Escherichia coli

Plant Physiol, 1994 May, 105(1), 269 - 77
Expression of lipoxygenase in wounded tubers of Solanum tuberosum L; Geerts A et al.; A lipoxygenase cDNA clone from Solanum tuberosum L . was analyzed to study the role of lipoxygenases in potato development and wound response . Sequence analysis and comparison of the deduced amino acid sequence revealed high homology to other plant lipoxygenases . Expression of the cDNA sequences in Escherichia coli and subsequent analysis of bacterial protein extracts showed lipoxygenase activity using linoleic, linolenic, or arachidonic acid as substrates . Transcripts encoding the potato lipoxygenase were most abundant in tuber tissue, lower in roots, and hardly detectable in leaves, petioles, and stems . The induction of lipoxygenase expression in tubers by wounding was dependent on various parameters . Whereas lipoxygenase transcript levels increased in discs from stored tubers incubated under aerobic conditions, tubers taken from a growing plant did not accumulate lipoxygenase transcripts in response to wounding . Incubation of tuber discs in buffer did not lead to an increase in lipoxygenase RNA levels; however, methyl jasmonate stimulated lipoxygenase expression after 24 h in stored tubers . Proteinase inhibitor II mRNAs decreased in stored tubers as well as in discs from growing tubers.

Free Radic Biol Med, 1994 May, 16(5), 555 - 9
Paraquat diaphorases in Escherichia coli; Liochev SI et al.; Extracts of E . coli contain at least three easily separable NAD(P)H:paraquat diaphorases . One of these is identified as thioredoxin reductase, which accounts for most of the PQ++ diaphorase in a thioredoxin reductase overproducer but is only 25% of this activity in a wild type . NADP+, but not NAD+, inhibited the diaphorase activity of thioredoxin reductase . All of the soluble PQ++ diaphorases of E . coli are stable during fractionation by HPLC and none depend upon the cooperative action of components separable by this technique . GSSG reductase is inhibited by PQ++ and is not, to any significant degree, a contributor to the diaphorase activity of E . coli.

Free Radic Biol Med, 1994 May, 16(5), 539 - 45
Reactive oxygen inducing vasoconstriction in the isolated perfused rat liver; Ikai I et al.; The effect of reactive oxygen generation on intact livers was studied . Production of reactive oxygen species in perfused livers isolated from normal and endotoxin-treated rats was measured using chemically enhanced chemiluminescence . The resting state chemiluminescence of the livers increased on endotoxin administration and was maximal about 6 h after treatment . Chemiluminescence from the livers was further stimulated severalfold by inclusion of phorbol myristate acetate in the perfusion medium, reaching maximum intensity 3 h after endotoxin treatment . Oxygen consumption by the endotoxin-treated liver showed a transient increase followed by a significant decrease on phorbol myristate acetate stimulation, which was inhibited by dexamethasone . These results are consistent with the occurrence of a respiratory burst followed by oxygen-radical-species-induced vasoconstriction in the intact perfused liver . The evaluation of reactive oxygen species by resident and accumulated macrophages in the intact liver is made possible by these studies, and related effects on the liver could be conveniently and quantitatively followed using this model.

Microbiology, 1994 May, 140 ( Pt 5), 1035 - 43
Molecular analysis of the operon which encodes the RNA polymerase sigma factor sigma 54 of Escherichia coli; Jones DH et al.; The rpoN gene (encoding the sigma factor sigma 54) of Escherichia coli was cloned and its nucleotide sequence determined . Promoter probe analysis confirmed the presence of a promoter in a 350 bp fragment covering the start of rpoN . The likely promoter was identified . The nucleotide sequence of the region extending 2.1 kb downstream of rpoN was also determined . This region contained four open reading frames encoding potential polypeptides of 10750, 17959, 32492 and 9810 Da; maxicell and T7 promoter studies showed that four polypeptides of similar molecular masses were expressed from this region . The amino acid sequence of the 17959 Da polypeptide showed homology to the enzyme IIA domains of several proteins of the bacterial sugar phosphotransferase system (PTS), and the 9810 Da polypeptide showed homology to the HPr proteins of the bacterial PTS . The proteins encoded downstream of rpoN are known to negatively regulate sigma 54 activity . The homologies therefore suggest that this effect on sigma 54 may be mediated by sequential protein phosphorylation and suggest that there is a link between signal transduction and transcription of sigma 54-dependent genes.

Microbiology, 1994 May, 140 ( Pt 5), 1027 - 34
The high-spin cytochrome o' component of the cytochrome bo-type quinol oxidase in membranes from Escherichia coli: formation of the primary oxygenated species at low temperatures is characterized by a slow 'on' rate and low dissociation constant; Poole RK et al.; Cytochromes b and o in membrane vesicles from aerobically grown Escherichia coli were readily reduced by succinate; one cytochrome, which we propose should be called cytochrome o', reacted with CO in the Fe(II) state to give a photodissociable CO adduct . The photodissociation spectrum (photolysed minus pre-photolysis) at sub-zero temperatures had a relatively high gamma/alpha absorbance ratio, indicating a high-spin haem, which, in the reduced state, probably contributes little to the sharp alpha absorbance of the oxidase complex in membranes . Reaction with oxygen of the unliganded high-spin haem between -132 degrees C and -95 degrees C following photolytic activation gave a product that is identified as the oxygenated form, being spectrally similar to, but not identical with, the CO adduct . In membranes, the forward velocity constant at -95 degrees C was 61 M-1s-1, and the dissociation constant was 1.6 x 10(-5) M O2, as it is in intact cells . These data clearly distinguish the oxygen-trapping strategy of the cytochrome o' in this oxidase from that of cytochrome a3 and also suggest that the presence of the soluble flavohaemoglobin (Hmp) in intact cells is without effect on such measurements of the primary oxygen reaction . In view of recent findings that this oxidase complex contains predominantly one mole of haem O and one of haem B, a revised nomenclature for the oxidase complex is proposed, namely, cytochrome bo'.

Biol Reprod, 1994 May, 50(5), 1108 - 12
Lipopolysaccharide-induced fetal death: the role of tumor-necrosis factor alpha; Silver RM et al.; Lipopolysaccharide (LPS) administration has been known to cause murine fetal death for over 50 years, but the responsible mechanism(s) remains unclear . We used the LPS-hyporesponsive murine strain, C3H/HeJ, to 1) establish whether LPS-induced fetal death is due to a maternal or fetal response to LPS and 2) to investigate the involvement of tumor necrosis factor alpha (TNF alpha) in fetal death caused by LPS . C3H/HeJ (LPS-hyporesponsive) or C3H/HeN (LPS-responsive) females were mated with C57B1/6 or C3H/HeN males (both LPS-responsive) . Administration of 10 micrograms LPS caused fetal death in C3H/HeN mothers . However, up to 1000 micrograms LPS did not result in the death of LPS-responsive fetuses when administered to C3H/HeJ mothers . Systemic administration of TNF alpha was able to cause fetal death in both C3H/HeN and C3H/HeJ strains of mice . Pretreatment of pregnant C3H/HeN mice with anti-TNF alpha antibodies significantly reduced fetal death caused by LPS administration . The administration of a sublethal dose of TNF alpha plus 10 micrograms LPS to pregnant C3H/HeJ mice restored abortifacient activity . These results indicate that LPS-induced fetal death is due to a maternal response as opposed to a direct fetal response to LPS and that TNF alpha appears to be an important mediator of fetal death caused by LPS.

DNA Cell Biol, 1994 May, 13(5), 521 - 30
Characterization of a Max:DNA complex by cross-linking to photoactive oligonucleotides; Mullen MA et al.; The structure of the Max:DNA complex was investigated by cross-linking Max to a series of photoactive oligonucleotides . A single photoactive aryl azide was introduced into oligonucleotides at defined positions downstream from the specific CACGTG binding site . Purified Max homodimers bound to and were cross-linked to oligonucleotides containing a photoactive group either 2, 5, 8, or 11 bp downstream from the binding site . Further analysis revealed that an amino-terminal chymotryptic peptide of Max was cross-linked to the oligonucleotide containing a photoactive probe 11 bp downstream from the specific binding site . This result is consistent with the recent crystal structure of the Max:DNA complex (Ferre-D'Amare et al., 1993) and further suggests that amino acid residues near the amino-terminus of Max are in close proximity to a region of DNA that is separated from the core binding site by one turn of the double helix.

Arzneimittelforschung, 1994 May, 44(5), 636 - 41
Comparative in vitro investigations of the influence of mofebutazone, phenylbutazone and diclofenac on phagocytosis and respiratory burst of human peripheral blood leucocytes; Neumuller J et al.; The phagocytosis and the release of oxidative products generated by the respiratory burst have been studied under the influence of the non-steroidal anti-inflammatories (NSAID) phenylbutazone (PB), mofebutazone (monophenylbutazone, MPB) and diclofenac (DF) using phagocytes of the peripheral blood from healthy human donors and patients with soft tissue rheumatism . The measurement of phagocytosis by flow cytometry (FC) was carried out in order to investigate the uptake of FITC-labelled bacteria (E . coli), separately by monocytes (MON) and polymorphonuclear leucocytes (PMN) . In addition the luminol-dependent chemiluminescence (CL) was measured using opsonized Zymosan on leucocytes of the peripheral blood that were purified by lysis of erythrocytes . In FC, PMNs and MONs could be identified by gating in the whole blood in which erythrocytes had been lysed . The NSAID were added to the in vitro tests for 30 min in concentrations of 10(-3) mol/l, 10(-4) mol/l, 10(-5) mol/l, 10(-6) mol/l, and 10(-8) mol/l . Using the FC phagocytosis test it was found that PB and MPB decreased the percentage of phagocytosing PMNs as well as MONs while DF increased it slightly in contrast to the controls . However, the fluorescence intensity of the phagocytes, which indicates the amount of ingested bacteria, was found to be unchanged . PB effected a significant concentration-dependent inhibition of CL in all of the concentrations used, with the exception of 10(-8) mol/l . MPB resulted in a borderline inhibition at 10(-3) mol/l although the measurement of every individual proband showed a concentration dependency . DF inhibited the luminol-dependent CL significantly only at a concentration of 10(-3) mol/l.

Plant J, 1994 May, 5(5), 613 - 23
Sequencing, processing, and localization of the petunia CMS-associated mitochondrial protein; Nivison HT et al.; The petunia mitochondrial fused gene (pcf), which is associated with cytoplasmic male sterility (CMS), is composed of sequences derived from atp9, coxII, and an unidentified reading frame termed urfS . Pcf transcripts are modified by editing at 11 sites . Codon usage and nearest neighbor analysis suggest that the urfS region is not derived originally from a plant mitochondrial coding region . Although the gene contains an open reading frame coding for a 43 kDa protein, a 25 kDa gene product has previously been identified (Nivison and Hanson, 1989) . N-terminal sequencing revealed that the 25 kDa protein is encoded within the urfS portion of pcf and that its actual molecular mass is 19.5 kDa . Through pulse-chase labeling of protein in isolated mitochondria, the 25 kDa protein was found to be processed from a 43 kDa precursor protein representing the entire pcf gene sequence . Antibodies to synthetic peptides encoded by the atp9 and coxII portions of pcf recognized petunia ATP9 or COXII but no other mitochondrial proteins on immunoblots . Controlled proteolysis experiments showed that both the 43 kDa precursor and the 25 kDa protein are soluble or loosely associated with membranes . Thus, the 25 kDa protein appears to be the only pcf-encoded protein that accumulates in mitochondria.

J Biomol NMR, 1994 May, 4(3), 433 - 54
1H, 15N and 13C resonance assignments for the first three zinc fingers of transcription factor IIIA; Liao X et al.; The first three zinc fingers (ZF1-3) of transcription factor IIIA (TFIIIA) from Xenopus have been shown to contribute the majority of the binding energy to the intact TFIIIA-DNA interaction {Liao et al . (1992) J . Mol . Biol., 223, 857-871} . We have expressed a 92-amino acid polypeptide containing the three N-terminal zinc fingers of TFIIIA . This three-fingered polypeptide has been isotopically labeled with 15N and 13C in E . coli and purified to homogeneity . Assignment of backbone 1H, 15N, aliphatic 1H and 13C and aromatic 1H and 13C resonances of delta NZF1-3 has been obtained using a combination of single-, double- and triple-resonance multidimensional NMR experiments . The secondary structures for each finger have been determined from NOE connectivities, 3JNH alpha values and chemical shifts . The results show that each finger folds into a canonical beta-sheet-helix zinc finger structural motif, while the linkers adopt an extended structure . The helix between the two histidine ligands in ZF3 is distorted by zinc coordination, to accommodate the presence of four intervening amino acids instead of three as in ZF1 and ZF2.

J Biomol NMR, 1994 May, 4(3), 411 - 32
Effect of disulfide bridge formation on the NMR spectrum of a protein: studies on oxidized and reduced Escherichia coli thioredoxin; Chandrasekhar K et al.; As a prelude to complete structure calculations of both the oxidized and reduced forms of Escherichia coli thioredoxin (M(r) 11,700), we have analyzed the NMR data obtained for the two proteins under identical conditions . The complete aliphatic 13C assignments for both oxidized and reduced thioredoxin are reported . Correlations previously noted between 13C chemical shifts and secondary structure are confirmed in this work, and significant differences are observed in the C beta and C gamma shifts between cis- and trans-proline, consistent with previous work that identifies this as a simple and unambiguous method of identifying cis-proline residues in proteins . Reduction of the disulfide bond in the active-site Cys32-Gly-Pro-Cys35 sequence causes changes in the 1H, 15N and 13C chemical shifts of residues close to the active site, some of them quite far distant in the amino acid sequence . Coupling constants, both backbone and side chain, show some differences between the two proteins, and the NOE connectivities and chemical shifts are consistent with small changes in the positions of several side chains, including the two tryptophan rings (Trp28 and Trp31) . These results show that, consistent with the biochemical behavior of thioredoxin, there are minimal differences in backbone configuration between the oxidized and reduced forms of the protein.

J Biomol NMR, 1994 May, 4(3), 385 - 410
Evaluation of an algorithm for the automated sequential assignment of protein backbone resonances: a demonstration of the connectivity tracing assignment tools (CONTRAST) software package; Olson JB Jr et al.; The peptide sequential assignment algorithm presented here was implemented as a macro within the CONnectivity TRacing ASsignment Tools (CONTRAST) computer software package . The algorithm provides a semi- or fully automated global means of sequentially assigning the NMR backbone resonances of proteins . The program's performance is demonstrated here by its analysis of realistic computer-generated data for IIIGlc, a 168-residue signal-transducing protein of Escherichia coli {Pelton et al . (1991) Biochemistry, 30, 10043-10057} . Missing experimental data (19 resonances) were generated so that a complete assignment set could be tested . The algorithm produces sequential assignments from appropriate peak lists of nD NMR data . It quantifies the ambiguity of each assignment and provides ranked alternatives . A 'best first' approach, in which high-scoring local assignments are made before and in preference to lower scoring assignments, is shown to be superior (in terms of the current set of CONTRAST scoring routines) to approaches such as simulated annealing that seek to maximize the combined scores of the individual assignments . The robustness of the algorithm was tested by evaluating the effects of imposed frequency imprecision (scatter), added false signals (noise), missing peaks (incomplete data), and variation in user-defined tolerances on the performance of the algorithm.

J Biomol NMR, 1994 May, 4(3), 349 - 66
1H, 15N and 13C resonance assignments, secondary structure, and the conformation of substrate in the binary folate complex of Escherichia coli dihydrofolate reductase; Falzone CJ et al.; By using fully 15N- and 15N/13C-labeled Escherichia coli dihydrofolate reductase, the sequence-specific 1H and 15N NMR assignments were achieved for 95% of the backbone resonances and for 90% of the 13C alpha resonances in the binary folate complex . These assignments were made through a variety of three-dimensional proton-detected 15N and 13C experiments . A smaller but significant subset of side-chain 1H and 13C assignments were also determined . In this complex, only one 15N or 13C resonance was detected per 15N or 13C protein nucleus, which indicated a single conformation . Proton-detected 13C experiments were also performed with unlabeled DHFR, complexed with 13C-7/13C-9 folate to probe for multiple conformations of the substrate in its binary complex . As was found for the protein resonances, only a single bound resonance corresponding to a productive conformation could be detected for C-7 . These results are consistent with an earlier report based on 1H NMR data {Falzone, C.J . et al . (1990) Biochemistry, 29, 9667-9677} and suggest that the E . coli enzyme is not involved in any catalytically unproductive binding modes in the binary complex . This feature of the E . coli enzyme seems to be unique among the bacterial forms of DHFR that have been studied to date.

Plant Mol Biol, 1994 May, 25(2), 167 - 77
The 24 kDa outer envelope membrane protein from spinach chloroplasts: molecular cloning, in vivo expression and import pathway of a protein with unusual properties; Fischer K et al.; The 24 kDa outer envelope membrane protein of spinach chloroplasts (omp24) represents a major constituent of this membrane . Sequences of tryptic and endoprotease Glu-C peptides derived from omp24 allowed the design of oligonucleotides which were used to generate a DNA fragment by polymerase chain reaction using spinach cDNA as template . This fragment served as a probe to screen a cDNA library for a full-length clone of the omp24 coding sequence . The protein predicted from the complete sequence only has 148 amino acids and a molecular mass of 16294 Da . It is an acidic protein (calculated isoelectric point 4.8) with a high content of proline residues . Expression of the coding sequence in Escherichia coli and characterization of the purified recombinant protein produced revealed that the overestimation of its molecular mass by SDS-PAGE (ca . 25 kDa) is due to its abnormal amino acid composition . Despite its rather low hydrophobicity (polarity index 49%), omp24 appears to be deeply embedded in the outer membrane . Insertion of omp24 into the membrane proceeds almost independently of surface receptors or targeting sequence but, in contrast to other known outer envelope membrane proteins, is stimulated by ATP.

FEMS Microbiol Lett, 1994 May 1, 118(1-2), 57 - 63
Identification, isolation and sequencing of the recA gene of Streptomyces lividans TK24; Nussbaumer B et al.; An internal fragment of the recA gene of Streptomyces cattleya was amplified by the polymerase chain reaction (PCR) employing degenerate oligonucleotide primers . Using this fragment as a hybridization probe, a recA homologous gene could be shown in each tested Streptomyces strain . A 4.4 kb BamHI fragment which carried the complete recA gene was isolated from Streptomyces lividans TK24 . Sequence analysis suggested that the coding region of the recA gene consists of 1122 bp . The highest similarity (approximately 78%) could be detected to the recA genes of Mycobacterium tuberculosis and Mycobacterium leprae . After fusion with an E . coli promoter the S . lividans recA gene could partially complement an Escherichia coli recA mutant.

FEMS Microbiol Lett, 1994 May 1, 118(1-2), 51 - 6
Cloning and sequence analysis of a recA-like gene from Streptomyces venezuelae ISP5230; Yao W et al.; When genomic DNA fragments from Streptomyces venezuelae ISP5230 were probed at moderate stringency with recA from Mycobacterium tuberculosis, a 2.0-kb SmaI fragment was identified . The fragment was isolated by cloning a BamHI digest of S . venezuelae DNA in pHJL400 and screening the plasmids in Escherichia coli by Southern hybridization using a sib-selection technique . Sequencing the hybridizing region located an open reading frame encoding 377 amino acids . Its deduced amino acid sequence resembled that of recA genes from other bacteria . The cloned S . venezuelae gene conferred partial resistance to ethyl methanesulfonate when expressed in E . coli from the lacZ promoter.

FEMS Microbiol Lett, 1994 May 1, 118(1-2), 167 - 73
Identification of an Escherichia coli periplasmic acid phosphatase containing of a 27 kDa-polypeptide component; Rossolini GM et al.; An acid phosphatase containing a 27-kDa polypeptide component has been identified in Escherichia coli by means of a zymogram technique . The enzyme is secreted in the periplasmic space and is able to hydrolyze several organic phosphate esters, but not diesters, showing preferential activity on p-nitrophenyl phosphate and other phenolic phosphate esters . Production of the enzyme apparently occurs only in cells growing on carbon sources other than glucose.

Biophys Chem, 1994 May, 50(1-2), 47 - 61
De novo design and creation of a stable artificial protein; Tanaka T et al.; Protein de novo design has been performed, as an exercise of the inverse folding problem . A beta/alpha-barrel protein was designed and synthesized using the Escherichia coli expression system for the structural characterization . A tertiary model with a two-fold symmetry was built, based upon the geometrical parameters extracted from X-ray crystal structures of several beta/alpha-barrel proteins . Amino acid frequencies at each position on the alpha- and beta-structures were investigated, and an amino acid sequence with 201 residues was designed . The associated gene was chemically synthesized and the fusion protein with human growth hormone was expressed in Escherichia coli . The purified protein after being cleaved and refolded was found to be stable and globular with the large amount of secondary structures . However, it has similar characteristics to the molten globules of natural proteins, with loose packing of side-chains . The approach for the tight packing is discussed.

Dev Comp Immunol, 1994 May-Jun, 18(3), 193 - 200
Mutation "white pupae" in the integument of Ceratitis capitata affects both defense and melanogenesis; Charalambidis ND et al.; Studying defense and melanogenesis processes in the cuticle of the "white pupae" (wp) and "dark pupae" (dp) mutant strains of Ceratitis capitata, we showed that both processes function equally well only in the cuticle of dp mutants, as in the wild-type cuticle . The cuticle of wp mutants lacks the ability to form Escherichia coli aggregates and to melanize in vivo . However, in this mutant, tyrosinase and dopachrome conversion factor activities, as well as melanin content and nonself-recognition proteins are expressed as in the wild strain . The present results indicate that the inability of wp mutant cuticle to immobilize E . coli seems to be due to lack of suitable site(s) on nonself-recognition proteins for adduct formation with tyrosine derivatives by the action of tyrosinase, and the inability to melanize, very probably due to deficiency of tyrosine derivatives (tanning precursors).

Res Microbiol, 1994 May, 145(4), 333 - 40
Easy-to-perform modified Elek test to identify Shiga-like toxin-producing diarrhoeogenic Escherichia coli; Germani Y et al.; We determined whether Shiga-like toxin I (SLT-I) -producing diarrhoeogenic Escherichia coli could be detected by a modified Elek tests . The test (SLT Elek test) is based on the principle of the Elek test and the Ouchterlony double-gel diffusion . The development of the SLT Elek test was preceded by a preliminary study; the purpose of the later was to establish whether a simplified purification procedure of SLT-I (involving bacterial sonic extract, "Affi-Gel Blue" chromatography and anion- and cation-exchange liquid chromatography) could be employed in the preparation of rabbit antisera to SLT-I . SLT-I-specific antisera were obtained after adsorption of sera with bacterial sonic extract from non-toxigenic E . coli . A total of 135 strains of E . colo were tested by the SLT Elek test (100 SLT-I-negative and 35 SLT-I-positive) . The results of the SLT Elek test and the Vero cell test correlated well: 30 strains gave positive results and 100 strains gave negative results in both tests . Only 5 strains gave discrepant results: they were weakly positive in the Vero cell assay, whereas 3 gave borderline reactions and 2 were negative in the SLT Elek test . Positive predictive value was 1, negative predictive value was 0.98; the SLT Elek test was 91% sensitive and 100% specific.

Res Microbiol, 1994 May, 145(4), 309 - 25
Ufr/s variation in Escherichia coli K12: a reversible double-mutation or alternate chromosome expression in non-complementing diploids?
Gratia JP.
A novel form of bacterial variation found in an FhuA- mutant of Escherichia coli K12 was characterized by the alternation of (1) simultaneous resistance to lipopolysaccharide-specific phage U3 and to FhuA-specific agents (Ufr phenotype); and (2) a return to the sensitivity pattern of the initial strain (Ufs) . In Ufr cells, loss of the U3 receptor permitted C21 adsorption without modifying the sensitivity to other tested phages or colicins . Genetic analysis revealed that Ufr variants were altered at two distinct loci . Ufr bacteria, though derived from a strain F- devoid of classical gene transfer mechanisms, were transiently able to promote mating between themselves and, to some extent, with other bacteria, including Rec- . Heterogenic matings resulted in the formation of persistent heterozygotes segregating Ufr- and Ufs-like bacteria . Pedigree analysis and subcloning of heterozygotic isolates indicate that they were diploids, as was the initial Ufr strain . Functional genetic complementation between these two genomes was only transient and the alternative forms were likely to result from the expression of a single chromosome of the heterozygotes . Mutation occurred in either form without causing any change in the alternative form.

Mikrobiologiia, 1994 May-Jun, 63(3), 450 - 6
{Effect of over-synthesis of cloned gene products on methanol metabolism in the recombinant methylotrophic yeast Hansenula polymorpha}; Gorlatova NV et al.; As is shown expression homologous (dihydroxyacetone kinase) and heterologous (HBsAg, beta-galactosidase) genes in methylotrophic yeasts Hansenula polymorpha DL1 negatively affects on the growth parameters of a host strain . The reducing of specific growth rate (mu max) and yield of biomass per the unit of a consumed substrate (Yx/s) were found in all recombinant strains grown on methanol . Overproduction of dihydroxyacetone kinase and beta-galactosidase in recombinant H . polymorpha was accompanied by two-fold increasing of the activity of alcohol oxidase, which is the first enzyme of methanol oxidation . Otherwise, the activity of formaldehyde dehydrogenase two-fold decreased in the recombinant strain overproducing HBsAg compared with the host strain . It is suggested that the over-synthesis of foreign proteins requiring an additional energetic and metabolic expenses might reduce the growth parameters and the activities of some enzymes of methanol metabolism in recombinant methylotrophic yeast H . polymorpha.

J Struct Biol, 1994 May-Jun, 112(3), 216 - 30
Transmission electron microscopy of GroEL, GroES, and the symmetrical GroEL/ES complex; Harris JR et al.; Two new 2-D crystal forms of the Escherichia coli chaperone GroEL (cpn60) 2 x 7-mer have been produced using the negative staining-carbon film (NS-CF) technique . These 2-D crystals, which contain the cylindrical GroEL in side-on and end-on orientations, both possess p21 symmetry, with two molecules in the respective unit cells . The crystallographically averaged images correlate well with those obtained by other authors from single particle analysis of GroEL and our own previous crystallographic analysis . 2-D crystallization of the smaller chaperone GroES (cpn10) 7-mer has also been achieved using the NS-CF technique . Crystallographically averaged images of GroES single particle images indicate considerable variation in molecular shape, which is most likely due to varying molecular orientation on the carbon support film . The quaternary structure of GroES does, nevertheless, approximate to a ring-like shape . The complex formed by GroEL and GroES in the presence of ATP at room temperature has been shown to possess a symmetrical hollow ellipsoidal conformation . This symmetrical complex forms in the presence of a 2:1 or greater molar ratio of GroES:GroEL . At lower molar ratios linear chains of GroEL form, apparently linked by GroES in a 1:1 manner, which provide supportive evidence for the ability of both ends of the GroEL cylinder to interact with GroES . The apparent discrepancy between our data and that of other groups who have described an asymmetrical "bullet-shaped" (holo-chaperone) GroEL/ES complex is discussed in detail.

Minerva Chir, 1994 May, 49(5), 429 - 31
{The conservative treatment of infected vascular prostheses in surgery to revascularize the lower limbs}; Celoria G et al.; Infected vascular grafts are associated with very high rates of limb loss and mortality . "Classic" treatment has invariably included graft excision . Recent reports have suggested that a more conservative approach may be indicated in selected cases, leaving the graft in place and using an aggressive local treatment associated with appropriate intravenous antibiotics . The authors report their experience with two patients with infected prosthetic vascular grafts in the groin . They both had purulent drainage from the groin wound, with the graft exposed close to the femoral anastomosis . They were both treated successfully without graft removal, and both graft maintained patency, with a follow-up of 22 and 19 months.

J Biochem (Tokyo), 1994 May, 115(5), 958 - 64
Hot spots for sulfhydryl inactivation of Cys mutants in the widely conserved sequence motifs of the metal-tetracycline/H+ antiporter of Escherichia coli; Yamaguchi A et al.; Unique hot spots for sulfhydryl inactivation of Cys mutants of the metal-tetracycline/H+ antiporter (TetA) were found at the fourth positions of the dual conserved sequence motifs, GKXXDRXGRR and GRXXXKXGEK {Yamaguchi, A., Kimura, T., Someya, Y., & Sawai, T . (1993) J . Biol . Chem . 268, 6496-6504} . The fourth positions of these motifs are occupied by Ser65 and Ala269, respectively . N-Ethylmaleimide (NEM) rapidly bound to and inactivated the S65C and A269C mutants . M64C and I268C showed low reactivity to NEM, probably due to the partial cripticity of these residues . In contrast, NEM rapidly bound to T270C but did not inactivate it . NEM completely inactivated S65C, whereas a small sulfhydryl reagent, methyl methanethiosulfonate (MMTS), caused only 40% inactivation . {14C}NEM binding to S65C was inhibited by tetracycline . These observations indicated that position 65 is located close to the substrate-protein interaction site and that the inactivation by sulfhydryl reagents comprises volume-dependent steric hindrance . The S65M mutant, having a side chain analogous to one of the thiomethyl cysteines of MMTS-modified S65C, showed about 30% of the Vmax value for the wild-type tetracycline transport, while the S65F mutant had completely lost the activity, confirming the idea of volume-dependent steric hindrance at position 65 . On the other hand, the A269C mutant was greatly inactivated by both NEM and MMTS . The degree of the inactivation by MMTS (90%) was higher than that of NEM (80%) . The synergetic inactivation observed for the S65C/A269C double mutant suggested different inactivation mechanisms for A269C and S65C.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1994 May, 115(5), 1021 - 6
Archae-opsin expressed in Escherichia coli and its conversion to purple pigment in vitro; Sugiyama Y et al.; Archae-opsin-1 (aO-1) has been expressed efficiently as a fusion protein with 13 heterologous amino acids at the amino terminus of the mature aO-1 in Escherichia coli under the control of T7 promoter . The E . coli-expressed aO-1, designated as aO-1002, which was located in the membrane fraction, was extracted with 8 M urea and partially purified by gel filtration chromatography in the presence of SDS . When all-trans retinal was added, aO-1002 in dimyristoylphosphatidylcholine and detergent-mixed micelles was converted to a purple pigment with lambda max at 558 nm at 20 degrees C via a 435/460 nm intermediate . Conversion of the intermediate to purple pigment was the rate-limiting step and proceeded as a two-state transition, because an isosbestic point was seen at 485 nm . Similar spectral changes were also observed in the regeneration process of hydroxylamine-bleached claret membranes and aO-1 isolated from claret membranes . Thus, the polypeptide of aO-1002 is considered to fold and form a retinal binding pocket in phospholipid and detergent micelles similarly to aO-1 isolated from the halobacterial membranes . Purple pigment showed a light-driven proton-pumping activity when reconstituted into phosphatidylcholine liposomes.

Biochem Mol Biol Int, 1994 May, 33(2), 377 - 84
Altered conformation and increased strand breaks in neuronal and astroglial DNA of aging rat brain; Bhaskar MS et al.; Melting temperatures (Tm) of the DNA isolated from young, adult, and old rat brain neurons and astrocytes were recorded under different conditions . There was a rise in Tm and decrease in hyperchromicity in the old when compared to the young and adult . Single and double strand breaks were assessed by using nick translation type incubation of DNA with E . coli Pol I and addition of nucleotides at the terminal 3'-OH by calf thymus terminal deoxynucleotidyl transferase . Results show that DNA from old brain cells is more compact in conformation . However, there is also an increase in the number of single and double strand breaks with age in both neuronal and astroglial DNA.

Appl Biochem Biotechnol, 1994 May-Jun, 47(2-3), 175 - 89; discussion 189-90
Phage display of enzymes and in vitro selection for catalytic activity; Soumillion P et al.; Despite recent progress, our understanding of enzymes remains limited: the prediction of the changes that should be introduced to alter their properties or catalytic activities in an expected direction remains difficult . An alternative to rational design is selection of mutants endowed with the anticipated properties from a large collection of possible solutions generated by random mutagenesis . We describe here a new technique of in vitro selection of genes on the basis of the catalytic activity of the encoded enzymes . The gene coding for the enzyme to be engineered is cloned into the genome of a filamentous phage, whereas the enzyme itself is displayed on its surface, creating a phage enzyme . A bifunctional organic label containing a suicide inhibitor of the enzyme and a ligand with high affinity for an immobilized receptor are constructed . On incubation of a mixture of phage enzymes, those phages showing an activity on the inhibitor under the conditions of the experiment are labeled . These phages can be recovered by affinity chromatography . The design of the label and the factors controlling the selectivity of the selection are analyzed . The advantages of the technique and its scope in terms of the enzymes that can be engineered are discussed.

J Biolumin Chemilumin, 1994 May-Jun, 9(3), 113 - 22
Low-light imaging technology in the life sciences; Hooper CE et al.; Photon imaging is an increasingly important technique for the measurement and analysis of chemiluminescence and bioluminescence . New high-performance low-light level imaging systems have recently become available for the life sciences . These systems use advances in camcera design and digital image processing and are now being used for a wide range of luminescence applications . They offer good sensitivity for photon detection and large dynamic range, and are suitable for quantitative analysis . This is achieved using a range of software techniques including image arithmetic, histogramming or summing regions of interest, feature extraction and multiple image processing for kinetics or assay screening . Improvements in image-processing hardware and software have increased the usefulness of these systems in the biosciences . Low-light imaging is a rapid and non-invasive method for the sensitive detection and analysis of luminescent assays . As such it offers a powerful and sensitive tool for investigating processes, both at the cellular level (luc and lux reporter genes, intracellular signalling) and for measurement of macro samples (immunoassays, gels and blots, tissue sections).

Somat Cell Mol Genet, 1994 May, 20(3), 233 - 42
Removal of cyclobutane pyrimidine dimers from a UV-irradiated shuttle vector introduced into human cells; Ganesan AK et al.; A shuttle vector (pZH-1) carrying the E . coli lacZ gene under control of the SV40 early promoter was irradiated with UV and introduced into repair-proficient or repair-deficient human cell lines . The expression of irradiated lacZ compared to unirradiated lacZ was greater in repair-proficient cells (HT-1080) than in repair-deficient cells (XP12RO-SV40) belonging to xeroderma pigmentosum complementation group A . To ascertain whether the expression of lacZ in the repair-proficient cells was correlated with the removal of cyclobutane pyrimidine dimers (CPDs), we purified DNA from the recipient cells and used the CPD-specific enzyme T4 endonuclease V to measure the frequency of CPDs remaining in the plasmid as a whole and in two restriction fragments derived from it . We found that removal of CPDs occurred in both fragments in the repair-proficient cells but not in the repair-deficient cells . Our results provide the first direct evidence for the removal of CPDs from UV irradiated plasmids introduced into human cells and support the notion that expression of the UV-damaged lacZ gene in repair-proficient human cells reflects the removal of transcription blocking lesions from the gene.

Mutagenesis, 1994 May, 9(3), 245 - 51
Induction of the SOS response and mutations by reactive oxygen-generating compounds in various Escherichia coli mutants defective in the mutM, mutY or soxRS loci; Kato T et al.; Derivatives of E . coli WP2s (uvrA trpE) defective in 7,8-dihydro-8-oxoguanine (8-OG) DNA glycosylase activity (mutM), MutY glycosylase activity on an A:8-OG mispair (mutY), and/or an adaptive response to oxidative stress by superoxide (soxRS) were constructed to compare the mutability to various reactive oxygen-generating compounds . Induction of Trp+ reversion was assayed both in the presence and absence of plasmid pKM101 . Phenazine methosulfate and phenazine ethosulfate showed mutagenic activity at a relatively low dose in soxRS mutants . In comparison to the parent strain WP2s, however, the introduction of mutM, mutY, or soxRS mutations, in any combination, did not make the strain hypersensitive in terms of mutability (i.e . mutation induction at relatively low doses) to hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, or phenylhydrazine . Mutagenicity of formaldehyde was detected only in the pKM101-carrying strains . On the other hand, bleomycin, menadione, plumbagin, paraquat, and diquat were not mutagenic to any strain, with or without pKM101 . The SOS-response inducing activity was measured by monitoring the expression of a umu'-'lacZ fusion gene, carried on the plasmid pSK1002 . The induction of the SOS response by hydrogen peroxide, cumene hydroperoxide, t-butyl hydroperoxide, formaldehyde, phenylhydrazine, and bleomycin was of almost the same magnitude between the parent strain and a mutM or soxRS mutant . Phenazine methosulfate and phenazine ethosulfate induced the SOS response only in the soxRS derivatives . No induction was detected by treatment with redox-cycling compounds, such as menadione, plumbagin, paraquat, or diquat.

Mutagenesis, 1994 May, 9(3), 183 - 5
Measurement of gene mutation in vivo using Muta Mouse and positive selection for lacZ- phage; Dean SW et al.; The use of transgenic animals for in vivo mutagenicity studies following rescue of the bacterial lacZ transgene has been hindered by the sheer scale of the experimental work involved . We describe here a new positive selection protocol which is based upon a modified E . coli bacterial host . This new system is potentially capable of generating mutation data much faster and more cheaply than previous methods.

Mol Microbiol, 1994 May, 12(4), 631 - 8
Novel growth rate control of dam gene expression in Escherichia coli; Rasmussen LJ et al.; Transcription of the dam gene in Escherichia coli is growth rate regulated by a mechanism distinct from that used for ribosomal RNA gene promoters . Single-copy operon fusions to lacZ indicated that the major promoter, P2, is responsible for most or all of the growth rate dependence . Promoter P2 is a typical sigma 70 promoter with 18 bp spacing between the -10 and -35 hexamers . Primer extension analysis was used to show that there was no inhibition of transcription from promoter P2 in cells induced for the stringent response . Beta-galactosidase specific activity from a single-copy dam::lacZ fusion was unaffected by either excess rrnB RNA or the level of Fis protein . Thus growth rate control of dam gene expression differs from that of the rRNA and tRNA genes by its lack of response to stringent control, ribosomal feedback and enhanced transcription by Fis protein . We devised a procedure for selection of mutant cells in which dam gene expression was unregulated . One such mutant (cde-4), obtained by miniTn10 insertion, showed the same level of beta-galactosidase activity at all growth rates tested . In contrast, growth rate-dependent expression of the rrnB gene was unaffected by cde-4 confirming the different modes of regulation . The cde-4::miniTn10 insertion is located close to kilobase 670 on the physical map in or near the lipB gene.

Mol Microbiol, 1994 May, 12(4), 621 - 9
Control of the LexA regulon by pH: evidence for a reversible inactivation of the LexA repressor during the growth cycle of Escherichia coli; Dri AM et al.; The LexA repressor controls the expression of several genes, including lexA, recA, and sfiA, which are induced when exponentially growing bacteria are exposed to DNA-damaging agents . Induction of this so-called SOS response takes place while LexA is cleaved in a reaction that requires the RecA protein and damaged DNA . We have shown that large fluctuations in the cellular concentration of the LexA repressor and in the rate of transcription of the sfiA gene also occur spontaneously during bacterial growth in complex medium such as LB . The possibility that changes in external or internal pH may explain these fluctuations has been explored . A consistent pattern was established whereby conditions leading to either increased or decreased pH were associated with altered expression of the lexA and sfiA genes . These data can be explained by a model in which the LexA repressor exists in either of two forms in equilibrium: a form favoured at homeostatic internal pH, which has a low affinity for the operators of LexA-controlled genes; and a form accumulated in response to a transient decrease in internal pH, which has a high affinity for operators.

Mol Microbiol, 1994 May, 12(4), 579 - 86
NarK is a nitrite-extrusion system involved in anaerobic nitrate respiration by Escherichia coli; Rowe JJ et al.; Escherichia coli can use nitrate as a terminal electron acceptor for anaerobic respiration . A polytopic membrane protein, termed NarK, has been implicated in nitrate uptake and nitrite excretion and is thought to function as a nitrate/nitrite antiporter . The longest-lived radioactive isotope of nitrogen, 13N-nitrate (half-life = 9.96 min) and the nitrite-sensitive fluorophore N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide have now been used to define the function of NarK . At low concentrations of nitrate, NarK mediates the electrogenic excretion of nitrite rather than nitrate/nitrite exchange . This process prevents intracellular accumulation of toxic levels of nitrite and allows further detoxification in the periplasm through the action of nitrite reductase.

Mol Microbiol, 1994 May, 12(4), 571 - 8
Role of rpoS (katF) in oxyR-independent regulation of hydroperoxidase I in Escherichia coli; Ivanova A et al.; We present evidence showing that rpoS (katF) is a regulator of katG gene transcription in an oxyR-independent manner . Mutation of the rpoS gene in several different Escherichia coli strains caused a significant reduction in catalase HPI activity . In rpoS-delta oxyR double mutants, the level of HPI was considerably lower compared to the delta oxyR parent strain, and was restored when transformed with an rpoS+ plasmid . Overproduction of HPI in oxyR- suppressor strains was greatly diminished after inactivation of the rpoS gene and was accompanied by a substantial increase in sensitivity to menadione . Beta-galactosidase expression from a katG::lacZ promoter was lower in rpoS strains compared to rpoS+ isogenic parents . Several delta oxyR strains had detectable levels of katG transcription that was significantly diminished after rpoS gene inactivation.

J Antimicrob Chemother, 1994 May, 33 Suppl A, 93 - 7
Bacteriuria of pregnancy--an update on significance, diagnosis and management; Bint AJ et al.; Bacteriuria of pregnancy is a common condition which is thought to be associated with serious sequelae in mother and fetus . A programme of screening, treatment and follow-up is likely to be cost-effective, but will depend on local prevalences of bacteriuria and pyelonephritis . If treatment is undertaken, nitrofurantoin and cephalexin are both safe and effective and have resistance rates of below 10% . Amoxycillin is also safe, but resistance rates in Escherichia coli are about 40% . A 7 day course of treatment remains advisable.

Exp Eye Res, 1994 May, 58(5), 573 - 84
Expression of recombinant bovine gamma B-, gamma C- and gamma D-crystallins and correlation with native proteins; Hay RE et al.; Despite the use of bovine gamma-crystallins in numerous biophysical and chemical studies, characterization of these proteins at the molecular level is incomplete . Bovine lenses have at least six gamma-crystallin protein fractions currently assigned as gamma s/I, gamma A/IVb, gamma B/II, gamma C/IIIb, gamma D/IIIa and gamma E/IVa . A lack of primary sequence data for corresponding gamma-crystallin genes and proteins, however, has made these assignments tenuous . To clarify these assignments, we have over-expressed recombinant bovine gamma-crystallin proteins in Escherichia coli using complementary DNAs corresponding to gamma B-, gamma C-, and gamma D-crystallin genes . The recombinant crystallins were characterized by chromatographic and spectroscopic comparisons with native bovine crystallin fractions gamma II, gamma IIIa and gamma IIIb . The elution of recombinant gamma B and native gamma II proteins was identical on cation-exchange chromatography as expected; however, recombinant gamma C coeluted with gamma IIIa and recombinant gamma D co-eluted with gamma IIIb . Sequential Edman degradation through the first 29 residues of the native gamma IIIa and gamma IIIb polypeptides confirmed the colinearity of their sequences with those predicted from the gamma C- and gamma D-crystallin genes, respectively . Absorption and UV circular dichroism (CD) spectra of the recombinant proteins were almost identical to those of their native counterparts, indicating that the secondary and tertiary structures of the recombinant proteins were characteristic for gamma-crystallins . Based on these data the bovine gamma-crystallins proteins and genes are correlated as follows: II/gamma B, IIIa/gamma C and IIIb/gamma D . The assignment of gamma IIIb (previously characterized as having a low critical temperature for phase separation) with gamma D rather than gamma C proves an exception to the hypothesis that the gamma ABC-crystallin group is more resistant to phase separation than the gamma DEF group . These corrected assignments should provide a more substantial base for investigations of residues responsible for phase separation and other biophysical characteristics . Additionally, expression of recombinant gamma-crystallins with structures similar to native proteins may prove to be useful in probing specific structure-function relationships of the gamma-crystallins.

Electrophoresis, 1994 May, 15(5), 647 - 53
Purification of human recombinant superoxide dismutase by isoelectric focusing in a multicompartment electrolyzer with zwitterionic membranes; Wenisch E et al.; Human recombinant superoxide dismutase (SOD), purified to homogeneity, is resolved by both conventional isoelectric focusing and immobilized pH gradients into three bands, with isoelectric points (pIs) in the pH range 4.8 to 5.1, the pI 4.80 form representing the minor component . Due to the fact that this enzyme is expressed in E . coli, N-terminal acetylation or glycosylation should be ruled out . When purified by small-scale preparative isoelectric focusing in immobilized pH gradient gels, it was found that, upon subsequent analysis, the pI 5.07 form would band in the same position, but the intermediate pI 4.92 band would split into the upper (pI 5.07) and the lower (pI 4.80) species, in nearly the same amounts, whereas the lowest pI component would always generate both the intermediate and upper forms . Enzymatic essays pointed out that these three isoforms had nearly the same specific activity, slightly higher than that of the starting material . Metal analysis indicated that all three forms contained the same metal/protein ratio, approaching the value Cu2Zn2-SOD, as reported in the literature . Circular dichroism spectra of the pI 4.80 and 5.07 forms showed the same profile in the 190-240 nm range, but marked differences in the 250-350 nm region . Treatment with EDTA produces 1-2 additional, slightly higher pI isoforms, whereas treatment with KCN generates a number of higher pI components, reaching pI values as high as pH 7, with nearly complete disappearance of the three major SOD isoforms . It is concluded that these three isoforms could represent interconvertible species, the highest pI component representing the most stable conformer.

Chin Med J (Engl), 1994 May, 107(5), 338 - 41
Detection of genes for heat-stable enterotoxin in Escherichia coli by biotinylated ST-DNA probes; Zhu QY et al.; Reference strains of enterotoxigenic Escherichia coli (ETEC), non-enterotoxigenic Escherichia coli (non-ETEC), enteropathogenic Escherichia coli (EPEC), enteroinvasive Escherichia coli (EIEC), and other enteropathogenic bacteria were used to prove the reliability of BIO-ST-DNA probe hybridization . In addition, 417 strains of E . coli isolated from children with diarrheal diseases in Shanxi Children's Hospital were examined for BIO-ST-DNA probe hybridization . In the test, BIO-ST-DNA hybridization was compared with suckling mouse assay in identifying ST-ETEC . The results obtained by both methods showed no significant difference . It was found that identification of ST-ETEC using hybridization is a simple, sensitive and more practical method.

Bioorg Med Chem, 1994 May, 2(5), 331 - 8
Functionalized 3,5-dihydroxybenzoates as potent novel inhibitors of EPSP synthase; Miller MJ et al.; Aromatic analogues of the EPSP synthase enzyme substrate (S3P), reaction intermediate (1), and product (EPSP) were synthesized from 3,5-dihydroxybenzoic acid and were evaluated as inhibitors of E . coli EPSP synthase . These simple, synthetically accessible aromatic analogues are highly effective competitive inhibitors versus S3P with an apparent Ki for the tetrahedral intermediate analogue 4 of 160 +/- 40 nM . This demonstrates that a simple benzene ring is a quite suitable substitute for the complex shikimate ring in the design of EPSP synthase inhibitors.

J Am Soc Nephrol, 1994 May, 4(11), 1890 - 5
The effect of uremia on tumor necrosis factor-alpha release after an in vitro whole-blood endotoxin challenge; Oettinger CW et al.; Uremia has been associated with immunologic aberrations, including anergy, increased susceptibility to infections, and reduced phagocytic activity of polymorphonuclear leukocytes . In this study, cytokine release in uremic and nonuremic blood after in vitro endotoxin stimulation was studied . Blood from nonuremic controls, chronic renal failure patients not on dialysis, and chronic hemodialysis patients predialysis and postdialysis was spiked with 10 ng/mL of Escherichia coli endotoxin and incubated for 2 and 26 h . Plasma tumor necrosis factor-alpha (TNF alpha) concentrations were determined by ELISA after each incubation period . To further study which uremic blood component may be responsible for enhanced release of TNF alpha, plasma and cellular components of chronic renal failure patients and controls were switched and then given an in vitro endotoxin stimulation (1 ng/mL) . It was found that (1) TNF alpha release is enhanced by uremia and is exacerbated with progressive declines in renal function, (2) enhanced TNF alpha release is related to a blood cellular phenomenon induced by uremia, and (3) enhanced TNF alpha release in hemodialysis patients is associated with a prolonged stimulation and/or reduced plasma elimination of TNF alpha.

J Invest Surg, 1994 May-Jun, 7(3), 175 - 86
Role of topical phospholipids in the prophylaxis of silicone elastomer-associated infection in the abdominal cavity; Guo W et al.; The present study evaluated the influence of phospholipids (phosphatidyl choline and phosphatidyl inositol) on the prevention of abdominal biomaterial-associated infection . Phospholipid-impregnated silicone elastomer (SE) fragments were either intraperitoneally implanted in rats or immersed in serum for 0, 4, and 14 days, and 3 x 10(9) cfu of 3H-labeled, live Escherichia coli were added in the peritoneal cavity or in vitro incubation medium . Three hours after incubation, the adherence of bacteria significantly decreased to phospholipid-impregnated SE fragments, which had been immersed/implanted for 0 and 4 days . However, the number of adhering bacteria did not differ between the impregnated and unimpregnated SE fragments after 14 days of immersion/implantation . A significantly lower number of adhering bacteria was noted on all unimpregnated SE fragments when phospholipid was supplemented in the peritoneal cavity or in vivo medium, compared with fragments with no supplement . The rate of bacterial DNA synthesis decreased significantly after incubation with phospholipid 2 h or more . Phospholipids did not further influence peritoneal morphology . Thus topical administration of phospholipids by impregnation to the surface of SE fragments or supplement in the incubation medium prevented bacterial adherence onto the SE fragments . This implies that the use of phospholipids might be a mode of preventing biomaterial-associated infections.

AIDS Res Hum Retroviruses, 1994 May, 10(5), 595 - 600
Identification and characterization of the Bel 3 protein of human foamy virus; Weissenberger J et al.; The human foamy virus (HFV) is a complex retrovirus that contains several regulatory and auxiliary bel genes besides the gag, pol, and env genes . In contrast to the gene products of bel 1 and bel 2/bet that were identified previously, the Bel 3 protein has not been described to date . Here we report the identification of Bel 3 in HFV-infected cells by immunoprecipitation, indirect immunofluorescence, and expression cloning under the control of a strong heterologous promoter . Bel 3 was immunoprecipitated with an antiserum directed against a bacterially expressed and purified form of recombinant Bel 3 antigen . Bel 3 was found to be expressed in low amounts in the cytoplasm of HFV-infected cells and to migrate with an apparent molecular mass of 19.4 kDa on electrophoresis in SDS-polyacrylamide gels, consistent with the calculated value of 18.2 kDa . Radioimmunoprecipitation of HFV-infected cell lysates with the hyperimmune serum against Bel 3 revealed at least two additional immunoreactive bands of 15.5 and 10.6 kDa . The results indicate that Bel 3 was labile, because it was partially degraded even at early time points after infection . On transfection and expression in transfected COS cells, recombinant Bel 3 was immunoprecipitated and migrated in three polypeptide bands of 18.7, 14.8, and 9.3 kDa under denaturing conditions . In the absence of reducing agents, the bacterially expressed and purified recombinant Bel 3 protein of 16.1 kDa can form homodimers of 30 kDa.

Protein Eng, 1994 May, 7(5), 689 - 96
Site-directed mutagenesis of a calcium binding site modifies specifically the different biochemical properties of annexin I; Trave G et al.; All the functions of annexins in vitro as well as in vivo are mediated and probably regulated by calcium . We have used recombinant annexin I, synthesized by Escherichia coli, and we have performed site-directed mutagenesis . We have mutated the endonexin fold of domain 2 that binds calcium . Mutations were performed in this domain of the molecule because it perfectly matches the calcium binding consensus sequence . The two glycines of this fold were mutated into glutamic acid . The helix content and the stability of the mutants are identical to those of the wild-type, suggesting that the mutations did not drastically affect the structure of the protein . The two mutants showed modified calcium binding affinities . However, the calcium binding affinity of the G131E mutant was far more altered than that of the G129E mutant . Furthermore, other biochemical properties of these mutants were modified to different extents . The binding to phospholipid was not seriously affected, whereas the self-association was lost by the G131E mutant . In the same way, liposome aggregation is conserved, but modified, while the calcium affinity measured by equilibrium dialysis is dramatically altered.

Mol Microbiol, 1994 May, 12(3), 423 - 32
Post-transcriptional regulation of the groEL1 gene of Streptomyces albus; Servant P et al.; Thermally induced expression of the heat-shock gene groEL is subject to post-transcriptional regulation in Streptomyces albus . When S . albus cells were shifted from 30 degrees C to 41 degrees C, synthesis of three GroEL-like proteins was induced from two genes transcribed from associated promoters P1 and P2 . Surprisingly, analyses of transcriptional fusions of these promoters with various reporter genes indicated constitutive expression independent of heat shock . In contrast, neo expression was thermally inducible as a GroEL1-APH translational fusion protein . Furthermore, expression of the groEL1-neo gene was heat inducible even after the groEL1 promoter region was replaced by a heterologous non-heat-inducible promoter such as the Escherichia coli lac promoter . Finally, synthesis of GroE proteins, as well as the GroEL-APH fusion protein, was heat inducible when their transcription was inhibited by rifampicin . Post-transcriptional regulatory signals needed for heat-induced GroEL1 synthesis were mapped within of the groEL1 structural gene.

Mol Microbiol, 1994 May, 12(3), 387 - 401
CooC and CooD are required for assembly of CS1 pili; Froehlich BJ et al.; Many strains of enterotoxigenic Escherichia coli (ETEC) isolated from patients with diarrhoeal disease exhibit CS1 pili on their surfaces . These appendages, which are thought to be important for colonization of the upper intestine, are composed largely of multiple identical protein subunits encoded by cooA . We have sequenced the DNA directly downstream of cooA and identified two open reading frames, cooC and cooD, transcribed in the same direction as cooB and cooA . Following cooD is DNA homologous to an insertion sequence, so cooB, A, C and D appear to encode all the information needed for E . coli K-12 to synthesize CS1 pili . Complementation analysis of mutants cloned in E . coli K-12 and constructed in an ETEC-derived strain indicates that cooC and cooD are not required for stability of the major CS1 pilin protein or for its transport to the periplasm, but, like cooB, both are needed for assembly of cooA into pili.

FEMS Microbiol Lett, 1994 May 1, 118(1-2), 83 - 8
Morphological appearances of K88ab fimbriae and optical diffraction analysis of K88 paracrystalline structures; Simons BL et al.; K88ab fimbriae are filamentous protein structures at the surface of certain enterotoxigenic Escherichia coli strains . Electron microscopy analysis of K88ab fimbriae showed that these structures have different morphological appearances dependent on the medium in which cells expressing these fimbriae or in which purified fimbriae were suspended . Thin and curled structures, thin and flexible fimbriae, a wider and rigid form of the fimbriae, and, in addition, paracrystalline structures were detected . Optical diffraction analysis of the paracrystalline structures indicated a helical conformation of K88ab fimbriae.

J Bacteriol, 1994 May, 176(10), 2938 - 45
Effects of transcriptional start site sequence and position on nucleotide-sensitive selection of alternative start sites at the pyrC promoter in Escherichia coli; Liu J et al.; In Escherichia coli, expression of the pyrC gene is regulated primarily by a translational control mechanism based on nucleotide-sensitive selection of transcriptional start sites at the pyrC promoter . When intracellular levels of CTP are high, pyrC transcripts are initiated predominantly with CTP at a site 7 bases downstream of the Pribnow box . These transcripts form a stable hairpin at their 5' ends that blocks ribosome binding . When the CTP level is low and the GTP level is high, conditions found in pyrimidine-limited cells, transcripts are initiated primarily with GTP at a site 9 bases downstream of the Pribnow box . These shorter transcripts are unable to form a hairpin at their 5' ends and are readily translated . In this study, we examined the effects of nucleotide sequence and position on the selection of transcriptional start sites at the pyrC promoter . We characterized promoter mutations that systematically alter the sequence at position 7 or 9 downstream of the Pribnow box or vary the spacing between the Pribnow box and wild-type transcriptional initiation region . The results reveal preferences for particular initiating nucleotides (ATP > or = GTP > UTP >> CTP) and for starting positions downstream of the Pribnow box (7 >> 6 and 8 > 9 > 10) . The results indicate that optimal nucleotide-sensitive start site switching at the wild-type pyrC promoter is the result of competition between the preferred start site (position 7) that uses the poorest initiating nucleotide (CTP) and a weak start site (position 9) that uses a good initiating nucleotide (GTP) . The sequence of the pyrC promoter also minimizes the synthesis of untranslatable transcripts and provides for maximum stability of the regulatory transcript hairpin . In addition, the results show that the effects of the mutations on pyrC expression and regulation are consistent with the current model for translational control . Possible effects of preferences for initiating nucleotides and start sites on the expression and regulation of other genes are discussed.

J Bacteriol, 1994 May, 176(10), 2814 - 21
Inhibition of fatty acid synthesis in Escherichia coli in the absence of phospholipid synthesis and release of inhibition by thioesterase action; Jiang P et al.; The effects of inhibition of Escherichia coli phospholipid synthesis on the accumulation of intermediates of the fatty acid synthetic pathway have been previously investigated with conflicting results . We report construction of an E . coli strain that allows valid {14C}acetate labeling of fatty acids under these conditions . In this strain, acetate is a specific precursor of fatty acid synthesis and the intracellular acetate pools are not altered by blockage of phospholipid synthesis . By use of this strain, we show that significant pools of fatty acid synthetic intermediates and free fatty acids accumulate during inhibition of phospholipid synthesis and that the rate of synthesis of these intermediates is 10 to 20% of the rate at which fatty acids are synthesized during normal growth . Free fatty acids of abnormal chain length (e.g., cis-13-eicosenoic acid) were found to accumulate in glycerol-starved cultures . Analysis of extracts of {35S}methionine-labeled cells showed that glycerol starvation resulted in the accumulation of several long-chain acyl-acyl carrier protein (ACP) species, with the major species being ACP acylated with cis-13-eicosenoic acid . Upon the restoration of phospholipid biosynthesis, the abnormally long-chain acyl-ACPs decreased, consistent with transfer of the acyl groups to phospholipid . The introduction of multicopy plasmids that greatly overproduced either E . coli thioesterase I or E . coli thioesterase II fully relieved the inhibition of fatty acid synthesis seen upon glycerol starvation, whereas overexpression of ACP had no effect . Thioesterase I overproduction also resulted in disappearance of the long-chain acyl-ACP species . The release of inhibition by thiosterase overproduction, together with the correlation between the inhibition of fatty acid synthesis and the presence of abnormally long-chain acyl-ACPs, suggests with that these acyl-ACP species may act as feedback inhibitors of a key fatty acid synthetic enzyme(s).

EMBO J, 1994 May 1, 13(9), 2089 - 96
Relaxation of replication control in chaperone-independent initiator mutants of plasmid P1; Mukhopadhyay G et al.; Escherichia coli chaperones DnaJ, DnaK and GrpE increase P1 plasmid initiator binding to the origin by promoting initiator folding . The binding allows initiation and also promotes pairing of origins which is believed to control initiation frequency . Chaperone-independent DNA binding mutants are often defective in replication control . We show here that these mutants have increased rates of association for DNA binding and defects in origin pairing . The increases in association rates were found to be due either to increased protein folding into active forms or to increases in the association rate constant, kon . Since the dissociation rate constants for DNA release with these mutants are not changed, it is unlikely that the DNA binding domain is affected . The pairing domain may thus control replication and modulate DNA binding . The role of the pairing domain in DNA binding can be significant in vivo as the selection for chaperone-independent binding favors pairing-defective mutants.

Am J Med Sci, 1994 May, 307(5), 335 - 9
Pigeon and dove eggwhite protect mice against renal infection due to P fimbriated Escherichia coli; Johnson JR et al.; Pigeon and dove eggwhite exhibit high level P1 antigenic activity and are potent and specific inhibitors of adherence mediated by P fimbriae of uropathogenic Escherichia coli . To evaluate pigeon and dove eggwhite as P fimbrial receptor analogues in the prevention of ascending renal infection, mice were challenged with a P fimbriated E . coli urosepsis isolate suspended in saline alone or in saline plus various inhibitors of adherence, including D-mannose, globoside, and chicken, dove, and pigeon eggwhite . D-mannose inhibited mannose-sensitive adherence but not P fimbrial adherence, and failed to prevent renal infection . Globoside and chicken eggwhite also failed to inhibit P fimbrial adherence; chicken eggwhite had little and globoside had no impact on renal infection . In contrast, dove and pigeon eggwhite eliminated P fimbrial adherence and significantly reduced the incidence and intensity of renal infection . These findings suggest that pigeon and dove eggwhite provide P1-antigen-specific protection against ascending renal infection in mice due to P fimbriated uropathogenic E . coli.

J Bacteriol, 1994 May, 176(9), 2513 - 6
Identification of the Shine-Dalgarno sequence required for expression and translational control of the pyrC gene in Escherichia coli K-12; Liu J et al.; Expression of the pyrC gene in Escherichia coli K-12 is regulated by a translational control mechanism in which CTP (and perhaps GTP) pool sizes determine the selection of alternative transcriptional start sites at the pyrC promoter . High CTP levels cause transcription to start primarily at a site that directs the synthesis of untranslatable pyrC transcripts . These transcripts form a hairpin at their 5' ends that blocks ribosome binding to the Shine-Dalgarno (SD) sequence . The pyrC ribosome binding site is unusual in that it contains two potential SD sequences, designated SD1 and SD2, which are located 11 and 4 nucleotides upstream of the translational initiation codon, respectively . In this study, we examined the functions of these two SD sequences in translational initiation . Mutations that inactivate either SD1 or SD2 were constructed and incorporated separately into a pyrC::lacZ protein fusion . The effects of the mutations on pyrC::lacZ expression, regulation, and transcript levels were determined . The results indicate that SD1 is the only functional pyrC SD sequence . The SD2 mutation did cause a small reduction in expression, but this effect appeared to be due to a decrease in transcript stability . In addition, we constructed a mutation that introduces a long spacer region between the hairpin at the 5' end of the pyrC transcript and a new pyrC SD sequence . As predicted by the model for translational control, this mutation caused constitutive expression of a pyrC::lacZ protein fusion.

Antibiot Khimioter, 1994 May, 39(5), 3 - 11
{Actinomycete plasmid and integrative vectors based on DNA of the temperate Phi C31 actinophage, determining limitation of lytic development of phage Phi C31, not dependent on repressor}; Sladkova IA et al.; Bireplicon plasmids were constructed . The plasmids consist of DNAs of the streptomycete plasmid pIJ702, the Escherichia coli plasmid pUC19 and the phi C31 actinophage genome fragment encoding the function of the site-specific integration into the chromosome . Part of the plasmids transformed Streptomyces lividans TK64 to thiostreptone resistance . The DNA transforming activity depended on the mutual orientations of the blocks used for the construction and probably depended on the structural stability of the plasmids in S . lividans . The integrative vectors consisting of the pUC19 plasmid DNA and the phage genome fragment with the integrative function efficiently transformed S . lividans . No phage plagues were detected with the standard procedure of integrants' infection by phi C31 phage, despite the absence of the phi C31 phage repressor gene in the integrated DNA . The phi C31 phage mutants including clear and virulent ones were not capable of lytic growth on the integrants . The region determining the limitation of the phi C31 phage lytic development was localized by the deletion analysis of the bireplicon plasmids . As a result actinomycete monoreplicon plasmids were formed . The region is the 1.3 kb phage fragment whose right end maps at 0.2 kb preceding the right end of the phi C31 phage genome linear map.

Zhonghua Yu Fang Yi Xue Za Zhi, 1994 May, 28(3), 147 - 50
{Inhibitory effects of fifteen kinds of Chinese herbal drugs, vegetables and chemicals on SOS response}; Jin ZC et al.; Effects of 15 kinds of herbal drugs, vegetables and chemicals on lex-dependent sfi-SOS response were determined by micropersistent and/or pulse models induced by 4-Nitroquinoline-N-oxide (4NQO) and Mitomycin C (MMC) in Escherichia coli(E . coli) PQ37 and PQ35, respectively . Results showed the water extract of Rhizoma Polygonati (RP), Fructus Chebulae (FC), Radix Polygoni Multiflori (RPM), Fructus Ligustri Lucidi (FLL), Bulbus Fritillariae Thunbergii (BFT), shell of water chestnut with a pedicle, Chinese chives juice, and solutions of 5-Fluorouracil, Tannic acid and garlicin could inhibit SOS responses with a dose-response relationship and suggested the inhibitory effects took place both inside and outside E . coli cells . Water extract of FC, FLL, BFT, shell of water chestnut with a pedicle, Chinese chives juice and solution of 5-Fluorouracil and Tannic acid could intracellularly inhibit SOS responses induced by MMC in E . coli PQ35, and acetone extract of Grifola Frondosa (GF) could extracellularly inhibit SOS responses in E . coli PQ37 and intracellularly in PQ35 induced by 4NQO or MMC . Water extract of raw hawthorn . Radix Angelicae Duhuricae (RAD), Radix Ophiopogonis (RO), and 5-Fluorodeoxyuridine could extracellularly inhibit SOS responses induced by 4NQO in E coli PQ37 . The possible mechanisms of intracellular inhibition and antidamage repair were discussed in the paper.

Biochem Cell Biol, 1994 May-Jun, 72(5-6), 175 - 81
Effect of pH and nonphysiological salt concentrations on human immunodeficiency virus-1 protease dimerization; Tyagi SC et al.; Human immunodeficiency virus-1 (HIV-1) protease is catalytically active as a dimer of identical subunits that associate through noncovalent interactions . To investigate the forces stabilizing HIV-1 protease in its active form, we have studied the effects of pH and salts on structure and function of the enzyme . Enzymatic activity was measured by following the hydrolysis of a fluorogenic substrate . Dissociation of the dimer into its subunits was monitored by gel filtration, while conformational changes in the enzyme were probed by measurements of intrinsic tryptophan fluorescence . Mg2+ ions were capable of dissociating the dimeric enzyme with a concomitant red shift and increase in quantum yield of the tryptophan fluorescence, indicating increased accessibility of tryptophan to the aqueous environment . These structural changes also were associated with a loss of catalytic activity which was insensitive to substrate concentration, consistent with noncompetitive inhibition . Both structural and functional changes could be attributed to binding of Mg2+ ions to a site with an apparent dissociation constant of approximately 2 M . In contrast, increasing concentrations of Na ions up to 5 M were without effect . Increasing pH had similar effects on HIV-1 protease as increasing Mg2+ ions concentration, with concomitant dissociation into subunits, increase in quantum yield and red shift in tryptophan fluorescence, and loss in catalytic activity . The apparent pKa for these structural and functional transitions was 6.95 +/- 0.08 . This value is consistent with that of an aspartic acid residue with an anomalously high pKa, which has been implicated in the catalytic activity of HIV-1 protease.

Microb Pathog, 1994 May, 16(5), 337 - 48
Cloning and expression of hemolysin genes from Treponema denticola strains ATCC 35404 (TD-4) and human clinical isolate GM-1 in Escherichia coli; Karunakaran T et al.; The oral spirochete, Treponema denticola is a putative etiologic agent in adult periodontitis, and acute necrotizing ulcerative gingivitis . In vitro, the oral treponeme produces several factors including proteases, hemolysins, hemin-binding proteins, which could potentially be involved in the virulence of this spirochete . Our laboratory has been investigating the pathobiology of T . denticola, and has demonstrated the production of several hemolysins by T . denticola . In this report two hemolysin genes from T . denticola strains ATCC 35404 (TD-4) and GM-1 were isolated by screening genomic DNA libraries of T . denticola on sheep blood agar plates . Physical maps of the insert fragments were not identical . Southern blot analyses suggested some degree of homology in the nucleotide sequence . Maxicell analyses of {35S}-methionine-labeled polypeptides from the recombinant plasmids have suggested the synthesis of an approximately 62.5 kDa polypeptide . Biochemical characterization of the T . denticola hemolysin genes indicated the activity to be inhibited by Mg2+, Ca2+ and Zn2+ but not by EDTA . Dithiothreitol and glutathione moderately enhanced the hemolytic activity of the recombinant plasmids . Iron partially inhibited the hemolytic activities . Addition of 2-2' bipyridyl moderately enhanced the activities, possibly by iron limitation . These results suggest the isolation of an identical hemolysin gene from T . denticola strains TD-4 and GM-1.

Vet Q, 1994 May, 16 Suppl 2, S117 - 21
Laboratory and clinical evaluation of a chromogenic endotoxin assay for horses with acute intestinal disorders; Steverink PJ et al.; In this study the laboratory and clinical performance of a chromogenic endotoxin assay for equine plasma was evaluated . The assay was sensitive (detection limit 3 ng LPS/L plasma), reproducible (within and between-assay CV at 50 ng LPS/L E . coli O111:B4 LPS standard addition was 5% and 7.5%, respectively), and not substantially affected by enhancement or inhibition phenomena (recovery of an in vitro spike was 75-125% in 80% of the samples) . LPS added to whole blood was rapidly inactivated upon incubation at 37 degrees C but not at 0 degrees C . A recently developed blood collection tube for LPS testing was found suitable, i.e . LPS-free and providing non-contaminated samples . In 48 horses suffering from acute abdominal diseases requiring surgical treatment, LPS levels were significantly higher in platelet-rich plasma (PRP) than in platelet-poor plasma (PPP), and the proportional difference was related to the PRP platelet count (r = 0.52, p < 0.001, mean difference 48%, range 8-77%) . LPS levels were also significantly higher in horses that died or were euthanized than in surviving horses (mean 16.5 and 7.1 ng/L PRP, respectively, p < 0.05) . We conclude that LPS can be measured in equine plasma with picogram sensitivity and recommend the use of PRP instead of PPP for clinical LPS testing . For clinical use a decision limit for endotoxaemia of 5 ng LPS/L PRP appeared to be inadequate . Analysis at a higher cut-off level for endotoxaemia and the evaluation of clinical, pathological, and laboratory parameters would be more meaningful.

Biosci Biotechnol Biochem, 1994 May, 58(5), 895 - 9
High level secretion of calf chymosin using a glucoamylase-prochymosin fusion gene in Aspergillus oryzae; Tsuchiya K et al.; A recombinant chymosin was secreted at high levels using fusion genes with A . oryzae glucoamylase gene (glaA) and a wheat bran solid-state culture system . Two portions of the A . oryzae glucoamylase, one with almost the entire glucoamylase (GA1-603) lacking 9 amino acids at the carboxyl terminal, and the other (GA1-511) lacking the starch binding-domain, were fused in frame with prochymosin cDNA . Western blot analysis indicated that the mature chymosin was released from the secreted fusion protein by autocatalytic processing . The transformant harboring the GA1-511-prochymosin construct showed about 5-fold chymosin production of the transformant in which the chymosin gene was directly expressed under the control of the glaA promoter in submerged culture . Moreover, wheat bran solid-state culture gave about 500-fold higher yield of the chymosin (approximately 150 mg/kg wheat bran) compared with the submerged culture.

Biotechnol Prog, 1994 May-Jun, 10(3), 314 - 9
Clonal variation in the Spodoptera frugiperda IPLB-SF21-AE insect cell population; Pasumarthy MK et al.; Clones have been isolated from the heterogeneous Spodoptera frugiperda IPLB-SF21-AE insect cell population . Five of these clones, in addition to the parent cell line and the SF9 cell line (another clonal isolate of the parent cell line), have been compared in regards to morphology, growth, budded virus synthesis, and recombinant protein synthesis . No significant differences in cell morphology were found among these cell lines . There was, however, a significant difference in the average cell size, with diameters ranging from 9.30 +/- 0.184 to 11.11 +/- 0.22 microns and from 9.17 +/- 0.05 to 11.25 +/- 0.24 microns for cells growing in Excell 401 serum-free medium in spinner flask cultures and in TNM-FH medium supplemented with 10% FBS in tissue flask cultures, respectively . While no significant differences in the growth rates were found in TNM-FH medium containing 10% calf serum, significant differences were found in Excell 401 serum-free medium, with population doubling times ranging from 38.5 +/- 6.6 to 64.5 +/- 6.4 h in spinner flask studies . Significant differences in expression levels of Escherichia coli beta-galactosidase (beta-gal) were also found in both 12-well plates and spinner flasks . In the 12-well plate studies, the peak levels of beta-galactosidase obtained by these cell lines ranged from 0.332 +/- 0.091 to 0.805 +/- 0.117 mg/10(6) cells and from 0.580 +/- 0.130 to 1.458 +/- 0.132 mg/10(6) cells in Excell 401 and Hyclone Hy-Q serum-free media, respectively . In the spinner flask studies, peak expression levels ranged from 0.128 +/- 0.053 to 0.573 +/- 0.215 mg/10(6) cells in Excell 401 serum-free medium.(ABSTRACT TRUNCATED AT 250 WORDS)

Biotechnol Prog, 1994 May-Jun, 10(3), 237 - 45
Characterization and polyelectrolyte precipitation of beta-galactosidase containing genetic fusions of charged polypeptides; Niederauer MQ et al.; Genetically engineered versions of beta-galactosidase were constructed through the addition of charged polypeptide fusion tails for the purpose of enhancing polyelectrolyte precipitation . Negatively charged aspartic acid tails and positively charged poly(arginine) tails were added to beta-galactosidase from Escherichia coli . These fusion proteins were all shown to possess specific activity equal to that of the native enzyme . Gel permeation and ion-exchange chromatography provided evidence concerning the integrity of the tails as well as their altered charge characteristics . All enzymes containing charged tails displayed enhanced polyelectrolyte precipitation over the native enzyme . An optimal number of charged residues, beyond which no further enhancement of precipitation was observed, was found to be approximately 10 residues for each type of tail . No interference from nucleic acids was observed in the precipitation of positively tailed beta-galactosidase.

Biotechnology (N Y), 1994 May, 12(5), 517 - 20
Efficient sampling of protein sequence space for multiple mutants; Caren R et al.; We describe here a method capable of generating a very large population of multiple mutants, the size of which is primarily limited by volume constraints . This method, referred to as recombination-enhanced mutagenesis, combines the power of in vitro mutagenesis with the high frequencies of in vivo recombination that can be achieved using single-stranded transduction systems . The recombination frequency between two mutations separated by as little as 19 amino acids is 0.02; this frequency approaches a value of 0.1 for mutations separated by more than 38 amino acids . Up to 10(8) independent recombinants were generated in 1 ml of an E . coli culture, and this number scales linearly (or better) with increasing volume . To prove the method's effectiveness, we applied it to the problem of reverting multiple mutants of mouse dihydrofolate reductase, which could not be reverted using mutagenesis alone . Thus, given an appropriate screen or selection scheme, recombination-enhanced mutagenesis is well-suited for addressing a range of combinatorially complex problems, such as antigen recognition, enzyme catalysis, protein folding, and transport/transduction across biomembranes.

Shock, 1994 May, 1(5), 359 - 61
Lack of alteration in cardiac beta-adrenoceptor site concentration in rats treated with an adenylate cyclase-stimulating dose of endotoxin; Gardey C et al.; In previous experiments on male rats we showed that 2 mg kg-1, intravenously, lipopolysaccharide from Escherichia coli, a sublethal nonhypotensive dose, induced circulating cardiodepressant activity, maximal alterations in steroid hormonal response, and myocardial adenylate cyclase hyperactivity 4 h later . The purpose of the present study was to investigate a possible relation between this adenylate cyclase hyperactivity and alterations in total ventricular beta-adrenoceptors . To this end, ventricular beta-adrenoceptor concentration and affinity were measured under the same conditions . Binding assays were performed using {3H}dihydroalprenolol . No significant change was observed in both parameters.

Shock, 1994 May, 1(5), 347 - 53
Effects of NW-nitro-L-arginine and dexamethasone on early events following lipopolysaccharide injection: observations in the hamster cheek pouch microcirculation; Bouskela E et al.; The effects of NW-nitro-L-arginine (L-NAG) and dexamethasone in the microcirculatory changes observed in early stages of endotoxemia was investigated in male hamsters treated with Escherichia coli lipopolysaccharide (LPS) . The cheek pouch was studied in vivo by means of intravital microscopy and mean arterial and venous pressures, mean arteriolar internal diameter, spontaneous arteriolar vasomotion, microvascular blood flow, macromolecular permeability, leukocyte adhesion, and mean survival time were evaluated in animals treated with either LPS alone or the combination of LPS with L-NAG, an inhibitor of both the constitutive and inducible NO synthases (NOs) . The intravenous injection of LPS (100 mg/kg) elicited a significant reduction in mean arterial blood pressure (MABP) and arteriolar blood flow . The observed arterioles dilated and the spontaneous vasomotion ceased . The combination LPS + L-NAG, both given intravenously, prevented the reduction of MABP and the vasodilation but did not help either the reduction of arteriolar blood flow or the cessation of vasomotion . In order to separate the effect of the two NOs, a group of hamsters was pretreated with dexamethasone (10 mg/kg, also intravenously) which inhibits the induction of the inducible NO synthase (iNOs) . In this group, the hypotension, vasodilation, and cessation of vasomotion were prevented but the decrease in arteriolar blood flow was not affected . The mean survival time was significantly decreased by the combination of LPS + L-NAG (35 +/- 6 h) and significantly increased by the pretreatment with dexamethasone (92 +/- 5 h) compared to LPS alone (56 +/- 7 h).(ABSTRACT TRUNCATED AT 250 WORDS)

Exp Appl Acarol, 1994 May, 18(5), 301 - 8
Transient expression of a Drosophila melanogaster hsp70 promoter/lacZ construct injected into larvae of two species of predatory mites (Acari: Phytoseiidae); Presnail JK et al.; The Drosophila melanogaster heat shock 70 promoter (hsp70) was used to regulate expression of the Escherichia coli beta-galactosidase gene (lacZ) in transiently-transformed predatory mite larvae . A construct containing the hsp70 promoter upstream of the D . melanogaster alcohol dehydrogenase (adh) translational start site and Escherichia coli lacZ gene fusion (adh/lacZ) was injected into larvae of Metaseiulus occidentalis and Amblyseius finlandicus . LacZ expression was compared to expression of a similar construct lacking any upstream regulatory sequence . Expression from the hsp70 promoter was strong and heat shock-dependent in both species . The Drosophila hsp70 promoter therefore appears useful for regulating expression of exogenous DNA in both phytoseiid species and may be broadly applicable in the Phytoseiidae . Furthermore, the lacZ gene is a useful gene for analysis of expression in both species . Larval microinjection provides a method of assessing transient expression and of examining native regulatory sequences in these two phytoseiids and will likely be useful in other phytoseiid mites with only minor modifications.

Fiziol Zh, 1994 May-Aug, 40(3-4), 101 - 3
{Interleukin-1 production by exudate and bone marrow macrophages in experimental acute infectious peritonitis}; Dygai AM et al.; The model of acute infectious peritonitis in mice has been used to show that inflammation is attended by a marked phasic increase in interleukin-1 (IL-1) production by macrophages of exudate and bone marrow . The increased IL-1 production by macrophages of exudate proceeds earlier than that by macrophages of the bone marrow . This indicates that activation of the bone marrow macrophages may be a result of the effect of OL-1 and other haemopoietic factors released by macrophages on the inflammatory focus.

Gene Ther, 1994 May, 1(3), 185 - 91
Folate receptor mediated DNA delivery into tumor cells: potosomal disruption results in enhanced gene expression; Gottschalk S et al.; We have used a particular folate receptor, which is overexpressed in tumor cells, for targeted DNA delivery into these cell types . This folate receptor internalizes folate through caveolae by a process named potocytosis, which is distinct from endocytosis, through clathrin-coated pits . When folate conjugated to poly-L-lysine was used to deliver the E . coli beta-galactosidase gene into tumor cells overexpressing the folate receptor, only low levels of beta-galactosidase activity were detectable . When a replication-defective adenovirus was coincubated with the DNA/folate complexes, 20 to 30% of the cells stained blue with X-gal and a 1000-fold increase of beta-galactosidase activity was observed . Thus, for high efficient DNA delivery and gene expression via the caveolae system, a potosomal disruption agent is needed . Furthermore, folate-mediated DNA delivery is restricted to tumor cells that highly overexpress the folate receptor, which will permit future development of tumor cell-specific delivery of toxic genes for cancer gene therapy.

Mol Microbiol, 1994 May, 12(3), 375 - 85
Synthesis of ribosomal proteins during growth of Streptomyces coelicolor; Blanco G et al.; Changes in expression of ribosomal protein genes during growth and stationary phase of Streptomyces coelicolor A3(2) in liquid medium were studied . Proteins being synthesized were pulse-labelled with {35S}-methionine, separated by two-dimensional polyacrylamide gel electrophoresis, and quantified using the BioImage computer software . Most of the ribosomal proteins were synthesized throughout the life cycle . Exceptions were two proteins whose synthesis drastically decreased at the approach of stationary phase . These two proteins were identified in purified ribosomes as homologues of Escherichia coli ribosomal proteins L10 and L7/L12, using antibodies raised against fusion proteins between these ribosomal proteins and Escherichia coli beta-galactosidase . The genes (rplJ and rplL) encoding the L10 and L7/L12 proteins were contained in a 1.2 kb BamHI fragment that was cloned and sequenced . The linkage and order of the genes coincide with other L10-L7/L12 operons . However, L11 and L1 genes were not present immediately upstream of the L10 gene, as is the case for E . coli and other bacteria . Instead, two open reading frames of unknown function were found immediately upstream of the L10 gene, in an adjacent 1.9 kb BamHI fragment.

Mol Cell Biol, 1994 May, 14(5), 2975 - 84
Developmental gene expression in Leishmania donovani: differential cloning and analysis of an amastigote-stage-specific gene; Charest H et al.; Leishmania protozoans are the causative agents of leishmaniasis, a major parasitic disease in humans . During their life cycle, Leishmania protozoans exist as flagellated promastigotes in the sand fly vector and as nonmotile amastigotes in the mammalian hosts . The promastigote-to-amastigote transformation occurs in the phagolysosomal compartment of the macrophage cell and is a critical step for the establishment of the infection . To study this cytodifferentiation process, we differentially screened an amastigote cDNA library with life cycle stage-specific cDNA probes and isolated seven cDNAs representing amastigote-specific transcripts . Five of these were closely related (A2 series) and recognized, by Northern (RNA) blot analyses, a 3.5-kb transcript in amastigotes and in amastigote-infected macrophages . Expression of the amastigote-specific A2 gene was induced in promastigotes when they were transferred from culture medium at 26 degrees C and pH 7.4 to medium at 37 degrees C and pH 4.5, conditions which mimic the macrophage phagolysosomal environment . A2 genes are clustered in tandem arrays, and a 6-kb fragment corresponding to a unit of the cluster was cloned and partially sequenced . An open reading frame found within the A2-transcribed region potentially encoded a 22-kDa protein containing repetitive sequences . The recombinant A2 protein produced in Escherichia coli cells was specifically recognized by immune serum from a patient with visceral leishmaniasis . The A2 protein repetitive element has strong homology with an S antigen of Plasmodium falciparum, the protozoan parasite responsible for malaria . Both the A2 protein of Leishmania donovani and the S antigen of P . falciparum are stage specific and developmentally expressed in mammalian hosts.

Circ Shock, 1994 May, 43(1), 18 - 25
Modulation of pyrogen-induced upregulation of endothelial cell adhesion molecules (CAMs) by interleukin-4: transcriptional mechanisms and CAM-shedding; Kapiotis S et al.; The pyrogens interleukin 1 (IL-1), tumor necrosis factor (TNF), and bacterial lipopolysaccharides (LPS) are known to increase endothelial cell (EC) adhesiveness for leukocytes by stimulating surface expression of various adhesion molecules . IL-4, a product of activated T-cells, was shown to affect pyrogen-mediated regulation of EC adhesion molecule surface expression . In the present study, we investigated the effect of IL-4 on pyrogen-induced upregulation of the cell adhesion molecules (CAMs) ICAM-1 (intercellular cell adhesion molecule-1), ELAM-1 (endothelial leucocyte adhesion molecule-1), and VCAM-1 (vascular cell adhesion molecule-1) in cultured human umbilical vein EC (HUVEC) . Surface expression of adhesion molecules was quantified by flow cytometry, HUVEC mRNA content was estimated by Northern blot analysis, and ICAM-1 antigen in conditioned media was measured by ELISA . Incubation of HUVEC with IL-1 (100 U/ml), TNF (500 U/ml), and LPS (10 micrograms/ml) caused significant increase in ICAM-1, ELAM-1, and VCAM-1 surface expression; IL-1 caused about an eightfold increase in ICAM-1 expression, about a 13-fold increase in ELAM-1 surface expression, and about a fourfold increase in VCAM-1 expression . Coincubation of pyrogens with IL-4 (500 U/ml) differentially influenced their proadhesive effects on the HUVEC surface . In the presence of IL-4, IL-1-induced ICAM-1 upregulation was reduced, ELAM-1 upregulation was not significantly influenced by IL-4, and induction of VCAM-1 was enhanced by IL-4.(ABSTRACT TRUNCATED AT 250 WORDS)

J Biochem (Tokyo), 1994 May, 115(5), 814 - 9
Modification of recombinant human granulocyte colony-stimulating factor (rhG-CSF) and its derivative ND 28 with polyethylene glycol; Yamasaki M et al.; Recombinant human granulocyte colony-stimulating factor (rhG-CSF) has been obtained from genetically engineered Escherichia coli as an unglycosylated protein . Both native glycosylated hG-CSF and rhG-CSF are rapidly cleared from the circulation, which may limit their effectiveness for clinical use . To improve this biological property, rhG-CSF and its derivative ND 28, which has a higher specific activity than does rhG-CSF, were modified with polyethylene glycol (PEG) . Modified rhG-CSF and ND 28 in which 1 to 3 mol of PEG were bound, were purified by two-step chromatography and characterized by several methods . The results of their physicochemical characterization suggest that PEG-modification does not appreciably change the conformation of rhG-CSF and ND 28 . As a result of the whole characterization, the PEG-modification of rhG-CSF and ND 28 enhanced the stability of rhG-CSF and ND 28 and decreased the plasma clearance rate, which led to more effective hemopoiesis.

Biol Pharm Bull, 1994 May, 17(5), 564 - 7
Measurement of staphylokinase by enzyme-linked immunosorbent assay using monoclonal antibodies; Mike A et al.; Hybridoma clones producing monoclonal antibodies specific for staphylokinase were isolated . A competitive assay revealed that the monoclonal antibodies studied could be divided into at least two groups . Representatives of these groups, AS22 and B3E6, recognized quite different epitopes on staphylokinase . This finding led us to develop an assay system for the quantitative analysis of staphylokinase by enzyme-linked immunosorbent assay using AS22 as the capturing antibody and biotinylated B3E6 as the "detector" . The lower limit of sensitivity of the assay was 20 pg of staphylokinase per ml . The assay exhibited good reproducibility, with values of 5.8 and 3.8% for the intra- and inter-assay coefficients of variation, respectively . Staphylokinase could be assayed in the presence of human plasma when the plasma was diluted more than 320-fold, and the measurement was unaffected by the presence of physiological concentrations of human plasminogen . Hence, this assay was considered useful for the detection and quantification of staphylokinase in clinical samples.

Biochem Mol Biol Int, 1994 May, 33(1), 187 - 94
cDNA and protein sequences coding for the precursor of phospholipase A2 from Taiwan cobra, Naja naja atra; Pan FM et al.; The cDNA sequence encoding phospholipase A2 (PLA2) was determined by analysis of polymerase-chain-reaction (PCR) product amplified from total cDNA mixture which had been constructed from the poly(A)+RNA of venom glands obtained from Taiwan cobras . Two oligonucleotide segments corresponding to the 5'- and 3'-noncoding regions of sea-snake PLA2 gene were used as primers for PCR-amplified reaction . Plasmids of transformed E . coli strain JM109 containing amplified PLA2 cDNA were purified and prepared for nucleotide sequencing by dideoxynucleotide chain-termination method . Sequencing more than five clones containing about 0.5 kb DNA inserts revealed two isoforms with complete reading frames of 468 base pairs each covering a precursor for phospholipase A2 with a deduced mature protein sequence of 119 amino acids and a 27 amino-acid signal peptide . These two enzymes of Group I PLA2 differ in six nucleotide residues at the gene level and three amino acids along the whole polypeptide chain, each consisting of 14 cysteine residues similar to all reported PLA2 of different snake venoms . The signal peptides and hydropathy profiles of Group I PLA2 reported here are distinctly different from those of Group II PLA2 in viperid snakes.

J Virol Methods, 1994 May, 47(3), 331 - 43
Use of TrpE fusion protein to identify antigenic domains within the BIV envelope protein; Chen P et al.; Nine different recombinant clones spanning various regions of the bovine immunodeficiency-like virus (BIV) envelope gene open reading frame were generated . These clones span the entire external glycoprotein as well as the transmembrane glycoprotein region . These proteins were expressed as fusions to the TrpE protein in E . coli . The levels of recombinant protein expressed varied, some clones expressed enough protein that can be detected in a Coomassie blue-stained gel, whereas other proteins could only be detected by Western blot analyses . A recombinant env protein representing the extracellular domain of the env protein was detected by BIV-infected bovine sera . In addition, a 134 amino acid peptide which may represent a major immunoreactive epitope was identified . This peptide is located at the amino terminus of the transmembrane glycoprotein and was specifically recognized by all BIV-infected calf sera tested . The identification of this epitope and the use of recombinant envelope protein will enable us to develop a more effective screening test to study the epidemiology of BIV infection.

Can J Microbiol, 1994 May, 40(5), 341 - 4
Survey of cytotoxin production among Escherichia coli strains characterized as enteropathogenic (EPEC) by serotyping and presence of EPEC adherence factor (EAF) sequences; Guth BE et al.; A total of 108 Escherichia coli strains characterized as enteropathogenic (EPEC) by serotyping and the presence of EPEC adherence factor (EAF) sequences were examined for cytotoxin production by cell line assays and colony hybridization with Shiga-like toxin (SLT) probes . Cytolethal distending toxin (CLDT) production was found in three (2.8%) strains belonging to serotype O86:H34, while one O111ab:NM strain hybridized with a SLT-II probe but did not express any cytotoxic activity . All four strains showed localized adherence to HeLa cells and hybridized to an E . coli attaching-effacing gene (eae) probe . The CLDT-producing strains had multiple plasmids and some were present in all strains, including a plasmid of approximately 54 MDa that hybridized with the EAF probe.

J Immunother Emphasis Tumor Immunol, 1994 May, 15(4), 292 - 302
A phase I trial of intravenous interleukin-6 in patients with advanced cancer; Weber J et al.; Eighteen patients were treated with escalating doses of recombinant, Escherichia coli-derived human interleukin-6 (IL-6) intravenously every 8 h . Therapy was given for two cycles of 7 days each separated by a week off therapy . Fevers and chills were observed in most patients . Mild renal and liver function abnormalities were noted at higher doses of IL-6 . Dose-limiting toxicity was reached at 30 micrograms/kg i.v . every 8 h due to reversible neurotoxicity, but significant rapidly reversible anemia and hyperglycemia were seen at lower doses . Platelet counts, white blood cell counts, and acute phase reactant levels were substantially elevated . No antitumor responses were seen . A maximum tolerated dose of 10 micrograms/kg i.v . every 8 h for two 7-day cycles is recommended for future phase II trials.

Biophys J, 1994 May, 66(5), 1388 - 97
Noise analysis of ion current through the open and the sugar-induced closed state of the LamB channel of Escherichia coli outer membrane: evaluation of the sugar binding kinetics to the channel interior; Nekolla S et al.; LamB, a sugar-specific channel of Escherichia coli outer membrane was reconstituted into lipid bilayer membranes and the current noise was investigated using fast Fourier transformation . The current noise through the open channels had a rather small spectral density, which was a function of the inverse frequency up to about 100 Hz . The spectral density of the noise of the open LamB channels was a quadratic function of the applied voltage . Its magnitude was not correlated to the number of channels in the lipid bilayer membrane . Upon addition of sugars to the aqueous phase the current decreased in a dose-dependent manner . Simultaneously, the spectral density of the current noise increased drastically, which indicated interaction of the sugars with the binding site inside the channel . The frequency dependence of the spectral density was of Lorentzian type, although the power of its frequency dependence was not identical to -2 . Analysis of the power density spectra using a previously proposed simple model (Benz, R., A . Schmid, and G . H . Vos-Scheperkeuter . 1987 . J . Membr . Biol . 100: 12-29), allowed the evaluation of the on- and the off-rate constants for the maltopentaose binding to the binding site inside the LamB channels . This means also that the maltopentaose flux through the LamB channel could be estimated by assuming a simple one-site, two-barrier model for the sugar transport from the results of the noise analysis.

Br J Pharmacol, 1994 May, 112(1), 289 - 91
The effect of nitric oxide synthase inhibition on the plasma fibrinolytic system in septic shock in rats; Korbut R et al.; 1 . We have investigated the effect of pretreatment of rats with nitric oxide (NO) synthase inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on the E . coli lipopolysaccharide (LPS)-induced changes in the plasma fibrinolytic system, platelet count, fibrinogen level, as well as in gross and microscopic pathophysiological changes indicative of disseminated intravascular coagulation (DIC) in rats . 2 . E . coli LPS (6 mg kg-1, i.p.) produced a decrease in the levels of plasma fibrinogen and a drop in the blood platelet count 6 h after administration . The decrease in fibrinogen but not the drop in platelet count was reversed by pretreatment with L-NAME (30 mg kg-1, i.p., 24 h and 15 min before administration of LPS) . 3 . Pretreatment with L-NAME antagonized the LPS-induced activation of fibrinolysis as measured by changes in the euglobulin clot lysis time (ECLT) and enhanced the LPS-induced rise in the plasma level of plasminogen activator inhibitor (PAI) . In animals pretreated with L-NAME there was also a marked reduction in the histological changes indicative of DIC . 4 . We propose that L-NAME can act as a protective agent in LPS-induced DIC, and this protection is due to an increased generation of PAI following inhibition of NO synthase.

Microbiology, 1994 May, 140 ( Pt 5), 1119 - 24
Frequencies of lipopolysaccharide core types in Escherichia coli strains from bacteraemic patients; Appelmelk BJ et al.; We have investigated the distribution of the various core types (R1, R2, R3, R4 and K-12) in 138 Escherichia coli isolates obtained from positive blood cultures . Rabbit antisera, raised against five rough strains expressing the respective core types, were made monospecific by extensive absorption . The reactivity of the antisera was tested in ELISA with bacterial cells that had been autoclaved for full exposure of core epitopes . One hundred and thirty strains could be typed directly, while eight strains required prior digestion with proteinase K for removal of cross-reactions . Ninety-four of the strains (68%) expressed the R1 type, and 9 (6.5%), 12 (8.7%), 7 (5.1%) and 3 (2.2%) strains expressed the R2, R3, R4 and K-12 core types, respectively . An R1R4 mixed core type, hitherto not yet described, was found in 13 (9.4%) strains . Results obtained with polyclonal antisera were in agreement with those obtained with monoclonal antibodies to the R1, R2 and R3 core types . Core typing may serve as an additional serological marker next to conventional typing of O-, H- and K-antigens.

Rinsho Byori, 1994 May, 42(5), 460 - 6
{Molecular cloning, expression and epitope mapping of nuclear antigen}; Yamamoto K; The antinuclear antibody is a major characteristic phenomenon in systemic type of autoimmune diseases . There is a good correlation between symptoms or diseases and the types of antinuclear antibodies in these patients . Therefore, the study of the mechanisms of antinuclear antibody production is thought to be important in the elucidation of the pathogenesis of such autoimmune diseases . The conventional methods to detect antinuclear antibodies have been useful to date in clinical studies . However, these methods are not sufficient for more precise investigations . Thus, we have applied the recently developed molecular biology to this field . cDNAs which encode nuclear antigens have been cloned and their protein products were expressed as recombinant fusion protein in E . coli . By this procedure, we could establish simple and reproducible method to detects antinuclear antibodies . Furthermore, epitope mapping of nuclear antigens could be performed using deletion mutants generated by enzymatic manipulations of the cDNAs . These epitope studies are facilitating a more precise understanding of the mechanisms of antinuclear antibody production.

FEMS Microbiol Lett, 1994 May 1, 118(1-2), 23 - 30
Characterization of Mip proteins of Legionella pneumophila; Ludwig B et al.; The Mip ('macrophage infectivity potentiator') protein of Legionella pneumophila has been shown to be an essential virulence factor, exhibiting peptidyl-prolyl cis/trans isomerase (PPIase) activity that can be inhibited by the immunosuppressant FK506 . The cloning and sequencing of mip genes from three different L . pneumophila strains revealed a single amino acid substitution which did not affect the isomerase property of the enzyme . Mip proteins isolated from two wild-type L . pneumophila strains and from two corresponding Escherichia coli K-12 recombinant clones derived from these strains exhibited identical enzymatic properties and the precursor proteins are processed at identical cleavage sites . The mature Mip proteins exist in an oligomeric form . Site-directed mutagenesis demonstrated that a substitution of an Asp residue at position 142 by a Leu residue affects PPIase activity of Mip.

J Bacteriol, 1994 May, 176(10), 2807 - 13
The RNA chain elongation rate in Escherichia coli depends on the growth rate; Vogel U et al.; We determined the rates of mRNA and protein chain elongation on the lacZ gene during exponential growth on different carbon sources . The RNA chain elongation rate was calculated from measurements of the time elapsing between induction of lacZ expression and detection of specific hybridization with a probe near the 3' end of the mRNA . The elongation rate for the transcripts decreased 40% when the growth rate decreased by a factor of 4, and it always correlated with the rate of translation elongation . A similar growth rate dependency was seen for transcription on the infB gene and on a part of the rrnB gene fused to a synthetic, inducible promoter . However, the untranslated RNA chain specified by the rrnB gene was elongated nearly twice as fast as the two mRNA species encoded by infB and lacZ.

Leukemia, 1994 May, 8(5), 780 - 5
Structural analysis of the deoxycytidine kinase gene in patients with acute myeloid leukemia and resistance to cytosine arabinoside; Flasshove M et al.; Deficiency of deoxycytidine kinase (dCK) activity represents one possible cause of resistance to cytosine arabinoside (ara-C) . Mutations of the dCK gene have recently been shown to be responsible for dCK deficiency and increased resistance in vitro . In order to define the relevance of this mechanism in vivo, we analyzed the dCK gene in 16 adult patients with relapsed/refractory acute myeloid leukemia (AML) and clinical resistance to standard-dose and/or high-dose ara-C . Southern blot analysis using genomic DNA from peripheral blood or bone marrow samples containing > or = 70% leukemic blasts and agarose gel electrophoresis of cDNA obtained by RT-PCR did not reveal gross rearrangements of the dCK gene . Sequencing of the dCK coding region showed point mutations in seven patients . Besides two silent mutations (or RFLPs) in codon 42 and 86, base pair mutations resulting in amino acid replacements were found in five patients affecting codon 20, 93, 98, 99, and 154, respectively . dCK cDNA clones from three patients with > or = 50% of sequenced clones revealing the specific base pair alteration were bacterially expressed in E . coli and analyzed for dCK activity . Normal enzyme activity was found in two patients (codon 20 and 98), and a complete loss of activity in one patient (codon 99) . We conclude that structural alteration of the coding region of the dCK gene represents one possible mechanism for ara-C resistance in vivo, but, considering the frequency of this event, other mechanisms may play a more important role for clinical resistance to ara-C in patients with AML.

Mutat Res, 1994 May 1, 307(1), 53 - 9
Enzyme-dependent pausing during in vitro replication of O4-methylthymine in a defined oligonucleotide sequence; Menichini P et al.; We had previously reported that an oligonucleotide containing a site-specifically incorporated O4-methylthymine (m4T) was replicated under kinetic conditions by the Klenow fragment of E . coli DNA polymerase I (Kf) (Dosanjh et al., 1993) . Using other polymerases for complete replication, but with limiting enzyme, a pause site before the m4T was observed . In order to investigate whether such a pause could be due to enzyme dissociation or stalling, trapping experiments were designed to aid in differentiating the two mechanisms . Rather than the generally used heparin or sheared DNA trap, these experiments utilized as the acceptor the same oligonucleotide containing unmodified thymine . It was observed that, under enzyme-limiting conditions, the nature of the enzyme played a major role in replication of m4T . With a running start, Kf and calf-thymus polymerase alpha-primase allowed replication beyond the m4T, while Sequenase and T7 showed a strong pause site at the base before m4T . When the oligonucleotide trap was added after different times of replication, it was found that Sequenase remained bound to the template-primer, regardless of whether T or m4T was present . In contrast, Kf dissociated and re-associated rapidly . Thus, m4T appears to be a strong replication block when using limiting amounts of a highly processive enzyme such as Sequenase or T7 . This may imply that such enzymes discriminate against forming a poor basepair but remain bound to the primer-template or become inactivated.

Mutat Res, 1994 May 1, 307(1), 335 - 44
Improved method for mutagenicity testing of gaseous compounds by using a gas sampling bag; Araki A et al.; A simple and safety gas exposure method was developed using a gas sampling bag as an exposure vessel and a preparation vessel of diluted gas . The gas exposure conditions such as amount of S9 in the plate, volume of gas for the plate, amount of top agar, exposure period and exposure temperature were examined by mutagenicity testing of 1,3-butadiene using the gas sampling bag . Mutagenicity tests of 14 compounds and 1,3-butadiene on S . typhimurium TA98, TA100, TA1535 and TA1537, and E . coli WP2 uvrA were also examined by the developed gas exposure method . 1,3-Butadiene, propyne (methyl acetylene), monochlorodifluoromethane, ethylchloride, diborane and silane were mutagenic . 1-Butene, 2-butene, 2-methylpropene, methyl vinyl ether, trichlorofluoromethane, dichlorodifluoromethane, 1,2-dichloro-1,1,2,2-tetrafluoroethane, 1,1-difluoroethane and phosphine were not mutagenic on S . typhimurium TA98, TA100, TA1535 and TA1537, and E . coli WP2 uvrA with or without metabolic activation . These results were compatible with a previous report, and this developed method has the advantage that it can be tested easily and safely for combustible and self-combustible substances such as 1,3-butadiene and silane.

Mutat Res, 1994 May 1, 307(1), 169 - 73
Response of the Muta mouse lacZ/galE- transgenic mutation assay to DMN: comparisons with the corresponding Big Blue (lacI) responses; Tinwell H et al.; The lacZ Muta mouse transgenic mutation assay was recently adapted into a selective assay based on use of E . coli galE(-) bacteria and phenyl galactoside (p-gal) . A preliminary assessment of this selective assay was undertaken using a single oral dose of 10 mg/kg of dimethyl nitrosamine (DMN) . The livers of treated male mice were assessed for UDS 2 h after dosing, and for lacZ- mutations 7, 11 and 20 days after dosing . A strong UDS response was recorded and a clear mutagenic response was observed at each of the 3 timepoints . Comparison of these data with earlier data derived using the Big Blue (lacI) mutation assay reveals a marginally greater sensitivity to DMN of the selective Muta mouse assay, an effect probably related to a biochemical difference between the strains of animal, as evidenced by the larger UDS response seen in the Muta mouse system . The original Muta mouse assay protocol was impractical . The galE- adaption makes the assay emminently practical and cost-effective . We are continuing to assess the true role of both the Big Blue assay and the galE- Muta mouse assay in mutagenicity/carcinogenicity prediction . The former assay enables access to B6C3F1 mice and F344 rats, the latter enables the rapid acquisition of data.

Mutat Res, 1994 May 1, 307(1), 149 - 56
Starvation-associated mutation in Escherichia coli: a spontaneous lesion hypothesis for "directed" mutation; Bridges BA; When stationary phase E . coli WU3610, carrying an ochre mutation in the tyrA gene, were incubated on plates lacking tyrosine, tyrosine-independent (Tyr+) mutants appeared from day 7 onwards in a time-dependent manner . These starvation-associated mutants did not contain either identifiable tRNA suppressors or reversions at the ochre site and are thus quite distinct from the mutants commonly found to arise during active growth . When an appropriate fluctuation assay protocol was employed slow growing Tyr+ mutants were also found to arise in growing cells, and their distribution was more characteristic of a replication-dependent than a time-dependent process . The rate of appearance of starvation-associated mutants at 37 degrees C was somewhat less than at 27 degrees C and this was attributed to a reduction in viability at the higher temperature . There was no evidence for the accumulation on the plates of mutations in other genes . Tyr+ mutants were, however, shown to arise during incubation of stationary phase cells under conditions where there was no selection for tyrosine independence, provided outgrowth was subsequently permitted on plates lacking tyrosine . This distinguishes the present system from those systems exhibiting genuine "directed" or "adaptive" mutation, should they exist . To explain the specificity which occurs, it is proposed that the appearance of stationary mutants in ochre strains reflects the time-dependent accumulation in the transcribed strand of a DNA lesion that has a high probability of miscoding during transcription and replication . A mutant RNA transcript permits protein synthesis which in turn triggers DNA replication . The mutation is then fixed in the DNA as a permanent heritable change . The apparent "directedness" of the process is, on this model, determined solely by the particular miscoding DNA lesion occurring in a transcribed strand at a site where a change in phenotype permits DNA replication to occur.

Mutat Res, 1994 May 1, 307(1), 115 - 20
Regulatory processes and the origins of spontaneous mutations; MacPhee DG; A review of information currently available about the origins of spontaneous mutational events suggests that there may be a role for known cellular control mechanisms in determining the frequencies with which such events can occur . Attention is also directed to recent findings with antimutator (dnaE) mutants of Escherichia coli which indicate that the final step involved in generating a spontaneous mutational event may be different from that involved in generating an SOS-dependent mutational event . Finally, the possible involvement of various sorts of treatments collectively referred to as stress responses (heat shock, cold shock or oxidative damage, etc.) in generating random mutations is discussed; if there is such an involvement, this may represent one way in which organisms are programmed to adapt to a wide variety of environmental challenges.

J Clin Endocrinol Metab, 1994 May, 78(5), 1108 - 12
Autoantibody epitope mapping of the 21-hydroxylase antigen in autoimmune Addison's disease; Song YH et al.; Patients with idiopathic Addison's disease are characterized by cytoplasmic adrenal autoantibodies, detectable by indirect immunofluorescence of cryocut sections of human adrenal cortex . Recently, autoantibodies that bind a 55-kilodalton protein in the microsomal fraction of adrenal gland extracts identified to be the cytochrome P450 enzyme 21-hydroxylase have been found in Addisonian patient sera . We confirm the finding and report here the autoantigenic epitopes involved in the autoantibody reactivity using recombinant DNA technology . Six cDNA fragments spanning different regions of the 21-hydroxylase gene were expressed as fusion proteins with glutathione S-transferase in Escherichia coli . Immunoblot analyses were used to evaluate the reactivity of the recombinant proteins with patients' sera to determine the autoepitopes involved . We found that a conserved region (amino acids 164-356) reacted with 25 of 30 adrenal autoantibody-positive sera tested . One serum sample reacted only with the amino portion of the 21-hydroxylase (amino acids 1-162) . In addition, 4 other enzymes important to steroid hormone biosynthesis, 11 beta-hydroxylase, 17 alpha-hydroxylase, side-chain cleavage enzyme P450, and 3 beta-hydroxysteroid dehydrogenase, were expressed in E . coli, but none of them gave positive autoantibody reactions by Western blot assays, even using sera from 5 patients with type I autoimmune polyglandular syndrome . The availability of recombinant antigens has permitted structural analysis of the autoepitopes involved in the autoimmune response to 21-hydroxylase in Addison's disease . Our findings should lead to the development of a simple and specific tool for immunodiagnosis of the disease.

J Infect Dis, 1994 May, 169(5), 1014 - 22
Borrelia burgdorferi and Escherichia coli lipopolysaccharides induce nitric oxide and interleukin-6 production in cultured rat brain cells; Tatro JB et al.; Borrelia burgdorferi, the spirochetal agent of Lyme disease, infects the central nervous system (CNS), but the factors that mediate inflammation and neurologic dysfunction are not known . Sonicated B . burgdorferi stimulated in a concentration-dependent manner the production of nitric oxide (NO) in glial-enriched primary cultures of neonatal rat brain cells via induction of NO synthase activity . Lipopolysaccharide (LPS) of Escherichia coli also stimulated nitrite accumulation in a concentration-dependent manner . Stimulation of NO production by B . burgdorferi sonicate and E . coli LPS was associated with increased levels of mRNA coding for the cytokine-inducible form of NO synthase . B . burgdorferi sonicate also stimulated release of interleukin-6, with a concentration-response relationship similar to that for its stimulation of nitrite production, as did E . coli LPS . A competitive antagonist of E . coli LPS, Rhodopseudomonas sphaeroides lipid A, inhibited LPS-induced stimulation of NO synthase activity but markedly potentiated that of B . burgdorferi, indicating that the initial triggering mechanism of B . burgdorferi is distinct from that of E . coli LPS . Induction of NO synthase by bacterial agents within the brain may represent a common pathway of CNS inflammation and neurotoxicity.

J Bacteriol, 1994 May, 176(9), 2677 - 88
Mechanism of binding of the antisense and target RNAs involved in the regulation of IncB plasmid replication; Siemering KR et al.; The replication frequency of the IncB miniplasmid pMU720 is dependent upon the expression of the repA gene . Binding of a small, highly structured, antisense RNA (RNA I) to its complementary target in the RepA mRNA (RNA II) inhibits repA expression and thus regulates replication . Analyses of binding of RNA I to RNA II indicated that the reaction consists of three major steps . The first step, initial kissing complex formation, involves base pairing between complementary sequences in the hairpin loops of RNA I and RNA II . The second step is facilitated by interior loop structures in the upper stems of RNA I and RNA II and involves intrastand melting and interstrand pairing of the upper stem regions to form an extended kissing complex . This complex was shown to be sufficient for inhibition of repA expression . The third step involves stabilization of the extended kissing complex by pairing between complementary single-stranded tail regions of RNA I and RNA II . Thus, the final product of RNA I-RNA II binding is not a full duplex between the two molecules.

J Bacteriol, 1994 May, 176(9), 2502 - 6
Effect of 4.5S RNA depletion on Escherichia coli protein synthesis and secretion; Jensen CG et al.; We examined the synthesis of individual proteins following depletion of 4.5S RNA by using a strain deficient in the induction of heat shock proteins . We found that initially the synthesis of all proteins was equally affected, and the peptide elongation rate was reduced by approximately 10% . For up to 1 generation time after the onset of inhibition of total protein synthesis, the processing of secreted proteins was unaffected . After further depletion of 4.5S RNA, accumulation of precursors of secreted proteins was observed under some growth conditions.

Infect Immun, 1994 May, 62(5), 2051 - 7
Characterization of a linear epitope on Chlamydia trachomatis serovar L2 DnaK-like protein; Birkelund S et al.; A cytoplasmic 75-kDa immunogen from Chlamydia trachomatis serovar L2 has previously been characterized as being similar to the Escherichia coli heat shock protein DnaK . We have localized a linear epitope for one monoclonal antibody specific for C . trachomatis DnaK . By use of a recombinant DNA technique, the epitope was limited to 14 amino acids . With synthetic peptides, the epitope was further limited to eight amino acids . Six of these amino acids are conserved in bovine HSP70, which has a known three-dimensional structure . The amino acid sequence homologous to the epitope is located in a linear part of the HSP70 molecule known as connect II.

Mutat Res, 1994 May, 314(3), 287 - 95
Endonuclease VII of phage T4 nicks N-2-acetylaminofluorene-induced DNA structures in vitro; Bertrand-Burggraf E et al.; We have tested in vitro the activity of T4 endonuclease VII on three different double-stranded oligonucleotides bearing a single N-2-acetylaminofluorene (AAF) adduct covalently bound to each of the three guanine residues located within the NarI site (G1G2CG3CC), a strong frameshift mutation hot spot in E . coli . With the oligonucleotides modified at G2 and G3 a specific cleavage pattern with T4 endonuclease VII was observed in the complementary strand while no cleavage was found in the adduct-bearing strand . On the other hand, when G1 was modified, only a very faint cleavage band was observed (< 1%) . These differences in nicking among the three AAF-modified DNA substrates are discussed in terms of the polymorphic nature in adduct-induced DNA structures as previously shown . This "non-physiological" activity of a DNA resolvase is discussed in terms of a potential role for such enzymes in the induction of frameshift mutations.

Mutat Res, 1994 May, 314(3), 273 - 85
Roles of transcription and repair in alkylation mutagenesis; Ito T et al.; Mutations occurring in Escherichia coli cells exposed to alkylating agents have been analyzed using an assay for forward mutations in the E . coli rpsL gene cloned on a high copy number plasmid . N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced mutations were recovered from wild-type and O6-methylguanine methyltransferase-deficient mutant (ada- ogt-) cells and their sequence alterations determined . We found that the mutations recovered from the wild-type strain were predominantly G:C to A:T transitions located at several hot spots in the rpsL sequence . A vast majority of the mutations were found at guanine residues preceded by thymine on the transcribed strand of the target gene . Although the methyltransferase mutant showed hypersensitivity to the alkylating reagent in terms of mutagenic effect and cell killing effects, the class and site distributions of the rpsL- mutations recovered from MNNG-treated ada- ogt- cells were similar to those observed with MNNG-treated wild-type cells . Therefore, the site preference of MNNG-induced rpsL- mutations seems to be due not to the specificity of methyl-transferring repair enzymes but probably to the distribution of the mutagenic lesions (O6-methylguanine) in the target sequence . Mutations induced by methyl methanesulfonate, an SN2 alkylating agent, showed similar class and site distributions in the rpsL system . The site preference of MNNG-induced mutations was significantly changed when the level of transcription of the rpsL gene was decreased to 120-fold lower than that promoted by the authentic rpsL promoter . Under these conditions, 78% of mutations were induced at the central guanine of 5'-GG(A or C)-3' and 2/3 of them were on the non-transcribed strand of the rpsL gene . These results suggested that the site preference of MNNG-induced mutations is determined by at least three factors: (i) a flanking-base effect on the chemical reactivity of a guanine residue, (ii) transcribed strand-specific repair, probably by the UvrABC system, and (iii) the effects of transcription of the target gene on the alkylation of DNA and the strand-specific repair.

J Histochem Cytochem, 1994 May, 42(5), 635 - 43
Immunostaining of DNA in electron microscopy: an amplification and staining procedure for thin sections as alternative to gold labeling; Bohrmann B et al.; We describe a new electron microscopic on-section staining technique with high specificity and sensitivity for DNA-containing structures . Lowicryl HM20 sections of specimens obtained by cryofixation and freeze-substitution are incubated in a first step with a primary IgM antibody specific for double-stranded DNA . The layer of bound antibodies at the section surface is amplified in a successive step by a secondary IgG antibody . Finally, electron scattering of the antibody layer produced is enhanced by staining with a mixture of uranyl acetate and potassium permanganate . The applicability of the method is exemplified by the detection of shape and distribution of various types of bacterial and eukaryotic chromatin.

J Virol, 1994 May, 68(5), 3421 - 4
Mutagenic study of codons 74 and 215 of the human immunodeficiency virus type 1 reverse transcriptase, which are significant in nucleoside analog resistance; Lacey SF et al.; Mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) at codon 215 has been shown to play a significant role in resistance to zidovudine (AZT) . Substitution of threonine with tyrosine or phenylalanine alone confers decreased susceptibility to the inhibitor . In this study we constructed a panel of 10 viruses with different amino acids at this codon, including 7 novel mutants, and assessed their susceptibilities to AZT . The majority of the new mutants were AZT sensitive, whereas the Thr-215-->Trp mutant was partially resistant (threefold less susceptible) . A combination of the Thr-215-->Trp with the other AZT resistance mutations Lys-70-->Arg and Met-41-->Leu gave additive resistance . The Thr-215-->Phe virus was less AZT resistant than the Thr-215-->Tyr mutant, both on its own and when each was combined with the Met-41-->Leu mutant . These observations confirm the general hypothesis that increased bulk of the amino acid side chains at this position confers decreased AZT sensitivity . A leucine-to-valine substitution at codon 74 has previously been found to confer dideoxynucleoside resistance . We constructed mutants with five novel amino acid substitutions (Ala, Gly, Glu, Met, and Asp) at codon 74 . Of these, only one (that with the Met substitution) retained enough RT activity to yield viable virus . It thus appears that there are severe structure-function constraints on the amino acid side chains at this position in the enzyme . The activities of the Leu-74-->Ala and Leu-74-->Met RT enzymes expressed in Escherichia coli appeared to have reduced susceptibility to ddGTP compared with the wild-type enzyme . The mutants described in this work may prove useful for correlation with structural studies of the human immunodeficiency virus type 1 RT.

Clin Diagn Lab Immunol, 1994 May, 1(3), 295 - 8
Lipopolysaccharide-reactive immunoglobulin E is associated with lower mortality and organ failure in traumatically injured patients; DiPiro JT et al.; Antilipopolysaccharide (anti-LPS) immunoglobulin G (IgG) and IgM have been associated with protection from LPS effects in vivo . We investigated the presence of IgE and anti-LPS in 32 patients that had experienced severe traumatic injury and in 35 healthy volunteers; we also investigated whether IgE anti-LPS was associated with important clinical events . Plasma samples were collected daily from patients in the intensive care unit and on one occasion from volunteers; the samples were assayed for IgE anti-LPS . IgE anti-LPS was assayed by enzyme-linked immunosorbent assay with monoclonal anti-human IgE as the capture antibody . Detection was accomplished with biotin-labeled LPS (Escherichia coli J5 mutant) followed by streptavidin-peroxidase with 2,2'-azino(3-ethylbenzthiazoline)sulfonic acid as the substrate . The assay was demonstrated to be specific for IgE and LPS-biotin by nonreactivity of control sera with high-titer anti-LPS IgG and IgM and by inhibition with unlabeled LPS . IgE anti-LPS was detected in 1 of 35 healthy controls (2.9%) and 25 of 32 traumatically injured patients (78%) (P < 0.001) . The presence of IgE anti-LPS was associated with a lower incidence of death (P = 0.026) and of renal failure (P = 0.0012) . There was no apparent temporal relationship between detection of IgE anti-LPS and clinical events . IgG anti-LPS was detected more frequently in patients that were positive for IgE anti-LPS (P = 0.06) but was not associated with clinical events . The inability to detect IgE anti-LPS may be related to adverse clinical events through depletion of specific IgE due to LPS exposure after trauma or through saturation of the assay by IgE with other specificities . We have reported increased total IgE concentrations in these patients . (J.T . DiPiro, R.G . Hamilton, T . R . Howdieshell, N . F . Adkinson, and A . R . Mansberger, Ann . Surg . 215:460-466, 1992).

J Biotechnol, 1994 Apr 30, 34(1), 79 - 86
High-level expression of a biologically active human interleukin-6 mutein; Skelly SM et al.; We have constructed two different muteins of interleukin-6 (IL-6) which were expressed in Escherichia coli . Both muteins lack the first 22 N-terminal amino acids of native IL-6 and lack one or the other of the two naturally occurring pairs of cysteines at either position 45 and 51 or position 74 and 84 of IL-6 . We found that there was a dramatic increase in the level of IL-6 produced from each mutein clone, compared to the level produced by the wild-type IL-6 clone . We also observed that the yield of soluble and properly refolded mutein IL-6 was highest when the cysteines at position 74 and 84 were left intact . The mutein IL-6 with cysteines at position 74 and 84 was as active as wild-type IL-6 and a lower concentration of the mutein IL-6 was required to reach maximal activity, compared to wild-type IL-6 . The mutein IL-6 with cysteines at position 45 and 51 had a much reduced biological activity.

Biochem Biophys Res Commun, 1994 Apr 29, 200(2), 877 - 83
Over-expression of the yeast ATP synthase subunit D in Escherichia coli: use of polyclonal antibodies directed against recombinant subunit D; Norais N et al.; The yeast ATP synthase subunit d was over-expressed in E . coli and formed inclusion bodies . It was purified by solubilization in urea and slow removal of the urea by stepwise dialysis in the presence of a non-ionic detergent . The resulting soluble subunit d was used to prepare polyclonal antibodies . Blots of yeast mitochondrial proteins were probed with these antibodies . The strain disrupted in ATP4 gene encoding the subunit 4 displayed only 8% of the wild type subunit d . Antibodies against subunit d did not inhibit the wild type ATPase activity.

Biochem Biophys Res Commun, 1994 Apr 29, 200(2), 844 - 51
Identification and characterization of the sec-A protein homologue in the cyanobacterium Synechococcus PCC7942; Nakai M et al.; The secA gene product mediates protein translocation across the cytoplasmic membrane in Escherichia coli . We have cloned a gene homologous to secA from the genome of the cyanobacterium Synechococcus PCC7942 . The deduced amino acid sequence, 948 amino acids long, shows 43% homology with that of the E . coli secA and 47-48% homology with those of the algal plastid secA genes . Upon subcellular fractionation, the cyanobacterial SecA protein was mainly found as soluble homodimer in the cytosol, but the remaining small but distinct fraction was associated with both the cytoplasmic and thylakoid membranes . The SecA protein likely participates in protein translocation across both the cytoplasmic and thylakoid membranes in cyanobacterial cells.

Biochem Biophys Res Commun, 1994 Apr 29, 200(2), 777 - 83
The purified COOH-terminal domain of the insulin receptor carries activity to stimulate protein kinase activity or autophosphorylation of the beta subunit domain of insulin receptor; Kasuya J et al.; Our previous study using a deletion mutant indicated that the COOH-terminal (CT) domain of the insulin receptor plays important roles in both catalytic efficiency and stability of the receptor kinase (Yan et al., J . Biol . Chem., 268 {1993} 22444) . In this study, we purified the CT domain of 98 amino acids from bacterial cells over-expressing the CT domain and examined its effect on insulin and IGF-I receptor protein kinases . The purified CT domain stimulated the kinase activities of purified insulin receptor-transmembrane/cytoplasmic domain (IRTMTPK) and its CT domain-deletion mutant (IRTMTPK delta CT), 3.3-fold and 2.3-fold, respectively, while it was less effective in stimulating the kinase activity of purified IGF-I receptor transmembrane/cytoplasmic domain (IGFIRTMTPK) (1.4-fold) . When the effect of the CT domain on autophosphorylation was examined, a marked increase in autophosphorylation was observed only with IRTMTPK delta CT . These results suggest that the CT domain specifically interacts with the insulin receptor cytoplasmic domain, thereby activating the kinase or autophosphorylation activity.

Biochem Biophys Res Commun, 1994 Apr 29, 200(2), 695 - 703
A novel mutation in Cu/Zn superoxide dismutase gene in Japanese familial amyotrophic lateral sclerosis; Nakano R et al.; Recently, several missense mutations in the Cu/Zn superoxide dismutase gene (SOD1) have been reported as a putative cause of chromosome-21q-linked familial amyotrophic lateral sclerosis (FALS) . We have discovered a novel missense mutation (substitution of Thr for Ala4) in exon 1 (GCC to ACC) in two FALS patients from one Japanese FALS family . No mutations were found in 17 cases of sporadic ALS . The enzyme activity of recombinant fusion protein containing the Cu/Zn superoxide dismutase (SOD) with the Ala4-to-Thr mutation was significantly reduced in E . coli . On the other hand, in the expression system in insect cells using Baculovirus, the mutant SOD expressed an enzyme activity as high as wild-type SOD . These results suggest that the stability of SOD with the Ala4-to-Thr mutation is disrupted especially in the fusion protein . Autopsy was carried out on one of the two patients, and the pathological findings were typical of FALS with posterior column involvement . These results raise the possibility that mutation of the SOD1 is responsible for FALS with broader pathological involvement.

J Biol Chem, 1994 Apr 29, 269(17), 12925 - 31
Cloning and expression of a novel, highly truncated phosphoinositide-specific phospholipase C cDNA from embryos of the brine shrimp, Artemia; Su X et al.; A novel, highly truncated form of a cDNA encoding Artemia phosphoinositide-specific phospholipase C (PLC), designated PLC-beta x, was isolated from a brine shrimp cDNA library . The full-length cDNA is of the beta-type, it is 2855 base pairs long, and it contains an open reading frame encoding 489 amino acids . The deduced amino acid sequence of PLC-beta c cDNA shows novel features . It lacks several hundred amino acids at the 5' end, as compared to PLC-beta s in the higher species . It contains conserved domains X and Y, but domain X is highly truncated at the 5' end (only 14-25 conserved amino acids as compared to about 150 amino acids in the higher eukaryotic organisms) . Northern blot hybridization showed that the PLC-beta x cDNA corresponds to a 4.4-kilobase mRNA . Northern blot hybridization with a cDNA probe from the 5' end and PCR performed upstream from domain Y showed that PLC-beta x is not a cloning artifact due to fusion of an unrelated clone into the coding region of the PLC-beta homologue . A functional PLC and new protein bands on SDS-PAGE were observed after subcloning full-length PLC-beta x cDNA, as well as a fragment containing the conserved regions, into expression plasmid vectors and transfecting into Escherichia coli . 1 mM lithium markedly stimulated expression in E . coli.

J Biol Chem, 1994 Apr 29, 269(17), 12840 - 5
Precursor-specific requirements for SecA, SecB, and delta muH+ during protein export of Escherichia coli; Ernst F et al.; We compare translocation into inside-out plasma membrane vesicles (INV) of the in vitro synthesized outer membrane proteins LamB and OmpA and the periplasmic protein Skp of Escherichia coli and demonstrate a precursor-specific dependence on the export factors SecA, SecB, and the proton-motive force (delta mu H+) . A partial reduction in soluble SecA caused a 50% decrease in translocation of preLamB . In contrast, removal of INV-bound SecA by urea extraction was required to see a decrease in translocation of preOmpA and preSkp, with 8% of preSkp still being translocated into urea-treated INV . Translocation of the three precursors into INV showed a corresponding differential sensitivity toward dissipation of delta mu H+ following removal of the F1-ATPase from the INV . While depletion of both F1 and SecA or simply lowering of the reaction temperature resulted in an inhibition of complete transmembrane translocation, it interfered less severely with signal sequence cleavage, indicating the formation of translocation intermediates under these conditions . The relative amounts of intermediate obtained were also different for the three preproteins correlating a low requirement for SecA and delta mu H+ with a facilitated initiation of translocation . Whereas preSkp was translocated independently of SecB, preLamB was not even targeted to the INV in its absence . Functional targeting of preOmpA required the presence of SecB during incubation of the precursor with INV and not during its synthesis . SecB, exogenously added during the period of synthesis, did not prevent the formation of translocation-incompetent preLamB . The latter results are consistent with an important targeting function of SecB, which so far has mostly been described as a molecular chaperone . The findings are discussed with respect to current models of bacterial protein export usually derived from the analysis of a single precursor.

J Biol Chem, 1994 Apr 29, 269(17), 12833 - 9
Identification of a soluble SecA/SecB complex by means of a subfractionated cell-free export system; Hoffschulte HK et al.; We have reconstituted the cell-free synthesis of the Escherichia coli precursor protein LamB from partially purified subfractions of an E . coli cell extract . PreLamB synthesized in this manner is translocated into salt-extracted plasma membrane vesicles only in the presence of SecA/SecB- or SecB-containing preparations of the E . coli cytosol . The most active preparations obtained upon purification were those containing a soluble SecA/SecB complex . Complex formation between SecA and SecB was verified by co-sedimentation and co-immunoprecipitation . When preLamB was synthesized in the presence of this material, a considerable amount of precursor was recovered from a soluble ternary complex consisting of preLamB, SecA, and SecB . Our results suggest that a soluble SecA/SecB complex participates in the export of preLamB and that this complex is functionally equivalent to a previously described 12 S (7 S) export factor (Muller, M., and Blobel, G . (1984) Proc . Natl . Acad . Sci . U.S.A . 81, 7737-7741; Watanabe, M., and Blobel, G . (1989) Proc . Natl . Acad . Sci . U.S.A . 86, 2728-2732).

J Biol Chem, 1994 Apr 29, 269(17), 12749 - 54
Repair of benzo(a)pyrene diol epoxide- and UV-induced DNA damage in dihydrofolate reductase and adenine phosphoribosyltransferase genes of CHO cells; Tang MS et al.; Using Uvr proteins we have quantified benzo(a)pyrene diol epoxide (BPDE)-DNA adduct formation and repair at the dihydrofolate reductase (DHFR) and adenine phosphoribosyltransferase (APRT) genes in two Chinese hamster ovary cell lines: B-11 cells, which are 50-fold amplified for DHFR, and AT3-2 cells, which are diploid for DHFR . We have found that: 1) BPDE-DNA adduct formation in different regions of the DHFR gene is proportional to the concentration of BPDE . 2) There is no significant difference in the repair of BPDE-DNA adducts between the coding and noncoding regions in either amplified or nonamplified DHFR gene domains . 3) Repair in the nonamplified DHFR gene is more efficient (30-40%) than in the amplified DHFR genes . 4) There are no significant differences of repair in the transcribed or nontranscribed strands of the DHFR gene . 5) BPDE-DNA adduct formation and repair in the APRT gene in B-11 and AT3-2 cells are the same . These results contrast those for the repair of cyclobutane pyrimidine dimers, which occurs preferentially in the transcribed strand of the DHFR gene and in which gene amplification appears to play no role.

J Biol Chem, 1994 Apr 29, 269(17), 12698 - 703
Hyperactive initiation of chromosomal replication in vivo and in vitro by a mutant initiator protein, DnaAcos, of Escherichia coli; Katayama T et al.; DnaA protein initiates genomic replication in Escherichia coli . A cold-sensitive dnaAcos mutant caused excessive initiation at a restrictive temperature without an increase in the level of DnaA protein . The chromosomal origin (oriC) was essential for the lethality caused by the dnaAcos product . Increased initiation activity was neutralized by multiple copies of oriC on a plasmid (pBR322) . OriC plasmids were replicated efficiently in vitro in a crude extract prepared from a dnaAcos mutant, with a specific activity for the DnaAcos protein 8-fold greater than that for the DnaA+ protein in a wild-type extract . OriC-dependent replication in the dnaAcos extract was inhibited by rifampicin and by gyrase inhibitors as was replication in the dnaA+ extract . As a control, replication of single-stranded phage phi X174 DNA, which did not require DnaA protein, was similar in extracts prepared from dnaA+ and dnaAcos cells . Thus, initiation at oriC by DnaAcos protein appears to be highly activated both in vivo and in vitro.

J Biol Chem, 1994 Apr 29, 269(17), 12600 - 5
The allosteric interaction between D-galactose and the Escherichia coli galactose repressor protein; Brown MP et al.; The Escherichia coli galactose repressor protein (GalR) inhibits transcription of the gal operon upon binding to two operator sites (1-7) . This DNA binding activity is inhibited when D-galactose or D-fucose binds to GalR (8-14) . Fluorescence spectroscopy was used to characterize the single tryptophan of GalR and to investigate the interaction between galactose and GalR . Fluorescence quenching experiments place both tryptophan residues of the GalR dimer in similar, solvent-exposed locations . Galactose is shown to enhance the intrinsic tryptophan fluorescence of GalR, the source of which is not explained by a change in decay times, but is due to an increase in the pre-exponential factor of the longest of the three fluorescence decay times . It is shown that the beta-anomer of D-galactose is the likely form that binds to GalR . An increase in pH from 6.3 to 9.5 causes the equilibrium association constant (K alpha) describing the galactose-GalR interaction to decrease 10-fold . The interaction is cooperative below pH 9.5 . Over the pH range of 6.3 to 9.5, the tryptophan solvent exposure of GalR increases . Galactose binding also induces an increase in exposure . These results, and others presented in this paper, show that both pH and galactose cause global alterations in the structure of GalR.

J Biol Chem, 1994 Apr 29, 269(17), 12559 - 66
The OmpR protein of Escherichia coli binds to sites in the ompF promoter region in a hierarchical manner determined by its degree of phosphorylation; Rampersaud A et al.; In Escherichia coli the ompF gene encodes a major outer membrane porin protein that is differentially regulated by the OmpR protein . OmpR acts as a positive as well as a negative regulator of ompF expression by binding to DNA sequences in the ompF promoter region . The DNA binding activity of OmpR is itself regulated by phosphorylation through the kinase protein EnvZ . Phosphorylation is believed to change the function of OmpR from an activator to a repressor molecule . By using purified OmpR and various regions of the ompF promoter we show that phosphorylation causes binding of OmpR to a DNA region between the -40 to -100 region of the ompF promoter previously shown to be important for ompF expression . As the amount of OmpR-phosphate increases, a binding site located at a further upstream -360 to -380 region was occupied . This latter site has been reported to be important for ompF repression . Further experiments indicate that the -70 to -100 region is a high affinity site, while the -45 to -60 and -360 to -380 regions are low affinity sites . We also provide evidence that OmpR binding at the -360 to -380 region requires previous binding at downstream sequences, which is indicative of long range interactions between OmpR molecules . We interpret our results in terms of a model for ompF regulation involving hierarchical binding by phosphorylated OmpR and potential DNA looping.

J Biol Chem, 1994 Apr 29, 269(17), 12552 - 8
Expression and activities of a recombinant basic fibroblast growth factor-saporin fusion protein; Lappi DA et al.; A fusion protein containing the full-length sequences of the mitogen, basic fibroblast growth factor (FGF-2), and the ribosome-inactivating protein, saporin (SAP), has been expressed in E . coli . As expected, it binds with high affinity to heparin-Sepharose like FGF-2 and can displace the binding of radiolabeled FGF-2 to its high affinity receptor . In contrast, the fusion protein only has much lower ribosome-inactivating activity than free saporin, although full ribosome-inactivating protein activity can be generated by proteolytic removal of the FGF-2 moiety . Cytotoxicity experiments with B16-F10 mouse melanoma cells establish that the fusion protein is active as a chemical conjugate against these intact cells . Presumably these cells have the ability to activate the SAP component of the fusion protein through an intra-cellular metabolism of the fusion protein . Because we also show the fusion protein has tumor growth inhibition properties and antimetastatic activity in in vivo models of melanoma, the findings support the hypothesis that FGF-based ligand-mediated cytotoxicity can serve to target cytotoxic agents in vivo.

J Biol Chem, 1994 Apr 29, 269(17), 12482 - 7
Wild-type operator binding and altered cooperativity for inducer binding of lac repressor dimer mutant R3; Chen J et al.; Substitution of the C-terminal leucine heptad repeat region of the normally tetrameric lactose repressor by the leucine heptad repeat dimerization domain of GCN4 protein resulted in cell extracts containing protein, designated R3, which behaved as a dimer based on gel retardation analysis of DNA binding (Alberti, S., Oehler, S., von Wilcken-Bergmann, B., and Muller-Hill, B . (1993) EMBO J . 12, 3227-3236) . We have purified this R3 protein and characterized its properties in comparison with the wild-type repressor . R3 protein elutes from a molecular sieve with a Stokes radius characteristic of a dimer and a deduced molecular mass of 66 kDa . Unlike other dimeric repressors, produced by deletion or mutation in the leucine heptad repeat region, which display reduced apparent operator affinity, R3 binds to operator DNA sequences with wild-type equilibrium and kinetic properties . Although inducer affinity at neutral pH is similar for R3 and wild-type protein, at elevated pH the R3 protein undergoes a slightly smaller decrease in affinity and exhibits minimal cooperativity in sugar binding compared with the wild-type protein . Interestingly, in the presence of operator DNA, a state in which inducer binding to wild-type repressor is also of reduced affinity and slightly cooperative, R3 binding affinity is decreased to a greater extent, and the protein displays higher cooperativity than wild-type repressor . Consistent with inducer binding data in the presence of operator, the release of operator from R3 protein requires a higher sugar concentration than wild-type protein . These results are interpreted in the context of alterations involving the subunit interface which affect the allosteric behavior of the repressor protein.

J Biol Chem, 1994 Apr 29, 269(17), 12432 - 7
Adenosylmethionine-dependent synthesis of the glycyl radical in pyruvate formate-lyase by abstraction of the glycine C-2 pro-S hydrogen atom . Studies of {2H}glycine-substituted enzyme and peptides homologous to the glycine 734 site; Frey M et al.; The active form of pyruvate formate-lyase (PFL) from Escherichia coli contains a glycyl radical in position 734 of the polypeptide chain which is produced post-translationally by pyruvate formate-lyase-activating enzyme (PFL activase) using S-adenosylmethionine (AdoMet) and dihydroflavodoxin as co-substrates (Wagner, A.F . V., Frey, M., Neugebauer, F.A., Schafer, W., and Knappe, J . (1992) Proc . Natl . Acad . Sci . U . S . A . 89, 996-1000) . Studying radical synthesis with {2-2H}glycine-labeled PFL, we have now found stoichiometric incorporation of a 2H atom into the 5'-deoxyadenosine (dAdo) co-product via mass and NMR spectroscopic analyses . Furthermore, a series of peptides homologous to the Gly-734 site of PFL have been synthesized for analyzing recognition determinants of PFL activase . Peptides that proved active as substrates (monitored by {14C}dAdo formation from {14C}AdoMet) were also competitive inhibitors of PFL conversion to the radical form . In the sequence of the standard peptide Arg-Val-Ser-Gly-Tyr-Ala-Val, which corresponds to amino acid residues 731-737 of PFL, the Gly residue was replaceable by D-Ala (actually displaying enhanced efficiency), whereas a normal Ala totally abolished the interaction with PFL activase . Our results show that the radical in pyruvate formatelyase is produced by stereospecific abstraction of the pro-S hydrogen of glycine 734 by the 5'-dAdo radical generated in the active center of PFL activase . Gly-734 is probably located in a beta-turn segment of the protein.

J Mol Biol, 1994 Apr 29, 238(2), 173 - 86
A mutant hook-associated protein (HAP3) facilitates torsionally induced transformations of the flagellar filament of Escherichia coli; Fahrner KA et al.; Two mutants with defects in hook-associated protein 3 (HAP3) were isolated that exhibit impaired swimming only when they interact with a solid surface or a semisolid matrix . Motility and chemotaxis were normal in liquid media, even in media containing viscous agents, but cells failed to swarm in 0.28% agar . Mutants appeared to carry a full complement of flagella of normal configuration and length . However, filaments rotating counterclockwise close to a glass surface transformed from normal to straight, while filaments rotating clockwise transformed from curly to straight . Both transformations propagated from base to tip, as expected if torsionally induced . The mutations mapped to the middle of flgL, to structural gene for HAP3, and sequence analysis revealed the same coding change in both mutants: a substitution of cysteine for arginine 168 . Our results show that the ability of a filament composed of normal flagellin subunits to resist mechanical stress depends on the structure of the protein (HAP3) to which it is attached at its base . The N-terminal sequence of HAP3 was found to be similar to the N-terminal sequence of flagellin, and the possibility that it provides a nucleation site for the C-terminal region of flagellin is discussed.

J Chromatogr A, 1994 Apr 29, 667(1-2), 125 - 30
Rapid purification method for human recombinant tumor necrosis factor alpha; Paquet A et al.; Human recombinant tumor necrosis factor alpha was purified in a single step to about 95% purity from Escherichia coli lysate by chromatography on hydroxyapatite . The last traces of contaminants were removed by fast protein liquid chromatography on a Mono Q column . The final product was found to be pure by gel electrophoresis with silver staining . A molecular mass of approximately 17,000 and a specific activity of 4.3 x 10(6) U/mg after a single purification step were found.

J Biol Chem, 1994 Apr 29, 269(17), 12447 - 55
Chaperone-assisted self-assembly of pili independent of cellular energy; Jacob-Dubuisson F et al.; Assembly of P pili on the surface of pyelonephritic Escherichia coli proceeds from periplasmic chaperone-subunit complexes . The outer membrane protein PapC, which has been termed a molecular usher, is thought to be the site of assembly, where the chaperone dissociates from the subunits as they are incorporated into the pilus across the outer membrane . The kinetics of assembly and the energy requirements of the "secretion" events at the outer membrane were investigated using a pulse-chase analysis in which preformed labeled periplasmic chaperone-subunit complexes were assembled into pili in synchrony by the induction of PapC . Provided that a sufficient amount of PapC was present and functional in the outer membrane, the incorporation of the major PapA subunit into pili was shown to be completed in less than 5 min . Our results also indicated that the targeting of PapC to the outer membrane may be a rate-limiting factor for pilus assembly . Following the arrival of PapC, the formation of pili seemed to proceed spontaneously and was not sensitive to a pH shift or an inhibitor of the electrochemical gradient across the cytoplasmic membrane . We suggest that the secretion of pili across the outer membrane may be independent of cellular energy and thermodynamically driven.

J Mol Biol, 1994 Apr 29, 238(2), 133 - 8
Prediction of an inter-residue interaction in the chaperonin GroEL from multiple sequence alignment is confirmed by double-mutant cycle analysis; Horovitz A et al.; A search for co-ordinated amino acid changes in the hsp60 family of chaperonins suggested that cysteine residues at positions 137 and 518 in the Escherichia coli chaperonin GroEL may interact with each other . In order to determine whether this interaction indeed exists we constructed a double-mutant cycle comprising wild-type GroEL, the single mutants Cys137-->Ser and Cys518-->Ser and the corresponding double mutant . The effects of the two mutations on the function of GroEL, in assisting the refolding of a non-folded protein substrate (rhodanese), are shown to be non-additive . It is also shown that ADP by itself specifically destabilizes the Cys518-->Ser mutant GroEL particle with this effect being suppressed in the double mutant . The observed pattern of co-ordinated mutations in the hsp60 family of chaperonins is thus shown to reflect a real interaction, though most likely indirect, between Cys137 and Cys518 in GroEL . Our study demonstrates that patterns of co-ordinated mutations combined with double-mutant cycle analysis can provide structural information on interactions in a protein without an available three-dimensional structure at atomic resolution.

Biochem Biophys Res Commun, 1994 Apr 29, 200(2), 1023 - 7
Chiral discrimination of enantiomeric 2'-deoxythymidine 5'-triphosphate by HIV-1 reverse transcriptase and eukaryotic DNA polymerases; Yamaguchi T et al.; Inhibitory effects of 2'-deoxy-L-thymidine 5'-triphosphate (L-dTTP), the enantiomer of the natural substrate D-dTTP, on the activity of mammalian DNA polymerases alpha, beta and gamma, Escherichia coli DNA polymerase I and human immunodeficiency virus 1 (HIV-1) reverse transcriptase were examined . When poly(rA)n-oligo(dT)12-18 was used as the template-primer, L-dTTP showed remarkable inhibitory effect on HIV-1 reverse transcriptase in competitive fashion with respect to the substrate dTTP . In contrast, L-dTTP did not inhibit DNA polymerases alpha and was slightly inhibitory to DNA polymerase beta . These results suggest that the nuclear DNA polymerases alpha and beta showed high specificity for the substrate with the natural configuration of the sugar moiety, D-dTTP, exhibiting little or no ability to recognize L-dTTP, whereas HIV-1 reverse transcriptase essentially lacked the ability to differentiate the D- and L-sugar moieties.

Nature, 1994 Apr 28, 368(6474), 867 - 71
An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis; Taylor SJ et al.; The tyrosine kinase activity of c-Src is stimulated during mitosis by dephosphorylation of its regulatory tyrosine residue . This is associated with increased accessibility of its Src homology-2 (SH2) domain for binding a phosphotyrosine-containing peptide . But physiological targets of activated c-Src in mitosis have not yet been identified . Here we report that a 68K protein (p68) becomes tyrosine-phosphorylated and physically associates with Src during mitosis in mouse fibroblasts . p68 independently binds the Src SH2 and SH3 domains in vitro and both domains are required for p68 phosphorylation and binding in vivo . p68 is closely related to the p62 protein that is associated with the Ras GTPase-activating protein (GAP) and selectively binds, directly or indirectly, polyribonucleotides . Because the Src SH3 domain also binds heterogeneous nuclear ribonucleoprotein K, these results raise the intriguing possibility that c-Src may regulate the processing, trafficking or translation of RNA in a cell-cycle-dependent manner.

Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3892 - 5
Peptide nucleic acid.DNA strand displacement loops as artificial transcription promoters; Mollegaard NE et al.; Homopyrimidine peptide nucleic acids (PNAs) form loop structures when binding to complementary double-stranded DNA by strand displacement, and we now show that RNA polymerase recognizes these and initiates RNA transcription from PNA/double-stranded DNA strand displacement complexes at an efficiency comparable to that of the strong Escherichia coli lacUV5 promoter . Thus PNA targets can be considered as artificial promoters controlled positively by the corresponding PNA as a transcription factor . Our results have implications for the mechanism of action of RNA polymerase and suggest the use of PNA as specific gene activating reagents and drugs.

Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3877 - 81
NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes; Spooner PJ et al.; NMR methods have been adopted to observe directly the characteristics of substrate binding to the galactose-H+ symport protein GalP, in its native environment, the inner membranes of Escherichia coli . Sedimented inner-membrane vesicles containing the GalP protein, overexpressed to levels above 50% of total protein, were analyzed by 13C magic-angle spinning NMR, when in their normal "fluid" state and with incorporated D-{1-13C}glucose . Using conditions of cross-polarization intended to discriminate bound substrate alone, it was possible to detect as little as 250 nmol of substrate added to the membranes containing about 0.5 mumol (approximately 26 mg) of GalP protein . Such high measuring sensitivity was possible from the fluid membranes by virtue of their motional contributions to rapid relaxation recovery of the observed nuclei and due to a high-resolution response that approached the static field inhomogeneity in these experiments . This good spectral resolution showed that the native state of the membranes presents a substrate binding environment with high structural homogeneity . Inhibitors of the GalP protein, cytochalasin B and forskolin, which are specific, and D-galactose, but not L-galactose, prevent or suppress detection of the 13C-labeled glucose substrate, confirming that the observed signal was due to specific interactions with the GalP protein . This specific substrate binding exhibits a preference for the beta-anomer of D-glucose and substrate translocation is determined to be slow, on the 10(-2) s time scale . The work describes a straightforward NMR approach, which achieves high sensitivity, selectivity, and resolution for nuclei associated with complex membrane proteins and which may be combined with other NMR methodologies to yield additional structural information on the binding site for the current transport system without isolating it from its native membrane environment.

Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3839 - 43
Replication initiator protein RepE of mini-F plasmid: functional differentiation between monomers (initiator) and dimers (autogenous repressor); Ishiai M et al.; Replication of mini-F plasmid requires the plasmid-encoded RepE initiator protein and several host factors including DnaJ, DnaK, and GrpE, heat shock proteins of Escherichia coli . The RepE protein plays a crucial role in replication and exhibits two major functions: initiation of replication from the origin, ori2, and autogenous repression of repE transcription . One of the mini-F plasmid mutants that can replicate in the dnaJ-defective host produces an altered RepE (RepE54) with a markedly enhanced initiator activity but little or no repressor activity . RepE54 has been purified from cell extracts primarily in monomeric form, unlike the wild-type RepE that is recovered in dimeric form . Gel-retardation assays revealed that RepE54 monomers bind to ori2 (direct repeats) with a very high efficiency but hardly bind to the repE operator (inverted repeat), in accordance with the properties of RepE54 in vivo . Furthermore, the treatment of wild-type RepE dimers with protein denaturants enhanced their binding to ori2 but reduced binding to the operator: RepE dimers were partially converted to monomers, and the ori2 binding activity was uniquely associated with monomers . These results strongly suggest that RepE monomers represent an active form by binding to ori2 to initiate replication, whereas dimers act as an autogenous repressor by binding to the operator . We propose that RepE is structurally and functionally differentiated and that monomerization of RepE dimers, presumably mediated by heat shock protein(s), activates the initiator function and participates in regulation of mini-F DNA replication.

Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3720 - 4
Mutants of Escherichia coli K-12 that are resistant to a selenium analog of lipoic acid identify unknown genes in lipoate metabolism; Reed KE et al.; Lipoic acid is a disulfide-containing cofactor required for the reactions catalyzed by alpha-ketoacid dehydrogenase enzyme complexes . We report the chemical synthesis and biological properties of lipoic acid analogs in which one or both sulfur atoms were replaced by selenium . Replacement of either the C-6 or the C-8 sulfur atom with selenium results in lipoic acid derivatives with apparently unaltered biological properties . However, simultaneous replacement of both sulfur atoms gave an analog (selenolipoic acid) that inhibited growth of wild-type Escherichia coli when present in minimal glucose medium at 50 ng/ml . This growth inhibition was reversed by the addition of either excess lipoic acid or acetate plus succinate . Labeling experiments with {75Se}selenolipoic acid showed that this compound was efficiently incorporated into the alpha-ketoacid dehydrogenase complexes of growing cells . Spontaneously arising selenolipoic acid-resistant (slr) mutants were isolated . Two of these isolates resistant to high levels of selenolipoic acid were studied in detail . The slr-1 mutation, which was mapped to min 99.6 of the E . coli chromosome, increased the lipoate requirement of lipA strains by 4-fold and appeared to define a gene encoding a lipoate-protein ligase . The slr-7 mutation, which was mapped to min 15.25 of the chromosome, completely suppressed the lipoate requirement of lipA strains and defined a gene of unknown function in the synthesis of lipoic acid.

Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3637 - 41
Functionally active targeting domain of the beta-adrenergic receptor kinase: an inhibitor of G beta gamma-mediated stimulation of type II adenylyl cyclase; Inglese J et al.; The beta-adrenergic receptor kinase (beta ARK) phosphorylates its membrane-associated receptor substrates, such as the beta-adrenergic receptor, triggering events leading to receptor desensitization . beta ARK activity is markedly stimulated by the isoprenylated beta gamma subunit complex of heterotrimeric guanine nucleotide-binding proteins (G beta gamma), which translocates the kinase to the plasma membrane and thereby targets it to its receptor substrate . The amino-terminal two-thirds of beta ARK1 composes the receptor recognition and catalytic domains, while the carboxyl third contains the G beta gamma binding sequences, the targeting domain . We prepared this domain as a recombinant His6 fusion protein from Escherichia coli and found that it had both independent secondary structure and functional activity . We demonstrated the inhibitory properties of this domain against G beta gamma activation of type II adenylyl cyclase both in a reconstituted system utilizing Sf9 insect cell membranes and in a permeabilized 293 human embryonic kidney cell system . Gi alpha-mediated inhibition of adenylyl cyclase was not affected . These data suggest that this His6 fusion protein derived from the carboxyl terminus of beta ARK1 provides a specific probe for defining G beta gamma-mediated processes and for studying the structural features of a G beta gamma-binding domain.

Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3544 - 8
Cation-promoted association of a regulatory and target protein is controlled by protein phosphorylation; Feese M et al.; A central question in molecular biology concerns the means by which a regulatory protein recognizes different targets . IIIGlc, the glucose-specific phosphocarrier protein of the bacterial phosphotransferase system, is also the central regulatory element of the PTS . Binding of unphosphorylated IIIGlc inhibits several non-PTS proteins, but there is little or no sequence similarity between IIIGlc binding sites on different target proteins . The crystal structure of Escherichia coli IIIGlc bound to one of its regulatory targets, glycerol kinase, has been refined at 2.6-A resolution in the presence of products, adenosine diphosphate and glycerol 3-phosphate . Structural and kinetic analyses show that the complex of IIIGlc with glycerol kinase creates an intermolecular Zn(II) binding site with ligation identical to that of the zinc peptidase thermolysin . The zinc is coordinated by the two active-site histidines of IIIGlc, a glutamate of glycerol kinase, and a water molecule . Zn(II) at 0.01 and 0.1 mM decreases the Ki of IIIGlc for glycerol kinase by factors of about 15 and 60, respectively . The phosphorylation of one of the histidines of IIIGlc, in its alternative role as phosphocarrier, provides an elegant means of controlling the cation-enhanced protein-protein regulatory interaction . The need for the target protein to supply only one metal ligand may account for the lack of sequence similarity among the regulatory targets of IIIGlc.

Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3510 - 4
An Escherichia coli homologue of eukaryotic potassium channel proteins; Milkman R; A DNA sequence in Escherichia coli K-12 contains an evident gene, kch, which predicts a protein 417 residues long with extensive similarity to a group of eukaryotic potassium channel proteins in amino acid sequence, in the presence of six apparent transmembrane (S) regions, and in the potassium-specific P (or H5) "pore" region found between S5 and S6 . Most of the kch gene, including all of these regions and the 5' flanking region, have been sequenced in 38 wild reference (ECOR) strains as well; variation is conservative, indicating the protein's importance to the species, possibly as a defense against osmotic shock . Since the major family of eukaryotic potassium channel proteins is thought to have evolved from a common ancestor, the evolutionary position of this evident bacterial homologue is of interest, particularly since its function may have changed less than those of eukaryotic channels in the last billion years . While cases of probable importation of eukaryotic genes into bacteria are known, there is no evidence that kch has been imported . The relevant properties of the Kch protein and further ways to investigate its evolutionary position are discussed.

Biochemistry, 1994 Apr 26, 33(16), 4933 - 9
Kinetic studies on a genetically engineered fused enzyme between rat cytochrome P4501A1 and yeast NADPH-P450 reductase; Sakaki T et al.; Three expression plasmids, pAMC1 for rat P4501A1, pAMR2 for P4501A1 and yeast NADPH-P450 reductase, and pAFCR1 for a fused enzyme between P4501A1 and the reductase, were constructed, and each was introduced into Saccharomyces cerevisiae AH22 cells . The microsomal fraction prepared from the recombinant yeast cells was subjected to kinetic studies of zoxazolamine 6-hydroxylation at 10 degrees C . The apparent Km and Vmax values for hydroxylation by the fused enzyme in AH22/pAFCR1 microsomes were 0.38 mM and 0.42 s-1, respectively . The rate constant for reduction of the fused enzyme with NADPH in the presence of 1 mM zoxazolamine was larger than 50 s-1 using a dual-wavelength stopped-flow spectrometer, indicating that electrons are rapidly transferred from NADPH through FAD and FMN to the heme iron of the fused enzyme . The rate constant kon for substrate binding to the fused enzyme was 25 mM-1.s-1, which is not much different from that of nonfused P4501A1 . These results together with spectral data measured during the hydroxylation reaction in the steady state suggest that the rate-limiting step of the reaction by the fused enzyme might be the release of product . On the other hand, the apparent Km and Vmax values for the hydroxylation of P4501A1 in AH22/pAMC1 and AH22/pAMR2 microsomes were 0.32 and 0.33 mM, and 0.015 and 0.29 s-1, respectively . The rate constants for the reduction of P4501A1 were 0.025 and 0.40 s-1, respectively, for AH22/pAMC1 and AH22/pAMR2 microsomes.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Apr 26, 33(16), 4794 - 9
Purification and characterization of bacterially expressed mammalian translation initiation factor 5 (eIF-5): demonstration that eIF-5 forms a specific complex with eIF-2; Chaudhuri J et al.; Eukaryotic translation initiation factor 5, eIF-5, has been purified to apparent electrophoretic homogeneity from overproducing Escherichia coli cells expressing the cDNA of the initiation factor under the control of the T7 promoter-T7 RNA polymerase system . Purified recombinant eIF-5 mimics natural eIF-5 isolated from mammalian cells in size, in specific activity, in its ability to catalyze the hydrolysis of GTP bound to the 40S initiation complex, and in the subsequent joining with 60S ribosomal subunits to form the 80S initiation complex . Further characterization of eIF-5 demonstrates that eIF-5 specifically associates with eIF-2, forming an eIF-2.eIF-5 complex . The protein complex sediments in glycerol gradients with an apparent M(r) of 160,000, suggesting that the two proteins associate in a 1:1 stoichiometry . The association between the two initiation factors is highly specific . Addition of 32P-labeled eIF-5 to a partially purified rabbit reticulocyte initiation factor preparation that contained, in addition to eIF-2 and eIF-5, other initiation factors and many other proteins resulted in the specific binding of labeled eIF-5 only to eIF-2, forming a 160-kDa protein complex . In agreement with these observations, we found that in crude initiation factor preparations derived from rabbit reticulocyte lysates, eIF-5 was present as an eIF-2.eIF-5 complex . The significance of eIF-2.eIF-5 complex formation in the overall mechanism of GTP hydrolysis in protein synthesis initiation is discussed.

Biochemistry, 1994 Apr 26, 33(16), 4762 - 8
Crystallographic analysis of the epimeric and anomeric specificity of the periplasmic transport/chemosensory protein receptor for D-glucose and D-galactose; Vyas MN et al.; The D-glucose/D-galactose-binding protein (M(r) = 33,000) found in the periplasm of bacterial cells serves as the primary high-affinity receptor of active transport for and chemotaxis toward both sugar epimers . This protein from Escherichia coli binds D-glucose with a Kd of 2 x 10(-7) M, which is about 2 times tighter than D-galactose . The 2.0-A resolution crystal structure of the binding protein complexed with D-galactose has been refined to a crystallographic R-factor of 0.167 . This structure, combined with that previously refined for the complex with D-glucose {Vyas, N.K., Vyas., M . N., & Quiocho, F . A . (1988) Science 242, 1290-1295}, provides understanding, in atomic detail, of recognition of sugar epimers and anomers . In the two complex structures, the sugar ring is positioned identically in the binding site, and each hydroxyl group common to both is involved in very similar cooperative hydrogen-bonding interactions with protein residues and ordered water molecules . Only the beta-anomer of both monosaccharides is bound, with Asp154 OD1 primarily responsible for accepting a hydrogen bond from the anomeric hydroxyl . Recognition of both sugar epimers is accomplished principally by hydrogen bonding of Asp14 OD1 with the equatorial OH4 of D-glucose and OD2 with the axial OH4 of D-galactose . These results are reconciled with equilibrium and fast kinetics data, which indicate binding of both anomers of the two sugars, and further compared with sugar recognition by other periplasmic sugar-binding proteins with specificities for arabinose/galactose/fucose, maltooligosaccharides, and ribose.

Proc Natl Acad Sci U S A, 1994 Apr 26, 91(9), 3784 - 8
RNA cleavage and chain elongation by Escherichia coli DNA-dependent RNA polymerase in a binary enzyme.RNA complex; Altmann CR et al.; In the absence of DNA, Escherichia coli RNA polymerase (EC 2.7.7.6) can bind RNA to form an equimolar binary complex with the concomitant release of the sigma factor . We show now that E . coli RNA polymerase binds at a region near the 3' terminus of the RNA and that an RNA in such RNA.RNA polymerase complexes undergoes reactions previously thought to be unique to nascent RNA in ternary complexes with DNA . These include GreA/GreB-dependent cleavage of the RNA and elongation by 3'-terminal addition of NMP from NTP . Both of these reactions are inhibited by rifampicin . Hence, by several criteria, the RNA in binary complexes is bound to the polymerase in a manner quite similar to that in ternary complexes . These findings can be explained by a model for the RNA polymerase ternary complex in which the RNA is bound at the 3' terminus through two protein binding sites located up to 10 nt apart . In this model, the stability of RNA binding to the polymerase in the ternary complex is due primarily to its interaction with the protein.

Nucleic Acids Res, 1994 Apr 25, 22(8), 1450 - 5
A simple in vivo footprinting method to examine DNA-protein interactions over the yeast PYK UAS element; Dumitru I et al.; In this report a modification to the in vivo footprinting assay is described . The method includes dimethyl sulfate treatment of whole yeast cells, followed by reiterative primer extension of the methylated genomic DNA using Taq DNA polymerase . Under appropriate reaction conditions chain extension terminates opposite a methylated purine when Taq DNA polymerase encounters a modified adenine or guanine . The procedure was used to examine, in vivo DNA-protein contacts over the upstream activation site (UAS) of the Saccharomyces cerevisiae PYK gene . In vivo analysis, using isogenic strains of yeast and Escherichia coli transformed with plasmid DNAs, confirmed the binding of both the trans-acting factor RAP1 and the transcriptional activator GCR1 to cis-acting recognition sites located within the PYK UAS element.

Nucleic Acids Res, 1994 Apr 25, 22(8), 1394 - 9
Contacts between the growing peptide chain and the 23S RNA in the 50S ribosomal subunit; Stade K et al.; Peptides of defined length carrying a diazirine photoaffinity label attached either to the alpha-NH2 group of the N-terminal methionine residue, or to the epsilon-NH2 group of an immediately adjacent lysine residue, were prepared in situ on Escherichia coli ribosomes in the presence of a synthetic mRNA analogue . Peptide growth was stopped simply by withholding the aminoacyl-tRNA cognate to an appropriate downstream codon . After photo-activation at 350 nm the sites of cross-linking to ribosomal RNA were determined by our standard procedures; the C-terminal amino acid of each peptide was labelled with tritium, in order to confirm whether the individual cross-linked complexes contained the expected 'full-length' peptide, as opposed to shorter products . The shortest peptides became cross-linked to sites within the 'peptidyl transferase ring' of the 23S RNA, namely to positions 2062, 2506, 2585 and 2609 . However, already when the peptide was three or four residues long, a new cross-link was observed several hundred nucleotides away in another secondary structural domain; this site, at position 1781, lies within one of several RNA regions which have been implicated in other studies as being located close to the peptidyl transferase ring . Further application of this approach, combined with model-building studies, should enable the path of the nascent peptide through the large ribosomal subunit to be definitively mapped.

Nucleic Acids Res, 1994 Apr 25, 22(8), 1342 - 5
DNA sequence specificity of mitoxantrone; Panousis C et al.; An in vitro transcription assay was used to determine the sequence specificity of binding of mitoxantrone to a 497 bp fragment of DNA containing the lac UV5 promoter . Transcriptional blockages of the E . coli RNA polymerase were observed dominantly prior to 5'-CpA sequences (64% occurrence), and to a lesser extent 5'-CpG sequences (29%) . Overall, 93% of all blockage sites were prior to pyrimidine (3'-5') purine sequences . An effect of flanking sequences was evident since the blockage sites contained an A/T base pair 5' prior to the consensus CpA and CpG intercalation sites . The consensus sequences for the preferred mitoxantrone intercalation sites are therefore 5'-(A/T)CA and 5'-(A/T)CG . The location of transcriptional blockages one base pair prior to the intercalation site is consistent with the fact that the mitoxantrone side chains lie in the major groove.

FEBS Lett, 1994 Apr 25, 343(2), 125 - 30
Human immunodeficiency virus type 1 (HIV-1) recombinant reverse transcriptase . Asymmetry in p66 subunits of the p66/p66 homodimer; Sharma SK et al.; A recombinant p66 form of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) can be obtained {(1991) Biotechnol . Appl . Biochem . 14, 69-81} from crude Escherichia coli extracts by immobilized metal affinity chromatography (IMAC) . We have analyzed the p66 HIV-1 RT, isolated in the presence of 0.3 M imidazole, by gel permeation HPLC on Superose 12 . The results show that it contains two major distinct p66 forms (24.1 min and 28.3 min peaks) which are distinguishable from the purified homodimeric (p66/p66) HIV-1 RT (22.2 min peak) . Protein peak 1 (24.1 min) is converted to a 22.3 min peak upon storage for 20 h at 4 degrees C . Under identical conditions, the isolated peak 2 (28.3 min) appeared as a conformationally heterogeneous mixture elaborated by peaks at 22.3 min and 25.9 min . The protein species thus obtained were active in the RNA-dependent DNA polymerase and RNase H activity assays and produced heterodimeric HIV-1 RT upon incubation with the HIV-1 protease . When the IMAC-purified, imidazole-free homodimeric (p66/p66) form of the enzyme was incubated with 0.3 M imidazole for 16 h at 4 degrees C, protein peaks at 28.3 min (peak A) and 30.5 min (peak B) were isolated by gel permeation HPLC . While both of these p66-containing species were stable and displayed identical RNA-dependent DNA polymerase activities, the protein in peak B was only 50% active in RNase H function compared with the protein from peak A . These imidazole-mediated dissociation studies support the hypothesis of partial unfolding of one of the RNase H domains of the p66/p66 homodimer, suggesting that the p66 subunits are asymmetric in the native enzyme.

J Biol Chem, 1994 Apr 22, 269(16), 12298 - 303
A novel class of FokI restriction endonuclease mutants that cleave hemi-methylated substrates; Waugh DS et al.; A genetic screen was used to identify amino acid substitutions that enable the FokI restriction endonuclease to cleave DNA in cells that express the cognate methyltransferase activity . Missense mutations that give rise to this phenotype were isolated at eight different positions (G188K, P196S, T343I, S388N, S395F, E407K, E410K, D421N), clustered in two regions of the polypeptide sequence of FokI . Two of the mutant endonucleases (P196S and D421N) were purified to homogeneity and analyzed in detail . Both mutants cleave FokI target sites (5'-GGATG-3') in a manner similar to the wild-type enzyme . Neither mutant cleaved noncanonical sequences, but both efficiently cleaved DNA substrates containing hemi-methylated FokI sites . This class of mutations has not been observed with other restriction enzymes.

J Biol Chem, 1994 Apr 22, 269(16), 12204 - 11
Chemical and genetic characterization of bacteriocins produced by Carnobacterium piscicola LV17B; Quadri LE et al.; Carnobacteriocins BM1 and B2 are thermostable class II bacteriocins produced by Carnobacterium piscicola LV17B . These bacteriocins were purified by a three-step procedure that included hydrophobic interaction, size exclusion, and reversed-phase high performance liquid chromatography . The purified peptides and fragments derived by enzymatic digestion were analyzed by Edman degradation, amino acid analysis, and mass spectrometry . An oxidized form of carnobacteriocin BM1 (carnobacteriocin B1) was also purified and characterized . Probes synthesized using information from the N-terminal amino acid sequences for the purified bacteriocins were used to locate structural genes for the carnobacteriocins . A 1.9-kilobase (kb) HindIII fragment from a 61-kb plasmid (pCP40) containing the carnobacteriocin B2 structural gene and a 4.0-kb EcoRI-PstI genomic fragment containing the carnobacteriocin BM1 structural gene were cloned and fully or partially sequenced, respectively . Expression of the chromosomal bacteriocin and its immunity function requires the presence of the 61-kb plasmid . The results indicate that both bacteriocins are synthesized as prebacteriocins . Post-translational cleavage of an 18-amino acid N-terminal extension at a Gly-Gly (positions -2 and -1) site takes place in each prepeptide to yield the mature 43-amino acid carnobacteriocin BM1 (molecular mass 4524.6) and the mature 48-amino acid carnobacteriocin B2 (molecular mass 4969.9) . These two peptides showed significant amino acid homology to each other and with those class II bacteriocins which contain the YGNGV amino acid motif near the N terminus.

J Biol Chem, 1994 Apr 22, 269(16), 12137 - 41
The invariant arginine in motif 2 of Escherichia coli alanyl-tRNA synthetase is important for catalysis but not for substrate binding; Lu Y et al.; Structural motifs 2 and 3, located in the active site of class II aminoacyl-tRNA synthetases, each contain an invariant arginine thought to participate in interactions with ATP . For Escherichia coli alanyl-tRNA synthetase (AlaRS), sequence comparisons indicate that Arg-69 should be aligned with the invariant arginine in motif 2 of other class II synthetases . Site-directed random mutagenesis has been employed to generate a set of proteins containing amino acid substitutions in a portion of motif 2 of AlaRS . In this set, only mutations at position 69 caused the enzyme to lose ability to complement growth of an alaS deletion strain, and proteins containing substitutions at position 69 alone are undetectable in a Western blot assay . A mutant protein containing the transposition of Arg-69 with Gly-71 does not complement growth but does accumulate in vivo and has thus been purified . Michaelis and dissociation constants for the interaction of this protein with ATP are indistinguishable from those of the wild-type enzyme . However, this two-position displacement of the arginine causes a decrease in the kcat for the ATP-PPi exchange reaction by 2 orders of magnitude . These data suggest a role for the invariant arginine of motif 2 in stabilization of the transition state during alanyladenylate synthesis.

J Biol Chem, 1994 Apr 22, 269(16), 12106 - 10
Semliki Forest virus 6K protein modifies membrane permeability after inducible expression in Escherichia coli cells; Sanz MA et al.; Semliki Forest virus encodes a small protein, known as 6K, that is associated with cellular membranes in the infected cells . This protein has been cloned and expressed in an inducible manner using pET vectors in Escherichia coli cells . Two different plasmids have been utilized; either the 6K gene is placed directly under the T7 promoter (pET3-6K) or the lac operator is located between the T7 promoter and the 6K gene (pET11-6K) . In both systems, efficient synthesis of the 6K protein is achieved by induction with isopropyl-1-thio-beta-D-galactopyranoside plus rifampicin . The synthesis of the 6K protein is very toxic for E . coli causing increased membrane permeability and cell lysis as shown by alterations in permeability to either choline or hygromycin B . These results indicate that the togavirus 6K is a membrane-active protein that shows structural and functional similarities to poliovirus 3A protein . The function that the 6K protein could play during the virus replication cycle is discussed in the light of these findings.

J Biol Chem, 1994 Apr 22, 269(16), 11912 - 20
Resonance Raman and Fourier transform infrared studies on the subunit I histidine mutants of the cytochrome bo complex in Escherichia coli . Molecular structure of redox metal centers; Uno T et al.; The cytochrome bo complex is a heme-copper terminal ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli . Site-directed mutagenesis studies were greatly successful in identifying ligands of the redox centers in the oxidase (Minagawa, J., Mogi, T., Gennis, R . B., and Anraku, Y . (1992) J . Biol . Chem . 267, 2096-2104) . In the present study, resonance Raman and Fourier transform infrared spectroscopies were applied to the subunit I histidine mutant oxidases, and the active-site structure was studied in detail . In the dithionite-reduced state, the wild-type oxidase and the His-284-->Ala and His-333-->Ala mutant oxidases showed two Raman lines (v3) at approximately 1492 and 1472 cm-1, which are attributable to low- and high-spin heme components, respectively . The Ala substitutions at His-106 and His-421 reduced the low-spin line, whereas the His-419-->Ala mutation eliminated the high-spin line . This indicates that His-106 and His-421 are the axial ligands of the low-spin heme B, while His-419 acts as a proximal ligand of the high-spin heme O . The v(Fe-CO) stretching frequency in the wild-type oxidase down-shifted from 523 to 498 cm-1 in the CuB-deficient mutant oxidase (His-333-->Ala), while the v(C-O) stretching frequency up-shifted from 1960 to 1970 cm-1 . These frequency differences were ascribed to the contribution from the reduced CuB at the binuclear center . It may indicate that delocalization of electrons at the CuB center toward the heme O iron is facilitated by a bridge ligand structure at the binuclear center and is essential for driving dioxygen reduction chemistry, and therefore, redox-coupled proton pumping.

J Biol Chem, 1994 Apr 22, 269(16), 11893 - 901
Specific recognition of mitochondrial preproteins by the cytosolic domain of the import receptor MOM72; Schlossmann J et al.; The import receptor MOM72 constitutes part of the protein translocation machinery of the outer mitochondrial membrane, the receptor-general insertion pore complex . The protein contains a membrane anchor at the NH2 terminus and a large cytosolic domain . In yeast and Neurospora crassa the cytosolic domain comprises about 570-580 amino acid residues . The cytosolic domain of yeast MOM72 was purified after expression in Escherichia coli as a homogeneous monomeric protein . It can recognize precursor proteins as demonstrated by its ability to compete for binding and import into the mitochondria and to physically interact with preproteins . A subset of preproteins including the ADP/ATP carrier and the phosphate carrier interact with very high affinity, precursors that are known to be targeted via MOM72 . Thus, the cytosolic domain of MOM72 plays a critical function in the recognition of preproteins by directly binding to precursor proteins and thereby facilitating their targeting to mitochondria.

J Biol Chem, 1994 Apr 22, 269(16), 11776 - 82
Differential effect of phosphorylation and substrate modulation on tau's ability to promote microtubule growth and nucleation; Brandt R et al.; The neuronal microtubule-associated protein tau promotes microtubule assembly and has been implicated in the development of axonal morphology . To study the effect of phosphorylation and substrate modulation on tau's distinct activities to promote growth of existing microtubules and nucleation of new ones, we phosphorylated bacterially expressed human tau by cAMP-dependent protein kinase in the absence or presence of heparin, an acidic substrate modulator . We found that heparin increased phosphorylation of tau by a factor of more than 2 and produced tau bands with decreased electrophoretic mobility . We demonstrate that phosphorylation of tau in the absence or presence of heparin similarly reduced tau's activity to promote microtubule growth, whereas tau's activity to promote microtubules was suppressed much more after phosphorylation in the presence of heparin . Using recombinant tau fragments we showed that heparin-induced phosphorylation caused a specific shift in electrophoretic mobility indicative of a change in tau's conformation . By aminoterminal sequencing of a tau fragment starting at residue 154 we provide evidence that phosphorylation of serine 156 is responsible for this mobility shift and for the effect on tau's nucleation activity . We conclude that tau's activities to promote growth of existing microtubules and nucleation of new ones are differentially affected by the phosphorylation of specific tau residues . Regulation of the phosphorylation state by substrate modulation may play an important role in regulating tau's function.

J Biol Chem, 1994 Apr 22, 269(16), 11760 - 8
Molecular cloning of cDNAs and genes coding for beta-methylcrotonyl-CoA carboxylase of tomato; Wang X et al.; Tomato cDNA and genomic clones were isolated by using as a probe a cDNA clone that had originally been identified by its ability to direct the synthesis of a biotin-containing polypeptide in Escherichia coli . The nucleotide sequences of the newly isolated cDNAs indicate that they are clones of a single mRNA molecule . However, one of the cDNA clones contains an insertion of a sequence which we identified as an unspliced intron . The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed similarity to regions of previously sequenced biotin enzymes, indicating that the isolated cDNAs code for a biotin-containing protein . Portions of the cDNAs were expressed in E . coli as glutathione S-transferase or beta-galactosidase fusion proteins . Each fusion protein was purified and used to immunize rabbits . The resulting antisera recognized a 78-kDa biotin-containing polypeptide in tomato leaf extracts . In addition, both antisera specifically inhibited beta-methylcrotonyl-CoA carboxylase activity in extracts from tomato leaves . These characterizations have identified the isolated tomato cDNAs and genes as coding for the 78-kDa biotin subunit of beta-methylcrotonyl-CoA carboxylase . Comparison of the deduced amino acid sequence of the biotin subunit of beta-methylcrotonyl-CoA carboxylase with other biotin enzymes suggest that this subunit contains the biotin carboxylase and biotin carboxyl-carrier domains.

J Biol Chem, 1994 Apr 22, 269(16), 11703 - 6
Thermal inactivation of tryptophan synthase . Stabilization by protein-protein interaction and protein-ligand interaction; Ruvinov SB et al.; This study investigates effects of ligands on thermal inactivation of the tryptophan synthase alpha and beta 2 subunits alone and in the alpha 2 beta 2 complex . Addition of pyridoxal phosphate to the apo-beta 2 subunit increases the temperature of one-half inactivation (Ti) from 52 to 77 degrees C . Ligands that promote association of the alpha and holo-beta 2 subunits markedly stabilize the more temperature-labile alpha subunit in the alpha 2 beta 2 complex from irreversible thermal denaturation . The combination of a beta 2 subunit ligand (L-serine) with an alpha subunit ligand (alpha-glycerol 3-phosphate) raises the inactivation temperature (Ti) of the alpha subunit in the holo-alpha 2 beta 2 complex from 54 to 66 degrees C . In contrast, values of Ti for inactivation of the alpha and beta subunits in the holo-alpha 2 beta 2 complex are more similar to respective values for the isolated alpha subunit (50 degrees C) and holo-beta 2 subunit (77 degrees C) . Surprisingly, the addition of L-serine results in a larger decrease in the Ti of the beta 2 subunit in the holo-alpha 2 beta 2 complex (78 degrees C-->64 degrees C) than in Ti of the holo-beta 2 subunit alone (77 degrees C-->71 degrees C) . The observation that ligands have different effects on the isolated and associated subunits provides evidence that the alpha and beta 2 subunits do not fully dissociate during thermal inactivation of the alpha 2 beta 2 complex at pH 7.8 and at approximately 0.1 ionic strength . Our results demonstrate that linkage between protein-ligand interactions and protein-protein interactions affects the conformational stability of the tryptophan synthase alpha 2 beta 2 complex.

J Biol Chem, 1994 Apr 22, 269(16), 11699 - 702
Tryptophan radicals formed by iron/oxygen reaction with Escherichia coli ribonucleotide reductase protein R2 mutant Y122F; Sahlin M et al.; The active state of the small subunit, protein R2, of ribonucleotide reductase is formed by the reaction of apoprotein with Fe2+ and O2, whereby the diferric site and a stable phenoxy free radical on a tyrosyl residue (Tyr122) is formed . The corresponding reaction was studied in the mutant Y122F R2 . It leads to a normal iron site, but the reduction equivalent from Tyr122 now has to be supplied from elsewhere . EPR spectroscopy shows formation of several paramagnetic species on different time scales . Using apoprotein with deuterium-labeled tryptophan residues, at least two species could be assigned to tryptophan free radicals . This is the first EPR observation of relatively stable protein-linked tryptophan radicals at room temperature and at 77 K . These tryptophan radicals may be involved as redox intermediates in long range electron transfer within the protein structure.

J Biol Chem, 1994 Apr 22, 269(16), 11695 - 8
Opposite stereospecificity of two tropinone reductases is conferred by the substrate-binding sites; Nakajima K et al.; Two tropinone reductases (TRs) catalyze opposite stereospecific reductions at a branching point in the biosynthetic pathway of tropane alkaloids . The two TRs, TR-I and TR-II, reduce the 3-keto group of the common substrate tropinone stereospecifically to 3 alpha- and 3 beta-hydroxy groups, to produce the stereoisomeric alkamines tropine and pseudotropine, respectively . Sixteen chimeric TR enzymes were expressed in Escherichia coli, and their stereospecificities, substrate specificities, and Km values for tropinone were compared with those of the wild-type enzymes . Stereospecificity and substrate specificity of the chimeric enzymes were closely correlated, and the carboxyl-terminal peptides of about 120 amino acid residues, in which 53 residues were different between TR-I and TR-II, were shown to determine both specificities . Further dissection of these peptide segments resulted in either enzymes with both TR activities or inactive enzymes . The substrate binding affinity of many chimeric enzymes was much lower than that of wild-type enzymes . These results indicate that the stereospecificity of TR is determined by the orientation of tropinone at the substrate-binding site, which is composed mainly of the carboxyl-terminal half region, and also that the amino-terminal half region constitutes the NADPH-binding site as postulated for short chain nonmetal dehydrogenases.

J Mol Biol, 1994 Apr 22, 238(1), 120 - 2
Crystallization of DNA polymerase II from Escherichia coli; Anderson WF et al.; DNA polymerase II of Escherichia coli, an alpha-like or group B polymerase, has been crystallized . The crystals are orthorhombic, space group P2(1)2(1)2, with cell dimensions a = 94.4 A, b = 118.2 A, c = 84.2 A and diffract to at least 3.0 A resolution . This is the first example of a group B polymerase to be crystallized.

J Mol Biol, 1994 Apr 22, 238(1), 1 - 8
A graphic approach to analyzing codon usage in 1562 Escherichia coli protein coding sequences; Zhang CT et al.; The occurrence frequencies of the four bases (adenine, cytosine, guanine and thymine) at each of the three codon positions for 1562 Escherichia coli protein coding sequences have been calculated . The 1562 x 4 x 3 = 18,744 data thus obtained have been analyzed by a graphic method in which the four base occurrence frequencies at each codon position for each coding sequence are represented by a point in a three-dimensional space . Thus, the 18,744 data, which would otherwise occupy several printed pages, can be intuitively displayed by a graphy . The point distribution pattern for each of the three codon positions has been analyzed . The results of our analysis indicate that the patterns for the first two codon positions reflect the origin for producing native folding structures of proteins . We thus come to the conclusion that the distribution patterns for the first two codon positions should be basically species-independent, as confirmed by studies for a number of other species . However, the distribution pattern for the third codon position is species-dependent . Based on the point distribution of the third codon position, six collective parameters have been defined to describe the overall feature of the pattern concerned . These collective parameters can be generally used to classify different species, and hence would be a useful vehicle for studies in taxonomy . In addition to E . coli, the collective parameters for a number of other species have been calculated and analyzed.

J Biol Chem, 1994 Apr 22, 269(16), 12233 - 9
Stable fiber-forming and nonfiber-forming chaperone-subunit complexes in pilus biogenesis; Striker R et al.; The P pilus is a composite fiber consisting of a thin adhesive tip fibrillum joined to the pilus rod that mediates specific adherence of uropathogenic Escherichia coli to human uroepithelial cells via the PapG tip adhesin . P pilus assembly depends upon the periplasmic chaperone PapD . The interaction of PapD with different pilus subunits was investigated to gain further insight into pilus assembly . PapA, the major subunit of the pilus rod, formed two periplasmic complexes (DA2 and DA) with PapD . PapK, an adaptor protein that joins the tip fibrillum to the pilus rod, formed only one complex with PapD (DK) . Only "fiber forming" or homopolymeric subunits, PapA in the rod and PapE in the tip fibrillum, were able to form subunit-subunit interactions in the periplasm . Subunits that are present in single or low copy in the pilus (PapK and PapG) did not form periplasmic intersubunit interactions . A pulse-chase analysis revealed that a chaperone-PapA complex is a true periplasmic intermediate in pilus assembly.

J Biol Chem, 1994 Apr 22, 269(16), 12254 - 62
RNA splicing regulates the activity of a SH2 domain-containing protein tyrosine phosphatase; Mei L et al.; A cDNA which encodes a protein tyrosine phosphatase with two src homology 2 (SH2) domains was isolated from a rat brain cDNA library . This phosphatase appears to be a rat homologue of PTP1D based on its amino acid sequence . The gene is expressed in a variety of tissues, and its mRNA is enriched in the brain, skeletal muscle, and lung . An RNA splice variant (PTP1Di) was also isolated which has four additional amino acid residues (Ala-Leu-Leu-Gln) in the catalytic domain . The catalytic domains of PTP1D and PTP1Di were expressed in Escherichia coli as glutathione S-transferase fusion proteins and purified to near homogeneity . Whereas both PTP1D and PTP1Di had catalytic activity, the Vmax of PTP1Di relative to that of PTP1D was 8-fold lower for para-nitrophenylphosphate, 20-fold lower for nicotinic acetylcholine receptor, and 14-fold lower for myelin basic protein . The Km values of PTP1Di were lower than those of PTP1D for both nicotinic acetylcholine receptor and myelin basic protein, suggesting a higher affinity of PTP1Di for a protein substrate . These two forms also differed in optimum pH for para-nitrophenylphosphate and sensitivity to the inhibitory effects of vanadate, molybdate, and spermidine . In order to see if this insert would affect the catalytic activity of other related phosphatases, the 4-amino acids were inserted in the corresponding region of the catalytic domain of PTP1C . Whereas both the wild type and PTP1Ci which contained the 4-amino acid insert dephosphorylated para-nitrophenylphosphate, nicotinic receptor, and myelin basic protein, the enzyme activity of PTP1Ci was only 11-24% of that of PTP1C wild type . These results demonstrate that the 4-amino acid insert in the catalytic domains of PTP1D down-regulates its phosphatase activity and suggests that RNA splicing may serve as a regulatory mechanism of protein tyrosine phosphatase activity.

J Biol Chem, 1994 Apr 22, 269(16), 12167 - 72
Immunoaffinity purification of the Escherichia coli rne gene product . Evidence that the rne gene encodes the processing endoribonuclease RNase E; Taraseviciene L et al.; The rne gene product was highly purified from Escherichia coli cells overproducing the protein by a procedure including immunoaffinity chromatography . Expression in vivo and in vitro of the cloned 6-kilobase pair DNA fragment containing the entire rne gene resulted in the synthesis of a protein migrating as a 180-kDa polypeptide in the SDS-polyacrylamide gel . The position of the protein on the two-dimensional polyacrylamide gel indicated that the protein is highly acidic . The enzymatic activity test which used as the substrate RNA I and 9 S RNA provided evidence that the rne gene is the structural gene for the RNA processing enzyme RNAse E . The Western blot analysis performed using a rabbit antiserum raised against a truncated 110-kDa protein fragment of RNase E (containing two-thirds of the sequence from the N terminus) revealed that the 180-kDa polypeptide is the only protein recognized by the antibodies in a wild type whole cell extract of E . coli . The antibodies cross-reacted with similar molecular weight proteins from a number of different bacteria, suggesting that the rne gene product is evolutionarily conserved in the bacterial world.

Biochem Pharmacol, 1994 Apr 20, 47(8), 1285 - 94
Effect of 3'-amino-2',3'-dideoxycytidine on DNA replicative intermediates; Williams MS et al.; 3'-Amino-2',3'-dideoxycytidine (3'-NH2-ddCyd) is a 3'-modified deoxycytidine analog that specifically inhibits DNA synthesis . Inhibition of chain elongation at the replication fork was examined utilizing a batch hydroxylapatite chromatography method . Exponentially growing cells were exposed to 3'-NH2-ddCyd and the diterpene aphidicolin for 9.5 hr at concentrations that inhibited DNA synthesis by approximately 60 and 90%, as determined by precursor uptake . Both agents demonstrated a concentration-dependent inhibition of pulse labeling of single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) generated by a limited alkaline lysis procedure . Upon removal of drug, the rate of elongation of pulse-labeled DNA was similar to that of untreated cells at both concentrations of aphidicolin and at the low concentration of the amino analog . Under these conditions, no reduction in cell survival was observed using the clonogenic assay technique . However, at the high concentration of 3'-NH2-ddCyd, the rate of elongation following drug removal was one-third that of untreated cultures, and a 50% loss in cell viability was observed . Furthermore, upon incubation of purified dsDNA with the Klenow fragment of Escherichia coli DNA polymerase I or purified ssDNA with calf thymus terminal deoxynucleotidyl transferase, only DNA from cells treated with the high concentration of 3'-NH2-ddCyd served as a poor template for further synthesis . The results indicate that 3'-NH2-ddCyd, in a concentration-dependent manner, inhibits DNA synthesis by reducing the rate of chain elongation at the replication fork, which subsequently leads to a functional blocking of 3'-ends in DNA . The data suggest that there may be a relationship between loss of cell viability and reduction in the number of 3'-ends available for DNA replication.

Gene, 1994 Apr 20, 141(2), 163 - 70
Production of enzymatically active rat protein disulfide isomerase in Escherichia coli; De Sutter K et al.; We report the development of a bacterial expression system allowing high-level synthesis of enzymatically active rat protein disulfide isomerase (rPDI) . After expression of the rpdi gene under control of the inducible trc promoter (Ptrc), a significant amount of soluble, active rPDI was detected in the periplasmic contents, which were released from the cells by cold osmotic shock . However, the exported molecules were incompletely or improperly processed, while the major amount of synthesized rPDI was in fact detected in the soluble cellular fraction . Substitution of the autologous eukaryotic export signal with the nucleotide (nt) sequence encoding the signal peptide (sOmpA) of the bacterial outer membrane protein A, and expression of the sompA::rpdi fusion gene under control of both the lpp promoter (Plpp) and the lac promoter-operator (POlac), resulted in high-level production of rPDI . Furthermore, the latter was efficiently exported into the periplasmic compartment, from where it was recovered as a soluble, fully active form with the sOmpA precisely removed . The synthesis of a small 21-kDa peptide accompanying the production of rPDI was also observed . This rPDI-related peptide (rPDIf), which represented a C-terminal fragment of rPDI including the second active site, arose by internal translation initiation within rpdi . Replacement of the presumed internal start codon by CTC completely eliminated the aforementioned phenomenon and resulted in the production of a slightly mutated, enzymatically active enzyme (rPDIm).

Biochemistry, 1994 Apr 19, 33(15), 4730 - 7
Immunoglobulin-type domains of titin: same fold, different stability?
Politou AS, Gautel M, Pfuhl M, Labeit S, Pastore A.
Titin is a 3-MDa protein thought to form a fibrous intracellular system in vertebrate striated muscle and to play an important role in sarcomere alignment during muscle contraction . It has also been implicated as a "molecular ruler", regulating the assembly and the precise length of the thick filaments {Whiting, A . J., Wardale, J., & Trinick, J . (1989) J . Mol . Biol . 205, 163-169} . Partial sequencing of titin-encoding cDNAs suggests that the protein is organized in a modular fashion, containing two classes of approximately 100-residue repeats {Labeit, S., Barlow, D . P., Gautel, M., Gibson, T., Holt, J., Hsieh, C . L., Francke, U., Leonard, K., Wardale, J., Whiting, A., & Trinick, J . (1990) Nature 345, 273-276} . These motifs, referred to as type I and type II modules, show sequence homology to the fibronectin III and immunoglobulin C2 superfamilies, respectively . Since the type II modules represent the most widely occurring motifs along the titin molecule, we expressed in Escherichia coli three domains of this type spanning different regions of the sarcomere (A-band and M-line) and studied their structure and stability . Using circular dichroism, nuclear magnetic resonance, and fluorescence spectroscopy, we showed that all the fragments examined are independently folded in solution and possess a beta-sheet conformation . Furthermore, employing NMR analysis, we identified an overall folding pattern present in all modules and related to the Ig fold, as previously suggested by theoretical predictions . The stability of the modules over a range of conditions was investigated by measuring key thermodynamic parameters for both thermal and chemical denaturation and by monitoring amide proton exchange as a function of time.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Apr 19, 33(15), 4682 - 94
Structural characteristics of the nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein . Studies with ribose and base-modified fluorescent nucleotide analogs; Bujalowski W et al.; Structural characteristics of the base- and ribose-binding regions of the high-affinity noninteracting nucleotide-binding site of Escherichia coli primary replicative helicase DnaB protein have been studied, using the base-modified fluorescent nucleotide analog 1, N6-ethenoadenosine diphosphate (epsilon ADP) and the ribose-modified fluorescent analogs 3'(2')-O-(N-methylantraniloyl)adenosine 5'-diphosphate (MANT-ADP), 3'-O-(N-methylantraniloyl)deoxyadenosine 5'-diphosphate (MANT-dADP), 3'-O-(N-methylantraniloyl)-deoxyadenosine 5'-triphosphate (MANT-dATP), and 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-ADP) . The obtained data indicate contrasting differences between these two regions . Binding of epsilon ADP to the DnaB helicase causes only approximately 21% increase of the nucleotide fluorescence intensity and no shift of the emission spectrum maximum . The fluorescence of bound epsilon ADP is characterized by a single lifetime of 24.2 +/- 0.6 ns, only slightly shorter than the fluorescent lifetime of the free epsilon ADP in solution (25.5 +/- 0.6 ns) . Solute-quenching studies of bound epsilon ADP, using different quenchers, acrylamide, I-, and Tl+, indicate limited accessibility of ethenoadenosine to the solvent . These results strongly suggest that the base-binding region of the DnaB nucleotide-binding site is located in the polar cleft on the enzyme's surface . Moreover, the limiting emission anisotropy of bound epsilon ADP is 0.21 +/- 0.02, compared to the anisotropy of 0.3 of completely immobilized epsilon ADP at the same excitation wavelength (lambda ex = 325 nm, lambda em = 410 nm), indicating that the adenine preserves substantial mobility when bound in the base-binding site . In contrast, fluorescence intensity at the emission maximum of TNP-ADP and MANT-ADP, which has modifying groups attached to the 2' and/or 3' oxygens of the ribose, increases upon binding to DnaB by factors of approximately 4.7 (lambda ex = 408 nm) and approximately 2.6 (lambda ex = 356 nm), respectively . Moreover, the maximum of emission spectrum of bound TNP-ADP is blue-shifted by approximately 11 nm and that of MANT-ADP by approximately 12 nm . Comparisons between spectral properties of TNP-ADP and MANT-ADP bound to DnaB and in different solvents suggest that the ribose-binding region of the DnaB nucleotide-binding site has relatively low polarity . Solute quenching studies of MANT-ADP fluorescence, using acrylamide, I-, and Tl+, indicate that the MANT group has very little accessibility to the solvent when bound to DnaB . Taken together, these results suggest that the ribose-binding region constitutes a hydrophobic cleft, or pocket, with very limited, if any, contact with the solvent.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1994 Apr 19, 33(15), 4677 - 81
Structural elements that contribute to an unusual tertiary interaction in a transfer RNA; Hou YM; Transfer RNAs (tRNAs) contain a set of defined tertiary hydrogen-bonding interactions that are established between conserved and semiconserved nucleotides . Although the crystal structures of tRNAs describe each of the tertiary interactions in detailed molecular terms, little is known about the underlying structural parameters that stabilize the tertiary interactions . Escherichia coli (E . coli) tRNA(Cys) has an unusual tertiary interaction between G15 in the dihydrouridine (D) loop and G48 in the variable loop that is critical for cysteine aminoacylation . All other tRNAs have a purine 15 and a complementary pyrimidine 48 that establish a tertiary interaction known as the Levitt base pair {Levitt, M . (1969) Nature 224, 759-763; Klug et al . (1974) J . Mol . Biol . 89, 511-516} . In this study, the G15.G48 tertiary interaction in E . coli tRNA(Cys) was used to investigate the structural elements that contribute to its variation from the Levitt base pair . Analysis with chemical probes showed that substitution of U21 with A21 in the D loop and formation of a Watson-Crick base pair between nucleotides 13 and 22 in the D stem switch the hydrogen-pairing of G15.G48 to a Levitt-like G15.G48 base pair . This switch was accompanied by a decrease of the catalytic efficiency of aminoacylation by 2 orders of magnitude . In contrast, insertion of additional nucleotides in the D or variable loops had little effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Chem Phys Lipids, 1994 Apr 19, 70(2), 133 - 45
The cryoprotectant trehalose destabilises the bilayer organisation of Escherichia coli-derived membrane systems at elevated temperatures as determined by 2H and 31P-NMR; Fabrie CH et al.; In this study, 2H and 31P-NMR techniques were used to study the effects of trehalose and glycerol on phase transitions and lipid acyl chain order of membrane systems derived from cells of E . coli unsaturated fatty acid auxotroph strain K1059, which was grown in the presence of {11,11-2H2}-oleic acid or {11,11-2H2}-elaidic acid . From an analysis of the temperature dependence of the quadrupolar splitting it could be concluded that neither 1 M trehalose or glycerol generally had any significant effect on the temperature of the lamellar gel to liquid-crystalline phase transition . In the case of the oleate-containing hydrated total lipid extract, glycerol but not trehalose caused a 5 degrees C increase of this transition temperature . In general, both cryoprotectants induced an ordering of the acyl chains in the liquid-crystalline state . Trehalose and glycerol both decrease the bilayer to non-bilayer transition temperature of the hydrated lipid extract of oleate-grown cells by about 5 degrees C, but only trehalose in addition induces an isotropic to hexagonal (HII) phase transition . In the biological membranes, trehalose and not glycerol destabilised the lipid bilayer, and in the case of the E . coli spheroplasts, part of the induced non-bilayer structures is ascribed to a hexagonal (HII) phase in analogy with the total lipids . Interestingly, 1 mM Mg2+ was a prerequisite for the destabilisation of the lipid bilayer . In the hydrated total lipid extract of E . coli grown on the more ordered elaidic acid, both transition temperatures were shifted about 20 degrees C upwards compared with the oleate-containing lipid, but the effect of trehalose on the lipid phase behaviour was similar . The bilayer destabilising ability of trehalose might have implications for the possible protection of biological systems by (cryo-)protectants during dehydration, in that protection is unlikely to be caused by preventing the occurrence of polymorphic phase transitions.

FEBS Lett, 1994 Apr 18, 343(1), 94 - 8
Histidine-118 of elongation factor Tu: its role in aminoacyl-tRNA binding and regulation of the GTPase activity; Jonak J et al.; The function of His118 in elongation factor (EF)-Tu from Escherichia coli was investigated by its substitution with glycine . The substitution had a differential effect on individual functions of the protein . The affinity for aminoacyl (aa)-tRNA and the intrinsic GTPase activity of the mutant EF-Tu were decreased whereas the response of its GTPase center to aa-tRNA was strongly increased . These results suggest that the region around His118 is involved in the binding of aa-tRNA and in the transmission of a turn-off signal generated by the interaction with aa-tRNA and directed to the GTPase center of EF-Tu.

FEBS Lett, 1994 Apr 18, 343(1), 70 - 4
Structural and enzymological analysis of the interaction of isolated domains of cytochrome P-450 BM3; Munro AW et al.; The interactions of the individually expressed haem- and flavin-containing domains of cytochrome P-450 BM3 have been analysed by enzymological and spectroscopic techniques . Electron transfer between the isolated domains occurs at a much lower rate than that occurring in the intact flavocytochrome . CD spectroscopic studies indicate that the linkage of the domains in intact P-450 BM3 creates haem and amino acid environments suitable for efficient electron transfer from its flavin domain.

FEBS Lett, 1994 Apr 18, 343(1), 65 - 9
A novel peptidyl-prolyl cis/trans isomerase from Escherichia coli; Rahfeld JU et al.; A novel peptidyl-prolyl cis/trans isomerase was isolated from Escherichia coli cell extract and characterized partially . Determination of the molecular mass by electrospray mass spectrometry indicated a protein of 10102 +/- 2 Da, smaller than cyclophilins or FK 506 binding proteins currently known . The specificity constant kcat/Km determined with Succinyl-Ala-Xaa-Pro-Phe-4-nitroanilide (Xaa = Leu) had a value comparable to those from cyclophilins from the same organism . However, the pattern of subsite specificity (Xaa = Gly, Ala, Val, Ile, Leu, Phe, Trp, His, Lys and Glu) was reminiscent of FK506 binding peptidyl-prolyl cis/trans isomerases . The enzyme activity was not inhibited by cyclosporin A or FK506 at inhibitor concentrations of < 5 microM, concentrations that affect most bacterial peptidyl-prolyl cis/trans isomerases . Computer-assisted analysis of 21 amino acid residues of the N-terminus determined by Edman degradation revealed no homology to known peptidyl-prolyl cis/trans isomerases.

FEBS Lett, 1994 Apr 18, 343(1), 47 - 50
Collagen-binding recombinant fibronectin fragments containing type II domains; Skorstengaard K et al.; Each of the two type II domains and four larger fragments, containing one or two type II domains of fibronectin, have been expressed in Escherichia coli . A special vector, containing a fragment encoding the cleavage site for Factor Xa, Ile-Glu-Gly-Arg, inserted immediately before the protein fragment of interest, was used . After treatment of the purified fusion proteins with reduced/oxidized glutathione, the correctly folded fibronectin fragments were released by proteolytic digestion with Factor Xa . The largest fragment, consisting of two type II and two type I domains, was the only fragment able to bind to immobilized gelatin.

FEMS Microbiol Lett, 1994 Apr 15, 117(3), 345 - 9
Phylogeny of human intestinal spirochaetes inferred from 16S rDNA sequence comparisons; Hookey JV et al.; The sequence of 1383 nucleotides of the DNA encoding 16S rDNA was determined for strains of human intestinal spirochaetes, comprising an unnamed isolate and "Brachyspira aalborgi" NCTC 11492 . A phylogenetic tree was inferred from aligned sequence comparisons between the intestinal spirochaetes, representatives of the Spirochaetales and Escherichia coli . The type strain of Brachyspira aalborgi, though related to the Serpulina spp . at approx . 96.5% sequence similarity was distinct and separated from the unnamed human intestinal isolate, HIS Oman, N26 . The latter formed a separated and novel lineage that bisected the Spirochaetales.

FEMS Microbiol Lett, 1994 Apr 15, 117(3), 319 - 25
The significance of enteroaggregative Escherichia coli in the etiology of hospitalized diarrhoea in Calcutta, India and the demonstration of a new honey-combed pattern of aggregative adherence; Paul M et al.; Previous studies have identified enteroadherent Escherichia coli that exhibit localized adherence, diffuse adherence and atypical diffuse adherence as diarrhoeagenic agents associated with infantile diarrhoea in Calcutta, India . In this study, a DNA probe specific for enteroaggregative adherence was used to determine the etiological significance of enteroaggregative E . coli in the causation of diarrhoea . From a total of 330 strains of E . coli recovered from 159 cases of acute secretory diarrhoea and 174 cases of invasive diarrhoea, 20 strains hybridized with the probe, whereas of the 25 E . coli strains recovered from 25 healthy controls only 1 strain hybridized with the probe . Of the 21 probe positive strains, 19 adhered to HeLa cells in the typical stacked-brick pattern while 2 strains recovered from 2 cases of secretory diarrhoea adhered to the glass surface in a hitherto undescribed formation which we have termed, based on the appearance, as the honey-comb pattern . The enteroaggregative E . coli strains identified in this study did not produce any conventional enterotoxins and were significantly associated with patients with secretory diarrhoea (10.7%) than with invasive diarrhoea (1.7%) . The results of this study indicate that enteroaggregative E . coli play a causal role in acute secretory diarrhoea in this part of the world which lends credence to the involvement of a potent toxin in the pathogenesis of EAggEC mediated infections.

Ann N Y Acad Sci, 1994 Apr 15, 718, 191 - 201; discussion 201-2
Structure-function relationships of the erythropoietin molecule; Lappin TR et al.; The tertiary structure of erythropoietin (EPO) remains to be elucidated by X-ray crystallography . Although the amino acid sequence of EPO is known, the specific features that confer its biological activity are not well understood . In order to study the structure-function relationships of EPO by in vitro mutagenesis, we have used the vector pGEX-2T to express human and murine EPO fused to the carboxyl terminus of glutathione S-transferase (GST) in E . coli . The fusion proteins were the predicted size (46 kDa) by SDS-PAGE . GST-huEPO eluted from glutathione-agarose using reduced glutathione (GSH) was tested by radioimmunoassay and in a mouse spleen cell assay (MSCA) . Dose-response curves parallel to recombinant human EPO (rHuEPO) were obtained in both assays . The ratio of immuno- to bioactivity was 4.7:1 . Thus the presence of the 26 kDa GST protein at the end terminus of EPO does not abrogate biological activity . GST-mEPO also gave dose-response curves parallel to rHuEPO in the MSCA but not in the RIA . The wild-type murine and three mutant GST-EPO fusion proteins (166 Des-Arg, Glu 159-->Val, and Arg 163-->Glu) were tested in the MSCA and assayed for GST activity . The ratio of bioactivity to enzyme activity for the Arg 163-->Glu mutant was approximately one third of the value obtained for each of the other fusion proteins, indicating that arginine at 163 is functionally important for EPO activity . The availability of these human and murine gene constructs in pGEX should facilitate site-directed mutagenesis and permit detailed studies of the structure-function relationships for the two erythropoietins.

Eur J Biochem, 1994 Apr 15, 221(2), 811 - 9
Stability and proteolytic domains of Nef protein from human immunodeficiency virus (HIV) type 1; Freund J et al.; Proteolytic experiments in conjunction with 1H-NMR spectroscopy show that the Nef (negative factor) protein from human immunodeficiency virus type 1 probably consists of two main domains, the N-terminal anchor domain at amino acid positions 2-65 and the C-terminal core domain at positions 66-206 . The N-terminal domain is likely to be located at the surface of the protein, while the C-terminal domain has a compactly folded core and is stable in the absence of the anchor domain . It is conceivable that the core domain represents a functional domain of the Nef protein, activated after the removal of the membrane anchor by the human-immunodeficiency-virus protease or cellular proteases . Nef is stable at pH 5-12 and denatures at 317-322 K . The Nef protein remains in its native conformation in dimethyl-sulfoxide/water mixtures up to 35% (by vol.), and in acetonitrile/water up to 14% (by vol.) . Nef refolds spontaneously after denaturation with urea or guanidinium hydrochloride . The 1H-NMR parameters and pKa values of five of the nine histidine residues and one of the seven tyrosine residues were determined and were found in four cases to be typical for residues which are not located in the interior of the protein.

Eur J Biochem, 1994 Apr 15, 221(2), 801 - 10
Primary structure and binding characteristics of locust and human muscle fatty-acid-binding proteins; Maatman RG et al.; The conservation between muscle fatty-acid-binding proteins (M-FABP) of Locusta migratoria flight muscle and human skeletal muscle was investigated . The locust M-FABP cDNA (632 bp) was isolated by 5' and 3' rapid amplification of cDNA ends . The identities of the locust and human M-FABP on the cDNA and protein levels were 54% and 42%, respectively . The predicted amino acid sequence of locust M-FABP indicated a molecular mass of 14935 Da and isoelectric point 6.1 . The locust M-FABP was expressed in Escherichia coli, purified by (NH4)2SO4 precipitation, anion-exchange and gel-filtration chromatographies and compared with the recombinant human M-FABP with respect to immunological and binding properties . In spite of the high sequence similarity, the proteins did not show immunological cross-reactivity . The binding parameters of locust M-FABP were analyzed with radiolabeled oleic acid by the Lipidex assay and titration microcalorimetry . Both methods revealed a Kd for oleic acid of 0.5 microM and a binding stoichiometry of 1 mol fatty acid/mol FABP . The delta H, delta G and delta S for oleic acid binding were -146 kJ.mol-1 and -36 J.mol-1 and -369 J.mol-1.K-1 respectively . All the information obtained from binding, fluorescence and displacement studies indicated that locust M-FABP has binding characteristics similar to human M-FABP . Finally the recombinant locust M-FABP was crystallized with and without oleic acid . All crystals were trigonal in the P3(1)21 space group . The unit cell dimensions were a = b = 5.89 nm and c = 14.42 nm.

Eur J Biochem, 1994 Apr 15, 221(2), 787 - 91
Structure/function relationship study of Gln156, Glu160 and Glu189 in the active site of trichosanthin; Wong KB et al.; Trichosanthin is a protein used medicinally in China for abortifacient purposes . It is also an RNA N-glycosidase which inactivates eukaryotic ribosomes by removing adenine4324 from 28S rRNA . Site-directed mutagenesis was performed to probe the role of Gln156, Glu160 and Glu189 in the active site of trichosanthin . The purified altered proteins were assayed for their potency in inhibiting in vitro protein synthesis . The data indicate Glu160 is involved in the catalytic reaction . Kinetics studies suggest the carboxylate group of Glu160 serves to stabilize the transition-state complex . Similar to ricin A, the variant {E160A}trichosanthin is more potent than {E160D}trichosanthin . This is because Glu189 serves as a back-up of the carboxylate group in case Glu160 is mutated to alanine . However, removal of Glu189 in the presence of Glu160 does not affect the ID50 value drastically . An activity of 1800-fold less than that of the wild-type protein was found when both Glu160 and Glu189 were changed to alanine, indicating that some other residues in the active site are also taken part in the lowering of energy barrier for the catalytic reaction . Although Gln156 is highly conserved in related proteins, its mutation to alanine only slightly decreases the activity, showing that this residue does not participate directly in catalysis.

Biochem J, 1994 Apr 15, 299 ( Pt 2), 561 - 7
Molecular cloning of cDNA for rat ovarian 20 alpha-hydroxysteroid dehydrogenase (HSD1); Miura R et al.; 20 alpha-Hydroxysteroid dehydrogenase (20 alpha-HSD, EC 1.1.1.149) catalyses the conversion of progesterone into 20 alpha-dihydroprogesterone (20 alpha-OHP) . Previously, we purified the enzyme (37 kDa) from rat ovary and determined its N-terminal amino acid sequence . In the present study we succeeded in cloning a full-length 20 alpha-HSD cDNA . mRNA was extracted from immature rat ovaries after successive treatment with equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) . A cDNA library was constructed in lambda ZAP . For screening, a 576 bp probe was amplified by the PCR using mixed primers based on the N-terminal sequence of 20 alpha-HSD, and labelled with {32P}dCTP . Eight positive clones were isolated from 1.2 x 10(4) recombinants . Analysis of the nucleotide sequence revealed that one clone of 1.2 kbp cDNA (pHSD12-07) contained a polyadenylation site and an open reading frame encoding 323 amino acids with the N-terminal sequence of 20 alpha-HSD . The fusion protein of pHSD12-07 produced by Escherichia coli reacted with a specific polyclonal antibody generated against rat ovarian 20 alpha-HSD . In addition, the in vitro transcription-translation product produced by Xenopus oocytes showed 20 alpha-HSD activity and Northern-blotting analysis revealed that the ovaries from normal adult rats contained a 1.2 kb mRNA . Thus we succeeded in isolating a clone encoding the full length of rat ovarian 20 alpha-HSD . The sequence showed high similarity with those of rat liver 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD), bovine lung prostaglandin F synthase (PGFS), human liver chlordecone reductase (CDR), frog lens rho-crystallin and aldose reductases, indicating that 20 alpha-HSD belongs to the aldo-keto reductase family.

Biochem J, 1994 Apr 15, 299 ( Pt 2), 381 - 7
Xylanase B from Neocallimastix patriciarum contains a non-catalytic 455-residue linker sequence comprised of 57 repeats of an octapeptide; Black GW et al.; A Neocallimastix patriciarum cDNA library was screened for xylanase-expressing clones, which were distinct from the previously characterized N . patriciarum xynA cDNA encoding xylanase A . A single cDNA, designated xynB, which did not exhibit homology with xynA, was isolated . Northern-blot analysis of mRNA from Avicel-grown N . patriciarum showed that xynB hybridized to a 3.4 kb mRNA species . The nucleotide sequence of xynB revealed a single open reading frame of 2580 bp coding for a protein designated xylanase B (XYLB), of M(r) 88,066 . The primary structure of XYLB was comprised of a 21-residue N-terminal signal peptide, followed by a 304-amino acid sequence that exhibited substantial homology with the catalytic domains of family F xylanases . The N-terminal domain was linked to a C-terminal 70-residue sequence by a putative linker region, comprising 12 tandem repeats of a sequence containing TLPG as the core sequence, followed by an octapeptide XSKTLPGG where X can be S, K or N, which was repeated in tandem 45 times . Truncated derivatives of xynB encoding the N-terminal 338 residues directed the synthesis of a functional xylanase, confirming that the region of XYLB, which exhibited homology with family F xylanases, constitutes the catalytic domain . To investigate the catalytic properties of XYLB, the catalytic domain was fused to the Escherichia coli maltose-binding protein, and the fusion protein purified by amylose affinity chromatography . The purified enzyme hydrolysed oat, rye and wheat arabinoxylan releasing primarily xylobiose, xylotriose and some xylose . The XYLB fusion did not cleave any cellulosic substrates . The data presented in this report suggest that the multiple xylanases of N . patriciarum arose, not through the duplication of a single gene, but by the transfer of distinct xylanase-encoding DNA sequences into the anaerobic fungus . The possible origin of the xynB gene is discussed.

EMBO J, 1994 Apr 15, 13(8), 2013 - 20
The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation; Missiakas D et al.; We have identified and functionally characterized a new Escherichia coli gene, dsbC, whose product is involved in disulfide bond formation in the periplasmic space . It corresponds to a previously sequenced open reading frame mapping upstream of recJ with no previously assigned function . Null mutations in dsbC were obtained using a screen for dithiothreitol (DTT)-sensitive mutants and were shown to result in the accumulation of reduced forms of a variety of disulfide bond-containing periplasmic proteins . This defect could be rescued by the addition of either oxidized DTT or cystine or by multicopy expression of dsbA, a known periplasmic disulfide oxidase . The DsbC protein is synthesized as a precursor form of 25.5 kDa which is processed to a 23.3 kDa mature species located in the periplasmic space . The DsbC protein was overexpressed, purified to homogeneity and shown to catalyse the reduction of insulin in a DTT-dependent manner at levels comparable with those of purified DsbA . The replacement of either cysteine residue of the predicted active site, F-(X4)-C-G-Y-C, completely inactivates DsbC protein function . We have further shown that in vivo overexpression of DsbC can functionally substitute for a loss of DsbA function . Taken together, all of our results demonstrate that DsbC acts in vivo as a disulfide oxidase.

EMBO J, 1994 Apr 15, 13(8), 1981 - 9
A single amino acid substitution (Glu134-->Ala) in NhaR1 increases the inducibility by Na+ of the product of nhaA, a Na+/H+ antiporter gene in Escherichia coli; Carmel O et al.; The mutation nhaAup (antup) has now been identified as a Glu134 to Ala substitution in NhaR and designated nhaR1 . This was demonstrated by sequence analysis showing that the mutant contains a wild-type nhaA but nhaR1 instead of nhaR and by the finding that nhaR1 cloned in a plasmid confers the NhaAup phenotype . Na+ (107 mM) increases by 5- to 10-fold the level of nhaA transcripts, similar to the effect on the NhaR-mediated expression of a nhaA'-'lacZ fusion . These results are in agreement with the notion that nhaR is a positive regulator which controls Na(+)-dependent transcription of nhaA . The promoter region of nhaR and nhaR1 was found to reside within the BglII-BamHI fragment of the C-terminal sequences of nhaA . The mutation nhaR1, while increasing dramatically the level of transcription, reduces the requirement for Na+ by 3- to 5-fold both for nhaA transcription and for the nhaR1-mediated expression of nhaA'-'lacZ fusion . NhaR1, like NhaR, binds specifically to the promoter region of nhaA . However, at equal protein concentration NhaR1 binds more DNA and the NhaR1-DNA complex shows higher mobility than that of NhaR-DNA, suggesting the existence of two different binding complexes . Yet in this assay the DNA binding pattern of neither NhaR nor NhaR1 was affected by the addition of Na+ . The possible relevance of these two DNA-binding complexes to the Na(+)-induced NhaR-mediated expression is discussed.

EMBO J, 1994 Apr 15, 13(8), 1960 - 8
Mechanism of post-segregational killing: Sok antisense RNA interacts with Hok mRNA via its 5'-end single-stranded leader and competes with the 3'-end of Hok mRNA for binding to the mok translational initiation region; Thisted T et al.; The hok/sok system of plasmid R1, which mediates plasmid stabilization by killing of plasmid-free segregants, codes for two RNA species, Hok mRNA and Sok antisense RNA . The lethal expression of hok is inhibited post-transcriptionally by the 67 nt Sok-RNA . In this paper, we analyse the secondary structure of Sok-RNA and the binding of Sok-RNA to Hok mRNA in vitro . The reaction between the two RNAs leads to the formation of a complete duplex in which Sok-RNA is hybridized over its entire length to Hok mRNA . The second-order rate constant of duplex formation was determined to be approximately 1 x 10(5) M-1s-1 . Mutations in the 5'-end single-stranded leader of Sok-RNA severely reduced the binding rate to wt Hok mRNA, whereas loop mutations in Sok-RNA had no such effect . The reduced binding rates were paralleled by abolished in vivo regulatory properties . These results suggest that, unlike in other well-characterized antisense/target RNA systems, the initial recognition reaction between Sok-RNA and Hok mRNA takes place between the single-stranded 5'-end of Sok-RNA and the complementary region in Hok mRNA, without the involvement of an antisense loop in the initial binding step . Furthermore, the finding that Sok-RNA competes with the 3'-end of full-length Hok mRNA for binding to the mok translational initiation region adds to the complexity of killer gene regulation.

EMBO J, 1994 Apr 15, 13(8), 1950 - 9
Mechanism of post-segregational killing: translation of Hok, SrnB and Pnd mRNAs of plasmids R1, F and R483 is activated by 3'-end processing; Thisted T et al.; The gene systems hok/sok of R1, srnB of F and pnd of R483 mediate plasmid maintenance by killing of plasmid-free segregants . Translation of the very stable mRNAs encoding the killer proteins is regulated by small unstable antisense RNAs . The differential decay rates of the inhibitory antisense RNAs and the mRNAs encoding the killer proteins is the basis for the onset of killer mRNA translation in newborn plasmid-free segregants and the killing of these cells . We have suggested previously that this requires that the killer mRNAs occur in two forms . A translationally inactive form was proposed to be converted into a 3'-truncated, translationally active mRNA . In the presence of the antisense RNA, translation from this killer mRNA should be inhibited . In this communication we present in vivo and in vitro evidence that support this model . The requirement for 3'-processing for killer gene expression is demonstrated . By using in vitro techniques it is shown that full-length Hok mRNA is translationally inactive, whereas a 3'-end truncated version of the Hok mRNA is translationally active . In vitro secondary structure probing suggests that the 3'-end of the full-length Hok mRNA folds back onto the translational initiation region of the mok gene and thereby inhibits translation of the mRNA . By inference we conclude that the Pnd and SrnB mRNAs are regulated by a similar mechanism.

EMBO J, 1994 Apr 15, 13(8), 1856 - 62
The initiation cascade for chromosome replication in wild-type and Dam methyltransferase deficient Escherichia coli cells; Lobner-Olesen A et al.; 'Newborn' Escherichia coli B/r cells, obtained by membrane elution, were used to study the cell cycles of wild-type and Dam methyltransferase mutants . In wild-type cells, initiation of chromosome replication was synchronous and tightly controlled . In dam mutants, initiation was altered, but not random . We propose that this is due to the absence of an initiation cascade caused by liberated DnaA molecules, and that this cascade normally synchronizes initiation . The dam- cells contained mainly two, three or four replication origins, and this affected nucleoid partitioning as well as cell division . In cultures growing with a 50 min doubling time, a variety of cell cycles were present and half the origins were used every 25 min . Some cells had a 25 min interdivision time, whereas others had an interdivision time longer than the generation time . Partitioning of nucleoids containing unequal numbers of replication origins could also be readily observed by fluorescence microscopy in the dam mutant . Based upon these observations we propose that the dam mutant is also an initiation cascade mutant.

EMBO J, 1994 Apr 15, 13(8), 1844 - 55
Xer-mediated site-specific recombination at cer generates Holliday junctions in vivo; McCulloch R et al.; Normal segregation of the Escherichia coli chromosome and stable inheritance of multicopy plasmids such as ColE1 requires the Xer site-specific recombination system . Two putative lambda integrase family recombinases, XerC and XerD, participate in the recombination reactions . We have constructed an E . coli strain in which the expression of xerC can be tightly regulated, thereby allowing the analysis of controlled recombination reactions in vivo . Xer-mediated recombination in this strain generates Holliday junction-containing DNA molecules in which a specific pair of strands has been exchanged in addition to complete recombinant products . This suggests that Xer site-specific recombination utilizes a strand exchange mechanism similar or identical to that of other members of the lambda integrase family of recombination systems . The controlled in vivo recombination reaction at cer requires recombinase and two accessory proteins, ArgR and PepA . Generation of Holliday junctions and recombinant products is equally efficient in RuvC- and RuvC+ cells, and in cells containing a multicopy RuvC+ plasmid . Controlled XerC expression is also used to analyse the efficiency of recombination between variant cer sites containing sequence alterations and heterologies within their central regions.

Biochem Biophys Res Commun, 1994 Apr 15, 200(1), 591 - 7
Involvement of ribonuclease III in the enhancement of expression of the speF-potE operon encoding inducible ornithine decarboxylase and polyamine transport protein; Kashiwagi K et al.; Since the speF-potE operon (pPT71 clone) encoding inducible ornithine decarboxylase (iODC) and polyamine transport potE protein is inducible at acidic pH, a gene encoding a protein involved in the enhancement of expression of the operon was searched for . Using the fused gene containing the upstream sequence of the speF-potE operon and the open reading frame of beta-galactosidase as a reporter gene, a clone (pPTS23) which causes the increase of beta-galactosidase activity at acidic pH was isolated . The clone also increased iODC activity at acidic pH and was identified as a gene encoding RNase III . This is the first example that RNase III increases the translational efficiency of mRNA derived from Escherichia coli gene by cutting the 5'-untranslated region of mRNA.

Biochem Biophys Res Commun, 1994 Apr 15, 200(1), 370 - 7
Modulation of the suppression efficiency and amino acid identity of an artificial yeast amber isoleucine transfer RNA in Escherichia coli by a G-U pair in the anticodon stem; Buttcher V et al.; The artificial amber suppressor corresponding to the major isoleucine tRNA from yeast (pVBt5), when expressed in E . coli, is a poor suppressor of the amber mutation lacIam181-Z . By analysing mutant forms, we could show that this was due to the presence of a U30-G40 wobble pair in the anticodon stem of the yeast tRNA and not to the level of the heterologously expressed tRNA . Efficient suppressors were obtained by restoring a normal U30-A40 or G30-C40 Watson-Crick pair . In vivo the mutant forms are exclusively charged by the bacterial lysyl-tRNA synthetase (LysRS), whereas the original yeast amber tRNA is charged at a low level by E . coli glutaminyl-tRNA synthetase (GlnRS) and LysRS . The inversion of the U30-G40 pair also induces a loss of the Gln identity . We conclude from these experiments that the U30-G40 base pair constitutes a negative determinant for LysRS interaction which operates either at the level of complex formation or at the catalytic step . As no direct contacts are seen between GlnRS and positions 30-40 of the complexed homologous tRNA, the U30-G40 pair of pVBt5 is believed to influence aminoacylation by GlnRS indirectly, probably at the level of the anticodon loop conformation by favouring an optimal apposition of the anticodon nucleotides with the protein.

Biochem Biophys Res Commun, 1994 Apr 15, 200(1), 268 - 74
Characterization and sequencing of an uncoupled lactose carrier mutant of Escherichia coli; Matos ME et al.; A lactose carrier mutant of Escherichia coli (ML308-22) showed a severe defect in thiomethylgalactoside accumulation but a faster than normal entry of o-nitrophenyl-galactoside . Sequencing of the mutant lacY gene revealed a point mutation resulting in the substitution of glycine-159 by a cysteine residue . The mutant showed an increased sensitivity to sulfhydryl reagents, a property that is consistent with the view that the Cysteine-159 is in or near the sugar recognition site and the energy coupling region of the carrier.

Blood, 1994 Apr 15, 83(8), 2334 - 44
Expression and characterization of recombinant anti-Rh(D) antibodies on filamentous phage: a model system for isolating human red blood cell antibodies by repertoire cloning; Siegel DL et al.; The production of human anti-red blood cell (RBC) Igs in vitro from immunized individuals would greatly facilitate the genetic analysis of the human immune response to RBC antigens and also provide useful serologic reagents . Technical difficulties inherent in human B-cell immortalization have led to the development of molecular approaches that bypass the need for cell transformation . By cloning human Ig gene segments into bacterial expression vectors, libraries are created of filamentous phage particles displaying Fab fragments on their surfaces . Libraries have been screened with purified, soluble antigen and selected clones genetically manipulated in Escherichia coli to produce soluble Fab fragments . Our goal has been to adapt this technique to the study of RBC autoantibodies and alloantibodies that have specificities against unpurifiable membrane-bound antigens . To test the feasibility of this approach, two sets of phage were created, one set expressing a human anti-Rh(D) Ig and the other expressing a human antitetanus toxoid Ig . After verifying the presence of functional phage-displayed Fabs through biochemical, flow cytometric, and electron microscopic analyses, a model library was constructed comprising one anti-Rh(D)-expressing phage per 10(4) antitetanus toxoid-expressing phage . A method was developed for screening the library with intact Rh(D)-positive RBCs . After four rounds of panning, anti-Rh(D) specificity was enriched more than 10,000-fold to a final frequency of approximately 100% . Plasmid DNA derived from anti-Rh(D) phage was used to produce milligram quantities of soluble recombinant anti-Rh(D) Fabs purified by nitrogen cavitation and nickel-chelation affinity chromatography . The authenticity of the Fabs was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting, which showed bands with molecular weights of approximately 50 kD and 26 kD under nonreducing and reducing conditions, respectively . Binding of recombinant anti-Rh(D) Fabs to Rh(D)-positive RBCs was demonstrated by flow cytometry and by an agglutination assay . Our results suggest that repertoire cloning by cell-surface enrichment may have broad application to the study of the human immune response to erythroid antigens in addition to membrane-bound antigens present on other hematopoietic cells.

J Immunol Methods, 1994 Apr 15, 170(2), 247 - 54
Distinction of highly homologous pregnancy-specific glycoprotein (PSG) isoforms by differential absorption of antisera with recombinant PSG fusion protein domains; Tschentscher P et al.; The N-terminal domains of two different highly homologous isoforms of pregnancy-specific beta 1 glycoproteins (PSGs) were expressed in bacteria . The N-terminal domain of PSG1 (PSG1-N) and PSG3 (PSG3-N) were chosen since PSG3-N, but not PSG1-N, contains an RGD sequence . Immunosorbents were prepared using bacterially expressed fusion proteins with the respective N domains . Antibodies from a polyclonal antiserum against native PSG were eluted from PSG1-N and were subsequently absorbed against PSG3-N . Using this procedure, antibodies were generated that were able to bind to native PSG and PSG1-N, but not to PSG3-N . These results show that the antiserum against native PSG crossreacts with PSG isoforms of two subgroups . From the PSG antiserum, antibodies can be isolated that differentially bind to V-like PSG domains which differ by eight non-conservative amino acid substitutions, three of which are clustered in a position corresponding to the CDR III of immunoglobulin V region domains . Purification of antibody populations by this technique should make it possible to distinguish rapidly between highly homologous PSG isoforms in tissues and body fluids.

J Biol Chem, 1994 Apr 15, 269(15), 11563 - 71
DNA distortion and nucleation of local DNA unwinding within sigma-54 (sigma N) holoenzyme closed promoter complexes; Morris L et al.; The sigma N (sigma 54) RNA polymerase holoenzyme has the distinctive property of binding to promoters to form a closed promoter complex, which only isomerizes to the open complex when acted upon by an enhancer binding activator protein . We probed promoter complexes that form between sigma N and its holoenzyme with the conformationally sensitive footprinting reagents ortho-copper phenanthroline, potassium permanganate, and diethylpyrocarbonate . Results from these experiments indicate that the contacts sigma N makes at the -12 promoter element are necessary to promote a local DNA distortion immediately adjacent to this promoter element when the holoenzyme but not sigma N alone binds promoter DNA . Complexes in which this local distortion is not detected are not activatable, and the altered DNA conformation is diminished in the activated complex . We propose that a barrier to open complex formation in the sigma N holoenzyme closed complex is at some step or steps after the initial nucleation of DNA strand separation, which is detected as an altered DNA conformation stably maintained within the closed complex . Thus the activator protein may promote a conformational change in the sigma N holoenzyme to allow propagation of the altered DNA conformation, probably local unwinding, which we propose is necessary for formation of the melted DNA state, characteristic of the open promoter complex.

J Biol Chem, 1994 Apr 15, 269(15), 11400 - 8
A partial structural repeat forms the heterodimer self-association site of all beta-spectrins; Kennedy SP et al.; The self-polymerization of alpha beta-spectrin heterodimers to form tetramers and higher oligomers is central to its role as a membrane stabilizer and organizer . Mutations near the amino terminus of alpha I sigma 1-spectrin or the COOH terminus of beta I sigma 1-spectrin often lead to profound impairment heterodimer polymerization and to hemolytic disease of varying severity . Previous studies using an 80-kDa univalent fragment of alpha I sigma 1-spectrin have established that the amino-terminal segment of alpha I sigma 1-spectrin mediates the association of the alpha subunit with either intact heterodimers or with isolated beta-spectrin (beta I sigma 1) . However, the nature of the self-association site in beta-spectrin has remained unclear . In the present study, native beta-spectrin and recombinant beta-spectrin peptides representing COOH-terminal portions of two alternative transcripts of the gene on chromosome 2 (beta I sigma 1 or "erythrocyte" spectrin and beta I sigma 2 or "muscle" spectrin), and one transcript of the gene on chromosome 14 (beta II sigma 1 or "beta G-fodrin") have been examined for their ability to bind either intact alpha beta-spectrin or the alpha I-spectrin 80-kDa univalent fragment . Deletion of the nonhomologous beta-spectrin sequence downstream of repeat 17 (spectrin domain III) had no discernible effect on binding . Truncations proximal to codon 2085 of beta I sigma 1-spectrin demonstrated a precipitous loss of activity, accounted for by a loss of both binding affinity and capacity . Further truncations to repeat 16 (codon 1979) restored binding activity to levels approximating that of the intact molecule . Repeat 16/17 and 17/16 chimeras displayed reduced binding activity . Collectively, these data indicate that the beta-subunit self-association site is highly sensitive to conformation, involves widespread interactions within the 17th repeat unit, is largely independent of sequences in domain III, and can be recreated by the deletion of all residues distal to the COOH end (codon 1979) of the 16th and presumably other spectrin sequence repeat units . All beta-spectrins appear to use this binding motif, regardless of the nature of the nonhomologous sequence in domain III.

J Biol Chem, 1994 Apr 15, 269(15), 11361 - 6
A hammerhead ribozyme inhibits the proliferation of an RNA coliphage SP in Escherichia coli; Inokuchi Y et al.; Ribozymes are potentially powerful tools for the suppression of intracellular gene expression . However, the few reports that exist of their activities in bacteria have described mixed success . Chuat and Galibert (Chuat, J.-C., and Galibert, F . (1989) Biochem . Biophys . Res . Commun . 162, 1025-1029) failed to detect any trans-activities of hammerhead ribozymes in Escherichia coli, while Sioud and Drlica (Sioud, M., and Drlica, K . (1991) Proc . Natl . Acad . Sci . U.S.A . 88, 7303-7307) reported complete inhibition of expression of the gene for a nonbacterial protein, HIV-1 integrase, by trans-acting hammerhead ribozymes in E . coli . It is of interest to determine whether ribozymes can really be used in natural bacterial systems (Altman, S . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 10898-10900) . We now report that a ribozyme designed to cleave the A2 gene of RNA coliphage SP, when transcribed from a plasmid in E . coli caused failure of the proliferation of progeny phage . Inactive ribozymes with altered catalytic sequences did not affect phage growth . These results indicate that it is mainly the catalytic activity of the ribozyme and not its function as an antisense molecule that is responsible for suppressing the proliferation of the RNA phage . Moreover, an analysis based on numbers of plaque-forming units and the function of the A2 protein indicated that antisense RNA may successfully compete with ribosomes in targeting mRNA while ribozymes in this study may not compete with ribosomes in naturally occurring bacterial transcription/translation-coupled systems.

J Biol Chem, 1994 Apr 15, 269(15), 11356 - 60
Reconstitution of recombinant 33-kDa subunit of the clathrin-coated vesicle H(+)-ATPase; Peng SB et al.; Evidence suggests that the ATP hydrolytic sector of the clathrin-coated vesicle proton-translocating ATPase is composed of four subunits of molecular masses of 70, 58, 40, and 33 kDa (Xie, X . S., and Stone, D . K . (1988) J . Biol . Chem . 263, 9859-9867) . We have now expressed recombinant 33-kDa polypeptide in Escherichia coli and in Spodoptera frugiperda (Sf9) cells . This subunit, renatured and purified from both sources, lacks intrinsic ATPase activity . Co-reconstitution of these recombinant 33-kDa polypeptides and recombinant 40-kDa subunit to a biochemically prepared 70-58-kDa subcomplex results in a 6-fold stimulation of calcium-activated, N-ethyl-maleimide-sensitive ATPase activity, documenting the essential role of the 33- and 40-kDa components in vacuolar type proton pump function and furthering the aim of reconstitution of a purely recombinant hydrolytic core.

J Biol Chem, 1994 Apr 15, 269(15), 11279 - 84
Critical nucleotides in the interaction of a LysR-type regulator with its target promoter region . catBC promoter activation by CatR; Parsek MR et al.; We have used site-directed mutagenesis to analyze the importance of nucleotides in the catBC promoter region for the binding of CatR, a member of the LysR family of DNA-binding proteins and for the in vivo activation of the catBC operon . The binding affinity of CatR for its binding site in the catBC promoter region was shown to increase about 2.2-fold in the presence of the inducer, cis,cis-muconate . Nucleotides were targeted for mutagenesis on the basis of previous footprinting data and sequence analysis of CatR binding sites . Critical nucleotides for CatR binding were found within an imperfect inverted repeat . Previous studies have indicated that the LysR family of DNA-binding proteins shares a consensus T-N11-A binding motif (Goethals, K., Van Montagu, M., and Holsters, M . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 1646-1650), but the CatR binding site sequence has located within the imperfect inverted repeat a G-N11-A instead of the predicted T . Mutagenesis of the G to a T resulted in an increase in both the binding of CatR to the catBC promoter and in the in vivo activation . Nucleotides within the -35 and -10 regions of the catBC promoter were found to be important for promoter activity but not for CatR binding.

J Biol Chem, 1994 Apr 15, 269(15), 11261 - 8
Tryptophan fluorescence provides a direct probe of nucleotide binding in the noncatalytic sites of Escherichia coli F1-ATPase; Weber J et al.; Tryptophan fluorescence was investigated as a tool to study the noncatalytic nucleotide-binding sites of Escherichia coli F1-ATPase . Site-directed mutagenesis, affinity labeling, and lin-benzo-ATP binding studies had shown that residues alpha R365 and beta Y354 are located close to the base moiety of bound nucleotide; here, we mutagenized each to tryptophan . The new tryptophans gave a fluorescence signal indicating an environment of high (alpha W365) or intermediate (beta W354) polarity in unoccupied sites . alpha W365 fluorescence was completely quenched by binding of ATP or ADP, providing a direct, specific probe of noncatalytic site nucleotide occupancy . Using this signal, we measured binding parameters for ATP and ADP, showed that nucleotide binding was magnesium-dependent, and showed that GTP and ITP did bind to some extent, but AMP, GDP, and IDP did not . It was possible to follow initial rates of MgATP hydrolysis and noncatalytic site binding under identical conditions; the results indicated that occupancy of noncatalytic sites was not required for catalysis . Fluorescence from beta W354 was quenched completely by lin-benzo-ATP, but only slightly by ATP or ADP . Probably, residue beta 354 is not as closely juxtaposed to the adenine ring of bound ATP and ADP as is residue alpha 365 . With either alpha W365 or beta W354 as donor and catalytic site-bound lin-benzo-ADP as acceptor, no fluorescence resonance energy transfer was detected, indicating that the distance between non-catalytic and catalytic sites is > or = 27 A.

J Biol Chem, 1994 Apr 15, 269(15), 11196 - 200
Identical mutations at corresponding positions in two homologous proteins with nonidentical effects; Bjorkman AJ et al.; The x-ray structure of a mutant (Gly72 to Asp) of the Escherichia coli ribose-binding protein with altered transport function has been solved and refined to 2.2-A resolution with a conventional R-factor (R-factor = {formula: see text}) of 16.0% and good stereochemistry . Comparison with the wild type ribose-binding protein shows that the structure is disturbed little at the actual mutation site, but quite appreciably in a neighboring loop . Changes in the surface of the protein at the site of mutation, however, seem to explain the functional effects . A corresponding mutation of the related glucose/galactose-binding protein has different structural and functional effects due to the different structural context of the mutation site in that protein . These results are consistent with the concept that these proteins have slightly different ways of interacting with the membrane components in transport and chemotaxis.

J Biol Chem, 1994 Apr 15, 269(15), 11192 - 5
Regulation of carboxypeptidase E . Effect of Ca2+ on enzyme activity and stability; Nalamachu SR et al.; Carboxypeptidase E (CPE), an enzyme that functions in the post-translational processing of bioactive peptides, is a member of the metallocarboxypeptidase gene family . A 12-residue region of CPE has 70% amino acid identity with the bacterial enzyme carboxypeptidase T (CPT); in CPT, this region has been identified previously as the Ca(2+)-binding region (Teplyakov, A., Polyakov, K., Obmolova, G., Strokopytov, B., Kuranova, I., Osterman, A., Grishin, N., Smulevitch, S., Zagnitko, O., Galperina, O., Matz, M., and Stepanov, V . (1992) Eur . J . Biochem . 208, 281-288) . Using 45Ca2+ binding, we determined that CPE binds Ca2+ . To investigate the potential function for the interaction of CPE with Ca2+, we investigated the effect of Ca2+ on aggregation, thermostability, and enzyme activity of CPE . CPE does not aggregate under a variety of Ca2+ concentrations at either pH 5.5 or 7.5, and with protein concentrations ranging from 10 to 100 micrograms/ml . Whereas Ca2+ generally stabilizes proteins to thermal denaturation, CPE was destabilized by Ca2+ and stabilized by low concentrations of EGTA . The Ca(2+)-induced destabilization of CPE was more pronounced at pH 8 than at lower pH values . At pH 8, CPE was unstable even at 37 degrees C, with approximately 40% loss of activity upon incubation for 30 min in the absence of added Ca2+ and 70% loss of activity upon incubation in the presence of 10 mM CaCl2 . Enzyme activity was not influenced by added Ca2+, but was stimulated by micromolar concentrations of EGTA; kinetic analysis showed this stimulation to be due to a change in Vmax, and not Km . Taken together, these data suggest that Ca2+ plays a role in the regulation of CPE activity.

J Biol Chem, 1994 Apr 15, 269(15), 11178 - 85
Functional expression and site-directed mutagenesis of a synthetic gene for alpha-bungarotoxin; Rosenthal JA et al.; In order to explore the structure-function relationships of the curare mimetic alpha-neurotoxins we have constructed and cloned a synthetic gene for Bungarus multicinctus alpha-bungarotoxin which is expressed in Escherichia coli . The recombinant alpha-bungarotoxin is expressed as a fusion protein with alpha-bungarotoxin linked to the COOH-terminal end of the T7 Gene 9-encoded coat protein . After treatment of the fusion protein with Factor Xa protease, a recombinant alpha-bungarotoxin is released that co-migrates with authentic alpha-bungarotoxin upon reverse-phase high performance liquid chromatography and ion-exchange chromatography . Final yields of active recombinant alpha-bungarotoxin were about 0.4 mg/liter of starting bacterial culture . The recombinant alpha-bungarotoxin contains 10 additional residues linked to the NH2-terminal Ile of the alpha-bungarotoxin sequence due apparently to the inaccessibility of the engineered cleavage site to Factor Xa . Nevertheless, the recombinant alpha-bungarotoxin is capable of binding to the nicotinic acetylcholine receptor with an apparent affinity that is only decreased approximately 1.7-fold from that of authentic alpha-bungarotoxin . Alanine substitution of a residue, Asp30, highly conserved among alpha-neurotoxins and previously suggested to play a key role in receptor recognition, resulted in a recombinant alpha-bungarotoxin whose receptor binding activity is indistinguishable from authentic alpha-bungarotoxin.

J Biol Chem, 1994 Apr 15, 269(15), 11147 - 54
Measurements of ATP binding on the large cytoplasmic loop of the sarcoplasmic reticulum Ca(2+)-ATPase overexpressed in Escherichia coli; Moutin MJ et al.; The large cytoplasmic loop of the sarcoplasmic reticulum Ca(2+)-ATPase (LCL), situated between Lys329 and Phe740, is believed to contain both its phosphorylation and ATP binding domains . A cDNA fragment coding for this amino acid sequence was generated in vitro and cloned in vector pQE8 which allowed the overexpression in Escherichia coli of this Ca(2+)-ATPase domain fused with a cluster of 6 histidines at its NH2 terminus . The fusion protein produced in an insoluble form within bacteria was solubilized in 4 M urea, purified on immobilized Ni2+, and then renatured by elimination of urea . More than 4 mg of purified renatured fusion protein was obtained from 500 ml of culture . ATP binding on the refolded protein was demonstrated by two methods: 1) detection of ATP-induced intrinsic fluorescence change and 2) binding of the fluorescent ATP analogue 2',3'-O-(2,4,6-trinitrophenyl)-adenosine-5'-triphosphate (TNP-ATP) and its chase by ATP . It is shown that the LCL protein has one single TNP-ATP binding site having a dissociation constant (Kd) of 1.6-1.9 microM . Both methods yielded a Kd for ATP around 200 microM . Binding of other nucleotides was detected with a sequence of Kd identical to that found for native Ca(2+)-ATPase: ATP < ADP < GTP < AMP < ITP . A Mg2+ binding site was also found on the LCL protein (Kd = 100 microM at pH 7.2) . The fluorescence of bound TNP-ATP was found to be highly dependent on Mg2+ binding on this site.

J Biol Chem, 1994 Apr 15, 269(15), 11138 - 46
Glycosphingolipid receptor function is modified by fatty acid content . Verotoxin 1 and verotoxin 2c preferentially recognize different globotriaosyl ceramide fatty acid homologues; Kiarash A et al.; Verotoxins (VT) are a family of Escherichia coli-derived toxins which have been associated with hemolytic uremic syndrome, the leading cause of acute pediatric renal failure, and hemorrhagic colitis . Verotoxins (VT1 and VT2c) both show terminal gal alpha 1-4gal-dependent binding to globotriaosylceramide (Gal alpha 1-4Gal beta 1-4Glc-Cer; Gb3), yet VT2c shows a thousandfold lower specific cytotoxic activity in vitro . Our previous studies have shown this discrepancy is a function of the receptor binding B subunit and that VT1/Gb3 binding in a lipid matrix is affected by heterogeneity in the ceramide fatty acid chain length . The influence of the fatty acid composition of Gb3 on the binding of VT1 and VT2c has now been compared using 14 homogeneous semisynthetic Gb3 molecular species of differing fatty acid chain length and degree of saturation from C12 to C24 . The binding of verotoxin was quantitated by Scatchard analysis using a solid-phase binding assay in the presence of auxiliary lipids, which may in some respects approximate to receptor function within the plasma membrane of sensitive cells . Differential binding was observed for several of these species in the lipid matrix, indicating that the fatty acid moiety of Gb3 is important for VT binding under such conditions . The short chain fatty acid containing Gb3 (C12 and C14) showed minimal binding . Middle and long chain fatty acid Gb3 homologues (C16, C18, C20, C22, and C24) were effectively recognized by VTs . The presence of an unsaturated fatty acid in Gb3 significantly increased VT binding in all cases . C20:0 and C22:1 containing Gb3 had the greatest capacity to bind VT1 . In contrast, C18:0 and C18:1 homologues showed the greatest capacity for VT2c binding (higher than VT1) . These results were, in general, reflected in cell cytotoxicity in that receptor-deficient cells reconstituted with C22:1Gb3 were maximally sensitive to VT1 in vitro whereas cells reconstituted with C18:1Gb3 were maximally sensitive to VT2c . VT2c was an ineffective inhibitor of 125I-VT1 binding to C22:1 Gb3 but in contrast, more effective than VT1 to compete binding to C18:1 Gb3 . Similarly, VT1 was less effective than VT2c to compete binding of 125I-VT2c to C18:1 but more effective than VT2c to compete for C22:1 Gb3 binding . These results suggest that VT1 and VT2c bind selectively to different but overlapping carbohydrate epitopes on the Gb3 molecule which are differentially available in these Gb3 fatty acid homologues in a lipid environment.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1994 Apr 15, 269(15), 11121 - 32
Recombinant replication protein A: expression, complex formation, and functional characterization; Henricksen LA et al.; Replication protein A (RPA) is a multisubunit, single-stranded DNA-binding protein that is absolutely required for replication of SV40 DNA . The three cDNAs encoding the subunits of human replication protein A (70, 32, and 14 kDa) have been expressed individually and in combination in Escherichia coli . When subunits were expressed individually, appropriately sized polypeptides were synthesized, but were found to be either insoluble or aggregated with other proteins . We examined the interactions between individual RPA subunits by expressing pairs of subunits and determining if they formed stable complexes . Only the 32- and 14-kDa subunits formed a soluble complex when coexpressed . This complex was purified and characterized . The 32-14 kDa subcomplex did not have any effect on DNA replication and was not phosphorylated efficiently in vitro . We believe that the 32.14-kDa subcomplex may be a precursor in the assembly of the complete RPA complex . Coexpression of all three subunits of RPA resulted in a significant portion of each polypeptide forming a soluble complex . We have purified recombinant RPA complex from E . coli and demonstrated that it has properties similar to those of human RPA . Recombinant human RPA has the same subunit composition and the same activities as the authentic complex from human cells . Recombinant human RPA binds single-stranded DNA and is capable of supporting SV40 DNA replication in vitro . In addition, recombinant RPA became phosphorylated when incubated under replication conditions.

J Biol Chem, 1994 Apr 15, 269(15), 11102 - 7
Probing the retinol-binding site of bovine beta-lactoglobulin; Cho Y et al.; The retinol-binding site of beta-lactoglobulin has been located by selective modification of amino acid residues which reside in the two putative binding sites . Based upon two separate crystallographic analyses of bovine beta-lactoglobulin, different binding sites for retinol have been proposed: one proposal favors an interior cavity, the other a surface cleft . To discriminate between these two models, we have made four separate site-directed mutations introducing a W19A or a K70M in the interior pocket and a F136A or K141M in the surface pocket . The K70M beta-lactoglobulin exhibited a marked decrease in its binding of retinoic acid compared to the F136A, K141M, and wild-type proteins . Retinyldenepropylamine, a retinyl Schiff base analog of retinol, was synthesized and its absorption spectrum when bound to the wild-type, K70M, and K141M proteins was examined to probe its interaction with the respective lysine residues . The retinylidenepropylamine bound in the K70M beta-lactoglobulin exhibited a kinetic red shift as distinct from the blue shift observed when it is bound to either the K141M or wild-type beta-lactoglobulins . The blue shift indicates protonation of the Schiff base . The resulting tagged peptide was isolated after cyanogen bromide cleavage and found to be the Ala25-Met107 peptide, consistent with the Lys70 being the residue which interacts with the bound retinol . These results support the proposal that retinol binds to an evolutionarily conserved interior cavity rather than the surface pocket.

J Biol Chem, 1994 Apr 15, 269(15), 11054 - 9
Mutagenic studies of the interaction between the aspartate receptor and methyltransferase from Escherichia coli; Shapiro MJ et al.; The 4 glutamate residues that are the sites of methylation in the aspartate receptor, which mediates bacterial chemotaxis, were systematically mutated to aspartate residues . None of the aspartate residues were methylated . In each case, the presence of the aspartate mutations altered the methylation rates at glutamate residues on the same receptor . Methylation was nearly eliminated at residues two turns of alpha helix, in the N-terminal direction, from a site of mutation, whereas less severe changes occurred at other positions . The methyltransferase apparently must contact a glutamate or glutamine residue two turns of helix in the C-terminal direction from the site of modification in order to methylate that position at a maximal rate . Mutations from glutamate to aspartate at any of the four sites also appear to alter the methylation rate at the other sites through a change in the structure of the receptor . The aspartate mutations did not substantially alter the affinity of the methyltransferase for the receptor.

J Biol Chem, 1994 Apr 15, 269(15), 11002 - 10
Mechanism of modulation of rat liver fructose-2,6-bisphosphatase by nucleoside triphosphates; Lee YH et al.; The mechanism of modulation of fructose-2,6-bisphosphatase of rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase by nucleoside triphosphates was studied by employing the Escherichia coli-expressed bisphosphatase domain and a COOH-terminal 30-amino acid truncated form . These forms had Km values for substrate and Ki values for products which were similar to those of the bisphosphatase of the intact bifunctional enzyme, but turnover numbers were 5-fold higher . All forms also exhibited substrate inhibition that was relieved by GTP and ATP . The nucleoside triphosphates bound to the active site, since they were competitive inhibitors at subsaturating substrate concentrations . Guanosine was also a competitive inhibitor at subsaturating substrate concentrations but did not activate at saturating substrate . ATP and GTP had Kd values of 467 and 110 microM, respectively, and 1 mol of nucleoside triphosphate/mol bound per mol of bisphosphatase . The Ki values for guanosine of two mutants, Lys356-->Ala and Arg360-->Ala, were unchanged from that of the wild-type enzyme . However, the Ki for GTP for Arg360-->Ala was 17-fold higher than that of the wild-type enzyme, whereas that for Lys356-->Ala was unchanged . It was concluded that 1) nucleoside triphosphate modulation of the bisphosphatase of the bifunctional enzyme involves a direct interaction with the active site of the bisphosphatase domain; and 2) the activation is caused by the phosphate moieties of GTP and ATP competing with the 2-phospho group of fructose-2,6-bisphosphate for the phosphoenzyme intermediate, thus relieving substrate inhibition.

Structure, 1994 Apr 15, 2(4), 309 - 16
Electrostatic activation of Escherichia coli methionine repressor; Phillips K et al.; BACKGROUND: The three-dimensional structure of the Escherichia coli methionine repressor (met repressor) is relatively unperturbed by the binding of its corepressor, S-adenosylmethionine (SAM), and of operator DNA . The positively charged corepressor binds to sites on the repressor remote from the DNA-binding site, and despite the lack of induced structural change is able to raise the affinity for operator DNA by a factor of up to 1000 . Neutral corepressor analogues also bind to the repressor, but do not increase operator affinity . These observations suggest that the corepressor effect may be electrostatic . RESULTS: Using the program DELPHI, we have calculated electrostatic potentials for the repressor and its complexes, and have obtained results consistent with an electrostatic model for repressor activation . The positive potential originating from the corepressor is propagated through the repressor-operator complex, and is significant at DNA phosphate groups buried in the protein-DNA interface . The rank order of calculated electrostatic interaction energies for complexes with SAM, and two closely-related analogues, is in agreement with experimental measurements of the corresponding repressor-operator affinities . CONCLUSION: Long-range (> 10 A) electrostatic interactions between bound corepressor and operator phosphate groups in the repressor-operator complex may be sufficient to explain repressor activation Met repressor could, therefore, be an electrostatically triggered genetic switch.

Biochem J, 1994 Apr 15, 299 ( Pt 2), 527 - 31
Substitutions for Glu-537 of beta-galactosidase from Escherichia coli cause large decreases in catalytic activity; Yuan J et al.; Glu-537 of beta-galactosidase (EC 3.2.1.23) was replaced by Asp, Gln and Val using synthetic oligonucleotides . The kcat values of the purified enzyme mixtures were reduced by about 100-fold for the Asp mutant, 30,000-60,000-fold for the Val mutant and 160,000-300,000-fold for the Gln mutant . The greatest differences in properties from the wild-type enzyme were found for the Asp-substituted enzyme: the Km values increased (from 0.12 to 0.42 mM for o-nitrophenyl beta-D-galactopyranoside), and from 0.04 to 0.37 mM for p-nitrophenyl beta-D-galactopyranoside), the Ki value for isopropyl beta-D-galactopyranoside increased (from 0.11 to 0.30 mM), the stability to heat decreased and methanol did not act as an acceptor . The enzymes with the other two substitutions had properties similar to those of the wild-type . For all three substituted enzymes, the inhibitory effects of the transition-state analogues (2-deoxy-2-amino-D-galactose and L-ribose) and the Mg2+ effects were similar to those of the normal enzyme . As all of the properties (except the kcat values) of the Gln- and Val-substituted enzyme preparations were similar to those of the wild-type enzyme, the activities in those preparations were probably due to the presence of a few wild-type enzyme molecules (formed from misreads) among the substituted enzymes . The enzymes with Gln and Val substitutions appear to be totally inactive . The results obtained support a recent suggestion that Glu-537 is an important catalytic residue of beta-galactosidase.

J Biol Chem, 1994 Apr 15, 269(15), 11030 - 6
D-type cyclin-binding regions of proliferating cell nuclear antigen; Matsuoka S et al.; Proliferating cell nuclear antigen (PCNA) is an auxiliary protein for DNA polymerase delta and is required for both DNA replication and DNA repair . PCNA forms complexes with D-type cyclins, candidate G1 cyclins in mammalian cells . To better understand the functions of the complexes, we examined interactions between PCNA and D-type cyclins, using in vitro-translated mouse PCNA and mouse cyclin D1 or D3 fused to glutathione S-transferase (GST) . Analysis of a set of deletion mutants of PCNA revealed that either the N-terminal (residues 2-64) or the C-terminal (residues 197-228) region is necessary for association with D-type cyclins . The cyclin binding of the chimeric protein of the N-terminal (residues 1-68) or the C-terminal (residues 195-261) region of PCNA and rat DNA polymerase beta which does not bind to the cyclins by itself supports this notion . The purified recombinant mouse PCNA expressed in Escherichia coli bound to the D-type cyclin-GST fusion proteins, thereby suggesting that PCNA binds directly to D-type cyclins, without the requirement of other cellular factors . This is apparently the first report on the structure-function relationship of PCNA which may link DNA replication and DNA repair with cell cycle control.

J Biotechnol, 1994 Apr 15, 33(3), 259 - 69
N-terminal variants of fatty acid-binding protein from bovine heart overexpressed in Escherichia coli; Specht B et al.; An expression vector for bovine heart fatty acid-binding protein (H-FABP) was constructed by introducing the coding part of the cDNA into the pET-3d vector . Transformed Escherichia coli strain BL21 (DE3)pLysS produced functional recombinant H-FABP up to 40% of the soluble proteins . The expression of fatty acid-binding protein was under the control of the T7-phi 10 promoter and the corresponding T7-RNA-polymerase in turn was induced by isopropyl beta-D-thiogalactopyranoside . By combination of cation exchange chromatography and gel filtration pure recombinant protein was obtained exhibiting isoelectric heterogeneity . Recombinant H-FABP was resolved into at least six variants with isoelectric points between 5.1 and 5.6 . After separation by preparative isoelectric focusing the four major variants were digested with trypsin and the resulting peptides were characterized by high performance liquid chromatography (HPLC), matrix assisted laser desorption/ionization (MALDI) mass spectrometry, amino acid sequencing and chemical modification . The structural differences were traced back to the N-termini beginning with either methionine, as expected from the cDNA, or methionine sulfoxide, valine and N-formyl methionine . The latter three arise from oxidation, cleavage of N-terminal methionine and incomplete deformylation, respectively.

J Mol Biol, 1994 Apr 15, 237(5), 560 - 76
Thermodynamics of RNA folding in a conserved ribosomal RNA domain; Laing LG et al.; A small, 58 nt domain of the large subunit ribosomal RNA (Escherichia coli sequence 1051 to 1108) is a highly conserved junction of three helices whose secondary structure has been established by phylogenetic comparisons . To detect any contributions of additional tertiary interactions, the thermal denaturation of the rRNA domain was followed by either UV hyperchromicity or calorimetry in buffers containing a wide range of Mg2+ concentrations . Several smaller fragments corresponding to two different hairpin stem-loop structures within the domain were also synthesized and melted for comparison with the larger molecule . A model of the secondary structure unfolding was devised, based on measured enthalpies and melting temperatures of the component hairpins and tabulated parameters of base-pair stacking and loop closure . The model closely simulates the observed melting data when three additional factors are included: two parameters to account for coaxial stackings within a junction of helices, and a set of undefined "tertiary" interactions that unfolds before the secondary structure and is preferentially stabilized by Mg2+ . A critical feature of this model is a conserved pair, U1082/A1086, that is within the junction loop and hypothesized to stack with an adjacent helix . The model correctly predicts the effects of disrupting this pair in a U1086 sequence variant . Although the set of "tertiary" interactions contributes a significant fraction of the RNA unfolding enthalpy (delta H approximately 25 kcal/mol, out of 180 kcal/mol total), its overall stability is marginal at 37 degrees C.

J Biol Chem, 1994 Apr 15, 269(15), 11299 - 305
The interferon-induced 67-kDa guanylate-binding protein (hGBP1) is a GTPase that converts GTP to GMP; Schwemmle M et al.; hGBP1 is an interferon-induced 67-kDa protein of human cells that readily binds to agarose-immobilized GTP, GDP, and GMP but not to other nucleotides . We cloned hGBP1 cDNA into a histidine-tagging vector, produced recombinant hGBP1 with 6 extra histidine residues at its N terminus in Escherichia coli, and purified this protein to near homogeneity from bacterial lysates . Purified hGBP1 hydrolyzed radiolabeled GTP but failed to hydrolyze ATP, UTP, or CTP at significant rates . Unexpectedly, the principal product of the GTP hydrolysis reaction was GMP rather than GDP . Although significant amounts of GDP were produced when the reaction was performed at 15 degrees C, GDP could not serve as substrate or as inhibitor of hGBP1 . hGBP1 lacked guanylate cyclase and guanylyltransferase activity . Degradation of GTP to GMP most likely occurred via two consecutive cleavages of single phosphate groups, because pyrophosphate was not a reaction product, and because hGBP1 failed to hydrolyze GTP gamma S . In vitro modification assays with radiolabeled mevalonic acid and farnesyl pyrophosphate showed that the CaaX motif at the C terminus of hGBP1 functions as an isoprenylation signal . Thus, hGBP1 is a GTPase with novel biochemical properties that may be membrane-associated in eukaryotic cells.

Mutat Res, 1994 Apr 15, 306(2), 143 - 51
Shuttle-vector mutagenesis by aflatoxin B1 in human cells: effects of sequence context on the supF mutational spectrum; Courtemanche C et al.; Rat-liver microsomes were used to activate aflatoxin B1 for in vitro modification of the pS189 shuttle vector and the related signature vector pSP189, both of which carry the Escherichia coli supF gene as a mutational target . Plasmid degradation was minimized by carrying out the in vitro incubations in the absence of Mg2+ ions . Modified plasmids were transfected into human Ad293 cells, then recovered and electroporated into E . coli MBM7070 for mutant identification . Point mutation frequencies for in vitro modified plasmids were dramatically increased over the spontaneous background level . Mutant plasmids were characterized by DNA-sequence analysis . The vast majority of aflatoxin B1-induced mutations were base substitutions, mostly G:C to T:A transversions . The spectrum of aflatoxin B1-induced mutations in the pS189 supF gene was very similar to that observed previously for the pS189 supF gene with aflatoxin B1 activated by cytochrome P450 1A2 (CYP1A2) synthesized from a cDNA expression vector within transfected Ad293 cells . However, the spectrum for pSP189, which carries the same supF gene as pS189, but with different surrounding sequences, exhibited some notable differences from that of pS189; this suggests that sequence context effects on mutagenic specificity can operate over distances of tens of base pairs.

Biochim Biophys Acta, 1994 Apr 14, 1212(1), 93 - 102
Characterization of polyprenyldiphosphate: 4-hydroxybenzoate polyprenyltransferase from Escherichia coli; Melzer M et al.; Polyprenyldiphosphate: 4-hydroxybenzoate polyprenyltransferase (4-HB polyprenyltransferase) is a key enzyme in ubiquinone biosynthesis in E . coli, encoded by the gene ubiA . By overexpression of ubiA and isolation of the membrane fraction, the enzyme was enriched approx . 3000-fold and characterized . The enzyme is membrane-bound and could not be solubilized by hypotonic buffer or detergent treatment . The enzymatic activity is optimal at pH 7.8 and depends on the presence of magnesium ions . Geranyldiphosphate (GPP), all-trans-farnesyldiphosphate (FPP) and all-trans-solanesyldiphosphate (SPP) are accepted as side chain precursors . The apparent Km values for these substances are are 254 microM, 22 microM and 31 microM, respectively . No reaction was observed with omega-t2-c5-octaprenyldiphosphate, in which five double bounds have cis-configuration . The reaction is stimulated by 0.01% CHAPS, but strongly inhibited by sodiumdeoxycholate, Tween 80 and Triton X-100 . The amino acid sequence shows striking similarities to 4-HB hexaprenyltransferase from yeast . Sequence homologies to other prenyltransferases are discussed.

Biochim Biophys Acta, 1994 Apr 14, 1212(1), 134 - 6
Cloning and expression in Escherichia coli of a cDNA coding for the oleoyl-acyl carrier protein thioesterase from coriander (Coriandrum sativum L.); Dormann P et al.; A cDNA for the oleoyl-acyl carrier protein (ACP) thioesterase (E.C . 3.1.2.14) from coriander seed endosperm (Coriandrum sativum) was isolated using a safflower oleoyl-ACP thioesterase cDNA probe . The coriander cDNA coded for a 42.3 kDa protein including a putative 40 amino acid plastid targeting transit peptide . The gene was expressed in Escherichia coli and the recombinant protein was isolated to homogeneity by alkyl-ACP affinity and anion-exchange chromatography . The pure protein showed a high thioesterase activity for oleoyl-ACP vs . other acyl-ACPs and therefore was identified as the coriander oleoyl-ACP thioesterase . Antibodies were raised against the recombinant protein and used to detect the coriander thioesterase in enriched endosperm fractions.

Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 3044 - 8
Yeast farnesyl-diphosphate synthase: site-directed mutagenesis of residues in highly conserved prenyltransferase domains I and II; Song L et al.; Prenyltransferases that catalyze the fundamental chain elongation reaction in the isoprenoid biosynthetic pathway contain several highly conserved amino acids, including two aspartate-rich regions thought to be involved in substrate binding and catalysis . We report a study of site-directed mutants for yeast farnesyl-diphosphate synthase (FPPSase; geranyl-diphosphate:isopentenyl-diphosphate, EC 2.5.1.10), a prenyltransferase that catalyzes the sequential 1'-4 coupling of isopentenyl diphosphate (IPP) with dimethylallyl diphosphate and geranyl diphosphate . A recombinant form of FPPSase extended by a C-terminal -Glu-Glu-Phe alpha-tubulin epitope (EEF in single-letter amino acid code) was engineered to facilitate rapid purification of the enzyme by immunoaffinity chromatography and to remove traces of contaminating activity from wild-type FPPSase in the Escherichia coli host . Ten site-directed mutants were constructed in FPPSase::EEF . The six aspartates in domain I (at positions 100, 101, and 104) and domain II (at positions 240, 241, and 244) were changed to alanine (mutants designated D100A, D101A, D104A, D240A, D241A, and D244A); three arginine residues were changed, Arg-109 and Arg-110 to glutamine and Arg-350 to alanine (mutants designated R109Q, R110Q, and R350A); and Lys-254 was converted to alanine (mutant designated K254A) . Mutations of the aspartatic residues and nearby arginine residues in domain I and Asp-240 and Asp-241 in domain II drastically lowered the catalytic activity of FPPSase::EEF . The D244A and K254A mutants were substantially less active, while kcat and the Michaelis constants for the R350A mutant were similar to those of FPPSase::EEF . Addition of an -EEF epitope to the C terminus of wild-type FPPSase resulted in a 14-fold increase of KmIPP and a 12-fold decrease of kcat, suggesting that the conserved hydrophilic C terminus of the enzyme may have a role in substrate binding and catalysis.

Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 3024 - 8
Molecular cloning, sequencing, and functional expression of a cDNA encoding human coproporphyrinogen oxidase; Martasek P et al.; Coproporphyrinogen oxidase (EC 1.3.3.3) catalyzes the sixth step in the heme biosynthetic pathway, the oxidation of coproporphyrinogen III to protoporphyrinogen IX . The activity of this enzyme is deficient in the disease hereditary coproporphyria . The sequence of the cDNA and predicted amino acid sequence of the human coproporphyrinogen oxidase are presented . The human protein sequence contains a region completely homologous to that we obtained by sequencing an 11-amino acid peptide fragment from purified murine liver coproporphyrinogen oxidase . Results of Southern blotting were consistent with the presence of a single human coproporphyrinogen oxidase gene, and Northern blotting demonstrated one transcript of similar size in erythroid and nonerythroid cell lines . Expression of the cDNA coding for the putative mature human coproporphyrinogen oxidase in Escherichia coli resulted in a 17-fold increase in coproporphyrinogen activity over endogenous activity.

Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 2989 - 93
Requirement for a conserved, tertiary interaction in the core of 23S ribosomal RNA; Aagaard C et al.; A putative base-pairing interaction that determines the folding of the central region of 23S rRNA has been investigated by mutagenesis . Each of the possible base substitutions has been made at the phylogenetically covariant positions adenine-1262 (A1262) and U2017 in Escherichia coli 23S rRNA . Every substitution that disrupts the potential for Watson-Crick base pairing between these positions reduces or abolishes the participation of 23S rRNA in protein synthesis . All mutant 23S rRNAs are assembled into 50S subunits, but the mutant subunits are less able to stably interact with 30S subunits to form translationally active ribosomes . The function of 23S rRNA is largely reestablished by introduction of an alternative G1262.C2017 or U1262.A2017 pair, although neither of these supports polysome formation quite as effectively as the wild-type pair . A 23S rRNA with a C1262.G2017 pair is nonfunctional . In contrast to the considerable effect the mutations have on function, they impart only slight structural changes on the naked rRNA, and these are limited to the immediate vicinity of the mutations . The data show that positions 1262 and 2017 pair in a Watson-Crick manner, but the data also indicate that these nucleotides engage in additional interactions within the ribosome and that these interactions determine what base pairs are acceptable there.

Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 2980 - 4
Reversible inactivation of the Escherichia coli RecBCD enzyme by the recombination hotspot chi in vitro: evidence for functional inactivation or loss of the RecD subunit; Dixon DA et al.; Genetic recombination in Escherichia coli is stimulated by a RecBCD enzyme-mediated event at DNA sequences known as Chi (chi) sites (5'-GCTGGTGG-3') . Previously, it was shown that chi acts to regulate the nuclease activity of RecBCD; here, we demonstrate that, under appropriate conditions, interaction with chi sites can also result in an inactivation of helicase activity of RecBCD . The unwinding of double-stranded DNA-containing chi sites, under conditions of limiting Mg2+ ion, results in the reversible inactivation of RecBCD; addition of excess Mg2+ to the reaction reactivates all activities of RecBCD . Inactivation is the consequence of a chi-dependent modification of RecBCD that appears to result from an inability of the chi-modified RecBCD to reinitiate unwinding of intact DNA molecules . This characteristic behavior of RecBCD and chi is displayed by the reconstituted RecBC (i.e., without the RecD subunit), except that it is not dependent on chi interaction . This biochemical similarity between the chi-modified RecBCD and RecBC enzymes implies that recognition of chi results in a dissociation or functional inactivation of RecD subunit and lends support to the hypothesis that interaction with chi results in ejection of the RecD subunit.

Biochemistry, 1994 Apr 12, 33(14), 4352 - 62
Incorporation of norleucine at methionine positions in recombinant human macrophage colony stimulating factor (M-CSF, 4-153) expressed in Escherichia coli: structural analysis; Randhawa ZI et al.; Expression of the 17.5-kDa truncated form of human recombinant macrophage colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is complicated by the replacement of methionine residues by norleucine . In order to detect and quantitate this mistranslational event, the intact and the S-carboxyamidomethylated proteins were analyzed by amino acid analysis, automated Edman amino acid sequencing, and electrospray mass spectrometry . In addition, the endoproteinase Glu-C generated peptides were subjected to amino acid sequencing, high-performance liquid chromatography, and electrospray ionization mass spectrometry . The extent of norleucine substitution in different batches of rM-CSF varied between 0% and 20% . The relative instability of methionine residues needs to be considered when calculating the extent of norleucine substitution at methionine positions . The mass spectrometry of the intact rM-CSF allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantitation of the norleucine incorporation down to the approximately 3% level . Selective ion chromatograms of molecular ions of interest obtained in reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry of proteolytic fragments offered a reliable and fast method of detection and quantitation of norleucine-containing peptides . Norleucine residues were uniformly distributed among all four methionine positions (10, 27, 61, and 65) . A substitution of methionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.

Biochemistry, 1994 Apr 12, 33(14), 4319 - 26
Analysis of the binding of 3,3',5-triiodo-L-thyronine and its analogues to mutant human beta 1 thyroid hormone receptors: a model of the hormone binding site; Cheng SY et al.; To understand the nature of the thyroid hormone binding site, we characterized the binding of 3,3',5-triiodo-L-thyronine (T3) and its analogues to eight naturally occurring mutated human beta 1 thyroid hormone receptors (h-TR beta 1) . The mutant receptors were derived from patients with the syndrome of generalized thyroid hormone resistance, and each has a point mutation in the hormone binding domain (KT, R338W; TP, L450H; IR, D322H; NN, G347E; AH, P453H; OK, M442V; RL, F459C; and ED, A317T) . Compared to the wild-type h-TR beta 1, binding of T3 was reduced by as much as 97% for the mutants . The order of binding affinity of wild-type h-TR beta 1 to the analogues is T3 > D-T3 > L-thyroxine > 3,5-diiodo-L-thyronine > 3,3',5'-triiodo-L-thyronine . The mutant receptors showed essentially the same order of reduced affinities for the analogues, but the amounts of the reductions varied in each case . These results suggest specific local interactions (interplay) of analogues with the mutated residues in the receptors . On the basis of these data and a putative structure of the hormone binding domains as an eight-stranded alpha/beta barrel, we propose the location of the hormone in the binding site of h-TR beta 1 . Ionic bonds anchor the hormone's alanine side chain to loop 4 of the 8-fold alpha/beta barrel . The phenyl ring lies across the amino-terminal face of the domain with the phenoxy ring pointing downward into the barrel interacting with beta-strand 8 on the opposite side.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Apr 12, 33(14), 4258 - 64
Identification of the catalytic base in long chain acyl-CoA dehydrogenase; Djordjevic S et al.; We have used molecular modeling and site-directed mutagenesis to identify the catalytic residues of human long chain acyl-CoA dehydrogenase . Among the acyl-CoA dehydrogenases, a family of flavoenzymes involved in beta-oxidation of fatty acids, only the three-dimensional structure of the medium chain fatty acid specific enzyme from pig liver has been determined (Kim, J.-J.P., Wang, M., & Paschke, R . (1993) Proc . Natl . Acad . Sci . U.S.A . 90, 7523-7527) . Despite the overall sequence homology, the catalytic residue (E376) of medium chain acyl-CoA dehydrogenase is not conserved in isovaleryl- and long chain acyl-CoA dehydrogenases . A molecular model of human long chain acyl-CoA dehydrogenase was derived using atomic coordinates determined by X-ray diffraction studies of the pig medium chain specific enzyme, interactive graphics, and molecular mechanics calculations . The model suggests that E261 functions as the catalytic base in the long-chain dehydrogenase . An altered dehydrogenase in which E261 was replaced by a glutamine was constructed, expressed, purified, and characterized . The mutant enzyme exhibited less than 0.02% of the wild-type activity . These data strongly suggest that E261 is the base that abstracts the alpha-proton of the acyl-CoA substrate in the catalytic pathway of this dehydrogenase.

Biochemistry, 1994 Apr 12, 33(14), 4231 - 6
Protein folding activities of Escherichia coli protein disulfide isomerase; Joly JC et al.; DsbA is an Escherichia coli periplasmic protein that mediates disulfide bond formation in newly secreted proteins in vivo . Addition of thiol reagents to purified dsbA reduces its disulfide bond and yields disulfide isomerase activity after removal of the thiol reagent . DsbA can catalyze the conversion of a stable misfolded protein, misfolded IGF-I (mis-IGF-I), to its correctly folded conformation under physiological conditions . This conversion is the result of breaking and re-forming two disulfide bonds . The uncatalyzed rate of this reaction is undetectable . Kinetic analysis of the reaction yielded a Km of 43 microM and a kcat of 0.2 min-1 . The oxidized form of dsbA stimulates the oxidative folding of completely reduced IGF-I at pH 7.0 . Thus, dsbA has two possible functions depending on its redox state . The reduced form of the protein is a disulfide isomerase while the oxidized protein can assist formation of disulfide bonds in reduced substrates under physiological conditions.

Biochemistry, 1994 Apr 12, 33(14), 4175 - 86
Construction of a synthetic gene for the metalloregulatory protein MerR and analysis of regionally mutated proteins for transcriptional regulation; Comess KM et al.; The transcriptional control protein MerR is a metalloregulatory switch, activating transcription of a mercury resistance operon in the presence of mercuric ions and repressing transcription in their absence . We report here the construction and utilization of a synthetic merR gene and a single-copy merT'-lacZ fusion reporter for mutagenic analysis of the MerR protein's function . Site-directed mutagenesis of clustered acidic residues within the central region of the MerR protein indicated that these residues are important to the protein's ability to repress transcription . Quadruple or sextuple mutations involving residues E83 and E84 and other nearby acidic residues result in a repression-deficient (RD) phenotype . One of the mutant proteins was purified and shown by gel shift assay to retain binding to its operator DNA with an affinity similar to wild-type protein, suggesting that transcriptional repression does not correlate with MerR binding affinity . A small region of merR corresponding to residues 81-92 also was mutagenized in a search for other RD mutants and for mutants displaying sufficient transcriptional activation in the absence of mercuric ion to be classified as constitutive activation (CA) mutants . In this case, oligonucleotide-directed randomization of the target region and a screening/selection protocol were employed . Sixteen different mutants with an RD phenotype were identified, as well as eight different mutants with a CA phenotype . A high frequency of S87C mutations is evident in the RD set of mutants . The CA mutants have a high incidence of S86C and A89V mutations . The CA double mutant S86C/A89V was purified and found to bind to its DNA site with an affinity similar to that of the wild-type protein . Chemical nuclease activity assays indicate that the nonmercurated S86C/A89V CA mutant has a DNA distortion activity identical to that of mercurated wild-type MerR . A unique disulfide bond bridging this CA mutant's dimer interface was found and is proposed to constrain protein conformation in a manner analogous to mercuric ion binding in the wild-type protein.

Proc Natl Acad Sci U S A, 1994 Apr 12, 91(8), 3443 - 7
C3G, a guanine nucleotide-releasing protein expressed ubiquitously, binds to the Src homology 3 domains of CRK and GRB2/ASH proteins; Tanaka S et al.; CRK protein, together with GRB2/ASH and Nck proteins, belongs to the adaptor-type Src homology (SH)2-containing molecules, which transduce signals from tyrosine kinases . Here another guanine nucleotide-releasing protein (GNRP), C3G, has been identified as a CRK SH3-binding protein . The nucleotide sequence of a 4.1-kb C3G cDNA contains a 3.2-kb open reading frame encoding a 121-kDa protein, and antibodies against C3G have been shown to detect a protein of 130-140 kDa . The carboxyl terminus of C3G has a peptide sequence homologous to GNRPs for Ras, and the expression of this carboxyl terminus region suppresses the loss of CDC25 function in the yeast Saccharomyces cerevisiae . The C3G protein expressed in Escherichia coli binds to CRK and GRB2/ASH proteins . Mutational analysis of C3G assigns the SH3 binding region to a 50-amino acid region containing a proline-rich sequence . The mRNAs of both the C3G and CRK proteins are expressed ubiquitously in human adult and fetal tissues . The results of these studies suggest that the complex of CRK and C3G, or GRB2/ASH and C3G, may transduce the signals from tyrosine kinases to Ras in a number of different tissues.

Am J Physiol, 1994 Apr, 266(4 Pt 2), H1451 - 6
Mediation of lung neutrophil uptake after endotoxin by CD18-integrin-dependent and -independent mechanisms; McCandless BK et al.; We studied polymorphonuclear neutrophil (PMN) uptake in lungs of endotoxemic rabbits using 111In-labeled PMN and isotope imaging by gamma scintigraphy . Rabbits were challenged intravenously with 100 micrograms Escherichia coli endotoxin either 4 or 24 h before an intravenous injection of 111In-labeled PMN, which was obtained from donor rabbits . The contribution of CD18 glycoprotein (beta 2-integrin) on PMN was examined using an anti-CD18 monoclonal antibody (MAb) IB4 infused 20 min before 111In-labeled PMN injection . In control rabbits, 111In-labeled PMN uptake in lungs was maximal within 5 min {36 +/- 2% increase above baseline (+/- SE)} and then fell exponentially with a disappearance half-time (t1/2) of 10 +/- 2 min . In rabbits challenged with endotoxin for either 4 or 24 h, maximum 111In-labeled PMN lung uptake and t1/2 values increased to 52 +/- 3 and 56 +/- 3% and to 26 +/- 2 and 31 +/- 6 min, respectively . Pretreatment with MAb IB4 (0.5 mg/kg iv) did not alter the PMN uptake response and t1/2 values in the 4-h endotoxin-challenged rabbits (i.e., maximum uptake of 52 +/- 3% above baseline and t1/2 of 26 +/- 2 min), whereas MAb IB4 prevented the increases in lung PMN uptake and t1/2 in 24-h endotoxin-challenged rabbits (maximum PMN uptake of 26 +/- 5% and t1/2 of 7 +/- 3 min; P < 0.001) . In contrast, the control MAb OKM-1 did not prevent lung PMN uptake and the disappearance of PMN from lungs at either times.(ABSTRACT TRUNCATED AT 250 WORDS)

Nucleic Acids Res, 1994 Apr 11, 22(7), 1179 - 85
Multimerization of the mouse TATA-binding protein (TBP) driven by its C-terminal conserved domain; Kato K et al.; The conformational states of the mouse TATA-binding protein (TBP) in solution were studied . A histidine tag and a factor Xa recognition site-carrying mouse TBP was expressed in E . coli, highly purified, and its fundamental functions as a TBP were demonstrated . We analyzed the molecular states of mouse TBP by gel filtration and glycerol gradient sedimentation, and found that TBP forms heterogeneous multimers in solution . Direct binding of TBP molecules to each other was proven by the far-Western procedure . Analyses using TBPs truncated at the N- and C-termini demonstrated that the functionally important C-terminal domain was responsible for homomultimer formation, and the N-terminal domain enhances multimerization . Furthermore, it was found that the TATA sequence dissociates homomultimers, and only monomeric TBP binds to the TATA-box . We suggest that TBP shares structural motifs in the C-terminal conserved domain for intermolecular interaction and TATA-binding.

Nucleic Acids Res, 1994 Apr 11, 22(7), 1155 - 60
Editing function of Escherichia coli cysteinyl-tRNA synthetase: cyclization of cysteine to cysteine thiolactone; Jakubowski H; A cyclic sulfur compound, identified as cysteine thiolactone by several chemical and enzymatic tests, is formed from cysteine during in vitro tRNA(Cys) aminoacylation catalyzed by Escherichia coli cysteinyl-tRNA synthetase . The mechanism of cysteine thiolactone formation involves enzymatic deacylation of Cys-tRNA(Cys) (k = 0.017 s-1) in which nucleophilic sulfur of the side chain of cysteine in Cys-tRNA(Cys) attacks its carboxyl carbon to yield cysteine thiolactone . Nonenzymatic deacylation of Cys-tRNA(Cys) (k = 0.0006 s-1) yields cysteine, as expected . Inhibition of enzymatic deacylation of Cys-tRNA(Cys) by cysteine and Cys-AMP, but not by ATP, indicates that both synthesis of Cys-tRNA(Cys) and cyclization of cysteine to the thiolactone occur in a single active site of the enzyme . The cyclization of cysteine is mechanistically similar to the editing reactions of methionyl-tRNA synthetase . However, in contrast to methionyl-tRNA synthetase which needs the editing function to reject misactivated homocysteine, cysteinyl-tRNA synthetase is highly selective and is not faced with a problem in rejecting noncognate amino acids . Despite this, the present day cysteinyl-tRNA synthetase, like methionyl-tRNA synthetase, still retains an editing activity toward the cognate product, the charged tRNA . This function may be a remnant of a chemistry used by an ancestral cysteinyl-tRNA synthetase.

FEBS Lett, 1994 Apr 11, 342(3), 329 - 33
Molecular cloning of the S-adenosylmethionine synthetase gene in Drosophila melanogaster; Larsson J et al.; We have isolated and sequenced cDNA clones encoding the Drosophila melanogaster S-adenosylmethionine synthetase . The deduced amino acid sequence contains 405 amino acid residues and shows high homology to rat, yeast, Arabidopsis and Escherichia coli counterparts . The gene is transcribed throughout Drosophila development but its main activity is seen in adult males and females . The highest transcription activity is seen in female ovaries . The transcript has an unusually long 5'-untranslated region, which might be of importance for translational regulation.

Gene, 1994 Apr 8, 141(1), 47 - 52
Sequence and characterization of three genes within the hydrogenase gene cluster of Bradyrhizobium japonicum; Fu C et al.; A 2.0-kb DNA fragment downstream from the hydrogenase-encoding structural genes within the hydrogenase gene cluster of Bradyrhizobium japonicum was sequenced . Analysis of the nucleotide (nt) sequence revealed three open reading frames (ORFs), designated hupC, hupD and hupF, which encode polypeptides of 28, 21 and 10.7 kDa, respectively . Based on analysis of the nt sequence and physiological studies, hupSL (hydrogenase structural genes) and hupCDF are organized as a single transcriptional unit . Plasmid pRY12 carrying hupSL genes did not complement (restore) hydrogenase activity of the hupSL deletion mutant strain (JHCS2), whereas the activity of the mutant was considerably restored by pLD22 harboring the entire hydrogenase operon (hupSLCDF genes) . Western blots revealed a very low level of hydrogenase protein in JHCS2 containing pRY12 . The results suggest that the products of the hupCDF genes may be involved in either stabilizing the hydrogenase peptides (i.e., from degradation) or in post-translational regulation of hydrogenase production . The products of hupC and hupD were successfully expressed in Escherichia coli by a phage T7 promoter system, although the apparent sizes of the gene products were slightly larger than those calculated from the deduced amino-acid sequences.

Gene, 1994 Apr 8, 141(1), 31 - 7
Sequencing, targeted mutagenesis and expression of a recA gene required for the extreme radioresistance of Deinococcus radiodurans; Gutman PD et al.; Deinococcus radiodurans and other members of the same genus share extreme resistance to ionizing radiation and many other agents that damage DNA . A DNA damage-sensitive and natural transformation-deficient strain generated by chemical mutagenesis (strain rec30) was found to be defective in a gene that has extended homology with recA of Escherichia coli . Upon transformation with a chromosomal DNA fragment that contained this deinococcal recA gene from wild-type (wt) D . radiodurans both DNA damage resistance and full transformation competence were restored in the rec30 mutant . Targeted insertional mutagenesis of the deinococcal recA gene was used to construct a mutant isogenic with the wt . The insertional mutant was phenotypically indistinguishable from strain rec30, indicating that the recA defect alone was responsible for observed phenotypic alterations . For example, in the case of ionizing radiation, the D37 of the wt was about 1.75 Mrad, while the D37 of rec30 and the insertional mutant were both 25 krad, a 70-fold decrease . Evidence is presented that expression of the deinococcal recA gene in E . coli is lethal, suggesting that the mode of interaction of the deinococcal RecA protein with nucleic acids or other cellular proteins differs at least in part from RecA of E . coli.

Gene, 1994 Apr 8, 141(1), 25 - 30
Cold-sensitive phenotype of Escherichia coli cells harboring a plasmid carrying the kil gene of phage lambda brought under control of cI857 gene promoters; Sugino Y et al.; A plasmid (pI-14), containing part of the phage lambda control region (EcoRI-D fragment of lambda cI857) with a large deletion between genes rex and kil, confers a cold-sensitive (cs) phenotype on the host bacteria, whereas the parent plasmid (pMM200) without the deletion made the host bacteria (Escherichia coli strain DOO) high-temperature sensitive . This phenomenon could be explained on the basis of the sequence analysis of the deletion . Upon this deletion, the lambda kil gene, which was originally under the control of the pL promoter, was brought under the control of the lambda cI promoters, resulting in the reversal of the host cell response to temperature . This example shows that gene circuits showing diametrically opposite responses to environmental factors (in this case, temperature) can be constructed from the same elements when the effector gene (here, kil) is connected in different ways to the sensor gene (here, cI857) . This cold-dependent killing activity was also dependent on the crp+ state of the host.

Gene, 1994 Apr 8, 141(1), 121 - 4
A gene encoding mycinamicin III O-methyltransferase from Micromonospora griseorubida; Inouye M et al.; A DNA fragment of 42 kb encompassing one of the mycinamicin II (Mm)-resistance-encoding genes, myrB, from a Mm-producing strain, Micromonospora griseorubida, was cloned in Escherichia coli using the cosmid vector pJB8 . Nucleotide sequencing of the neighboring region of myrB and a computer-aided analysis of the sequence predicted the presence of an open reading frame (ORF) with 254 amino acids which showed great similarity to the macrocin O-methyltransferase (tylF gene product) in tylosin (Ty)-producing Streptomyces fradiae . When a 1.0-kb AluI fragment containing the complete ORF was fused to the lacZ promoter in the correct orientation and expressed in E . coli, a mycinamicin III (MIII) O-methyltransferase (MOMT) activity was detected only upon induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) . All these data indicate that this ORF codes for the structural gene of MOMT, and it is designated mycF.

J Mol Biol, 1994 Apr 8, 237(4), 378 - 87
Linking an easily detectable phenotype to the folding of a common structural motif . Selection of rare turn mutations that prevent the folding of Rop; Castagnoli L et al.; Rop is the simplest and most regular member of a family of proteins characterized by a bundle of four antiparallel helices . Rop is dimeric, each monomer being formed by two helices connected by a sharp bend . In this work we have extensively mutagenized three residues that form the connection between the two alpha-helices to ask whether the bend region contains any important folding information . The characterization of a collection of random mutants indicated that this structure is rather insensitive to amino acid substitutions and that most amino acids are tolerated in these positions by the Rop native structure . In order to identify the rare amino acid sequences that would prevent Rop from folding and/or dimerizing, we exploited the observation that Rop can functionally substitute the dimerization domain of the lambda repressor . In fact plasmids expressing a hybrid protein formed by the amino-terminal domain of the lambda repressor covalently linked to Rop, confer immunity to lambda infection on their hosts . We have shown that this property depends on the ability of the Rop moiety to fold and dimerize . The analysis of 380 Rop mutants containing random amino acid sequences at positions 30, 31 and 32 allowed us to identify three mutant Rop proteins that are defective in dimerization, probably as a consequence of their inability to fold . In these mutants the tripeptides VED, VPD and YPD substitute the wild-type DAD at positions 30, 31 and 32 . Other combinations of amino acids are found resulting in levels of immunity that are lower than the wild-type but still sufficient to prevent single plaque formation . This result suggests that a smaller proportion of the corresponding Rop protein reaches a thermodynamic and proteolytically stable dimeric state.

J Mol Biol, 1994 Apr 8, 237(4), 368 - 77
Specialized ribosomes allow for the study of mutations in functionally important regions in 16 S rRNA, without affecting cell growth . The identification of functional regions in the central domain of 16S rRNA; Brink MF et al.; In order to identify the sequences in the central domain of 16 S rRNA of Escherichia coli that are important for ribosome function, we have generated random mutations using a PCR-based mutagenesis technique . We show that the effects of such mutations on ribosomal activity can be analyzed in vivo utilizing the specialized ribosome system . With this system the effect of rRNA mutations on ribosomal activity can be studied by measuring the translation of a modified CAT-mRNA by specialized ribosomes . Specialized ribosomes do not translate the endogenous mRNAs and, therefore, are expected to constitute a non-essential pool of ribosomes within the cell . In total, we have isolated 28 different clones harboring specialized ribosomes with single or multiple point mutations . We demonstrate that for none of these clones was cell growth retarded, even though some of the mutations severely impaired the activity of the specialized ribosomes, to as low as 3% of the wild-type level . For all mutants, their individual activities ranged between 3% and 100% of that of the wild-type activity . Comparison of several mutants indicates that mutations within the hairpin loops 787-795 and 898-901 strongly reduce the ribosomal activity . We also present evidence that the single-stranded region centered around residue A815 may be involved in maintaining translational accuracy.

Science, 1994 Apr 8, 264(5156), 265 - 7
Mutational isolation of a sieve for editing in a transfer RNA synthetase; Schmidt E et al.; Editing reactions are essential for the high fidelity of information transfer in processes such as replication, RNA splicing, and protein synthesis . The accuracy of interpretation of the genetic code is enhanced by the editing reactions of aminoacyl transfer RNA (tRNA) synthetases, whereby amino acids are prevented from being attached to the wrong tRNAs . Amino acid discrimination is achieved through sieves that may overlap with or coincide with the amino acid binding site . With the class I Escherichia coli isoleucine tRNA synthetase, which activates isoleucine and occasionally misactivates valine, as an example, a rationally chosen mutant enzyme was constructed that lacks entirely its normal strong ability to distinguish valine from isoleucine by the initial amino acid recognition sieve . The misactivated valine, however, is still eliminated by hydrolytic editing reactions . These data suggest that there is a distinct sieve for editing that is functionally independent of the amino acid binding site.

Science, 1994 Apr 8, 264(5156), 258 - 60
Recombination in adaptive mutation; Harris RS et al.; The genetic requirements for adaptive mutation in Escherichia coli parallel those for homologous recombination in the RecBCD pathway . Recombination-deficient recA and recB null mutant strains are deficient in adaptive reversion . A hyper-recombinagenic recD strain is hypermutable, and its hypermutation depends on functional recA and recB genes . Genes of subsidiary recombination systems are not required . These results indicate that the molecular mechanism by which adaptive mutation occurs includes recombination . No such association is seen for spontaneous mutation in growing cells.

J Biol Chem, 1994 Apr 8, 269(14), 10883 - 90
The crystal structure of succinyl-CoA synthetase from Escherichia coli at 2.5-A resolution; Wolodko WT et al.; The x-ray crystal structure of succinyl-CoA synthetase (SCS) from Escherichia coli has been determined by the method of multiple isomorphous replacement to a resolution of 2.5 A . Crystals of SCS are tetragonal with a space group of P4(3)22 and unit cell dimensions of a = b = 98.47 A and c = 400.6 A . One molecule of SCS (142 kDa) is contained in the asymmetric unit . The current model has been refined to a conventional R factor of 21.6% with root mean square deviations from ideal stereochemistry of 0.022 A for bond lengths and 3.25 degrees for bond angles . The quaternary organization of the E . coli enzyme is an alpha 2 beta 2 heterotetramer . In this tetramer, the alpha-subunits interact only with the beta-subunits, whereas the beta-subunits interact to form the dimer of alpha beta-dimers . The two active site pockets are located at regions of contact between alpha- and beta-subunits . One molecule of coenzyme A is bound to each alpha-subunit at a typical nucleotide-binding motif, and His-246 of each alpha-subunit is phosphorylated . This phosphohistidine, a catalytic intermediate, is stabilized by two helix dipoles (the "power" helices), one from each of the two subunit types . A short segment of the beta-subunit from one alpha beta-dimer is in close proximity to the CoA-binding site of the other alpha beta-dimer, providing a possible rationale for the overall tetrameric structure.

J Biol Chem, 1994 Apr 8, 269(14), 10771 - 5
A mutational study of the C-terminal zinc-finger motif of the Escherichia coli UvrA protein; Wang J et al.; The cysteine 763 residue in the C-terminal zinc-finger region of Escherichia coli UvrA protein was subjected to random mutagenesis, and the results suggested that the UvrA mutants with a small amino acid (Ser, Ala, or Gly) substituting for the cysteine 763 were almost as active as the wild-type in supporting nucleotide excision repair, but its replacement with a large, bulky amino acid (Tyr, Trp, or Phe) rendered the mutants inactive . The C763F mutant UvrA protein was purified for further characterization, and it was found this mutant UvrA protein lost its DNA binding (single-stranded or double-stranded DNA) activity and those other activities dependent on DNA binding, such as formation of damage-specific UvrA2B complexes and the supercoiling reaction . However, this mutant protein retained vigorous ATPase activity and was capable of negatively complementing the wild-type UvrA in JM109 strain . The purified C763F mutant UvrA protein contains a single zinc ion/molecule, half that of the wild-type . It appears that the C763F mutation destabilizes the zinc-anchored structure in the C-terminal zinc finger region, and as a result, the C763F mutant UvrA protein lost its ability to bind DNA.

J Biol Chem, 1994 Apr 8, 269(14), 10713 - 9
High affinity interaction of dipteran high mobility group (HMG) proteins 1 with DNA is modulated by COOH-terminal regions flanking the HMG box domain; Wisniewski JR et al.; The cells of the dipteran insects Chironomus and Drosophila contain high mobility group (HMG) 1 proteins that are homologous to the HMG1 protein of mammals but comprise one instead of two DNA-binding HMG boxes . Mobility shift assays have revealed that Chironomus cHMG1a and cHMG1b bind double strand and four-way junction DNA in a similar way at apparent dissociation constants in the range of 7.5-20 x 10(-9) M . Both proteins are monomeric and highly asymmetric molecules in solution . cHMG1a and cHMG1b exhibit Stokes' radii of 2.4 and 2.3 nm, respectively, and both show a frictional ratio of 1.5 . Despite these similarities in their hydrodynamic properties, the binding site of cHMG1a on DNA is approximately 1.5 of the size found for the cHMG1b . Enzymatically and chemically prepared peptides of cHMG1a as well as bacterially expressed cHMG1a with terminal deletions and point substitutions showed that sequences flanking the folded domain that constitutes the HMG box are essential for the interaction of the HMG box with DNA . In particular, changes in the number of positive and negative charges, respectively, within basic and acidic domains modulated the DNA binding affinity of the cHMG1a protein . The alteration of fluorescence of the Trp residues suggest that this modulation is due to interaction of the acidic domain with the positively charged HMG box.

J Biol Chem, 1994 Apr 8, 269(14), 10660 - 7
GATA-type zinc finger motif-containing sequences and chorion gene transcription factors of the silkworm Bombyx mori; Drevet JR et al.; To characterize a DNA-binding protein, BCFI, which regulates the expression of silkmoth chorion genes through binding to gene promoter elements identical to those recognized by the GATA family of transcription factors, we have carried out polymerase chain reaction amplifications of Bombyx mori genomic DNA using degenerate primers derived from the conserved DNA binding domain of mammalian GATA factors . Two single copy genes, BmGATA alpha and BmGATA beta, were identified, which encode sequences containing GATA-type zinc finger motifs . The BmGATA beta gene is expressed in follicular and Bm5 tissue culture cells, the two cell types that contain BCFI . No BmGATA alpha gene transcripts were detectable in the tissues that were tested . Upon overexpression in Escherichia coli, a peptide encompassing the BmGATA beta zinc finger motif was able to bind specifically to the BCFI recognition motif of the chorion gene promoters . A polyclonal antibody directed against the zinc finger domain of BmGATA beta was also used in gel retardation assays to confirm that factor BCFI is indeed encoded by the BmGATA beta gene . Conceptual translation of a complete cDNA clone encoding the BmGATA beta protein revealed that this protein has a size similar to that of an immunoreactive protein, presumably BCFI, which is present in follicular cell extracts.

J Biol Chem, 1994 Apr 8, 269(14), 10461 - 6
Functional alpha-tropomyosin produced in Escherichia coli . A dipeptide extension can substitute the amino-terminal acetyl group; Monteiro PB et al.; Unlike the muscle protein, alpha-tropomyosin expressed in Escherichia coli does not bind actin, does not exhibit head-to-tail polymerization, and does not inhibit actomyosin ATPase activity in the absence of troponin . The only chemical difference between recombinant and muscle tropomyosins is that the first methionine is not acetylated in the recombinant protein (Hitchcock-De-Gregori, S.E., and Heald, R . W . (1987) J . Biol . Chem . 262, 9730-9735) . We expressed three fusion tropomyosins in E . coli with 2, 3, and 17 amino acids fused to its amino terminus . All three fusions restored actin binding, head-to-tail polymerization, and the capacity to inhibit the actomyosin ATPase to these unacetylated tropomyosins . Unlike larger fusions, the small fusions of 2 and 3 amino acids do not interfere with regulatory function . Therefore the presence of a fused dipeptide at the amino terminus of unacetylated tropomyosin is sufficient to replace the function of the N-acetyl group present in muscle tropomyosin . A structural interpretation for the function of the acetyl group, based on our results and the coiled coil structure of tropomyosin, is presented.

J Biol Chem, 1994 Apr 8, 269(14), 10370 - 7
Mutational analysis of the traffic ATPase (ABC) transporters involved in uptake of eye pigment precursors in Drosophila melanogaster . Implications for structure-function relationships; Ewart GD et al.; The white, brown, and scarlet genes of Drosophila melanogaster encode three proteins that belong to the Traffic ATPase superfamily of transmembrane permeases and are involved in the transport of guanine and tryptophan (precursors of the red and brown eye pigments) . We have determined the nucleotide sequences of two mutant white alleles (wco2 and wBwx) that cause reduced red pigmentation but have no effect on brown pigmentation . In wco2 the effect is only observed when interacting with the bw6 allele or a newly isolated allele (bwT50) . These alleles of the brown gene were cloned and sequenced . In wco2 the codon for glycine 588 is changed to encode serine; in wBwx the triplet ATC encoding isoleucine 581 is deleted; asparagine 638 is changed to threonine in bw6, and glycine 578 is changed to aspartate in bwT50 . No other relevant changes to the gene structures were detected . P-element-mediated germline transduction was used to construct a fly strain containing a white gene with a mutation of the nucleotide binding domain . Such flies had white eyes, indicating that the mutated white gene was unable to support either guanine or tryptophan transport . The implications of these mutations are discussed in terms of a model of the Drosophila pigment precursor transport system.

J Biol Chem, 1994 Apr 8, 269(14), 10341 - 51
Identification of a novel determinant for basic domain-leucine zipper DNA binding activity in the acute-phase inducible nuclear factor-interleukin-6 transcription factor; Brasier AR et al.; Nuclear factor-interleukin-6 (NF-IL6), a member of the CCAAT box/enhancer-binding protein (C/EBP) family, contains a basic domain-leucine zipper (bZIP) DNA binding motif . Controlled protease digestion was used to probe free and DNA-complexed NF-IL6 protein . Digestion with trypsin in the absence of DNA produced the leucine zipper domain (containing residues 303-345) . In contrast, digestion of NF-IL6.DNA complexes produced a stable domain, spanning residues 266-345, termed the tryptic core domain (TCD) . The NH2-terminal boundary of the TCD is longer than tryptic peptides reported from C/EBP alpha.DNA complexes . Digestion of NF-IL6 with endoprotease Asp-N produced a domain smaller than the TCD (NF-IL6 bZIP domains (NFBD) (272-345)), a domain identified either in the absence or the presence of DNA . Both recombinant peptides bind acute-phase response element DNA in a sequence-specific fashion . The equilibrium disassociation constant (Kd) for the TCD was 36 +/- 8 nM, whereas the Kd for NFBD (272-345) was 283 +/- 160 nM . Moreover, in comparison with the TCD, NFBD (272-345) formed unstable DNA complexes with a 15-fold faster off-rate . We conclude that the amino acids represented between 266 and 272 termed the complex stabilizing subdomain, influences DNA complex formation independent of DNA binding specificity, and may be one mechanism for heterogeneity of DNA interaction by C/EBP family members.

J Biol Chem, 1994 Apr 8, 269(14), 10265 - 9
The alpha/beta subunit interaction in H(+)-ATPase (ATP synthase) . An Escherichia coli alpha subunit mutation (Arg-alpha 296-->Cys) restores coupling efficiency to the deleterious beta subunit mutant (Ser-beta 174-->Phe); Omote H et al.; The Ser-beta 174 residue of the Escherichia coli H(+)-ATPase beta subunit has been shown to be near the catalytic site together with Gly-beta 149, Gly-beta 172, Glu-beta 192, and Val-beta 198 (Iwamoto, A., Park, M.-Y., Maeda, M., and Futai, M . (1993) J . Biol . Chem . 268, 3156-3160) . In this study, we introduced various residues at position 174 and found that the larger the side chain volume of the residue introduced, the lower the enzyme activity became . The Phe-beta 174 mutant was defective in energy coupling between catalysis and transport, whereas the Leu-beta 174 mutant could couple efficiently, although both mutants had essentially the same ATPase activities (approximately 10% of the wild type) . The defective energy coupling of the Phe-beta 174 mutant was suppressed by the second mutation (Arg-alpha 296-->Cys) in the alpha subunit . The Cys-alpha 296/Phe-beta 174 mutant had essentially the same membrane ATPase activity as the Phe-beta 174 single mutant when assayed under the conditions that stabilize the double mutant enzyme . These results indicate the importance of the alpha/beta interaction, especially that between the regions near Arg-alpha 296 and Ser-beta 174, for energy coupling in the H(+)-ATPase . The 2 residues (Ser-beta 174 and Arg-alpha 296) may be located nearby at the interface of the two subunits . About 1 mol of N-{14C}ethylmaleimide could bind to 1 mol of the alpha subunit of Cys-alpha 296/Phe-beta 174 or Cys-alpha 296 mutant ATPase, but could not inhibit the enzyme activity . This is the first intersubunit mutation/suppression approach to ATPase catalysis and its energy coupling.

J Biol Chem, 1994 Apr 8, 269(14), 10225 - 8
DNA polymerase alpha overcomes an error-prone pause site in the presence of replication protein-A; Suzuki M et al.; Eukaryotic DNA polymerase alpha pauses at some sites on the natural DNA template of M13mp2 . Terminal misincorporations of dA or dG, in place of dT, by DNA polymerase alpha have been reported to be within one of the pause sites, pause site II (positions 6269 and 6270 (Fry, M., and Loeb, L.A . (1992) Proc . Natl . Acad . Sci . U.S.A . 89, 763-767)) . The DNA products arrested within pause site II (position 6270) were separated, annealed with synthetic templates, and further elongated by DNA polymerase alpha . It was confirmed that a considerable amount of terminal misincorporation of dG in place of dT occurred at this position . When M13mp2 DNA was coated with various amounts of replication protein-A (RP-A), however, DNA polymerase alpha was able to overcome the pause site II, whereas pause bands at other sites barely decreased . In contrast, Escherichia coli single-stranded DNA-binding protein did not specifically abolish the arrested band at pause site II, though it generally suppressed the reaction . Since RP-A hardly increased the elongation frequency from the primer carrying a 3'-mismatched terminal deoxynucleotide, the reduction of arrested products by RP-A may be attributed to the change in the incorporation mode from noncomplementary to complementary deoxynucleotides within pause site II and may not be due to the reinitiation from the mismatched 3'-terminal deoxynucleotide . To confirm this, we amplified the reaction products at pause site III by means of a polymerase chain reaction method and showed that the complementary strand to pause site II, which was elongated in the presence of RP-A, did not carry any detectable misinsertion . Therefore, the errorprone step of the DNA synthesis catalyzed by DNA polymerase alpha may be readily avoided by RP-A.

J Biol Chem, 1994 Apr 8, 269(14), 10221 - 4
Suppressor mutations in F1 subunit epsilon recouple ATP-driven H+ translocation in uncoupled Q42E subunit c mutant of Escherichia coli F1F0 ATP synthase; Zhang Y et al.; The Q42E mutation in the polar loop of subunit c of the Escherichia coli F1F0 ATP synthase leads to an uncoupling of H+ translocation through F0 and ATP synthesis/hydrolysis in F1 . We have isolated four second-site suppressor mutants in which the coupling defect is corrected . Substitutions for Glu31 in F1 subunit epsilon were found in each suppressor mutant, where the substitutions were E31G, E31V, and E31K (the last being found twice) . The different substitutions vary in effectiveness in restoring wild type growth properties in the order epsilon E31G > epsilon E31V > epsilon E31K . Biochemical properties of epsilon E31G/cQ42E and epsilon E31K/cQ42E membranes were compared . In epsilon E31G/cQ42E mutant membranes, ATP-driven H+ translocation by F1F0 and the binding and coupling of F1 to F0 showed a striking pH dependence . Near normal function was observed at pH 7.0, but function was lost at pH 7.8 . The function of epsilon E31K/cQ42E membranes was much less affected by changes in pH . Relative to epsilon E31G/cQ42E membranes, the ATP-driven H+ transport function of epsilon E31K/cQ42E membranes was approximately the same at pH 7.5, greater at pH 7.8, and less at pH 7.0 . The differences between mutants could be explained if cGlu42 ionized at pH 7.8 with loss of function in epsilon E31G/cQ42E membrane and a similar ionization were compensated for by the positively charged Lys in the epsilon E31K/cQ42E membrane.

J Biol Chem, 1994 Apr 8, 269(14), 10201 - 4
Cloning and characterization of cDNA encoding rat hemin-sensitive initiation factor-2 alpha (eIF-2 alpha) kinase . Evidence for multitissue expression; Mellor H et al.; Reticulocytes contain an eukaryotic initiation factor-2 alpha (eIF-2 alpha) kinase that is negatively regulated by hemin . This protein kinase, which has been termed heme-regulated inhibitor and heme-controlled repressor (HCR), has a strong inhibitory effect on the initiation of protein synthesis and plays an important role in translational control in these cells . Previous evidence has suggested that HCR may be an erythroid-specific enzyme . As reported herein, we have cloned and characterized the cDNA encoding an eIF-2 alpha kinase from rat brain . The predicted amino acid sequence of the kinase from rat brain shows 82% homology to rabbit reticulocyte HCR with the greatest variation concentrated in a central region of approximately 135 amino acids located between protein kinase subdomains IV and VI . We have expressed the rat brain cDNA in Escherichia coli and have demonstrated that the recombinant enzyme is a hemin-sensitive eIF-2 alpha kinase . We have also shown that mRNA recognized by the rat brain cDNA is expressed in a wide range of non-erythroid rat tissues at a lower but significant level compared with reticulocytes . These findings raise the possibility of additional, uncharacterized roles for HCR in non-erythroid cells.

Gene, 1994 Apr 8, 141(1), 17 - 23
IHF supresses the inhibitory effect of H-NS on HU function in the hin inversion system; Goshima N et al.; In the hin-mediated DNA inversion system, HU facilitates formation of the synaptic complex composed of two recombination sites spaced 996 bp apart and of the enhancer situated between them, by looping the DNA as to promote interaction of Hin invertase with the Fis enhancer factor {Johnson et al., Nature 329 (1987) 462-465} . The HU requirement for the in vivo hin-mediated inversion was demonstrated previously {Wada et al., Gene 76 (1989) 345-352; Hillyard et al., J . Bacteriol . 172 (1990) 5402-5407; Haykinson and Johnson, EMBO J . 12 (1993) 2503-2512} and in the current experiments . This HU action, however, required IHF when H-NS was present in the cell; i.e., the inversion reaction of the hin-invertible DNA fragment carried by the pKK1202R plasmid proceeded efficiently in host cells either deficient in H-NS or in the presence of both H-NS and IHF, but not in the cells depleted for IHF alone . The level of hin mRNA in mutant cells lacking HU or IHF, in which hin inversion did not occur, was normal or slightly increased . When IHF was absent, the stimulating effect of HU on in vitro DNA circle formation of a 125-bp hin fragment between hixL and the enhancer where Fis binds was inhibited by H-NS . The present study provides an example of a multi-component interaction between HU, H-NS and IHF on the hin DNA region, which contains three characteristic sites, a d(A/T)4 stretch and bent DNA site, and two putative IHF-binding sites.

J Biol Chem, 1994 Apr 8, 269(14), 10797 - 803
Effects of nucleotide sequence on the specificity of rne-dependent and RNase E-mediated cleavages of RNA I encoded by the pBR322 plasmid; Lin-Chao S et al.; RNase E, an endoribonuclease encoded by the Escherichia coli ams/rne/hmp1 locus, cleaves RNA I, an antisense regulator of the replication of ColE1 type plasmids, in a single-stranded region near its 5' end . The rne-3071 mutation prolongs the RNA 1 half-life in cells cultured at an elevated temperature and imparts temperature sensitivity on RNase E isolated from the mutant strain . Here we report the effects of specific sequence changes introduced by site-directed mutagenesis on the location of ribonucleolytic cleavage near the 5' end of pBR322 RNA I in rne-3071 and congenic rne+ E . coli and on cleavage of RNA I by RNase E in vitro . Primer extension analyses showed that the occurrence and position of cleavages in vivo and in vitro are altered highly specifically by sequence changes but that the site of cleavage bears no simple relationship to a particular nucleotide order . Our results do not support either the notion that cleavage by RNase E is determined by a consensus sequence or the contrary view that RNase E is a virtually nonspecific single-stranded endonuclease with a preference for cutting 5' to an AU dinucleotide.

J Biol Chem, 1994 Apr 8, 269(14), 10790 - 6
A+U content rather than a particular nucleotide order determines the specificity of RNase E cleavage; McDowall KJ et al.; Ribonuclease E has a central role in Escherichia coli mRNA decay and is dependent on a functional product of the rne (also called ams or hmp1) gene . We investigated the requirements for RNase E cleavage by introducing random mutations into the decanucleotide region at the 5' end of pACYC184 RNA I and studying the effects of these mutations on the position of rne-dependent cleavage in vivo and RNase E-mediated cutting in vitro . We find that the precise point of RNase E cleavage can be altered specifically and reproducibly by sequence changes in the region cleaved and, therefore, is not determined by a distance measured in nucleotides from any other sequence or region of secondary structure in RNA I . Although cleavage by RNase E occurs within sequences rich in A and/or U nucleotides and is affected by the extent of continuity of A and U nucleotides in the regions cleaved, there is no simple relationship between the order of nucleotides and the phosphodiester bond cleaved . Thus, our results are not consistent with either the notion that RNase E cleavages are determined by a simple consensus sequence or the contrary view that RNase E has few primary structural constraints other than a preference for cleaving 5' to an AU dinucleotide.

Nature, 1994 Apr 7, 368(6471), 520 - 5
Promoter targeting by adenovirus E1a through interaction with different cellular DNA-binding domains; Liu F et al.; A puzzling property of the transcriptional activators encoded by several animal viruses is their ability to function promiscuously . The adenovirus E1a protein, for example, stimulates transcription of adenoviral genes as well as a wide variety of other viral and cellular genes . We show that E1a can interact with several classes of cellular DNA-binding domains and thereby be recruited to diverse promoters . Our results explain how a single protein can regulate transcription of multiple genes that lack a common promoter element.

Biochemistry, 1994 Apr 5, 33(13), 3980 - 5
Dynamics of lactose permease of Escherichia coli determined by site-directed fluorescence labeling; Jung K et al.; Recently we described the use of site-directed pyrene labeling of engineered lactose permease containing paired Cys residues to obtain proximity relationships between helices in the C-terminal half of the molecule {Jung, K., Jung, H., Wu, J., Prive, G . G., & Kaback, H.R . (1993) Biochemistry 32, 12273} . Pyrene excimer fluorescence was detected for the double Cys mutants His322-->Cys/Glu325-->Cys, Arg302-->Cys/Glu325-->Cys, and Glu269-->Cys/His322-->Cys, indicating that helix X (His322-->Cys/Glu325-->Cys) is in an alpha-helical conformation and that helices VIII (Glu269-->Cys) and IX (Arg302-->Cys) are close to helix X (His322-->Cys and Glu325-->Cys) . In this report, these interactions are used to study dynamic aspects of the permease . Excimer fluorescence between helices VIII and X or helices IX and X is markedly diminished by sodium dodecyl sulfate, while the excimer observed within helix X is unaffected, suggesting that tertiary interactions are disrupted by the denaturant with little effect on secondary structure . Furthermore, excimer fluorescence observed between helices VIII (Glu269-->Cys) and helix X (His322-->Cys) is quenched by Tl+, and the effect is markedly and specifically attenuated by ligands of the permease, suggesting that the pyrene becomes less accessible to the aqueous phase . The reactivity of single Cys residues at positions 269 or 322 was also examined by studying the rate of increase in fluorescence with N-(l-pyrenyl)maleimide . With both mutants, ligands of the permease cause a dramatic increase in reactivity which is consistent with the notion that these positions are transferred into a more hydrophobic environment.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Apr 5, 33(13), 3949 - 58
Ecotin is a potent anticoagulant and reversible tight-binding inhibitor of factor Xa; Seymour JL et al.; Ecotin, a serine protease inhibitor found in the periplasm of Escherichia coli, has been characterized as an extremely potent anticoagulant and reversible tight-binding inhibitor of human factor Xa (FXa) . The ecotin gene was cloned by PCR, highly expressed in E . coli, and purified from the E . coli periplasm . The binding of ecotin to FXa was stoichiometric with an equilibrium dissociation constant Ki of 54 pM . The association rate constant was 1.35 x 10(6) M-1 s-1, and the dissociation rate constant, measured in the presence of human leukocyte elastase (HLE) to prevent reassociation of ecotin with FXa, was 6.5 x 10(-5) s-1 . Ecotin prolonged clotting time ca . 10-fold at 0.3 microM and at 2 microM in activated partial thromboplastin time and prothrombin time assays, respectively . Ecotin did not effectively inhibit the human plasma proteases thrombin, tissue factor.factor VIIa, factor XIa, activated protein C, plasmin, or tissue plasminogen activator (t-PA); however, it did potently inhibit factor XIIa, plasma kallikrein, HLE, and bovine trypsin and chymotrypsin . Coincubation of ecotin and FXa at 10 microM each resulted in a (ecotin)2.(FXa)2 complex as determined by gel filtration . Dimerization of ecotin alone was measured by fluorescence titration which yielded a Kd of ca . 390 nM . FXa cleaved ecotin slowly at pH 4.0 between M84 and M85 . Replacement of the P1 Met84 residue with Arg and Lys led to FXa inhibitors with Ki values of 11 and 21 pM, respectively . The P1 Arg and Lys mutants also significantly inhibited thrombin, factor XIa, activated protein C, plasmin, factor XIIa, kallikrein, and bovine trypsin and chymotrypsin but did not inhibit tissue factor.factor VIIa, t-PA, or HLE.

Biochemistry, 1994 Apr 5, 33(13), 3913 - 8
Involvement of the gamma-phosphate of UTP in the synergistic inhibition of Escherichia coli aspartate transcarbamylase by CTP and UTP; England P et al.; The allosteric control of Escherichia coli aspartate transcarbamylase (ATCase) involves synergistic feedback inhibition by CTP and UTP . Previously reported results {England, P., & Herve, G . (1992) Biochemistry 31, 9725-9732} suggest that this phenomenon relies entirely on interactions between the two neighboring allosteric sites, which belong to the same regulatory dimer . Furthermore, it has been demonstrated that UTP alone binds to the enzyme, but that it is only in the presence of CTP that this binding inhibits the catalytic activity . The properties of mutants in which the synergistic inhibition is totally abolished suggested that the terminal gamma-phosphate of the pyrimidine triphosphate nucleotides may play a crucial role in promoting site-site interactions within the regulatory dimer . In the present work, kinetic studies and binding experiments by continuous-flow dialysis were performed, using combinations of diphosphate and triphosphate nucleotides . The results obtained show that the gamma-phosphate lf UTP is indeed essential for synergistic inhibition to occur, as UDP is unable to inhibit ATCase activity, whether alone or in combination with CTP . On the contrary, the gamma-phosphate of CTP can be suppressed without modifying the inhibitory properties of this nucleotide and its synergy of action with UTP . These results indicate that the mutual effects of CTP and UTP on their respective binding are not symmetrical and that the signals emitted upon binding of the two triphosphate pyrimidine nucleotides to the regulatory sites do not follow the same pathway and involve different mechanisms.

Biochemistry, 1994 Apr 5, 33(13), 3878 - 84
Cross-linking of mRNA analogues containing 4-thiouridine residues on the 3'- or 5'-side of the coding triplet to the mRNA binding center of the human ribosome; Graifer DM et al.; The interaction between mRNA and 18S rRNA within complexes of human placenta 80S ribosomes has been investigated by photochemical cross-linking experiments using mRNA analogues substituted with 4-thiouridine at specific locations . mRNA analogues 51 or 54 nucleotides long were prepared from synthetic DNA templates . These mRNA analogues contained either the sequence GGGACC (coding for glycine and threonine, respectively) or the single triplet GGG together with 2-4 4-thiouridine residues located at various positions with respect to the coding triplets . The products of cross-linking of the mRNA analogues to 18S rRNA within different model complexes without tRNA or in the presence of cognate tRNAs were analyzed by reverse transcription . Two cross-linking sites in the 18S rRNA were detected . The first site, U630, was cross-linked by mRNA 8' (s4U at +20, +22, +24, and +26), mRNA 9e' (s4U at -16, -18, and -20), and mRNA 10 (s4U at +4, +6, -1, and -3) but, unexpectedly, not with either mRNA 10b (s4U at +4 and +6) or mRNA 10c (s4U at -1 and -3) . The second site, U1111/A1112, was cross-linked by mRNA 10 and mRNA 10c but not by any of the other mRNA analogues . There is significant tRNA dependence on cross-linking only for mRNA analogue 9e' . Both of the sites detected correspond to sites of mRNA cross-linking in Escherichia coli 16S rRNA.

Biochemistry, 1994 Apr 5, 33(13), 3872 - 7
Site-directed mutagenesis of kappa-bungarotoxin: implications for neuronal receptor specificity; Fiordalisi JJ et al.; Postsynaptic polypeptide neurotoxins isolated from the venoms of elapid and hydrophid snakes exhibit the ability to bind selectively to and inhibit different types of receptors that function in nerve signal transmission . On the basis of their amino acid sequences and three-dimensional structures, these neurotoxins are clearly related, but nothing is yet known about the basis for their physiological receptor specificity . In this report, site-directed mutants of kappa-bungarotoxin, produced by an Escherichia coli expression system, are tested to determine the function of selected amino acid side chains in the interaction between toxin and receptor . Highly conserved residues at the bottom of the second loop (a region that has been shown to be a major point of contact with the receptor), particularly those residues at the junction between the beta-sheet and the end of the loop, were selected . The results demonstrate that a single amino acid substitution of the invariant arginine residue (Arg-40 to Ala-40) renders the toxin unable to inhibit nerve transmission in the chick ciliary ganglion up to a concentration of 10 microM . Significantly, the results also show that conversion to alanine of the nearby proline residue (Pro-42) found to be invariant in all kappa-neurotoxins, but not found in any potent alpha-neurotoxin, produces a toxin with full inhibitory capacity . However, the introduction of a lysine residue at this position (P-42-K), like that found in alpha-bungarotoxin, reduces activity significantly.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1994 Apr 5, 33(13), 3848 - 54
Hybridization of a complementary ribooligonucleotide to the transcription start site of the lacUV-5-Escherichia coli RNA polymerase open complex . Potential for gene-specific inactivation reagents; Perrin DM et al.; An ribooligonucleotide, UGGAA, complementary to the template strand of the lacUV-5 promoter can hybridize to the transcription "bubble" of the open complex formed by Escherichia coli RNA polymerase . Its site-specific binding, measured by gel retardation, enzyme inhibition, and chemical nuclease footprinting, is dependent on catalysis by RNA polymerase and the sequence of the hybridizing ribooligonucleotide . When UGGAA is linked to the chemical nuclease 1,10-phenanthroline copper, site-specific scission of the template strand of the transcriptionally active gene is observed . The formation of single-stranded DNA at transcription start sites by RNA polymerases provides a target for antigene strategies.

Biochemistry, 1994 Apr 5, 33(13), 3841 - 7
Xenopus laevis ovarian DNA helicase I: A 3' to 5' helicase that unwinds short duplexes; Poll EH et al.; A novel DNA helicase isolated from Xenopus laevis ovaries {Poll, E . H . A., & Benbow, R . M . (1988) Biochemistry 27, 8701-8706} was characterized biochemically . The directionality of DNA unwinding was determined to be 3' to 5' . A short 3' ssDNA tail adjacent to duplex DNA was required for DNA unwinding; the minimum length of this tail was between four and nine bases . Only short duplex DNA regions were unwound: duplex DNA of 16 base pairs was readily unwound, whereas a 26 base pair duplex was not . Longer duplex regions were unwound in the presence of Escherichia coli single-strand DNA binding protein if, in addition, the duplex region was flanked by an unpaired 3' or 5' tail and the substrate resembled a branched replicative intermediate . X . laevis DNA helicase I exhibited high affinity for ssDNA, moderate affinity for dsDNA, and no affinity for RNA . DNA unwinding activity was stimulated by monovalent cations, with an optimal concentration of 150 mM for NaCl or KCl or 125 mM for Na chi PO4 or K chi PO4 . The ATP analog ATP gamma S inhibited the DNA unwinding and copurifying DNA-dependent ATPase activity, whereas AMPPCP and AMPPNP moderately inhibited DNA unwinding activity and had little effect on the copurifying DNA-dependent ATPase activity . CTP was a relatively strong inhibitor of DNA unwinding activity, but GTP, UTP, dCTP, dGTP, or TTP showed moderate or no inhibition . The copurifying DNA-dependent ATPase activity was not inhibited by CTP, GTP, UTP, dCTP, dGTP, or TTP.

Eur J Pharmacol, 1994 Apr 4, 270(2-3), 143 - 9
Lipopolysaccharide-induced pleural neutrophil accumulation depends on marrow neutrophils and platelet-activating factor; Bozza PT et al.; The involvement of platelet-activating factor (PAF) in lipopolysaccharide (LPS)-induced leukocyte accumulation in the rat pleural cavity was investigated . Intrathoracic (i.t.) injection of LPS (250 ng/cavity) induced a marked increase in the number of neutrophils at 1 h, which was maximum within 6-12 h, reducing after 24 h . In parallel, an increase in blood neutrophil counts within 1-6 h, concomitantly with a reduction in the number of these cells in the bone marrow, was observed . The number of eosinophils recovered from LPS-injected pleural cavity increased at 12 h and was maximum within 24-48 h . No change in blood or bone marrow eosinophil counts was detected . The pretreatment with WEB 2086 or PCA 4248 (20 mg/kg) significantly inhibited pleural neutrophil accumulation, blood neutrophilia and the decrease in the marrow neutrophil content, but not eosinophil accumulation . The blood neutrophilia and the decrease in marrow neutrophil counts induced by the intravenous (i.v.) injection of LPS (250 ng) were significantly lower than those observed after i.t . injection . Furthermore, WEB 2086 and PCA 4248 were ineffective against the systemic alteration induced by i.v . LPS . it was concluded that LPS-induced neutrophil, but not eosinophil, accumulation in the pleural cavity is related to the mobilization of neutrophils from the bone marrow and involves PAF dependent mechanisms.

Proteins, 1994 Apr, 18(4), 394 - 403
Preliminary X-ray analysis of Escherichia coli GMP synthetase: determination of anomalous scattering factors for a cysteinyl mercury derivative; Tesmer JJ et al.; We have initiated a project to determine the three-dimensional structure of GMP synthetase (GMPS) from Escherichia coli . GMPS catalyzes the conversion of XMP to GMP in the final step of de novo guanine nucleotide biosynthesis, and is a member of the glutamine amidotransferase family: a group of enzymes responsible for the assimilation of nitrogen into compounds such as amino acids, purine and pyrimidine bases, amino sugars, and antibiotics . The E . coli guaA gene encoding GMPS was cloned into a tac expression vector, overexpressed, and its gene product purified . Conditions for the growth of protein crystals were developed using recombinant GMPS in the presence of MgCl2, ATP, and XMP . The crystals are monoclinic, space group P2(1), with cell parameters of a = 156.0 A, b = 102.0 A, c = 78.8 A, beta = 96.7 degrees . Diffraction data to 2.8 A spacings were collected on a Xuong-Hamlin area detector with an overall Rsym of 5.2% . Both the volume of the unit cell and the peaks in the self-rotation function are consistent with one GMPS tetramer of D2 symmetry in the crystallographic asymmetric unit . Previously, GMPS has been observed only as a dimer in solution . GMPS was covalently modified with p-chloromercuribenzylsulfonic acid (PCMBS), and its X-ray fluorescence spectrum was measured through the LIII absorption edge of mercury . Anomalous scattering factors for cysteinyl mercury were derived from this spectrum, and the feasibility of structure determination by multi-wavelength anomalous diffraction was evaluated . The optimal MAD dispersive signal is 4.5% of magnitude of F, and the optimal MAD Bijvoet signal is 7.5% of magnitude of F at a concentration of approximately 1 mercury per 10-kDa protein . The anomalous scattering factors tabulated here should be transferable to cysteinyl mercury in other proteins.

Plant Cell, 1994 Apr, 6(4), 501 - 10
Phosphorylation and calcium binding properties of an Arabidopsis GF14 brain protein homolog; Lu G et al.; Arabidopsis GF14 omega was originally described because of its apparent association with a DNA-protein complex; it is a member of the 14-3-3 kinase regulatory protein family that is conserved throughout eukaryotes . Here, we demonstrated that recombinant GF14 omega is expressed in Escherichia coli as a dimer . Blot binding and electrophoretic mobility shift analyses indicated that GF14 omega binds calcium . Equilibrium dialysis further demonstrated that GF14 omega binds an equimolar amount of calcium with an apparent binding constant of 5.5 x 10(4) M-1 under physiological conditions . The C-terminal domain, which contains a potential EF hand motif, is responsible for the calcium binding . The C-terminal domain also cross-reacted with the anti-GF14 omega monoclonal antibody . In addition, GF14 omega is phosphorylated by Arabidopsis protein kinase activity at a serine residue(s) in vitro . Therefore, GF14 omega protein has biochemical properties consistent with potential signaling roles in plants . The presence of a potential EF hand-like motif in the highly conserved C terminus of 14-3-3 proteins together with the calcium-dependent multiple functions attributed to the 14-3-3 proteins indicate that the C terminus EF hand is a common functional element of this family of proteins.

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