 1991 Aug, 20(3), 205 - 10
Isolation of transcripts preferentially expressed during fruit body primordia differentiation in the basidiomycete Agrocybe aegerita; Salvado JC et al.; An Agrocybe aegerita cDNA library, constructed from fruit body primordia poly(A)+ RNAs, was screened by differential colony hybridization . Clones which preferentially hybridized to poly(A)+ RNA sequences from fruit body primordia, versus poly(A)+ RNAs from mycelium, were isolated . Eight of these clones (EMAa-1 to EMAa-8) encoded eight different poly(A)+ RNAs which were demonstrated to be undetectable in the four stages preceding primordia formation and to be concomitantly accumulated when primordia differentiate, suggesting that EMAa gene products are closely involved in the morphogenesis of primordia . The eight EMAa cDNAs hybridize to at least seven unique regions distributed randomly in the A . aegerita genome . The expression of two EMAa cDNA sequences in E . coli led to the isolation of their gene products as fusion proteins.

Biotechnol Appl Biochem, 1991 Aug, 14(1), 69 - 81
Metal affinity chromatography of recombinant HIV-1 reverse transcriptase containing a human renin cleavable metal binding domain; Sharma SK et al.; A metal binding peptide, hexahistidine, preceding a renin cleavage sequence (Pro-Phe-His-Leu-Val-Ile-His-) was engineered on to the N-terminus of HIV-1 reverse transcriptase (RT) . The chimeric protein was expressed in Escherichia coli and characterized after purification by DEAE chromatography and HPLC . Amino-terminal sequencing confirmed the presence of the first 15 amino acids of the chimeric protein . The chimeric exhibited RT activity like that of HIV-1 RT and was cleaved by human renin at the expected site . The potential of a hexa-histidine fusion in the purification of recombinant HIV-1 RT by immobilized metal affinity chromatography (IMAC) on the commonly used resin (IDA-Ni2+) was investigated . The chimeric gene product from a crude E . coli extract was strongly retarded on a immobilized nickel column, while most of the contaminating E . coli proteins were eliminated after elution with 20-35 mM imidazole . The bound chimeric protein was eluted with 300 mM imidazole and appeared predominantly as a single band on an SDS-polyacrylamide gel . The remarkable specificity of this affinity tail was further demonstrated by separating the chimeric protein from HIV-1 RT in a crude extract prepared by mixing extracts from cells expressing HIV-1 RT and the hexahistidine recombinant chimeric protein . The usefulness of a enzymatically cleavable metal binding peptide in the rapid purification and production of HIV-1 RT without proteolysis to a heterodimer is discussed.

Jpn J Cancer Res, 1991 Aug, 82(8), 879 - 82
Detection of hepatitis C virus infection by enzyme-linked immunosorbent assay system using core protein expressed in Escherichia coli; Muraiso K et al.; Enzyme-linked immunosorbent assay of the core protein of hepatitis C virus (HCV) expressed in E . coli led to detection of the antibody against this virus in patients with chronic hepatitis . Some of the negative results obtained using a different viral protein became positive with this E . coli-expressed viral protein, and were also positive for the viral RNA . Thus, use of the core protein of HCV facilitates accurate detection of HCV infection.

Mol Gen Genet, 1991 Aug, 228(1-2), 49 - 54
The rne gene is the structural gene for the processing endoribonuclease RNase E of Escherichia coli; Chauhan AK et al.; Using T7 RNA polymerase and specific constructs derived from 5S rRNA and RNA I genes, we generated substrates for the RNA processing enzyme RNase E . Using these substrates we have shown that a 3.2 kb DNA fragment that complements the rne-3071 mutation can express RNase E activity . We also found that T7 RNA polymerase terminates within the 5S rRNA gene.

Eur J Biochem, 1991 Aug 1, 199(3), 671 - 6
Localization on the mitochondrial F1 ATPase alpha subunit of an epitope masked in the membrane-bound enzyme using a monoclonal antibody and synthetic peptides; Moradi-Ameli M et al.; The epitope of the monoclonal antibody 20D6 was localized by N-terminal sequencing of the smallest immunoreactive peptides obtained after CNBr and trypsin cleavage of the F1 alpha subunit of the mitochondrial ATPase/ATP synthase . Immunochemical analysis of overlapping synthetic octapeptides, covering the immunoreactive peptide sequence, has defined the seven-amino-acid sequence recognized by 20D6 as 84EGDIVKR90 . The binding of 20D6 was lost after substituting either I87 by K or S, or R90 by C or A as it occurs in the alpha subunit sequence of Escherichia coli or chloroplast ATPase, respectively . This explained the lack of immunoreactivity of 20D6 to these species and indicated the importance of charged as well as hydrophobic residues in the epitope . Immunochemical analysis of synthetic peptides by polyclonal anti-F1 antisera showed that this region is highly immunodominant . In a competitive ELISA, the monoclonal antibody bound with similar affinity to F1 in the presence and absence of substrate as well as to cold dissociated F1, indicating that the epitope was located on the surface of the alpha subunit and not buried between F1 subunits . The lack of binding of 20D6 when F1 is bound to the membrane showed that the epitope exposed at the surface of purified soluble F1 became masked after binding to the membrane . This suggests that it is located at the interface between F1 and the membrane.

Am J Pathol, 1991 Aug, 139(2), 461 - 6
Expression of endothelial leukocyte adhesion molecule-1 in septic but not traumatic/hypovolemic shock in the baboon; Redl H et al.; Baboons were subjected to septic or traumatic/hypovolemic shock and their tissues were examined for the de novo expression of endothelial leukocyte adhesion molecule 1 (ELAM-1), using immunohistochemical techniques . In animals with septic shock induced with live Escherichia coli, there was widespread expression of ELAM-1, recognized by monoclonal antibodies H4/18 or ENA-1 in most tissues examined with strong staining in the lung, liver, and kidneys . Endothelial leukocyte adhesion molecule 1 expression was evident in capillaries, venules, small veins, arterioles, and arteries . In contrast, baboons with traumatic/hypovolemic shock had minimal levels of focal ELAM expression in all organs studied . Similarly evidence of neutrophil activation, measured by granulocyte elastase levels in the plasma was much more pronounced in animals with septic shock . The study documents that lipopolysaccharide (LPS)- and cytokine-induced endothelial activation occurs in vivo in septic shock . Much higher levels of ELAM-1 expression and plasma granulocyte-elastase titer in septic shock, as contrasted with traumatic/hypovolemic shock, are consistent with the higher levels of circulating tumor necrosis factor, other cytokines, and LPS in sepsis.

Proc Natl Acad Sci U S A, 1991 Aug 1, 88(15), 6711 - 5
Stable transformation of Leptomonas seymouri by circular extrachromosomal elements; Bellofatto V et al.; To define the cis-acting sequences necessary for gene expression and DNA replication in trypanosomatids, we have developed a selectable vector that can be grown in Escherichia coli and maintained stably in the insect trypanosomatid Leptomonas seymouri . The vector is relatively small (6 kilobase pairs) and contains a portion of the L . seymouri alpha-tubulin gene positioned in-frame with a truncated neomycin phosphotransferase gene that confers resistance to the aminoglycoside G418 . This construct is maintained in cells as a high-copy-number circular extrachromosomal element containing several head-to-tail copies of the transforming plasmid . In L . seymouri, alpha-tubulin-neomycin phosphotransferase fusion RNAs are polyadenylylated and possess a trans-spliced mini-exon . Additional DNA sequences can be inserted into the vector, propagated, and expressed in transformed cells.

Mol Cell Biol, 1991 Aug, 11(8), 3842 - 9
Ribosomal protein genes are overexpressed in colorectal cancer: isolation of a cDNA clone encoding the human S3 ribosomal protein; Pogue-Geile K et al.; We have isolated a cDNA clone encoding the human S3 ribosomal protein from a normal human colon cDNA library . The clone was identified as one of many that detected genes whose level of expression was increased in adenocarcinoma of the colon relative to normal colonic mucosa . Increased levels of the S3 transcript were present in the tumors of all eight patients examined . Moreover, the S3 mRNA was also more abundant in 7 of 10 adenomatous polyps, the presumed precursor of carcinoma . Additional studies demonstrated that increased levels of mRNAs encoding several other ribosomal proteins, including S6, S8, S12, L5, and P0, were present in colorectal tumors and polyps . These results suggest that there is increased synthesis of ribosomes in colorectal tumors and that this increase is an early event in colon neoplasia.

J Virol, 1991 Aug, 65(8), 4051 - 6
An HLA-C-restricted CD8+ cytotoxic T-lymphocyte clone recognizes a highly conserved epitope on human immunodeficiency virus type 1 gag; Littaua RA et al.; A unique epitope on the gag protein of human immunodeficiency virus type 1 (HIV-1), located at amino acid 145 to 150, has been mapped by using a CD8+ cytotoxic T-lymphocyte (CTL) clone . This epitope is highly conserved among 18 HIV-1 strains . The HIV-1 gag-specific human leukocyte antigen (HLA) class I-restricted CD8+ CTL clone was generated from fresh peripheral blood mononuclear cells of an HIV-seropositive donor by stimulation with gamma-irradiated allogeneic peripheral blood mononuclear cells in the presence of an anti-CD3 monoclonal antibody and recombinant interleukin-2 . This gag-specific CTL clone killed autologous target cells infected with a recombinant vaccinia virus containing the gag gene of HIV-1 and target cells pulsed with an authentic p24gag construct expressed in Escherichia coli . Fine specificity was determined by using a panel of overlapping 30-amino-acid-long synthetic peptides and subsequently using smaller peptides to precisely map the CTL domain on p24 . The epitope is on a highly conserved region, and it overlaps with a major B-cell epitope of gag . This CD8+ T-cell epitope is restricted by HLA-Cw3, which has not been previously identified as a restricting element for human CTL responses.

J Virol, 1991 Aug, 65(8), 3986 - 94
Monoclonal antibody-defined epitope map of expressed rubella virus protein domains; Wolinsky JS et al.; An expanded library of murine monoclonal antibodies (MAbs) was generated by infecting BALB/C mice with the Therien strain of rubella virus (RV) and selecting secreting hybrids by enzyme-linked immunosorbent assay (ELISA) using purified virion targets . A panel of plasmids containing specified RV cDNA fragments was also constructed by using a variety of strategies with pGE374- and pGE374-derived expression vectors . Hybrid RecA-RV-beta-galactosidase (LacZ)- or RecA-RV-truncated LacZ-containing proteins collectively representing the entire open reading frame of the structural proteins of RV were overexpressed in Escherichia coli . Bacterial lysates were then probed by ELISA with selected MAbs and by immunoblot following separation by electrophoresis under denaturing conditions . With this approach, MAbs that appeared to react with linear determinants defined epitopes localized within the following domains: MAbs C-1, C-2, and C-8 bind epitopes within the predicted amino-terminal 21 amino acids of the capsid region C9 to C29; MAb C-9 binds to a domain bounded by C64 and C97; MAbs E2-1 through E2-6 bind to the E2 glycoprotein backbone region from E2(1) to E2(115); MAbs E1-18 and E1-20 bind to the E1 glycoprotein region from E1(202) to E1(283) . MAb E1-18 neutralizes RV infectivity; MAb E1-20 neutralizes infectivity and modestly inhibits hemagglutination . Analyses with selected synthetic peptides have confirmed several of the molecular domains deduced with the expressed proteins . These plasmid constructions and peptides have proven useful in beginning to unravel the molecular organization of several antigenic sites of this human pathogen.

J Neurochem, 1991 Aug, 57(2), 483 - 90
Molecular cloning and expression of biologically active human glia maturation factor-beta; Kaplan R et al.; Glia maturation factor-beta, a protein found in the brains of all vertebrates thus far examined, appears to play a role in the differentiation, maintenance, and regeneration of the nervous system . Using oligonucleotide probes based on the sequences of three tryptic peptides derived from bovine glia maturation factor-beta, we screened a human brainstem cDNA library in lambda gt11 . A 0.7-kb clone was isolated, sequenced in its entirety, and found to encode a polypeptide of 142 amino acids which contained regions identical to the three bovine peptides . This polypeptide, human recombinant glia maturation factor-beta, has been expressed in Escherichia coli and found to possess structural characteristics and biological activity indistinguishable from those of the native bovine protein.

EMBO J, 1991 Aug, 10(8), 2195 - 202
The path of mRNA through the Escherichia coli ribosome; site-directed cross-linking of mRNA analogues carrying a photo-reactive label at various points 3' to the decoding site; Rinke-Appel J et al.; mRNA analogues approximately 40 bases long were prepared by T7 transcription from synthetic DNA templates . Each message contained the sequence ACC-GCG (coding for threonine and alanine, respectively), together with a single thio-U residue located at a variable position on the 3'-side of these coding triplets . The thio-U residue was either substituted with 4-azidophenacyl bromide to introduce a photo-reactive group, or was left unsubstituted for direct UV cross-linking . After binding to Escherichia coli 70S ribosomes in the presence of tRNA-Thr or tRNA-Ala, the thio-U residue or azidophenyl group was photo-activated and the products of cross-linking (which was exclusively to the 30S subunit) were analysed . Immunological analysis of the cross-linked proteins showed that S5 and S3, together with S1, were the targets of cross-linking at positions close to the decoding site, with the cross-linking to S3 and S1 persisting at positions further away . Analysis of the 16S RNA showed cross-links to the region of bases 1390-1400 in all cases, but in one instance (with the reactive nucleotide 11 bases from the decoding site) simultaneous cross-linking was observed to the latter region and to position 532; these two RNA regions are far apart in current three-dimensional models of the 30S subunit.

Acta Virol, 1991 Aug, 35(4), 383 - 90
Recombinant proteins derived from immunodominant regions of the gag and env genes of HIV-1 and their potential for use in serologic diagnosis; Kovac J; Based on the published sequences of human immunodeficiency virus (HIV-1) isolates highly conserved regions of the gag and env genes containing immunodominant epitopes were selected and expressed in E . coli . The expression vectors pKK24 and pEX41 produced viral proteins recognized by sera obtained from HIV-1 seropositive individuals . A testing system was designed to determine the practical value of bacterially synthesized proteins of HIV-1 gag and env genes for serodiagnosis of HIV-1 infection.

Microb Pathog, 1991 Aug, 11(2), 143 - 7
Influence of raffinose on the relative synthesis rate of K88 fimbriae and the adhesive capacity of Escherichia coli K88; Blomberg L et al.; The synthesis rate of K88 fimbriae relative to the rate of total protein synthesis was estimated during growth of a wild-type Escherichia coli Bd 1107/7508 (K88ac) in M9 minimal medium supplemented with glucose or raffinose as the carbon source . Rates of synthesis of fimbriae and total protein were analysed by immunoprecipitation and TCA precipitation of radioactive pulse labelled K88 fimbriae and E . coli K88 cells, respectively . In addition, the effect of raffinose on the in vitro adhesion of E . coli K88 cells to immobilized piglet ileal mucus was studied . The relative rate of K88 fimbriae synthesis increased gradually during log phase, reached maximum at late log phase, and thereafter decreased continuously . The use of raffinose as the carbon source, did not alter the relative rate of K88 fimbrial synthesis or the adhesive capacity of E . coli K88 cells, as compared to glucose.

FEMS Microbiol Lett, 1991 Aug 1, 66(2), 183 - 8
Characterization of highly virulent Escherichia coli strains by ribosomal DNA restriction fragment length polymorphism; Picard B et al.; Patterns of ribosomal DNA polymorphism were examined to compare carboxylesterase B type B1 strains and B2 strains of Escherichia coli isolated from extra-intestinal infections . DNA from 14 type B2 strains showing the presence of alpha-haemolysin and mannose-resistant haemagglutinin and lethality to mice and 14 type B1 strains lacking these characteristics, was digested with HindIII, EcoRI, BamHI or BglII restriction enzymes and analysed by Southern blotting . The obtained ribotypes clearly differentiated the B2 strains from the B1 strains . These results indicate that genotypes of the highly virulent B2 strains are different from that of the less virulent B1 strains.

FEMS Microbiol Lett, 1991 Aug 1, 66(2), 177 - 82
Occurrence of pap-, sfa-, and afa-related sequences among F165-positive Escherichia coli from diseased animals; Harel J et al.; A total of 160 Escherichia coli positive for F165 fimbrial antigen and isolated from diarrheic and septicemic animals, were examined for the presence of the pap, afa, and sfa/foc operons or related nucleotide sequences using colony hybridization . Most isolates shared DNA sequences with the pap operon sequences alone or in association with afa or sfa . Thus, our results indicate that F165-positive E . coli from diseased animals share DNA sequences with operons coding for adhesins important in human extra-intestinal disease and that multiple adhesin systems are often found in single isolates . However, 20% of the F165-positive isolates did not show any homology with the probes representing the three adhesin systems, suggesting that one of the operons responsible for F165 production could be different from the pap, sfa/foc, and afa operons.

FEMS Microbiol Lett, 1991 Aug 1, 66(2), 125 - 9
Cloning of regulator genes controlling fimbrial production by enterotoxigenic Escherichia coli; Willshaw GA et al.; Sequences regulating production of fimbriae were cloned from two enterotoxigenic Escherichia coli strains . One cloned region, from E . coli 0.25.H42, controlled expression of coli surface-associated (CS) antigen 4, whereas the function of the other, from E . coli 0167.H5, was unclear . Both regulators were related to the cfaD gene that controls expression of colonization factor antigen I (CFA/I) although low stringency conditions were required to show significant hybridization between cfaD and the regulatory fragment from E . coli 0167 . The cloned regulatory genes promoted expression of CFA/I, CS1, CS2 and CS4 antigens but the levels of production in the presence of the 0167 regulator were lower than those promoted by the CS4 regulator or cfaD.

Development, 1991 Aug, 112(4), 1077 - 93
Phosphorylation, expression and function of the Ultrabithorax protein family in Drosophila melanogaster; Gavis ER et al.; Alternative splicing of the Ultrabithorax homeotic gene transcript generates a family of five proteins (UBX isoforms) that function as transcription factors . All isoforms contain a homeodomain within a common 99 aa C-terminal region (C-constant region) which is jointed to a common 247 aa N-terminal (N-constant) region by different combinations of three small optional elements . Unlike the UBX proteins expressed in E . coli, UBX isoforms expressed in D . melanogaster cells are phosphorylated on serine and threonine residues, located primarily within a 53 aa region near the middle of the N-constant region, to form at least five phosphorylated states per isoform . Similar, if not identical states can be generated in vitro from purified E . coli UBX protein by a kinase activity in nuclear extracts from D . melanogaster cells . Temporal developmental profiles of UBX isoforms parallel those for the respective mRNAs, and all isoforms are similarly phosphorylated throughout embryogenesis . Analysis by cotransfection assays of the promoter activation and repression functions of mutant UBX proteins with various deletions in the N-constant region shows that repression is generally insensitive to deletion and, hence, presumably to phosphorylation . By contrast, the activation function is differentially sensitive to the different deletions in a manner indicating the absence of a discrete activating domain and instead, the presence of multiple activating sequences spread throughout the region.

Mol Gen Genet, 1991 Aug, 228(1-2), 209 - 14
Mechanisms of deletion formation in Escherichia coli plasmids . II . Deletions mediated by short direct repeats; Mazin AV et al.; A set of plasmids containing 42, 21 and 31 bp direct repeats was used to analyze the effect of repeat length on the frequencies of deletion formation and the structure of the deleted derivatives of different recombination-deficient Escherichia coli strains . Agarose gel electrophoresis of plasmid DNA demonstrated that the formation of deletions in these plasmids was associated with dimerization of plasmid DNA . Restriction analysis of the dimers showed that deletions at short direct repeats arose non-conservatively, that is, the formation of a deletion in one monomeric plasmid unit was not associated with a duplication in the other . Mutations in the recA, recF, recJ and recO genes had no marked effect on either the frequencies of deletion formation or the structure of dimers . In contrast, recB recC mutations greatly increased the frequencies of deletion formation, 6-fold for 42 bp, and 115-fold for 21 bp direct repeats . Conversion of DNA replication to the rolling circle mode in a recB recC strain, resulting in the formation of double-stranded ends, is suggested as the stimulatory effector.

Mol Gen Genet, 1991 Aug, 228(1-2), 153 - 9
Molecular mechanisms of deletion formation in Escherichia coli plasmids . I . Deletion formation mediated by long direct repeats; Dianov GL et al.; Derivatives of plasmid pBR327 with the tet gene interrupted by 165 pb or 401 bp direct repeats were constructed . In cells harboring these plasmids, deletions which restored the wild-type tet gene gave rise to tetracycline-resistant colonies, thereby allowing a simple phenotypic test for deletion formation . The frequencies of deletions in these plasmids were measured in Escherichia coli strains proficient or deficient in general recombination . The structure of plasmid DNA isolated from tetracycline-resistant transformants was analyzed by agarose gel electrophoresis, restriction mapping and sequencing . The data presented here demonstrate that deletion formation is always associated with dimerization of plasmid DNA . Dimeric plasmids were of two types . Those which carried both a deletion and a compensating duplication were the major type in a Rec+ background and were rare in recA, recF, recJ and recO backgrounds . Dimers of the second type contained deletions, but no compensating duplications, and their formation was RecA-independent . The data presented demonstrate that deletion formation mediated by long direct repeats is mainly the result of unequal crossing-over between two plasmid molecules.

Am J Gastroenterol, 1991 Aug, 86(8), 976 - 80
An increased number of gamma/delta T-cells and gastric epithelial cell expression of the groEL stress-protein homologue in Helicobacter pylori-associated chronic gastritis of the antrum; Engstrand L et al.; Numerous studies have shown that the presence of Helicobacter pylori in the stomach is linked to the development of chronic gastritis most commonly seen in the antrum . However, the pathogenic mechanisms are unclear . In 23 of 31 patients, examined due to symptoms from the upper gastrointestinal tract, H . pylori-associated chronic gastritis of the antrum was diagnosed histologically and by growth of H . pylori . Immunoperoxidase staining on gastric biopsy specimens from these patients showed an increased number of gamma/delta T-cells within the epithelium . Furthermore, the Mab ML30 (raised against the 65 kDa heat shock protein of mycobacteria) demonstrated positive staining in the gastric epithelial cells in all H . pylori-positive but not in H . pylori-negative biopsy specimens . H . pylori also reacted with ML30, as detected by immunoperoxidase staining as well as by immunoblotting . Intraepithelial gamma/delta T cells may play a role in host defense against invading H . pylori, and the bacteria may trigger an autoimmune response to stress proteins expressed by the gastric epithelial cells.

Infect Immun, 1991 Aug, 59(8), 2664 - 72
Virulence patterns and long-range genetic mapping of extraintestinal Escherichia coli K1, K5, and K100 isolates: use of pulsed-field gel electrophoresis; Ott M et al.; A total of 127 extraintestinal Escherichia coli strains of the capsule serotypes K1, K5, and K100 from human and animal sources were analyzed for DNA sequences specific for the genes for various adhesins (P fimbriae {pap} and P-related sequences {prs}, S fimbriae {sfa}/F1C fimbriae {foc}, and type I fimbriae {fim}), aerobactin (aer), and hemolysin (hly) . The expression of corresponding virulence factors was also tested . Twenty-four selected strains were analyzed by long-range DNA mapping to evaluate their genetic relationships . DNA sequences for the adhesins were often found in strains not expressing them, while strains with hemolysin and aerobactin genes usually did express them . Different isolates of the same serotype often expressed different virulence patterns . The use of virulence-associated gene probes for Southern hybridization with genomic DNA fragments separated by pulsed-field gel electrophoresis revealed that a highly heterogeneous restriction fragment length and hybridization pattern existed even within strains of the same serotype . Long-range DNA mapping is therefore useful for the evaluation of genetic relatedness among individual isolates and facilitates the performance of precise molecular epidemiology.

Virus Res, 1991 Aug, 20(3), 205 - 21
Elimination of UL56 gene by insertion of LacZ cassette between nucleotide position 116030 to 121753 of the herpes simplex virus type 1 genome abrogates intraperitoneal pathogenicity in tree shrews and mice; Rosen-Wolff A et al.; In order to investigate whether or not the UL56 gene is involved in those processes determining the viral pathogenicity and latency, a recombinant virus HSV-1-M-LacZ was constructed in which the DNA sequences between nucleotide position (np) 116030 and 121753 were replaced by the E . coli beta-galactosidase (LacZ) gene . This deletion spans from the carboxyterminus of UL55 (np 116030) to the second exon of IE110 (np 121753) eliminating UL56 and the variable region of the BamHI DNA fragment B which were implicated in intraperitoneal pathogenicity and latency . The host range and growth kinetics of the recombinant virus HSV-1 M-LacZ were comparable to the parental strain HSV-1 F . As expected it was found that HSV-1-M-LacZ lost its virulent phenotype and was not able to develop acute infection in animals . The state of the UL56 gene was investigated by determining the cDNA sequence of the UL56 gene transcript of HSV-1 F using PCR products obtained after amplification of the cDNA with oligonucleotide primers corresponding to the translational start and stop codons of this gene . This analysis revealed that the DNA sequence of the UL56 gene of HSV-1 F differed from those DNA sequences determined for the genomic DNA of HSV-1 strain 17 . Between nucleotide position 116343 and 116344 two nucleotides -AG- are inserted which prolong the ORF of the UL56 gene to 233 amino acids with a predicted molecular weight of 30 kDa.

Mol Microbiol, 1991 Aug, 5(8), 1941 - 59
Site-specific insertion of genes into integrons: role of the 59-base element and determination of the recombination cross-over point; Hall RM et al.; From examination of published DNA sequences of genes found inserted at a specific site in integrons, all genes are shown to be associated, at their 3' ends, with a short imperfect inverted repeat sequence, a 59-base element or relative of this element . The similarity of the arrangement of gene inserts in the integron and in the Tn7 transposon family is described . A refined consensus for the 59-base element is reported . Members of this family are highly diverged and the relationship of a group of longer elements to the 59-base elements is demonstrated . The ability of 59-base elements of different length and sequence to act as sites for recombination catalysed by the integron-encoded DNA integrase is demonstrated, confirming that elements of this family have a common function . The ability of elements located between gene pairs to act as recombination sites has also been demonstrated . The recombination cross-over point has been localized to the GTT triplet which is conserved in the core sites, GTTRRRY, found at the 3' end of 59-base elements . Recombination at the core site found in inverse orientation at the 5' end of the 59-base elements was not detected, and the sequences responsible for orientation of the recombination event appear to reside within the 59-base element . A model for site-specific insertion of genes into integrons and Tn7-like transposons is proposed . Circular units consisting of a gene associated with a 59-base element are inserted into an ancestral element which contains neither a gene nor a 59-base element.(ABSTRACT TRUNCATED AT 250 WORDS)

J Gen Microbiol, 1991 Aug, 137 ( Pt 8), 1775 - 82
Mutants of Escherichia coli producing pyrroloquinoline quinone; Biville F et al.; In glucose minimal medium a PTS- strain of Escherichia coli {delta (ptsH ptsI crr)} could grow slowly (doubling time, d = 10 h) . When the population reached 5 x 10(6) to 2 x 10(7) cells ml-1, mutants growing rapidly (d = 1.5 h) appeared and rapidly outgrew the initial population . These mutants (EF mutants) do not use a constitutive galactose permease for glucose translocation . They synthesize sufficient pyrroloquinoline quinone (PQQ) to yield a specific activity of glucose dehydrogenase (GDH) equivalent to that found in the parent strain grown in glucose minimal medium supplemented with 1 nM-PQQ . Membrane preparations containing an active GDH oxidized glucose to gluconic acid, which was also present in the culture supernatant of EF strains in glucose minimal medium . Glucose utilization is the only phenotypic trait distinguishing EF mutants from the parent strain . Glucose utilization by EF mutants was strictly aerobic as expected from a PQQ-dependent catabolism . The regulation of PQQ production by E . coli is discussed.

Mech Dev, 1991 Aug, 35(1), 25 - 31
Unusual cell specific expression of a major human cytomegalovirus immediate early gene promoter-lacZ hybrid gene in transgenic mouse embryos; Kothary R et al.; Transgenic mice carrying the human cytomegalovirus immediate early gene promoter driving the E . coli lacZ gene displayed an unusual cell specific expression of beta-galactosidase during development . LacZ expression was first detected in cells lining the apex of the neural fold of day 8.5 embryos . By day 10 of gestation, expression was prominent in the spinal ganglia, the ganglia of cranial nerves V, VII, VIII, IX, and X, in a line of cells marking the ventrolateral pathway adjacent to the dermamyotome, and in a column of differentiated cells in the entire ventrolateral neural tube posterior to the mesencephalon . Expression was also found in the myotomes . Neural tube explants from day 8.5 embryos cultured in vitro showed lacZ expression in cells migrating away from the explant . We conclude that the HCMV-IEP-lacZ transgene is expressed in a subpopulation of neural crest cells and its early derivatives.

Protein Eng, 1991 Aug, 4(6), 701 - 8
Engineering proteins, subcloning and hyperexpressing oxidoreductase genes; Darwish K et al.; A very efficient system for subcloning and studying protein sequences, combining previously established elements for hyperexpression, replication and screening, was used to hyperproduce and characterize seven different products . It expedited the cloning of genes, in a multipurpose recombinant DNA construct, for all the requirements to study and engineer proteins with a strain of Escherichia coli . Genes encoding six heme proteins and a flavoprotein have been subcloned and expressed to 13-30% of the total cell protein, greatly facilitating purification and analyses . Three of the heme proteins and the flavoprotein incorporated prosthetic groups in E . coli, and exhibited the expected activities . Four of the enzymes have been purified to homogeneity and two of these crystallized for X-ray diffraction analysis . A rapid mutagenesis protocol, based on polymerase chain reactions, was successfully applied to clone derivatives of one of these enzymes, cytochrome c peroxidase . Thus, this system fulfills all criteria for engineering proteins in an efficient and concerted manner.

New Biol, 1991 Aug, 3(8), 799 - 811
dif, a recA-independent recombination site in the terminus region of the chromosome of Escherichia coli; Kuempel PL et al.; dif (deletion induced filamentation) is a newly identified locus that lies within the terminus region of the Escherichia coli chromosome . The Dif phenotype was characterized by a subpopulation of filamentous cells with abnormal nucleoids and induction of the SOS repair system . Interactions between dif-carrying plasmids as well as between such plasmids and the bacterial chromosome demonstrated that dif is a cis-acting, recA-independent recombination site . Filamentation continued in dif mutants in which SOS-associated division inhibitors were inoperative, which showed that induction of these inhibitors was not the primary cause of filamentation . Filamentation was not observed in dif recA or dif recBC mutants, which were unable to carry out homologous recombination . The dif site shows homology with the cer site of plasmid ColE1, which resolves plasmid multimers to monomers . It is proposed that dif functions to resolve dimeric chromosomes produced by sister chromatid exchange, and that the Dif phenotype is due to the inability of these mutants to resolve multimers prior to cell division.

Biotechniques, 1991 Aug, 11(2), 256 - 61
Protein recovery from the all-agarose ProSieve gel system; Morgan JH et al.; Electrophoresis in polyacrylamide gels is one of the most powerful tools used for the analysis of proteins . However, this technique is not widely used for protein purification for a variety of reasons such as the following: less than quantitative recoveries; involved, time-consuming methodologies; and impurities in the protein preparations from gel-polymerization by-products that can modify the proteins and interfere with subsequent experiments . As an alternative, we have developed a simple and quantitative recovery procedure for proteins separated by electrophoresis in the all-agarose ProSieve gel system . Using this procedure, greater than 90% of each protein examined was recovered, and these proteins were unaffected by the recovery procedure.

Antimicrob Agents Chemother, 1991 Aug, 35(8), 1647 - 50
Quinolone resistance-determining region in the DNA gyrase gyrB gene of Escherichia coli; Yoshida H et al.; Thirteen spontaneous quinolone-resistant gyrB mutants of Escherichia coli KL16, including two that were examined previously, were divided into two types according to their quinolone resistance patterns . Type 1 mutants were resistant to all the quinolones tested, while type 2 mutants were resistant to acidic quinolones and were hypersusceptible to amphoteric quinolones . Nucleotide sequence analysis disclosed that all nine type 1 mutants had a point mutation from aspartic acid to asparagine at amino acid 426 and that all four type 2 mutants had a point mutation from lysine to glutamic acid at amino acid 447.

Genetics, 1991 Aug, 128(4), 687 - 94
Genetic evidence against intramolecular rejoining of the donor DNA molecule following IS10 transposition; Bender J et al.; Tn10 and IS10 transpose by a nonreplicative mechanism in which the transposon is excised from the donor molecule and integrated into a target DNA site, leaving behind a break at the original donor site . The fate of this broken donor DNA molecule is not known . We describe here two experiments that address this issue . One experiment demonstrates that a polar IS10 element gives rise to polarity-relief revertants at less than 1% the frequency of transposition of the same element in the same culture . In a second experiment, transpositions of an IS10 element from one site in the bacterial genome to another are selected and the resulting isolates examined for alterations at the donor site; none of 1088 such isolates exhibited a detectable change at the donor locus . These results are compatible with two possible fates of the transposon donor molecule: degradation ("donor suicide"), or restoration of the original information at the donor site by a recombinational repair mechanism analogous to double-strand break repair . These results argue against the possibility that the donor molecule gap is simply resealed by intramolecular rejoining.

Mol Gen Genet, 1991 Aug, 228(1-2), 312 - 5
Tn5 insertion specificity is not influenced by IS50 end sequences in target DNA; Lodge JK et al.; The bacterial transposon Tn5 inserts into dozens of sites in a gene, some of which are used preferentially (hotspots) . Features of certain sites and precedents provided by several other transposons had suggested that sequences in target DNA corresponding to the ends of Tn5 or of its component IS50 elements might facilitate transposition to these sites . We tested this possibility using derivatives of plasmid pBR322 carrying IS50 I or O end sequences . Tn5 inserted frequently into an IS50 I end at the major hotspot in pBR322, but not into either an I end or an O end 230 bp away from this hotspot . Adenine (dam) methylation at GATC sequences in the I end segment interferes with its use as the end of a transposon, but a dam- mutation did not affect Tn5 insertion relative to an I end sequence in target DNA . These results support models in which the ability of Tn5 to find its preferred sites depends on several features of DNA sequence and conformation, and in which target selection is distinct from recognition of the element ends during transposition.

Mol Gen Genet, 1991 Aug, 228(1-2), 136 - 42
Interaction of RecBCD enzyme with DNA damaged by gamma radiation; Brcic-Kostic K et al.; The DNA of a gene 2 mutant (T4 2-) of phage T4 is degraded by RecBCD enzyme in the bacterial cytoplasm . Under normal conditions, recBCD+ cells are therefore incapable of supporting the growth of phage T4 2- . Only if the nucleolytic activity of RecBCD enzyme is absent from the cytoplasm are T4 2(-)-infected bacteria able to form plaques . We found that recBCD+ cells can form plaques if, before infection with T4 2-, they have been exposed to gamma radiation . It is suggested that gamma ray-induced lesions of the bacterial DNA (e.g., double-strand breaks) bind RecBCD enzyme . This binding enables the enzyme to begin to degrade the bacterial chromosome, but simultaneously prevents its degradative action on the ends of minor DNA species, such as unprotected infecting phage chromosomes . Degradation of the chromosomal DNA, which occurs during the early postirradiation period, ceases about 60 min after gamma ray exposure . The reappearance of the nucleolytic action of RecBCD enzyme on T4 2- DNA accompanies the cessation of degradation of bacterial DNA . Both, this cessation and the reappearance of the nucleolytic action of ReCBCD enzyme on T4 2- DNA depend on a functional recA gene product . These results suggest that postirradiation DNA degradation is controlled by the recA-dependent removal of RecBCD enzyme from the damaged chromosome . By making use of the temperature-sensitive mutant recB270, we showed that RecBCD-mediated repair of gamma ray-induced lesions occurs during the early postirradiation period, i.e . during postirradiation DNA degradation . It is shown that the RecD subunit of RecBCD enzyme also participates in this repair.

Gan To Kagaku Ryoho, 1991 Aug, 18(10), 1543 - 9
{Biological functions of DNA topoisomerases}; Andoh T; DNA topoisomerases are enzymes involved in various aspects of genetic processes by catalyzing topological changes of DNA . In DNA replication of closed circular DNA, for example, the parental strands are unwound as the synthesis of daughter strands proceed and overwinding and hence positive supercoils are introduced and accumulated in the unreplicated portions of the molecules, which retard and inhibit progression of replication machinery . Analogous situation may often prevail in transcription, recombination etc . within the cells . In order to solve this problem inherent in the metabolism of circular or looped double stranded DNA, living organisms may have evolved the exquisite device of DNA topoisomerases . In the present article biological functions of DNA topoisomerase are briefly reviewed.

Am J Physiol, 1991 Aug, 261(2 Pt 1), L126 - 32
Dibutyryl cAMP, prostaglandin E2, and antioxidants protect cultured bovine bronchial epithelial cells from endotoxin; Koyama S et al.; Acute bronchitis secondary to bacterial infection in the airway is accompanied by an acute inflammatory response composed predominantly of neutrophils . Mucosal injury with denudation of the airway epithelium to basement membrane frequently occurs . We postulated that endotoxin might explain this cytotoxicity and neutrophil influx . To test this hypothesis, bovine bronchial epithelial cells were cultured, and the culture supernatant fluids were evaluated for neutrophil chemotactic activity (NCA) and lactate dehydrogenase (LDH) after exposure to endotoxin . Escherichia coli endotoxin stimulated the release of NCA and LDH in a dose-dependent manner . Because intracellular augmentation of adenosine 3',5'-cyclic monophosphate (cAMP) has anti-inflammatory effects, we postulated that dibutyryl cAMP (DBcAMP) and prostaglandin E2 (PGE2) might modulate the effect of endotoxin . DBcAMP and PGE2 decreased the release of NCA and LDH . Because cAMP might exert its effect by decreasing intracellular release of oxidants, we investigated the capacity of the antioxidants dimethyl sulfoxide (DMSO) and allopurinol to attenuate the effects of endotoxin . DMSO and allopurinol alone or in combination attenuated the effects of endotoxin-induced NCA and LDH release . These data suggest that endotoxin may account for the pathophysiological changes seen with bronchial bacterial infection or endotoxin inhalation and that the inflammatory responses may be attenuated by DBcAMP, PGE2, and antioxidants.

Am J Physiol, 1991 Aug, 261(2 Pt 1), G229 - 38
Kinetics and regulation of a polarized Na(+)-H+ exchanger from Caco-2 cells, a human intestinal cell line; Watson AJ et al.; The kinetics and regulation of Na(+)-H+ exchange were studied using BCECF to measure pHi in Caco-2 cells grown on membrane filters . Na(+)-H+ exchange was defined as a Na(+)-dependent H+ efflux in response to an acid load imposed by an NH4Cl prepulse in the absence of added CO2 . Na(+)-H+ exchange was present exclusively on the basolateral membrane, had a Kt (Na+) of 21 +/- 2 mM, and an ID50 for amiloride dependent on medium {Na+} with an apparent Ki for amiloride of 3 microM . Na(+)-H+ exchange rates had a greater than first-order dependence on intracellular {H+}, suggesting the presence of an internal proton modifier site . Results also suggest that Na(+)-H+ exchange is kinetically inactivated at resting pHi (7.35 +/- 0.02), since neither removal of Na+ nor addition of amiloride affected resting pHi, although monensin alkalinized cells to pHi 7.6 . To evaluate regulation of Na(+)-H+ exchange, cells were exposed to either forskolin, 1,9-dideoxyforskolin (a noncyclase-activating forskolin derivative), 8-BrcAMP, E . coli STa toxin, ionomycin, phorbol dibutyrate, or cellular shrinkage in hypertonic medium . Only forskolin and 1,9-dideoxyforskolin caused a significant change (inhibition) in Na(+)-H+ exchange rate . Experiments performed with the Ussing chamber-voltage clamp technique verified that forskolin, 8-BrcAMP, E . coli STa toxin, ionomycin, and phorbol dibutyrate increased transepithelial Isc, verifying that all the regulatory pathways tested were functional and responsive to agonists . Results suggested that the Isc was due to Cl- secretion, since no net transcellular Na+ or Cl- flux was detected in basal conditions, and the Isc response to forskolin was abolished by omission of serosal Cl- . Because forskolin, but not 1,9-dideoxyforskolin, increased both cellular cAMP and Isc, the inhibition of Na(+)-H+ exchange by forskolin derivatives was mediated by a mechanism not involving activation of adenylyl cyclase . In conclusion, Caco-2 cells use a basolateral Na(+)-H+ exchanger to regulate pHi, but this exchanger is not affected by cell shrinkage or second messenger pathways that regulate Na(+)-H+ exchangers in other cell systems.

Proc Natl Acad Sci U S A, 1991 Aug 1, 88(15), 6873 - 7
Replacement of leucine-93 by alanine or threonine slows down the decay of the N and O intermediates in the photocycle of bacteriorhodopsin: implications for proton uptake and 13-cis-retinal----all-trans-retinal reisomerization; Subramaniam S et al.; We report that the replacement of Leu-93 in bacteriorhodopsin by Ala (L93A) or Thr (L93T) slows down the photocycle by approximately 100-fold relative to wild-type bacteriorhodopsin . Time-resolved visible absorption spectroscopy and resonance Raman experiments, respectively, show the presence of long-lived O-like and N-like intermediates in the photocycles of the above mutants . We infer the existence of an equilibrium between the N and O intermediates in the photocycles of these mutants . The L93A and L93T mutants exhibit normal proton pumping under continuous illumination, suggesting that the decay of the N and/or O intermediate, and consequently, proton translocation, can be accelerated by the absorption of a second photon . Since the 13-cis----all-trans reisomerization of retinal is completed during the decay of the N and O intermediates, we conclude that the interaction of Leu-93 with retinal is important in this phase of the photocycle . This conclusion is supported by a recent structural model of bacteriorhodopsin that suggests that Leu-93 is near the C-13 methyl group of retinal.

J Immunol, 1991 Aug 1, 147(3), 959 - 64
Immune-related intestinal chloride secretion . III . Acute and chronic effects of mast cell mediators on chloride secretion by a human colonic epithelial cell line; Barrett KE; Excessive fluid and electrolyte secretion, resulting symptomatically in diarrhea, has been associated with mast cell activation in a variety of experimental and clinical settings . The present study has used a human colonic epithelial cell line to examine mechanisms underlying this phenomenon . Acute addition of mixed mast cell mediators (as a lysate of rat basophilic leukemia cells) to epithelial cells led to prompt and sustained chloride secretion . The response was partially inhibitable by an antihistaminic drug and an adenosine antagonist, suggesting that histamine, adenosine, and possibly other mediators are responsible for producing the acute effect . Supernatants from immunologically activated rat basophilic leukemia cells had similar effects . Chronic exposure of epithelial cells to the lysate mediator preparation, followed by washing, had no effect on their basal electrical or electrolyte-transporting properties . However, the chloride secretory response of the cells to subsequent addition of vasoactive intestinal peptide, carbachol, and heat-stable enterotoxin of Escherichia coli was significantly enhanced, whereas responses to an adenosine agonist or PGE1 were unaffected . This study has, therefore, demonstrated two ways in which mast cell mediators can directly influence intestinal epithelial cells to secrete more chloride and, hence, to enhance fluid secretion in the gut . The findings may be of relevance to our understanding of inflammatory diarrhea and may aid the development of novel therapies for this disorder.

J Bacteriol, 1991 Aug, 173(16), 5207 - 19
Characterization and expression of the Escherichia coli Mrr restriction system; Waite-Rees PA et al.; The mrr gene of Escherichia coli K-12 is involved in the acceptance of foreign DNA which is modified . The introduction of plasmids carrying the HincII, HpaI, and TaqI R and M genes is severely restricted in E . coli strains that are Mrr+ . A 2-kb EcoRI fragment from the plasmid pBg3 (B . Sain and N . E . Murray, Mol . Gen . Genet . 180:35-46, 1980) was cloned . The resulting plasmid restores Mrr function to mrr strains of E . coli . The boundaries of the mrr gene were determined from an analysis of subclones, and plasmids with a functional mrr gene produce a polypeptide of 33.5 kDa . The nucleotide sequence of the entire fragment was determined; in addition to mrr, it includes two open reading frames, one of which encodes part of the hsdR . By using Southern blot analysis, E . coli RR1 and HB101 were found to lack the region containing mrr . The acceptance of various cloned methylases in E . coli containing the cloned mrr gene was tested . Plasmid constructs containing the AccI, CviRI, HincII, Hinfl (HhaII), HpaI, NlaIII, PstI, and TaqI N6-adenine methylases and SssI and HhaI C5-cytosine methylases were found to be restricted . Plasmid constructs containing 16 other adenine methylases and 12 cytosine methylases were not restricted . No simple consensus sequence causing restriction has been determined . The Mrr protein has been overproduced, an antibody has been prepared, and the expression of mrr under various conditions has been examined . The use of mrr strains of E . coli is suggested for the cloning of N6-adenine and C5-cytosine methyl-containing DNA.

J Bacteriol, 1991 Aug, 173(16), 5144 - 50
L-lyxose metabolism employs the L-rhamnose pathway in mutant cells of Escherichia coli adapted to grow on L-lyxose; Badia J et al.; Escherichia coli cannot grow on L-lyxose, a pentose analog of the 6-deoxyhexose L-rhamnose, which supports the growth of this and other enteric bacteria . L-Rhamnose is metabolized in E . coli by a system that consists of a rhamnose permease, rhamnose isomerase, rhamnulose kinase, and rhamnulose-1-phosphate aldolase, which yields the degradation products dihydroxyacetone phosphate and L-lactaldehyde . This aldehyde is oxidized to L-lactate by lactaldehyde dehydrogenase . All enzymes of the rhamnose system were found to be inducible not only by L-rhamnose but also by L-lyxose . L-Lyxose competed with L-rhamnose for the rhamnose transport system, and purified rhamnose isomerase catalyzed the conversion of L-lyxose into L-xylulose . However, rhamnulose kinase did not phosphorylate L-xylulose sufficiently to support the growth of wild-type E . coli on L-lyxose . Mutants able to grow on L-lyxose were analyzed and found to have a mutated rhamnulose kinase which phosphorylated L-xylulose as efficiently as the wild-type enzyme phosphorylated L-rhamnulose . Thus, the mutated kinase, mapped in the rha locus, enabled the growth of the mutant cells on L-lyxose . The glycolaldehyde generated in the cleavage of L-xylulose 1-phosphate by the rhamnulose-1-phosphate aldolase was oxidized by lactaldehyde dehydrogenase to glycolate, a compound normally utilized by E . coli.

J Bacteriol, 1991 Aug, 173(16), 5097 - 104
Analysis and possible role of hyperrecombination in the termination region of the Escherichia coli chromosome; Louarn JM et al.; The frequency of excisive homologous recombination has been measured at various positions along the Escherichia coli chromosome . The reporter system makes use of a lambda cI857 prophage integrated by homologous recombination within Tn5 or Tn10 transposons already installed at known positions in the E . coli chromosome . The excision frequency per cell and per generation was determined by monitoring the evolution of the relative number of temperature-resistant (cured) bacteria is a function of the age of the cultures . Excisions, due to RecA-dependent homologous exchanges, appeared to occur more frequently in the preferential termination zone for chromosome replication . The highest frequency of excision observed is compatible with a recombination event at each replication cycle in this region . On the basis of these data, we propose a model involving homologous recombination in the final steps of bacterial chromosome replication and separation.

J Bacteriol, 1991 Aug, 173(16), 5024 - 9
Escherichia coli catabolite gene activator protein mutants defective in positive control of lac operon transcription; Eschenlauer AC et al.; We isolated three Escherichia coli catabolite gene activator protein mutants that are defective in the positive control of transcription initiation from the lac operon promoter region yet retain negative control of transcription from other promoters . One mutant has a substitution of valine for glutamate at residue 72, which lies in the cyclic AMP binding domain and contacts cyclic AMP . The other two mutants have substitutions of asparagine and cysteine for glycine 162, which lies in a surface-exposed turn of the DNA-binding domain . Surprisingly, although all three mutants can repress the lacP2/P3 promoters through the catabolite gene activator protein target site of lac, none displays strong dominance over the ability of wild-type catabolite gene activator protein to stimulate the lacP1 promoter.

J Bacteriol, 1991 Aug, 173(16), 4983 - 93
Expression and operon structure of the sel genes of Escherichia coli and identification of a third selenium-containing formate dehydrogenase isoenzyme; Sawers G et al.; A detailed analysis of the expression of the sel genes, the products of which are necessary for the specific incorporation of selenium into macromolecules in Escherichia coli, showed that transcription was constitutive, being influenced neither by aerobiosis or anaerobiosis nor by the intracellular selenium concentration . The gene encoding the tRNA molecule which is specifically aminoacylated with selenocysteine (selC) proved to be monocistronic . In contrast, the other three sel genes (selA, -B, and -D) were shown to be constituents of two unlinked operons . The selA and selB genes formed one transcriptional unit (sel vector AB), while selD was shown to be the central gene in an operon including two other genes, the promoter distal of which (topB) encodes topoisomerase III . The promoter proximal gene (orf183) was sequenced and shown to encode a protein consisting of 183 amino acids (Mr, 20,059), the amino acid sequence of which revealed no similarity to any currently known protein . The products of the orf183 and topB genes were required neither for selenoprotein biosynthesis nor for selenation of tRNAs . selAB transcription was driven by a single, weak promoter; however, two major selD operon transcripts were identified . The longer initiated just upstream of the orf183 gene, whereas the 5' end of the other mapped in a 116-bp nontranslated region between orf183 and selD . Aerobic synthesis of all four sel gene products incited a reexamination of a weak 110-kDa selenopolypeptide which is produced under these conditions . The aerobic appearance of this 110-kDa selenopolypeptide was not a consequence of residual expression of the gene encoding the 110-kDa selenopolypeptide of the anaerobically inducible formate dehydrogenase N (FDHN) enzyme, as previously surmised, but rather resulted from the expression of a gene encoding a third, distinct selenopolypeptide in E . coli . A mutant strain no longer capable of synthesizing the 80- and 110-kDa selenopolypeptides of FDHH and FDHN, respectively, still synthesized this alternative 110-kDa selenopolypeptide which was present at equivalent levels in cells grown aerobically and anaerobically with nitrate . Furthermore, this strain exhibited a formate- and sel gene-dependent respiratory activity, indicating that it is probable that this selenopolypeptide constitutes a major component of the formate oxidase, an enzyme activity initially discovered in aerobically grown E . coli more than 30 years ago.

J Bacteriol, 1991 Aug, 173(15), 4707 - 16
Identification and characterization of a gene responsible for inhibiting propagation of methylated DNA sequences in mcrA mcrB1 Escherichia coli strains; Kretz PL et al.; Identifying and eliminating endogenous bacterial enzyme systems can significantly increase the efficiency of propagation of eukaryotic DNA in Escherichia coli . We have recently examined one such system which inhibits the propagation of lambda DNA rescued from transgenic mouse tissues . This rescue procedure utilizes lambda packaging extracts for excision of the lambda DNA from the transgenic mouse genome, as well as E . coli cells for subsequent infection and propagation . This assay, in combination with conjugal mating, P1 transduction, and gene cloning, was used to identify and characterize the E . coli locus responsible for this difference in efficiency . It was determined that the E . coli K-12 mcrB gene when expressed on a high-copy-number plasmid can cause a decrease in rescue efficiency despite the presence of the mcrB1 mutation, which inactivates the classic McrB restriction activity . (This mutation was verified by sequence analysis.) However, this McrB1 activity is not observed when the cloned mcrB1 gene is inserted into the E . coli genome at one copy per chromosome . A second locus was identified which causes a decrease in rescue efficiency both when expressed on a high-copy-number plasmid and when inserted into the genome . The data presented here suggest that this locus is mrr and that the mrr gene product can recognize and restrict cytosine-methylated sequences . Removal of this DNA region including the mrr gene from E . coli K-12 strains allows high rescue efficiencies equal to those of E . coli C strains . These modified E . coli K-12 plating strains and lambda packaging extract strains should also allow a significant improvement in the efficiency and representation of eukaryotic genomic and cDNA libraries.

J Bacteriol, 1991 Aug, 173(15), 4625 - 36
The murG gene of Escherichia coli codes for the UDP-N-acetylglucosamine: N-acetylmuramyl-(pentapeptide) pyrophosphoryl-undecaprenol N-acetylglucosamine transferase involved in the membrane steps of peptidoglycan synthesis; Mengin-Lecreulx D et al.; Physiological properties of the murG gene product of Escherichia coli were investigated . The inactivation of the murG gene rapidly inhibits peptidoglycan synthesis in exponentially growing cells . As a result, various alterations of cell shape are observed, and cell lysis finally occurs when the peptidoglycan content is 40% lower than that of normally growing cells . Analysis of the pools of peptidoglycan precursors reveals the concomitant accumulation of UDP-N-acetylglucosamine (UDP-GlcNAc) and UDP-N-acetylmuramyl-pentapeptide (UDP-MurNAc-pentapeptide) and, to a lesser extent, that of undecaprenyl-pyrophosphoryl-MurNAc-pentapeptide (lipid intermediate I), indicating that inhibition of peptidoglycan synthesis occurs after formation of the cytoplasmic precursors . The relative depletion of the second lipid intermediate, undecaprenyl-pyrophosphoryl-MurNAc-(pentapeptide)GlcNAc, shows that inactivation of the murG gene product does not prevent the formation of lipid intermediate I but inhibits the next reaction in which GlcNAc is transferred to lipid intermediate I . In vitro assays for phospho-MurNAc-pentapeptide translocase and N-acetylglucosaminyl transferase activities finally confirm the identification of the murG gene product as the transferase that catalyzes the conversion of lipid intermediate I to lipid intermediate II in the peptidoglycan synthesis pathway . Plasmids allowing for a high overproduction of the transferase and the determination of its N-terminal amino acid sequence were constructed . In cell fractionation experiments, the transferase is essentially associated with membranes when it is recovered.

J Bacteriol, 1991 Aug, 173(15), 4570 - 7
Downstream deletion analysis of the lac promoter; Xiong XF et al.; We have generated a series of deletions in the downstream region of the lac promoter . The promoter activities of these mutations were compared by measuring the levels of beta-galactosidase gene expression in vivo . Our results show that deletion of downstream lac promoter sequences changes the promoter strength only two- to threefold . The effects of these deletions on transcription initiation site location were studied through primer extension assay of in vivo mRNAs . We found that the transcription start sites are primarily chosen as an approximate distance from the -10 region of the lac promoter; however, starts are sometimes manifested at a GAATT(C) sequence, which is identical to the wild-type preferred start site . lac promoter P2 and a newly identified promoter, P3, are transcribed in vivo at low levels . Catabolite activator protein complexed with cyclic AMP represses P2 and P3 expression in vivo . The secondary catabolite activator protein binding site plays at most a modest role in catabolite repression in vivo.

Mol Cell Biol, 1991 Aug, 11(8), 3860 - 7
Phosphorylation of Xenopus cyclins B1 and B2 is not required for cell cycle transitions; Izumi T et al.; The cdc2 kinase and B-type cyclins are known to be components of maturation- or M-phase-promoting factor (MPF) . Phosphorylation of cyclin B has been reported previously and may regulate entry into and exit from mitosis and meiosis . To investigate the role of cyclin B phosphorylation, we replaced putative cdc2 kinase phosphorylation sites in Xenopus cyclins B1 and B2 by using oligonucleotide site-directed mutagenesis . We found that Ser-90 of cyclin B2 and Ser-94 or Ser-96 of cyclin B1 are the main phosphorylation sites both in functional Xenopus egg extracts and after phosphorylation with purified MPF in vitro . Microtubule-associated protein (MAP) kinase from Xenopus eggs phosphorylated cyclin B1 significantly at Ser-94 or Ser-96, whereas it was largely inactive against cyclin B2 . The substitutions that ablated phosphorylation at these sites, however, resulted in no functional differences between mutant and wild-type cyclin, as judged by the kinetics of M-phase degradation, induction of mitosis in egg extracts, or induction of oocyte maturation . These results indicate that the phosphorylation of Xenopus B-type cyclins by cdc2 kinase or MAP kinase is not required for the hallmark functions of cyclin.

Appl Microbiol Biotechnol, 1991 Aug, 35(5), 611 - 4
Positive identification of a lambda gt11 clone containing a region of fungal phytase gene by immunoprobe and sequence verification; Mullaney EJ et al.; As the initial step in a project to provide a more cost-effective source of the phytase enzyme, this paper reports on the use of a polyclonal antibody raised to phytase purified from an isolate of Aspergillus niger (A . ficuum) to screen an A . niger lambda gt11 expression library and the use of amino acid sequencing to identify a clone containing part of the fungal phytase gene . The described use of amino acid sequence fragments to verify the cloning of a gene has potential applications in other cloning projects.

Biotechnology (N Y), 1991 Aug, 9(8), 738 - 42
Enhancing the thermostability of glucose isomerase by protein engineering; Quax WJ et al.; We have engineered recombinant glucose isomerase (GI) from Actinoplanes missouriensis by site-directed mutagenesis to enhance its thermal stability in both the soluble and immobilized forms . Substitution of arginine for lysine at position 253, which lies at the dimer/dimer interface of the GI tetramer, produced the largest stabilization under model industrial conditions . We discuss our results in terms of a model in which chemical glycation of lysines by sugars in the industrial corn syrup substrate represents a major pathway of destabilization.

Biotechnology (N Y), 1991 Aug, 9(8), 731 - 7
Mutations in human interferon gamma affecting inclusion body formation identified by a general immunochemical screen; Wetzel R et al.; High level expression of the gene for human interferon-gamma (HuIFN-gamma) in E . coli JM101 cultured at 37 degrees C results in the distribution of over 90 percent of the total accumulated gene product into inclusion bodies (IBs) . We have identified mutations throughout the molecule that alter the distribution between the soluble and inclusion body fractions without greatly affecting total expression level . Some mutants retain high biological activity but are localized almost entirely in the soluble fraction . Mutations affecting IB distribution as well as stability to intracellular proteolysis were detected by immunochemical screens and verified by gel assays . Immunochemical screens such as those employed here may allow identification of folding and stability mutants in heterologously expressed proteins when there is no other basis for selection or screening . These results also suggest that one solution to production problems arising from IB formation may be to identify mutations in the target protein that favor expression of soluble protein while retaining biological activity.

J Biotechnol, 1991 Aug, 20(1), 95 - 104
On-line determination of intracellular beta-galactosidase activity in recombinant Escherichia coli using flow injection analysis (FIA); Kracke-Helm HA et al.; A flow injection analysis (FIA) system was developed for the determination of cytoplasmic beta-galactosidase activity in recombinant Escherichia coli . The FIA system and its application for on-line monitoring of beta-galactosidase production during cultivation of recombinant E . coli in a 60-l airlift tower loop reactor is described . The results demonstrate that an FIA assay in conjunction with a cell disintegration step can be applied successfully for on-line monitoring of intracellular protein formation.

J Biotechnol, 1991 Aug, 20(1), 17 - 27
High cell density cultivation of Escherichia coli at controlled specific growth rate; Riesenberg D et al.; A high cell density cultivation (HCDC) for growth of Escherichia coli in an especially designed glucose/mineral salt medium is proposed . The HCDC essentially starts as a batch process which is followed by a two-phase fed-batch cultivation . After unlimited growth at mu max = 0.45 h-1 in the batch part, growth was controlled at a reduced specific growth rate (mu = 0.11 h-1 less than mu max) over a period of 3 doubling times in which the biomass concentration increased from 12 to 95 g 1(-1) (phase 1 of fed-batch cultivation) . Control of growth (mu) was realized by a PO2 control loop (by variation of glucose feeding) and a mu control loop (by variation of agitation speed N) while the actual mu was calculated from the off-gas composition . If the agitation rate cannot be increased anymore the mu controller is switched off (end of phase 1) . In the following phase 2, mu declines, however, the still acting pO2 (glucose) controller guarantees sufficient O2 supply till the end of the cultivation with a biomass concentration of 110 g 1(-1) (dry mass) . The proposed HCDC suppresses generation of inhibitory by-products and the high yield coefficients indicate the economy of the process.

Gene, 1991 Jul 31, 104(1), 85 - 90
Sequence and conservation of genes at the distal end of the transfer region on plasmids F and R6-5; Cram DS et al.; The nucleotide sequence of the region downstream of transfer gene traI, including fertility inhibition gene finO, on the conjugative plasmids F and R6-5, has been determined . Analysis of the F sequence revealed two open reading frames (ORF's), ORF248 and ORF186; ORF186 (finO) is interrupted by the insertion of IS3 . The R6-5 sequence also contained ORF248 and an intact ORF186, although an additional ORF (ORF286) was located between the two genes . ORF248, which we have designated traX, and ORF186 (finO) are highly conserved on both plasmids . The organisation of these genes indicates that traI and traX on F, and traI, traX and ORF286 on R6-5 are co-transcribed from their respective promoters upstream of traI . Sequences homologous to traX were detected on a range of conjugative F-like plasmids, whereas sequences homologous to ORF286 were only found on plasmids R6-5, R100 and R1 . The conservation of traX sequences suggests a functional importance for that gene and/or its product.

Gene, 1991 Jul 31, 104(1), 75 - 80
DNA synthesis on discontinuous templates by DNA polymerase I of Escherichia coli; Clark JM; DNA polymerases normally catalyze DNA synthesis in a template-directed manner . Generally, the continuity of the phosphodiester backbone of the template strand was thought to be an absolute requirement for DNA synthesis . Here, I demonstrate that a 3'-exonuclease-deficient derivative of the Klenow (large) fragment of Escherichia coli DNA polymerase I (PolIk) can carry out DNA synthesis on discontinuous templates in vitro . Addition of multiple nucleotides (nt) to the 3' end of a blunt-end duplex, templated by unlinked single-stranded oligodeoxyribonucleotides (oligos), was monitored electrophoretically . The reaction was demonstrable with either homopolymers or mixed-sequence oligos, but showed a requirement for complementarity between the first nt added to the duplex and the 3' nt of the unlinked oligo . These results demonstrate that continuity of the phosphodiester backbone of the template strand is not absolutely required for in vitro DNA synthesis by a 3'-exonuclease-deficient form of PolIk.

Gene, 1991 Jul 31, 104(1), 19 - 24
High-level synthesis of active adenylate cyclase toxin of Bordetella pertussis in a reconstructed Escherichia coli system; Sebo P et al.; The Bordetella pertussis adenylate cyclase(Cya) toxin-encoding locus (cya) is composed of five genes . The cyaA gene encodes a virulence factor (CyaA), exhibiting adenylate cyclase, hemolytic and invasive activities . The cyaB, D and E gene products are necessary for CyaA transport, and the cyaC gene product is required to activate CyaA . We reconstructed, in Escherichia coli, the cya locus of B . pertussis by cloning the different genes on appropriate vectors under the control of strong promoters and E . coli-specific translation initiation signals . We show that in the absence of additional gene products, CyaA is synthesized at high levels, is endowed with adenylate cyclase activity, but is devoid of invasive and hemolytic activities . CyaC is sufficient to confer upon the adenylate cyclase holotoxin full invasive and partial hemolytic activities . Coexpression of the cyaB, D and E genes neither stimulates nor potentiates the activation brought about by CyaC . This reconstructed system should help to elucidate both the mechanism and the structural requirements of holotoxin activation.

Gene, 1991 Jul 31, 104(1), 119 - 22
A twin-reporter vector for simultaneous analysis of expression signals of divergently transcribed, contiguous genes in filamentous fungi; Punt PJ et al.; To analyze the promoter region(s) of divergently transcribed fungal genes, a twin-reporter vector was constructed . This vector contains two divergently oriented reported genes, encoding Escherichia coli beta-glucuronidase (uidA) and E . coli beta-galactosidase (lacZ) . Terminator regions of the Aspergillus nidulans nitrate and nitrite reductase-encoding genes, niaD and niiA, respectively, have been cloned 3' to the reporter genes to ensure proficient transcription termination of the reporter genes . The reporter genes have been separated by a unique NotI restriction site, which can be used for the insertion of expression signals . A mutant argB selection marker has been introduced in order to obtain A . nidulans transformants with a single copy of the vector integrated at the argB locus . The use of the vector was demonstrated by insertion of the A . nidulans niaD-niiA intergenic region and analysis of A . nidulans transformants obtained with this construct . Control of expression of both reporter genes was found to be in accordance with previously published data on control of nitrate assimilation {Cove, Biol . Rev . 54 (1979) 291-327}.

Gene, 1991 Jul 31, 104(1), 107 - 11
Improved shuttle vectors for cloning and high-level Cu(2+)-mediated expression of foreign genes in yeast; Macreadie IG et al.; New yeast episomal vectors having a high degree of utility for cloning and expression in Saccharomyces cerevisiae are described . One vector, pYEULlacZ, is based on pUC19 and employs the pUC19 multiple cloning site for the selection of recombinants in Escherichia coli by lacZ inactivation . In addition, the vector contains two genes, URA3 and leu2-d, for selection of the plasmid in ura3 or leu2 yeast strains . The presence of the leu2-d gene appears to promote replication at high copy numbers . The introduction of CUP1 cassettes allows these plasmids to direct Cu(2+)-regulated production of foreign proteins in yeast . We show the production of a helminth antigen as an example of the vector application.

Gene, 1991 Jul 31, 104(1), 47 - 54
Expression of lacZ gene fusions affects downstream transcription in yeast; Barnes CA et al.; Chimeric genes containing Escherichia coli lacZ sequences are often used to characterize gene expression in yeast cells . By Northern analysis, we found that such genes produce multiple transcripts due to inefficient 3'-end formation . The same transcript pattern was found for two related chimeric genes when these genes were cloned separately into the commonly used vector, YIp5, and integrated into the yeast genome at two different locations . Each chimeric gene was composed of promoter and N-terminal coding regions from the yeast SSA1 or SSA2 genes fused in-frame to the lac operon . Transcripts were shown to initiate within the yeast promoter fragment, but transcript size indicated that 3' ends were localized to three different regions: within the lac operon near the 3' end of the lacZ gene; near a terminator region previously identified upstream of the URA3 gene in YIp5; and at the URA3 terminator region . Readthrough transcription of the URA3 promoter from upstream lac sequences decreased the basal activity of the URA3 promoter, although induced URA3 transcription levels were unaffected . This readthrough transcription also resulted in a novel, longer URA3 transcript.

Biochem Biophys Res Commun, 1991 Jul 31, 178(2), 664 - 71
Expression of mouse tyrosine hydroxylase in Escherichia coli; Ichikawa S et al.; Enzymatically active mouse tyrosine hydroxylase (TH) was successfully expressed at a high level in Escherichia coli using a T7 RNA polymerase directed expression system . The specific activity of mouse TH in E . coli cell lysate was 7.5 nmol/mg protein/min . Kinetic characteristics of recombinant TH were examined . Km for tyrosine and (6R)-tetrahydrobiopterin (6R-BH4) cofactor were determined to be 7.2 microM (420 microM 6R-BH4), 19 microM {( 6R-BH4} less than 55 microM, 20 microM tyrosine) and 54 microM {( 6R-BH4} greater than 55 microM, 20 microM tyrosine), respectively . These were in good agreement with previously reported values for this enzyme.

Gene, 1991 Jul 31, 104(1), 113 - 8
Vectors for the expression and analysis of DNA-binding proteins in yeast; Bonner JJ; A series of 13 vectors is described . All are yeast centromere plasmids with the LEU2 gene for selection in yeast, and pUC19 sequences for growth in Escherichia coli . All contain the GAL1 promoter directing transcription into a multiple cloning site (MCS) . For twelve of the plasmids, synthetic oligodeoxyribonucleotides create an ATG start codon, in a productive context for yeast, prior to the MCS . Spacing between the ATG and the MCS is variable, to facilitate the cloning of gene fragments in the appropriate reading frame . Nine of the plasmids also contain the strong transcriptional activator from the herpes simplex virus VP16 gene, joined downstream from the MCS . In these nine vectors, all possible combinations of reading frames are available . The suitability of these plasmids for the expression and analysis of DNA-binding domains is tested by cloning into them fragments of the yeast HSF1 gene, encoding the heat shock transcription factor (HSF) . The regulation of reporter gene expression by the chimeric HSF-VP16 fusions is described, as is the utility of these vectors for other applications.

Biochemistry, 1991 Jul 30, 30(30), 7609 - 14
Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa; Langsetmo K et al.; The highly conserved, buried, Asp 26 in Escherichia coli thioredoxin has a pKa = 7.5, and its titration is associated with a sizable destabilization of the protein {Langsetmo, K., Fuchs, J., & Woodward, C . (1991) Biochemistry (preceding paper in this issue)} . A fit of the experimental pH dependence of thioredoxin stability to a theoretical expression for the pH/stability relation in proteins agrees closely with a pKa value of 7.5 for Asp 26 . The agreement between the experimental and theoretical changes in protein stability due to substitution of Asp 26 by alanine is also good . The local structure in the vicinity of Asp 26 in the low-pH crystal structure (with uncharged Asp 26) is hydrophobic, indicating that the aspartate would be highly destabilized . In theoretical calculations, the desolvation penalty for deprotonating Asp 26 in this environment is similar to the total protein folding energy . As a consequence, the Asp 26 pKa would be much greater than 7.5, and/or the protein might not fold . This suggests that a compensating process partially stabilizes the Asp 26 carboxyl group when it is charged . A simple model for this proposed, whereby the Lys 57 side chain rotates to form a salt bridge with Asp 26 when it is deprotonated.

Biochemistry, 1991 Jul 30, 30(30), 7603 - 9
The conserved, buried aspartic acid in oxidized Escherichia coli thioredoxin has a pKa of 7.5 . Its titration produces a related shift in global stability; Langsetmo K et al.; Aspartic acid 26 in Escherichia coli thioredoxin is located at the bottom of a hydrophobic cavity, near the redox-active disulfide of the active site . Asp 26 is embedded in the protein except for part of the surface of one carboxyl oxygen . The high degree of evolutionary conversion of Asp 26 suggests that it plays a critical role in thioredoxin function . We have determined the pKa of Asp 26 by a novel electrophoretic method based on the relative electrophoretic mobilities of wild-type thioredoxin and of D26A thioredoxin (with Asp 26 replaced by alanine) . The pKa of Asp 26 determined by this technique is 7.5, more than 3 units above the pKa of a solvated carboxyl side chain . The titration of Asp 26 is thermodynamically linked to the stability of thioredoxin . As expected if thioredoxin stability depends on the ionization state of Asp 26, delta Go WT, the free energy of the cooperative denaturation reaction of wild-type thioredoxin by guanidine hydrochloride, varies with pH in a sigmoidal fashion in the vicinity of pH 7.5 . Over the same pH range, the free energy for D26A folding, delta Go D26A, is pH independent and D26A is highly stabilized compared to wild type . From the thermodynamic cycle describing the linkage of Asp 26 titration to thioredoxin stability, the difference in free energy between wild-type thioredoxin with protonated Asp 26 and wild-type thioredoxin with deprotonated Asp 26, delta delta Go (COOH----COO-), is calculated to be 4.9 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Jul 30, 30(30), 7527 - 34
HIV-1 Rev expressed in recombinant Escherichia coli: purification, polymerization, and conformational properties; Wingfield PT et al.; The high-level expression of HIV-1 Rev in Escherichia coli is described . Protein in crude bacterial extracts was dissociated from bound nucleic acid with urea . A simple purification and renaturation protocol, monitored by circular dichroism, is described which results in high yields of pure protein . The purified protein binds with high affinity to the Rev-responsive element mRNA and has nativelike spectroscopic properties . The protein exhibits concentration-dependent self-association as judged by analytical ultracentrifugation and gel filtration measurements . Purified Rev showed reversible heat-induced aggregation over the temperature range 0-30 degrees C . This hydrophobic-driven and nonspecific protein association was inhibited by low concentrations of sulfate ions . Rev solutions at greater than 80 micrograms/mL, incubated at 0-4 degrees C, slowly polymerized to form long hollow fibers of 20-nm diameter . Filament formation occurs at a lower protein concentration and more rapidly in the presence of Rev-responsive mRNA . The nucleic acid containing filaments are about 8 nm in diameter and up to 0.4 micron in length . On the basis of physical properties of the purified protein, we have suggested that in the nucleus of infected cells, Rev binding to the Rev-responsive region of env mRNA may be followed by helical polymerization of the protein which results in coating of the nucleic acid . Coated nucleic acid could be protected from splicing in the nucleus and exported to the cytoplasm.

Biochemistry, 1991 Jul 30, 30(30), 7451 - 6
Substrate-decreased modification by diethyl pyrocarbonate of two histidines in isocitrate lyase from Escherichia coli; Ko YH et al.; The inactivation of tetrameric 188-kDa isocitrate lyase from Escherichia coli at pH 6.8 (37 degrees C) by diethyl pyrocarbonate, exhibiting saturation kinetics, is accompanied by modification of histidine residues 266 and 306 . Substrates isocitrate, glyoxylate, or glyoxylate plus succinate protect the enzyme from inactivation, but succinate alone does not . Removal of the carbethoxy groups from inactivated enzyme by treatment with hydroxylamine restores activity of isocitrate lyase . The present results suggest that the group-specific modifying reagent diethyl pyrocarbonate may be generally useful in determining the position of active site histidine residues in enzymes.

Biochemistry, 1991 Jul 30, 30(30), 7461 - 6
Kinetics and equilibria for the reactions of coenzymes with wild type and the Y70F mutant of Escherichia coli aspartate aminotransferase; Toney MD et al.; The Y70F mutant of aspartate aminotransferase has reduced affinity for coenzymes compared to the wild type . The equilibrium dissociation constants for pyridoxamine phosphate (PMP) holoenzymes, KPMPdiss, were determined from the association and dissociation rate constants to be 1.3 nM and 30 nM for the wild type and mutant, respectively . This increase in KPMPdiss for Y70F is due to a 27-fold increase in the dissociation rate constant . Pyridoxal phosphate (PLP) association kinetics are complex, with three kinetic processes detectable for wild type and two for Y70F . A directly determined, accurate value of KPLPdiss for wild type enzyme has been difficult to obtain because of the low value of this constant . The values of KPLPdiss for the holoenzymes were determined indirectly through the measured values for KPMPdiss, glutamate-alpha-ketoglutarate half-reaction equilibrium constants, and the equilibrium constant for the transamination of PLP by glutamate catalyzed by Y70F . The values of KPLPdiss obtained by this procedure are 0.4 pM for wild type and 40 pM for Y70F . The increases in KPMPdiss and KPLPdiss for Y70F correspond to delta delta G values of 1.9 and 2.7 kcal/mol, respectively, and are directly attributed to the loss of the hydrogen bond from the phenolic hydroxyl group of Tyr70 to the coenzyme phosphate . The delta G for association of PLP with wild type enzyme is 4.7 kcal/mol more favorable than that for PMP.

Biochemistry, 1991 Jul 30, 30(30), 7456 - 61
Tyrosine 70 fine-tunes the catalytic efficiency of aspartate aminotransferase; Toney MD et al.; The aspartate aminotransferase mutant Y70F exhibits kcat = 8% and kcat/KM = 2% of the wild type values for the transamination of aspartate and alpha-ketoglutarate . The affinity of the enzyme for the noncovalently bound inhibitor maleate is reduced 17-fold by the mutation, while only a 2.5-fold reduction is observed for alpha-methylaspartate, which forms a stable, covalent external aldimine . The high population of the quinonoid intermediate formed in the reaction of the wild type with beta-hydroxyaspartate is more than 75% diminished by the mutation . The values of the Y70F C alpha-H kinetic isotope effects for the aspartate reaction are larger than those of wild type (DV = 2.4 vs 1.52; D(V/K) = 2.5 vs 1.7) . Conversely, the Y70F value of D(V/K) for the glutamate reaction is decreased compared to wild type (1.75 vs 2.5) . These results, combined with previous studies of Lys258 mutants, eliminate Tyr70 as an essential component of the catalytic apparatus, with the caveat that the functionally of the deleted hydroxyl group is possibly replaced by a water molecule.

Biochemistry, 1991 Jul 30, 30(30), 7359 - 62
The chaperonin GroEL binds a polypeptide in an alpha-helical conformation; Landry SJ et al.; Chaperones facilitate folding and assembly of nascent polypeptides in vivo and prevent aggregation in refolding assays in vitro . A given chaperone acts on a number of different proteins . Thus, chaperones must recognize features present in incompletely folded polypeptide chains and not strictly dependent on primary structural information . We have used transferred nuclear Overhauser effects to demonstrate that the Escherichia coli chaperonin GroEL binds to a peptide corresponding to the N-terminal alpha-helix in rhodanese, a mitochondrial protein whose in vitro refolding is facilitated by addition of GroEL, GroES, and ATP . Furthermore, the peptide, which is unstructured when free in aqueous solution, adopts an alpha-helical conformation upon binding to GroEL . Modification of the peptide to reduce its intrinsic propensity to take up alpha-helical structure lowered its affinity for GroEL, but, nonetheless, it could be bound and took up a helical conformation when bound . We propose that GroEL interacts with sequences in an incompletely folded chain that have the potential to adopt an amphipathic alpha-helix and that the chaperonin binding site promotes formation of a helix.

Int J Cancer, 1991 Jul 30, 48(6), 879 - 88
Immunological characterization of the Epstein-Barr virus phosphoprotein PP58 and deoxyribonuclease expressed in the baculovirus expression system; Chen HF et al.; The open reading frames of the phosphoprotein pp58 (BMRFI) and the deoxyribonuclease (BGLF5) of the Epstein-Barr-virus (EBV) strain M-ABA were cloned in the baculovirus expression vectors pAc373 and pAc360 and expressed in the Spodoptera frugiperda (SF158) insect cells . The recombinant phosphoprotein pp58 expressed in SF158 cells was recognized by the anti-pp58 rabbit anti-sera which were generated by immunizing rabbits with a TrpE-BMRFI fusion protein expressed in E . coli . DNA-cellulose chromatography showed that the recombinant pp58 exhibited DNA-binding activities . Immunofluorescence, immunoblot and ELISA analysis indicated that sera from patients with nasopharyngeal carcinoma (NPC) contained antibodies against pp58 . The recombinant EBV DNase expressed in SF158 cells was recognized by the anti-EBV DNase rabbit anti-sera which were generated by immunizing rabbits with a TrpE-C-terminal part of BGLF5 fusion protein expressed in E . coli . The anti-EBV DNase rabbit anti-sera recognized also a protein of about 52 kDa in the EBV-harboring human B-cell lines Raji, Jijoye, B95-8, M-ABA and BL74 induced by TPA and n-butyrate . The recombinant EBV DNase exhibited exonuclease and endonuclease activities, a requirement for magnesium, and a high pH optimum (8.0) . Its enzyme activities could be inhibited by sera from NPC patients and anti-EBV DNase rabbit anti-sera . Comparable studies of Raji EBV-DNase and recombinant EBV-DNase implied that recombinant EBV-DNase could also be used in the enzyme activity assay for the detection of NPC . In contrast to the enzyme inhibition test, immunofluorescence and immunoblot analysis demonstrated that the recombinant EBV DNase exhibited only a weak immunological reaction with NPC sera.

Science, 1991 Jul 19, 253(5017), 292 - 8
Mechanism of assembly of the tyrosyl radical-dinuclear iron cluster cofactor of ribonucleotide reductase; Bollinger JM Jr et al.; Incubation of the apoB2 subunit of Escherichia coli ribonucleotide reductase with Fe2+ and O2 produces native B2, which contains the tyrosyl radical-dinuclear iron cluster cofactor required for nucleotide reduction . The chemical mechanism of this reconstitution reaction was investigated by stopped-flow absorption spectroscopy and by rapid freeze-quench EPR (electron paramagnetic resonance) spectroscopy . Two novel intermediates have been detected in the reaction . The first exhibits a broad absorption band centered at 565 nanometers . Based on known model chemistry, this intermediate is proposed to be a mu-peroxodiferric complex . The second intermediate exhibits a broad absorption band centered at 360 nanometers and a sharp, isotropic EPR signal with g = 2.00 . When the reaction is carried out with 57Fe2+, this EPR signal is broadened, demonstrating that the intermediate is an iron-coupled radical . Variation of the ratio of Fe2+ to B2 in the reaction and comparison of the rates of formation and decay of the intermediates to the rate of formation of the tyrosyl radical (.Y122) suggest that both intermediates can generate .Y122 . This conclusion is supported by the fact that both intermediates exhibit an increased lifetime in a mutant B2 subunit (B2-Y122F) lacking the oxidizable Y122 . Based on these kinetic and spectroscopic data, a mechanism for the reaction is proposed . Unlike reactions catalyzed by heme-iron peroxidases, oxygenases, and model complexes, the reconstitution reaction appears not to involve high-valent iron intermediates.

FEBS Lett, 1991 Jul 29, 286(1-2), 121 - 4
Presence of an acyl carrier protein in NADH:ubiquinone oxidoreductase from bovine heart mitochondria; Runswick MJ et al.; The amino-acid sequence of a subunit of NADH:ubiquinone oxidoreductase from bovine heart mitochondria has been determined and is closely related to those of acyl carrier proteins that are involved in fatty acid biosynthesis in Escherichia coli and plants . Evidence for the presence of covalently attached pantetheine-4'-phosphate in the bovine protein has been obtained by determination of the molecular mass of the isolated subunit by electrospray mass spectrometry, before and after incubation of the protein at alkaline pH under reducing conditions . This decreased the molecular mass from 10,751.6 to 10,449.4, a difference of 302.2 mass units; the value calculated from the protein sequence with one covalently attached pantetheine-4'-phosphate is 10,449.8 . The acyl group which is removed by alkaline reduction, appears to be attached via a thioester linkage . By analogy with the bacterial protein it is likely that the attachment site of the pantetheine-4-phosphate is serine-44, which is found in a highly conserved region of the sequence . At present the function of the acyl carrier protein in mitochondrial complex I is not understood.

FEBS Lett, 1991 Jul 29, 286(1-2), 71 - 5
Evidence for a ribosome-associated thiol protease cleaving wheat germ methionyl-tRNA synthetase; Archambault de Vencay J et al.; A wheat germ protease is responsible for Mr 105,000 methionyl-tRNA synthetase hydrolysis, generating two fragments of Mr 82,000 (harbouring the catalytic domain) and 20,000, respectively . Specificity of the protease was sought for using different kinds of protein substrates . It turned out that charged peptides were preferentially cleaved and that no proteolysis occurred when proteins were replaced by small synthetic substrates, harbouring target sites similar to those cleaved in proteins . The protease could be a ribosomal protein, since it remained associated to ribosomal structure, even after treatment by deoxycholate, Triton X-100, 800 mM KC1 and puromycin . Nevertheless, it was still active after ribonuclease treatment of the ribosomes . An identical protease activity was found in rat liver, but not in E . coli.

FEBS Lett, 1991 Jul 29, 286(1-2), 58 - 60
Site-directed mutagenesis reveals the importance of disulfide bridges and aromatic residues for structure and proliferative activity of human interleukin-4; Kruse N et al.; Mutant proteins (muteins) of human Interleukin-4 (IL4) were constructed by means of in vitro mutagenesis . The muteins were expressed in E . coli, submitted to a renaturation and purification protocol and analysed for biological activity . Exchange of the cysteines at either position 46 or 99 which form one of the three disulfide bridges resulted in a nearly complete loss of biological activity and an unstable protein . The exchange of tyrosine 124 also inactivated the protein, while a mutation of tyrosine 56 left some residual activity . Exchange of the other four cysteines or of the single tryptophane had smaller effects.

FEBS Lett, 1991 Jul 29, 286(1-2), 125 - 8
The phosphate recognition site of Escherichia coli maltodextrin phosphorylase; Schinzel R et al.; The role of two positively charged amino acid residues located at the active site of Escherichia coli maltodextrin phosphorylase was investigated by site-directed mutagenesis . Substitution of Lys539 by an arginine caused a 600-fold reduction, substitution of Arg534 by a glutamine caused an even larger 7000-fold reduction of the catalytic rate while substrate binding remained essentially unaffected . Since the Arg534----Gln exchange reduces the catalytic rate near to inactivity and even the conservative Lys534----Arg exchange caused a marked decrease of activity, the central functional role of both positively charged residues in phosphorylase catalysis anticipated by the crystallographic analysis of the corresponding amino acid residues Arg569 and Lys574 in the catalytic site of phosphorylase b was confirmed.

FEBS Lett, 1991 Jul 29, 286(1-2), 201 - 3
Chemical synthesis of 10 kDa chaperonin . Biological activity suggests chaperonins do not require other molecular chaperones; Mascagni P et al.; Molecular chaperones are required for the correct folding and assembly of certain other polypeptides . It is not known whether molecular chaperones themselves require other chaperones to become functional . A 97-amino acid chaperone, the chaperonin 10 protein was chemically synthesised so that during synthesis and purification there was no contact of the chaperone with any other protein . The purified, synthetic chaperonin 10 protein formed oligomeric structures spontaneously and was biologically active as a chaperonin . This is the first description of a chemically synthesised chaperonin, and suggests that no other chaperones are required for correct folding, polymerisation and biological activity of this chaperone.

FEBS Lett, 1991 Jul 29, 286(1-2), 44 - 6
Differential expression of GABAA receptor alpha-subunits in rat brain during development; McKernan RM et al.; Unique cytoplasmic loop regions of the alpha 1, alpha 2, alpha 3, and alpha 5 subunits of the GABAA receptor have been expressed in E . coli and used to generate polyclonal antisera specific for these subunits . The antibodies identify proteins by SDS-polyacrylamide gel electrophoresis and western blotting of molecular size 51 kDa, 53 kDa, 59 kDa and 55 kDa, respectively, which show differential patterns of expression during development . Whereas the alpha 2 and alpha 3 subunits are present at early stages, the expression of alpha 1 and alpha 3 subunits is low at birth and increases with age . This differential expression could be correlated with previous studies examining the developmental expression of BZ1 and BZ2 benzodiazepine binding sites.

Cell, 1991 Jul 26, 66(2), 373 - 82
Primary structure of the human splicing factor ASF reveals similarities with Drosophila regulators; Ge H et al.; We described previously the purification of a human protein, called alternative splicing factor (ASF), that can switch utilization of alternative 5' splice sites in an SV40 early pre-mRNA . We now report the isolation of a cDNA, designated ASF-1, that encodes this protein . ASF-1 consists of 248 amino acid residues, including an 80 residue RNA-binding domain at its N-terminus and a 50 residue C-terminal region that is 80% serine plus arginine . ASF-1 produced in E . coli can activate splicing in vitro and switch 5' splice-site utilization, establishing that the recombinant protein is sufficient to supply these activities . Analysis of additional cDNAs revealed that ASF pre-mRNA can itself be alternatively spliced, surprisingly, by utilization of a shared 5' splice site and two closely spaced 3' splice sites . Use of the upstream site results in a second mRNA (ASF-2) in which translation of the downstream exon occurs extensively in an alternative reading frame distinct from ASF-1.

Cell, 1991 Jul 26, 66(2), 361 - 71
Homologous pairing in vitro stimulated by the recombination hotspot, Chi; Dixon DA et al.; Genetic recombination in Escherichia coli is stimulated at DNA sequences known as Chi sites, 5'-GCT-GGTGG-3' . We describe the in vitro formation of homologously paired joint molecules that is dependent upon this recombination hotspot . Chi-dependent joint molecule formation requires RecA, RecBCD, and SSB proteins and a Chi site in the donor linear dsDNA . The donor dsDNA is unwound by RecBCD enzyme, and the invasive strand is generated by nicking at Chi . This Chi-dependent invading strand must contain homology to the recipient supercoiled DNA substrate at its newly formed 3' end for efficient joint molecule formation . Action at Chi generates invasive ssDNA from the 5' but not the 3' side of Chi, suggesting that the nuclease activity of RecBCD enzyme is attenuated upon encountering a Chi site . These results support the view that RecBCD enzyme action can precede RecA protein action and reconcile the seemingly opposing degradative and recombination functions of RecBCD enzyme.

J Chromatogr, 1991 Jul 26, 550(1-2), 823 - 30
High-performance capillary electrophoresis of proteins . Sodium dodecyl sulphate-polyacrylamide gel-filled capillary column for the determination of recombinant biotechnology-derived proteins; Tsuji K; Fused-silica capillary columns were filled with sodium dodecyl sulfate-polyacrylamide gel and the column effluent was monitored at 214 nm using a commercially available high-performance capillary electrophoresis (HPCE) instrument to separate and rapidly quantify recombinant biotechnology-derived proteins . An excellent linear relationship (r greater than 0.999) exists between the peak migration time and the molecular weights of reference proteins in the range 10,000-100,000 and 40,000-200,000 dalton by use of the capillary columns filled with acrylamide gel at a T composition of 5% and 3%, respectively . The relative standard deviation (R.S.D.) of the peak migration time is ca . 1% . Theoretical plates of 5 X 10(5)-1 X 10(6) per metre are routinely being obtained . Calibration graphs of peak area versus weight of recombinant biotechnology-derived proteins are linear (r greater than 0.999) and the proteins may be quantified with an R.S.D . of ca . 3-7% . As little as 50 nmol of a protein may be quantified and an impurity peak of molecular weight ca . 1500 less than that of the parent compound (ca . 60,000 dalton) may be differentiated by HPCE with a gel-filled capillary column.

J Biol Chem, 1991 Jul 25, 266(21), 13731 - 6
Kinetics for formate dehydrogenase of Escherichia coli formate-hydrogenlyase; Axley MJ et al.; Kinetic parameters of the selenium-containing, formate dehydrogenase component of the Escherichia coli formate-hydrogenlyase complex have been determined with purified enzyme . A ping-pong Bi Bi kinetic mechanism was observed . The Km for formate is 26 mM, and the Km for the electron-accepting dye, benzyl viologen, is in the range 1-5 mM . The maximal turnover rate for the formate-dependent catalysis of benzyl viologen reduction was calculated to be 1.7 x 10(5) min-1 . Isotope exchange analysis showed that the enzyme catalyzes carbon exchange between carbon dioxide and formate in the absence of other electron acceptors, confirming the ping-pong reaction mechanism . Dissociation constants for formate (12.2 mM) and CO2 (8.3 mM) were derived from analysis of the isotope exchange data . The enzyme catalyzes oxidation of the alternative substrate deuterioformate with little change in the Vmax, but the Km for deuterioformate is approximately three times that of protioformate . This implies formate oxidation is not rate-limiting in the overall coupled reaction of formate oxidation and benzyl viologen reduction . The deuterium isotope effect on Vmax/Km was observed to be approximately 4.2-4.5 . Sodium nitrate was found to inhibit enzyme activity in a competitive manner with respect to formate, with a Ki of 7.1 mM . Sodium azide is a noncompetitive inhibitor with a Ki of about 80 microM.

Nucleic Acids Res, 1991 Jul 25, 19(14), 3821 - 7
DNA binding properties of the integrase proteins of human immunodeficiency viruses types 1 and 2; van Gent DC et al.; Integration of retroviral DNA into the host chromosome requires the integrase protein (IN) . We overexpressed the IN proteins of human immunodeficiency viruses types 1 and 2 (HIV-1 and HIV-2) in E . coli and purified them . Both proteins were found to specifically cut two nucleotides off the ends of linear viral DNA, and to integrate viral DNA into target DNA . This demonstrates that HIV IN is the only protein required for integration of HIV DNA . Although the two types of IN proteins have only 53% amino acid sequence similarity, they act with equal efficiency on both type 1 and type 2 viral DNA . Binding of IN to DNA was tested: purified IN does not bind very specifically to viral DNA ends . Nevertheless, only viral DNA ends are cleaved and integrated . We interpret this as follows: in vitro quick aspecific binding to DNA is followed by slow specific cutting and integration . IN can not find viral DNA ends in the presence of an excess of aspecific DNA; in vivo this is not required since the IN protein is in constant proximity of viral DNA in the viral core particle.

Nucleic Acids Res, 1991 Jul 25, 19(14), 3811 - 9
The htrM gene, whose product is essential for Escherichia coli viability only at elevated temperatures, is identical to the rfaD gene; Raina S et al.; We have identified a new E . coli gene, htrM . The htrM gene was identified because its insertional inactivation by the Tn5 transposon results in E . coli's inability to form colonies at temperatures above 43 degrees C . The corresponding htrM+ gene was cloned on the basis of its ability to correct the temperature-sensitive phenotype of the htrM::Tn5 insertion mutations . The htrM gene has been mapped to 81.2 min on the conventional E . coli genetic map . It was sequenced and shown to code for an acidic, 34,893-Da polypeptide . Three transcriptional starts were located 48, 90 and 123 nucleotides upstream of the ATG, initiation codon referred to as the P1, P2 and P3(hs) promoters, respectively . The -10 and -35 regions of the P1 promoter bear a close similarity to the E sigma 70-recognized consensus sequences, while the -12 region of the P2 promoter resembles the consensus promoter sequence transcribed by the rpoN gene product . Transcripts of the htrM gene accumulate with increasing temperature . The -10 and -35 regions of the P3(hs) promoter, represented by nucleotides 160 to 130 upstream of the ATG initation codon, are similar to the E sigma 32-recognized consensus sequences . The sigma 32 transcription factor is essential for maximal htrM gene transcription, since htrM RNA transcripts are made at reduced rates in a rpoH null mutant background . Surprisingly, the htrM gene turns out to be identical to rfaD, whose product is required for the biosynthesis of the ADP-L-glycero-D manoheptose lipopolyaccharide precursor {Pegues et al . (1990) J . Bacteriol . 172, 4652-4660}.

J Biol Chem, 1991 Jul 25, 266(21), 14113 - 8
Maltose transacetylase of Escherichia coli . Mapping and cloning of its structural, gene, mac, and characterization of the enzyme as a dimer of identical polypeptides with a molecular weight of 20,000; Brand B et al.; malQ mutants, lacking amylomaltase, cannot grown on maltose . However, when maltose is present in the medium, it can be accumulated to high internal levels . In a subsequent slow reaction, accumulated maltose becomes acetylated and leaks back into the medium . The enzyme responsible for this acetylation uses acetyl-CoA as acetyl donor and can be measured in crude extracts (Boos, W., Ferenci, T., and Shuman, H . A . (1981) J . Bacteriol . 146, 725-732) . The structural gene for the enzyme, which we named mac, was mapped at 10.4 min on the Escherichia coli linkage map . We cloned a 3.4-kilobase pair PstI-EcoRI DNA fragment containing the mac gene . Cell-free extracts of a strain harboring the multicopy plasmid were used to purify the maltose-transacetylating activity to apparent homogeneity . On sodium dodecyl sulfate-polyacrylamide gels the enzyme exhibited a molecular weight of 20,000 . Using molecular sieve chromatography, a molecular weight of 40,000 was determined for the native enzyme . Therefore, the enzyme is a dimer of two identical subunits . At a sugar concentration of 100 mM the enzyme acetylates glucose, maltose, mannose, galactose, and fructose in decreasing relative rate of 1, 0.55, 0.20, 0.07, 0.04 . Maltotriose and other oligosaccharides were acetylated with 2% of the rate determined for glucose . The Km for glucose and maltose were 62 and 90 mM, and the Vmax was 0.20 and 0.11 mmol/min x mg enzyme, respectively.

J Biol Chem, 1991 Jul 25, 266(21), 13842 - 8
Outer membrane translocation of the extracellular enzyme pullulanase in Escherichia coli K12 does not require a fatty acylated N-terminal cysteine; Kornacker MG et al.; Site-directed mutagenesis was used to construct three mutant derivatives of the extracellular, cell surface lipoprotein pullulanase (PulA) in which the normally fatty acylated cysteine of the signal peptide-bearing precursor was replaced by other amino acids . When produced in Escherichia coli expressing all genes required for pullulanase secretion, approximately 90% of the PulA derivatives persisted as cell-associated precursors, indicating inefficient signal peptide processing . Processed (intermediate-sized) forms of the two derivatives that were studied in detail were found to result from proteolytic cleavage at different sites within the signal peptide . Both were further processed to smaller polypeptides by cleavage at an undetermined site that is presumably close to their C termini . The intermediate-sized pullulanase derived from prepullulanase in which Cys+1 had been replaced by Leu and Gly-1 by Glu (PulA:C1L/G-1E) appeared rapidly, was apparently entirely extracellular, and accounted for approximately 10% of synthesized PulA . Prolonged incubation did not result in further conversion of the precursor to the intermediate form, and the precursor remained anchored to the cytoplasmic membrane . The smaller processed form was also found extracellularly . The active form of the extracellular enzyme was monomeric, which is again in contrast to the fatty acylated, wild-type enzyme . Taken together, these results indicate that replacement of Cys+1 of prePulA eliminates processing by lipoprotein signal peptidase and does not permit processing by leader peptidase, but allows inefficient, aberrant processing by an unknown peptidase and immediate secretion of the resulting polypeptide, which retains most of its signal peptide . Processing and secretion only occur when the pullulanase secretion functions are expressed.

J Biol Chem, 1991 Jul 25, 266(21), 13811 - 4
Secretion in yeast . Purification and in vitro translocation of chemical amounts of prepro-alpha-factor; Bush GL et al.; The Saccharomyces cerevisiae mating pheromone precursor, prepro-alpha-factor, can be translocated across yeast endoplasmic reticulum membranes post-translationally in an in vitro system . This characteristic makes prepro-alpha-factor potentially useful as a probe in the biochemical dissection of the mechanism of this basic cellular process . Efforts have been limited by the inability to isolate sufficient quantities of such secretory protein precursors in a translocation-competent form . We report here the one-step purification of chemical amounts of translocation-competent prepro-alpha-factor using nickel ion affinity chromatography on nitrilotriacetate resin . An oligonucleotide encoding 6 histidine residues was inserted into a genomic clone encoding prepro-alpha-factor 5' of the naturally occurring translational stop codon by site-directed mutagenesis . The construct was expressed at high levels in a SecY- strain of Escherichia coli . The produced preprotein was solubilized in 6 M guanidine hydrochloride and bound to nitrilotriacetate resin . Prepro-alpha-factor was recovered at a purity in excess of 95% by elution with 0.25 M imidazole, 8 M urea, which competitively displaced the histidine affinity tag from the nickel column . The chemical amounts of prepro-alpha-factor obtained in this way were determined to be competent for translocation across yeast microsomal membranes and for subsequent modifications such as signal sequence cleavage and N-linked glycosylation.

J Biol Chem, 1991 Jul 25, 266(21), 13661 - 71
Determination of binding constants for cooperative site-specific protein-DNA interactions using the gel mobility-shift assay; Senear DF et al.; We have investigated the question of whether the gel mobility-shift assay can provide data that are useful to the demonstration of cooperativity in the site-specific binding of proteins to DNA . Three common patterns of protein-DNA interaction were considered: (i) the cooperative binding of a protein to two sites (illustrated by the Escherichia coli Gal repressor); (ii) the cooperative binding of a bidentate protein to two sites (illustrated by the E . coli Lac repressor); and (iii) the cooperative binding of a protein to three sites (illustrated by the lambda cI repressor) . A simple, rigorous, and easily extendable statistical mechanical approach to the derivation of the binding equations for the different patterns is presented . Both simulated and experimental data for each case are analyzed . The mobility-shift assay provides estimates of the macroscopic binding constants for each step of ligation based on its separation of liganded species by the number of ligands bound . Resolution of the binding constants depends on the precision with which the equilibrium distribution of liganded species is determined over the entire range of titration of each of the sites . However, the evaluation of cooperativity from the macroscopic binding constants is meaningful only for data that are also accurate . Some criteria that are useful in evaluating accuracy are introduced and illustrated . Resolution of cooperative effects is robust only for the simplest case, in which there are two identical protein binding sites . In this case, cooperative effects of up to 1,000-fold are precisely determined . For heterogeneous sites, cooperative effects of greater than 1,000-fold are resolvable, but weak cooperativity is masked by the heterogeneity . For three-site systems, only averaged pair-wise cooperative effects are resolvable.

J Biol Chem, 1991 Jul 25, 266(21), 13640 - 5
Secretion of the cell surface lipoprotein pullulanase in Escherichia coli . Cooperation or competition between the specific secretion pathway and the lipoprotein sorting pathway; Pugsley AP et al.; The fatty acid-acylated enzyme pullulanase is normally found in either of two locations in Escherichia coli, depending on whether or not the producing strains also express the genes specifically required for the second step in pullulanase secretion . When they are expressed, the enzyme is localized to the cell surface, while in their absence, it is directed to an unidentified location in the cell envelope which, upon lysis, forms vesicles whose density is intermediate between those of outer and cytoplasmic membrane vesicles . In order to test the role of the putative lipoprotein sorting signal, Asp2, in pullulanase sorting and secretion, the structural gene (pulA) was subjected to site-directed mutagenesis . Replacement of the Asp2 residue by Asn, Glu, or Ser caused the enzyme to fractionate with outer membrane-derived vesicles rather than with intermediate density vesicles from E . coli cells devoid of pullulanase secretion genes . A pronounced secretion defect was observed in a two-step secretion assay in which the first (sec gene-dependent) and second (pul gene-dependent) secretion steps were uncoupled . We propose that the Asp residue increases the efficiency of pullulanase secretion by allowing the enzyme to be initially sorted to a region of the cell envelope wherein most of the pullulase-specific secretion factors are located.

J Biol Chem, 1991 Jul 25, 266(21), 13592 - 7
A genetic approach for analyzing the pathway of LamB assembly into the outer membrane of Escherichia coli; Misra R et al.; Results presented in this study demonstrate that a mutation which inserts an additional tyrosine between the 2 tyrosines at residues 118 and 119 of mature LamB protein results in a temperature-dependent assembly defect . This defect leads to the accumulation of an intermediate at the restrictive temperature that is most likely an assembly-defective monomer . These monomers are rapidly degraded in the wild type (htrA+) strain, and the biphasic kinetics of this degradation indicate that the mutation affects the assembly process and not the final product, i.e . stable trimers . In addition, our data show that the temperature-dependent assembly defect in the mutant strain is reversible, and therefore the accumulated monomers represent a true assembly intermediate . Fractionation studies show that the monomers, which can be accumulated in htrA (degP) mutants at the restrictive temperature, are associated with the outer membrane, indicating that trimerization of LamB is not a prerequisite for localization.

J Biol Chem, 1991 Jul 25, 266(21), 13572 - 9
Identification of active site residues of Escherichia coli fumarate reductase by site-directed mutagenesis; Schroder I et al.; Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor . The enzyme is structurally and catalytically similar to succinate dehydrogenase (succinate-ubiquinone oxidoreductase) from both procaryotes and eucaryotes . Both enzymes have been proposed to contain an essential cysteine residue at the active site based on studies with thiol-specific reagents . Chemical modification studies have also suggested roles for essential histidine and arginine residues in catalysis by succinate dehydrogenase . In the present study, a combination of site-directed mutagenesis and chemical modification techniques have been used to investigate the role(s) of the conserved histidine 232, cysteine 247, and arginine 248 residues of the flavorprotein subunit (FrdA) in active site function . A role for His-232 and Arg-248 of FrdA is shown by loss of both fumarate reductase and succino-oxidase activities following site-directed substitution of these particular amino acids . Evidence is also presented that suggests a second arginine residue may form part of the active site . Potential catalytic and substrate-binding roles for arginine are discussed . The effects of removing histidine-232 of FrdA are consistent with its proposed role as a general acid-base catalyst . The fact that succinate oxidation but not fumarate reduction was completely lost, however, might suggest that alternate proton donors substitute for His-232 . The data confirm that cysteine 247 of FrdA is responsible for the N-ethylmaleimide sensitivity shown by fumarate reductase but is not required for catalytic activity or the tight-binding of oxalacetate, as previously thought.

J Biol Chem, 1991 Jul 25, 266(21), 13492 - 4
Acyl carrier protein-derived sequence encoded by the chloroplast genome in the marine diatom Cylindrotheca sp . strain N1; Hwang SR et al.; The chloroplast genome of chromophytic and rhodophytic algae differs from the plastid genome of plants and green algae in that it encodes the gene for the small subunit (rbcS) of ribulose 1,5-bisphosphate carboxylase/oxygenase . Hybridization studies indicated that there was a second region of chloroplast DNA from the marine diatom Cylindrotheca sp . strain N1 that strongly hybridized to a previously isolated Cylindrotheca fragment that contained the rbcS gene and flanking sequences . Subsequent determination of the oligonucleotide sequence of this second chloroplast DNA fragment, however, indicated that hybridization was due to identical sequences 3' to the previously cloned Cylindrotheca chloroplast rbcL rbcS genes . Sequences derived from the 5' end of the second chloroplast DNA fragment contained a short open reading frame of 80 amino acids which was found to be highly homologous to bacterial acyl carrier protein and nuclear-encoded acyl carrier protein from plants . Amino acid residues in the environment of Ser-36 of the Escherichia coli protein, which is bound to a 4'-phosphopantetheine moiety, are virtually identical in the Cylindrotheca deduced sequence and all other sources of this protein . Unlike plant acyl carrier-deduced amino acid sequences, there was no leader peptide sequence found for the presumptive Cylindrotheca protein, consistent with the location of this DNA fragment on the chloroplast genome of this organism . DNA encoding the putative acyl carrier protein gene and rbcS thus represent two genes that are chloroplast-encoded in the chromophytic marine diatom Cylindrotheca, a significant departure from the organization of such genes in plants.

Nature, 1991 Jul 25, 352(6333), 349 - 51
Was the loss of the D helix in alpha globin a functionally neutral mutation?
Komiyama NH, Shih DT, Looker D, Tame J, Nagai K.
Proteins in the globin family are found in a variety of species from bacteria to man . From the many globin sequences known, evolutionary trees have been constructed showing that alpha and beta globins diverged from a common ancestor between 425 and 500 million years ago, after vertebrate species had appeared and roughly when sharks and bony vertebrates diverged . The alpha and beta globins assemble to form tetrameric haemoglobin, alpha 2 beta 2, which can switch between quaternary states having high and low oxygen affinity . This allows the protein to bind oxygen cooperatively and therefore efficiently transport oxygen from the lungs to respiring tissues . The alpha and beta globins have closely related tertiary structures, being alpha-helical proteins with similar haem-binding sites . Most globins consist of eight helices, designated A to H from the N terminus, connected by short nonhelical segments, but all known vertebrate alpha globins lack a D helix . Because the loss of this helix by alpha globin occurred shortly before tetrameric haemoglobin appeared, it might be a functionally important mutation required for a tetramer assembly or allostery . We have now tested this idea by engineering human haemoglobins containing beta subunits without a D helix and alpha subunits with a D helix . Both of these mutations have little effect on the oxygen-binding properties of the molecule . Thus it is possible that deletion of the D helix in the alpha subunit was caused by a neutral mutation.

J Biol Chem, 1991 Jul 25, 266(21), 14018 - 23
Recombinant human pim-1 protein exhibits serine/threonine kinase activity; Hoover D et al.; The protein predicted by the sequence of the human pim-1 proto-oncogene shares extensive homology with known serine/threonine protein kinases, and yet the human Pim-1 enzyme has previously been reported to exhibit protein tyrosine kinase activity both in vitro and in vivo . Recently a new class of protein kinases has been identified which exhibits both protein-serine/threonine and protein-tyrosine kinase activities . We therefore investigated the possibility that the human Pim-1 kinase likewise possesses such bifunctional enzymatic phosphorylating activities . A full-length human pim-1 cDNA was subcloned into the bacterial vector pGEX-2T and the Pim-1 protein expressed as a fusion product with bacterial glutathione S-transferase (GST) . The hybrid GST-Pim-1 fusion protein was affinity purified on a glutathione-Sepharose column prior to treatment with thrombin for cleavage of the Pim-1 protein from the transferase . Pim-1 was purified and the identity of recombinant protein confirmed by amino-terminal sequence analysis . Pim-1 was tested for kinase activity with a variety of proteins and peptides known to be substrates for either mammalian protein-serine/threonine or protein-tyrosine kinases and was found to phosphorylate serine/threonine residues exclusively in vitro . Both the Pim-1-GST fusion protein and the isolated Pim-1 protein exhibited only serine/threonine phosphorylating activity under all in vitro conditions tested . Pim-1 phosphorylated purified mammalian histone H1 with a Km of approximately 51 microM . Additionally, Pim-1 exhibited low levels of serine/threonine autophosphorylating activity . These observations place the human Pim-1 in a small select group of cytoplasmic transforming oncogenic kinases, including the protein kinase C, the Raf/Mil, and the Mos subfamilies, exhibiting serine/threonine phosphorylating activity.

Nucleic Acids Res, 1991 Jul 25, 19(14), 3943 - 51
The DNA unwinding reaction catalyzed by Rep protein is facilitated by an RHSP-DNA interaction; Yancey JE et al.; The unwinding reaction catalyzed by the Escherichia coli Rep protein is stimulated by a small 15 kDa protein called Rep helicase stimulatory protein (RHSP)(1) . The RHSP-stimulated unwinding reaction catalyzed by Rep protein proceeded at a rapid rate after a time lag of 1-2 min at 37 degrees C . This time lag was eliminated by preincubating RHSP with the DNA substrate, indicating that stimulation resulted from an interaction between RHSP and DNA . RHSP was shown to increase the rate as well as the extent of the unwinding reaction catalyzed by Rep protein . RHSP bound both single- and double-stranded DNA with apparent equal affinity, forming an unusually stable complex . Electron microscopy illustrated that the RHSP-DNA complex consisted of large protein aggregates bound to DNA forming a highly condensed, aggregated DNA-protein complex . The protein aggregates were not observed in the absence of DNA and appeared to form cooperatively in the presence of DNA . NH2-terminal amino acid sequence analysis suggested that RHSP was identical to E . coli ribosomal-protein L14 . Binding assays showed that the interaction between RHSP and rRNA was similar to the RHSP-DNA interaction . Several models are put forth to explain the stimulation of the unwinding reaction catalyzed by Rep protein . In addition, the potential physiological significance of the RHSP-stimulated Rep protein unwinding reaction is discussed.

J Biol Chem, 1991 Jul 25, 266(21), 13783 - 8
Isolation and characterization of Escherichia coli K-12 mutants lacking both 2-acyl-glycerophosphoethanolamine acyltransferase and acyl-acyl carrier protein synthetase activity; Hsu L et al.; 2-Acyl-glycerophosphoethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase are thought to be dual catalytic activities of a single inner membrane enzyme . A filter disc replica print method for the detection of acyl-ACP synthetase activity by colony fluorography was used to screen a mutagenized population of cells for acyl-ACP synthetase mutants (aas) . All aas mutants lacked both acyl-ACP synthetase and 2-acyl-GPE acyltransferase activities in vitro . There was no detectable acyl-CoA-independent incorporation of exogenous fatty acids into phosphatidylethanolamine or the major outer membrane lipoprotein in aas mutants . Exogenous lysophospholipid uptake and acylation was also lacking in aas mutants . Lipoprotein acylation by phospholipids synthesized by the de novo biosynthetic pathway was not affected in aas mutants showing that this gene product was not directly involved in lipoprotein biogenesis . The aas mutants had an altered membrane phospholipid composition and accumulated both 2-acyl-GPE and acylphosphatidylglycerol . Acylphosphatidylglycerol accumulation was due to the transacylase activity of lysophospholipase L2 (the pldB gene product) since aas pldB double mutants accumulated 2-acyl-GPE, but not acylphosphatidylglycerol . The aas allele was mapped to 61 min of the Escherichia coli chromosome, and the deduced gene order in this region was thyA-aas-lysA . The biochemical, physiological, and genetic analyses of aas mutants support the conclusion that 2-acyl-GPE acyltransferase and acyl-ACP synthetase are two activities of the same protein and confirm that this enzyme system participates in membrane phospholipid turnover and governs the acyl-CoA independent incorporation of exogenous fatty acids and lysophospholipids into the membrane.

Biochim Biophys Acta, 1991 Jul 23, 1089(3), 331 - 8
Construction and expression of synthetic wild-type and mutant genes encoding porcine pancreatic colipase: tryptophan fluorescence studies; Ernst EG et al.; Based on the known (95-residue) amino acid (aa) sequence of porcine pancreatic colipase (CLP), a cofactor of pancreatic lipase, a 297 bp gene was designed and assembled from eight synthetic, overlapping DNA fragments . Optimized for expression in bacteria, the CLP-encoding gene (CLP) was inserted into the lacZ gene fragment contained in the small expression vector, pUC8, and cloned in Escherichia coli JM109 . Expression of this construct yielded a protein approx . 11 kDa in size, equivalent to CLP, with an Mr of 10,336, plus ten additional amino acids at the N-terminus . The recombinant CLP (reCLP) was solubilized from bacterial inclusion bodies and then purified and refolded . A mutant CLP gene, changing Tyr-55 to Trp, was then constructed by site-directed mutagenesis . Since porcine CLP contains no Trp, this strategy provided a protein with an internal fluorescent probe for biophysical studies . The presence of Trp in the mutant protein was confirmed using fluorescence spectroscopy . Both wild-type (wt) and mutant reCLP reacted on Western blots with an affinity-purified rabbit anti-CLP antibody, raised against native CLP . The Tyr-55 to Trp exchange did not affect the activity of reCLP . Fluorescence studies of the interaction between reCLP and the bile salt, taurodeoxycholate (TDOC), showed that Trp-55 in the hydrophobic binding site of mutant reCLP inserted into the interior of the bile salt micelle.

Biochim Biophys Acta, 1991 Jul 23, 1089(3), 325 - 30
Exonuclease III promotes in vitro binding of the replication initiator protein of plasmid pSC101 to the repeated sequences in the ori region; Fueki T et al.; Purified Rep protein, a replication initiator protein of plasmid pSC101, has less binding affinity for the direct repeats (DR) in the replication origin region (ori) than that for the inverted repeats (IR) in the promoter region of the structure gene of Rep (rep) (Sugiura, S . et al . (1990) J . Biochem . 107, 369-376) . We found a protein factor that promotes binding of purified Rep to the DR sequence in the cell extract of Escherichia coli . In the presence of the factor, DNA fragments containing the DR sequence can form a specific DNA-protein complex by the addition of low concentrations of Rep . On the contrary, IR-containing DNA loses its binding activity for Rep by preincubation with the factor . We purified extensively the factor and identified it as exonuclease III (exo III) . Enzymatic action of the factor or authentic exo III at 37 degrees C is necessary for binding of Rep to DR-DNA . This binding of Rep to duplex DNA treated with exo III is DR-sequence specific . Since Rep cannot bind to the single stranded DR sequence, the present finding suggests that partial single-stranded regions around the DR sequence are required for binding of Rep.

Biochim Biophys Acta, 1991 Jul 23, 1089(3), 320 - 4
Restructuring the translation initiation region of the human parathyroid hormone gene for improved expression in Escherichia coli; Morelle G et al.; Overexpression of native human parathyroid hormone in Escherichia coli was achieved by a modification of the 5' end of the genomic gene sequence, thereby adapting this part of the translation initiation region to the bacterial host . Some simple rules abstracted from optimization studies of translation initiation of a beta-interferon gene were applied . These included (a) extending complementarity of the mRNA to the anticodon loop of tRNAfMet by use of a codon with a purine nucleotide directly following the ATG, (b) avoidance of stable secondary structure in the mRNA by use of synonymous A/U-rich codons, (c) elimination of a potential second Shine-Dalgarno sequence . The appropriate silent changes led to a 20-fold increase in parathyroid hormone production resulting in 4.3% of total soluble protein . This result proves the validity of our simple approach for optimization of foreign gene expression in E . coli.

Biochemistry, 1991 Jul 23, 30(29), 7105 - 11
An in vitro novel mechanism of regulating the activity of pyruvate kinase M2 by thyroid hormone and fructose 1, 6-bisphosphate; Ashizawa K et al.; We have recently shown that the cytosolic thyroid hormone binding protein (p58-M2) in human epidermoid carcinoma A431 cells is a monomer of pyruvate kinase, subtype M2 (PKM2) . To characterize further the molecular properties of p58-M2, we overexpressed p58-M2 in Escherichia coli and purified it to homogeneity . At 22 degrees C, the monomeric p58-M2, exhibited kinase activity with an apparent Vmax of 22 +/- 9 units/mg . The Km for adenosine diphosphate (ADP) and phosphoenolpyruvate (PEP) are 3.85 +/- 2.4 and 1.55 +/- 0.73 mM, respectively . Upon activation by fructose 1,6-bisphosphate (Fru-1,6-P2), Vmax and Km for ADP and PEP were changed to 490 +/- 27 units/mg and 0.63 +/- 0.09 and 0.13 +/- 0.01 mM, respectively . These results indicated that p58-M2 has intrinsic kinase activity . Analysis of the molecular size indicated that the activation of p58-M2, by Fru-1,6-P2 resulted in the association of the monomeric p58-M2 to the tetrameric PKM2 . p58-M2 bound to 3,3',5-triiodo-L-thyronine (T3) (Ka = 1.7 x 10(7) M-1) and exhibited analogue specificity, whereas PKM2 did not bind thyroid hormone . The order of binding affinity was L-T3 greater than L-thyroxine greater than 3,3',5-triiodothyropropionic acid greater than 3'-isopropyl-3,5-triiodo-L-thyronine greater than 3'5',3-triiodo-L-thyronine . Binding of T3 and its analogues resulted in the inhibition of the kinase activity of p58-M2 . The order of kinase inhibitory activity and preventing its association to tetrameric PKM2 was parallel to that of binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1991 Jul 23, 30(29), 7072 - 80
Nucleotide positions responsible for the processivity of the reaction of exonuclease I with oligodeoxyribonucleotides; Brody RS; The processive hydrolysis of single-stranded oligodeoxyribonucleotides by exonuclease I from Escherichia coli has been investigated . Oligodeoxyribonucleotides and their analogues, which contain either an abasic site or a methylphosphonate internucleotide linkage, were partially hydrolyzed by exonuclease I . The relative dissociation constant for the enzyme and each oligomeric product was calculated from the concentration of that oligomer found in solution and hence released by the enzyme before complete hydrolysis . The results have led to a characterization of the two oligodeoxyribonucleotide domains that bind to exonuclease I . The first domain, which begins at the reactive 3'-terminal phosphodiester and extends to the 7th nucleoside base, requires both phosphodiester monoanions and base residues for its interaction with the enzyme . The second domain includes phosphodiester monoanions in positions 9-13 from the 3'-terminus but does not require nucleoside bases . Methylphosphonate substitutions indicate that only two or three of these phosphodiesters, in variable positions, must remain anionic in order to obtain full enzyme binding . The residues between the two binding domains do not play a significant role in the enzyme-oligomer interaction.

Biochemistry, 1991 Jul 23, 30(29), 7089 - 96
Analysis of the regulatory and structural defects of troponin C central helix mutants; Dobrowolski Z et al.; Five deletion mutants of the D/E linker region of the troponin C central helix were tested for conformational and functional differences from wild-type troponin C . The mutants were in the region 87KEDAKGKSEEE97: dEDA, dKG, dKGK, dKEDAKGK, and dSEEE, designed to change the length of the central helix and the orientation of the Ca(2+)-binding domains relative to each other {Dobrowolski, Z., Xu, G.-Q., & Hitchcock-DeGregori, S.E . (1991) J . Biol . Chem . 266, 5703-5710} . Previous work showed that all mutants except dSEEE are partially defective in one part of the Ca2+ switch or the other . All mutants undergo Ca(2+)-dependent conformational changes as detected by changes in electrophoretic mobility, alpha-helix content, and hydrophobic exposure . Deletions of the central helix do not extensively alter the thermal stability of troponin C, as determined by temperature-dependent loss of alpha-helix . There are differences among the mutants that do not correlate with function . All troponin C mutants show Ca(2+)-dependent interaction with troponin I and T in polyacrylamide gels . Troponin I-troponin C interaction was also analyzed by Ca(2+)-dependent increase in the monomer/excimer ratio of tropinin I and relief of inhibition of the actomyosin S1 ATPase . While all mutants retain basic function, dKGK, dKEDAKGK, and dEDA have altered interaction with troponin I in the absence of Ca2+ . dSEEE differs in conformation from wild type, but it is normal in functional assays . This conserved region of the D/E linker is not required for interaction with troponin I in the presence or absence of urea.

Biochemistry, 1991 Jul 23, 30(29), 7080 - 8
Formation of cyclobutane thymine dimers photosensitized by pyridopsoralens: quantitative and qualitative distribution within DNA; Moysan A et al.; As after irradiation with 254-nm UV light, exposure of thymidine and three isomeric pyridopsoralen derivatives to UVA radiation, in the dry state, leads to the formation of the six diastereomers of cyclobutadithymidine as the predominant reaction . This unexpected photosensitized reaction, which also gives rise to both 5R* and 5S* diastereomers of 5,6-dihydro-5-(alpha-thymidylyl)thymidine (or "spore" photoproduct), is selective since {2 + 2} dimerization of 2'-deoxycytidine was not detected under the same experimental conditions . The cis-syn isomer of cyclobutadithymine was also found to be produced within isolated DNA following UVA irradiation in aqueous solutions containing 7-methylpyrido{3,4-c}psoralen . Quantitatively, this photoproduct represents about one-fifth of the overall yield of the furan-side pyridopsoralen {2 + 2} photocycloadducts to thymine . DNA sequencing methodology was used to demonstrate that pyridopsoralen-photosensitized DNA is a substrate for T4 endonuclease V and Escherichia coli photoreactivating enzyme, two enzymes acting specifically on cyclobutane pyrimidine dimers . Furthermore, the dimerization reaction of thymine is sequence dependent, with a different specificity from that mediated by far-UV irradiation as inferred from gel sequencing experiments . Interestingly, adjacent thymine residues are excellent targets for 7-methylpyrido{3,4-c}psoralen-mediated formation of cyclobutadithymine in TTTTA and TTAAT sites, which are also the strongest sites for photoaddition . The formation of cyclobutane thymine dimers concomitant to that of thymine-furocoumarin photoadducts and their eventual implication in the photobiological effects of the pyridopsoralens are discussed.

Biochim Biophys Acta, 1991 Jul 22, 1066(2), 131 - 43
Cytochrome b5 and a recombinant protein containing the cytochrome b5 hydrophobic domain spontaneously associate with the plasma membranes of cells; George SK et al.; Both cytochrome b5, isolated from rabbit liver microsomes, and LacZ:HP, a recombinant protein consisting of enzymatically active Escherichia coli beta-galactosidase coupled to the C-terminal membrane-anchoring hydrophobic domain of cytochrome b5, were shown to spontaneously associate with the plasma membranes of erythrocytes and 3T3 cells . Association was promoted by low pH values, but proceeded satisfactorily over several hours at physiological pH and temperature . About 150,000 cytochrome b5 molecules or 100,000 LacZ:HP molecules could be associated per erythrocyte . These proteins were not removed from the membrane by extensive washing, even at high ionic strength . After incubation with fluorescently labeled cytochrome b5 or LacZ:HP, cells displayed fluorescent membranes . The lateral mobility of fluorescently labeled cytochrome b5 and LacZ:HP was measured by photo-bleaching techniques . In the plasma membrane of erythrocytes and 3T3 cells, the apparent lateral diffusion coefficient D ranged from 1.0.10(-9) to 8.10(-9) cm2 s-1 with a mobile fraction M between 0.4 and 0.6 . The lateral mobility of these proteins closely resembled that reported for lipid-anchored proteins and was much higher than that reported for Band 3, an erythrocyte membrane-spanning protein with a large cytoplasmic domain . These results suggest that the hydrophobic domain of cytochrome b5 could be employed as a universal, laterally mobile membrane anchor to associate a variety of diagnostically and therapeutically useful recombinant proteins with cells.

FEBS Lett, 1991 Jul 22, 285(2), 165 - 9
Codon bias and gene expression; Kurland CG; The frequencies with which individual synonymous codons are used to code their cognate amino acids is quite variable from genome to genome and within genomes, from gene to gene . One particularly well documented codon bias is that associated with highly expressed genes in bacteria as well as in yeast; this is the so-called major codon bias . Here, it is suggested that the major codon bias is not an arrangement for regulating individual gene expression . Instead, the data suggest that this codon bias, which is correlated with a corresponding bias of tRNA abundance, is a global arrangement for optimizing the growth efficiency of cells . On the practical side, it is suggested that heterologous gene expression is not as sensitive to codon bias as previously thought, but that it is quite sensitive to other characteristics of the heterologous gene.

FEBS Lett, 1991 Jul 22, 285(2), 160 - 4
On the origins of genetic variants; Wintersberger U; Two contrasting mechanisms responsible for the creation of genetic variants are described: one is the manifestation of the limited accuracy of the cellular machinery for DNA replication, the other results from the ability of cells to repair damaged DNA . Replication-dependent variants and those caused by episodical DNA damage enhance the probability that a small fraction of a cell population may survive a sudden (physical or biological) change of environmental conditions . Replication-independent variants arise during persistent but not immediately lethal stress (e.g . starvation) of a non-dividing population . The variants observed under these conditions are of selective advantage because they are able to cope with the particular stress situation . The molecular basis of their creation is a matter of intensive debate.

Gene, 1991 Jul 22, 103(2), 163 - 9
Characterization of nucleotide sequences from European hepatitis C virus isolates; Fuchs K et al.; We characterized the 5' end and parts of the structural genes of European isolates of hepatitis C virus (HCV) and compared them with recently published RNA sequences of American and Japanese HCV isolates . The cDNA, obtained by reverse transcription of viral RNA extracted from different sera, was amplified by nested PCR, cloned and sequenced . Within 239 nucleotides (nt) of the 5' end, we found only three single-nt exchanges compared to two sequences of Japanese origin and one exchange to the prototype HCV sequence (ptHCV) (homology greater than 99%) . The sequence of the core region (534 nt) in two European isolates showed a homology of about 97-98% on the nt level, as compared to ptHCV and one Japanese isolate, and 90% to other Japanese isolates . The amino acid (aa) homology was between 98-99% among all published sequences . A greater discrepancy was found in the European isolates within the 434 nt sequenced from the N-terminus of the putative envelope region, where the nt homology to ptHCV and one Japanese isolate was 90-93% (aa homology 93-95%), and to other Japanese isolates was 72-73% (aa homology 77-78%), indicating that the European isolates may be more closely related to the ptHCV and one Japanese isolate than to the other Japanese isolates . Amplified genes encoding structural proteins (core, envelope) were expressed in Escherichia coli . Sera from chronically infected patients reacted strongly with the recombinant core protein, but no specific immunoreactivity occurred with the putative envelope protein.

J Mol Biol, 1991 Jul 20, 220(2), 271 - 9
Regulation of the F plasmid traY promoter in Escherichia coli K12 as a function of sequence context; Silverman PM et al.; TraJ and SfrA are, respectively, plasmid and host (Escherichia coli)-encoded proteins normally required for F plasmid traY promoter function . Beginning with plasmids in which a traY-lacZ fusion gene, designated phi (traY'-'lacZ)hyb, and lacY are expressed from the F plasmid traY promoter, we isolated mutants in which lac gene expression was SfrA or TraJ-independent . A total of 45 of 50 SfrA-independent isolates obtained after 2-aminopurine mutagenesis proved to have chromosomal mutations, whereas four out of four isolates obtained without mutagenesis had plasmid mutations . All of 17 isolates selected for TraJ-independent expression after mutagenesis had plasmid mutations . By restriction endonuclease digestions, 25 of 26 SfrA-independent and TraJ-independent plasmid mutations were insertions . Four of the former and three of the latter were examined further . By sequence analysis, all seven proved to be IS1 or IS2 insertions defining five insertion sites between base-pairs -49 and -82 with respect to the major traY transcription initiation site . In two cases, the same insertion allele was obtained from the two selection schemes . All three of the mutants selected for TraJ-independent gene expression manifested SfrA-independent expression as well, and levels of beta-galactosidase in different plasmid mutant strains lacking TraJ and SfrA were indistinguishable . By primer extension analysis, transcription initiation sites for traY mRNA synthesis were unaltered by the mutations . Replacing the tra sequence upstream from base-pair -78, without genetic selection, increased beta-galactosidase activity in the absence of TraJ and SfrA greater than tenfold . Activity increased two- to threefold more in a traJ+ sfrA mutant strain, and fivefold more in a traJ+ sfrA+ strain . Activity was unaltered in an sfrA+ strain without TraJ . By primer extension analysis, the traY promoter was utilized under all conditions . The data indicate that regulation of traY promoter activity is strongly dependent on sequence context.

J Mol Biol, 1991 Jul 20, 220(2), 325 - 33
Escherichia coli sigma 70 and NusA proteins . II . Physical properties and self-association states; Gill SC et al.; In this paper we examine the physical properties and potential for self-association of the Escherichia coli transcription factors, sigma 70 and NusA . We show, by a combination of chemical crosslinking, equilibrium and velocity sedimentation, quasi-elastic light scattering, and small-angle X-ray scattering that NusA exists as a monomer at KCl concentrations between 0.01 and 1.5 M, and that sigma 70 exists as a monomer at KCl concentrations between 0.1 and 1.5 M . The shape and hydration characteristics of each of these monomeric proteins are also examined . The results serve as background for the companion paper in which a thermodynamic analysis is made of the interactions of these transcription factor with E . coli core RNA polymerase in solution and as a component of the functional transcription complex.

J Mol Biol, 1991 Jul 20, 220(2), 307 - 24
Escherichia coli sigma 70 and NusA proteins . I . Binding interactions with core RNA polymerase in solution and within the transcription complex; Gill SC et al.; This paper describes the binding interactions of Escherichia coli transcription factors sigma 70 and NusA with core RNA polymerase, both free in solution and as a part of the functional transcription complex . High pressure liquid chromatography gel filtration and fluorescence techniques have been used to monitor the binding of these factors to core polymerase in solution at salt concentrations roughly comparable to the in vivo environment (250 mM-KCl, 50 mM-potassium phosphate (pH 7.5}; under these conditions all the interacting species exist separately as protein monomers . We find that sigma 70 and NusA binds competitively to core polymerase with a 1:1 binding stoichiometry in this milieu, and that NusA does not bind to the polymerase holoenzyme . Association constants of approximately 2 x 10(9) and 1 x 10(7) M-1 have been measured for the sigma 70-core polymerase interaction and for the NusA-core polymerase interaction, respectively . These findings are consistent with the original formulation of the NusA-sigma 70 cycle put forward by Greenblatt & Li, and provide the basis for a further (and preliminary) quantitative examination of these same interactions within the transcription complex . We use a number of molecular biological techniques, together with data from the literature, to estimate these binding constants in various phases of the transcription cycle . In keeping with our results in solution, we find that the effective binding affinity of sigma 70 for core polymerase within the "open" promoter-polymerase complex is at least 500-fold greater than that of NusA . As the transcription complex moves from the initiation to the elongation phase these relative binding affinities are reversed; the average association constant of NusA for the core polymerase in the elongation complex remains practically the same as in free solution (approx . 3 x 10(7) M-1), while the affinity of sigma 70 for core polymerase in this complex drops to less than 5 x 10(5) M-1 . These results are used to begin to define the basic conformational states and interaction potentials of core polymerase in the various stages of the transcription cycle.

J Mol Biol, 1991 Jul 20, 220(2), 209 - 16
Determination of the positions of bound water molecules in the solution structure of reduced human thioredoxin by heteronuclear three-dimensional nuclear magnetic resonance spectroscopy; Forman-Kay JD et al.; The presence of bound water molecules in the solution structure of reduced human thioredoxin has been investigated using three-dimensional 1H rotating frame Overhauser 1H-15N multiple quantum coherence spectroscopy . It is demonstrated that the backbone amide protons of Lys21, Lys39, Lys82, Gly83 and Asn102, as well as the side-chain amide group of Asn102, are in close proximity to bound water molecules . Examination of the high-resolution solution structure of reduced human thioredoxin reveals that these results are best accounted for by four bound water molecules . Subsequent simulated annealing calculations carried out on the basis of interproton distance and hydrogen bonding restraints to the bound water molecules, supplemented by the original set of experimental restraints used in the calculation of the three-dimensional structure of human thioredoxin, permit a more precise localization of the bound water positions . Potential hydrogen bonds to these water molecules are described and a comparison is made to corresponding bound water molecules in the crystal structure of oxidized Escherichia coli thioredoxin.

J Mol Biol, 1991 Jul 20, 220(2), 205 - 8
Binding of the anticodon domain of tRNA(fMet) to Escherichia coli methionyl-tRNA synthetase; Meinnel T et al.; A stem and loop RNA domain carrying the methionine anticodon (CAU) was designed from the tRNA(fMet) sequence and produced in vitro . This domain makes a complex with methionyl-tRNA synthetase (Kd = 38(+/- 5) microM; 25 degrees C, pH 7.6, 7 mM-MgCl2) . The formation of this complex is dependent on the presence of the cognate CAU anticodon sequence . Recognition of this RNA domain is abolished by a methionyl-tRNA synthetase mutation known to alter the binding of tRNA(Met).

J Mol Biol, 1991 Jul 20, 220(2), 335 - 49
A severely truncated form of translational initiation factor 2 supports growth of Escherichia coli; Laalami S et al.; We have constructed strains carrying null mutations in the chromosomal copy of the gene for translational initiation factor (IF) 2 (infB) . A functional copy of the infB gene is supplied in trans by a thermosensitive lysogenic lambda phage integrated at att lambda . These strains enabled us to test in vivo the importance of different structural elements of IF2 expressed from genetically engineered plasmid constructs . We found that, as expected, the gene for IF2 is essential . However, a protein consisting of the C-terminal 55,000 Mr fragment of the wild-type IF2 protein is sufficient to allow growth when supplied in excess . This result suggests that the catalytic properties are localized in the C-terminal half of the protein, which includes the G-domain, and that this fragment is sufficient to complement the IF2 deficiency in the infB deletion strain.

J Mol Biol, 1991 Jul 20, 220(2), 193 - 8
Deletions induced by gamma rays in the genome of Escherichia coli; Raha M et al.; An Escherichia coli lysogen was constructed with a lambda phage bearing a lacZ gene surrounded by about 100 x 10(3) base-pairs of dispensable DNA . The lacZ mutants induced by gamma rays in this lysogen were more than 10% large deletions, ranging in size from 0.6 x 10(-3) to 70 x 10(3) base-pairs . These deletions were centered, not on lacZ, but on a ColE1 origin of DNA replication located 1.2 x 10(3) bases downstream from lacZ . This suggested that this origin of replication was involved in the process by which the deletions were formed . In agreement with this hypothesis, a lysogen of the same phage without the ColE1 origin showed a very much lower percentage of radiation-induced deletions, as did a second lysogen of a lambda phage without any known plasmid origin of replication . Indirect evidence is presented for radiation-induced deletions centered on the lambda origin of DNA replication in a lysogen . It is suggested that high percentages of large deletions may occur among radiation-induced mutations in mammalian cells because deletions centered on some of the thousands of origins of replication in these genomes do not kill the cells.

J Mol Biol, 1991 Jul 20, 220(2), 227 - 39
Sigma subunit of Escherichia coli RNA polymerase loses contacts with the 3' end of the nascent RNA after synthesis of a tetranucleotide; Bowser CA et al.; We have used photocrosslinking to analyze the contacts between the 3' end of the RNA and Escherichia coli RNA polymerase during the early steps of RNA synthesis using the nucleotide analog 8-azido-ATP (8-N3-ATP) . The crosslinking group on 8-N3-ATP contacts the beta, beta' and sigma subunits when the analog is bound to the holoenzyme . We show here that 8-N3-ATP is a substrate for E . coli RNA polymerase and acts as an RNA chain terminator when incorporated into the 3' end of nascent RNA . 8-N3-AMP was incorporated uniquely at the 3' end of tri-, tetra- and pentanucleotides synthesized from a poly{d(A-T)} template and at the 3' end of pentanucleotides from two promoters (lambda PR' and E . coli rrnB P1) . The oligonucleotides were covalently attached to the RNA polymerase by irradiation of transcription complexes with ultraviolet light . All RNAs labeled the beta and beta' subunits, but sigma was contacted only by the trinucleotide and tetranucleotide on poly{d(A-T)} . Sigma is still present in transcription complexes containing the pentanucleotide on poly{d(A-T)}, despite the lack of labeling . Neither pentanucleotide from the authentic promoters contacted sigma . We conclude that as holoenzyme moves downstream, either two separate conformational changes occur, after synthesis of the trinucleotide and tetranucleotide, which result in movement of sigma away from the nucleotide binding site or, alternatively, sigma remains fixed relative to the DNA while the domain on core polymerase forming the nucleotide binding site moves downstream.

Carbohydr Res, 1991 Jul 18, 214(1), 35 - 41
A bireactant, irreversible, active-site-directed inhibitor of beta-D-galactosidase (Escherichia coli) . Synthesis and properties of (1/2,5,6)-2-(3-azibutylthio)-5,6-epoxy-3-cyclohexen-1-ol; Huber RE et al.; (1/2,5,6)-2-(3-Azibutylthio)-5,6-epoxy-3-cyclohexen-1-ol (1) was synthesized and was found to irreversibly inactivate beta-D-galactosidase (Escherichia coli) . The inactivation was prevented by the presence of isopropyl 1-thio-beta-D-galactopyranoside (IPTG) . The vinyloxirane group of 1 reacted with water and other nucleophiles, especially at higher pH values . Reaction of 1 with beta-D-galactosidase was slow enough so that a competitive-inhibition constant (Ki) of 29mM could be determined . The inhibition constant for (1,2/3,6)-6-(3-azibutylthio)-2-bromo-4-cyclohexene-1,3-diol (2), the precursor of the bireactant inhibitor 1, was 13 mM, while that of (1,3/2,4)-3-(3-azibutylthio)-5-cyclohexene-1,2,4-triol (3), the product formed when the reactant is allowed to react with water, was 23mM . After irradiation by light, beta-D-galactosidase that had initially been treated with the bireactant compound and then digested with trypsin, showed a new pattern of elution from h.p.l.c., indicating that there was reaction at two regions of the beta-D-galactosidase molecule.

Nature, 1991 Jul 18, 352(6332), 213 - 8
Structural basis of anticodon loop recognition by glutaminyl-tRNA synthetase; Rould MA et al.; The refined crystal structure of Escherichia coli glutaminyl transfer RNA synthetase complexed with transfer RNA(Gln) and ATP reveals that the structure of the anticodon loop of the enzyme-bound tRNA(Gln) differs extensively from that of the known crystal structures of uncomplexed tRNA molecules . The anticodon stem is extended by two non-Watson-Crick base pairs, leaving the three anti-codon bases unpaired and splayed out to bind snugly into three separate complementary pockets in the protein . These interactions suggest that the entire anticodon loop provides essential sites for glutaminyl tRNA synthetase discrimination among tRNA molecules.

Biochemistry, 1991 Jul 16, 30(28), 7027 - 33
Comparative mutagenesis of O6-methylguanine and O4-methylthymine in Escherichia coli; Dosanjh MK et al.; The qualitative and quantitative features of mutagenesis by two DNA adducts of carcinogenic alkylating agents, O6-methylguanine (m6G) and O4-methylthymine (m4T), were examined in vivo . The deoxyhexanucleotides 5'-GCTAGC-3' and 5'-GCTAGC-3' were synthesized, where the underlined bases are the positions of m4T or m6G, respectively . By use of recombinant DNA techniques, the respective hexanucleotides or an unmodified control were inserted into a six-base gap in the otherwise duplex genome of the Escherichia coli virus M13mp19-NheI . The duplex adducted genome was converted to single-stranded form and introduced into an E . coli strain that was phenotypically normal with regard to m6G/m4T repair, a strain deficient in repair by virtue of an insertion in the gene encoding the Ada-m6G/m4T DNA methyltransferase, or the same two cell lines after challenge with N-methyl-N'-nitro-N-nitrosoguanidine . Treatment with this alkylating agent chemically compromises alkyl-DNA repair functions . The mutation efficiency of m6G was low or undetectable (0-1.7%) in all cell systems tested, owing, we believe, to rapid repair . In striking contrast, the mutagenicity of m4T was high (12%) in cells fully competent to repair alkylation damage and was roughly doubled when those cells were pretreated with N-methyl-N'-nitro-N-nitrosoguanidine to suppress repair . Taken together, these data suggest that m4T is potentially more mutagenic than m6G and, if formed by a DNA methylating agent, may pose a significant threat to the genetic integrity of an organism.

Biochemistry, 1991 Jul 16, 30(28), 6861 - 6
Sugar-binding and crystallographic studies of an arabinose-binding protein mutant (Met108Leu) that exhibits enhanced affinity and altered specificity; Vermersch PS et al.; In addition to hydrogen bonds, van der Waals forces contribute to the affinity of protein-carbohydrate interactions . Nonpolar van der Waals contacts in the complexes of the L-arabinose-binding protein (ABP) with monosaccharides have been studied by means of site-directed mutagenesis, equilibrium and rapid kinetic binding techniques, and X-ray crystallography . ABP, a periplasmic transport receptor of Escherichia coli, binds L-arabinose, D-galactose, and D-fucose with preferential affinity in the order of Ara greater than Gal much greater than Fuc . Well-refined, high-resolution structures of ABP complexed with the three sugars revealed that the structural differences in the ABP-sugar complexes are localized around C5 of the sugars, where the equatorial H of Ara has been substituted for CH3 (Fuc) or CH2OH (Gal) . The side chain of Met108 undergoes a sterically dictated, ligand-specific, conformational change to optimize nonpolar interactions between its methyl group and the sugar . We found that the Met108Leu ABP binds Gal tighter than wild-type ABP binds Ara and exhibits a preference for ligand in the order of Gal much greater than Fuc greater than Ara . The differences in affinity can be attributed to differences in the dissociation rates of the ABP-sugar complexes . We have refined at better than 1.7-A resolution the crystal structures of the Met108Leu ABP complexed with each of the sugars and offer a molecular explanation for the altered binding properties.

Biochemistry, 1991 Jul 16, 30(28), 6842 - 7
Characterization of Escherichia coli ATP synthase beta-subunit mutations using a chromosomal deletion strain; Lee RS et al.; (1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene . JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit . Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes . JP17 was shown to be efficacious for studies of beta-subunit mutations . Site-directed mutants were studied directly in JP17 . Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17 . (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure . The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism . The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1 . However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis . (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site . Neither beta R398C or beta R398W impaired catalysis significantly.

Biochemistry, 1991 Jul 16, 30(28), 6970 - 6
Evidence for a "cysteine-histidine box" metal-binding site in an Escherichia coli aminoacyl-tRNA synthetase; Miller WT et al.; Escherichia coli alanyl-tRNA synthetase contains the sequence Cys-X2-Cys-X6-His-X2-His . This motif is distinct from the zinc fingers of DNA-binding proteins but has some similarity to the Cys-X2-Cys-X4-His-X4-Cys zinc-binding motif of retroviral gag proteins, where it has a role in RNA packaging . In Ala-tRNA synthetase, this sequence is located in an amino-terminal domain which has the site for docking the acceptor end of the tRNA near the bound aminoacyl adenylate and is immediately adjacent in the sequence to the location of a mutation that affects the specificity of tRNA recognition . We show here that Ala-tRNA synthetase contains approximately 1 mol of zinc/mol of polypeptide and that addition of the zinc chelator 1,10-phenanthroline inhibits its aminoacylation activity . Conservative mutations of specific cysteine or histidine residues in the "Cys-His box" destabilize and inactivate the enzyme, whereas mutations of intervening amino acids do not inactivate . The possibility that this motif can bind zinc (or cobalt) was demonstrated with a synthetic 22 amino acid peptide that is based on the sequence of the alanine enzyme . The peptide-cobalt complex has the spectral characteristics of tetrahedral coordination geometry . The results establish that the Cys-His box motif of Ala-tRNA synthetase has the potential to form a specific complex with zinc (at least in the context of a synthetic peptide analogue) and suggest that this motif is important for enzyme stability/activity.

J Biol Chem, 1991 Jul 15, 266(20), 12971 - 5
p-Aminobenzoate biosynthesis in Escherichia coli . Purification of aminodeoxychorismate lyase and cloning of pabC; Green JM et al.; p-Aminobenzoate, a component of the vitamin folate, is one of seven compounds derived from the aromatic precursor chorismate in Escherichia coli . Historically the gene products of pabA and pabB were assumed to be sufficient for de novo p-aminobenzoate biosynthesis . Recent studies, however, have shown that these proteins, as nonidentical subunits of a single enzyme, act on chorismate to form a diffusible intermediate, most likely 4-amino-4-deoxychorismate . This intermediate is then converted to p-aminodeoxychorismate lyase (Nichols, B . P., Seibold, A . S., and Doktor, S . Z . (1989) J . Biol . Chem . 264, 8597-8601) . Here we describe partial characterization of the intermediate and the purification of aminodeoxychorismate lyase 4100-fold to near homogeneity . Further purification of this enzyme by high pressure liquid chromatography permitted isolation of a pure sample that yielded N-terminal sequence . A 64-fold redundant oligonucleotide probe was used to identify a lambda clone containing the gene encoding aminodeoxychorismate lyase . The aminodeoxychorismate lyase gene, designated pabC, was mapped to 25 min on the E . coli chromosome and lies on a 7.5-kilobase pair EcoRI fragment . A strain harboring a pACYC184 recombinant containing pabC overproduced aminodeoxychorismate lyase activity 77-fold.

J Biol Chem, 1991 Jul 15, 266(20), 12884 - 8
Mutagenesis of Cerebratulus lacteus neurotoxin B-IV identifies NH2-terminal sequences important for biological activity; Howell ML et al.; We have previously demonstrated that a synthetic gene encoding Cerebratulus lacteus neurotoxin B-IV can be expressed in bacteria, and the recombinant toxin purified and refolded (Howell, M . L., and Blumenthal, K . M . (1989) J . Biol . Chem . 264, 15268-15273) . This toxin, which contains an NH2-terminal methionine residue not present in authentic B-IV, has a specific toxicity 35-40% that of the naturally occurring form . Deletion of the codon for the NH2-terminal methionine allows expression of fully active recombinant B-IV, demonstrating that hydroxylation of Pro-10 is not important for biological activity . Site-directed mutagenesis of the des-Met(-1) form has been employed to analyze the contribution of NH2-terminal sequences of this toxin to its activity . We have emphasized replacement of helix-favoring residues by helix-destabilizing ones which are otherwise sterically similar . When Ala-3 or Ala-8 is replaced by serine, little or no effect on specific toxicity is observed . However, the double mutant in which both alanines are substituted with serine is more than twice as active as natural B-IV, although the secondary structures and conformational stabilities of the wild-type and mutant forms are the same . When Ala-3 and 8 are simultaneously replaced with glycine, the resulting toxin displays an activity similar to that of the wild-type form . The conformational properties of this mutant are unchanged from that of either wild-type or the serine double mutant . These data indicate that insertion into the NH2-terminal region of toxin B-IV of residues which can participate in hydrogen bond formation enhances biological activity of the protein.

Eur J Biochem, 1991 Jul 15, 199(2), 361 - 9
Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli; Louis JM et al.; Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli . The full-length fusion proteins did not exhibit self-processing in E . coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns . Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease . Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture . Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease . Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site . Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).

Blood, 1991 Jul 15, 78(2), 364 - 8
DEGR-factor Xa blocks disseminated intravascular coagulation initiated by Escherichia coli without preventing shock or organ damage; Taylor FB Jr et al.; One of the aims of research in the area of thrombosis has been to design an effective anticoagulant that would function in a predictable and direct manner . In evaluating the role of coagulation in sepsis we used factor Xa blocked in the active center with {5-(dimethylamino)1-naphthalenesulfonyl}-glutamylglycylarginyl+ ++ chloromethyl ketone (DEGR-Xa) . We infused 1 mg/kg of DEGR-Xa together with LD100 concentrations of Escherichia coli (4 x 10(10) organisms/kg) into five baboons . As controls, we infused E coli alone into five baboons . The inflammatory, coagulant, and cell injury responses to E coli of both the treated and control groups were lethal and were similar in every respect except for the complete inhibition of the consumption of fibrinogen in the DEGR-Xa group . The half life of DEGR-Xa was approximately 10 hours and 2 hours, as determined by isotopic and enzyme-linked immunosorbent assays, respectively . These results for the first time demonstrate that, although coagulation occurs in E coli sepsis, fibrin formation per se did not influence the lethal outcome in this model . These results also show the effectiveness of DEGR-Xa as an anticoagulant and raise the possibility that it could serve as an alternative to anticoagulants currently in use.

Biochem Biophys Res Commun, 1991 Jul 15, 178(1), 54 - 9
NAD(P)H oxidation by hydrogen peroxide in Escherichia coli; Coves J et al.; A protein fraction from Escherichia Coli soluble extracts contain a NAD(P)H:hydrogen peroxide oxidoreductase activity . This activity is compared to and found to be distinct from well-known E . Coli enzymes involved in the protection from peroxides: hydroperoxidase I (HPI) and its o-dianisidine peroxidase component and the alkyl hydroperoxide reductase.

Biochem Biophys Res Commun, 1991 Jul 15, 178(1), 385 - 92
NMR spectra of exchangeable protons of pyridoxal phosphate-dependent enzymes; Metzler CM et al.; We have recorded 1H NMR spectra in H2O for exchangeable protons of four pyridoxal phosphate-dependent enzymes: D-serine dehydratase, aspartate aminotransferase, tryptophan: indole-lyase and glutamate decarboxylase . The molecular masses range from 48-250 kDa . In every case there are downfield peaks which are lost when the apoenzyme is formed . In most cases some peaks shift in response to interactions with substrates and inhibitors and with changes in pH . We associate one downfield resonance with the proton on the ring nitrogen of the coenzyme and others with imidazole groups that interact with coenzyme or substrates . The chemical shift for the coenzyme-bound proton differs for free enzyme, substrate Schiff base or quinonoid forms.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6147 - 51
Rapid determination of nucleotides that define tRNA(Gly) acceptor identity; McClain WH et al.; Expression of the genetic code depends on the recognition of specific tRNAs by the enzymes that aminoacylate them . A computer comparison of tRNA sequences coupled with analysis of mutant nonsense-suppressor tRNAs has revealed the structural features that distinguish the acceptor identity of Escherichia coli tRNA(Gly) from tRNAs that accept phenylalanine, arginine, lysine, and glutamine . On replacement of several nucleotides in the acceptor stem and anticodon of the latter tRNAs with tRNA(Gly)-derived residues, the resulting molecules acquired a tRNA(Gly) identity.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6132 - 6
Interaction of Escherichia coli tRNA(Ser) with its cognate aminoacyl-tRNA synthetase as determined by footprinting with phosphorothioate-containing tRNA transcripts; Schatz D et al.; A footprinting technique using phosphorothioate-containing RNA transcripts has been developed and applied to identify contacts between Escherichia coli tRNA(Ser) and its cognate aminoacyl-tRNA synthetase . The cloned gene for the tRNA was transcribed in four reactions in which a different NTP was complemented by 5% of the corresponding nucleoside 5'-O-(1-thiotriphosphate) . The phosphorothioate groups of such transcripts are cleaved by reaction with iodine to permit sequencing of the transcripts . Footprinting was achieved by performing the same reaction with the phosphorothioate-tRNA-enzyme complex . At 1 mM iodine, selective protection of the tRNA transcripts in the cognate system was observed, with strong protection at positions 52 and 68 and weak protection at positions 46, 53, 67, 69, and 70 . It is suggested that these regions of the tRNA interact with the helical arm of the synthetase.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6127 - 31
A copper-thiolate polynuclear cluster in the ACE1 transcription factor; Dameron CT et al.; ACE1 is the transcriptional activator of the metallothionein (CUP1 locus) gene in Saccharomyces cerevisiae . Previous data had implicated the N-terminal domain of ACE1 as responsible for the Cu-dependent specific DNA binding . An expression system in Escherichia coli was constructed to enable the isolation of an ACE1 domain containing the DNA and Cu-binding regions . Here we report the purification and characterization of the Cu-ACE1 truncated molecule . Spectroscopic techniques showed that ACE1 contains an unusual type of DNA binding structure that is based on a polynuclear Cu(I)-cysteinyl thiolate cluster . The cluster consists of six or seven Cu(I) ions coordinated to cysteinyl thiolates in a trigonal geometry distorted from planarity . The Cu(I)-cysteine cluster of Cu-ACE1 exhibits structural properties analogous to the Cu(I)-thiolate polynuclear cluster in yeast Cu-metallothionein itself, suggesting an unusual mechanism for the evolution of this regulatory factor . The Cu cluster organizes and stabilizes the conformation of the N-terminal domain of ACE1 for specific DNA binding.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6122 - 6
The heme groups of cytochrome o from Escherichia coli; Puustinen A et al.; Cytochrome o, one of the two terminal ubiquinol oxidases of Escherichia coli, is structurally and functionally related to cytochrome c oxidase of mitochondria and some bacteria . It has two heme groups, one of which binds CO and forms a binuclear oxygen reaction center with copper . The other heme is unreactive toward ligands, exhibits strong interactions with the binuclear center, and is mainly responsible for the reduced-minus-oxidized alpha band . Protoheme has been thought to be the prosthetic group of b-type cytochromes, including cytochrome o . However, the hemes of cytochrome o are of a different kind, for which we propose the name heme O . Its pyridine hemochrome spectrum is blue-shifted by 4 nm relative to that of protoheme, and chromatographic behavior showed that it is much more hydrophobic than protoheme . Fast atom bombardment mass spectrometry yielded a molecular mass of 839 Da . Heme O is proposed to be a heme A-like molecule, containing a 17-carbon hydroxyethylfarnesyl side chain, but with a methyl residue replacing the formyl group.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6038 - 42
Molecular cloning and characterization of the yeast gene for squalene synthetase; Jennings SM et al.; Squalene synthetase (farnesyl-diphosphate: farnesyl-diphosphate farnesyltransferase, EC 2.5.1.21) is a critical branch point enzyme of isoprenoid biosynthesis that is thought to regulate the flux of isoprene intermediates through the sterol pathway . The structural gene for this enzyme was cloned from the yeast Saccharomyces cerevisiae by functional complementation of a squalene synthetase-deficient erg9 mutant . Identification of this ERG9 clone was confirmed by genetic linkage analysis in yeast and expression of enzyme activity in Escherichia coli . The predicted squalene synthetase polypeptide of 444 amino acids (Mr, 51,753) lacks significant homology to known protein sequences, except within a region that may represent a prenyl diphosphate (substrate) binding site . The ERG9-encoded protein contains a PEST consensus motif (rich in proline, glutamic acid, serine, and threonine) present in many proteins with short cellular half-lives . Modeling of the protein suggests that it contains at least one, and possibly two, membrane-spanning domains . Disruption of the chromosomal squalene synthetase coding region by insertional mutagenesis indicates that ERG9 is a single copy gene that is essential for cell growth in yeast.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6018 - 22
A beta subunit mutation disrupting the catalytic function of Escherichia coli RNA polymerase; Lee J et al.; The substitution of the evolutionarily conserved Glu-813 for lysine in the beta subunit of RNA polymerase (RNAP) causes a partial loss of function in the assembled RNAP . In the presence of the four ribonucleoside triphosphates, the mutant RNAP displayed a decreased frequency of promoter clearance and diminished elongation rate . Both defects could be compensated by raising the ribonucleoside triphosphate concentration . In the abortive initiation reaction limited by the incomplete set of ribonucleoside triphosphates, the mutant RNAP generated aberrant patterns of products indicative of their enhanced loss from the RNAP-promoter complex . A model is proposed, attributing the multiple effect of the mutation to the malfunctioning of the RNAP active center.

Gene, 1991 Jul 15, 103(1), 119 - 23
Simultaneous transient expression assays of the trypanosomatid parasite Leishmania using beta-galactosidase and beta-glucuronidase as reporter enzymes; LeBowitz JH et al.; We describe a transient transfection protocol for cultured Leishmania major promastigotes, utilizing Escherichia coli genes encoding beta-galactosidase and beta-glucuronidase inserted into an expression vector derived from the dihydrofolate reductase-thymidylate synthase locus . Less than 0.1 pg of either reporter enzyme can be detected with a simple fluorimetric assay, and transfection of 10 micrograms of either reporter construct yields activities at least 100-fold over background . Simultaneous introduction of both constructs showed that the activity of each reporter gene was unaffected by the presence of the other, allowing one reporter construct to serve as a control for experimental variability in test gene constructs containing the second reporter gene . These results show that it is feasible to apply transient expression assays to the identification of cis-acting elements of genes encoding nonabundant mRNAs in the genus Leishmania.

Gene, 1991 Jul 15, 103(1), 1 - 9
SOS induction as an in vivo assay of enzyme-DNA interactions; Heitman J et al.; We have constructed strains which are convenient and sensitive indicators of DNA damage and describe their use . These strains utilize an SOS::lac Z fusion constructed by Kenyon and Walker {Proc . Natl . Acad . Sci . USA 77 (1980) 2819-2823} and respond to DNA damage by producing beta-galactosidase . They can be used to characterize restriction systems and screen for restriction endonuclease mutants . Applications include the study of other enzymes involved in DNA metabolism, such as DNA methyltransferases, topoisomerases, recombinases, and DNA replication and repair enzymes.

J Biol Chem, 1991 Jul 15, 266(20), 13311 - 7
Inhibition of degradation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in vivo by cysteine protease inhibitors; Inoue S et al.; 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase is a key regulatory enzyme of cholesterol biosynthesis and is located in the endoplasmic reticulum (ER) . A fusion protein, HMGal, consisting of the membrane domain of HMG-CoA reductase fused to Escherichia coli beta-galactosidase and expressed in Chinese hamster ovary (CHO) cells from the SV40 promoter, was previously constructed and was found to respond to regulatory signals for degradation in a similar fashion to the intact HMG-CoA reductase . Degradation of both HMG-CoA reductase and HMGal in CHO cells was enhanced by addition of mevalonate or low density lipoprotein (LDL) . In this report we show that 2 cysteine protease inhibitors, N-acetyl-leucyl-leucyl-norleucinal (ALLN) and N-acetyl-leucyl-leucyl-methioninal (ALLM), completely inhibit the mevalonate- or LDL-accelerated degradation of HMG-CoA reductase and HMGal and also block the basal degradation of these enzymes . It has been shown that in vitro these protease inhibitors inhibit the activities of Ca(2+)-dependent neutral proteases as well as lysosomal proteases, including cathepsin L, cathepsin b, and cathepsin D . However, the mevalonate-accelerated degradation of HMG-CoA reductase and HMGal is not affected by lysosomotropic agents, suggesting that the site of action of these inhibitor peptides in preventing the degradation is not the cathepsins . In brefeldin A-treated cells, where protein export from the ER is blocked, ALLN is still effective in inhibiting the degradation of HMG-CoA reductase and HMGal . These results indicate the involvement of non-lysosomal Ca(2+)-dependent proteases in the basal and the accelerated degradation of HMG-CoA reductase and HMGal . Enzymatic assays in vitro and immunoblot analyses have revealed calpain- and calpastatin-like proteins in CHO cells . The activities and the amount of these proteins do not change under conditions of enhanced degradation, indicating that the levels of these proteins are not subject to mevalonate regulation.

Gene, 1991 Jul 15, 103(1), 79 - 82
A procedure for amino acid sequencing in internal regions of proteins; Benhar I et al.; We describe a novel procedure for determining the amino acid (aa) sequence of the internal regions of proteins . This procedure has been implemented by directly determining the sequence of aa 65-75 of the product of the trpR gene of Escherichia coli, the trp repressor . This method is based on the insertion of the cleavage site of a specific protease (factor Xa) into the protein immediately before the region to be sequenced by Edman degradation . The simplicity of the procedure makes it appealing for studies of protein structure-function relationships, and of the expression of genetic information . The method is particularly useful when there is ambiguity concerning the co-linearity of the aa and nucleotide sequences.

Gene, 1991 Jul 15, 103(1), 73 - 7
A marker-coupled method for site-directed mutagenesis; Shen TJ et al.; A marker-coupled method for site-directed mutagenesis (SDM) has been developed . In this method, target DNA is first cloned into a plasmid vector which carries an inactivated tetracycline-resistance (TcR)-encoding tet gene . Using this cloned plasmid as template, polymerase chain reaction (PCR) is performed with a mutagenic primer and a marker primer . The mutagenic primer contains the desired mutations to be introduced into the target DNA, and the marker primer contains a mutation for restoring the activity of the inactivated tet gene . The PCR product is annealed with a gapped duplex plasmid template, extended and ligated in vitro . The resulting uni-strand-mutated plasmid is converted into the gapped duplex form, transformed into Escherichia coli JM109 and spread on yeast extract/tryptone culture medium + Tc plates . The TcR colonies grown on these plates all carry active tet genes . Due to the 'tight coupling' between the marker primer and the mutagenic primer formed in the PCR product, these TcR colonies should also carry the mutagenic primer, e.g., the desired mutations in the target DNA . In fact, practically all of the TcR colonies have been found to be the desired mutants in the present experiments . Therefore, this method provides a very efficient approach for SDM.

Gene, 1991 Jul 15, 103(1), 25 - 30
Participation of the histone-like protein HU and of IHF in minichromosomal maintenance in Escherichia coli; Kano Y et al.; The closely related Escherichia coli genes, hupA, hupB, himA and himD (hip), encode the bacterial histone-like protein subunits, HU-2, HU-1, IHF chi and IHF beta, respectively . We report here that E . coli minichromosomes {plasmids (2.7-12.2 kb) with oriC} carrying the intact mioC region were unable to transform mutants deficient in both HU and integration host factor (IHF), whereas they could transform mutants deficient in either HU or IHF as efficiently as the wild-type strain . Minichromosomes carrying a deletion of the proximal part of mioC or a DnaA box just upstream from mioC could not transform cells deficient in IHF, but could transform cells deficient in HU . These results suggested that HU and IHF participate in minichromosomal replication from oriC in E . coli.

Gene, 1991 Jul 15, 103(1), 125 - 30
An improved transformation vector for the lignin-degrading white rot basidiomycete Phanerochaete chrysosporium; Randall T et al.; In this study, a lignin peroxidase-encoding gene (LIP) of Phanerochaete chrysosporium was disrupted by inserting into its coding region the kanamycin-resistance determinant from Tn903 . The resulting recombinant plasmid, pUGLG1: kan, was transformed into P . chrysosporium with the expectation that the disrupted gene might replace the homologous LIP gene in the chromosome . However, the results showed that pUGLG1: kan sequences do not integrate into the chromosome; instead, the plasmid is maintained intact in the transformants in an extrachromosomal state . Our data also show that pUGLG1: kan undergoes replication in P . chrysosporium, is maintained as a circular element, is recoverable from meiotic and mitotic progeny, although at a low frequency, and can be recovered intact by Escherichia coli transformation . These results suggest that the GLG1 component of pUGLG1: kan contains as yet unidentified sequences that allow its autonomous replication in P . chrysosporium transformants.

Gene, 1991 Jul 15, 103(1), 113 - 8
Analysis of the cbhE' plasmid gene from acute disease-causing isolates of Coxiella burnetii; Minnick MF et al.; A gene termed cbhE' was cloned from the QpH1 plasmid of Coxiella burnetii . Expression of recombinants containing cbhE' in vitro and in Escherichia coli maxicells, produced an insert-encoded polypeptide of approx . 42 kDa . The CbhE protein was not cleaved when intact maxicells were treated with trypsin . Hybridizations of total DNA isolated from the six strains of C . burnetii indicate that this gene is unique to C . burnetii strains associated with acute disease, i.e., Hamilton{I}, Vacca{II}, and Rasche{III} . The cbhE' gene was not detected in strains associated with chronic disease (Biotzere{IV} and Corazon{V}) or the Dod{VI} strain . The cbhE' open reading frame (ORF) is 1022 bp in length and is preceded by a predicted promoter/Shine-Dalgarno (SD) region of TCAACT(-35)-N16-TAAAAT(-10)-N14-AGAAGGA (SD) located 10 nucleotides (nt) before the presumed AUG start codon . The ORF ends with a single UAA stop codon and has no apparent Rho-factor-independent terminator following it . The cbhE' gene codes for the CbhE protein of 341 amino acid (aa) residues with a deduced Mr of 39,442 . CbhE is predominantly hydrophilic with a predicted pI of 4.43 . The function of CbhE is unknown . No nt or aa sequences with homology to cbhE' or CbhE, respectively, were found in searches of a number of data bases.

Blood, 1991 Jul 15, 78(2), 357 - 63
C4b-binding protein exacerbates the host response to Escherichia coli; Taylor F et al.; Activated protein C is a plasma anticoagulant . For activated protein C to function as an anticoagulant, it must form a complex with protein S . Protein S anticoagulant activity is neutralized by formation of a reversible complex with C4b binding protein (C4bBP) . C4bBP is an acute-phase plasma protein . When C4bBP levels increase, mass action forces the level of free protein S to decrease, giving rise to an acquired functional protein S deficiency . It has been proposed that these elevated C4bBP levels and the resultant acquired deficiency of protein S that occurs in inflammation could contribute to a hypercoagulable state . An experimental model to test this hypothesis was suggested by our previous studies that demonstrated that inhibition of protein C activation rendered baboons hypercoagulable in response to sublethal Escherichia coli infusion (J Clin Invest 79:918, 1987) . We have extended these studies to examine the effect of inhibition of protein S activity with C4bBP in the host (baboon) response to infusion of sublethal concentrations of E coli organisms . Five sets of animals were studied: (1) those challenged with sublethal concentrations of E coli alone (0.4 x 10(10)/kg); (2) those supplemented only with C4bBP (20 mg/kg); (3) those challenged with the same level of E coli but supplemented with C4bBP (20 mg/kg); (4) those challenged with sublethal E coli and supplemented with C4bBP (20 mg/kg) and sufficient protein S (2.3 mg/kg) to fill the protein S binding sites on C4bBP; and (5) those challenged with lethal concentrations of E coli . Sublethal E coli infusion (group 1 animals) caused only an acute-phase response with no consumption of fibrinogen, detectable organ damage, or detectable tumor necrosis factor (TNF) in the plasma . C4bBP infusion (group 2 animals) resulted in no significant physiologic changes, no detectable plasma TNF, and little change in fibrinogen level . The group 3 animals, receiving both sublethal E coli and C4bBP, exhibited rapid consumption of fibrinogen, systemic organ damage, and detectable circulating TNF ultimately leading to death . The overall response of this group was very similar to the response of the group 5 animals receiving an LD100 dose of E coli . The group 4 animals, which were treated exactly as above except that C4bBP was supplemented with a slight excess of protein S, responded essentially like those that received sublethal E coli alone . These studies suggest that the elevation of C4bBP during an inflammatory response can contribute to fibrinogen consumption and vascular damage . This vascular damage may be associated with enhanced elaboration of cytokines like TNF.(ABSTRACT TRUNCATED AT 400 WORDS)

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6063 - 7
Resolution of Holliday junctions in vitro requires the Escherichia coli ruvC gene product; Connolly B et al.; In previous studies, Holliday junctions generated during RecA-mediated strand-exchange reactions were resolved by fractionated Escherichia coli extracts . We now report the specific binding and cleavage of synthetic Holliday junctions (50 base pairs long) by a fraction purified by chromatography on DEAE-cellulose, phosphocellulose, and single-stranded DNA-cellulose . The cleavage reaction provided a sensitive assay with which to screen extracts prepared from recombination/repair-deficient mutants . Cells with mutations in ruvC lack the nuclease activity that cleaves synthetic Holliday junctions in vitro . This deficiency was restored by a multicopy plasmid carrying a ruvC+ gene that overexpressed junction-resolving activity . The UV sensitivity and deficiency in recombinational repair of DNA exhibited by ruv mutants lead us to suggest that RuvC resolves Holliday junctions in vivo.

Gene, 1991 Jul 15, 103(1), 31 - 6
Construction and characterization of an Escherichia coli mutant with a deletion of the metZ gene encoding tRNA (f1Met); Kenri T et al.; The Escherichia coli metZ gene encoding tRNA (f1Met) was replaced by the chloramphenicol-resistance-encoding gene . The resulting mutant exhibited slightly lower growth rates as compared to the wild type at 37 degrees C or 42 degrees C, but grew apparently slower than the latter at 30 degrees C, indicating a slight cold sensitivity of growth . beta-Galactosidase was produced efficiently from the start codon AUG of the intact lacZ gene or trpA'::lac' Z fusion gene, in the metZ deletion mutant . The lac repressor from the lacI gene and the chimeric protein from a hupB' ::lac'Z fusion gene, whose start codons are GUG, were also synthesised in the deletion mutant . These results provide evidence that tRNA (f1Met) is not essential for growth of E . coli and that the start codons, AUG and GUG, are both recognized by tRNA (f1Met), a minor N-formyl methionine-specific tRNA, in the tRNA (f1Met)-depleted cells.

Biochem J, 1991 Jul 15, 277 ( Pt 2), 469 - 75
Isolation, characterization and sequence analysis of a full-length cDNA clone encoding acetohydroxy acid reductoisomerase from spinach chloroplasts; Dumas R et al.; Acetohydroxy acid reductoisomerase (AHRI), the second enzyme in the parallel isoleucine/valine-biosynthetic pathway, catalyses an unusual two-step reaction in which the substrate, either 2-acetolactate or 2-aceto-2-hydroxybutyrate, is converted via an alkyl migration and an NADPH-dependent reduction to give 2,3-dihydroxy-3-methylbutyrate or 2,3-dihydroxy-3-methylvalerate respectively . We have isolated and characterized a full-length cDNA from a lambda gt11 spinach library encoding the complete acetohydroxy acid reductoisomerase protein precursor . The 2050-nucleotide sequence contains a 1785-nucleotide open reading frame . The derived amino acid sequence indicates that the protein precursor consists of 595 amino acid residues including a presequence peptide of 72 amino acid residues . The N-terminal sequence of the first 16 amino acid residues of the purified AHRI confirms the identity of the cDNA . The derived amino acid sequence from this open reading frame shows 23% identity with the deduced amino acid sequences of the Escherichia coli and Saccharomyces cerevisiae AHRI proteins . There are two blocks of conserved amino acid residues in these three proteins . One of these is a sequence similar to the 'fingerprint' region of the NAD(P)H-binding site found in a large number of NAD(P)H-dependent oxidoreductases . The other, a short sequence (Lys-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Ser-His-Gly-Phe) containing the amino acids lysine and histidine, could well be the catalytic site of the first step of the AHRI reaction . Southern-blot analysis indicated that AHRI is encoded by a single gene per haploid genome of about 7.5 kbp containing at least four introns.

J Biol Chem, 1991 Jul 15, 266(20), 13318 - 23
Molecular cloning of cDNA for lipopolysaccharide-binding protein from the hemolymph of the American cockroach, Periplaneta americana . Similarity of the protein with animal lectins and its acute phase expression; Jomori T et al.; A previous paper described the purification of a calcium-dependent lipopolysaccharide-binding protein from the hemolymph of Periplaneta americana (Jomori, T., Kubo, T., and Natori, S . (1990) Eur . J . Biochem . 190, 201-206) . This paper describes the molecular cloning and characterization of cDNA for the LPS-binding protein . This protein was found to have a carbohydrate-recognition domain at its carboxyl terminus containing amino acid sequences that are conserved in various mammalian C-type lectins . It was also shown to contain an N-linked carbohydrate chain, and the amino acid residue carrying this chain was assigned as Asn at position 56 (23rd amino acid residue from the amino terminus) . Northern blot analysis revealed the presence of multiple mRNAs that hybridized with this cDNA and transient increases in their content after injection of Escherichia coli into adult Periplaneta, suggesting that the LPS-binding protein plays a role in the acute phase response of this insect.

J Biol Chem, 1991 Jul 15, 266(20), 13303 - 10
Discrete functional stages of vaccinia virus early transcription during a single round of RNA synthesis in vitro; Luo Y et al.; We have developed a system for analysis of discrete steps in vaccinia virus early mRNA synthesis during a single round of transcription in vitro . A synthetic early promoter is used to direct transcription by vaccinia RNA polymerase of a G-less cassette in linear duplex DNA . Omission of GTP from transcription reactions leads to the formation of ternary elongation complexes paused stably at the end of the G-less cassette . These complexes can be induced to elongate by provision of GTP . While initiation of transcription is sensitive to low concentrations of salt and Sarkosyl, elongation is relatively resistant to these agents . Termination can be studied in a single synthetic cycle by forming transcription complexes paused just proximal to the termination signal TTTTTNT that can subsequently elongate and terminate . By selectively incorporating the termination-inhibiting analog BrUMP into proximal and distal portions of the nascent transcript, we localize the termination signal within or near the sequence UUUUUNU in the nascent RNA . We show that access of the vaccinia termination factor (VTF/capping enzyme) to the transcriptional apparatus can occur subsequent to initiation and synthesis of a 390-nucleotide nascent RNA . Termination is more sensitive to inhibition by salt and Sarkosyl than in elongation . This sensitivity is not reversed by preincubation of VTF with the transcription complex . Finally, we confirm the identity of VTF and vaccinia mRNA capping enzyme by demonstration of VTF activity associated with capping enzyme expressed in Escherichia coli.

J Biol Chem, 1991 Jul 15, 266(20), 13296 - 302
Isoenzymes of horse liver alcohol dehydrogenase active on ethanol and steroids . cDNA cloning, expression, and comparison of active sites; Park DH et al.; Horse liver alcohol dehydrogenase occurs as isoenzymes: E is active on ethanol but not steroids; S is active on ethanol and steroids . The cDNAs for these isoenzymes were cloned; both were 1.8-kilobase long and contained complete coding sequences . Both enzymes were expressed in Escherichia coli, and the purified proteins had properties similar to those of the natural enzymes . The amino acid sequence deduced from the open reading frame of the E-type cDNA agreed with the amino acid sequence of the E isoenzyme determined by protein sequencing and x-ray crystallography . When compared with the E-type cDNA, the coding region of the S-type cDNA contains 24 substitutions and 3 deletions, giving rise to an amino acid sequence for the S . isoenzyme that differs from that of the E isoenzyme at 10 positions: nine conservative substitutions and one deletion, of Asp-115 . These changes can be accommodated in the three-dimensional structure of the E isoenzyme, and models of the E and S isoenzymes complexed with a 3 beta-hydroxy-5 beta-steroid were built . The modeling shows that Leu-116 apparently sterically hinders binding of steroids in the E isoenzyme, and deletion in the S isoenzyme of Asp-115 moves Leu-116 and relieves the hindrance . The human gamma and rat liver enzymes are also active on steroids, but they have a different constellation of amino acid residues in the substrate pocket . Thus, there are multiple bases for the activity on steroids.

J Biol Chem, 1991 Jul 15, 266(20), 13103 - 9
Requirements for the catalysis of strand transfer synthesis by retroviral DNA polymerases; Buiser RG et al.; We have examined the properties of reverse transcriptases (RTs) required for strand transfer synthesis on poly(rA) . In this process, a primer is elongated on one template and then switches to other templates for additional elongation until it is much longer than the templates on which it was made . Models of retrovirus replication require the RT to catalyze two distinct strand transfers . Additionally, they propose that the RT ribonuclease H (RNase H) activity is involved in both transfers . RTs from human immunodeficiency virus (HIV), avian myeloblastosis virus, and murine leukemia virus differ in molecular mass and subunit composition . However, they all catalyzed strand transfer synthesis on (rA)300, generating characteristically long products . An RNase H-deficient enzyme, HIV-RTRD, catalyzed strand transfer synthesis to the same degree as native HIV-RT, indicating that a functional RNase H activity is not required . Additionally, N-ethylmaleimide, which inhibits RNase H but not polymerase activity of HIV-RT, did not diminish strand transfer synthesis . Highly processive DNA synthesis by each RT was found to be required for the strand transfer reaction . RNase H- murine leukemic virus RT has a structural modification that not only eradicates RNase H, but also makes the polymerase much less processive for DNA synthesis . However, conditions that allow this modified enzyme to bind repeatedly to the same primer during synthesis, i.e . conditions that simulate higher processivity, allow strand transfer synthesis . Catalysis of strand transfer synthesis is not a property of all DNA polymerases, since the Klenow fragment of Escherichia coli DNA polymerase I is unable to catalyze this reaction even if high processivity is simulated . These results suggest that strand transfer synthesis relies on an unidentified functional activity present in RTs.

Blood, 1991 Jul 15, 78(2), 382 - 6
The stable prostacyclin analog, iloprost, and prostaglandin E1 inhibit monocyte procoagulant activity in vitro; Crutchley DJ et al.; Exposure of human peripheral blood to 100 ng/mL of bacterial endotoxin for 2 hours resulted in a 20-fold increase in monocyte procoagulant activity . The activity was functionally identified as tissue factor, because it was not expressed in plasma deficient in factor VII and was specifically inhibited by a monoclonal antibody directed against human tissue factor . When the stable prostacyclin analog, iloprost, was added to blood 30 minutes before endotoxin, a dose-dependent inhibition of monocyte procoagulant activity occurred, with an I50 of 20 nmol/L . Prostaglandin E1 (PGE1) produced similar effects, with an I50 of 150 nmol/L . Exposure of THP-1 monocytic cells to 100 ng/mL endotoxin resulted in a threefold increase in procoagulant activity after 2 hours and a 20-fold increase after 6 hours . A 30-minute pretreatment with iloprost or PGE1 again inhibited development of procoagulant activity, with I50 values of 5 nmol/L and 150 nmol/L, respectively . Treatment of THP-1 cells with iloprost 2 hours after exposure to endotoxin significantly inhibited further increases in procoagulant activity . Iloprost was less potent under these conditions, 30% inhibition being obtained at 100 nmol/L and 70% at 1 mumol/L . These results suggest that prostacyclin may be a physiologic modulator of monocyte tissue factor expression; in addition, its stable analog, iloprost, may have clinical potential for the treatment of thrombotic disorders in which elevated monocyte procoagulant activity plays a role.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6142 - 6
Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation; Seger R et al.; Microtubule-associated protein 2 kinase (MAP kinase), which exists in several forms, is a protein serine/threonine kinase that participates in a growth factor-activated protein kinase cascade in which it activates a ribosomal protein S6 kinase (pp90rsk) while being regulated itself by a cytoplasmic factor (MAP kinase activator) . Experiments with recombinant MAP kinase, ERK2, purified from Escherichia coli in a nonactivated form revealed a self-catalyzed phosphate incorporation into both tyrosine and threonine residues . Another MAP kinase, ERK1, purified from insulin-stimulated cells also autophosphorylated on tyrosine and threonine residues . Autophosphorylation of ERK2 correlated with its autoactivation, although both autophosphorylation and autoactivation were slow compared to that occurring in the presence of MAP kinase activator . Therefore, we propose that autophosphorylation is probably involved in the MAP kinase activation process in vitro, but it may not be sufficient for full activation . The specificity toward tyrosine and threonine residues indicates that the MAP kinases ERK1 and ERK2 are members of a group of kinases with specificity for tyrosine as well as serine and threonine residues.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 6013 - 7
Substrate inhibition of the human immunodeficiency virus type 1 reverse transcriptase; Furman PA et al.; Substrate inhibition was observed with the heterodimeric (p66/p51) and the homodimeric (p66/p66, p51/p51) forms of human immunodeficiency virus type 1 reverse transcriptase (RNA-dependent DNA polymerase, EC 2.7.7.49) . An apparent Ki value of 195 +/- 37 microM was determined for dTTP using the bacterial cloned and expressed heterodimer . Similar values were obtained with the homodimeric and the virus-encoded enzymes . When poly-(rC).p(dG)10 was used as template-primer, dGTP exhibited substrate inhibition with an apparent Ki value of 189 +/- 32 microM . Substrate inhibition was not observed with dTTP when DNA.DNA template-primers were used . Hill coefficients for substrate binding determined in the presence of saturating concentrations of template-primer were equal to 1.0, suggesting that substrate inhibition of the heterodimer is not the result of an allosteric mechanism involving the p51 subunit . Furthermore, UV crosslinking experiments with {gamma-32P}dTTP showed crosslinking only to the p66 subunit . Substrate inhibition was not as pronounced with other retroviral reverse transcriptases as it was with human immunodeficiency type 1 reverse transcriptase.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 5993 - 7
Molecular mimicry by Trypanosoma cruzi: the F1-160 epitope that mimics mammalian nerve can be mapped to a 12-amino acid peptide; Van Voorhis WC et al.; Antigenic mimicry by Trypanosoma cruzi antigens that share epitopes with mammalian tissues may drive autoreactive B- or T-cell clones to expand and cause autoimmune pathogenesis . We have been studying one of these antigens, F1-160, a 160-kDa protein on the surface of T . cruzi that antigenically mimics a 48-kDa protein found in mammalian axonal and myenteric plexus cells . The F1-160 antigen has been characterized by cloning and expression of T . cruzi DNA encoding F1-160 in Escherichia coli . Recombinant peptides from various regions of the F1-160 gene were expressed and used to compete with affinity-purified polyclonal anti-F1-160 antibodies binding to nerve . Recombinant 48-amino acid peptide (48X) derived from expression of base pairs 611-761 of the DNA sequence completely inhibited anti-F1-160 binding to nerve . Recombinant peptides expressed from DNA lacking this region did not inhibit anti-F1-160 binding to nerve . Three peptides were synthesized to encompass the 48X peptide, a 12-amino acid peptide and two 18-amino acid peptides . The 12-amino acid peptide TPQRKTTEDRPQ (12X), corresponding to bases 615-651, completely inhibited the binding of anti-F1-160 antibodies to nerve at a concentration of 80 ng/ml (30 microM) . The two 18-residue peptides did not inhibit, even at 10 micrograms/ml . Thus, the epitope of F1-160 crossreactive with nervous tissue can be mapped to a 12-amino acid peptide . Some humans with T . cruzi infection make antibodies to F1-160 and to the 48X and 12X peptides . Control sera from uninfected persons did not react with these antigens . Anti-48X antibodies, immunoselected from human serum with 48X peptide, bind to human nerve axons . This demonstrates that some individuals infected with T . cruzi make antibodies to the F1-160 epitope crossreactive with nervous tissues.

Proc Natl Acad Sci U S A, 1991 Jul 15, 88(14), 5954 - 8
Heparin stimulates epidermal growth factor receptor-mediated phosphorylation of tyrosine and threonine residues; Revis-Gupta S et al.; We have described previously that in extracts of A431 cells epidermal growth factor (EGF) stimulates the phosphorylation of tyrosine as well as of threonine residues in the EGF receptor and in lipocortin 1 . We now report that heparin at low concentrations also stimulates the autophosphorylation of the EGF receptor and of the recombinant 56-kDa domain of the EGF receptor that lacks the EGF binding site . To study the stimulations of phosphorylation of threonine residues, a fusion protein was prepared with glutathione S-transferase (GST) and an EGF receptor fragment, TK8 (residues 647-688), that contains the threonine phosphorylation site but no tyrosine . We show that the phosphorylation of threonine residues in GST-TK8 by extracts of A431 cells is stimulated by heparin but not by EGF . These and other results suggest that heparin acts as a chaperone, a substrate modulator, that enhances the susceptibility of the substrate to phosphorylation by protein kinases.

J Biol Chem, 1991 Jul 15, 266(20), 13044 - 9
Chaperonins facilitate the in vitro folding of monomeric mitochondrial rhodanese; Mendoza JA et al.; In vitro refolding of the monomeric mitochondrial enzyme, rhodanese (thiosulfate sulfurtransferase; EC 2.8.1.1) is facilitated by molecular chaperonins . The four components: two proteins from Escherichia coli, chaperonin 60 (groEL) and chaperonin 10 (groES), MgATP, and K+, are necessary for the in vitro folding of rhodanese . These were previously shown to be necessary for the in vitro folding of ribulose-1,5-bisphosphate carboxylase at temperatures in excess of 25 degrees C (Viitanen, P . V., Lubben, T . H., Reed, J., Goloubinoff, P., O'Keefe, D . P., and Lorimer, G . H . (1990) Biochemistry 29, 5665-5671) . The labile folding intermediate, rhodanese-I, which rapidly aggregates at 37 degrees C in the absence of the chaperonins, can be stabilized by forming a binary complex with chaperonin 60 . The discharge of the binary chaperonin 60-rhodanese-I complex, results in the formation of active rhodanese, and requires the presence of chaperonin 10 . Optimal refolding is associated with a K(+)-dependent hydrolysis of ATP . At lower protein concentrations and 25 degrees C, where aggregation is reduced, a fraction of the rhodanese refolds to an active form in the absence of the chaperonins . This spontaneous refolding can be arrested by chaperonin 60 . There is some refolding (approximately equal to 20%) when ATP is replaced by nonhydrolyzable analogs, but there is no refolding in the presence of ADP or AMP . ATP analogs may interfere with the interaction of rhodanese-I with the chaperonins . Nondenaturing detergents facilitate rhodanese refolding by interacting with exposed hydrophobic surfaces of folding intermediates and thereby prevent aggregation (Tandon, S., and Horowitz, P . (1986) J . Biol . Chem . 261, 15615-15618) . The chaperonin proteins appear to play a similar role in as much as they can replace the detergents . Consistent with this view, chaperonin 60, but not chaperonin 10, binds 2-3 molecules of the hydrophobic fluorescent reporter, 1,1'-bi(4-anilino)naphthalene-S,5'-disulfonic acid, indicating the presence of hydrophobic surfaces on chaperonin 60 . The number of bound probe molecules is reduced to 1-2 molecules when chaperonin 10 and MgATP are added . The results support a model in which chaperonins facilitate folding, at least in part, by interacting with partly folded intermediates, thus preventing the interactions of hydrophobic surfaces that lead to aggregation.

Eur J Biochem, 1991 Jul 15, 199(2), 371 - 8
Recombinant human tyrosine hydroxylase isozymes . Reconstitution with iron and inhibitory effect of other metal ions; Haavik J et al.; Human tyrosine 3-monooxygenase (tyrosine hydroxylase) exists as four different isozymes (TH1-TH4), generated by alternative splicing of pre-mRNA . Recombinant TH1, TH2 and TH4 were expressed in high yield in Escherichia coli . The purified isozymes revealed high catalytic activity {when reconstituted with Fe(II)} and stability at neutral pH . The isozymes as isolated contained 0.04-0.1 atom iron and 0.02-0.06 atom zinc/enzyme subunit . All three isozymes were rapidly activated (13-40-fold) by incubation with Fe(II) salts (concentration of iron at half-maximal activation = 6-14 microM), and were inhibited by other divalent metal ions, e.g . Zn(II), Co(II) and Ni(II) . They all bind stoichiometric amounts of Fe(II) and Zn(II) with high affinity (Kd = 0.2-3 microM at pH 5.4-6.5) . Similar time courses were observed for binding of Fe(II) and enzyme activation . In the absence of any free Fe(II) or Zn(II), the metal ions were released from the reconstituted isozymes . The dissociation was favoured by acidic pH, as well as by the presence of metal chelators and dithiothreitol . The potency of metal chelators to remove iron from the hydroxylase correlated with their ability to inhibit the enzyme activity . These studies show that tyrosine hydroxylase binds iron reversibly and that its catalytic activity is strictly dependent on the presence of this metal.

J Biol Chem, 1991 Jul 15, 266(20), 13161 - 70
Isolation of a DNA helicase from HeLa cells requiring the multisubunit human single-stranded DNA-binding protein for activity; Seo YS et al.; A DNA helicase, dependent on the multisubunit human single-stranded DNA binding protein (HSSB), was isolated from HeLa cells . At low levels of helicase, only the multisubunit SSBs, HSSB and yeast SSB, stimulated DNA helicase activity . At high levels of the helicase Escherichia coli SSB partially substituted for HSSB whereas other SSBs such as T4 gene 32 and adenovirus DNA binding protein did not stimulate the enzyme activity . Maximal activation of helicase activity occurred in the presence of one molecule of HSSB for every 20 nucleotides of single-stranded DNA . The addition of E . coli SSB significantly lowered the amount of HSSB required for strand displacement, suggesting that the HSSB plays at least two roles in the activation of the helicase . One is to bind single-stranded DNA, thereby preventing sequestration of the helicase, the other involves the interaction of the HSSB with the helicase . Monoclonal antibodies that interact with the 70- and 34-kDa subunits of HSSB inhibited its stimulation of the helicase activity . The DNA helicase acted catalytically in displacing duplex DNA and translocated in the 3' to 5' direction . The helicase displaced fragments from both ends of a DNA substrate that contained duplex region at both termini, but the 3' to 5' fragment was displaced 20 times faster than the 5' to 3' fragment . Since this helicase also displaced fully duplex DNA, the release of the 5' to 3' fragment may have occurred by entry of the helicase through the duplex end in a 3' to 5' direction.

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