Escherichia coli

Nature, 1987 Nov 5-11, 330(6143), 41 - 6
Contributions of hydrogen bonds of Thr 157 to the thermodynamic stability of phage T4 lysozyme; Alber T et al.; Measurements of changes in structure and stability caused by 13 different substitutions for threonine 157 in phage T4 lysozyme show that the most stable lysozyme variants contain hydrogen bonds analogous to those in the wild-type enzyme and that structural adjustments allow the protein to be surprisingly tolerant of amino-acid substitutions.

J Biol Chem, 1987 Nov 5, 262(31), 14978 - 82
Escherichia coli H+-ATPase . Glutamic acid 185 in beta subunit is essential for its structure and assembly; Noumi T et al.; The uncD gene for the beta subunit of Escherichia coli H+-ATPase was cloned downstream of the lac promoter and mutagenized (Glu-185----Gln or Lys) by an oligonucleotide-directed procedure . The recombinant plasmid was introduced into a strain in which the unc operon for subunits of H+-ATPase was deleted . The wild-type or mutant beta subunit synthesized amounted to about 10% total cell protein and was mainly found in the cytoplasmic fraction . These subunits could be purified to almost homogeneity by conventional procedures . The wild-type and two mutant beta subunits had essentially the same Kd values for 8-anilinonaphthalene-1-sulfonate, aurovertin, and ATP, although the fluorescence intensities of 8-anilinonaphthalene-1-sulfonate and aurovertin were significantly less when bound to the two mutant beta subunits than when bound to the wild-type subunit . The three beta subunits showed essentially the same circular dichroism spectra, indicating alpha-helical contents of about 16-18% . Thus, the mutations did not cause marked change of the secondary structure of the subunit . However, measurements of theta 208 during linear increase in temperature suggested that replacement of Glu-185 by Gln or Lys slightly changed the stability of the secondary structure . Only trace amounts of alpha beta gamma complexes could be reconstituted using the two mutant beta subunits . These results suggest that Glu-185 or the region in its vicinity may be essential for subunit assembly . The methods developed in this study should be useful for further studies on the beta subunit.

J Biol Chem, 1987 Nov 5, 262(31), 15269 - 76
Purification and characterization of a new DNA-dependent ATPase with helicase activity from Escherichia coli; Wood ER et al.; A previously unreported single-stranded DNA-dependent nucleoside 5'-triphosphatase with DNA unwinding activity has been purified from extracts of Escherichia coli lacking the F factor . Fractions of the purified enzyme contain a major polypeptide of Mr = 75,000 which contains the active site(s) for both ATP hydrolysis and helicase activity . This is consistent with the results of gel filtration chromatography which indicate a native molecular mass of 75 kDa . The 75-kDa helicase has a preference for ATP (dATP) as a substrate in the hydrolysis reaction and requires the presence of a single-stranded DNA cofactor . The helicase reaction catalyzed by the enzyme has been characterized using an in vitro strand displacement assay . The 75-kDa helicase displaces a 71-nucleotide DNA fragment in an enzyme concentration-dependent and time-dependent reaction . The helicase reaction depends on the presence of a hydrolyzable nucleoside 5'-triphosphate (NTP) suggesting that NTP hydrolysis is required for the unwinding activity . In addition, the enzyme can displace a 343-nucleotide DNA fragment albeit less efficiently . The direction of the unwinding reaction is 3' to 5' with respect to the strand of DNA on which the enzyme is bound . The molecular size of this helicase and the direction of the unwinding reaction are similar to both helicase II and Rep protein . However, the 75-kDa helicase has been shown to be distinct from both helicase II and Rep protein using immunological, physical, and genetic criteria . The discovery of a new helicase brings the total number of helicases found in E . coli cell extracts (lacking F factor) to five.

J Biol Chem, 1987 Nov 5, 262(31), 15251 - 5
Nucleotide sequence, promoter analysis, and linkage mapping of the unusually organized operon encoding ribosomal proteins S7 and S12 in maize chloroplast; Giese K et al.; The nucleotide sequence of the operon encoding maize chloroplast ribosomal protein genes S7 and S12 and the promoter activity of a chimeric construct of the -10/-35 sequence of this operon (attached to a promoterless chloramphenicol acetyltransferase gene) have been determined . This operon occurs in the chloroplast genome divided in two parts: part A contains exon 1 of rpS12 (encoding the N-terminal 38 amino acid residues), whereas part B has the following structure: promoter-rpS12 (exon 2 + intron + exon 3)-spacer-rpS7-terminator . Part A is located at the approximate coordinate position 41000, whereas two copies of part B are located at two distant locations in the genome at coordinate positions 18700 and 120200 . This unusual organization of the S12 operon in maize (a monocot plant) is similar to that reported in a dicot and a lower plant . The deduced amino acid sequence of maize chloroplast S7 shows 43, 38, 71, and 85% and of S12 shows 66, 72, 91 and 90% sequence identity to the corresponding sequences of Escherichia coli, Euglena gracilis, Marchantia polymorpha, and Nicotiana tabacum, respectively . The promoter upstream of rpS12 (part B) is transcriptionally active in E . coli.

J Biol Chem, 1987 Nov 5, 262(31), 14867 - 70
Nucleotide and deduced amino acid sequences of two distinct cDNAs for rat phosphoribosylpyrophosphate synthetase; Taira M et al.; The rat Yoshida sarcoma (YS) cDNA library was screened using oligonucleotide probes designed from peptide sequences of rat phosphoribosylpyrophosphate synthetase, and two distinct clones were obtained . Nucleotide sequencing revealed that both clones encode 317 amino acids containing the peptide sequences . The deduced amino acid sequences of the two differ only by 13 residues (96%) identity), whereas the nucleotide sequences are relatively divergent (81% identity in the coding regions) . These results, together with N-terminal amino acid sequencing data, suggest the existence of two different subunits of this enzyme, designated as PRS I and II (their genes as PRPS1 and PRPS2) . Transcripts of 2.3 and 3.7 kilobases were detected in YS cells and rat liver by Northern blot analysis, using PRS I and II cDNAs respectively, as probes . In the liver, the expression of both genes increased after partial hepatectomy.

J Mol Biol, 1987 Nov 5, 198(1), 133 - 6
Shielding of the D loop of ribosome-bound tRNA by elongation factor G; Robertson JM et al.; The topography of the complex of elongation factor G with post-translocative ribosomes has been studied in the Escherichia coli system using fluorescence spectroscopy . We find that a fluorophore attached to the D loop of tRNA is shielded from solvent access by the presence of the factor, and this effect is dependent on factor-promoted GTP hydrolysis . The shielding result suggests that (1) the factor could bind to the tRNA during translocation and (2) the tRNA binding site may be close to that of the factor . The alternative explanation, that the factor affects the conformation of the tRNA bound at a distant site, seems less likely.

Biochemistry, 1987 Nov 3, 26(22), 6950 - 7
Prediction of a common structural domain in aminoacyl-tRNA synthetases through use of a new pattern-directed inference system; Webster TA et al.; The aminoacyl-tRNA synthetases are united by a common function with little evidence of a common structural relationship . Outside of an 11 amino acid stretch called the "signature sequence", no global primary sequence similarity exists . The signature sequence matches 4-11 amino acids in several aminoacyl-tRNA synthetases . High-resolution X-ray data are available for two of these enzymes, revealing that their signature sequence regions are small segments of a common mononucleotide binding foldlike structure . A new methodology for the analysis of dissimilar primary sequences supports the expectation that all of the signature sequence regions form a common structure . In our analysis, two complex pattern descriptors were constructed to describe the synthetase mononucleotide binding fold . These were compared to primary sequences annotated with predicted secondary structures and hydropathy profiles . Regions in 8 out of 12 (67%) heterologous aminoacyl-tRNA synthetase groups (where each group is specific for the same amino acid) match the first descriptor, and 7 of these (58%) also match the second descriptor . In contrast, only 4 regions in a set of 54 control proteins (7.4%) match the first descriptor, and only 2 regions (3.7%) match both . Alignment of these 8 regions to the descriptor (1) positions all known signature sequence regions as the first loop of a mononucleotide binding foldlike structure, (2) extends the previous alignments by another 40-odd amino acids, and (3) identifies potential sites in 3 out of 6 heterologous aminoacyl-tRNA synthetases with no previous alignments . Potential sites are also proposed for two additional heterologous synthetases on the basis of matches to less specific descriptors.

Biochemistry, 1987 Nov 3, 26(22), 7107 - 13
Labeling of the ATP synthase of Escherichia coli from the head-group region of the lipid bilayer; Aggeler R et al.; The isolated and membrane-bound forms of the adenosinetriphosphatase of Escherichia coli (ECF1 and ECF1F0, respectively) have been reacted with two lysine-specific reagents, sodium hexadecyl 4-{3H}formylphenyl phosphate (HFPP) and sodium methyl 4-{3H}formylphenyl phosphate (MFPP), and with the photoreactive reagent 1,2-{3H}dipalmitoyl-sn-glycerol 3-{{{(4-azido-2-nitrophenyl)amino}ethyl}-phosphate} (arylazidoPE) . HFPP and arylazidoPE are amphipathic molecules, inserting by their hexadecyl moieties (one and two chains, respectively) into the lipid bilayer, with the reactive groups intercalated among the phospholipid head groups . MFPP is the water-soluble analogue of HFPP . The labeling patterns of ECF1F0 obtained with HFPP and arylazidoPE were very similar; in both cases the a and b subunits of the F0 part were the most heavily labeled polypeptides of the complex . Models of subunit a, arranged in six transmembrane helices, place most of the lysines in the head-group region, available for reaction with HFPP . Subunits alpha and beta of the ECF1 part were very poorly labeled in comparison to the a and b subunits, together incorporating only 4% as much HFPP and 7.5% as much arylazidoPE as the two F0 subunits together on a protein mass basis . Trypsin cleavage studies localized any labeling of the alpha subunit by arylazidoPE to the N-terminal 15 residues of this polypeptide . When MFPP was used, the alpha and beta subunits were very much more reacted than the F0 subunits . This implies that most of the mass of the alpha and beta subunits in ECF1F0 is above the membrane and not in contact with the bilayer surface.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Nov 3, 26(22), 7121 - 7
The active form of Escherichia coli DNA photolyase contains a fully reduced flavin and not a flavin radical, both in vivo and in vitro; Payne G et al.; Escherichia coli DNA photolyase is a flavoprotein that when purified is blue in color and contains a stable neutral radical FAD (E-FADH) . In the presence of a suitable electron donor (i.e., thiols, tyrosine, or NADH) the radical FAD adsorbs visible light and undergoes photoreduction to the fully reduced FAD (E-FADH2) . The in vitro quantum yield of dimer repair for E-FADH is 0.07 while that of E-FADH2 approaches the in vivo value of 1 . Electron paramagnetic resonance studies on whole cells indicate that the in vivo form of photolyase is E-FADH2 with enzyme containing radical FAD generated predominantly during the ammonium sulfate precipitation step of the purification . Activity measurements of E-FADH using long-wavelength photoreactivating light indicate that enzyme containing FAD in the radical form is not active in dimer repair . Dimer repair observed with E-FADH at shorter wavelengths is probably photoreduction of E-FADH followed by dimer repair by E-FADH2.

Klin Wochenschr, 1987 Nov 2, 65(21), 1042 - 7
{Serologic AIDS diagnosis with polypeptides obtained by genetic technics of the human immunodeficiency virus (HIV-1)}; Lenz A et al.; Serologic testing for human immunodeficiency virus type 1 (HIV-1) is currently based on enzyme linked immunosorbent assay (ELISA) as screening method . Positive ELISA-results have to be confirmed by at least one second procedure such as Western blotting or immunofluorescence . To obtain new diagnostic reagents for confirmatory testing, we expressed viral antigens in procaryotic systems . Peptides representing epitopes of structural core (gag)- and envelope (env)-proteins of HIV were produced in E . coli as stable immunogenic beta-galactosidase fusion proteins . Recombinant proteins were taken for immunoblot-assays . The results of Western blotting with those fusion proteins were in general comparable with conventional ELISA, immunofluorescence, immunoblot with cell-culture derived virus and commercially available ELISA tests based on recombinant proteins . Immunoblots using recombinant transmembrane protein (gp41) derived polypeptide were more sensitive than the conventional procedure with purified virion proteins . Western blotting with recombinant fusionproteins provide reliable and inexpensive serodiagnostics without handling of infectious cell cultures.

FEBS Lett, 1987 Nov 2, 223(2), 387 - 90
A unique amino acid substitution in the outer membrane protein OmpA causes conjugation deficiency in Escherichia coli K-12; Ried G et al.; The outer membrane protein OmpA of E . coli K-12 can serve as a receptor for phages and is required for stabilizing mating aggregates during F'-mediated conjugation . Selection for resistance to OmpA-specific phages yields mutants with alterations in the protein at four cell surface exposed sites . It is shown that conjugation deficiency can be caused by apparently only one type of amino acid substitution at one of these sites, the replacement of glycine-154 by aspartic acid . This suggests that, in contrast to binding of phages, a ligand of the donor cell recognizes only a very small area of the protein.

Eur J Biochem, 1987 Nov 2, 168(3), 687 - 94
Cyclic nucleotide binding to cAMP receptor protein from Escherichia coli . Optical and ligand-binding studies; Donoso-Pardo JL et al.; cAMP receptor protein from Escherichia coli has been purified on a large scale . Analogues of cAMP modified on the 6-NH2 group of the adenosine ring, the ribose 2'OH group or the cyclic phosphate are able to displace cAMP from its binding site with dissociation constants of similar magnitude to that of cAMP . More extensive modification produces weaker binding . Ultraviolet/visible difference spectroscopy and fluorescence spectroscopy show that the environment of the bound adenosine moiety is considerably less polar than that in aqueous solvent, while an anthraniloyl group substituted on the 2'OH position remains accessible to solvent . The 2-NH2 group of cGMP appears to be protonated in the bound form, while no change in the charge state of cAMP is apparent.

FEBS Lett, 1987 Nov 2, 223(2), 395 - 401
Catalytic and noncatalytic nucleotide binding sites of the Escherichia coli F1 ATPase . Amino acid sequences of beta-subunit tryptic peptides labeled with 2-azido-ATP; Wise JG et al.; Under appropriate conditions tight, noncovalent binding of 2-azido-adenine nucleotides to either catalytic or noncatalytic binding sites on the E . coli F1-ATPase occurs . After removal of unbound ligands, UV-irradiation results primarily in the covalent incorporation of nucleotide moieties into the beta-subunit in both catalytic and noncatalytic site labeling experiments . Minor labeling of the alpha-subunit was also observed . After trypsin digestion and purification of the labeled peptides, microsequencing studies identified two adjacent beta-subunit tryptic peptides labeled by 2-azido-ADP or -ATP . These beta-subunit peptides were labeled on tyrosine-331 (catalytic sites) and tyrosine-354 (noncatalytic sites) in homology with the labeling patterns of the mitochondrial and chloroplast enzymes.

Nature, 1987 Nov 26-Dec 2, 330(6146), 381 - 4
Improved immunogenicity of a peptide epitope after fusion to hepatitis B core protein; Clarke BE et al.; Synthetic vaccines for viral diseases can use defined regions of viral proteins as immunogens: the peptide sequence of amino acids 141-160 of the VP1 protein of foot and mouth disease virus (FMDV) elicits virus-neutralizing antibodies to protect guinea pigs, cattle and pigs either when coupled to a carrier protein or when administered in liposomes or in incomplete Freund's adjuvant . The immune response to these peptides is much lower than that to complete virus particles and the same sequence fused to the N terminus of beta-galactosidase did not produce a more potent immunogen than synthetic peptide alone . We report here an expression system for immunogenic epitopes linked to a carrier protein, hepatitis B core antigen, to form part of a virus-like complex which can present these epitopes to the immune system at high density . The immunogenicity of these structures approaches that of FMDV particles.

J Bacteriol, 1987 Nov, 169(11), 5216 - 23
Nucleotide sequence analysis of the gene encoding the Deinococcus radiodurans surface protein, derived amino acid sequence, and complementary protein chemical studies; Peters J et al.; The complete nucleotide sequence of the gene encoding the surface (hexagonally packed intermediate {HPI})-layer polypeptide of Deinococcus radiodurans Sark was determined and found to encode a polypeptide of 1,036 amino acids . Amino acid sequence analysis of about 30% of the residues revealed that the mature polypeptide consists of at least 978 amino acids . The N terminus was blocked to Edman degradation . The results of proteolytic modification of the HPI layer in situ and Mr estimations of the HPI polypeptide expressed in Escherichia coli indicated that there is a leader sequence . The N-terminal region contained a very high percentage (29%) of threonine and serine, including a cluster of nine consecutive serine or threonine residues, whereas a stretch near the C terminus was extremely rich in aromatic amino acids (29%) . The protein contained at least two disulfide bridges, as well as tightly bound reducing sugars and fatty acids.

Proc Natl Acad Sci U S A, 1987 Nov, 84(21), 7557 - 61
Platelet-activating factor (PAF) stimulates the PAF-synthesizing enzyme acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase and PAF synthesis in neutrophils; Doebber TW et al.; Platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF) induced in isolated rat peritoneal and human peripheral neutrophils a rapid and potent activation of the PAF biosynthetic enzyme acetyl-CoA:1-alkyl-sn-glycero-3-phosphocholine O2-acetyltransferase (EC 2.3.1.67) . The PAF-induced activation of the neutrophil acetyltransferase (8-10 times basal neutrophil activity) was maximal within 30 sec after PAF addition, as was the PAF-stimulated degranulation . After 1 min of PAF stimulation, the elevated acetyltransferase activity steadily decreased . Within 2 min of stimulation of neutrophils with 10(-6) M PAF, the 7-fold increase in acetyltransferase activity was coincident with substantial PAF synthesis (as measured by {3H}acetate incorporation into PAF), which was 14% of the PAF synthesis induced by the Ca2+ ionophore A23187 at 10(-5) M . PAF activation of the acetyltransferase and PAF synthesis required intact neutrophils as they did not occur in cells broken by sonication . The neutrophil acetyltransferase was 10-30 times more sensitive to activation by PAF than was degranulation as the acetyltransferase activation was evident with 10(-9) M PAF and was about maximal with 3 x 10(-8) M PAF . The unstimulated and PAF-induced acetyltransferase exhibited the same Km for acetyl-CoA (67 microM), but the Vmax for the PAF-induced enzyme (1667 pmol/min per 10(7) cells) was 10 times that of the unstimulated enzyme (175 pmol/min per 10(7) cells) . The PAF induction of the acetyltransferase was less sensitive to inhibition by the specific PAF receptor antagonist L-652,731 than was PAF-induced degranulation . This, along with the differing sensitivities to PAF, suggests that acetyltransferase activation and degranulation induced by PAF either involve two different PAF receptors or involve one receptor type with different receptor occupancy requirements . Escherichia coli alkaline phosphatase, which greatly decreased the activity of the acetyltransferase in spleen microsomes, had little or no effect on the basal or PAF-induced neutrophil acetyltransferase . Thus, by stimulating the activity of acetyltransferase, PAF induces in neutrophils the synthesis of more PAF, thereby probably augmenting the neutrophil response to the initial PAF.

Res Vet Sci, 1987 Nov, 43(3), 405 - 6
Attempted induction of local Shwartzman reaction in the chicken; Katiyar AK et al.; Chickens and rabbits were injected intradermally with an endotoxin, namely Escherichia coli lipopolysaccharide (LPS) . Twenty-four hours later, LPS was again administered intravenously to induce a local Shwartzman reaction . A typical cutaneous inflammatory reaction developed in rabbits, but not in chickens . Even very high doses of LPS, that made the birds visibly sick, failed to elicit the reaction . The results suggest that chickens are refractory to the Shwartzman reaction . A noteworthy feature of the chickens' response to intradermal endotoxin was the formation of prominent perivascular lymphoid aggregates.

Plasmid, 1987 Nov, 18(3), 205 - 14
Characterization and sequence of a plasmid from the trachoma biovar of Chlamydia trachomatis; Sriprakash KS et al.; The 7.5-kb plasmid of Chlamydia trachomatis, trachoma biovar, was mapped for restriction enzyme sites and sequenced . The complete nucleotide sequence, the first reported for any chlamydial plasmid, revealed nine open reading frames which could code for polypeptides greater than 10 kDa . These putative polypeptides contain 35-47% hydrophobic amino acid residues . Two putative polypeptides of 30 kDa are highly basic and one of 23 kDa is acidic . A region composed of four 22-bp repeats, AT-rich clusters, an inverted repeat, and a 30-kDa basic protein is similar in organization to the origins of replication in a number of Escherichia coli plasmids . Northern blot analysis of the plasmid RNA showed transcripts longer than the length of the plasmid, suggesting a single transcription initiation signal.

J Comp Pathol, 1987 Nov, 97(6), 637 - 43
Depression of lymphocyte response to mitogens in sheep infected with tick-borne fever; Woldehiwet Z; Lymphocytes obtained from sheep experimentally infected with Cytoecetes phagocytophila, the causative agent of tick-borne fever, showed reduced blastogenesis induced by the mitogens phytohaemagglutinin and E . coli lipopolysaccharide . The period of reduced lymphocyte reactivity coincided with the period of parasitaemia and leucopenia.

Am Ind Hyg Assoc J, 1987 Nov, 48(11), 933 - 4
The presence of free-living amoebae in portable and stationary eye wash stations; Tyndall RL et al.; Portable and stationary eye wash stations were tested for the presence of free-living amoebae . Such amoebae may be found in potable waters, and at least one genera, the Acanthamoebae, can cause severe infections when introduced into traumatized eyes . Concentrates of filtrates of water from eye wash stations were placed on nonnutrient agar plates seeded with Escherichia coli . Resultant outgrowths of free-living amoebae, which were morphologically identified as mixtures of Hartmannella and Acanthamoebae, were inoculated intranasally into weanling mice . Subsequently, brain and lung tissues from injected mice were tested for amoebae as an indication of persistent infection . Acanthamoebae and Hartmannella were detected in some eye wash stations at each of four test sites . Both portable and stationary stations harbored Acanthamoebae . Some of the isolates caused persistent pulmonary infection but were not isolated from brain tissue . Flushing stationary eye wash stations temporarily reduced the number of stations positive for amoebae . Treatment of portable stations with 25 ppm of free chlorine also reduced the number of stations harboring amoebae but caused corrosion in some of the stations.

Gene Anal Tech, 1987 Nov-Dec, 4(6), 111 - 8
Induction of multiple replacement mutations by oligonucleotide-directed mutagenesis with extended mismatch primers; Lichtler A et al.; We have developed a method called oligo-scanning mutagenesis that uses oligonucleotides to mutate up to 12 contiguous bases in a single step . Some advantages of this procedure are that the position and sequence of the replacement mutations are completely specified by the investigator, and combinations of mutations can easily be generated . The technique uses a gapped substrate and the Escherichia coli dam methylation error-correcting mechanism to increase the yield of mutants.

Ann Inst Pasteur Microbiol, 1987 Nov-Dec, 138(6), 657 - 66
Evaluation of the Biken test to detect heat-labile (LT) enterotoxin produced by porcine and human Escherichia coli strains; Said AC et al.; Fifty-seven strains of enterotoxigenic Escherichia coli isolated from humans and pigs and producing thermolabile (LT) enterotoxin were used to ascertain the efficiency of the Biken test compared to the passive immune haemolysis test (PIH), considered as very sensitive for detecting that enterotoxin . The two assays were carried out using anti-porcine (anti-LTp), anti-human (anti-LTh), anti-cholera toxin (anti-CT) and anti-choleragenoid (anti-Cg) antisera . Our results showed that the Biken test was very irregular, with many false-negative results . Positive results (ranging from 78.9 to 22.8) were dependent upon the antiserum used . Conversely, the PIH test was much more efficient in the detection of LT, since 100% of the LT+ strains were positive in this test whatever the antiserum used.

Mol Microbiol, 1987 Nov, 1(3), 327 - 34
Effects of deletions in the spacer region of the rrnB operon on the transcription of the large ribosomal RNAs from Escherichia coli; Szymkowiak C et al.; A series of deletions was constructed within the spacer region of the genes for the 16S and 23S RNA on plasmids bearing the rrnB operon . The accumulation and synthesis rates for the 16S and 23S RNAs were determined from normal growing cells and maxicells after transformation with the mutated plasmids . A marked difference in the transcription efficiency of the plasmid-encoded ribosomal 16S and 23S RNAs was observed with cells carrying plasmids, where a sequence motif analogous to the antitermination recognition sequence (Box A) had been deleted . The overall synthesis rate of ribosomal RNAs of such cells was not altered, however, indicating that the difference in transcription rates from the plasmid genes is compensated by altered transcription rates of the corresponding chromosomal genes . In addition, the accumulation of various tRNA species encoded on rRNA operons and non rRNA operons was quantitated and compared . From these results we infer that the regulation of ribosomal RNA transcription does not only occur at the promoter sites but sequence regions possibly involved in antitermination within the operon are crucial for a coordinated synthesis of all ribosomal RNAs.

Mol Microbiol, 1987 Nov, 1(3), 251 - 8
Nucleotide sequence and transcriptional startpoint of the glpT gene of Escherichia coli: extensive sequence homology of the glycerol-3-phosphate transport protein with components of the hexose-6-phosphate transport system; Eiglmeier K et al.; The nucleotide sequences of the glpT gene of Escherichia coli and its regulatory region have been elucidated and the primary structure of the glycerol-3-phosphate transport protein deduced . Extensive amino acid sequence homology was found with two other cytoplasmic membrane proteins: the functionally related hexose-6-phosphate transport protein, and the UHPC protein involved in regulating hexose-6-phosphate uptake . Although no significant amino acid sequence homology was found with other transport proteins, such as the arabinose, citrate, glucose, melibiose, lactose or xylose transporters, all of these proteins share a common secondary structure arrangement with the GLP T protein as they apparently contain twelve membrane-spanning alpha-helical segments . The promoter for glpT was located by transcript mapping and shown to overlap a site to which catabolite activator protein binds in vitro . These findings indicate how catabolite repression may be mediated but do not explain its physiological significance in glycerol metabolism.

Mol Biol (Mosk), 1987 Nov-Dec, 21(6), 1560 - 5
{Structural and functional organization of the colicin operon in the ColD-CA23 plasmid}; Pshennikova ES et al.; The organization of the genes involved in colicin D synthesis was studied . These are colicin, immunity and lysis genes . The nucleotide sequence of the immunity gene, its structural and regulatory regions were determined . This gene was shown to be located next to the colicin gene on the same strand and followed by the lysis gene . When colicin synthesis is induced with mitomycin C the immunity gene is transcribed from the general SOS-dependent promotor as a part of the colicin operon . However it has its own SOS-independent promotor in normal growth conditions . A high homology in amino acid sequences of Co1D lysis protein and that of Co1E1, Co1E2, Co1E3, Co1DF13, Co1A was revealed . A detailed scheme of Co1D-CA23 colicin operon structural organization is suggested.

Mutagenesis, 1987 Nov, 2(6), 491 - 6
Protection of Chinese hamster cells against the cytotoxic and mutagenic effects of alkylating agents by transfection of the Escherichia coli alkyltransferase gene and a truncated derivative; Fox M et al.; The cytotoxic and mutagenic effects of various monofunctional and bifunctional alkylating agents have been assessed in V79 Chinese hamster cells that express either the entire O6-alkylguanine (O6AG) and alkylphosphotriester alkyltransferase (ATase) gene (clone 8 cells) or a truncated form that codes only for O6AG ATase activity (clone SB cells) . Protection ratios, as determined by D37 values, were greater for clone 8 cells than for SB cells . Significant protection against the mutagenic effects of N-methyl-N-nitrosourea and ethylmethanesulphonate at the hypoxanthine phosphoribosyltransferase (HPRT) locus was observed in clone 8 and SB cells . Streptozotocin and the haloethyl nitrosoureas, chlorozotocin and bis-chloroethylnitrosourea were less efficient in inducing HPRT-deficient mutants and a smaller degree of protection was afforded by the transfected genes . This is possibly due to the propensity of these compounds to induce multi-locus deletions . Southern analysis of DNA from clone 8 and SB cells indicated the presence of multiple copies of the plasmid integrated into clone 8 cells but few copies in clone SB cells . The copy number did not change but ATase levels fell when cells were grown in the absence of G418.

Mutagenesis, 1987 Nov, 2(6), 433 - 9
Study of the genotoxic potential of 17 mycotoxins with the SOS Chromotest; Krivobok S et al.; Seventeen mycotoxins {aflatoxins B1 (AFB1), B2 (AFB2), G1 (AFG1), G2 (AFG2), aflatoxicol, sterigmatocystin, patulin, citrinin, penicillic acid, T-2 toxin, diacetoxyscirpenol, zearalenone, zearalenol (alpha and beta isomers 1:1), ochratoxin A, norsolorinic acid, averufin, versicolorin A} were tested using the SOS Chromotest (PQ37 and PQ35) . Six of the mycotoxins (AFB1, AFG1, AFB2, aflatoxicol, sterigmatocystin and versicolorin A) were genotoxic on PQ37 strain with and without metabolic activation . The results obtained with metabolic activation are in agreement with positive results obtained in other tests of genotoxicity . Except for AFB2, the presence of a double bond C8-C9 in the dihydrobenzofurane (DHBF) ring explained the activity due to the formation of an epoxide, but the coumarin cyclopentenone ring also plays a role in the qualitative differences of genotoxic activity . The wild-type uvrB gene in PQ35 decreases the genotoxic response with and without metabolic activation . Without metabolic activation, only mycotoxins possessing the DHBF ring group and double linkage C8-C9 exhibit a genotoxic effect.

Eur J Respir Dis, 1987 Nov, 71(5), 410 - 8
Morphology and function of blood monocytes after incubation with lung surfactant; Wiernik A et al.; Human blood monocytes were incubated for different periods of time with lung surfactant (phospholipid concentration 1-2.5 mg/ml) . After short-term (30 min) incubation, there was an increase in the nitroblue tetrazolium (NBT) reduction of the monocytes both at rest and during stimulation with E . coli bacteria, and enhanced ingestion of fluorescein-labelled yeast particles . Electron microscopic examination of the same monocytes showed an active cell surface with numerous protrusions . Long-term (24 h) incubation with surfactant resulted in a reduced ability of the cells to adhere to plastic dishes . Although the NBT-reduction of resting monocytes was increased after long-term incubation with surfactant, the additional enhancement of NBT-reduction after stimulation with bacteria was decreased . These cells were rounded, usually devoid of surface structures, their nuclei were condensed, and their cytoplasm filled with surfactant material . Thus, monocytes are initially activated in the presence of surfactant, but if the cells become overfed with surfactant lipids their functional capacity decreases.

J Biochem (Tokyo), 1987 Nov, 102(5), 975 - 83
Molecular assembly of the lipoprotein trimer on the peptidoglycan layer of Escherichia coli; Choi DS et al.; The molecular assembly of the major outer membrane lipoprotein on the peptidoglycan layer was studied using two hybrid genes coding for different OmpF-lipoprotein hybrid proteins . One gene codes for a "lipoprotein" in which the diacylglyceryl cysteine residue is replaced with the Ala-Glu residue of the NH2 terminus of the OmpF protein (hybrid protein I) . The other gene codes for the lipid-free "lipoprotein" from which the COOH-terminal lysine residue was further deleted (hybrid protein II) . Hybrid protein I existed as a trimer . A significant portion of it was found to be composed of only the free form, which was noncovalently associated with the peptidoglycan layer . The purified hybrid protein I trimer was dissociated into the subunit in the presence of guanidine-HCl and reassociated on dialysis . Both the native and reassociated trimers were bound to the lipoprotein-free peptidoglycan layer . No enhancement of the binding was observed when the reassociation reaction was carried out simultaneously . Hybrid protein II, on the other hand, did not exhibit association with peptidoglycan in both the cellular fractionation and in vitro binding experiments, although it existed as a trimer . It is concluded that 1) the protein domain of the lipoprotein exists as a trimer which is noncovalently as well as covalently associated with the peptidoglycan layer and 2) although the deletion of the COOH terminal lysine residue did not interfere with the trimerization, it interfered with the noncovalent interaction with the peptidoglycan layer.

J Assoc Off Anal Chem, 1987 Nov-Dec, 70(6), 991 - 3
Rapid fluorogenic enumeration of Escherichia coli in selected, naturally contaminated high moisture foods; Poelma PL et al.; An assay for the enzyme glucuronidase was used to determine the presence of Escherichia coli in selected, naturally contaminated high moisture foods . Raw pork sausage, ground turkey, and ground beef were inoculated into tubes containing the substrate 4-methylumbelliferyl beta-D-glucuronide (MUG) in lauryl tryptose (LT) medium . After incubation at 35 degrees C for 24 h, the inoculated LT-MUG tubes were examined under longwave ultraviolet light for the presence of a fluorogenic glucuronidase end product . A fluorescing tube indicated the presumptive presence of E . coli . The 10 day most probable number method of the AOAC and the LT-MUG procedure gave comparable recoveries of E . coli.

Isr J Med Sci, 1987 Nov, 23(11), 1128 - 31
Prevalence of postenteritis cow's milk protein intolerance; Hager C et al.; The object of this study was to ascertain the frequency of postinfectious cow's milk protein hypersensitivity (CMPH) . Twenty-four infants less than 3 months old were included in the study . Following hospitalization for acute gastroenteritis, the infants were given a protein hydrolysate formula for a period of 6 weeks, after which an intestinal biopsy was performed . Thereafter, a milk challenge was given . The existence of CMPH was defined as a postchallenge reduction of one or more of the mucosal disaccharidases below the normal levels for our laboratory . A bacterial etiology of the gastroenteritis was found in 10 . Nineteen infants had no adverse reaction to cow's milk after 6 weeks on a hypoallergenic formula . Only two could be confidently diagnosed as having developed secondary CMPH; both had been infected by Escherichia coli 0 111 . One infant had primary CMPH and one extra-intestinal CMPH . The incidence of secondary CMPH with gastrointestinal manifestations in this series was considerably less than described elsewhere.

Biochem Int, 1987 Nov, 15(5), 915 - 24
Characterization of the substrate-binding sites of the mitochondrial nicotinamide nucleotide transhydrogenase; Wakabayashi S et al.; The mitochondrial energy-linked nicotinamide nucleotide transhydrogenase (TH) is modified and inhibited by p-fluorosulfonylbenzoyl-5'-adenosine (FSBA) . The modification appears to occur at the NAD(H)-binding site when TH alone or TH in the presence of NADPH is incubated with FSBA . However, when this site is protected by NADH, then FSBA inhibits TH more slowly and modifies a different, though specific, site . This second site could be the NADP(H)-binding site . Using {3H}FSBA in the presence of NADPH, the NAD(H)-binding site was modified, and a single tryptic peptide carrying the label was isolated and sequenced . The amino acid sequence of this peptide was Glu-Ser-Gly-Glu-Gly-Gln-Gly-Gly-Tyr*-Ala-Lys . The modified residue was Tyr . The labeled peptide isolated after incubating TH with {3H}FSBA in the presence of NADH could not be completely purified . However, amino acid analysis and partial sequencing made it possible to identify this segment on the amino acid sequence of bovine TH as derived from its cDNA by Yamaguchi et al . (private communication).

Vet Immunol Immunopathol, 1987 Nov, 16(3-4), 235 - 50
K88 variants K88ab, K88ac and K88ad in oral vaccination of different porcine adhesive phenotypes . Immunological aspects; Bijlsma IG et al.; Sows of different adhesive phenotypes were vaccinated orally during the last 4 weeks of gestation with K88-positive Escherichia coli . Sows susceptible to adhesion by the K88 variant of the vaccination strain produced a significant IgA-class specific anti-K88 response in colostrum and milk and post-farrowing serum . Indications for an IgM and IgG-class specific anti-K88 response were also found in this group but only in milk . In sows resistant to adhesion by the K88 variant of the vaccination strain only an IgA-class specific anti-K88 antibody response was found in mammary secretions and in post-farrowing sera, but titres did not reach the high values of the former group . The response in the second group was attributed to the frequent administration of large quantities of K88-positive E . coli which to some extent can be compared with a colonization effect . Specificity for the serological components of the K88 variants was detectable in colostral IgA of sows susceptible to the vaccination strain only.

Vet Microbiol, 1987 Nov, 15(3), 201 - 7
In vitro adhesion of K88ab-, K88ac- and K88ad-positive Escherichia coli to intestinal villi, to buccal cells and to erythrocytes of weaned piglets; Cox E et al.; The phenotype of 21 weaned piglets, concerning adhesion of Escherichia coli possessing K88ab, K88ac or K88ad fimbriae to pig cells, was determined in an in vitro assay . Comparison was made with adhesion of these three K88 variant strains to buccal mucosal epithelial cells and to erythrocytes (haemagglutination) in the same piglets . Whereas adhesion of the three K88 variant strains to intestinal villi was piglet specific, buccal cell adhesion (BCA) and haemagglutination (HA) were not . The K88ab strain was weakly adhesive or non-adhesive in the BCA and negative in the HA test . K88ac strains consistently gave negative and K88ad consistently gave positive results in both assays . After washing the bacteria with phosphate-buffered saline, the K88ab strain revealed a positive HA test . Neither the BCA, nor HA test can be used to determine the pig intestinal adhesive phenotype.

Mol Cell Biol, 1987 Nov, 7(11), 4139 - 41
Selection against expression of the Escherichia coli gene gpt in hprt+ mouse teratocarcinoma and hybrid cells; Besnard C et al.; Thioxanthine is toxic for mammalian cells transformed by the dominant selectable marker gpt . It allowed us to select, in the presence of the endogenous hypoxanthine-guanine phosphoribosyltransferase gene, mutants that did not express gpt any more and also hybrid cells that had lost the chromosome carrying it . The gpt marker is thus dominant in negative as well as in positive selection, which makes it potentially very useful for genetic studies of mammalian cells.

Mol Cell Biol, 1987 Nov, 7(11), 4010 - 6
Yeast pre-mRNA splicing requires a minimum distance between the 5' splice site and the internal branch acceptor site; Thompson-Jager S et al.; We have generated several deletions within the intron of a yeast actin gene construct which have lead to different splicing efficiencies as measured by Northern blot (RNA blot) and primer extension analyses . Our data especially demonstrate that a minimum distance from the 5' splice site to the internal branch acceptor site is required for accurate and efficient splicing . In a construct in which splicing was completely abolished, splicing could be restored by expanding the distance from the 5' splice site to the internal branch acceptor site with heterologous sequences . Alternative splicing, i.e., exon skipping and the use of a cryptic 5' splice site, was observed when the mRNA precursor was derived from a tandem repeat of a truncated intron with flanking exon sequences.

Mol Gen Genet, 1987 Nov, 210(1), 5 - 9
RNA polymerase sigma-related proteins in Escherichia coli: detection by antibodies against a synthetic peptide; Fujita N et al.; Antibodies were raised against a synthetic tetradecameric peptide with an amino acid sequence, DLIQEGNIGLMKAV, which corresponds to the most highly conserved region of bacterial RNA polymerase sigma factors . In a Western-blot analysis of total Escherichia coli proteins, the antiserum reacted specifically with at least three proteins with apparent molecular weights of 75 kDa, 27 kDa and 23 kDa, in addition to the known sigma factors (sigma 70 and sigma 32) . The majorities of sigma 70 and sigma 32 were recovered as associated forms with the RNA polymerase on glycerol gradient centrifugation, while the other cross-reacting proteins were not . Unambiguous evidence was obtained which indicated that the intracellular level of sigma 32 increased rapidly upon heat-shock, at least in the strain containing high copy numbers of the rpoH gene.

Mol Gen Genet, 1987 Nov, 210(1), 101 - 10
Identification of components of a new stability system of plasmid R1, ParD, that is close to the origin of replication of this plasmid; Bravo A et al.; We provide evidence that a mutation which derepresses an autoregulated system that is located in the vicinity of the basic replicon of R1, stabilizes the ParA- and ParB- miniplasmid of R1 pKN1562, without increasing its copy number . The system, which we have called ParD, maps inside the 1.45-kb PstI-EcoRI fragment that is adjacent to the origin of replication of the plasmid . Two proteins whose expression is coordinated are components of the system . The sequence of the PstI-EcoRI fragment was obtained . The wild-type ParD system determines in cis a basal but detectable stability.

Mol Gen Genet, 1987 Nov, 210(1), 10 - 5
Heat-shock induction of RNA polymerase sigma-32 synthesis in Escherichia coli: transcriptional control and a multiple promoter system; Fujita N et al.; Transcriptional start sites of the rpoH gene which codes for a minor sigma factor (sigma 32) of Escherichia coli RNA polymerase were determined . The rpoH gene is transcribed, both in vivo and in vitro, from two major (P1 and P2) and one minor (P2*) promoters . In vitro synthesis of the rpoH mRNAs is dependent on the major species of RNA polymerase holoenzyme (E sigma 70) but not on the minor one (E sigma 32) . S1 nuclease analysis of the in vivo RNA showed that the level of rpoH transcript from the downstream P2 promoter increases rapidly when E . coli cells are transferred from 30 degrees C to 42 degrees C, while the transcript from the upstream P1 promoter remains at a constant level . Under these conditions, the metabolic stabilities of rpoH mRNAs are virtually unaffected, suggesting that the synthesis of rpoH mRNA from the P2 promoter is specifically enhanced upon heat-shock.

Mol Gen Genet, 1987 Nov, 210(1), 1 - 4
Hierarchy of the strength of Escherichia coli stringent control signals; Glass RE et al.; The quantitative effect of ppGpp, the effector of stringent control, on various Escherichia coli promoters was measured in an in vitro mixed transcription system . This allowed us to determine, among these promoters, the hierarchy of promoters according to their ppGpp susceptibility . The strength of the stringent control signal, however, was found to be altered when the test promoters were transcribed by ppGpp-insensitive RNA polymerases from relaxed mutants of E . coli, which carry substitutions in the beta subunit gene (rpoB) . Thus, it was concluded that the activity of the stringent control signal depends on the nature of the RNA polymerase as well as that of the promoter.

J Pediatr Surg, 1987 Nov, 22(11), 1041 - 4
Indium 111 labeled white blood cell scintigraphy for the diagnosis of upper abdominal abscesses in a child with Wiscott-Aldrich syndrome; Weinreb BD et al.; We report on a case of multiple hepatic abscesses in an immunodeficient patient where initial radiologic evaluation by ultrasonography and computed tomography confused early management by failing to demonstrate the abscesses and by suggesting other diagnoses . Indium 111 (In-111) white blood cell (WBC) scanning with Tc-99 liver-spleen scan subtraction accurately demonstrated subcapsular hepatic abscesses in four out of four sequential studies, and later confirmed resolution of the abscesses . We suggest that In-111 WBC scanning may be used as a highly specific method of diagnosing suspected upper abdominal abscesses in children.

Gut, 1987 Nov, 28(11), 1460 - 6
Absence of complement fixing antibodies against lipopolysaccharides from Escherichia coli in a subgroup of patients with Crohn's disease; Zeitz M et al.; Complement fixing antibodies against different Escherichia coli lipopolysaccharides were determined in patients with Crohn's disease and in healthy individuals and compared with antitetanus toxoid antibodies . All healthy individuals had antilipopolysaccharide antibodies, 10 of 27 patients with Crohn's disease had no antibodies and six had rapidly changing antibody titres . These abnormalities were found in patients with disease in the colon, with arthropathy and fistula . Antilipid A was found at lower titres in Crohn's disease . Neither antitetanus toxoid antibodies, nor immunoglobulin concentrations were different in patients with or without antilipopolysaccharide antibodies . There was no evidence for circulating immune complexes in patients lacking antilipopolysaccharide antibodies . Certain subgroups of patients with Crohn's disease have altered antibody levels to typical enteral antigens which most likely can be explained by local antibody binding to lipopolysaccharides at inflammatory sites, or by changes in immunoregulation in this disease.

Genes Dev, 1987 Nov, 1(9), 1028 - 37
Activation of a cryptic 5' splice site in the upstream exon of the phage T4 td transcript: exon context, missplicing, and mRNA deletion in a fidelity mutant; Chandry PS et al.; A collection of 100 td mutants defective in phage T4 thymidylate synthase (TS) production was screened for splicing impairments . Splicing-defective mutants were identified by a rapid assay developed to detect imbalances in the td protein products (TS, the exon ligation product, and NH2TS, encoded by the pre-mRNA) . Thirteen selected mutants, confirmed to be splicing defective by an RNA-oligodeoxynucleotide hybridization assay, were all shown to be inhibited in the first step of the group I splicing pathway, cleavage at the 5' splice site . Of these, only one, SC99, appeared to be a specificity mutant . Whereas the 12 other mutants had sequence changes within the functionally important 5' and 3' domains of the intron, SC99 was shown to be an exon mutant . The G----A change at residue -3 of the upstream exon of SC99 resulted in loss of normal 5' splice site recognition . Furthermore, activation of a remote cryptic splice site at residue -29 of the upstream exon and missplicing of mRNA that is deleted for 29 nucleotides of the 5' exon are characteristic for this mutant . These results underscore the role of exon sequences in guiding the fidelity of the splicing reaction and they raise provocative questions about the alignment of introns within exon contexts that are consistent with accurate splicing and synthesis of an intact gene product.

EMBO J, 1987 Nov, 6(11), 3507 - 14
p13suc1 acts in the fission yeast cell division cycle as a component of the p34cdc2 protein kinase; Brizuela L et al.; cdc2+ encodes a protein kinase that is required during both G1 and G2 phases of the cell division cycle in fission yeast . suc1+ is an essential gene that was originally identified as a plasmid-borne sequence that could rescue certain temperature-sensitive cdc2 mutants . To investigate the role of the suc1+ gene product in the cell cycle p13suc1 has been expressed in Escherichia coli and purified . An immunoaffinity purified anti-p13suc1 polyclonal serum has been prepared and used to identify p13suc1 in fission yeast . The abundance of this protein did not alter either during the cell cycle or during entry into stationary phase . p13suc1 was found in yeast lysates in a complex with the cdc2+ gene product . Approximately 5% of cellular p34cdc2 was associated with p13suc1, and this fraction of p34cdc2 was active as a protein kinase . The stability of the complex was disrupted in yeast strains carrying temperature-sensitive alleles of cdc2 that are suppressible by overexpression of suc1+ . The level of association between p13suc1 and p34cdc2 was not affected by cell cycle arrest in adverse nutritional conditions . p13suc1 is not a substrate of the p34cdc2 protein kinase . We propose instead that it acts as a regulatory component of p34cdc2 that facilitates interaction with other proteins.

Biophys J, 1987 Nov, 52(5), 867 - 72
Triplet state sublevel kinetics of tryptophan 54 in the complex of Escherichia coli single-stranded DNA binding protein with single-stranded poly(deoxythymidylic) acid; Zang LH et al.; The individual sublevel kinetics of the lowest triplet state of tryptophan 54 (Trp 54) which is highly perturbed in the complex of Escherichia coli single-stranded DNA binding protein (Eco SSB) with poly(deoxythymidylic) acid (poly{dT}) have been studied by optically detected magnetic resonance (ODMR) spectroscopy . The triplet sublevel decay constants of Trp 54, kx, ky, kz, are 0.99, 0.072, and 0.045 s-1, respectively, in the poly(dT) complex of a point-mutated Eco SSB in which Trp 88 is substituted by phenylalanine . Tx is the only radiative triplet sublevel . Negative polarity of the Tx----Tz and Tx----Ty phosphorescence-detected ODMR signals results from the steady state population pattern, nx greater than ny, nz, and implies that the relations, px greater than or equal to 14py, and px greater than or equal to 22pz exist for the relative populating rates . Spin-orbit coupling between radiative singlet states and the Tx sublevel of the lowest triplet state of Trp 54 is enhanced selectively upon complexing of Eco SSB with poly(dT).

Biochem J, 1987 Nov 1, 247(3), 641 - 9
Segmental structure and protein domains in the pyruvate dehydrogenase multienzyme complex of Escherichia coli . Genetic reconstruction in vitro and 1H-n.m.r . spectroscopy; Radford SE et al.; A deletion in vitro can be made in the aceEF-lpd operon encoding the pyruvate dehydrogenase multienzyme complex of Escherichia coli, which causes deletion of two of the three homologous lipoyl domains that comprise the N-terminal half of each dihydrolipoamide acetyltransferase (E2p) polypeptide chain . An active complex is still formed and 1H-n.m.r . spectroscopy of this modified complex revealed that many of the unusually sharp resonances previously attributed to conformationally mobile segments in the wild-type E2p polypeptide chains had correspondingly disappeared . A further deletion was engineered in the long (alanine + proline)-rich segment of polypeptide chain that linked the one remaining lipoyl domain to the C-terminal half of the E2p chain . 1H-n.m.r . spectroscopy of the resulting enzyme complex, which was also active, revealed a further corresponding loss in the unusually sharp resonances observed in the spectrum . These experiments strongly support the view that the sharp resonances derive, principally at least, from the three long (alanine + proline)-rich sequences which separate the three lipoyl domains and link them to the C-terminal half of the E2p chain . Closer examination of the 400 MHz 1H-n.m.r . spectra of the wild-type and restructured complexes, and of the products of limited proteolysis, revealed another sharp but smaller resonance . This was tentatively attributed to another, but smaller, (alanine + proline)-rich sequence that separates the dihydrolipoamide dehydrogenase-binding domain from the inner core domain in the C-terminal half of the E2p chain . If this sequence is also conformationally flexible, it may explain previous fluorescence data which suggest that dihydrolipoamide dehydrogenase bound to the enzyme complex is quite mobile . The acetyltransferase active site in the E2p chain was shown to reside in the inner core domain, between residues 370 and 629.

Acta Endocrinol (Copenh), 1987 Nov, 116(3), 381 - 6
Serum profiles and short-term metabolic effect of pituitary and authentic biosynthetic human growth hormone in man . A double-blind cross-over study; Jorgensen JO et al.; In a double-blind cross-over study we compared pituitary and methionine-free biosynthetic human growth hormone (P-hGH and B-hGH) with respect to pharmacokinetics and short-term metabolic effects in 9 hypopituitary children . They treated themselves for 4 weeks with 2 IU sc daily at 20.00 h . After admittance to hospital 2 IU was given: im the first day, and sc the second . They then switched over to the alternative preparation . The serum profiles of B- and P-hGH were identical . Comparing im and sc absorption, the latter was slower and resulted in smaller areas under the curves, indicating greater local degradation . Both preparations caused identical increases in somatomedin-C, but slightly more sustained after sc injection . Plasma glucose, plasma glucagon, and serum insulin fluctuated within normal ranges . The glucose profile pointed at a modest anti-insulin effect of hGH when given in the morning . The concentration in the blood of lactate, alanine, glycerol and B-OH-butyrate, and in serum of triglyceride, cholesterol and carbamide revealed no abnormalities with either hGH preparation . Finally, no development of anti-GH or E . coli polypeptide antibodies was seen . In conclusion, the pharmacokinetics and short-term metabolic effects of B-hGH and P-hGH were identical.

Radiat Res, 1987 Nov, 112(2), 398 - 402
The effect of cosmic-ray shielding on the ultraweak bioluminescence emitted by cultures of Escherichia coli; Tilbury RN et al.; Neither the growth of Escherichia coli nor its associated luminescence was significantly affected when cultures were shielded from the soft component of cosmic rays . The study included experiments in which the cultures were shielded intermittently during their two periods of luminescence emission and experiments in which the cultures were continuously shielded throughout their entire growth cycle . These results do not support previous suggestions that the ultraweak bioluminescences from living organisms might be cosmic-ray-excited fluorescences induced in certain biological molecules synthesized during the various stages of growth.

Mutat Res, 1987 Nov, 192(3), 175 - 80
Expression of a novel R-plasmid pEB017 compared to pKM101 in Escherichia coli wild-type, recA and uvrA strains; Obaseiki-Ebor EE et al.; The expression of bacterial resistance to UV irradiation and nitrofurantoin by a novel R-plasmid pEB017 in DNA-repair-proficient (wild-type) and -deficient (recA; uvrA) host strains was compared to the effects of plasmid pKM101 in the isogenic strains . pEB017 partially protected the uvrA strain, and completely protected the wild-type and recA strains from the killing effect of UV irradiation; pKM101 had no effect on the recA strain and only enhanced the survival of the wild-type and the uvrA strains after UV irradiation . pEB017 conferred nitrofurantoin resistance 10-fold on the wild-type and the recA strains and 4-fold on the uvrA strain; pKM101 did not confer nitrofurantoin resistance on the wild-type and recA strains but gave 4-fold resistance in the uvrA strain.

Proc Soc Exp Biol Med, 1987 Nov, 186(2), 218 - 22
Lack of direct coronary vascular effects of Escherichia coli endotoxin in dogs; Buffington CW et al.; This study explored the hypothesis that coronary vascular injury and dysfunction result from intracoronary administration of Escherichia coli endotoxin (0.025 to 0.025 to 0.4 mg/kg) in dogs . Peak hyperemic coronary flow following a 15-sec period of stopped flow and the maximum flow in response to adenosine were used to estimate coronary vascular reserve . The wet-to-dry ratio of myocardial tissue was used to estimate extravascular water content as an indicator of vascular leak due to endothelial injury . Intracoronary saline was used as a control . Peak reactive hyperemia and maximum flow at constant coronary pressure were not different in the animals receiving intracoronary endotoxin (n = 6) and the animals receiving saline (n = 5) during 4 hr following treatment . In addition, wet-to-dry ratios were similar in these two groups . These data fail to support the hypothesis that endotoxin, per se, produces coronary vascular injury of sufficient magnitude to produce myocardial dysfunction.

J Bacteriol, 1987 Nov, 169(11), 5317 - 9
Distribution of shufflon among IncI plasmids; Komano T et al.; A shufflon or clustered inversion is a novel type of DNA rearrangement originally discovered in the IncI1 plasmid R64 (T . Komano, A . Kubo, and T . Nisioka, Nucleic Acids Res . 15:1165-1172, 1987) . In a 1.95-kilobase region of R64 DNA, four DNA segments inverted independently or in groups, resulting in a complex DNA rearrangement . We found similar types of shufflon in other IncI1 plasmids, including delta, pIP111, pIP565, pIP112, pIP186, R144, R163, R483, and R621a . A variant type of shufflon occurs in the IncI1 plasmid ColIb.

J Bacteriol, 1987 Nov, 169(11), 5314 - 6
Effects of segregation and selection on instability of plasmid pACYC184 in Escherichia coli B; Lenski RE et al.; We use a mathematical model to analyze the dynamics of loss of nonconjugative pACYC184 from populations of Escherichia coli B in glucose-limited continuous culture . This model incorporates both plasmid segregation and selection against plasmid carriage . It is concluded that there is intense selection against plasmid carriage (s = 0.3 per culture generation), which amplifies the frequency of segregants arising de novo.

J Bacteriol, 1987 Nov, 169(11), 5311 - 3
Mapping of the constitutive lysyl-tRNA synthetase gene of Escherichia coli K-12; Emmerich RV et al.; The constitutive lysyl-tRNA synthetase gene (lysS) was mapped at 62.1 min on the Escherichia coli chromosome by a combination of conjugation and transduction, with physical confirmation by two-dimensional gel electrophoresis . Revertant analysis suggests that the altered isoelectric point and the low amount of the mutant LysS protein may be due to a single mutational event.

J Bacteriol, 1987 Nov, 169(11), 5304 - 7
Escherichia coli strains carrying the cloned cytochrome d terminal oxidase complex are sensitive to near-UV inactivation; Sammartano LJ et al.; To determine if membrane-bound cytochromes function as endogenous near-UV photosensitizers, strains containing the cloned cydA and cydB genes were tested for near-UV sensitivity . A strain containing both cloned genes overproduced cytochromes b558, b595, and d . Another strain containing only cloned cydB overproduced cytochrome b558 . Both cytochrome-overproducing strains were hypersensitive to broad-spectrum near-UV inactivation . The presence of excess cytochromes did not affect sensitivity to far-UV radiation and provided protection against H2O2 inactivation.

J Bacteriol, 1987 Nov, 169(11), 5201 - 8
Fusion of Escherichia coli heat-stable enterotoxin and heat-labile enterotoxin B subunit; Guzman-Verduzco LM et al.; The 3' terminus of the DNA coding for the extracellular Escherichia coli heat-stable enterotoxin (ST) devoid of transcription and translation stop signals was fused to the 5' terminus of the DNA coding for the periplasmic B subunit of the heat-labile enterotoxin (LTB) deleted of ribosomal binding sites and leader peptide . By RNA-DNA hybridization analysis, it was shown that the fused DNA was transcribed in vivo into an RNA species in close agreement with the expected molecular weight inferred from the nucleotide sequence . The translation products of the fused DNA resulted in a hybrid molecule recognized in Western blots (immunoblots) with antibodies directed against the heat-labile moiety . Anti-LTB antibodies coupled to a solid support bound ST and LTB simultaneously when incubated with ST-LTB cellular extracts . By {35S}cysteine pulse-chase experiments, it was shown that the fused ST-LTB polypeptide was converted from a precursor with an equivalent electrophoretic mobility of 20,800 daltons to an approximately 18,500-dalton species, which accumulated within the cell . The data suggest that wild-type ST undergoes at least two processing steps during its export to the culture supernatant . Blocking the natural carboxy terminus of ST inhibited the second proteolytic step and extracellular delivery of the hybrid molecule.

J Bacteriol, 1987 Nov, 169(11), 5152 - 6
Cloning, expression, and primary structure of a Chlamydia trachomatis binding protein; Kaul R et al.; The gene encoding an 18,000-dalton eucaryotic cell-binding protein of Chlamydia trachomatis serovar L2 was cloned into Escherichia coli, and the nucleotide sequence of a 1,658-base-pair PstI restriction endonuclease fragment encoding this protein was determined . The recombinant chlamydial gene consists of a 486-base-pair open reading frame encoding a polypeptide of molecular weight 18,314 . The resultant polypeptide, comprising 162 amino acids, possesses a highly charged carboxy-terminal end . The expression of this recombinant protein is under the control of a vector promoter . The recombinant 18,000-dalton protein possessed the same eucaryotic cell-binding characteristics as did the native chlamydial 18,000-dalton protein when electrophoresed and transferred to nitrocellulose . Polyclonal antibodies to the recombinant protein exhibited neutralizing activity.

J Bacteriol, 1987 Nov, 169(11), 5140 - 51
High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli; Bishai WR et al.; ABM508 is a recombinant fusion protein consisting of the N-terminal 485 amino acids of diphtheria toxin joined to alpha-melanocyte-stimulating hormone . When expressed in Escherichia coli under the control of the tox promoter and signal sequence, ABM508 is severely degraded . When overexpressed from a thermoinducible lambda pR promoter fusion, ABM508 is largely insoluble . We compared the expression of ABM508 (501 amino acids) to a full-length mutant form of the toxin (CRM197; 535 amino acids) and found that CRM197 showed minimal proteolysis . Thus, the removal of the C-terminal 50 amino acids of the toxin destabilizes the protein, making it a target for proteases . Proteolysis of ABM508 could be reduced by removal of the tox signal sequence (thereby directing the protein to the cytoplasm) and growth in lon and htpR mutant strains of E . coli . We also showed that the solubility of tox gene products expressed in E . coli was directly related to the growth temperature of the culture . Thus, a fragment A fusion protein (223 amino acids), ABM508, and CRM197 were found in soluble extracts when expressed at 30 degrees C but could not be released by the same procedures after growth at 42 degrees C . On the basis of these observations, we fused the coding sequences for mature ABM508 to the trc promoter (inducible at 30 degrees C by isopropyl-beta-D-thiogalactoside) and expressed this construct in a lon htpR strain of E . coli . This plasmid made 10 mg of soluble tox protein per liter of culture (7.7% of the total cell protein) or 14 times more than our previous maximal level . Extracts from lon htpR cells harboring this plasmid had high levels of ADP-ribosyltransferase activity, and although proteolysis still occurred, the major tox product corresponded to full-length ABM508.

J Bacteriol, 1987 Nov, 169(11), 5087 - 94
Mutations in Escherichia coli that effect sensitivity to oxygen; Jamison CS et al.; Fifteen oxygen-sensitive (Oxys) mutants of Escherichia coli were isolated after exposure to UV light . The mutants did not form macroscopic colonies when plated aerobically . They did form macroscopic colonies anaerobically . Oxygen, introduced during log phase, inhibited the growth of liquid cultures . The degree of inhibition was used to separate the mutants into three classes . Class I mutants did not grow after exposure to oxygen . Class II mutants were able to grow, but at a reduced rate and to a reduced final titer, when compared with the wild-type parent . Class III mutants formed filaments in response to oxygen . Genetic experiments indicated that the mutations map to six different chromosomal regions . The results of enzymatic assays indicated that 7 of the 10 class I mutants have low levels of catalase, peroxidase, superoxide dismutase, and respiratory enzymes when compared with the wild-type parent . Mutations in five of the seven class I mutants which have the low enzyme activities mapped within the region 8 to 13.5 min . P1 transduction data indicated that mutations in three of these five mutants, Oxys-6, Oxys-14, and Oxys-17, mapped to 8.4 min . The correlation of low enzyme levels and mapping data suggests that a single gene may regulate several enzymes in response to oxygen . The remaining three class I mutants had wild-type levels of catalase, peroxidase, and superoxide dismutase, but decreased respiratory activity . The class II and III mutants had enzyme activities similar to those of the wild-type parent . Our results demonstrate that mutations in at least six genes can be expressed as oxygen sensitivity . Some of these genes may be involved in respiration or cell division or may regulate the expression of several enzymes.

J Bacteriol, 1987 Nov, 169(11), 4907 - 11
Role of phenylalanine 150 in the receptor-binding domain of the K88 fibrillar subunit; Jacobs AA et al.; Recently, we reported the isolation of three peptides, Ile-83-Ala-Phe-85, Ser-148-Leu-Phe-150, and Ala-156-Ile-Phe-158, derived from the K88 fibrillar subunit and found to inhibit the binding of K88 fibrillae to cavia erythrocytes or pig intestinal epithelial cells (A . A . C . Jacobs, J . Venema, R . Leeven, H . van Pelt-Heerschap, and F . K . de Graaf, J . Bacteriol . 169:735-741, 1987) . The gene encoding the K88 fibrillar adhesin was modified by oligonucleotide-directed site-specific mutagenesis such that each of the phenylalanine residues at positions 85, 150, and 158 were replaced by serine . Replacement of phenylalanine 85 or 158 had no apparent effect on the biosynthesis of the fibrillae or on their adhesive capacity . In contrast, substitution of phenylalanine 150 with serine resulted in a dramatic decrease in adhesive capacity of the K88 fibrillae . Apparently, phenylalanine 150 plays an essential role in the interaction of the adhesin with receptor molecules present on eucaryotic cells.

Infect Immun, 1987 Nov, 55(11), 2541 - 5
Immunization of mice against tetanus with fragments of tetanus toxin synthesized in Escherichia coli; Fairweather NF et al.; Two recombinant plasmids, pTet11 and pTet18, which express nontoxic protein fragments of tetanus toxin in Escherichia coli, were constructed . pTet11 protein (86 kilodaltons) is a fusion between part of the E . coli trpE protein and 441 amino acids of tetanus fragment C, and pTet18 (63 kilodaltons) consists of part of fragment B and all of fragment C of tetanus toxin . The synthesis of these proteins was induced in E . coli cultures, and the proteins were partially purified . Mice were immunized with these proteins, and dose-dependent titers of anti-tetanus toxoid antibodies were obtained . The proteins were able to induce neutralizing antibodies in mice, as demonstrated by the ability of mice immunized with 1 microgram or more of protein to survive challenge with 10 50% lethal doses of tetanus toxin.

Biochimie, 1987 Nov-Dec, 69(11-12), 1161 - 8
Properties of gamma-aminobutyraldehyde dehydrogenase from Escherichia coli; Prieto MI et al.; gamma-Aminobutyraldehyde dehydrogenase from Escherichia coli K-12 has been purified and characterized from cell mutants able to grow in putrescine as the sole carbon and nitrogen source . The enzyme has an Mr of 195,000 +/- 10,000 in its dimeric form with an Mr of 95,000 +/- 1,000 for each subunit, a pH optimum at 5.4 in sodium citrate buffer, and does not require bivalent cations for its activity . Km values are 31.3 +/- 6.8 microM and 53.8 +/- 7.4 microM for delta-1-pyrroline and NAD+, respectively . An inhibitory capacity for NADH is also shown using the purified enzyme.

J Nat Prod, 1987 Nov-Dec, 50(6), 1118 - 25
Traditional medicinal plants of Thailand, IX . 10-Hydroxy-11-methoxydracaenone and 7,10-dihydroxy-11-methoxydracaenone from Dracaena loureiri; Meksuriyen D et al.; Examination of Dracaena loureiri, a Thai medicinal plant possessing anti-bacterial activity, has led to the isolation of two new representatives, 1 and 2, of a rare skeleton of homoisoflavans . Proton assignments of the two isolates were aided by extensive 2D-homonuclear chemical shift correlation and nOe difference spectroscopy . Carbon assignments were completed through the utilization of simple and sensitive one-dimensional techniques such as selective population transfer via one-bond (CSCM 1D) and selective polarization transfer via long range coupling (selective INEPT) experiments . Conformational assignments were proposed through nOe difference spectroscopy and have been established by X-ray crystallography . The absolute configuration is proposed based on the octant rule and a biogenetic pathway for this type of homoisoflavan is briefly discussed.

Biokhimiia, 1987 Nov, 52(11), 1770 - 6
{Hexamere purine nucleoside phosphorylase from Escherichia coli K-12 . Kinetic analysis and mechanism of reaction}; Bezirdzhian KhO et al.; A kinetic analysis of the phosphorolytic reaction catalyzed by hexameric purine nucleoside phosphorylase II from E . coli K-12 in the presence and absence of reaction products was carried out . The results of the kinetic analysis are consistent with a rapid equilibrium random Bi-Bi mechanism, in which a dead-end ternary (enzyme.purine base.phosphate) complex is formed.

J Biochem (Tokyo), 1987 Nov, 102(5), 1231 - 40
Preparation and characterization of monoclonal antibodies against phosphoenolpyruvate carboxylase of Escherichia coli; Ishijima S et al.; Twelve hybridoma clones which secrete monoclonal antibodies (mAb) against purified phosphoenolpyruvate carboxylase {EC 4.1.1.31} from Escherichia coli K-12 were obtained . These 12 mAb were prepared from the ascites fluids of mice . Six among the 12 mAb formed precipitin lines with the enzyme on immunodiffusion . Four mAb inhibited the activity of the enzyme and 2 mAb enhanced it . Four mAb altered the sensitivity of the enzyme to allosteric effectors . Competitive enzyme-binding experiments among the 12 different mAb were also performed . The results showed that the 12 mAb can be classified into at least 8 groups.

Mol Cell Biol, 1987 Nov, 7(11), 4048 - 57
Amino acid sequences that determine the nuclear localization of yeast histone 2B; Moreland RB et al.; Histone-beta-galactosidase protein fusions were used to identify the domain of yeast histone 2B, which targets this protein to the nucleus . Amino acids 28 to 33 in H2B were required for nuclear localization of such fusion proteins and thus constitute a nuclear localization sequence . The amino acid sequence in this region (Gly-29 Lys Lys Arg Ser Lys Ala) is similar to the nuclear location signal in simian virus 40 large T antigen (Pro-126 Lys Lys Lys Arg Lys Val) (D . Kalderon, B.L . Roberts, W.D . Richardson, and A.E . Smith, Cell 39:499-509, 1984) . A point mutation changing lysine 31 to methionine abolished nuclear localization of an H2B-beta-galactosidase fusion protein containing amino acids 1 to 33 of H2B . However, an H2B-beta-galactosidase fusion protein containing both this point mutation and the H2A interaction domain of H2B was nuclear localized . These results suggest that H2A and H2B may be cotransported to the nucleus as a heterodimer.

EMBO J, 1987 Nov, 6(11), 3539 - 42
gal4 transcription activator protein of yeast can function as a repressor in Escherichia coli; Paulmier N et al.; The chromosomal lac operator of Escherichia coli was replaced by a 22 bp oligonucleotide containing the binding site of the yeast gal4 protein . Induction of gal4 protein synthesis in these bacteria repressed beta-galactosidase synthesis at least 30-fold . These results show that it is possible to detect in bacteria with a simple assay the DNA binding activity of a eukaryotic protein with a defined sequence specificity . This opens new avenues for the isolation in E . coli of mutants of DNA binding proteins unable to bind to their DNA targets, and for direct cloning in bacteria of cDNA coding for DNA binding proteins with defined sequence specificity.

Mutat Res, 1987 Nov, 189(3), 263 - 9
Study of the genotoxic potential of 48 inorganic derivatives with the SOS chromotest; Olivier P et al.; The genotoxic potential of 48 inorganic derivatives was studied using the bacterial colorimetric assay: the SOS Chromotest . Some of these compounds are known as carcinogens (As, CR(VI), Cd, Ni) or suspected carcinogens for human beings (Hg, Pb), others are known as non-carcinogens . Among these 48 derivatives, only the two Cr(VI) compounds and the Sn(II) compounds gave positive results.

J Bacteriol, 1987 Nov, 169(11), 5028 - 34
Temporal control of colicin E1 induction; Salles B et al.; The expression of the gene encoding colicin E1, cea, was studied in Escherichia coli by using cea-lacZ gene fusions . Expression of the fusions showed the same characteristics as those of the wild-type cea gene: induction by treatments that damage DNA and regulation by the SOS response, sensitivity to catabolite repression, and a low basal level of expression, despite the presence of the fusion in a multicopy plasmid . Induction of expression by DNA-damaging treatments was found to differ from other genes involved in the SOS response (exemplified by recA), in that higher levels of DNA damage were required and expression occurred only after a pronounced delay . The delay in expression following an inducing treatment was more pronounced under conditions of catabolite repression, indicating that the cyclic AMP-cyclic AMP receptor protein complex may play a role in induction . These observations also suggest a biological rationale for the control of cea expression by the SOS response and the cyclic AMP-cyclic AMP receptor protein catabolite repression system.

Infect Immun, 1987 Nov, 55(11), 2546 - 53
Activities of complete and truncated forms of pertussis toxin subunits S1 and S2 synthesized by Escherichia coli; Locht C et al.; The genes encoding the S1 and S2 subunits of pertussis toxin were expressed in Escherichia coli under lac operon transcription and translation control with pUC8 and pUC18 as the expression vectors . Various versions of the subunits were detected with anti-S1 or anti-S2 monoclonal antibodies . Recombinant S1, but not S2, subunit contained the enzymatic NAD-glycohydrolase and NAD:Gi ADP-ribosyltransferase activities . Both activities were also expressed by a truncated version of the S1 subunit in which the 48 carboxy-terminal amino acid residues, including a predicted Rossman structure and one of the two cysteines, had been deleted . The epitope for an anti-S2 monoclonal antibody was localized to the N-terminal 40-amino-acid region of the S2 subunit . Both the S1 and S2 subunits expressed in E . coli reacted with human hyperimmune serum . The full length and the truncated recombinant S1 subunit also reacted in Western blots with a neutralizing and protective monoclonal anti-S1 antibody . The different versions of S1 and S2 subunits expressed in E . coli are useful for mapping active sites, epitopes, and regions that interact with receptors or the other subunits in the holotoxin . These recombinant subunits will also facilitate the development of a safer, new-generation vaccine against whooping cough.

J Biochem (Tokyo), 1987 Nov, 102(5), 1261 - 73
Structure and expression of cDNA for an inhibitor of blood coagulation isolated from human placenta: a new lipocortin-like protein; Iwasaki A et al.; An inhibitor of blood coagulation, a new protein with an apparent molecular weight of 34,000 and an isoelectric point of 4.9, was purified from human placental tissue by EDTA extraction . Five cDNA clones were isolated from the human placental lambda gt11 cDNA library using the mouse monoclonal antibody raised against the coagulation inhibitor as the probe . The longest insert consists of 1,566 nucleotides, and contains 960 nucleotides entirely encoding the 320 amino acids of the inhibitor, and a poly A tail . The deduced amino acid sequence was corroborated by chemical analyses of the protein . The entire amino acid sequence shows homology to those of lipocortin I, lipocortin II, and endonexin-related proteins . The cDNA for the inhibitor was expressed in Escherichia coli under the regulation of the trc promotor of the plasmid pKK233-2 . The resulting recombinant protein manifested inhibitory activities against both blood coagulation and phospholipase A2 activity, as did the coagulation inhibitor isolated from human placenta.

Am J Med Sci, 1987 Nov, 294(5), 388 - 94
Mithramycin selectively inhibits transcription of G-C containing DNA; Miller DM et al.; Mithramycin induces a reversible inhibition of cellular RNA synthesis without affecting DNA synthesis . The authors have shown this drug induces myeloid differentiation of HL-60 promyelocytic leukemia cells and is an effective agent in certain patients with chronic granulocytic leukemia . In order to investigate the mechanism by which this drug inhibits RNA synthesis we have compared the effect of mithramycin on RNA synthesis by whole cells, isolated nuclei, and RNA synthesis by isolated E . coli RNA polymerase and eukaryotic RNA polymerase II . Exposure of HL-60 cells to mithramycin at concentrations of 4.6 X 10(-7) m or higher for 48 hours causes an almost immediate inhibition of RNA synthesis (up to 85% at 4 hours) with only modest cytotoxicity at these concentrations . Endogenous RNA synthesis by isolated nuclei can be inhibited by mithramycin only at high concentrations (greater than 10(-5) m), suggesting that mithramycin primarily may inhibit initiation, rather than elongation . Mithramycin inhibits in vitro transcription of salmon sperm DNA by E . coli RNA polymerase at DNA:drug ratios similar to those required for RNA synthesis inhibition in whole cells . Similar DNA binding studies with synthetic oligonucleotides demonstrate that mithramycin is a potent inhibitor of transcription of Poly dG.dC by E . coli RNA polymerase but has no effect on transcription of Poly dA.dT . The rapid inhibition of whole cell and isolated RNA polymerase transcription, and the relative insensitivity of isolated nuclei, suggest mithramycin may interact with specific DNA sequences in order to inhibit the initiation of RNA synthesis in intact cells.

Microb Pathog, 1987 Nov, 3(5), 387 - 91
Nucleotide sequence analysis of a P fimbrial regulatory element of the uropathogenic Escherichia coli strain KS71 (04:K12); Rhen M et al.; The nucleotide sequence of a trans-acting P-fimbrial regulatory element obtained from the uropathogenic Escherichia coli strain KS71 (04:K12) was determined . The regulatory element was found to contain an open reading frame of 231 nucleotide residues that showed 95.2% homology with papl, a functionally analogous regulatory gene of E . coli strain J 96.

Biochem Int, 1987 Nov, 15(5), 881 - 6
Prevention of alterations in the transport of nutrients in pyelonephritic rats by immunization with pili; Garg UC et al.; The uptake of D-glucose, L-aspartate, L-lysine and L-proline was studied in renal brush border membrane vesicles prepared from control, infected and actively immunized-infected rats . The uptake of D-glucose, L-lysine and L-proline was decreased significantly (p less than 0.05) during the course of infection in the infected animals . However, the uptake of L-aspartate was increased significantly (p less than 0.05) in early stages and decreased significantly (p less than 0.05) in later stages of infection in the infected animals . When the animals were actively immunized with pili, still there were changes in the uptake of D-glucose and L-aspartate, but the changes appeared later and less pronounced . No change in the uptake of L-lysine and L-proline was observed in the immunized-infected animals . The findings demonstrated that active immunization with pili prevents alterations in the uptake of nutrients in pyelonephritic rats.

Am J Vet Res, 1987 Nov, 48(11), 1574 - 6
Influence of Bordetella avium infection on association of Escherichia coli with turkey trachea; Van Alstine WG et al.; Four-week-old Bordetella avium-infected and B avium-free turkeys were inoculated intratracheally with a suspension of fimbriated or nonfimbriated Escherichia coli . Numbers of E coli associated with tracheal sections were determined at postinoculation hour (PIH) 1 or 6 . Significantly (P less than 0.05) greater numbers of E coli were isolated from the tracheas of B avium-infected turkeys compared with numbers in B avium-free turkeys . In B avium-free turkeys, tracheal associated E coli were 90% less at PIH 6 compared with that at PIH 1 . However, in B avium-infected turkeys, numbers of E coli were not affected by postinoculation time . Seemingly, B avium-infected turkeys had reduced capacity to clear E coli from the trachea.

J Med Chem, 1987 Nov, 30(11), 2062 - 7
Design and synthesis of phosphonate inhibitors of glutamine synthetase; Farrington GK et al.; Inhibitors 1-4 have been shown previously to undergo enzymatic phosphorylation by glutamine synthetase (GS) . Phosphonates 6-9 were designed as chemically stable analogues of these phosphorylated inhibitors, incorporating either a tetrahedral sulfur group (6-8) (-S-, -SO-, -SO2-) or phosphinate (9) adjacent to methylphosphonic acid . Phosphonates 6-8 resemble the transiently stable phosphorylated methionine sulfone (2), whereas 9 resembles phosphorylated 2-amino-4-phosphonobutyric acid (4) . When tested as inhibitors of glutamine synthetase from bacteria, mammals, and plants, analogue 9 proved to be the most potent, with a Ki value of 7.5 X 10(-5) M vs . the Escherichia coli enzyme . Analysis of the inhibition data for 6-9 suggests that a replacement of the oxygen bridging the tetrahedral sulfur (6-8) or phosphinate (9) and the terminal phosphate with a hydrophobic methylene drastically reduces the enzyme's affinity for inhibitors . Enhanced affinity of GS for phosphonate 9 may result from interaction of the negative charge on the phosphinate with Mn2+ at the active site.

J Bacteriol, 1987 Nov, 169(11), 5119 - 24
Location of F plasmid transfer operon genes traC and traW and identification of the traW product; Maneewannakul S et al.; As part of an analysis of the conjugative transfer genes associated with the expression of F pili by plasmid F, we have investigated the physical location of the traC and traW genes . We found that plasmid clones carrying a 2.95-kilobase EcoRI-EcoRV F transfer operon fragment were able to complement transfer of F lac traC mutants and expressed an approximately 92,000-dalton product that comigrates with TraC . We also found that traW-complementing activity was expressed from plasmids carrying a 900-base-pair SmaI-HincII fragment . The traW product was identified as an approximately 23,000-dalton protein . The two different F DNA fragments that expressed traC and traW activities do not overlap . Our data indicate that the traC gene is located in a more-tra operon promoter-proximal position than suggested on earlier maps and that traW is distal to traC . These results resolve a long-standing question concerning the relationship of traW to traC . The clones we have constructed are expected to be useful in elucidating the role of proteins TraC and TraW in F-pilus assembly.

J Bacteriol, 1987 Nov, 169(11), 4984 - 90
Proton leakiness caused by cloned genes for the F0 sector of the proton-translocating ATPase of Escherichia coli: requirement for F1 genes; Brusilow WS; To study expression of uncG, the gene coding for the gamma subunit of the Escherichia coli proton-translocating ATPase, deletions were made in the intergenic region between uncA, the gene coding for the alpha subunit, and uncG . Two deletions which fused uncA and uncG coded for alpha-gamma fusion polypeptides which were synthesized well both in vitro and in vivo, demonstrating that uncG expression is normally controlled by nucleotides in the intergenic region . Multicopy plasmids carrying these fusion genes and the genes for the other subunits of the ATPase had a harmful effect on the growth of E . coli . The effect was overcome by N,N'-dicyclohexylcarbodiimide, indicating that the cells probably leaked protons . The deleterious effect was eliminated by making a nonpolar deletion in the upstream F0 gene uncB, or by cloning each of the uncA-uncG fusion genes onto a separate plasmid, removed from the F0 genes, thus demonstrating that the fusion genes were not primarily responsible for the proton permeability . A plasmid which carried F0 genes and the gene for the delta subunit caused deleterious proton leakiness in unc+ cells but not in cells from which the unc operon was deleted . The proton leakiness caused by these different plasmids was therefore due to the production of a leaky F0 proton channel and required the presence of F1 genes . The results support a model for ATPase assembly in which F1 genes or polypeptides are involved in the formation or opening of the F0 proton channel.

Mol Microbiol, 1987 Nov, 1(3), 317 - 25
Nucleotide sequencing of the structural gene for colicin N reveals homology between the catalytic, C-terminal domains of colicins A and N; Pugsley AP; An 1800 bp fragment of DNA from a natural ColN plasmid (pCHAP4) encompassing the colicin N structural gene (cna) and its regulatory region was subjected to nucleotide sequencing and deletion analysis . The region of DNA immediately upstream from cna contains two tandemly-arranged and overlapping potential LexA binding sites (SOS boxes), in line with the previous demonstration that cna expression is repressed by LexA protein . Deletion of the LexA binding site allowed efficient transcription of cna from an upstream lacZ promoter, whereas its presence reduced lacZ-promoted cna expression to varying extents depending on the proximity of lacZp and the SOS boxes . The molecular weight of colicin N, as deduced from the nucleotide sequence, is 41,696, which is close to the experimentally determined molecular weight of 39,000 . Colicin N has a glycine-rich amino terminus similar to that found in many other colicins . Part of the glycine-rich domain of colicin N could be replaced by an unrelated sequence devoid of glycine residues without affecting either colicin release or activity . The carboxy-terminal half of colicin N exhibits significant homology to the C-terminus of colicin A . The latter colicin forms pores in the cytoplasmic membrane of Escherichia coli, thereby depolarizing the membrane and causing cell death . The C-terminus of colicin A is endowed with this catalytic activity . Although colicin N was previously found to cause lysis of Escherichia coli cells, a more detailed investigation revealed that it too depolarizes the Escherichia coli cytoplasmic membrane and that lysis is a secondary effect.

Mol Microbiol, 1987 Nov, 1(3), 259 - 73
The parD- mutant of Escherichia coli also carries a gyrAam mutation . The complete sequence of gyrA; Hussain K et al.; The phenotype of a recently-described mutant (OV6), conditionally defective in chromosome partitioning and septal positioning, was originally thought to be due to a new gene (parD) mapping at 88.4 min . We have now shown that, in addition to the parD mutation, OV6 carries a gyrAam mutation and that this mutation is probably responsible for the gross phenotype of the mutant . We have cloned the gyrA gene, identified the GyrA protein, sequenced the gyrA gene and flanking genes, cloned and sequenced the gyrAam mutation, and identified its truncated product . In addition, we have identified the transcriptional start point of the gyrA gene . The E . coli GyrA protein has extensive homologies with Gyrase proteins of other organisms and weak sequence homologies with some eukaryotic cytoskeletal proteins.

J Gen Microbiol, 1987 Nov, 133 ( Pt 11), 3247 - 55
The respiratory chain of anaerobically grown Escherichia coli: reactions with nitrite and oxygen; Rothery RA et al.; The reactions of nitrite and oxygen with the cytochrome d oxidase of Escherichia coli were studied, following growth of cells on glycerol with fumarate as respiratory oxidant . Optical difference spectroscopy was used to investigate the kinetics of product formation during the reaction of the respiratory chain with nitrite . Two kinetically distinct species were formed in the reaction with nitrite; these had spectral features at 438 nm and 630 nm . These observations indicate that the cytochrome d does not contribute significantly to absorbance in the Soret region, and that changes elicited by ligand binding in the Soret region are largely attributable to haemoprotein b-590 . Inhibition of respiratory oxidase activity by nitrite was also investigated . The inhibition was competitive with oxygen (Ki 0.83 mM, pH 7), which allowed analysis of the reaction of the oxidase with oxygen itself . The reaction with oxygen was cooperative with an apparent number of oxygen-binding sites, n, of 1.26 at pH 7, increasing to 1.72 at pH 6 . We propose a model for the oxidase in which there are two ligand-binding sites.

Plasmid, 1987 Nov, 18(3), 237 - 45
Molecular rearrangements between two plasmids of the FII incompatibility group in different recombination--deficient Escherichia coli strains; Martinez-Salazar JM et al.; The frequencies and types of plasmid molecular rearrangements generated in different recombinant mutants which carried two plasmids of the FII incompatibility group were studied . The wild-type cells generated molecular rearrangements mainly by interplasmidic recombination with a frequency of 2.4 x 10(-6) per cell per cell doubling . Cells in which RecF was the principal recombination pathway generated different types of molecular rearrangements that involved either both plasmids or one of the plasmids and the chromosome . The frequencies of molecular rearrangements for these cells were 50-fold greater than those of wild-type cells . The recA- cells, even when the RecE pathway was derepressed, generated rearrangements only between one of the plasmids and the chromosome, at very low frequencies (10(-9} . In wild-type cells and in RecF cells, interplasmidic recombination generated mainly cointegrates carrying DNA deletions . These cointegrates were stable in recA- or recA- RecE+ cells, but unstable in wild-type or RecF+ cells . In the latter, the cointegrates generated smaller plasmids with different molecular structures at relatively low frequencies.

Mutagenesis, 1987 Nov, 2(6), 445 - 53
DNA sequence analysis of the mutational specificity of u.v . light in the SUP4-o gene of yeast; Kunz BA et al.; We have characterized mutations induced in the SUP4-o gene of Saccharomyces cerevisiae by u.v . irradiation . Mutants were selected following treatment with 60 J/m2 u.v . light which reduced cell survival to 10% and increased the SUP4-o mutation frequency 100-fold above background . DNA sequence analysis of 120 mutants revealed that u.v . induced all types of base substitutions, although transitions, in particular G:C----A:T events predominated . In addition, a small number of single base pair deletions and double mutations, occurring in tandem or separated by a few base pairs, were recovered . The base pair substitutions were not distributed randomly in the SUP4-o gene and, with one exception, were all located at sites of adjacent pyrimidines, suggesting that they were targeted by u.v . photolesions . A substantial fraction of the mutations were detected at hotspots for u.v . mutagenesis . The majority of changes occurred at the 3' base of dipyrimidine sequences where both cyclobutane dimers and {6-4}-photoproducts could form . Approximately one-third of the induced base substitutions were found at potential pyrimidine dimer sites where {6-4}-photoproducts would be expected to occur rarely . The possible origins of the induced mutations and the role of cyclobutane dimers as premutational u.v . lesions in yeast are considered.

Biochem Cell Biol, 1987 Nov, 65(11), 997 - 1000
Construction of cosmid genomic libraries for the normal and myopathic Syrian hamsters; McCully JD et al.; Cosmid genomic libraries from both normal and myopathic Syrian hamsters have been constructed . MboI was used to generate 35- to 50-kilobase DNA fragments which were isolated from a 5-25% NaCl gradient . The 35- to 50-kilobase DNA fragments were ligated to the cosmid vector pCV108 and packaged into Escherichia coli DK1 . Approximately 3 X 10(5) - 4 X 10(5) clones were obtained per microgram of ligated DNA . Thirteen clones have been isolated from 2 X 10(5) colonies using a cardiac myosin heavy chain clone as a probe . Restriction maps of two of these clones are presented here.

Am J Vet Res, 1987 Nov, 48(11), 1577 - 83
A vaccine strain of pseudorabies virus with deletions in the thymidine kinase and glycoprotein X genes; Marchioli CC et al.; A pseudorabies virus (PRV) mutant with deletions in genes for glycoprotein X (gX) and thymidine kinase, designated delta GX delta TK, was constructed and evaluated as a vaccine for protecting swine against PRV-induced mortality . Doses greater than or equal to 10(3) plaque-forming units (PFU) of this strain given to mice provided protection from challenge exposure with virulent PRV . Sera tested from mice inoculated with delta GX delta TK had high titers of neutralizing antibody to PRV, but reactivity in the same sera was not significantly different from that in sera from noninoculated mice (controls) when sera from both groups were evaluated by use of an ELISA with gX antigen produced in Escherichia coli . Compared with noninoculated pigs (controls), those given delta GX delta TK (greater than or equal to 10(2) PFU) were protected completely from lethal challenge exposure, without experiencing adverse effects on weight gain and with reduction of shedding of virulent challenge virus . Serotest results indicated that, although inoculated pigs responded with strong neutralizing antibody titers, the response of delta GX delta TK-inoculated pigs to gX, as determined by ELISA before challenge exposure, was not significantly greater than the ELISA values obtained from control pigs . The ELISA values from a group of pigs inoculated with a commercially available vaccine were significantly (P less than 0.05) higher than those of control pigs . The experimental vaccine, delta GX delta TK, was avirulent for mice, swine, and sheep, but was mildly virulent for calves (mortality, 1 of 12) and more virulent for dogs (mortality, 3 of 6) and cats (mortality, 2 of 6).(ABSTRACT TRUNCATED AT 250 WORDS)

Anal Biochem, 1987 Nov 1, 166(2), 284 - 6
Assay of nucleoside-diphosphate kinase activity by the coupled nucleotidyltransferase; Sato NL; A method coupled with Escherichia coli RNA polymerase is described for assaying nucleoside-diphosphate kinase activity . The principle of the procedure is indirect but allows processing of a large number of samples by employing filter-paper disk techniques.

EMBO J, 1987 Nov, 6(11), 3465 - 70
Topology analysis of the SecY protein, an integral membrane protein involved in protein export in Escherichia coli; Akiyama Y et al.; The secY (prlA) gene product is an essential component of the Escherichia coli cytoplasmic membrane, and its function is required for the translocation of exocytoplasmic proteins across the membrane . We have analyzed the orientation of the SecY protein in the membrane by examining the hydropathic character of its amino acid sequence, by testing its susceptibility to proteases added to each side of the membrane, and by characterizing SecY-PhoA (alkaline phosphatase) hybrid proteins constructed by TnphoA transpositions . The orientation of the PhoA portion of the hybrid protein with respect to the membrane was inferred from its enzymatic activity as well as sensitivity to external proteases . The results suggest that SecY contains 10 transmembrane segments, five periplasmically exposed parts, and six cytoplasmic regions including the amino- and carboxyterminal regions.

Proc Natl Acad Sci U S A, 1987 Nov, 84(22), 8049 - 53
Genetic analysis of the interaction of the insertion sequence IS903 transposase with its terminal inverted repeats; Derbyshire KM et al.; The insertion sequence IS903 has perfect, 18-base-pair terminal repeats that are the presumed binding sites of its transposase . We have isolated mutations throughout this inverted repeat and analyzed their effect on transposition . We show that every position in the inverted repeat (with the possible exception of position 4) is important for efficient transposition . Furthermore, various substitutions at a single position can have a wide range of effects . Analysis of these hierarchical effects suggests that transposase contacts the minor groove in the region from position 13 to position 16 but makes major groove (or more complex) interactions with the outer portion of the inverted repeat . Our data indicate that the transposase exhibits relaxed specificity for the "second" end of a transposed segment; the defect in transposition of virtually all mutant inverted repeats can be rescued by a wild-type end . However, this rescue exhibits a pronounced position effect; in most cases, it is efficient only when the wild-type end is close to the 3' end of the transposase gene . This confirms the cis-acting nature of the transposase protein and suggests the initial transposase-inverted repeat interaction is the rate-limiting step in transposition . From the behavior of transposons with one mutant and one wild-type end, we infer that the inverted repeat contains two functional domains--one for initial complex formation with transposase and the other for effective completion of transpositional recombination . To support this hypothesis we show that an end with a mutation in one domain can significantly rescue an end with a mutation in the other domain.

Proc Natl Acad Sci U S A, 1987 Nov, 84(22), 8021 - 5
Isolation of temperature-sensitive Abelson virus mutants by site-directed mutagenesis; Engelman A et al.; Mutants of Abelson virus encoding temperature-sensitive protein-tyrosine kinase (EC 2.7.1.112) were created by site-directed mutagenesis using sequence information from temperature-sensitive mutants of the related v-src oncogene . Expression of these two independent mutations in Escherichia coli resulted in reduced phosphorylation of the mutant proteins at high temperature . Viruses containing one of the mutations induced conditional transformation of both NIH 3T3 and lymphoid cells when expressed in the context of a truncated transforming protein . These results underscore the functional homology between protein-tyrosine kinases and suggest that transfer of mutations within a related gene family may provide a rapid method to create mutants.

Proc Natl Acad Sci U S A, 1987 Nov, 84(22), 7876 - 80
Efficient Tn10 transposition into a DNA insertion hot spot in vivo requires the 5-methyl groups of symmetrically disposed thymines within the hot-spot consensus sequence; Lee SY et al.; Transposon Tn10 inserts preferentially at particular insertion "hot spots" that share a symmetrical 6-base-pair consensus sequence: 5' GCTNAGC 3' . The protein that recognizes this sequence is not known but is likely to be the Tn10-encoded transposase protein . We present evidence that the 5-methyl groups of the two thymines in this sequence are essential for efficient transposon insertion; in their absence the sequence is still recognized, but at lower efficiency . We have reached this conclusion by examination of a specific hot spot whose sequence is 5' GCCAGGC 3' . The innermost cytosines of this sequence happen to be substrates for methylation at their 5 positions by the bacterial dcm-encoded methylase . We find that Tn10 transposes into this site 15 times more frequently in a Dcm+ host than in a Dcm- host; in the Dcm- host, insertions still occur, but at a low frequency . Thus, at this site, the absence of pyrimidine 5-methyl groups at the third positions of the consensus sequence is sufficient to convert a strong insertion hot spot into a weaker but still recognizable hot spot . This observation supports the general proposition, suggested previously by comparisons among consensus sequences, that the presence or absence of these 5-methyl groups is one major feature that can make the difference between a strong and a weak Tn10 insertion hot spot.

Proc Natl Acad Sci U S A, 1987 Nov, 84(22), 7807 - 11
Identification of a transposon Tn7-dependent DNA-binding activity that recognizes the ends of Tn7; McKown RL et al.; The bacterial transposon Tn7 is distinguished by its capacity for high-frequency transposition to a specific site in the Escherichia coli chromosome . tnsB is one of the five Tn7-encoded transposition genes . We have identified in vitro a tnsB-dependent DNA binding activity that interacts specifically with cis-acting transposition sequences at the Tn7 termini . Although the left and right termini of Tn7 are structurally distinct, each end contains several copies of a closely homologous 22-base-pair sequence . We present results indicating that this 22-base-pair repeat sequence is recognized by the tnsB-dependent binding activity.

J Gen Virol, 1987 Nov, 68 ( Pt 11), 2975 - 80
Expression of bovine rotavirus neutralization antigen in Escherichia coli; Francavilla M et al.; A 646 bp fragment derived from a full length cDNA clone of genomic segment 9 of bovine rotavirus (NCDV strain) was inserted into Escherichia coli expression plasmid pEX1 . The fragment encodes amino acids 50 to 265 of the major vital neutralization antigen VP7, a 326 amino acid long outer shell glycoprotein . Several transformed bacterial clones were isolated in which the recombinant plasmid directed the synthesis of a cro-beta-galactosidase-VP7 fusion protein that was recognized by rabbit polyclonal antibodies against NCDV rotavirus . Sera from rabbits immunized with the fusion protein specifically reacted with VP7 among NCDV virion polypeptides . The chimeric polypeptide was also specifically recognized by two monoclonal antibodies against UK strain rotavirus VP7 that exhibited virus-neutralizing activity . However, immune sera to the chimeric polypeptide showed no neutralizing activity against bovine rotavirus . These results are discussed in view of a recent report that a fusion VP7-beta-galactosidase polypeptide comprising 35 more amino acids at the carboxy terminus was able to induce neutralizing antibodies in mice to simian rotavirus SA11.

J Gen Virol, 1987 Nov, 68 ( Pt 11), 2933 - 8
Identification of human papillomavirus type 16 E7 protein by monoclonal antibodies; Oltersdorf T et al.; A number of human papillomavirus (HPV) type 16 proteins have recently been identified in human cervical carcinoma cell lines using polyclonal antisera against papillomavirus gene products expressed in Escherichia coli . E7 protein has been found to be the most abundant papillomavirus protein in these cells . Here we describe a panel of monoclonal antibodies recognizing a 15K Mr non-glycosylated cytoplasmic HPV-16 E7 protein . One of the antibodies cross-reacted with HPV-18 E7 protein.

Virology, 1987 Nov, 161(1), 138 - 44
Nucleotide sequence and expression in Escherichia coli of the gene encoding the nonstructural protein NCVP2 of bovine rotavirus; Bremont M et al.; Cloned DNA copy of rotavirus genome segment 5 from bovine rotavirus RF strain has been used to determine the nucleotide sequence of the gene that encodes for the nonstructural viral protein NCVP2 . The sequence data indicated that segment 5 consists of 1581 base pairs and is A + T rich (66%) . The positive strand of segment 5 contains a single open reading frame that extends 491 codons and possesses 5'- and 3'-terminal untranslated regions of 32 and 73 base pairs, respectively . The first AUG conforms to the Kozak consensus sequence and if utilized, would yield a protein having a calculated molecular weight of 58,654, slightly higher than the apparent molecular weight of NCVP2 (MW 54,000) . Although it is not evident whether the gene product is glycosylated, four potential glycosylation sites were found at positions 50, 168, 403, and 438 . NCVP2 has been expressed in Escherichia coli using the inducible expression vector pKK233-2 . Following IPTG induction high levels of full-length nonfused proteins were synthesized and accumulated in induced cells.

Proc Natl Acad Sci U S A, 1987 Nov, 84(21), 7448 - 52
Biochemical evidence for the secY24 defect in Escherichia coli protein translocation and its suppression by soluble cytoplasmic factors; Fandl JP et al.; The secY (prlA) gene product is an integral membrane protein that has been identified genetically as one of the central components of the Escherichia coli protein translocation machinery . We have examined the effect of the secY24 (temperature-sensitive) mutation on the protein translocation activity of E . coli inverted membrane vesicles . Vesicles isolated from cells carrying this allele and grown at the nonpermissive temperature (42 degrees C) were less than 1% as active in translocation as vesicles isolated from an isogenic secY+ strain under the same conditions . Vesicles from the mutant strain grown at the permissive temperature (32 degrees C) were partially active, but those vesicles preincubated at 40 degrees C lost 90% of their activity . Moreover, the secY24 translocation defect on in vivo- or in vitro-inactivated vesicles was suppressed, or compensated, by an S300 soluble fraction from wild-type cells or from secY24 cells grown at nonpermissive temperature . The suppressing factor(s) was heat-labile and sensitive to proteinase K . These results provide biochemical evidence for the essential role of SecY in the translocation process and indicate that the translocation defect of SecY24 membranes can be compensated for by supplementing with additional soluble cytoplasmic proteins.

Mutat Res, 1987 Nov, 184(3), 181 - 6
Effects of the Escherichia coli recF suppressor mutation, recA801, on recF-dependent DNA-repair associated phenomena; Volkert MR et al.; In recb recC sbcB mutants genetic recombination is dependent upon the recF gene . recA801, recA802 and recA803 (formerly called srfA mutations) were originally isolated as mutations that suppress recombination deficiency caused by a recF mutation in a recB recC sbcB genetic background . Since the recA801 mutation also suppressed some of the UV sensitivity due to recF143, we sought to determine what DNA-repair pathways were actually being restored by the recA801 mutation in this genetic background . In this paper we show that the suppression of recF143 by recA801 does not extend to the recF143-mediated defects in induced repair of UV-damaged phages . In addition, we show that recA801 suppresses only slightly the recF143-associated defect in induced expression of the SOS-regulated muc genes of pKM101 . These results suggest that recA801 suppresses primarily the RecF pathway of recombinational repair.

J Virol, 1987 Nov, 61(11), 3645 - 7
Expression of hepatitis A virus cDNA in Escherichia coli: antigenic VP1 recombinant protein; Ostermayr R et al.; The genome of hepatitis A virus (HAV) was reverse transcribed into cDNA and molecularly cloned . cDNA clones coding for the capsid protein VP1 that carries the major HAV antigen were cloned into the expression vector pUR290 and expressed in Escherichia coli . The recombinant fusion protein reacted in an immunoblot with rabbit anti-HAV serum, suggesting that it possesses HAV antigenicity.

J Bacteriol, 1987 Nov, 169(11), 5224 - 30
Characterization of high-level expression and sequencing of the Escherichia coli K-12 cynS gene encoding cyanase; Sung YC et al.; Restriction fragments containing the gene encoding cyanase, cynS, without its transcriptional regulatory sequences were placed downstream of lac and tac promoters in various pUC derivatives to maximize production of cyanase . Plasmid pSJ105, which contains the cynS gene and an upstream open reading frame, gave the highest expression of cyanase . Approximately 50% of the total soluble protein in stationary-phase cultures of a lac-deleted strain containing plasmid pSJ105 was cyanase . The inserted DNA fragment of pSJ105 was transferred into pUC18 derivatives that contain a hybrid tac promoter, instead of the lac promoter, and a strong terminator to generate pSJ124 . Stationary-phase cultures of JM101 containing plasmid pSJ124 overexpressed a similar level of cyanase . In JM101(pSJ124), maximum production of cyanase could be obtained either by induction with isopropyl-beta-D-thiogalactopyranoside (IPTG) for 3 h or by growth without IPTG into late stationary phase . The latter conditions resulted in a 10- to 20-fold increase in plasmid content and presumably titration of the lac repressor . The nucleotide sequence of the cloned cynS gene from Escherichia coli K-12 was determined . The predicted amino acid sequence differed from the known amino acid sequence of cyanase isolated from a B strain by four residues . However, overexpressed cyanase was purified to homogeneity, and a comparison of the enzymes from the two sources indicated that they did not differ with respect to physical and kinetic properties . The cynS gene was located next to the lac operon, and the direction of cynS transcription was opposite that of lac.

J Bacteriol, 1987 Nov, 169(11), 5180 - 7
Genetics of type IIa heat-labile enterotoxin of Escherichia coli: operon fusions, nucleotide sequence, and hybridization studies; Pickett CL et al.; Operon fusions for the Escherichia coli heat-labile enterotoxin type IIa (LT-IIa) operon were isolated and characterized . The LT-IIa genes are organized in a transcriptional unit similar to those of cholera toxin (CT) and the closely related E . coli heat-labile toxin type I (LT-I, with subtypes LTh-I and LTp-I) . The nucleotide sequence of the LT-IIa genes was determined and compared with the sequences of LTh-I and CT . The A subunit gene of LT-IIa was found to be 57% homologous with the A subunit gene of LTh-I and 55% homologous with the A gene of CT . Most of the homology derived from the region of the A gene which encodes the A1 fragment . The B gene of LT-IIa was not homologous with the B gene of LTh-I or CT . DNA probes containing various portions of the LT-IIa genes and adjacent sequences were used for hybridization studies with restriction endonuclease fragments of DNA from a collection of LT-II-producing strains . These studies showed that a probe containing much of the A subunit gene hybridized well to DNA from the various strains, but a probe for the B subunit gene did not.

J Bacteriol, 1987 Nov, 169(11), 5078 - 86
Novel incompatibility and partition loci for the REPI replication region of plasmid ColV-K30; Perez-Casal JF et al.; The minimum pColV-K30 REPI region necessary for replication was located within a ca . 1.3-kilobase DNA segment . Adjacent to the essential replication sequences, there are two DNA regions that express incompatibility with plasmids containing the F secondary replicon of the F EcoRI fragment f7 . One of these regions corresponds to incE, already described in that F plasmid fragment which expresses incompatibility with f7-containing plasmids . The other is a novel sequence that we designated incF, which confers incompatibility with REPI, P307, and f7 derivatives, cis-acting pColV-K30 sequences conferring stability to REPI-containing plasmids were also identified and localized noncontiguous to REPI, ca . 20 kilobases downstream from the aerobactin iron transport genes, which were thus flanked by REPI and its partition (par) sequences.

Ann Inst Pasteur Microbiol, 1987 Nov-Dec, 138(6), 681 - 92
{Specific detection of the thermolabile enterotoxin of Escherichia coli in human feces by competitive immunoenzyme tests in the presence of protease inhibitors}; Germani Y et al.; Two simple two-step competitive enzyme-linked immunoassays for human E . coli heat-labile enterotoxin (LTh) employing microtitration plates coated with rabbit anti-LTh antibody (ELISA) or GM1 ganglioside (GM1-ELISA) are described . LTh of the test sample competed with the same toxin coupled with horse-radish peroxidase . ELISA and GM1-ELISA were able to detect, respectively, as low as 5 ng and 6.5 ng TLh/ml and up to 9 and 11 micrograms TLh/ml . Both techniques were applied to the study of 167 infant diarrhoeas; ETEC producing LTh were identified in 17 diarrhoeal stools . When the faeces were diluted in phosphate buffer, only 17.6% (3 stools) and 29% (5 stools) of LTh-positive faeces were identified in ELISA and GM1-ELISA . When the stools were diluted with phenylmethylsulphonyl fluoride (PMSF), a synthetic protease inhibitor, 82% (14 stools) and 88% (15 stools) of LTh-positive stool supernatants were detected . Aprotinin, another protease inhibitor, was without effect and foetal calf serum, horse serum and bovine serum albumin enabled detection of only a low percentage of LTh-positive stools.

Ann Inst Pasteur Microbiol, 1987 Nov-Dec, 138(6), 667 - 80
{Direct detection in diarrheal stools of Escherichia coli adhesion factors CFA/I, CFA/II and E8775 by the india ink immune reaction}; Germani Y et al.; An India ink immune reaction was used for the direct detection, in diarrhoeal stools, of Escherichia coli possessing the CFA/I, CFA/II and E8775 fimbriae . With this method, a presumptive diagnosis of enterotoxigenic E . coli (ETEC) can rapidly be made . Staining required the bivalency of rabbit anti-fimbriae IgG; the F(ab')2 fragment was necessary . The reaction was impossible with purified Fab fragment . A comparative study of this technique and detection by culture of ETEC strains showed good correlation when the India ink immune reaction was performed at the beginning of infection.

Arch Int Physiol Biochim, 1987 Nov, 95(4), 361 - 71
Effect of Escherichia coli heat-stable enterotoxin on cell volume and intracellular inorganic ions of rat intestinal cells; Gilles-Baillien M et al.; 1.--Electron micrographs of rat jejunum mucosa incubated for 1 h in the presence of Escheria coli heat-stable enterotoxin (STa) in the lumen shows alterations of villous cells as well as of crypt cells . The brush border of mature enterocytes is partially desintegrated and covered with a thick mucus . Crypts are occupied on half of their height by cells very similar to Paneth cells, loaded with numerous large dark inclusions . 2.--Cell volume and intracellular inorganic ion concentrations have been estimated in mucosal scrapings of jejunum sacs, incubated in vitro for 1 or 3 h . The quick action (1 h of incubation) of STa is a swelling of the intestinal calls accompanied by an increase in Na+, Cl- and Ca2+ intracellular concentrations and a decrease in the K+ and Mg2+ ones . The delayed action (3 h of incubation) is an increase of extracellular space and a decrease in cell volume; and at the same time the intracellular concentration of Na+, Cl-, K+, Ca2+ and Mg2+ is augmented . 3.--After 3 h of incubation intestinal cells from the other levels of intestine (duodenum, ileum and colon) show the same variations in cell volume and intracellular inorganic ion concentrations under the influence of STa, as those recorded in the jejunum . 4.--The present work favours the hypothesis that all intestinal cells, villous or cryptic, are involved in the alteration of fluid ion transport ending in diarrhea.

Mol Biol (Mosk), 1987 Nov-Dec, 21(6), 1664 - 70
{Degradation of nucleic acids during generation of superoxide-anion in the presence of copper ions}; Vodolazkin DI et al.; The efficiency of Escherichia coli nucleic acids samples: covalently closed circular DNA, linear chromosomal DNA, total RNA degradation mediated by the action of high oxygen pressure; hydrochloric hydroxylamine in alkaline conditions in the presence of cooper ions and in analogous conditions without cooper ions was studied . The nativity of nucleic acids was determined by means of fluorometric analysis of nucleic acids/ethidium bromide complexes . Experiments revealed, that the destructive effect of active oxygen species decreased in the following order: NH2OH.HCl in alkaline conditions in the presence of copper ions-NH2.HCl in alkaline conditions--high pressure of pure oxygen . The stability of nucleic acids decreased in the following order: covalently closed circular DNA-linear DNA-RNA.

Mol Biol (Mosk), 1987 Nov-Dec, 21(6), 1504 - 12
{Correlation between the effectiveness of translation initiation and secondary structure of mRNA in the hybrid gene cro-lacIZ}; Khudiakov IuE et al.; A set of plasmids containing short DNA deletions in the N-part of cro-lacIZ coding zone as constructed using Bal31 nuclease . Development of models of mRNA secondary structure was carried out stepwise beginning from the 5'-end by taking into consideration the hairpins first formed during mRNA synthesis . Comparison of the results of mRNA secondary structure determination and protein production analysis demonstrated a correlation between the efficiency of translation initiation and the appearance of a single-stranded region upon disruption of the mRNA local secondary structure in the translation initiation zone generated by ribosomes . These results confirm the suggestion of the central role played by the Shine-Dalgarno sequence in the generation of a single-stranded region . Some plasmids from the set are supposed to determine protein synthesis by a translation reinitiation mechanism in the absence of Shine--Dalgarno interaction . In this case, correlation between reinitiation efficiency and local disruption of the mRNA secondary structure by the terminating ribosome was also observed . The terminating ribosome that forms the single-stranded region near the initiation codon fulfils the major function of the Shine--Dalgarno interaction . In addition, the possible effect of another mRNA secondary structure region on the translation initiation efficiency is discussed.

Antibiot Med Biotekhnol, 1987 Nov, 32(11), 846 - 9
{Immunochemical study of modified forms of L-asparaginase}; Iulaev MF et al.; Certain catalytic and immunological properties of L-asparaginase modified by polyglucin were studied . It was shown that the modified forms of L-asparaginase maintained high catalytic activity (Km 0.80.10(-5)-1.89.10(-5) M) and at the same time appeared to be more resistant to inactivation under the effect of antibodies.

Biochem Int, 1987 Nov, 15(5), 1051 - 6
Molecular cloning of cDNA for rat liver general acyl CoA dehydrogenase and homology between the rat liver and pig kidney enzymes; Inagaki T et al.; cDNA clone for general acyl CoA dehydrogenase (GAD) was isolated from a rat liver cDNA expression library in lambda gt11 using anti-pig kidney GAD antibody . Size of the isolated cDNA was estimated to be 1.5-1.6 kb . By immunological analysis of fusion protein and epitope selection, the cDNA clone was identified as that containing the GAD gene . Partial amino acid sequence deduced from nucleotide sequence of the cDNA coincided with that of the pig kidney enzyme . The antibody cross-reacted with rat liver enzyme and molecular weights of these enzyme proteins were shown to be almost the same . All these results indicate that rat liver GAD shares a common structure with pig kidney enzyme.

Mol Biochem Parasitol, 1987 Nov, 26(1-2), 203 - 14
Structure and expression of the knob-associated histidine-rich protein of Plasmodium falciparum; Ellis J et al.; cDNA clones encoding 473 amino acids of the knob-associated histidine-rich protein (PfHRPI) of Plasmodium falciparum clone FCR-3/A2 (Gambia) have been isolated and sequenced . Although a short region close to the amino terminus of the predicted sequence contains three blocks of six, seven or nine consecutive histidine residues, the most abundant amino acid is lysine . The predicted sequence contains a putative amino-terminal signal sequence and two potential asparagine glycosylation sites . A 1284 bp Sau3A cDNA fragment was expressed in Escherichia coli as a fusion protein that was recognized by an anti-PfHRPI monoclonal antibody . Pulsed field gradient electrophoresis indicated that the PfHRPI gene is located on chromosome 2 . The PfHRPI gene was present, apparently intact, in knobless parasites derived from one uncloned P . falciparum isolate (St . Lucia) . In a knobless derivative of another uncloned isolate (Malayan Camp) and in a cloned knobless line (FCR-3/D4) of a third isolate (Gambian), that part of the gene covered by the cDNA clone has been deleted . Loss of PfHRPI expression may therefore arise via several different mechanisms of gene alteration.

Jpn J Cancer Res, 1987 Nov, 78(11), 1179 - 81
Cloning of granulocyte colony-stimulating factor cDNA from human macrophages and its expression in Escherichia coli; Komatsu Y et al.; Human granulocyte colony-stimulating factor (hG-CSF) cDNA was cloned, by using a synthetic oligonucleotide probe, from an Okayama-Berg cDNA library of lipopolysaccharide-stimulated human peripheral blood macrophages . The cDNA encodes a polypeptide with an amino acid sequence which completely matches that of the known polypeptide with hG-CSF activity derived from human tumor cell lines . Expression in E . coli of high levels of the protein (about 10% of total cellular proteins) was accomplished under control of the trp promoter, and the purified protein was proved to have hG-CSF activity . Our data provide evidence that human peripheral blood macrophages do produce hG-CSF mRNA when stimulated exogenously, suggesting they are the producer of naturally occurring hG-CSF.

Proc Natl Acad Sci U S A, 1987 Nov, 84(22), 7851 - 5
pH-sensitive immunoliposomes mediate target-cell-specific delivery and controlled expression of a foreign gene in mouse; Wang CY et al.; A plasmid containing the Escherichia coli chloramphenicol acetyltransferase (CAT) gene under the control of a mammalian cAMP-regulated promoter was entrapped in H-2Kk antibody-coated liposomes composed of dioleoyl phosphatidylethanolamine, cholesterol, and oleic acid (pH-sensitive immunoliposomes) . The entrapped or free DNA was injected intraperitoneally into immunodeficient (nude) BALB/c mice bearing ascites tumor generated by H-2Kk-positive RDM-4 lymphoma cells . About 20% of the injected immunoliposomes were taken up by the target RDM-4 cells . Uptake was much less when liposomes without antibody were used . The presence of the targeting antibody on liposomes also significantly decreased the nonspecific uptake of liposomes by the spleen . Significant CAT enzyme activity was detected in RDM-4 cells from mice treated with DNA entrapped in the pH-sensitive immunoliposomes . Furthermore, CAT expression in RDM-4 cells was under the control of cAMP, as only the cells from mice injected with 8-bromo-cAMP and 3-isobutyl-1-methylxanthine showed CAT activity . CAT activity in liver and spleen was much lower (by factors of 12 and 5, respectively) than in the RDM-4 cells, and the activities in these reticuloendothelial organs were not regulated by cAMP . CAT activity in RDM-4 cells from mice injected with DNA entrapped in pH-insensitive immunoliposomes (containing phosphatidylcholine in place of phosphatidylethanolamine) was approximately one-fourth that in RDM-4 cells from mice injected with pH-sensitive immunoliposomes, indicating the superior delivery efficiency of the pH-sensitive liposomes . These results are discussed in terms of the DNA-carrier potential of immunoliposomes in therapy of cancer and genetic diseases.

J Neurosci, 1987 Nov, 7(11), 3554 - 60
Phosphatase and carbocyanine dye binding define different types of phosphate groups in mammalian neurofilaments; Ksiezak-Reding H et al.; The phosphorylation state of human and bovine spinal cord neurofilaments (NF) was studied by direct phosphate analysis and carbocyanine dye ("Stains-all") binding to NF polypeptides resolved on SDS-polyacrylamide gels . Electrophoretically purified NF-H (200 kDa), NF-M (160 kDa), and NF-L (68 kDa) of human origin contained 24, 18, and 4 mol phosphate/mol protein, whereas bovine NF contained 53, 23, and 5 mol phosphate/mol protein, respectively . Incubation of NF preparations with E . coli alkaline phosphatase removed about 55% of the phosphate from NF-H, about 30% of the phosphate from both human and bovine NF-M, but did not change the phosphate content of NF-L . This treatment also inhibited or substantially reduced the binding of electroblotted NF-H and NF-M to 2 anti-NF monoclonal antibodies known to recognize phosphorylated sites on projection side arms . "Stains-all" was found to be a very sensitive probe for detection of phosphorylated cytoskeletal proteins . Without the phosphatase treatment, NF and other phosphoproteins, MAP1, MAP2, tubulin, and tau, all bound the carbocyanine dye on SDS gels, forming blue dye-protein complexes . Measured densitometrically at 615 nm, the staining intensity (relative units/mol protein) was 9, 9, and 3 for human and 10, 13, and 6 for bovine NF-H, NF-M, and NF-L, respectively . NF-H bound the dye less efficiently than was expected from its phosphate content . After phosphatase treatment, NF-H, with half of its phosphate residues remaining, no longer formed blue complex with "Stains-all," the staining intensity of NF-M decreased by 20-40%, and the staining of NF-L was not changed.(ABSTRACT TRUNCATED AT 250 WORDS)

Arch Dermatol, 1987 Nov, 123(11), 1494 - 8
Anthralin inhibits elevated levels of thioredoxin reductase in psoriasis . A new mode of action for this drug; Schallreuter KU et al.; Membrane-associated thioredoxin reductase (TR) activity has been measured in 3-mm punch biopsy specimens from psoriatic and uninvolved skin in eight patients with chronic plaque-type psoriasis vulgaris . The mean specific activity for this free radical reducing enzyme in psoriatic vs uninvolved skin was 28.5 +/- 8.0 U vs 16.8 +/- 4.25 U . Because TR contains two reactive thiolate groups at its active site, this enzyme reacts with anthralin to form a covalent anthralin-TR complex causing irreversible enzyme deactivation . This mode of action for anthralin was confirmed by using pure TR from Escherichia coli . Keratinocyte cell cultures, grown from normal and psoriatic skin of one donor, revealed 24% and 42% inhibition of cell surface TR activity, respectively, in the presence of 2 X 10(-5)M anthralin . Time-dependent topical application of anthralin on guinea pig skin gave 70% inhibition of TR with concentrations of 0.25% to 1.0% after 24 hours in open and occlusive applications of the drug . Short contact with 1% anthralin showed 70% inhibition after 120 minutes.

Virology, 1987 Nov, 161(1), 145 - 52
Deletion mapping and expression in Escherichia coli of the large genomic segment of a birnavirus; Azad AA et al.; The large genomic segment of infectious bursal disease virus encodes a polyprotein in which the viral polypeptides are present in the following order: N-VP2-VP4-VP3-C . Expression in Escherichia coli of the large segment results in the processing of the polyprotein . The expression product reacts with a virus neutralizing and protective monoclonal antibody that recognizes a conformational epitope on the surface of the virus . Different regions of the large genomic segment were deleted at defined restriction sites and the truncated fragments were ligated to various expression vectors for high-level expression in E . coli . The expressed proteins were probed with three different monoclonal antibodies that recognize epitopes encoded by different regions of the large genomic segment . These deletion mapping studies suggest that VP4 is involved in the processing of the precursor polyprotein, and the conformational epitope recognized by the virus neutralizing monoclonal antibody is present within VP2.

Proc Natl Acad Sci U S A, 1987 Nov, 84(21), 7363 - 7
Transcriptional control of the endogenous MYC protooncogene by antisense RNA; Yokoyama K et al.; A plasmid carrying antisense human MYC DNA and the gene encoding Escherichia coli xanthine/guanine phosphoribosyltransferase (Ecogpt) was introduced into human promyelocytic leukemia cell line HL-60 by protoplast fusion . High-level expression of antisense MYC RNA was obtained by selecting cells resistant to progressively higher levels of mycophenolic acid over a period of greater than 6 months . The constitutive production of MYC protein in clones producing high levels of antisense MYC RNA was reduced by 70% compared to parental HL-60 cells . Inhibition of MYC expression was observed not only at the translational but also at the transcriptional level, implying that antisense RNA can regulate transcription of the MYC gene . The Pst I-Pvu II fragment (920 base pairs) of the MYC leader sequence is the primary transcriptional target of the antisense RNA . The suppression of endogenous MYC gene expression by antisense RNA decreases cell proliferation and triggers monocytic differentiation.

Biochem Biophys Res Commun, 1987 Oct 29, 148(2), 546 - 52
Cloning and sequence of cDNA encoding a peptide C-terminal alpha-amidating enzyme from Xenopus laevis; Mizuno K et al.; The C-terminal alpha-amide formation of the peptides is one of the most important events of prohormone processing . We have recently isolated an alpha-amidating enzyme, AE-I, from Xenopus laevis skin, which is the only enzyme ever purified to homogeneity . In this study, we report cloning and sequence of cDNA encoding AE-I . Our results indicate that enzyme AE-I is initially synthesized as a precursor with 400 amino acid residues, which is further processed to the mature enzyme consisting of 344 residues . Preliminary expression in E . coli of the cDNA corresponding to AE-I was found to produce an enzyme with appreciable alpha-amidating activity.

Biochem Biophys Res Commun, 1987 Oct 29, 148(2), 629 - 35
Improvement of cytotoxicity of tumor necrosis factor (TNF) by increase in basicity of its N-terminal region; Soma G et al.; Two new recombinant TNFs (named rTNF-Scw1 and -Scw2) with higher basicity than conventional recombinant human TNF-alpha (rTNF-alpha) in the N-terminal region were constructed . Their sequences were constructed based on those of partially purified cytotoxic factors from the culture supernatant of acute monocytic leukemia cells THP-1, which unlike rTNF-alpha are cytotoxic to T24 bladder carcinoma cells in vitro . These new rTNF-Ss showed a broader cytotoxicity to tumor cells than rTNF-alpha . This increase in the basicity of the N-terminal region over that of conventional TNF significantly increased the cytotoxicity on tumor cells in vivo as well as in vitro.

Nucleic Acids Res, 1987 Oct 26, 15(20), 8479 - 99
The symbiotic nitrogen fixation regulatory operon (fixRnifA) of Bradyrhizobium japonicum is expressed aerobically and is subject to a novel, nifA-independent type of activation; Thony B et al.; The Bradyrhizobium japonicum N2 fixation regulatory gene, nifA, was sequenced and its transcription start site determined . Between the start of transcription and the nifA gene an open reading frame of 278 codons was found and named fixR . A deletion in fixR which allowed transcription into nifA resulted in a 50% reduced Fix activity . The fixRnifA operon was expressed in soybean root nodules, in cultures grown anaerobically with nitrate as terminal electron acceptor, in microaerobic cultures, and in aerobic cultures . The transcription start site (+1) was preceded by a characteristic nif(-24/-12)-type promoter consensus sequence . Double base-pair exchanges in the -12 but not in the -24 region resulted in a 'promoter-down' phenotype . A promoter-upstream DNA region between -50 and -148 was essential for maximal promoter activity . Expression from the promoter was not dependent on nifA . We conclude that the fixRnifA promoter is positively controlled, and that it requires a newly postulated transcriptional factor in order to become activated.

Nucleic Acids Res, 1987 Oct 26, 15(20), 8319 - 31
DNA synthesis is initiated at two positions within the origin of replication of plasmid R1162; Lin LS et al.; DNA synthesis of broad host-range plasmid R1162 is initiated from two positions, flanking a large (40 bp stem, 40 bp loop) inverted repeat . Each start-point is located within a highly conserved, but oppositely oriented, 10 base-pair sequence . Synthesis from the two positions converges within the intervening inverted repeat . An analysis of deletions suggests that both start positions must be present for synthesis . A model describing possible early events in replication of plasmid R1162 is presented.

Nucleic Acids Res, 1987 Oct 26, 15(20), 8205 - 15
Mutation spectrum in Escherichia coli DNA mismatch repair deficient (mutH) strain; Rewinski C et al.; The Dam-directed post-replicative mismatch repair system of Escherichia coli removes base pair mismatches from DNA . The products of the mutH, mutL and mutS genes, among others, are required for efficient mismatch repair . Absence of any of these gene products leads to persistence of mismatches in DNA with a resultant increase in spontaneous mutation rate . To determine the specificity of the mismatch repair system in vivo we have isolated and characterized 47 independent mutations from a mutH strain in the plasmid borne mnt repressor gene . The major class of mutations comprises AT to GC transitions that occur within six base pairs of the only two 5'-GATC-3' sequences in the mnt gene . In the wild type control strain, insertion of the IS1 element was the major spontaneous mutational event . A prediction of the Dam-directed mismatch repair model, that the mutation spectra of dam and mutH strains should be the same, was confirmed.

Nucleic Acids Res, 1987 Oct 26, 15(20), 8249 - 66
Nucleotide sequence of the DdeI restriction-modification system and characterization of the methylase protein; Sznyter LA et al.; The DdeI restriction-modification system was previously cloned and has been maintained in E . coli on two separate and compatible plasmids (1) . The nucleotide sequence of the endonuclease and methylase genes has now been determined; it predicts proteins of 240 amino acids, Mr = 27,808, and 415 amino acids, Mr = 47,081, respectively . Inspection of the DNA sequence shows that the 3' end of the methylase gene had been deleted during cloning . The clone containing the complete methylase gene was made and compared to that containing the truncated gene; only clones containing the truncated form support the endonuclease gene in E . coli . Bal-31 deletion studies show that methylase expression in the Dde clones is also dependent upon orientation of the gene with respect to pBR322 . The truncated and complete forms of the methylase protein were purified and compared; the truncated form appears to be more stable and active in vitro . Finally, comparison of the deduced amino acid sequence of M . DdeI with that of other known cytosine methylases shows significant regions of homology.

J Biol Chem, 1987 Oct 25, 262(30), 14648 - 54
Evidence for in vitro translesion DNA synthesis past a site-specific aminofluorene adduct; Michaels ML et al.; The ability of Escherichia coli DNA polymerase I and T7 DNA polymerase to bypass bulky C-8 guanyl-2-aminofluorene adducts in DNA was studied by in vitro DNA synthesis reactions on a site-specific aminofluorene-modified M13mp9 template . This site-specifically modified DNA was prepared by ligating an oligonucleotide containing a single aminofluorene adduct into a gapped heteroduplex of M13mp9 DNA (Johnson, D . L., Reid, T . M., Lee, M.-S., King, C . M., and Romano, L . J . (1986) Biochemistry 25, 449-456) . The resulting covalently closed duplex DNA molecule was then cleaved with a restriction endonuclease, denatured, and annealed to a primer on the 3' side of the adduct to form a template specifically designed to study bypass . In this system, any synthesis that was not blocked by the bulky aminofluorene adduct would proceed to the 5' terminus of the single-stranded template, while synthesis interrupted by the adduct would terminate at or near the adduct location . We have measured DNA synthesis on this template and find that the amount of radiolabeled nucleotide incorporated by either E . coli DNA polymerase I (large fragment) or T7 DNA polymerase was much greater than would be predicted if the aminofluorene adduct were an absolute block to DNA synthesis . Furthermore, the products of similar reactions electrophoresed on polyacrylamide gels showed conclusively that the majority of the DNA synthesized by either the T7 DNA polymerase or E . coli DNA polymerase I bypassed the aminofluorene lesion . Substitution of Mn2+ for Mg2+ as the divalent cation resulted in even higher levels of translesion synthesis.

J Biol Chem, 1987 Oct 25, 262(30), 14697 - 701
Human copper-zinc superoxide dismutase complements superoxide dismutase-deficient Escherichia coli mutants; Natvig DO et al.; An Escherichia coli double mutant, sodAsodB, that is deficient in both bacterial superoxide dismutases (Mn superoxide dismutase and iron superoxide dismutase) is unable to grow on minimal medium in the presence of oxygen and exhibits increased sensitivity to paraquat and hydrogen peroxide . Expression of the evolutionarily unrelated eukaryotic CuZn superoxide dismutase in the sodAsodB E . coli mutant results in a wild-type phenotype with respect to aerobic growth on minimal medium and in resistance to paraquat and hydrogen peroxide . This supports the hypothesis that superoxide dismutation is the in vivo function of these proteins . Analysis of the growth of sodAsodB cells containing plasmids encoding partially active CuZn superoxide dismutases, produced by in vitro mutagenesis, shows a correlation between cell growth and enzyme activity . Thus, the sodAsodB strain provides a controlled selection for varying levels of superoxide dismutase activity.

J Biol Chem, 1987 Oct 25, 262(30), 14576 - 82
Influence of negative supercoiling and of the proximity of left-handed Z-DNA on the Escherichia coli lactose repressor-operator interaction; Hsieh WT et al.; The influence of negative supercoiling and of flanking (dC-dG) tracts in either right-handed B- or left-handed Z-structures on the interaction of the Escherichia coli lac repressor was investigated . The operator was embodied within the lac control sequence, which was 95, 59, or 29 base pairs in length . Thus, the (dC-dG) regions (in either B- or Z-conformations) were at different distances from the repressor-binding site . Surprisingly, the presence of the promoter sequence (-59 to -20 relative to the +1 transcription start site) of the lac operator region increases the binding affinity of lactose repressor to the operator at high negative supercoil densities . This influence of the promoter region on the binding was abolished when the flanking (dC-dG) tracts were in the left-handed Z-DNA conformation . In contrast, minimal differences in the binding affinities were observed between plasmids containing shorter operator fragments (59 or 29 base pairs), whether the flanking (dC-dG) tracts were in right-handed B- or left-handed Z-forms . The promoter region may be directly involved in the repressor-operator complex in a previously unrecognized manner or may exert structural influence on the operator region . In general, increasing the amount of negative supercoiling increases the binding affinity and decreases the dissociation rate constants for the three operator-containing fragments, both with and without flanking Z-DNA tracts . Thus, the lac operator region possesses a previously unrecognized structural pliability, as influenced by negative supercoiling and neighboring sequences and/or conformations, which modulates its biological properties.

J Biol Chem, 1987 Oct 25, 262(30), 14441 - 7
Purification, properties, and N-terminal amino acid sequence of homogeneous Escherichia coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme; Mukherjee JJ et al.; Starting with 100 g (wet weight) of a mutant of Escherichia coli K-12 forced to grow on L-threonine as sole carbon source, we developed a 6-step procedure that provides 30-40 mg of homogeneous 2-amino-3-ketobutyrate CoA ligase (also called aminoacetone synthetase or synthase) . This ligase, which catalyzes the cleavage/condensation reaction between 2-amino-3-ketobutyrate (the presumed product of the L-threonine dehydrogenase-catalyzed reaction) and glycine + acetyl-CoA, has an apparent molecular weight approximately equal to 85,000 and consists of two identical (or nearly identical) subunits with Mr = 42,000 . Computer analysis of amino acid composition data, which gives the best fit nearest integer ratio for each residue, indicates a total of 387 amino acids/subunit with a calculated Mr = 42,093 . Stepwise Edman degradation provided the N-terminal sequence of the first 21 amino acids . It is a pyridoxal phosphate-dependent enzyme since (a) several carbonyl reagents caused greater than 90% loss of activity, (b) dialysis against buffer containing hydroxylamine resulted in 89% loss of activity coincident with an 86% decrease in absorptivity at 428 nm, (c) incubation of the apoenzyme with 20 microM pyridoxal phosphate showed a parallel recovery (greater than 90%) of activity and 428-nm absorptivity, and (d) reduction of the holoenzyme with NaBH4 resulted in complete inactivation, disappearance of a new absorption maximum at 333 nm . Strict specificity for glycine is shown but acetyl-CoA (100%), n-propionyl-CoA (127%), or n-butyryl-CoA (16%) is utilized in the condensation reaction . Apparent Km values for acetyl-CoA, n-propionyl-CoA, and glycine are 59 microM, 80 microM, and 12 mM, respectively; the pH optimum = 7.5 . Added divalent metal ions or sulfhydryl compounds inhibited catalysis of the condensation reaction.

J Immunol Methods, 1987 Oct 23, 103(1), 131 - 9
Antibodies against synthetic carboxy-terminal peptides distinguish H-ras and K-ras oncogene products p21; Ishihara H et al.; Synthetic peptides corresponding to the carboxy-terminal region of H-ras, K-ras, and N-ras oncogene product p21 proteins are used to obtain antibodies specific to each ras oncogene product . The synthetic peptides of 32 amino acids are immunogenic in rabbits without being coupled to carriers . Specific antibodies are purified by absorption of the antisera with the other peptides coupled to CH-Sepharose 4B, and antibodies reacting with all three peptides are obtained by affinity chromatography . These findings imply that antibodies specific to each peptide recognize the variable carboxy-terminal region while antibodies reacting with all three peptides recognize the constant region of the carboxy-terminal amino acid sequence of p21 proteins . The affinity-purified antibodies against H-ras and K-ras peptides are shown to react specifically with c-H-ras and v-K-ras p21 proteins expressed in E . coli and eukaryotic cells, respectively . These antibodies may be useful tools to study the functional roles of p21 carboxy-terminal domain and to detect differential expression of the family of ras oncogenes in cancerous tissues . The affinity-purified anti-N-ras peptide antibody, however, fails to react with N-ras p21 in spite of its positive reactivity with the N-ras peptide.

Science, 1987 Oct 23, 238(4826), 527 - 30
Model substrates for an RNA enzyme; McClain WH et al.; M1 RNA, the catalytic RNA subunit of Escherichia coli ribonuclease P, can cleave novel transfer RNA (tRNA) precursors that lack specific domains of the normal tRNA sequence . The smallest tRNA precursor that was cleaved efficiently retained only the domain of the amino acid acceptor stem and the T stem and loop . The importance of the 3' terminal CCA nucleotide residues in the processing of both novel and normal tRNA precursors implies that the same enzymatic function of M1 RNA is involved.

Biochemistry, 1987 Oct 20, 26(21), 6825 - 31
Does tryptophan intercalate in DNA? A comparative study of peptide binding to alternating and nonalternating A.T sequences; Rajeswari MR et al.; The interactions of a tetrapeptide, lysyltryptophylglycyllysine tert-butyl ester (KWGK), with synthetic double-stranded polynucleotides {poly(dA).poly(dT), poly{d(A-T)}, poly(rA).poly(dT), and poly(rA).poly(rU)}, Escherichia coli DNA, and single-stranded polynucleotides {poly(rA), poly(rU), poly(dA), and poly(dT)} were studied in a low-salt buffer by absorption and fluorescence spectroscopy . From fluorescence quenching data, we determined the two binding constants K1 and K2 of the two-step mechanism previously proposed for lysyltryptophyllysine binding to polynucleotides {Brun, F., Toulme, J.J., & Helene, C . (1975) Biochemistry 14, 558-563} . The first complex (PN)1 is purely due to electrostatic interactions between the lysyl residues and the phosphate groups . The second complex (PN)2 involves an additional stacking of the indole moiety of the tryptophyl residue with the bases (or base pairs) of the polynucleotide and is in equilibrium with (PN)1 . K2 measures the ratio of the concentrations of stacked and unstacked complexes . The fluorescence decay of the tryptophyl residue in KWGK was not significantly different in the presence and in the absence of double-stranded polynucleotides in agreement with the previous model, which assumes total quenching of tryptophan fluorescence in complex (PN)2 and identical fluorescence characteristics for free KWGK and complex (PN)1 . The stacking of the tryptophyl residue with A.T base pairs in alternating poly{d(A-T)} was found to be 10 times more efficient than that with nonalternating poly(dA).poly(dT) . Among A-T-containing double-stranded polynucleotides, poly(rA).poly(dT) was found to be the most favorable for tryptophan stacking . A similar behavior was previously demonstrated for several intercalating agents such as ethidium bromide, propidium iodide, and daunomycin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Oct 20, 26(21), 6810 - 6
DNA repair catalyzed by Escherichia coli DNA photolyase containing only reduced flavin: elimination of the enzyme's second chromophore by reduction with sodium borohydride; Jorns MS et al.; DNA photolyase from Escherichia coli contains FAD plus a partially characterized, second chromophore . In vivo, the flavin is fully reduced (FADH2), but oxidation to a stable, blue radical (FADH.) occurs during enzyme isolation . The second chromophore is irreversibly reduced by reaction of the enzyme with sodium borohydride or by photoreduction in the presence of dithiothreitol . A similar reaction occurs with the protein-free chromophore and sodium cyanoborohydride . Reduction of the second chromophore is accompanied by a complete loss of the chromophore's visible absorption and fluorescence but does not significantly affect catalytic activity . The results show that the enzyme can repair dimers by a pathway involving only FADH2 . Enzyme-bound FADH2 is fluorescent and exhibits emission (505 nm) and absorption (360 nm) maxima similar to that expected for a 1,5-dihydroflavin derivative . It is proposed that dimer cleavage via the second chromophore independent pathway involves electron donation from excited FADH2 to pyrimidine dimer . Pyrimidine dimer radicals are unstable and spontaneously monomerize . Unmodified second chromophore can also act as a sensitizer in a pathway that requires FADH2 . This pathway may be similar to that proposed for the second chromophore independent reaction except that excited FADH2 would be produced via energy transfer from the excited second chromophore . The fluorescence observed for enzyme-bound, unmodified second chromophore is quenched by FADH . and increases 6-fold when the latter is reduced, but the absorption spectrum (lambda max = 390 nm epsilon 390 = 12.7 x 10(3) M-1 cm-1) is independent of the redox state of the flavin.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemistry, 1987 Oct 20, 26(21), 6801 - 10
Evidence for nucleotide-mediated changes in the domain structure of the recA protein of Escherichia coli; Kobayashi N et al.; We have used limited trypsin digestion as a means of investigating changes in the structural properties of recA protein accompanying the binding of different nucleoside triphosphates . The levels of four partial digestion products are greatly increased in digests of recA protein complexed with dTTP, dATP, ATP, or the ATP analogue adenosine 5'-O-(3-thiotriphosphate) (ATP gamma S) . These bands (22, 19, and 17.5 kilodaltons) are absent or present at reduced levels in digests of recA protein alone . Unlike these nucleotides, all of which bind tightly to recA protein, nucleotides and analogues that bind poorly produce little or no change in the digestion pattern of recA protein . We have compared the rates of fragment accumulation in the presence of dTTP and show a saturable dependence on nucleotide concentration . Binding of single-stranded DNA to recA protein does not alter the pattern of digestion products compared to protein alone, and the digestion pattern of recA protein-DNA-ATP gamma S ternary complexes is similar to that of uncomplexed enzyme . We have used monoclonal antibody binding, high-performance liquid chromatography separation of peptides, and amino acid composition analyses to localize the regions of recA protein which are altered in their susceptibility to trypsin when nucleoside triphosphates are present . The results of these analyses indicate that the fragments arise from trypsin cutting at two or more sites near the middle of the primary sequence . These cleavage sites are more than 80-110 residues away from the site of photoaffinity labeling by 8-N3ATP (Tyr-264) . Our results suggest that, in the presence of certain nucleotides, recA protein is organized into two stable structural domains.

Biochemistry, 1987 Oct 20, 26(21), 6765 - 74
Replacement of proline-76 with alanine eliminates the slowest kinetic phase in thioredoxin folding; Kelley RF et al.; The conformational transition observed upon addition of guanidine hydrochloride (Gdn-HCl) to solutions of oxidized Escherichia coli thioredoxin is dominated by a slow kinetic phase (time constant tau 1 = 300-800 s) that has features appropriate to a proline peptide isomerization . This observation has been interpreted as reflecting a folding pathway involving an obligatory isomerization of the imide peptide bond between isoleucine-75 and proline-76 {Kelley, R . F., & Stellwagen, E . (1984) Biochemistry 23, 5095-5102}; this peptide bond is known to have the cis configuration in the folded state {Eklund, H., Cambillan, C., Sjoberg, B.-M., Holmgren, A., Jornvall, H., Hoog, J.-O., & Branden, C.-I . (1984) EMBO J . 3, 1443-1449} . We have tested this hypothesis by examining the conformational transitions of two thioredoxin mutants, trxA76 having an alanine substituted for proline-76 and trxA2 {Russel, M., & Model, P . (1983) J . Bacteriol . 154, 1064-1070} having proline-34 replaced with serine . Both mutant proteins display far-ultraviolet circular dichroic spectra similar to that of native wild-type thioredoxin . The tryptophan fluorescence emission of native trxA2 is equivalent to that of wild-type thioredoxin, while the emission intensity of native trxA76 at 350 nm is 2-fold greater . Tryptophan fluorescence and peptide ellipticity measurements indicate that the mutant proteins undergo two-state and reversible equilibrium unfolding transitions upon addition of guanidine hydrochloride (Gdn-HCl) . These transitions are centered at 2.4 and 1.5 M Gdn-HCl for trxA2 and trxA76, respectively, as compared to a midpoint of 2.5 M denaturant for wild-type thioredoxin . As observed for wild-type thioredoxin, fluorescence measurements reveal monophasic unfolding kinetics for trxA2 at a variety of final denaturant concentrations . The tau for unfolding varies monotonically from 210 s in 2.4 M Gdn-HCl to 7 s for a final Gdn-HCl concentration of 3.5 M . Refolding of denatured trxA2 in 1.5 M Gdn-HCl detected by fluorescence measurements is described by three kinetic phases with time constants and fractional amplitudes (alpha) similar to those of wild-type thioredoxins . The fractional amplitude (alpha 1) of the slowest of these phases, tau 1 = 430 +/- 38 s in 2.0 M Gdn-HCl, decreases with final Gdn-HCl concentration . Multimixing experiments suggest that this phase results from an equilibration between denatured forms and has a tau of 34 s in 4 M denaturant, features previously observed for the wild-type protein.(ABSTRACT TRUNCATED AT 400 WORDS)

Biochemistry, 1987 Oct 20, 26(21), 6723 - 35
Structure-function relationships in EF-hand Ca2+-binding proteins . Protein engineering and biophysical studies of calbindin D9k; Linse S et al.; Genes encoding the minor A component of bovine calbindins D9k--the smallest protein known with a pair of EF-hand calcium-binding sites--with amino acid substitutions and/or deletions have been synthesized and expressed in Escherichia coli and characterized with different biophysical techniques . The mutations are confined to the N-terminal Ca2+-binding site and constitute Pro-20----Gly (M1), Pro-20----Gly and Asn-21 deleted (M2), Pro-20 deleted (M3), and Tyr-13----Phe (M4) . 1H, 43Ca, and 113Cd NMR studies show that the structural changes induced are primarily localized in the modified region, with hardly any effects on the C-terminal Ca2+-binding site . The Ca2+ exchange rate for the N-terminal site changes from 3 s-1 in the wild-type protein (M0) and M4 to 5000 s-1 in M2 and M3, whereas there is no detectable variation in the Ca2+ exchange from the C-terminal site . The macroscopic Ca2+-binding constants have been obtained from equilibration in the presence of the fluorescent chelator 2-{{2-{bis(carboxymethyl)-amino}- 5-methylphenoxy}methyl}-6-methoxy-8-{bis(carboxymethyl)amino}quinoline or by using a Ca2+-selective electrode . The Ca2+ affinity of M4 was similar to that of M0, whereas the largest differences were found for the second stoichiometric step in M2 and M3 . Microcalorimetric data show that the enthalpy of Ca2+ binding is negative (-8 to -13 kJ.mol-1) for all sites except the N-terminal site in M2 and M3 (+5 kJ.mol-1) . The binding entropy is strongly positive in all cases . Cooperative Ca2+ binding in M0 and M4 was established through the values of the macroscopic Ca2+-binding constants . Through the observed changes in the 1H NMR spectra during Ca2+ titrations we could obtain ratios between site binding constants in M0 and M4 . These ratios in combination with the macroscopic binding constants yielded the interaction free energy between the sites delta delta G as -5.1 +/- 0.4 kJ.mol-1 (M0) and less than -3.9 kJ.mol-1 (M4) . There is evidence (from 113Cd NMR) for site-site interactions also in M1, M2, and M3, but the magnitude of delta delta G could not be determined because of sequential Ca2+ binding.

Biochemistry, 1987 Oct 20, 26(21), 6638 - 44
lac permease of Escherichia coli: arginine-302 as a component of the postulated proton relay; Menick DR et al.; The lac permease of Escherichia coli was modified by site-directed mutagenesis such that Arg-302 in putative helix IX was replaced with Leu . In addition, Ser-300 (helix IX) was replaced with Ala, and Lys-319 in putative helix X was replaced with Leu . Permease with Leu at position 302 manifests properties that are similar to those of permease with Arg in place of His-322 {Puttner, I . B., Sarkar, H . K., Poonian, M . S., & Kaback, H . R . (1986) Biochemistry 25, 4483} . Thus, permease with Leu-302 is markedly defective in active lactose transport, efflux, exchange, and counterflow but catalyzes downhill influx of lactose at high substrate concentrations without H+ translocation . In contrast, permease molecules with Ala at position 300 or Leu at position 319 catalyze lactose/H+ symport in a manner indistinguishable from that of wild-type permease . By molecular modeling, Arg-302 may be positioned in helix IX so that it faces the postulated His-322/Glu-325 ion pair in helix X . In this manner, the guanidino group in Arg-302 may interact with the imidazole of His-322 and thereby play a role in the H+ relay suggested to be involved in lactose/H+ symport {Carrasco, N., Antes, L . M., Poonian, M . S., & Kaback, H . R . (1986) Biochemistry 25, 4486}.

J Mol Biol, 1987 Oct 20, 197(4), 723 - 8
A new algorithm for best subsequence alignments with application to tRNA-rRNA comparisons; Waterman MS et al.; The algorithm of Smith & Waterman for identification of maximally similar subsequences is extended to allow identification of all non-intersecting similar subsequences with similarity score at or above some preset level . The resulting alignments are found in order of score, with the highest scoring alignment first . In the case of single gaps or multiple gaps weighted linear with gap length, the algorithm is extremely efficient, taking very little time beyond that of the initial calculation of the matrix . The algorithm is applied to comparisons of tRNA-rRNA sequences from Escherichia coli . A statistical analysis is important for proper evaluation of the results, which differ substantially from the results of an earlier analysis of the same sequences by Bloch and colleagues.

J Mol Biol, 1987 Oct 20, 197(4), 659 - 70
Expression of the leuX gene in Escherichia coli . Regulation at transcription and tRNA processing steps; Nomura T et al.; The leuX (supP) gene of Escherichia coli codes for a suppressor tRNA (tRNA(6Leu} that inserts leucine at the amber codon . Analysis of both in-vitro and in-vivo transcripts indicated that the gene is organized into a single gene operon, carrying its own promoter and rho-independent terminator, and its primary transcript accumulates in cells of wild-type E . coli with respect to tRNA processing . Systematic and quantitative measurements of both the unprocessed primary transcript and mature tRNA(Leu6) indicated that: (1) transcription of the leuX gene is under stringent control in vivo and is repressed in vitro by ppGpp; (2) transcription of the leuX gene is under growth rate-dependent control; but (3) the level of mature tRNA stays constant under various growth conditions . A model is proposed, which assumes that the enzyme catalyzing the first-step reaction in the leuX tRNA processing is limited, thereby keeping the level of mature tRNA(Leu6) at a constant level irrespective of changes in the level of the unprocessed primary transcript.

J Mol Biol, 1987 Oct 20, 197(4), 647 - 57
The tRNA-tufB operon transcription termination and processing upstream from tufB; Van Delft JH et al.; Two genes, tufA and tufB, located at 73 and 88 minutes of the Escherichia coli linkage map, code for the polypeptide chain elongation factor EF-Tu . tufB is transcribed with four upstream tRNA genes, thrU, tyrU, glyT and thrT, into a cotranscript of approximately 1800 nucleotides . Here we show that in vivo processing yields a 1320 nucleotide transcript of tufB . S1 nuclease fine mapping reveals that the processing site is located in the intergenic region at about 72 to 74 nucleotides upstream from the initiation codon of the tufB cistron . A deletion in the cloned tRNA-tufB operon, encompassing the 3' half of thrU, the complete tyrU, glyT, thrT genes and ten nucleotides of the intergenic region, causes a threefold increase of the rate of plasmid tufB transcription, a fourfold increase of plasmid-borne tufB RNA and a twofold increase of plasmid-borne EF-TuB . We conclude that the deletion has eliminated a transcription termination site probably located after the thrT gene . Termination at this site uncouples tRNA synthesis from tufB transcription.

FEBS Lett, 1987 Oct 19, 223(1), 191 - 6
Chiral beta and random fractional deuteration for the determination of protein sidechain conformation by NMR; LeMaster DM; Stereospecific assignments of the aspartic acid and asparagine beta-protons of the 108 residue protein E . coli thioredoxin have been obtained by the use of chiral deuteration . In addition protein samples have been prepared in which all carbon bound hydrogen positions are substituted to an extent of 75% with deuterium . These random fractionally deuterated samples significantly facilitate the measurement of coupling constants and intraresidue NOE intensities which combined with the stereospecific assignments have provided determination of the first sidechain dihedral angle chi 1 for all four asparagine residues and eight of the ten assigned aspartic acid residues.

FEBS Lett, 1987 Oct 19, 223(1), 127 - 30
Heat shock inactivates a supernatant factor(s) specifically required for efficient expression of the amp gene in Escherichia coli; Kuriki Y; A 160,000 x g supernatant of E . coli extract prepared from cells grown at 30 degrees C stimulated specifically the expression of the amp gene on pBR322 and pBR328 in an in vitro gene expression system from E . coli . This activity of the supernatant was markedly reduced when the cells were exposed to 42 degrees C for 30 min prior to preparing the supernatant . These results are consistent with the view that heat shock-induced repression of the amp gene expression is due to inactivation of a supernatant factor(s) required for effective expression of the amp gene.

FEBS Lett, 1987 Oct 19, 223(1), 174 - 80
All four internally repetitive domains of pig calpastatin possess inhibitory activities against calpains I and II; Maki M et al.; Complementary DNA portions coding for each domain (domain L and internally repetitive domains, domains 1-4, each composed of approximately 140 amino acid residues) of pig calpastatin were subcloned into E . coli plasmids to express the respective portions of the proteinase inhibitor gene in bacteria . Cell extracts of E . coli harboring recombinant plasmids were assayed for calpain inhibition . All four internally repetitive domains showed inhibitory activities, essentially similar to one another, against calpains I and II . No inhibition was observed in the case of the N-terminal non-homologous domain (domain L) . These results support our previous conclusion that the repetitive region is a functional unit of the proteinase inhibitor.

Biochim Biophys Acta, 1987 Oct 16, 903(3), 465 - 72
Perturbation of the lipid bilayer of model membranes by synthetic signal peptides; Nagaraj R et al.; The interaction of synthetic peptides corresponding to the signal sequences of Escherichia coli alkaline phosphatase: Lys-Gln-Ser-Thr-Ile-Ala-Leu-Ala-Leu-Leu-Pro-Leu-Leu-Phe-Thr-Pro-Val-Thr- Lys-Ala - OCH3, chicken lysozyme: Met-Lys-Ser-Leu-Leu-Ile-Leu-Val-Leu-Cys(Bzl)-Phe-Leu-Pro-Leu- Ala-Ala-Leu-Gly-OCH2-C6H5 and variant of the chicken lysozyme signal sequence with a charged residue in the hydrophobic region: Lys-Leu-Leu-Ile-Ala-Leu-Val-Leu-Lys-Phe-Leu-Pro-Leu-Ala-Ala- Leu-Gly-OCH3 with model membranes of brain phosphatidylserine (PS) and egg phosphatidylcholine (PC) have been investigated by 90 degrees light scattering and fluorescence spectroscopy . Our results indicate that the association of signal peptides with model membranes results in extensive perturbation of the lipid bilayer so as to cause fusion of PS vesicles and aggregation of PC vesicles . The vesicles are also rendered permeable to hydrophilic molecules like carboxyfluorescein . The variant peptide with the lysine residue in the hydrophobic region also has the ability to perturb lipid bilayers of model membranes.

Cell Immunol, 1987 Oct 15, 109(2), 384 - 96
Effect of antisera against recombinant tumor necrosis factor and the monocyte-derived cytotoxin(s) on monocyte-mediated killing of various tumor cells; Nissen-Meyer J et al.; Antisera raised against recombinant tumor necrosis factor (TNF) and against the monocyte-derived cytotoxic/cytostatic protein factor (CF), which is related to recombinant TNF, have been compared with respect to their ability to inhibit monocyte-mediated killing of various types of cells which differ in their sensitivity to recombinant TNF . During 6 hr of coculturing monocytes and target cells, the recombinant TNF antiserum inhibited killing of the extremely TNF-sensitive WEHI 164 clone 13 cells and actinomycin D-treated WEHI 164 cells from which the clone 13 cells were derived (parental WEHI 164 cells (P-WEHI 164 cells} . The CF antiserum also inhibited monocyte-mediated killing of these cells during 6 hr of coculturing with monocytes, but on a per volume basis it was less potent than the recombinant TNF antiserum, consistent with the fact that the CF antiserum also was much less potent in inhibiting the cytotoxic activity of recombinant TNF . However, during 72 hr of coculturing with monocytes and target cells, the CF antiserum inhibited monocyte-mediated killing of P-WEHI 164 cells more efficiently than the recombinant TNF antiserum . Moreover, during 72 hr of coculturing with monocytes, only the CF antiserum was able to significantly inhibit monocyte-mediated killing of the relatively recombinant TNF-resistant K562 cells . This suggests that a factor immunologically different from recombinant TNF, perhaps a form of natural TNF differing somewhat immunologically from recombinant TNF, was involved in the killing of K562 cells, and possibly in the killing of P-WEHI 164 cells, during 72 hr of coculturing with monocytes . Although this factor was present extracellularly, it appears that it may act as a monocyte-associated factor in monocyte-mediated killing of K562 cells, since exposure to recombinant interferon-gamma (rIFN-gamma) in the absence of Escherichia coli endotoxin (lipopolysaccharide, LPS) activated the monocytes to mediate killing of K562 cells more efficiently than exposure to LPS alone, despite the fact that only little cytotoxic/cytostatic activity was released from the monocytes without the addition of LPS . The ability of rIFN-gamma and LPS to activate monocytes to produce and release CF has also been studied.

Eur J Biochem, 1987 Oct 15, 168(2), 451 - 9
Biochemical and immunological evidence for a second nitrate reductase in Escherichia coli K12; Iobbi C et al.; Genes different from those of the narGHI operon and encoding a nitrate reductase activity have been cloned by Bonnefoy et al . (unpublished results) . We have shown by the use of well-known assay methods that the encoded enzyme activity is catalyzed by a true nitrate reductase and not by trimethylamine-N-oxide reductase or formate dehydrogenase . The biochemical and immunological study, employing anti-(nitrate reductase) serum raised against the known enzyme, revealed that Escherichia coli contains a second nitrate reductase (nitrate reductase Z) which shares some similarities as well as differences with the known enzyme . By using a strain with a deletion of the narGHI operon and carrying a multicopy plasmid having the nitrate reductase Z genes, we have shown that nitrate reductase Z is a membrane-bound molybdoenzyme able to couple formate oxidation with nitrate reduction . Like the known nitrate reductase, this enzyme has chlorate reductase activity . The molecular mass and pH and temperature dependence of enzyme Z are similar to these of the known enzyme . On the other hand the two enzymes have significant difference in their electrophoretic mobility on polyacrylamide gels . Unlike the known enzyme, enzyme Z is synthesized in small amounts; the expression of its structural genes does not seem to be induced by nitrate, repressed by oxygen or activated by the product of the fnr gene . The immunological comparison of the two enzymes was performed by rocket immunoelectrophoresis, double diffusion on agar plates and immunoblots . These techniques disclosed a difference between the two enzymes in their recognition by the antiserum and showed that E . coli has two types of nitrate reductase.

Eur J Biochem, 1987 Oct 15, 168(2), 385 - 91
Studies on the sequence and structure of the Escherichia coli K-12 nupG gene, encoding a nucleoside-transport system; Westh Hansen SE et al.; The nupG gene, encoding one of the two active nucleoside-transport systems in Escherichia coli K-12, has been cloned on the multicopy plasmid pBR322 and derivatives thereof . The recombinant plasmids complemented a chromosomal nupG mutation . A genetic map was determined by digestion with restriction endonucleases and the nucleotide sequence of a 3-kb stretch of DNA has been determined on fragments cloned into M13 phages . An open reading frame of 1254 bp, encoding a protein with a calculated molecular mass of 45.333 kDa, was deduced to be the coding region of nupG . Minicell-forming strains carrying plasmids containing this gene were shown to produce a hydrophobic, membrane-bound polypeptide with an apparent molecular mass of approximately 43 kDa.

Eur J Biochem, 1987 Oct 15, 168(2), 365 - 9
Mutants affecting tRNA(Phe) from Escherichia coli . Studies of the suppression of thermosensitive phenylalanyl-tRNA synthetase; Delamarche C et al.; Four mutants of pheV, a gene coding for tRNA(Phe) in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild-type pheV to complement the thermosensitive defect in a mutant of phenylalanyl-tRNA synthetase . One of these mutants, leading to the change C2----U2 in tRNA(Phe), is expressed about 10-fold lower in transformed cells than wild-type pheV . This mutant, unlike the remaining three (G15----A15, G44----A44, m7G46----A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number . The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity . A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl-tRNA synthetase . The mutant gene pheV (U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second-site mutation outside the tRNA structural gene, in the discriminator region . This mutation, C(-6)----T(-6), restores expression of the mutant U2 to about the level of wild-type tRNA(Phe).

J Biol Chem, 1987 Oct 15, 262(29), 13991 - 6
Involvement of lactaldehyde dehydrogenase in several metabolic pathways of Escherichia coli K12; Baldoma L et al.; Lactaldehyde dehydrogenase (E.C . 1.2.1.22) of Escherichia coli has been purified to homogeneity . It has four apparently equal subunits (molecular weight 55,000 each) and four NAD binding sites per molecule of native enzyme . The enzyme is inducible, only under aerobic conditions, by at least three different types of molecules, the sugars fucose and rhamnose, the diol ethylene glycol and the amino acid glutamate . The enzyme catalyzes the irreversible oxidation of several aldehydes with a Km in the micromolar range for alpha-hydroxyaldehydes (lactaldehyde, glyceraldehyde, or glycolaldehyde) and a higher Km, in the millimolar range, for the alpha-ketoaldehyde methylglyoxal . It displays substrate inhibition with all these substrates . NAD is the preferential cofactor . The functional and structural features of the enzyme indicate that it is not an isozyme of other E . coli aldehyde dehydrogenases such as glyceraldehyde phosphate dehydrogenase, glycolaldehyde dehydrogenase, or acetaldehyde dehydrogenase . The enzyme, previously described as specific for lactaldehyde, is thus identified as a dehydrogenase with a fairly general role in aldehyde oxidation, and it is probably involved in several metabolic pathways.

J Biol Chem, 1987 Oct 15, 262(29), 14100 - 4
Construction and properties of bifunctionally active membrane-bound fusion proteins . Escherichia coli proline carrier linked with beta-galactosidase; Hanada K et al.; For construction of bifunctionally active membrane-bound fusion proteins, we designed plasmids encoding fusion proteins in which the carboxyl terminus of Escherichia coli proline carrier was joined to the amino terminus of E . coli beta-galactosidase directly or with a collagen linker inserted between the two . The expressions of these fusion proteins complemented deficiencies in both proline transport and beta-galactosidase activity in E . coli cells . The fusion proteins were stable and mostly localized in the cytoplasmic membrane . The proline transport activities of the fusion proteins were kinetically similar to that of the wild type proline carrier . The beta-galactosidase moiety of the collagen-linked fusion protein was liberated from membrane vesicles by collagenase treatment . The Km value of released beta-galactosidase for o-nitrophenyl beta-D-galactopyranoside hydrolysis was similar to that of membrane-bound beta-galactosidase in the fusion protein . These results indicated that the fusion proteins are bifunctionally active and exhibit normal proline transport and beta-galactosidase activities . The crypticity of the beta-galactosidase activity associated with the fusion proteins indicated that the carboxyl terminus of the proline carrier was located on the cytoplasmic side of the membrane.

J Biol Chem, 1987 Oct 15, 262(29), 14213 - 21
Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin; Ecker DJ et al.; To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight "mutagenesis modules." A series of site-specific mutations were constructed to selectively perturb various regions of the molecule . The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified . To determine the structural effects of the amino acid substitutions, the solution structure of ubiquitin was investigated by two-dimensional NMR and each of the mutant proteins were screened for structural perturbations . With one exception, virtually no changes were seen other than at the point of mutation . Functional studies of the mutant proteins with the ubiquitin-activating enzyme E1 and in the reticulocyte protein degradation assay were used to identify regions of the molecule important to ubiquitin's activity in intracellular proteolysis.

J Biol Chem, 1987 Oct 15, 262(29), 14030 - 5
Engineered Tet repressor mutants with single tryptophan residues as fluorescent probes . Solvent accessibilities of DNA and inducer binding sites and interaction with tetracycline; Hansen D et al.; Mutants of the Tn10-encoded Tet repressor containing single or no tryptophan residues were constructed by oligonucleotide-directed mutagenesis . The Trp-75 to Phe exchange reduces the dissociation rate of the complex with the inducer tetracycline by a factor of 2 . The Trp-43 to Phe exchange has no effect on inducer binding . The fluorescence emission spectra of both tryptophan residues are quenched to a different extent by binding of tetracycline: Trp-75 is quenched to zero and Trp-43 to only 50% . It is concluded that Trp-75 is in the vicinity of the inducer binding site . The different fluorescence emission spectra of both tryptophan residues depend on the native structure of Tet repressor . Quenching studies with iodide indicate that the DNA binding motif is solvent exposed in free repressor and moves towards the interior of the protein upon inducer binding . The inducer binding site is in the interior of the protein . The fluorescence of tetracycline is enhanced upon binding to Tet repressor . The excitation at 280 nm results mainly from the change in environment and in part from energy transfer from tryptophan to the drug.

J Biol Chem, 1987 Oct 15, 262(29), 13928 - 32
The cloning and DNA sequence of the gene xylE for xylose-proton symport in Escherichia coli K12; Davis EO et al.; The gene xylE, coding for xylose-proton symport in Escherichia coli, was cloned and its DNA sequence determined . The cloning strategy utilized lambda placMu insertions and exploited the proximity of xylE to malB . A 2.8-kilobase HincII fragment of cloned DNA restored {14C}xylose transport and xylose-proton symport activities to a xylose transport-negative strain . The xylE gene was identified as a 1473-base pair open reading frame, located 373 base pairs downstream of malG, encoding a hydrophobic protein of Mr 53,607 . The amino acid sequence of XylE bore little resemblance to the lactose-proton LacY symporter or melibiose-sodium MelB symporter, but a high degree of homology was found with the arabinose-proton AraE symporter of E . coli and glucose transport proteins of mammals . Structural analyses and comparisons suggest that 12 membrane-spanning segments may occur in the XylE protein.

Hosp Pract (Off Ed), 1987 Oct 15, 22(10), 171 - 3, 177-9, 182-3 passim
I.v . immune globulin: efficacy and safety; Minnefor AB et al.; Experimental studies suggest that IGIV therapy can be highly effective in the prevention and treatment of infectious diseases in a number of different patient groups . Several intravenous immune globulins are available in the United States (Table 5) . For the most part, these preparations are well tolerated, but adverse reactions have been observed . They may be due to a rapid rate of infusion, the presence of circulating immune complexes, or the production of anti-IgA by IgA-deficient individuals . With respect to HIV transmission, clinical experience strongly suggests that IGIV preparations are safe.

J Biol Chem, 1987 Oct 15, 262(29), 14273 - 81
On the explanation of the acidic pH requirement for in vitro activity of colicin E1 . Site-directed mutagenesis at Glu-468; Shiver JW et al.; The in vitro acidic pH dependence of colicin E1 channel activity was investigated by directed mutagenesis of Glu-468 in the colicin E1 channel domain, a residue conserved in the sequences of the four channel-forming colicins examined so far . Mutations were made to the amino acids leucine, serine, glutamine, or lysine, residues of different polarity and charge . All of the mutant polypeptides possessed high cytotoxic activity in vivo, although in vitro activity, especially with planar membranes, was lower than that of the wild type protein . A change in the in vitro acidic pH dependence of activity could be readily detected in the mutation to the hydrophobic leucine residue . The dependence of mutant activity on pH in the interval 3.5-5.0 was markedly smaller than that of the wild type, whether assayed on membrane vesicles or membrane bilayers . Differences in pH dependence between the wild type and the polar serine and glutamine mutants were small or of marginal statistical significance . No change in pH dependence could be detected with the charged lysine mutant . The residual pH dependence in all cases indicated that more than one carboxylic residue must be protonated to account for the increased activity at acidic pH values . A role of Glu-468 in the mechanism of channel formation or function was implied by the changes determined in vitro of channel parameters relative to the wild type: (i) the relatively small rates of current increase measured for colicin COOH-terminal peptide derived from the mutants, (ii) the small values of steady-state conductance of mutant peptide at pH 3.5, and (iii) the reduced anion selectivity of peptide from the serine mutant.

Nucleic Acids Res, 1987 Oct 12, 15(19), 7663 - 75
Relation of the Escherichia coli dnaX gene to its two products--the tau and gamma subunits of DNA polymerase III holoenzyme; Lee SH et al.; The Escherichia coli DNA polymerase III holoenzyme 71.1 kDa tau subunit is a 643 amino acid protein encoded by the dnaX gene . This gene also encodes the holoenzyme 56.5 kDa gamma subunit . The tau factor (as a tau'-LacZ' fusion protein) has been isolated and shown to be cleaved in vitro to form gamma and a 135 kda C-terminal cleavage product . The tau'-LacZ' fusion protein, gamma, and the C-terminal cleavage product have been isolated . N-terminal sequencing has demonstrated that tau and gamma share the same N-terminal sequences and that tau is proteolytically cleaved in vitro between residues 498 and 499 to form gamma . In addition, residues 420-440 were shown to be present in both tau and gamma by use of antibody specific for a synthetic peptide corresponding to that sequence . Some mechanism functions in vivo to ensure that tau and gamma are synthesized in a ratio of about one-to-one, as shown by radioimmune precipitation of tau and gamma from cellular extracts.

Biochim Biophys Acta, 1987 Oct 9, 910(1), 89 - 92
Cloning and sequencing of a carp beta s-crystallin cDNA; Chang T et al.; The mRNAs were extracted from common carp (Cyprinus carpio) lenses, purified, reverse transcribed, dC tailed and cloned into Escherichia coli with pBR322 as vector . The cloning efficiency was around 1 X 10(7) colonies per micrograms of mRNA . A clone (pC20) was found by hybrid-arrested to contain the cDNA related to carp crystallins . However, comparison of the derived amino-acid sequence with bovine gamma-II and beta s-crystallins indicates that this carp crystallin sequence resembles closely the bovine beta s-crystallin and should be better classified as such except that this fish sequence does not contain the N-terminal 'arm' of four amino-acid residues present in bovine beta s-crystallin.

Cell, 1987 Oct 9, 51(1), 121 - 6
Mutants of GAL4 protein altered in an activation function; Gill G et al.; Activating region I of GAL4 has been defined as a region 48 amino acids long which, when attached to GAL4's DNA-binding domain, activates transcription in yeast . Here we describe mutants bearing changes in and around this highly acidic activating region . We find mutations that increase the activation function invariably increase the acidity of the region and some but not all of the mutations that decrease the activation function decrease the acidity of the region.

Cell, 1987 Oct 9, 51(1), 113 - 9
A new class of yeast transcriptional activators; Ma J et al.; We describe yeast transcriptional activators encoded by E . coli genomic DNA fragments fused to the coding sequence of the DNA-binding portion of GAL4 . All of the new activating sequences that we have analyzed, like those of GAL4 and GCN4, are acidic; most of these sequences show no obvious sequence homology when compared with the identified activating regions of GAL4 and GCN4 or among themselves . We also describe a fusion protein that contains no yeast protein sequence but activates transcription in yeast.

Cell, 1987 Oct 9, 51(1), 101 - 11
Tn10 transposition and circle formation in vitro; Morisato D et al.; We describe a cell-free system that promotes Tn10 transposition and transposon circle formation, a related intramolecular event . Tn10 circle formation in vitro has been characterized in detail, and is shown to require a supercoiled substrate and to proceed in the absence of ATP . The reaction requires Tn10 transposase protein, and either of two E . coli proteins, integration host factor (IHF) and HU, which are small DNA binding proteins that change the conformation of DNA . Tn10 is composed of inverted repeats of insertion sequence IS10 . Pair-wise combinations of the IS10 "outside" and "inside" ends mediate distinct classes of rearrangements in vivo, and they exhibit different reaction requirements in vitro . In contrast to the Tn10 reaction, which involves two outside ends, circle formation with two inside ends proceeds with a transposase fraction alone, in the absence of added host factors, and is inhibited by methylation of the dam site within each terminus.

Biochim Biophys Acta, 1987 Oct 9, 910(1), 63 - 71
An amylase gene from Drosophila pseudoobscura is expressed in Escherichia coli . Functional selection and biochemical comparisons of the fly- and clone-produced amylases; Chernin MI et al.; An amylase gene from Drosophila pseudoobscura was isolated from a genomic library constructed in pBR322 and cloned in Escherichia coli by selecting for the ability of its product to hydrolyze starch, a carbon source not normally utilized by E . coli . Hybridization of pAMY17F to D . pseudoobscura polytene chromosomes shows a positive signal at the amylase pseudogene locus (bank 78, chromosome 3) . The chimeric plasmid pAMY17F, has been altered in such a way as to increase amylase expression . Southern and Northern hybridizations to the cloned amylase DNA indicate that the source of the gene is from D . pseudoobscura . Biochemical properties such as pH optima, substrate specificities, electrophoretic analyses, inhibitor sensitivities, heat stabilities, temperature responsiveness and molecular weights indicate that the amylases produced by the fly and bacterial clone are similar and have similar properties . It appears that E . coli/pAMY17F is producing an amylase like that found in D . pseudoobscura.

Biochemistry, 1987 Oct 6, 26(20), 6531 - 8
NMR analyses of the conformations of L-isoleucine and L-valine bound to Escherichia coli isoleucyl-tRNA synthetase; Kohda D et al.; The 400-MHz 1H NMR spectra of L-isoleucine and L-valine were measured in the presence of Escherichia coli isoleucyl-tRNA synthetase (IleRS) . Because of chemical exchange of L-isoleucine or L-valine between the free state and the IleRS-bound state, a transferred nuclear Overhauser effect (TRNOE) was observed among proton resonances of L-isoleucine or L-valine . However, in the presence of isoleucyl adenylate tightly bound to the amino acid activation site of IleRS, no TRNOE for L-isoleucine or L-valine was observed . This indicates that the observed TRNOE is due to the interaction of L-isoleucine or L-valine with the amino acid activation site of IleRS . The conformations of these amino acids in the amino acid activation site of IleRS were determined by the analyses of time dependences of TRNOEs and TRNOE action spectra . The IleRS-bound L-isoleucine takes the gauche+ form about the C alpha-C beta bond and the trans form about the C beta-C gamma 1 bond . The IleRS-bound L-valine takes the gauche- form about the C alpha-C beta bond . Thus, the conformation of IleRS-bound L-valine is the same as that of IleRS-bound L-isoleucine except for the delta-methyl group . The side chain of L-isoleucine or L-valine lies in an aliphatic hydrophobic pocket of the active site of IleRS . Such hydrophobic interaction with IleRS is more significant for L-isoleucine than for L-valine . The TRNOE analysis is useful for studying the amino acid discrimination mechanism of aminoacyl-tRNA synthetases.

Biochemistry, 1987 Oct 6, 26(20), 6502 - 7
Chemical modification of arginine residues in the lactose repressor; Whitson PA et al.; The lactose repressor protein was chemically modified with 2,3-butanedione and phenylglyoxal . Arginine reaction was quantitated by either amino acid analysis or incorporation of 14C-labeled phenylglyoxal . Inducer binding activity was unaffected by the modification of arginine residues, while both operator and nonspecific DNA binding activities were diminished, although to differing degrees . The correlation of the decrease in DNA binding activities with the modification of approximately 1-2 equiv of arginine per monomer suggests increased reactivity of a functionally essential residue(s) . For both reagents, operator DNA binding activity was protected by the presence of calf thymus DNA, and the extent of reaction with phenylglyoxal was simultaneously diminished . This protection presumably results from steric restriction of reagent access to an arginine(s) that is (are) essential for DNA binding interactions . These experiments suggest that there is (are) an essential reactive arginine(s) critical for repressor binding to DNA.

Biochemistry, 1987 Oct 6, 26(20), 6472 - 8
Ligand binding and protein dynamics: a fluorescence depolarization study of aspartate transcarbamylase from Escherichia coli; Royer CA et al.; The polarization of the fluorescence and the real-time fluorescence intensity decay of the two tryptophan residues of aspartate transcarbamylase from Escherichia coli were studied as a function of temperature . The protein was dissolved in an 80% glycerol/buffer mixture, and temperatures were varied between -40 and 20 degrees C in order to limit the depolarization to local rotations of the tryptophans . Two fluorescent species contribute to over 95% of the emission . They differ in their fluorescence lifetimes by approximately 4 ns depending upon the temperature observed and their fractional contributions to the total intensity . The Y-plot analysis of the polarization and lifetime data allows for the distinction of two rotational species by their critical amplitude of rotation, the first being component 1 and the second being component 2 . We suggest that these two species correspond to the two tryptophan residues of the protein . The polarization and lifetime experiments were carried out for ATCase in presence of the bisubstrate analogue N-(phosphonoacetyl)-L-aspartate (PALA) and in presence of the nucleotide effector molecules ATP and CTP . The binding of PALA results in an increase in the thermal coefficient of frictional resistance to rotation of tryptophan 1 and a decrease in that of tryptophan 2 . ATP binding does not affect the degree to which the protein hinders tryptophan rotation but does result in a change in the critical amplitude of rotation of tryptophan 2 . The results obtained in the presence of CTP are similar to those obtained with PALA.

Biochemistry, 1987 Oct 6, 26(20), 6444 - 7
Mechanism of substrate inactivation of Escherichia coli S-adenosylmethionine decarboxylase; Anton DL et al.; S-Adenosylmethionine decarboxylase, a pyruvoyl-containing decarboxylase, is inactivated in a time-dependent process under turnover conditions . The inactivation is dependent on the presence of both substrate and Mg2+, which is also required for enzyme activity . The rate of inactivation is dependent on the concentration of substrate and appears to be saturable . Inactivation by {methionyl-3,4-14C}-adenosylmethionine results in stoichiometric labeling of the protein . In contrast, when either S-{methyl-3H}adenosylmethionine or {8-14C}adenosylmethionine is used, there is virtually no incorporation of radioactivity . Automated Edman degradation of the alpha (pyruvoyl-containing) subunit reveals that substrate inactivation results in the conversion of the pyruvoyl group to an alanyl residue . These data suggest a mechanism of inactivation which involves the transamination of the nascent product to the pyruvoyl group, followed by the elimination of methylthioadenosine and the generation of a 2-propenal equivalent which could undergo a Michael addition to the enzyme . This is the first evidence for a transamination mechanism for substrate inactivation of a pyruvoyl enzyme.

Biochemistry, 1987 Oct 6, 26(20), 6387 - 92
Electron microscopy and biochemical properties of polyamine-compacted DNA; Baeza I et al.; We have obtained polyamine-compacted DNA and analyzed it by electron microscopy employing the method described by Dubochet, suitable for the study of complexes in which the main interactions are of ionic character . In addition, we have developed a simple biochemical method, based on the action of pancreatic DNase I, to demonstrate the condensation of DNA with spermidine . DNA-spermidine complexes are resistant to the action of DNase I, and there is a strong correlation between the presence of condensed DNA forms, both as toroids and as cylinders, and the insensitivity to DNase I activity . We have also shown that pBR322 DNA-spermidine complexes are transcriptionally active in the presence of Escherichia coli RNA polymerase . This supports the data concerning the biological activity of spermidine-condensed DNA.

J Mol Biol, 1987 Oct 5, 197(3), 439 - 51
Translational control of phage f1 gene expression by differential activities of the gene V, VII, IX and VIII initiation sites; Blumer KJ et al.; Phage-specific transcription and subsequent RNA processing in Escherichia coli infected with the filamentous phage (f1, M13, fd) generate a pool of abundant and relatively long-lived phage mRNA species encoding the four adjacent genes V, VII, IX and VIII . Yet the products of gene V and gene VIII are synthesized at much higher levels than the gene VII and gene IX proteins . To ask if the translational initiation sites heading these genes show corresponding differences in activity and/or functional properties, we have purified a number of the phage mRNAs from cells infected with f1 and examined them in in vitro initiation reactions . The ribosome binding patterns obtained for the phage mRNA species and for smaller defined RNA fragments containing selected initiator regions reveal a large range in apparent ribosome binding strengths . The gene V and gene VIII sites are recognized efficiently in each mRNA species in which they are present . Gene IX site activity appears to be limited by local mRNA structure: the site has undetectable or low ribosome binding activity in all of the phage mRNA species, but is at least tenfold more active if the RNA sequences required to form a potential hairpin stem-and-loop 15 nucleotides upstream from the initiator AUG have been removed . The gene VII site shows no evidence of interaction with ribosomes in any phage mRNA or RNA fragment tested . The same striking differences in initiation activity were observed in vivo by cloning small f1 DNA fragments containing gene V or gene VII initiation site sequences to drive beta-galactosidase synthesis . High levels of a gene V-beta-galactosidase fusion protein are initiated at the V site, but no detectable synthesis occurs from the VII site . If the VII site is preceded by all of the information encoding the upstream gene V, however, modest amounts of a fusion protein initiated at the VII site are produced . The overall results, in accord with the observed yields of proteins in the phage-infected cell, provide strong evidence that the properties of these translational initiation sites determine in a significant way the differential expression of phage f1 genes V, VII, IX and VIII.

J Mol Biol, 1987 Oct 5, 197(3), 453 - 70
Dual level control of the Escherichia coli pheST-himA operon expression . tRNA(Phe)-dependent attenuation and transcriptional operator-repressor control by himA and the SOS network; Mechulam Y et al.; Previous studies of phenylalanyl-tRNA synthetase expression in Escherichia coli have established that the pheST operon transcription is controlled by a Phe-tRNA(Phe)-mediated attenuation mechanism . More recently, the himA gene, encoding the alpha-subunit of integration host factor, was recognized immediately downstream from pheT, possibly forming part of the same transcriptional unit . By using the in-vitro transcription and S1 mapping techniques, transcription termination after pheT could be excluded, indicating that himA can be expressed from polycistronic messenger RNAs encompassing the pheST region . However, the presence of a secondary promoter able to express himA and located within pheT is demonstrated . To further investigate the regulation of the pheST-himA operon expression, genetic fusions between various parts of this operon and the lacZ gene were constructed and studied . Our results confirm the autoregulation of himA previously described, and demonstrate that it occurs through the modulation of the secondary promoter activity within pheT . Surprisingly, it is found that the pheST promoter is also submitted to the same control . Consistent with this, DNA sequences homologous to the integration host factor binding site consensus are present at the level of both promoters . However, evidence in favor of two different repressor complexes is provided . Previously observed SOS induction of the himA expression is shown to occur through the modulation of both promoter activities . Contrasting with the other genes under SOS control, the LexA protein binding site consensus sequence could not be found in the two promoter regions . This suggests that either the LexA protein directly participates in the formation of an active holorepressor, or that the product of an SOS gene is able to inhibit the formation or the binding of such a repressor . Finally, our results indicate that the pheST-himA operon expression is controlled by two different mechanisms acting independently . (1) The phenylalanyl-tRNA synthetase and the himA product expressions are controlled by an operator-repressor type mechanism, in which the himA product and the SOS network are involved . (2) Through its partial cotranscription with pheST, himA expression is also under attenuation control . The latter control may provide a way to couple the intracellular concentration of the himA product to the functional state of the translational apparatus.

J Biol Chem, 1987 Oct 5, 262(28), 13773 - 9
Characterization of rat cellular retinol-binding protein II expressed in Escherichia coli; Li E et al.; Rat cellular retinol-binding protein II (CRBP II) is a small (15.6 kDa) intracellular protein that binds all-trans-retinol . In the adult rat, expression of the CRBP II gene is essentially limited to the small intestinal lining cells (enterocytes), suggesting that CRBP II may be uniquely adapted for intestinal metabolism of newly absorbed retinol . Functional and structural analysis of this protein has been hampered by difficulties in freeing rat intestinal CRBP II from its ligand without denaturation . To circumvent this problem, we have obtained efficient expression of rat apoCRBP II in Escherichia coli . The purified E . coli-derived apoprotein, when complexed with all-trans-retinol, demonstrates fluorescence excitation-emission spectra and absorption spectra indistinguishable from that of CRBP II-retinol isolated from rat intestine . Quantitative ligand binding studies were performed by monitoring either the fluorescence of bound retinol or the quenching of protein fluorescence . They revealed that E . coli-derived CRBP II binds retinol tightly (the apparent dissociation constant is estimated to be 10(-7)-10(-8) M), with a stoichiometry of 1:1 . Fluorescence quenching studies used acrylamide as a probe for the exposure of the 4 tryptophan residues to solvent . The results indicate that although there is heterogeneity in the exposure of these 4 tryptophan residues to solvent, they are situated in a relatively nonpolar environment . These studies suggest that E . coli-derived apoCRBP II will serve as a useful model for studying retinol-protein interactions.

J Biol Chem, 1987 Oct 5, 262(28), 13654 - 61
The 0 degree C closed complexes between Escherichia coli RNA polymerase and two promoters, T7-A3 and lacUV5; Kovacic RT; The promoter-specific binding of Escherichia coli RNA polymerase to the T7-A3 and the lacUV5 promoters at 0 degrees C was analyzed by DNase I footprinting . At 37 degrees C, the footprint from RNA polymerase bound to the A3 promoter is essentially the same as that reported by Galas, D.J., and Schmitz, A., (1978) Nucleic Acids Res . 5, 3157-3170 for the lacUV5 promoter . At 0 degrees C, the footprint for the A3 promoter is well defined but reduced in size . The principal difference between the 0 and 37 degrees C footprints is a region from -2 to +18 which is protected by polymerase at the higher but not at the lower temperature . In contrast, the 0 degree C footprint for the lacUV5 promoter differs substantially in character from the footprint for A3 at 0 degree C . The footprint is similar to the pattern of DNase I digestion of DNA bound to a surface; alternating regions of sensitive and protected DNA are spaced at intervals of about 10 base pairs . This region of DNase I-sensitive and -resistant DNA has the same boundaries as the 0 degree C footprint on T7-A3 . Temperature shift experiments confirmed the sequence specificity of the RNA polymerase interaction with UV5 at 0 degree C . These results indicate that RNA polymerase binds specifically to each promoter sequence in a closed complex . The increased time and amounts of RNA polymerase required to form the 0 degree C footprint on the lacUV5 promoter indicate that it binds RNA polymerase more weakly than does the T7-A3 promoter . Therefore there is a correlation between the binding constant for closed complex formation estimated from kinetic measurements and the formation of the 0 degree C footprint . The -35 region of the promoter may be more important in establishing the 0 degree C footprint because the T7-A3 promoter is a better match to the consensus sequence . Conversely, the -10 region seems less important because lacUV5 is a perfect match to the consensus, whereas the T7-A3 promoter matches at only five out of seven positions . The 0 degree C footprints encompass both regions along with the spacer; the combination of these regions rather than an individual region may determine the character of the footprint and the magnitude of the binding constant.

FEBS Lett, 1987 Oct 5, 222(2), 353 - 7
Vitamin K-dependent carboxylase . Possible role for thioredoxin in the reduction of vitamin K metabolites in liver; Johan L et al.; In the liver vitamin K epoxide, which is produced during the posttranslational carboxylation of protein-bound glutamic acid residues, is recycled by the action of one or more dithiol-dependent reductases . In vitro synthetic dithiols may serve as a cofactor for these enzymes, but the physiological reductant has not yet been found . In this paper we report that in vitro the commercially available thioredoxin/thioredoxin reductase from E . coli can replace the synthetic dithiols during the various reactions of the vitamin K cycle . Based on the assumption that in vivo thioredoxin also plays a role in the regeneration of vitamin K hydroquinone from the epoxide, an extension of the generally accepted vitamin K cycle is proposed.

J Mol Biol, 1987 Oct 5, 197(3), 571 - 84
19F nuclear magnetic resonance as a probe of anticodon structure in 5-fluorouracil-substituted Escherichia coli transfer RNA; Gollnick P et al.; The use of 19F nuclear magnetic resonance (n.m.r.) spectroscopy as a probe of anticodon structure has been extended by investigating the effects of tetranucleotide binding to 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 (anticodon FAC) . 19F n.m.r . spectra were obtained in the absence and presence of different concentrations of oligonucleotides having the sequence GpUpApX (X = A,G,C,U), which contain the valine codon GpUpA . Structural changes in the tRNA were monitored via the 5-fluorouracil residues located at positions 33 and 34 in the anticodon loop, as well as in all other loops and stems of the molecule . Binding of GpUpApA, which is complementary to the anticodon and the 5'-adjacent FUra 33, shifts two resonances in the 19F spectrum . One, peak H (3.90 p.p.m.), is also shifted by GpUpA and was previously assigned to FUra 34 at the wobble position of the anticodon . The effects of GpUpApA differ from those of GpUpA in that the tetranucleotide induces the downfield shift of a second resonance, peak F (4.5 p.p.m.), in the 19F spectrum of 19F-labeled tRNA(Val)1 . Evidence that the codon-containing oligonucleotides bind to the anticodon was obtained from shifts in the methyl proton spectrum of the 6-methyladenosine residue adjacent to the anticodon and from cleavage of the tRNA at the anticodon by RNase H after binding dGpTpApA, a deoxy analog of the ribonucleotide codon . The association constant for the binding of GpUpApA to fluorinated tRNA(Val)1, obtained by Scatchard analysis of the n.m.r . results, is in good agreement with values obtained by other methods . On the basis of these results, we assign peak F in the 19F n.m.r . spectrum of 19F-labeled tRNA(Val)1 to FUra 33 . This assignment and the previous assignment of peak H to FUra 34 are supported by the observation that the intensities of peaks F and H in the 19F spectrum of fluorinated tRNA(Val)1 are specifically decreased after partial hydrolysis with nucleass S1 under conditions leading to cleavage in the anticodon loop . The downfield shift of peak F occurs only with adenosine in the 3'-position of the tetranucleotide; binding of GpUpApG, GpUpApC, or GpUpApU results only in the upfield shift of peak H . The possibility is discussed that this base-specific interaction between the 3'-terminal adenosine and the 5-fluorouracil residue at position 33 involves a 5'-stacked conformation of the anticodon loop . Evidence also is presented for a temperature-dependent conformational change in the anticodon loop below the melting temperature of the tRNA.

J Mol Biol, 1987 Oct 5, 197(3), 555 - 69
Mobility of individual 5-fluorouridine residues in 5-fluorouracil-substituted Escherichia coli valine transfer RNA . A 19F nuclear magnetic resonance relaxation study; Hardin CC et al.; 19F nuclear magnetic resonance (n.m.r.) relaxation parameters of 5-fluorouracil-substituted Escherichia coli tRNA(Val)1 were measured and used to characterize the internal mobility of individual 5-fluorouridine (FUrd) residues in terms of several models of molecular motion . Measured relaxation parameters include the spin-lattice (T1) relaxation time at 282 MHz, the 19F(1H) NOE at 282 MHz, and the spin-spin (T2) relaxation time, estimated from linewidth data at 338 MHz, 282 MHz and 84 MHz . Dipolar and chemical shift anisotropy contributions to the 19F relaxation parameters were determined from the field-dependence of T2 . The results demonstrate a large chemical shift anisotropy contribution to the 19F linewidths at 282 and 338 MHz . Analysis of chemical shift anisotropy relaxation data shows that, relative to overall tumbling of the macromolecule, negligible torsional motion occurs about the glycosidic bond of FUrd residues in 19F-labeled tRNA(Val)1, consistent with the maintenance of base-base hydrogen-bond and/or stacking interactions at all fluorouracil residues in the molecule . The dipolar relaxation data are analyzed by using the "two-state jump" and "diffusion in a cone" formalisms . Motional amplitudes (theta) are interpreted as being due to pseudorotational fluctuations within the ribose ring of the fluorinated nucleoside . These amplitudes range from approximately 30 degrees to 60 degrees, assuming a correlation time (tau i,2) of 1.6 ns . By using available 19F n.m.r . assignment data for the 14 FUrd residues in 5-fluorouracil-substituted tRNA(Val)1, these motional amplitudes can be correlated directly with the environmental domain of the residue . Residues located in tertiary and helical structural domains show markedly less motion (theta approximately equal to 30 to 35 degrees) than residues located in loops (theta approximately equal to 45 to 60 degrees) . A correlation between residue mobility and solvent exposure is also demonstrated . The amplitudes of internal motion for specific residues agree quite well with those derived from X-ray diffraction and molecular dynamics data for yeast tRNA(Phe).

Nature, 1987 Oct 29-Nov 4, 329(6142), 858 - 60
Distal residues in the oxygen binding site of haemoglobin studied by protein engineering; Nagai K et al.; The geometries of the Fe-O2 and Fe-CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds . It has been suggested that steric hindrance by Val-E11 and His-E7 and a hydrogen bond between His-E7 and oxygen affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity . We have produced mutant haemoglobins in E . coli having Val(67 beta)E11 replaced by Ala, Met, Leu or Ile and His(58 beta)E7 by Gln, Val or Gly . We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding . The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe-CO bond observed by resonance Raman spectroscopy . We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the beta subunit are critical for fine-tuning ligand affinity.

Science, 1987 Oct 2, 238(4823), 48 - 53
A portable signal causing faithful DNA methylation de novo in Neurospora crassa; Selker EU et al.; Methylation of cytosine residues in eukaryotic DNA is common, but poorly understood . Typically several percent of the cytosines are methylated; however, it is unclear what governs which sequences eventually become modified . Neurospora crassa DNA containing the "zeta-eta" (zeta-eta) region, which is a region of unusually heavy methylation, was tested for its ability to direct DNA methylation de novo . DNA stripped of its methylation by propagation in Escherichia coli was reintroduced into Neurospora crassa by transformation . The zeta-eta region reproducibly became "properly" methylated whether inserted at its native chromosomal position or at ectopic sites . Adjacent Neurospora and bacterial sequences in the transforming DNA rarely became methylated . A model is presented that accounts for position-independent faithful methylation as observed in the zeta-eta region, as well as position-dependent methylation, as occasionally observed, especially with sequences not native to Neurospora.

J Interferon Res, 1987 Oct, 7(5), 641 - 6
Expression of a pseudogene for interferon-alpha L; Liu XY et al.; The human leukocyte interferon-alpha L (IFN-alpha L) appears to be a pseudogene because it contains a termination codon in the DNA coding for the precursor peptide (signal peptide), but the remainder of the gene codes for an interferon protein of normal length . To determine if this gene codes for an active interferon molecule, an expression plasmid was constructed for IFN-alpha L that was produced in Escherichia coli . The IFN-alpha L is active on human and bovine cells, and exhibits a trace of activity on mouse cells.

DNA, 1987 Oct, 6(5), 497 - 504
Enzymatic synthesis of oligoribonucleotides of defined sequence; Ebe K et al.; A plasmid DNA vector is described that is suitable for cloning synthetic DNA sequences . These cloned synthetic DNA sequences can be transcribed in vitro to produce oligoribonucleotides of defined sequence . Transcription is directed by a promoter based on the consensus sequence for Escherichia coli promoters and uses E . coli RNA polymerase . The vector is useful for cloning oligodeoxyribonucleotides of mixed sequences, the individual sequences being resolved by transformation and colony selection . Oligoribonucleotide synthesis from the vector is highly specific . Application of these sequences in hybridization experiments is demonstrated.

J Bacteriol, 1987 Oct, 169(10), 4750 - 8
Kinetically resolved states of the Halobacterium halobium flagellar motor switch and modulation of the switch by sensory rhodopsin I; McCain DA et al.; Spontaneous switching of the rotation sense of the flagellar motor of the archaebacterium Halobacterium halobium and modulation of the switch by attractant and repellent photostimuli were analyzed by using a computerized cell-tracking system with 67-ms resolution coupled to electronic shutters . The data fit a three-state model of the switch, in which a Poisson process governs the transition from state N (nonreversing) to state R (reversing) . After a reversal, the switch returns to state N, passing through an intermediate state I (inactive), which produces a ca . 2-s period of low reversal frequency before the state N Poisson rate is restored . The stochastic nature of the H . halobium switch reveals a close similarity to Escherichia coli flagellar motor properties as elucidated previously . Sensory modulation of the switch by both photoattractant and photorepellent signals can be interpreted in terms of modulation of the single forward rate constant of the N to R transition . Insight into the mechanism of modulation by the phototaxis receptor sensory rhodopsin I (SR-I) was gained by increasing the lifetime of the principal photointermediate of the SR-I photochemical reaction cycle, S373, by replacing the native chromophore, all-trans-retinal, with the acyclic analog, 3,7,11-trimethyl-2,4,6,8-dodecapentaenal . Flash photolysis of analog-containing cells revealed an eightfold decrease in the rate of thermal decay of S373, and behavioral analysis showed longer periods of reversal suppression than that of cells with the native chromophore over similar ranges of illumination intensities . This indicates that attractant signaling is governed by the lifetime of the S373 intermediate rather than by the frequency of photocycling . In this sense, SR-I is similar to rhodopsin, whose function depends on an active photoproduct (Meta-II).

Blood, 1987 Oct, 70(4), 1006 - 13
Carrier testing in hemophilia B with an immunoassay that distinguishes a prevalent factor IX dimorphism; Smith KJ et al.; Immunoassays with a monoclonal antibody (A-1) detect a prevalent dimorphism in plasma coagulation factor IX . The antibody was shown to react with a dimorphic segment of the normal factor IX sequence as follows . First, A-1 bound to isolated activation peptide (residues 146 through 180) prepared from activated factor IX from a normal plasma pool . Second, binding of recombinant factor IXs with A-1 or factor IX from normal individuals was strong only when they had Threonine (Thr) at position 148; factor IXs with the Alanine (Ala) allele at that position were far less reactive . Third, immunoblot reactivity of Escherichia coli fusion proteins containing known segments of the factor IX sequence restricted the epitope to residues 147 through 153 . In 120 hemophilia B pedigrees, the Ala immunoassay allele frequency was 0.19 and did not differ from the Ala frequency in normal males . In 22 of 49 families, immunoassay testing was informative for classification of obligate or possible carriers . In one large family, 4 obligate carriers were heterozygous for the dimorphism and 3 of their 7 daughters were classified as carriers . In other families, when the affected member had less than 1 nmol/L factor IX antigen, heterozygosity for Thr/Ala alleles excluded the carrier state even when DNA studies were not informative . Strong linkage disequilibrium of Thr/Ala alleles with the common TaqI DNA polymorphism was found . Nineteen of 75 normal and hemophilic factor IX genes had the 1.3-kilobase (kb) fragment and coded for the Ala allele; the rest had the 1.8-kb fragment and coded for Thr . In selected families, the A-1 immunoassay is an inexpensive and rapid method to confirm and supplement restriction fragment length polymorphism analyses of DNA for carrier testing.

Protein Eng, 1987 Oct-Nov, 1(5), 413 - 7
Recombinant-derived interleukin-1 alpha stabilized against specific deamidation; Wingfield PT et al.; Recombinant-derived human interleukin-1 alpha (IL-1 alpha), purified from Escherichia coli, was resolved by isoelectric focusing on polyacrylamide gels into two species of isoelectric points (pI) 5.45 and 5.20, which constituted approximately 75% and approximately 25% of the total IL-1 alpha protein respectively . The pI 5.45 and pI 5.20 species were separated by chromatofocusing and subjected to N-terminal sequence analysis . The pI 5.45 species contained the expected Asn residue at position 36 of the mature protein sequence whereas the pI 5.20 species contained an Asp residue at the same position . A mutant protein in which Asn-36 was substituted for a Ser residue was isolated from E . coli and shown to be homogeneous on isoelectric focusing analysis with a pI = 5.45 . 1H-n.m.r . and circular dichroism analyses of wild-type and the mutant IL-1 alpha indicated a similar conformation which was also indicated by the identical receptor binding affinities of IL-1 alpha with Asn, Asp or Ser in position 36 . The mutant protein was stabilized against specific base-catalysed and temperature-induced deamidation, and may be more suitable than the wild-type position for physical and structural studies.

Immunology, 1987 Oct, 62(2), 285 - 90
The effect of corticosteroids upon murine B cells in vivo and in vitro as determined in the LPS-culture system; Sabbele NR et al.; The influence of the synthetic corticosteroid dexamethasone sodium phosphate (DEXA) upon mouse B cells was studied . This was done by in vivo treatment of mice with a single or multiple injection of DEXA, and by culturing murine spleen cells and bone marrow cells in vitro in the presence of different concentrations of DEXA . The effect of DEXA on the B-cell compartment was assayed by polyclonal stimulation of the B cells by Escherichia coli lipopolysaccharide (LPS) in vitro and subsequent measurement of the Ig-secreting cell response in the protein A plaque assay . DEXA treatment could greatly reduce the number of B cells in the spleen, but the bone marrow B-cell compartment was quite resistant to DEXA . The in vitro LPS-induced IgM response of the residual B cells from both spleen and bone marrow and their capacity to switch from IgM to IgG and IgA secretion were not affected . These data indicate that DEXA can decrease the total number of B cells but not the functional capacity of the residual LPS-reactive B cells . This was confirmed at the clonal level by limiting dilution culture experiments . The contrasting effects of DEXA on splenic and bone marrow B cells was also found when the cells were exposed to the drug in vitro . It was found that 10(-8) M DEXA in vitro reduced the response of splenic B cells to LPS by more than 80%, while a similar reduction of the response by bone marrow B cells required a 1000-fold higher concentration.

Proc Natl Acad Sci U S A, 1987 Oct, 84(20), 7290 - 4
Recombinant hydrophilic region of murine retroviral protein p15E inhibits stimulated T-lymphocyte proliferation; Schmidt DM et al.; Retroviral envelope protein p15E and antigenically related proteins have been implicated as potential mediators of immune dysfunction associated with retroviral infections and with neoplasia . Due to its extreme hydrophobicity, purified p15E has not been available in a nondenatured form or in sufficient quantities for detailed studies on the mechanisms of its immunosuppressive effects . Therefore, a plasmid was constructed to direct the synthesis in Escherichia coli of the major hydrophilic region of murine p15E . The purified recombinant p15E derivative, soluble under physiological conditions, inhibited by up to 60% (EC50 = 7.5 nM) the anti-CD3-driven proliferation of human T lymphocytes but had no effect on the proliferation of the transformed T-cell line Jurkat . The recombinant protein also inhibited, by up to an average of 92% (EC50 = 2.1 microM), the proliferation of the murine T-cell line CTLL-2 . These data (i) provide direct evidence that a retroviral envelope protein can itself inhibit lymphoproliferative function and (ii) map the inhibitory activity to a specific region of p15E . The availability of soluble, recombinant p15E should facilitate studies of the pathogenesis of the immunosuppression accompanying retroviral infections and neoplastic diseases.

Proc Natl Acad Sci U S A, 1987 Oct, 84(19), 6707 - 11
Identification and biochemical characterization of p70TRK, product of the human TRK oncogene; Mitra G et al.; TRK is a human transforming gene generated in a colon carcinoma by a somatic rearrangement that fused a nonmuscle tropomyosin gene to sequences that shared extensive homology with members of the tyrosine-protein kinase supergene family . These sequences are likely to be derived from a transmembrane receptor gene whose putative ligand binding domain has been replaced by tropomyosin . In the present studies, we have expressed the entire coding sequences of the TRK oncogene as well as its protein kinase-related carboxyl-terminal domain in Escherichia coli . Antisera raised against these bacteria-synthesized TRK polypeptides has allowed us to identify the gene product of the TRK oncogene as a 70-kDa protein . Immunoprecipitates containing p70TRK have an associated protein kinase activity specific for tyrosine residues . Moreover, p70TRK is phosphorylated in vivo in serine (75%), threonine (20%), and tyrosine (5%) residues . Finally, immunofluorescence and cellular fractionation studies indicate that p70TRK is preferentially located in the cytoplasmic fraction.

Avian Dis, 1987 Oct-Dec, 31(4), 907 - 9
A Mycoplasma gallisepticum strain-specific DNA probe; Khan MI et al.; Total DNA from the vaccine F strain (K810) and the reference S6-strain of Mycoplasma gallisepticum (MG) was cloned in Escherichia coli using the plasmid pUC8 . A 6-kilobase fragment, specific for the vaccine strain, was identified by colony dot and Southern hybridization analyses . When labeled and used as a probe, this fragment hybridized with the homologous and one other vaccine F-strain (F2F10), but it did not hybridize with other MG strains (Fg38, S6, A5969, V503) or with three other species of avian mycoplasmas.

Bioorg Khim, 1987 Oct, 13(10), 1375 - 81
{Analogs of deoxyribonucleoside-5'-triphosphates modified in the base and sugar moieties: substrate properties in DNA biosynthesis in cell-free systems}; Atrazhev AM et al.; Incorporation of 2'-deoxyribonucleotide 5'-triphosphate derivatives, chemically modified both in the base and at 3'-position, into DNA by four different DNA polymerases was investigated . It is shown that 3'-azido- and 3'-amino-2',3'-dideoxy-(E)-5-(2-bromovinyl)-uridine 5'-triphosphates effectively terminate DNA synthesis catalyzed by E . coli DNA polymerase I, rat liver DNA polymerase beta, and AMV reverse transcriptase . Calf thymus DNA polymerase alpha incorporates only the 3'-amino derivative . DNA polymerases I and beta catalyse DNA synthesis in the presence of beta-D-(2'-deoxyribofuranosyl)-1-benzimidazol 5'-triphosphate, inserting the corresponding monophosphate in place of -dGTP, whereas 3'-substituted analogues of this compound were inactive in the reactions.

Bioorg Khim, 1987 Oct, 13(10), 1366 - 74
{A new terminator of DNA biosynthesis--possible conformation analog of the substrate in a DNA-synthesizing complex}; Diatkina NB et al.; 2',3'-Dideoxy-2',3'-dehydrothymidine 5'-triphosphate (dddTTP) reveals the termination substrate properties in the DNA synthesis catalyzed by E . coli polymerase I (Klenow fragment), rat liver DNA polymerase beta, calf thymus terminal deoxynucleotidyl transferase, and reverse transcriptase of avian myeloblastosis virus but does not affect calf thymus DNA polymerase alpha . For DNA polymerase I, dddTTP by an order of magnitude is more effective than any known termination substrate . It is supposed that dddTTP models the conformational state of the substrate's carbohydrate moiety in the complex DNA polymerase + template-primer.

Microb Pathog, 1987 Oct, 3(4), 269 - 78
Analysis of the plasmids of Escherichia coli O148:H28 from travellers with diarrhea; Danbara H et al.; 98 Escherichia coli strains of serotype O148:H28 isolated from diarrheal patients from 10 Asian countries and Mexico at Osaka Airport Quarantine were analyzed for enterotoxigenicity and plasmid profile . They were classified into three groups . The first group contained 44 strains that were non-enterotoxigenic and carried 3.9 kb and 50 kb non-enterotoxin plasmids . The second group contained 9 strains that produced LT and ST . They carried a 45 kb enterotoxin plasmid, and 4.6 kb and 9.2 kb non-enterotoxin plasmids . The third group contained 45 strains that produced ST . They carried a 40 kb enterotoxin plasmid, and non-enterotoxin plasmids other than 3.9 kb, 4.6 kb, 9.2 kb and 50 kb . Southern blot hybridization demonstrated that all the non-enterotoxin or enterotoxin plasmids carried by the strains of the same group were identical or similar . These results suggested that the 98 E . coli strains with O148:H28 serotype were derived from three clones, and that the individual strains among each group were derived from a single clonal strain.

Vestn Khir Im I I Grek, 1987 Oct, 139(10), 75 - 8
{Metronidazole in the complex treatment of peritonitis of appendicular etiology in children}; Sleptsov VP et al.; Metronidazole was used for treatment of 212 patients with peritonitis of appendicular origin . It resulted in lowered leukocytosis, elimination of the stab neutrophil shift, earlier reduction of the leukocyte intoxication index . The amount of suppurations of postoperative wounds decreased from 17% to 4.3% . The amount of reoperations was 2.4% instead of 5.4% . Metronidazole is recommended for treatment of acute appendicitis complicated by peritonitis.

J Gen Microbiol, 1987 Oct, 133 ( Pt 10), 2759 - 66
Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation; Jones MR et al.; Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated . These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S . The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6 . This pH dependence was not shown by the resistant strains . We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains . We conclude that uncoupler resistance in these strains of E . coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.

J Gen Microbiol, 1987 Oct, 133 ( Pt 10), 2733 - 5
Transformation frequency increases with increase in agitation rate of chemostat-cultivated Escherichia coli K12, strain C-600; Wase DA et al.; The transformation frequency of Escherichia coli C-600, continuously cultivated in a chemostat operated at constant dilution rate, increased with increase in agitation rate (impeller speed) . Cell counts at each impeller speed remained approximately constant . The phenomenon correlated with changes in mean cell volume associated with the changes in agitation rate.

J Gen Microbiol, 1987 Oct, 133 ( Pt 10), 2719 - 25
Identification of the Escherichia coli cysM gene encoding O-acetylserine sulphydrylase B by cloning with mini-Mu-lac containing a plasmid replicon; Sirko AE et al.; A region located at around 52' on the Escherichia coli chromosome was cloned by use of mini-Mu-lac containing a plasmid replicon and recloned into pBR322 . Enzyme assays on transformants carrying the cloned fragments indicated the presence in the latter of the cysA and cysM genes coding for sulphate permease and O-acetylserine sulphydrylase B, respectively.

Pathology, 1987 Oct, 19(4), 361 - 3
Cysteine requirements of naturally occurring cysteine auxotrophs of Escherichia coli; McIver CJ et al.; The requirements for cysteine of naturally occurring cysteine auxotrophs of Escherichia coli were determined in a defined liquid medium . Maximal growth was obtained in the presence of cysteine concentrations between 20 and 250 mg/l . At concentrations below 20 mg/l growth of the auxotrophs, but not the prototrophic control, was suboptimal in this system . In the presence of cysteine concentrations in excess of 250 mg/l, growth of both auxotrophic and prototrophic E . coli was inhibited with lower growth yields, a decreased specific growth rate and an extended lag phase being observed . These effects were minimised in the presence of 2 mM L-leucine, L-isoleucine and L-valine.

Avian Dis, 1987 Oct-Dec, 31(4), 787 - 91
Immunologic effects of low levels of ochratoxin A in ovo: utilization of a chicken embryo model; Harvey RB et al.; Ochratoxin A (OA) was administered to 13-day-old chicken embryos via the chorioallantoic membrane . The 7-day LD50 value (day 20 incubation) of OA was calculated at 7.9 micrograms of OA . Ochratoxin-treated embryos (2.5 micrograms) had slight but significant changes in numbers of immunoglobulin-bearing cells in the bursa but not in the spleen . Chicks hatched from in ovo-treated eggs were challenged with 9 X 10(4) colony-forming units (CFU) of beta-hemolytic Escherichia coli (O1:K1) at 7 days of age via the thoracic air sac . Lesion scores of OA-treated chicks were equal to or less severe than those of controls . Hatchmates of the above chicks were vaccinated with a homologous killed E . coli bacterin (O1:K1) at both 2 and 4 weeks of age and challenged with 10(4) CFU of E . coli at 7 weeks . Post-challenge lesions were present in three vaccinated untreated controls and no OA-treated chicks . We conclude that although in ovo exposure to OA may marginally suppress immunoglobulin-bearing cells of bursa, chicks hatched from OA-treated eggs respond as well as controls to an antigen and resist infection by a virulent organism.

Vet Microbiol, 1987 Oct, 15(1-2), 137 - 50
Cytotoxins in non-enterotoxigenic strains of Escherichia coli isolated from feces of diarrheic calves; De Rycke J et al.; We have examined the cytotoxic responses produced in HeLa and Vero cell cultures by sonicates from 15 non-enterotoxigenic (STa-, LT-) strains of E . coli, highly lethal for mice parenterally LD50 less than 3 X 10(7) CFU), which had been isolated from feces of diarrheic calves . Three types of cytotoxic responses were observed . Type 1 (five strains) consisted of enlargement, rounding and polynucleation of HeLa cells, an effect previously reported with cytotoxic necrotizing factor (CNF) in E . coli from infant and piglet enteritis . Type 2 toxicity (three strains and the control Vir strain S5) was also characterized by enlargement and polynucleation of HeLa cells, but in contrast to Type 2 effect, cells were elongated . Sonicates from the latter strains were lethal for chickens, producing the lesions previously described with Vir strains . Type 3 toxicity (two strains and the control VT strain H19), produced an extensive destruction of both Vero and HeLa cell cultures . Cytotoxic effects were completely abolished upon heating for 1 h at 60 degrees C for Type 1 and 2 extracts and at 80 degrees C for Type 3 extracts . Seroneutralization assays showed that cytotoxins of the same type were closely related antigenically . In addition, a slight cross-neutralization was observed between Type 1 (CNF) and Type 2 (Vir) toxins.

Vet Microbiol, 1987 Oct, 15(1-2), 129 - 36
Binding of fibronectin to Escherichia coli isolated from bovine mastitis from different geographical regions; Faris A et al.; Seventy strains of Escherichia coli, isolated from bovine mastitis in Australia, Denmark, Norway and the U.S.A., were tested for their ability to bind fibronectin . Fifty-three strains (76%) interacted with iodinated fibronectin at a level exceeding 5% of the total radioactivity added . Binding of the amino-terminal (29 kD) fragment of fibronectin was tested for 15 strains, and 6 strains (40%) bound greater than 5% . Bacteria binding the 29 kD fragment at greater than or equal to 19% of the added protein, consistently showed "high" attachment to bovine skin fibroblasts . These cells were shown by immunofluorescence to produce extracellular matrix containing fibronectin . Strains binding lower amounts of fibronectin or 29 kD fragment adhered poorly to these fibroblasts.

J Biochem (Tokyo), 1987 Oct, 102(4), 725 - 32
Role of unique consecutive glutamine repeats in active murine interleukin-2 molecule; Kashima N et al.; Murine interleukin-2 (MIL-2) cDNA deleted of 11 repeats of a CAG sequence, and that encoded from 33Met to 149Gln were inserted into an expression vector carrying an Escherichia coli tryptophan promoter and were expressed in E . coli, respectively . Recombinant MIL-2 deleted of 11 glutamine repeats (MIL-2(-Gln} supported the growth of murine CTLL-2 cells but recombinant MIL-2 initiated from 34Asp (34Asp-MIL-2) did not . The growth of human T-cell blasts was not supported by MIL-2(-Gln) or 34Asp-MIL-2 . MIL-2(-Gln) had identical biological and immunological activities with intact MIL-2, but 34Asp-MIL-2 did not . These results suggest that the consecutive glutamine repeats do not play a role in the biological and immunological activities of MIL-2, but that the peptide sequence around them does, and the species hierarchy that MIL-2 does not act on human lymphocyte is not due to the presence of glutamine repeats in MIL-2.

Mol Gen Mikrobiol Virusol, 1987 Oct, (10), 19 - 23
{Expression in Escherichia coli cells of the nucleotide sequence of DNA coding for the bovine lipotropic hormone}; Beklemishev AB et al.; A cDNA fragment of bovine proopiomelanocortin coding for beta-lipotropic hormone was joined with a promoter and ribosome binding site of B . amyloliquefaciens and cloned in E . coli in pBR 327 plasmid . The level of beta-lipotropin synthesis in bacterial cells transformed by the obtained plasmid was estimated immunochemically . The level of beta-lipotropin production was shown to be 5 mg per liter of bacterial culture.

Mol Gen Genet, 1987 Oct, 209(3), 612 - 4
The relative rate of synthesis and levels of single-stranded DNA binding protein during induction of SOS repair in Escherichia coli; Perrino FW et al.; Induction of the SOS response in Escherichia coli results in an increase in the relative rate of synthesis of single-stranded DNA binding protein (SSB) . In contrast to RecA protein, this increase is slow and does not lead to higher SSB levels . The significance of ssb induction to SOS repair is discussed.

Mol Gen Genet, 1987 Oct, 209(3), 526 - 32
DNA base changes induced following in vivo exposure of unadapted, adapted or ada- Escherichia coli to N-methyl-N'-nitro-N-nitrosoguanidine; Richardson KK et al.; The adaptive response is one of the major repair pathways in Escherichia coli that removes DNA alkylation damage . To investigate the role of the adaptive response in mutagenesis, the E . coli gpt forward mutation assay system was used to determine the mutation spectrum of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) in MNNG-adapted and unadapted GP120 (wild-type) and unadapted PJ5 (ada-5) cells . We observed that 34/37 mutations in the unadapted GP120 cells, 38/40 mutations in the adapted GP120 cells, and 10/10 mutations in the PJ5 cells were GC----AT transitions . The remaining 3/37 mutations in the unadapted GP120 cells were large insertions . The remaining 2/40 mutations in the adapted GP120 cells were transversions with one a GC----CG and the other an AT----CG . A surrounding sequence specificity of mutagenesis was observed for the GC----AT transitions in both the unadapted (GP120 and PJ5) and adapted (GP120) cells, with 70% of the unadapted PJ5, 68% of the unadapted GP120, and 61% of the adapted GP120 mutations occurring at the middle G of the sequence 5'--GG(A or T)--3' . Both strains also displayed a statistically significant preference for mutagenesis at guanine bases in the non-transcribed strand . The overall distribution of mutated sites in the gpt gene in adapted and unadapted cells was similar, although the rate of mutations at certain sites appeared different . These minor differences could result from either non-uniform repair of alkylation damage at different sites on the DNA, or altered processing of the alkylated bases to mutations in the adapted state.(ABSTRACT TRUNCATED AT 250 WORDS)

Mol Gen Genet, 1987 Oct, 209(3), 508 - 17
Regulation of MAL gene expression in yeast: gene dosage effects; Goldenthal MJ et al.; Both the MAL1 and MAL6 loci in Saccharomyces strains have been shown by functional and structural studies to comprise a cluster of at least three genes necessary for maltose utilization . They include regulatory, maltose transport and maltase genes designated MALR, MALT and MALS, respectively . Subclones of each gene derived from the MAL6 locus were inserted into the multicopy shuttle plasmid YEp13, introduced into MAL1 and mal1 strains and the effects of altered gene dosage of each gene, or a combination of them, on MAL gene expression investigated . MAL1 strains transformed with a plasmid carrying the MAL6S gene showed coordinate four to five fold increases in both maltase enzyme activity and its mRNA, whereas no increase in maltose transport activity or of MALT mRNA was observed when MAL6T was present on multicopy plasmids . The presence of the MAL6R gene on a multicopy plasmid led to greatly increased transcription of both inducible and constitutive mRNAs with homology to the regulatory gene; it also gave rise to two fold increases in both induced maltase mRNA levels and enzyme activity, but only in the presence of maltose . However, it had no apparent effect on the accumulation of MALT mRNA . Finally, the induction kinetics of plasmid-borne and chromosomal MALS and MALT gene expression were examined under conditions of altered gene dosage of the MAL6 regulatory and structural genes . The results of these experiments indicate that MALR encodes a trans-acting positive activator that requires maltose for induction of MALS and MALT transcription even when the regulatory gene is present on a multicopy plasmid . Maltose transport can be a rate-limiting factor in MAL gene expression, at least in the early stages of induction . The regulation of the MALS and MALT genes, whose activities are coordinately induced in MAL1 strains by maltose, may in fact exhibit some important differences.

Mol Gen Genet, 1987 Oct, 209(3), 489 - 93
Nucleotide sequences from the colicin E8 operon: homology with plasmid ColE2-P9; Uchimura T et al.; The primary structures of the immunity (Imm) and lysis (Lys) proteins, and the C-terminal 205 amino acid residues of colicin E8 were deduced from nucleotide sequencing of the 1,265 bp ClaI-PvuI DNA fragment of plasmid ColE8-J . The gene order is col-imm-lys confirming previous genetic data . A comparison of the colicin E8 peptide sequence with the available colicin E2-P9 sequence shows an identical receptor-binding domain but 20 amino acid replacements and a clustering of synonymous codon usage in the nuclease-active region . Sequence homology of the two colicins indicates that they are descended from a common ancestral gene and that colicin E8, like colicin E2, may also function as a DNA endonuclease . The native ColE8 imm (resident copy) is 258 bp long and is predicted to encode an acidic protein of 9,604 mol . wt . The six amino acid replacements between the resident imm and the previously reported non-resident copy of the ColE8 imm ({E8 imm}) found in the ribonuclease-producing ColE3-CA38 plasmid offer an explanation for the incomplete protection conferred by {E8 Imm} to exogenously added colicin E8 . Except for one nucleotide and amino acid change in the putative signal peptide sequence, the ColE8 lys structure is identical to that present in ColE2-P9 and ColE3-CA38.

Antibiot Med Biotekhnol, 1987 Oct, 32(10), 723 - 30
{Design of hybrid plasmids based on the actinomycete plasmid pSB24.1 and plasmid prBR325 and a study of their stability in the Streptomyces-E . coli system}; Podgornova GP et al.; Actinomycete plasmid pSB24.1 was cloned on vector of the E . coli pBR325 system . The following bireplicon plasmids were obtained: pSU501 and pSU502 (by XhoI site of pSB24.1 and SalGI site of pBR325), pSU503 (by Bg1II (c) site of pSB24.1 and BamHI site of pBR325) and pSU504 (by Bg1II(b) site of pSB24.1 and BamHI site of pBR325) . In the cells of E . coli C600 plasmids pSU501-504 determined phenotype AprCmrTcs and were stable . In the cells of Str . lividans the initial structure pSU501 selected by Ltz+ phenotype was maintained at a frequency of 12.5 per cent . Analysis of the deletion variants of pSU501 isolated from Str . lividans showed that the deletions were induced by both the pBR325 region and the pSB24.1 DNA fragment near XhoI site . The region from SacII(a) site to Bg1II(b) site clockwise in the map of plasmid pSB24.1 was not significant for its replication and maintenance in Str . lividans . There were detected unique sites of pSB24.1 and its derivatives useful for cloning . Possible shortening of plasmid pSB24.1 by 567 kb (the length between the terminator of the open frame reading translation and XhoI site) was revealed.

Mol Biol Med, 1987 Oct, 4(5), 307 - 22
Selection of mutations that increase alpha 1-antitrypsin gene expression in Escherichia coli; Sutiphong J et al.; The gene encoding human alpha-1-antitrypsin (A1AT), when cloned and expressed as a full-length, non-fusion gene product in Escherichia coli, accumulates to levels up to 0.1% of total cellular protein . Truncation of the gene at its 5' end or synthesis as a fusion protein increases expression up to 200-fold . Extensive mutagenesis in vitro within this same 5'-terminal region aimed at improving codon usage and disrupting potential secondary structure increased expression only 10 to 20-fold . We have developed a translational fusion system for selecting mutations and applied it to the study of A1AT expression in E . coli . With this methodology, we have obtained single base-pair mutations having up to a 20-fold effect on A1AT expression . When we combined these multiple single base-pair mutations, we achieve up to a 200-fold increase in A1AT expression . The resulting gene product is of authentic size (394 amino acid residues) and contains two amino acid substitutions (Asn in place of Asp) in codons 2 and 6 . This protein is primarily in the soluble fraction of the E . coli lysate and has identical activity to A1AT purified from human sera . The methodology used to generate these mutations may be generally applicable to the study of genes that do not express well in E . coli initially, and provides an alternative to secondary structure analysis in the redesign of such genes.

Genomics, 1987 Oct, 1(2), 153 - 8
Microdissection and microcloning from the proximal region of mouse chromosome 7: isolation of clones genetically linked to the pudgy locus; Greenfield AJ et al.; Microdissection and microcloning have been utilized in order to create a bank of clones from the proximal region of mouse chromosome 7 . Several important loci map to this area, including the albino locus (c), pink-eye dilution (p), and the developmental mutant, pudgy (pu) . By use of interspecific crosses between Mus musculus domesticus and Mus spretus, we have generated backcross progeny segregating for the mutations chinchilla (cch) and pink-eye dilution (p) . Exploiting the evolutionary divergence between the two species, we have analyzed the inheritance of restriction fragment length variants of three microclones and their linkage to the two markers cch and p, respectively . All three clones studied map to the dissected region, and as such also show genetic linkage to the pudgy locus . This bank of chromosome 7-derived microclones should provide molecular start points for the isolation of a variety of developmental loci of unknown gene product, including the pudgy locus.

Eur J Respir Dis, 1987 Oct, 71(4), 250 - 8
Surfactant abnormality after endotoxin-induced lung injury in guinea-pigs; Tahvanainen J et al.; Endotoxin (30 mg/kg) or saline was given endotracheally to guinea-pigs in order to investigate surfactant function in respiratory failure . Six hours later, bronchoalveolar lavage was performed . The lavage was analyzed for protein, phospholipids and surface activity, and fractioned into the phospholipid-rich sediment and the phospholipid-poor supernatant . The latter fraction was analyzed for surfactant inhibitor activity . After endotoxin, PaO2 and static lung-thorax compliance decreased . The lavage from endotoxin-treated animals revealed a 180% increase in protein, a 52 67% decrease in surfactant phospholipids, and increased minimum surface tension, as compared to the controls . After endotoxin, the supernatant contained a 58% higher activity of surfactant inhibitor, and the sediment had slower surface adsorption than after saline . We propose that abnormal surfactant function is important in the pathogenesis of respiratory failure in high-permeability pulmonary edema.

EMBO J, 1987 Oct, 6(10), 3177 - 83
Formamidopyrimidine-DNA glycosylase of Escherichia coli: cloning and sequencing of the fpg structural gene and overproduction of the protein; Boiteux S et al.; An Escherichia coli genomic library composed of large DNA fragments (10-15 kb) was constructed using the plasmid pBR322 as vector . From it 700 clones were individually screened for increased excision of the ring-opened form of N7-methylguanine (2-6-diamino-4-hydroxy-5N-methyl-formamidopyrimidine) or Fapy . One clone overproduced the Fapy-DNA glycosylase activity by a factor of 10-fold as compared with the wild-type strain . The Fapy-DNA glycosylase overproducer character was associated with a 15-kb recombinant plasmid (pFPG10) . After subcloning a 1.4-kb fragment which contained the Fapy-DNA glycosylase gene (fpg+) was inserted in the plasmids pUC18 and pUC19 yielding pFPG50 and pFPG60 respectively . The cells harbouring pFPG60 displayed a 50- to 100-fold increase in glycosylase activity and overexpressed a 31-kd protein . From these cells the Fapy-DNA glycosylase was purified to apparent physical homogeneity as evidenced by a single protein band at 31 kd on SDS-polyacrylamide gels . The amino acid composition of the protein and the amino acid sequence deduced from the nucleotide sequence demonstrate that the cloned fragment contains the structural gene coding for the Fapy-DNA glycosylase . The nucleotide sequence of the fpg gene is composed of 809 base pairs and codes for a protein of 269 amino acids with a calculated mol . wt of 30.2 kd.

Biochem J, 1987 Oct 1, 247(1), 195 - 9
Characterization of human and mouse granulocyte-macrophage-colony-stimulating factors derived from Escherichia coli; Schrimsher JL et al.; Human and mouse granulocyte-macrophage-colony-stimulating factors (hGM-CSF and mGM-CSF, respectively), isolated from Escherichia coli cells expressing the corresponding human and mouse genes, have been characterized . The observed properties of the proteins have been compared with those properties which can be deduced from the DNA sequence alone and the published properties of natural GM-CSFs . The purified E . coli-derived proteins were found to have the expected molecular masses, amino acid compositions and N- and C-terminal amino acid sequences . The finding of 70-90% unprocessed N-terminal methionine for both proteins is discussed . The four Cys residues were found to be involved in two intramolecular disulphide bonds, linking the first and third, and second and fourth Cys residues . This disulphide bond arrangement is probably the one existing in natural material, since, although not glycosylated, both E . coli-derived proteins showed biological activity (colony stimulating assay for hGM-CSF, and cell proliferation assay for mGM-CSF) comparable with that reported for the respective proteins purified from animal cells.

Arch Microbiol, 1987 Oct, 148(4), 298 - 304
The Streptomyces ghanaensis low copy plasmid pSG2 and its use for vector construction; Wohlleben W et al.; A plasmid, pSG2, was isolated from Streptomyces ghanaensis and characterized by electron microscopy, buoyant density measurement, and restriction enzyme analysis . The length of 13.8 kb, single restriction sites for HindIII, EcoRV and PvuII and the possibility of deleting non-essential regions of the plasmid made pSG2 a suitable basic replicon for vector development . pSG2 has a copy number of about four . Plasmid pSG2 was fused to a pACYC184 derivative modified to harbour a thiostrepton resistance gene . The resulting plasmid, designated pSW1, is a 16.6 kb shuttle plasmid which replicates in Escherichia coli and in several Streptomyces strains, including S . ghanaensis, S . lividans and S . viridochromogenes . Replacement of a BglII-fragment of plasmid pSG2 by a fragment encoding thiostrepton resistance resulted in a low copy 12.2 kb Streptomyces plasmid . This plasmid, designated pSW2, is a Streptomyces broad host range plasmid.

Acta Med Okayama, 1987 Oct, 41(5), 187 - 93
Radioprotective effects of thiomethylhydantoin derivatives on Escherichia coli and mice; Nishimura A et al.; Protection of Escherichia coli NIHJ and C57BL mice from the effects of 60Co gamma-rays provided by S-alk(en)yl-L-cysteines and their hydantoin derivatives was examined . E . coli (10(6) cells/ml) suspended in a 20 mM aqueous solution of one of the drugs was irradiated with 60 Gy of gamma-rays . Five week-old male mice were exposed to 5.0-9.5 Gy of gamma-rays after a single intraperitoneal injection of 0.75 mmol/kg body weight of each compound . In both E . coli and mice, S-allyl compounds afforded more effective radioprotection than S-propyl compounds . The replacement of the alpha-hydrogen of S-substituted cysteines by methyl groups decreased the radioprotective effect . Hydantoin derivatives were much more radioprotective than the original sulfur-containing amino acids . Especially, DL-5-allylthiomethyl-5-methylhydantoin had a remarkable radioprotective effect in mice . The gamma-radiolysis mechanism of thiomethylhydantoin derivatives was discussed in connection with the radioprotective effect of the drugs.

Eur J Immunol, 1987 Oct, 17(10), 1527 - 30
In vitro production of human interleukin 1 alpha and interleukin 1 beta by peripheral blood mononuclear cells examined by sensitive sandwich enzyme immunoassay; Tanaka K et al.; The in vitro production of human interleukin 1 alpha (hIL 1 alpha) and interleukin 1 beta (hIL 1 beta) by peripheral blood mononuclear cells was examined by sensitive sandwich enzyme immunoassays which could discriminate hIL 1 alpha and hIL 1 beta without cross-reaction with human IL2 . In culture supernatants of mononuclear cells, two components were detected by sandwich enzyme immunoassay for hIL 1 alpha or hIL 1 beta . The molecular weight of one component was shown to be equal to that of recombinant hIL 1 alpha or hIL 1 beta by gel filtration . The elution volume of the other component corresponded to a molecular weight of about 30,000 . The sum of the two components for both hIL 1 alpha and hIL 1 beta in culture supernatants of peripheral blood mononuclear cells from healthy subjects increased 1.7 to 38-fold by Escherichia coli lipopolysaccharide . The sum of the two components for hIL 1 beta was 13 to 97-fold larger than that for hIL 1 alpha.

Biull Eksp Biol Med, 1987 Oct, 104(10), 497 - 9
{Activation of the production of the tumor-necrosis factor by the combined action of lipopolysaccharide and muramyl dipeptide in vitro and in vivo}; Fuks BB et al.; The effect of bacterial lipopolysaccharide (LPS), muramyl dipeptide (MDP) and their combination on the production of tumour necrosis factor by spleen cells in vitro and on tumour regression in vivo has been studied . TNF activity was detected in spleen cell supernatants and serum of mice treated with drugs, using L929 cells as targets . The combination of LPS and MDP was more effective in TNF production than each of the drugs used alone in vitro and in vivo . The injection of LPS and MDP to A/Sn mice with subcutaneous nodes of sarcoma SA-I resulted in total tumour necrosis . The treatment of mice with these drugs in water solutions was more effective, however, more toxic than the administration of LPS-treated splenocytes in MDP solution.

Biotechnol Appl Biochem, 1987 Oct, 9(5), 368 - 79
Partial purification of an oxygen scavenging cell membrane fraction for use in anaerobic biochemical reactions; Jacobson KB et al.; Anaerobic conditions are necessary to prevent the autoxidation of tetrahydrobiopterin . An enzymatic method for achieving such an anaerobic condition is shown to be obtained by a lactate oxidase activity in the membrane of Escherichia coli when the enzyme concentration exceeds 0.05 unit/ml . A procedure is described for partial purification of the membrane fragment that involves salt precipitation and gel sieve chromatography on Bio-Gel A-50m . The rate of removal of oxygen is used to define the enzyme activity, to determine stability during storage, and to define conditions for stabilization of tetrahydrobiopterin . A second assay procedure that measures the rate of reduction of resazurin is described and is useful when the enzyme concentration is low.

Acta Physiol Scand, 1987 Oct, 131(2), 297 - 301
Endotoxin-induced prostaglandin (PGF2 alpha) biosynthesis, fever and miosis in dexamethasone-treated goats; Jonasson H et al.; Prostaglandin-releasing, adrenocortical, febrile and miotic responses to endotoxin (ET) (E . coli lipopolysaccharide; 0.25 microgram kg-1) were studied in goats with and without prolonged dexamethasone influence . The i.v . injection of ET induced a three-fold peak elevation in plasma 15-ketodihydro-PGF2 alpha at 1.5 h post-injection, that is, between the first and second phase of the temperature elevation . During the latter phase, the plasma concentration of this primary PGF 2 alpha metabolite gradually returned to basal level, which implies that the second phase of ET fever is not PG dependent . The PG response exhibited a similar pattern, but was less pronounced in the dexamethasone-ET experiments, where the duration of maximum temperature elevation and of the miosis became shortened by about 20 min, and the typical biphasic pattern of ET fever was no longer seen . The ET-induced rise in plasma aldosterone concentration was completely blocked by dexamethasone . The corresponding rise in plasma cortisol concentration was prevented for 2 h, but was later only partially inhibited in spite of the repeated dexamethasone treatment.

Proc Natl Acad Sci U S A, 1987 Oct, 84(20), 7144 - 8
Proposed mechanism for generation and localization of new cell division sites during the division cycle of Escherichia coli; Cook WR et al.; The earliest detectable event at future sites of cell division in Escherichia coli is the appearance of paired periseptal annuli that flank the site of formation of the division septum . The development and localization of these structures were followed as the cell progressed through the division cycle . The data suggest that (i) new periseptal annuli are generated from annuli already in position at the midpoint of the newborn cell; (ii) the nascent annuli are then displaced laterally during cell elongation to positions at 1/4 and 3/4 cell length; and (iii) the annuli at 1/4 and 3/4 cell length are retained during division, becoming the midpoint annuli of the newborn cells at the sites of the forthcoming division septum . The results indicate that the sites of future divisions can be identified and committed to the division process prior to the division cycle in which these sites are utilized for septum formation, and they suggest a model in which preexisting sites of cell division generate future division sites by a replication/displacement mechanism.

Proc Natl Acad Sci U S A, 1987 Oct, 84(20), 7139 - 43
Primary structure and subcellular localization of the knob-associated histidine-rich protein of Plasmodium falciparum; Pologe LG et al.; Plasmodium falciparum-infected erythrocytes bind to venular endothelial cells by means of electron-dense deformations (knobs) on the parasitized erythrocyte surface . The primary structure of a parasite-derived histidine-rich protein associated with the knob structure was deduced from cDNA sequence analysis . The 634 amino acid sequence is rich in lysine and histidine and contains three distinct, tandemly repeated domains . Indirect immunofluorescence, using affinity-purified monospecific antibodies directed against recombinant protein synthesized in Escherichia coli, localized the knob-associated histidine-rich protein to the membrane of knobby infected erythrocytes . Immunoelectron microscopy established that the protein is clustered on the cytoplasmic side of the erythrocyte membrane and is associated with the electron-dense knobs . A role for this histidine-rich protein in knob structure and cytoadherence is suggested based upon these data.

J Clin Microbiol, 1987 Oct, 25(10), 2025 - 7
DNA probes for identification of enteroinvasive Escherichia coli; Gomes TA et al.; Eighty-one Escherichia coli strains belonging to all known invasive O serogroups were tested with two distinct invasiveness probes (pMR17 and pSF55) . All 54 Sereny test-positive strains and 5 strains that lost Sereny positivity during storage hybridized with both probes . Probe-positive strains carried a 120- to 140-megadalton plasmid, did not produce lysine decarboxylase, and, with the exception of certain serotypes, were nonmotile . Motile strains of serotype O144:H25 were for the first time characterized as invasive by hybridization with the probes.

J Clin Microbiol, 1987 Oct, 25(10), 1917 - 9
Enteroadherent Escherichia coli as a cause of diarrhea among children in Mexico; Mathewson JJ et al.; Enteropathogenic Escherichia coli (EPEC) often exhibits localized adherence or diffuse adherence to HEp-2 cells . We recently provided evidence that HEp-2 cell-adherent or enteroadherent E . coli (EAEC) not belonging to EPEC serogroups was the cause of diarrhea among U.S . travelers to Mexico . In the present study, we looked for EAEC and EPEC in stool specimens from 154 children with acute diarrhea and 137 well children seen at several outpatient clinics in Guadalajara, Mexico . EAEC showing localized adherence (EAEC-L) was isolated from 13.0% of the patients and 0.7% of the controls (P less than 0.0001) . EAEC showing diffuse adherence (EAEC-D) was recovered from 20.8% of the patients and 7.3% of the controls (P less than 0.001) . EPEC was isolated from 4.5 and 6.7% of the patients and controls, respectively . Among all enteropathogens, only enterotoxigenic E . coli occurred as commonly (21.4%) as EAEC-D and EAEC-L did in children with diarrhea . Of the EAEC-L strains isolated from children with diarrhea, 20% belonged to recognized EPEC serogroups, and 3.1% of EAEC-D strains belonged to recognized EPEC serogroups . This study suggests that EAEC may be an important pediatric enteropathogen in Mexican children with diarrhea and further supports the observation that adherence to HEp-2 cells may be a marker of virulence independent of EPEC serogroup among E . coli strains.

Eur J Biochem, 1987 Oct 1, 168(1), 227 - 31
Co-expression of both the maize large and wheat small subunit genes of ribulose-bisphosphate carboxylase in Escherichia coli; Gatenby AA et al.; A cDNA clone for the precursor form of the small subunit of wheat ribulose-bisphosphate carboxylase has been modified to allow the expression in Escherichia coli of a mature form of small subunit that lacks the transit peptide . Synthesis of the protein is controlled by a lac promoter, and translation is initiated from a lacZ ribosome binding site, giving rise to a small subunit with several beta-galactosidase amino acids fused to its N-terminus . A plasmid has been constructed that enables both wheat small subunits and maize large subunits to be synthesized in the bacterial cell, but using different promoters to allow independent expression of the rbcS and rbcL genes . When the small subunit is synthesized in the absence of the large subunit, it is found in the soluble fraction but the polypeptide is unstable and has a half-life of less than 15 min . Its size on sucrose gradients indicates a monomeric or dimeric form . When large subunit synthesis is induced in cells containing the small subunit, both subunits are found predominantly in the insoluble fraction and are fully stable for more than 120 min, suggesting that aggregation of the subunits may occur . The two subunits do not assemble together to form an active holoenzyme in vivo, even when nascent large subunits ware synthesized in a pool of mature small subunits . This indicates that other factors may be required to mediate the assembly of the higher plant enzyme.

Arch Biochem Biophys, 1987 Oct, 258(1), 233 - 9
Reaction of 5-enol-pyruvoylshikimate-3-phosphate synthase with diethyl pyrocarbonate: evidence for an essential histidine residue; Huynh QK; 5-enol-Pyruvoylshikimate-3-phosphate synthase catalyzes the reversible condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 5-enol-pyruvoylshikimate 3-phosphate and inorganic phosphate . The enzyme is a target for the nonselective herbicide glyphosate (N-phosphonomethylglycine) . Diethyl pyrocarbonate inactivated this enzyme with a second-order rate constant of 220 M-1 min-1 at pH 7.0 and 0 degrees C . The rate of inactivation is pH dependent and the pH inactivation rate data show the involvement of a group with a pKa of 6.8 . Almost all of the original activity was recovered by treatment of the inactivated enzyme with hydroxylamine . The difference spectrum of the inactivated and native enzyme reveals a single peak at 242 nm but no trough at around 278 nm is observed . Complete inactivation required the modification of four histidine residues per molecule of the enzyme . However, statistical analysis of the residual activity and the extent of modification shows that among the four modifiable residues, only one is critical for activity . Furthermore, this inactivation is prevented by the substrates of the enzyme . The above results indicated that one histidine is located within or very close to the active site and may play an important role in catalysis.

Virology, 1987 Oct, 160(2), 389 - 99
Immunological analysis of 140-kDa adenovirus-encoded DNA polymerase in adenovirus type 2-infected HeLa cells using antibodies raised against the protein expressed in Escherichia coli; Sasaguri Y et al.; The E2B region of adenovirus genome contains a long open reading frame (ORF) extending from 24 to 14.2 map units which encodes most of the 140-kDa DNA polymerase . It was cloned at the polylinker region of pUC18 vector with Escherichia coli JM109 as the host . A clone was serendipitously isolated that expressed in E . coli a protein of approximately 120 kDa in size at high levels . DNA sequence analysis of this clone showed the presence of an in-frame fusion of a region, encoding 13 amino acids located upstream, to the first ATG of the ORF . Polyclonal antibodies raised against this protein purified from E . coli were used for immunological analysis . The antibodies were able to detect a 140- and a 66-kDa polypeptide from the adenovirus type 2-infected HeLa cells on Western blots . In addition, the antibodies showed evidence of cross-reactivity with partially purified DNA polymerase alpha from uninfected HeLa cells . The subcellular localization of the viral polymerase in the infected HeLa cells by using indirect immunofluorescence showed that the viral protein is associated with globular structures in the nucleus . The replicating viral DNA and the polymerase were colocalized in these globular sites . Furthermore, HeLa cells infected with Ad5ts149, a temperature-sensitive mutant defective in DNA replication, showed the presence of these globular sites only at the permissive temperature, suggesting that these sites are probably involved in viral DNA replication.

Proc Natl Acad Sci U S A, 1987 Oct, 84(19), 6805 - 9
Recovery from ultraviolet light-induced inhibition of DNA synthesis requires umuDC gene products in recA718 mutant strains but not in recA+ strains of Escherichia coli; Witkin EM et al.; Ultraviolet light (UV) inhibits DNA replication in Eschericia coli and induces the SOS response, a set of survival-enhancing phenotypes due to derepression of DNA damage-inducible genes, including recA and umuDC . Recovery of DNA synthesis after UV irradiation ("induced replisome reactivation," or IRR) is an SOS function requiring RecA protein and postirradiation synthesis of additional protein(s), but this recovery does not require UmuDC protein {Khidhir, M . A., Casaregola, S . & Holland, I . B . (1985) Mol . Gen . Genet . 199, 133-140} . IRR occurs in strains carrying either recA718 (which does not reduce recombination, SOS inducibility, or UV mutagenesis) or umuC36 (which eliminates UV mutability), but not in recA718 umuC36 double mutants . In recA430 mutant strains, IRR does not occur whether or not functional UmuDC protein is present . IRR occurs in lexA-(Ind-) (SOS noninducible) strains if they carry an operator-constitutive recA allele and are allowed to synthesize proteins after irradiation . We conclude the following: (i) that UmuDC protein corrects or complements a defect in the ability of RecA718 protein (but not of RecA430 protein) to promote IRR and (ii) that in lexA(Ind-) mutant strains, IRR requires amplification of RecA+ protein (but not of any other LexA-repressed protein) plus post-UV synthesis of at least one other protein not controlled by LexA protein . We discuss the results in relation to the essential, but unidentified, roles of RecA and UmuDC proteins in UV mutagenesis.

Mutat Res, 1987 Oct, 192(2), 105 - 8
Induction of the SOS response by hydroxyurea in Escherichia coli K12; Barbe J et al.; Hydroxyurea at concentrations higher than 10(-2) M induced the recA and sfiA genes of E . coli as well as the lambda prophage by a pathway independent of the recBC genes . In addition, the hydroxyurea-mediated induction of the SOS response is accompanied by a recA-dependent decrease on the cellular ATP pool . The presence of the multicopy plasmid pPS2, harboring the nrdAB genes (encoding the ribonucleoside reductase enzyme), abolished the hydroxyurea-induced expression of the recA gene . These data lead us to suggest that induction of the SOS response by hydroxyurea is due to the blocking of DNA replication by the inhibition of the ribonucleoside reductase complex activity.

J Bacteriol, 1987 Oct, 169(10), 4854 - 6
Spontaneous missense mutations in the rplX gene for ribosomal protein L24 from Escherichia coli; Nishi K et al.; Temperature-resistant pseudorevertants of the temperature-sensitive Escherichia coli mutant KNS19, harboring a mutation in rplX, the gene for ribosomal protein L24, were isolated, cloned, and sequenced . The codon GAC for the amino acid Asp in the temperature-sensitive mutant corresponding to position 84 in the protein chain mutated either back to the wild type (Gly) or to codons for the amino acids Tyr and Glu . Furthermore, rplX genes from two other mutants with an altered protein L24 were cloned and sequenced . The mutations were localized at position 56 (Gly to Asp) and at position 62 (Glu to Lys) in the rplX gene . The latter two mutants lacked a conditional lethal phenotype . The results suggest that the amino acid Gly at positions 56 and 84 in the protein might be involved in loop formations.

J Bacteriol, 1987 Oct, 169(10), 4852 - 3
Genetic separability of the chorismate mutase and prephenate dehydrogenase components of the Escherichia coli tyrA gene product; Maruya A et al.; Fragments of the tyrA gene of Escherichia coli, when suitably engineered, can express either the chorismate mutase activity or the prephenate dehydrogenase activity without the other.

J Bacteriol, 1987 Oct, 169(10), 4841 - 4
Mutations in the recD gene of Escherichia coli that raise the copy number of certain plasmids; Seelke R et al.; Chromosomal mutants were isolated in which, for several small plasmids, there was an increased amount of either covalently closed circular plasmid DNA or total plasmid DNA or both . The mutations were mapped to recD, which has been shown to affect exonuclease V activity and a variety of plasmid maintenance and replication functions . Our results suggest that rolling-circle plasmid replication can occur in recD mutants and that site-specific recombination can resolve the resulting linear multimers into covalently closed circular plasmid forms.

J Bacteriol, 1987 Oct, 169(10), 4834 - 6
recA gene of Escherichia coli complements defects in DNA repair and mutagenesis in Streptomyces fradiae JS6 (mcr-6); Matsushima P et al.; Streptomyces fradiae JS6 (mcr-6) is a mutant which is defective in repair of DNA damage induced by a variety of chemical mutagens and UV light . JS6 is also defective in error-prone (mutagenic) DNA repair (J . Stonesifer and R . H . Baltz, Proc . Natl . Acad . Sci . USA 82:1180-1183, 1985) . The recA gene of Escherichia coli, cloned in a bifunctional vector that replicates in E . coli and Streptomyces spp., complemented the mutation in S . fradiae JS6, indicating that E . coli and S . fradiae express similar SOS responses and that the mcr+ gene product of S . fradiae is functionally analogous to the protein encoded by the recA gene of E . coli.

J Bacteriol, 1987 Oct, 169(10), 4710 - 5
Molecular analysis of the regulatory region of the Escherichia coli K-12 tyrB gene; Yang J et al.; The tyrB gene from Escherichia coli K-12 was cloned and sequenced, and the transcriptional start point of tyrB was determined by primer extension . By using a fusion plasmid in which the lacZ structural gene is transcribed from the tyrB promoter, it was shown that the expression of tyrB is controlled at the transcriptional level by the TyrR protein, with tyrosine as corepressor . The fusion plasmid was used to isolate mutants in which the repression of tyrB had been abolished . The tyrB promoter-operator region of these mutants was sequenced, and the tyrB operator was identified . A comparison between the tyrB operator and those of the other genes belonging to the tyrR regulon is presented.

J Bacteriol, 1987 Oct, 169(10), 4686 - 91
Signal sequence mutations that alter coupling of secretion and translation of an Escherichia coli outer membrane protein; Benson SA et al.; The lamB701-708 signal sequence mutation reduces expression of LamB, an outer membrane protein of Escherichia coli . To investigate the possibility that synthesis and export of LamB are coupled, as suggested by the expression defect of the lamB701-708 mutation, we isolated intragenic suppressors of the lamB701-708 mutation . The expression defect imposed by the lamB701-708 mutation is suppressed by an export-defective signal sequence mutation, suggesting that translation and export are coupled . The additional observation that not all export-defective signal sequence mutations suppressed the lamB701-708 expression defect suggests that translational arrest can be uncoupled from export.

J Bacteriol, 1987 Oct, 169(10), 4678 - 85
Involvement of a low-molecular-weight substance in in vitro activation of the molybdoenzyme respiratory nitrate reductase from a chlB mutant of Escherichia coli; Boxer DH et al.; The soluble subcellular fraction of a chlB mutant contains an inactive precursor form of the molybdoenzyme nitrate reductase, which can be activated by the addition to the soluble fraction of protein FA, which is thought to be the active product of the chlB locus . Dialysis or desalting of the chlB soluble fraction leads to the loss of nitrate reductase activation, indicating that some low-molecular-weight material is required for the activation . The protein FA-dependent activation of nitrate reductase can be restored to the desalted chlB soluble fraction by the addition of a clarified extract obtained after heating the chlB soluble fraction at 100 degrees C for 8 min . The heat-stable substance present in this preparation has a molecular weight of approximately 1,000 . This substance is distinct from the active molybdenum cofactor since its activity is unimpaired in heat-treated extracts prepared from the organism grown in the presence of tungstate, which leads to loss of cofactor activity . Mutations at the chlA or chlE locus, which are required for molybdenum cofactor biosynthesis, similarly do not affect the activity of the heat-treated extract in the in vitro activation process . Moreover, the active material can be separated from the molybdenum cofactor activity by gel filtration . None of the other known pleiotropic chlorate resistance loci (chlD, chlG) are required for the expression of its activity . Magnesium ATP appears to have a role in the formation of the active substance . We conclude that a low-molecular-weight substance, distinct from the active molybdenum cofactor, is required to bestow activity on the molybdoenzyme nitrate reductase during its biosynthesis.

J Bacteriol, 1987 Oct, 169(10), 4614 - 20
Promoter region of the nar operon of Escherichia coli: nucleotide sequence and transcription initiation signals; Li SF et al.; The nar operon, which encodes the three subunits of nitrate reductase in Escherichia coli, is fully induced under anaerobic conditions with nitrate . Two distinct regulatory domains have been delineated in the 5' region of the operon which respond respectively to positive induction by the fnr gene product under anaerobic conditions and to positive induction by the narL gene product in the presence of nitrate (S.F . Li, T . Rabi, and J.A . DeMoss, J . Bacteriol . 164:25-32) . To characterize these two regulatory regions, we determined the DNA sequence for a 500-base-pair (bp) region extending upstream from the first structural gene of the nar operon . Analysis of subsequent subclones of the operon established that the 5' limit of the nar operon lies between 215 and 260 bp upstream from the translational start site of the first structural gene . The region required for induction by the fnr gene product is located within 160 bp from the translation start site, while the region responding to induction by nitrate extends an additional 100 bp upstream . Protein fusions of lacZ with the N-terminal sequence of the narG gene were constructed so that beta-galactosidase formation was under the control of the nar promoter and one or both regulatory domains . Analysis of strains bearing these fusion plasmids indicated that the expression of the hybrid proteins paralleled that of nitrate reductase by the parent plasmids, demonstrating that the regulatory signals did not extend significantly into the first structural gene . The transcriptional start site and the level of the transcription were determined by the S1 mapping procedure . One major transcript was identified which initiated -50 bp from the translational start site of the first structural gene . The synthesis of the transcript was repressed aerobically, was fully induced by nitrate anaerobically, and was greatly reduced in an Fnr- mutant . Possible regulatory sequences were identified in the 200-bp regulatory region extending upstream from the transcription start site.

J Bacteriol, 1987 Oct, 169(10), 4559 - 64
Role of DNA polymerase I in postreplication repair: a reexamination with Escherichia coli delta polA; Sharma RC et al.; Using strains of Escherichia coli K-12 that are deleted for the polA gene, we have reexamined the role of DNA polymerase I (encoded by polA) in postreplication repair after UV irradiation . The polA deletion (in contrast to the polA1 mutation) made uvrA cells very sensitive to UV radiation; the UV radiation sensitivity of a uvrA delta polA strain was about the same as that of a uvrA recF strain, a strain known to be grossly deficient in postreplication repair . The delta polA mutation interacted synergistically with a recF mutation in UV radiation sensitization, suggesting that the polA gene functions in pathways of postreplication repair that are largely independent of the recF gene . When compared to a uvrA strain, a uvrA delta polA strain was deficient in the repair of DNA daughter strand gaps, but not as deficient as a uvrA recF strain . Introduction of the delta polA mutation into uvrA recF cells made them deficient in the repair of DNA double-strand breaks after UV irradiation . The UV radiation sensitivity of a uvrA polA546(Ts) strain (defective in the 5'----3' exonuclease of DNA polymerase I) determined at the restrictive temperature was very close to that of a uvrA delta polA strain . These results suggest a major role for the 5'----3' exonuclease activity of DNA polymerase I in postreplication repair, in the repair of both DNA daughter strand gaps and double-strand breaks.

J Bacteriol, 1987 Oct, 169(10), 4499 - 506
Changes in the linking number of supercoiled DNA accompany growth transitions in Escherichia coli; Balke VL et al.; The supercoiling levels of plasmid DNA were determined from Escherichia coli which was grown in ways that are known to alter global patterns of gene expression and metabolism . Changes in DNA supercoiling were shown to occur during several types of these nutrient upshifts and downshifts . The most dramatic change in supercoiling was seen in starved cells, in which two populations of differentially relaxed plasmids were shown to coexist . Thus, some changes in the external nutritional environment that cause the cells to reorganize their global metabolism also cause accompanying changes in DNA supercoiling . Results of experiments with dinitrophenol suggested that the observed relaxations were probably not due to reduced pools of ATP . When rifampin was used to release supercoils restrained by RNA polymerase, the cellular topoisomerases responded by removing these new, unrestrained supercoils . We interpret these results as implying that the cellular topological machinery maintains a constant superhelical energy in the DNA except during certain growth transitions, when changes in metabolism and gene expression are accompanied by changes in DNA supercoiling.

Infect Immun, 1987 Oct, 55(10), 2471 - 6
Lipid X ameliorates pulmonary hypertension and protects sheep from death due to endotoxin; Golenbock DT et al.; Lipid X (2,3-diacylglucosamine-1-phosphate) is a novel monosaccharide precursor of lipid A that has some of the physiologic activities of endotoxin but little toxicity . To determine whether lipid X would interfere with the toxic effects of endotoxin, we pretreated sheep with either 100 or 200 micrograms of lipid X per kg of body weight and then challenged them with a potentially fatal dose of Escherichia coli endotoxin (20 micrograms/kg) . Twenty-one sheep underwent pulmonary artery catheterization and were monitored for changes in pulmonary artery pressure, temperature, pH, partial O2 pressure, partial CO2 pressure, blood pressure, and cell counts over 7 h . Overall mortality for control animals was 37% versus 5.3% for pretreated animals . None of the 13 animals pretreated with 100 micrograms of lipid X per kg died . These differences in survival were significant (P less than 0.05) . Animals pretreated with 100 micrograms of lipid X per kg had significantly lower pulmonary artery pressure during both phases 1 and 2 of endotoxin-induced pulmonary artery hypertension . A higher dose of lipid X, 200 micrograms/kg, produced pulmonary hypertension . Perhaps because lipid X is a subunit of lipid A, lipid X shows a partial pyrogenic effect while also decreasing the pyrogenic activity of complete lipopolysaccharide (LPS) . Lipid X did not prevent endotoxin-induced neutropenia or moderate hypotension in response to LPS . Lipid X is a potential prototype compound for a new type of chemotherapy directed at blocking the harmful effects of LPS during bacterial septicemia.

Blood, 1987 Oct, 70(4), 915 - 20
Cloning and nucleotide sequence of a mouse erythrocyte beta-spectrin cDNA; Cioe L et al.; A rabbit monospecific antibody for mouse beta-spectrin was used to screen a mouse anemic spleen cDNA expression library . A mouse beta-spectrin cDNA clone was isolated and identified by its ability to make mouse beta-spectrin-like antigens in Escherichia coli . This clone was used to probe total RNA from various mouse tissues . Anemic spleen RNA showed two strongly hybridizing RNA species of approximately 6 and 8 kb . Two very faintly hybridizing bands of about 6 kb and 10 kb could also be seen in total mouse brain RNA . All of these bands could be detected after hybridization under both stringent and nonstringent conditions . This suggests that erythroid beta-spectrin may also be expressed in the brain . No bands could be detected in kidney, liver, or spleen RNA . Southern blot analysis of mouse genomic DNA showed a single hybridizing band after digestion with several restriction endonucleases even under nonstringent conditions . Nucleotide sequencing of the cDNA insert revealed almost complete identity between the N-terminus of the deduced amino acid sequence of the cDNA clone and the C-terminal 15 amino acids of a peptide derived from the beta-8 repeat unit of human erythrocyte beta-spectrin . The deduced amino acid sequence contained most of the conserved amino acids characteristic of the 106 amino acid repeat unit first found in human alpha-spectrin and thus provides the first evidence for a complete 106 amino acid repeat unit structure in beta-spectrin.

Gastroenterology, 1987 Oct, 93(4), 765 - 73
Evidence for platelet-activating factor as a mediator of endotoxin-induced gastrointestinal damage in the rat . Effects of three platelet-activating factor antagonists; Wallace JL et al.; The potential role of platelet-activating factor (PAF) as a mediator of gastrointestinal ulceration associated with septic shock was examined in the rat . The damaging effects of both PAF and Escherichia coli endotoxin in the stomach and small intestine were compared, as were their effects on plasma leakage into the lumen of the gastrointestinal tract . Intravenous administration of either endotoxin or PAF produced extensive necrosis and vascular congestion in the stomach and small intestine, but not the distal colon . With either agent, the duodenum and jejunum were the tissues most susceptible to damage and in which the greatest plasma leakage was observed . The prolonged hypotension and gastrointestinal damage induced by PAF or endotoxin were significantly inhibited by three structurally dissimilar PAF antagonists (CV-3988, BN-52021, and Ro-193704) . CV-3988 (10 mg/kg) significantly (p less than 0.05) reduced both endotoxin- and PAF-induced plasma leakage in the stomach and small intestine . Of the three antagonists, only CV-3988 significantly reduced ethanol-induced gastric mucosal damage, perhaps reflecting actions of this compound unrelated to antagonism of PAF receptors . These studies support the hypothesis that PAF is an important mediator of the hypotension and plasma leakage observed during endotoxic shock and its endogenous release may contribute to the gastrointestinal ulceration associated with this syndrome . Thus, PAF receptor antagonists may be useful for prevention of such ulceration.

Biochimie, 1987 Oct, 69(10), 1081 - 96
Higher-order structure of domain III in Escherichia coli 16S ribosomal RNA, 30S subunit and 70S ribosome; Baudin F et al.; We have investigated in detail the conformation of domain III of 16S rRNA (nucleotides 913-1408), using a variety of chemical and enzymatic structure probes . The sites of reaction were identified by primer extension with reverse transcriptase using appropriate oligodeoxyribonucleotide primers . This study has been done on 16S rRNA in its naked form, in the 30S subunit and in the 70S ribosome . Data obtained with naked RNA broadly confirm the secondary structure model proposed essentially by comparative sequence analysis, and allow identification of nucleotides involved in tertiary interactions . Our results are in reasonably good agreement with structure probing experiments of Moazed et al . {1} . However, several discrepancies have been observed . Within the 30S subunit, a high number of nucleotides become unreactive whereas other nucleotides show an enhanced reactivity . This probably reflects local conformational changes . Interestingly, they are located in strategic regions of the RNA, e.g . around C1400 (involved in tRNA binding) and C1192 (involved in spectinomycin recognition) . Results are also discussed together with the topographical localization of the ribosomal proteins in this area . The study on the 70S particle allows identification of regions at the interface of subunits or exposed at the surface of the ribosome.

Biochimie, 1987 Oct, 69(10), 1049 - 64
The topography of ribosomal proteins on the surface of the 30S subunit of Escherichia coli; Stoffler-Meilicke M et al.; Eight ribosomal proteins, S6, S10, S11, S15, S16, S18, S19 and S21 have been localized on the surface of the 30S subunit from Escherichia coli by immuno electron microscopy . The specificity of the antibody binding sites was demonstrated by stringent absorption experiments . In addition we have reinvestigated and refined the locations of proteins S5, S13 and S14 on the ribosomal surface which had previously been localized in our laboratory (Tischendorf et al., Mol . Gen . Genet . 134, 209-223, 1974) . Thus altogether 16 out of the 21 ribosomal proteins of the small subunit from E . coli have been mapped in our laboratory.

Biochem Int, 1987 Oct, 15(4), 745 - 52
Role of Ca2+/calmodulin in the regulation of sugar uptake in Escherichia coli heat-stable enterotoxin induced diarrhoea in mice; Goyal J et al.; The mucosal-to-serosal fluxes of 3-O-methyl-D-glucose, a non-metabolizable analogue of D-glucose, were carried out in control and heat-stable enterotoxin treated mice in the presence or absence of Ca2+-ionophore, Ca2+-channel blocker, calmodulin inhibitor and Na+-K+-ATPase inhibitor . The transport of the sugar was significantly decreased (p less than 0.01) in the experimental animals . In the presence of Ca2+-ionophore, the uptake of the sugar decreased significantly (p less than 0.01) only in the control group while experimental group remained unaffected . Ca2+ channel blocker and calmodulin inhibitor significantly increased (p less than 0.01) the uptake of sugar in both the groups, however, the changes were more pronounced in the experimental group . Ouabain blocked the uptake of the sugar in both the groups . These studies indicated that heat-stable enterotoxin inhibit Na+-K+-ATPase by increasing Ca2+ uptake and calmodulin activity, thus resulting in decreased uptake of 3-O-methyl-D-glucose in heat-stable enterotoxin treated mice.

Prostaglandins, 1987 Oct, 34(4), 493 - 503
Hydroxyeicosatetraenoic acids are increased in bronchoalveolar lavage fluid of endotoxemic pigs; Olson NC et al.; We hypothesized that lipoxygenase metabolites of arachidonic acid might be produced during endotoxin-induced acute respiratory failure (ARF) observed in young pigs . We used radioimmunoassay (RIA) to determine the presence of 5-hydroxyeicosatetraenoic acid (5-HETE), 12-HETE, and 15-HETE in bronchoalveolar lavage fluid (BALF) of saline (n = 12)- and endotoxin (n = 18)-treated pigs . Endotoxin, infused at 5 micrograms/kg for 1 hr followed by 2 micrograms/kg/hr for an average of 3 hrs, caused pulmonary hypertension, a biphasic increase in pulmonary vascular resistance, hypoxemia, bronchoconstriction, leukopenia, and thrombocytopenia . Relative to saline controls, the levels of immunoreactive (i)-5-HETE (816 +/- 209 pg/ml), i-12-HETE (1589 +/- 517 pg/ml), and i-15-HETE (448 +/- 78 pg/ml) were significantly (P less than 0.05) increased in BALF recovered from endotoxemic pigs at postmortem . Relative to control BALF i-HETE concentrations, the endotoxin values were 3.5x, 5.1x, and 2.8x higher for i-5-HETE, i-12-HETE, and i-15-HETE, respectively . We conclude that during porcine endotoxemia, the 5-, 12-, and 15-lipoxygenase pathways are activated and that HETES might be involved in the pathophysiology of endotoxin-induced ARF.

Immunol Lett, 1987 Oct, 16(1), 31 - 8
The heterogeneity of bovine IgG2 . II . The identification of IgG2b; Butler JE et al.; Goat and rabbit polyclonal reagents can be raised which recognize a new isotype of bovine antibodies . The polyclonal goat reagent was raised against a preparation enriched in the major IgG2 isotype (IgG2a) which contained the new isotype as a contaminant . The polyclonal rabbit reagent was prepared against a trypsin-derived Fc fraction of bovine IgG1 which contained the Fc of the new isotype as a contaminant . This new isotype is present in the sera of the cattle of all breeds tested regardless of their IgG2a allotype and is antigenically distinct from IgG2a, IgG1, IgA, IgM and IgE . The new isotype coelutes from DEAE anion exchangers with IgG1 and the more acidic populations of IgG2a . The isotype is tentatively designated IgG2b . The distribution of IgG2b antibody activity to E . coli K99 and phosphorylcholine among 15 cattle of different A allotypes is not correlated with the IgG2a or IgG1 antibody activity in these animals.

Biokhimiia, 1987 Oct, 52(10), 1624 - 31
{Hexameric purine nucleoside phosphorylase II from Escherichia coli K-12 . Physico-chemical and catalytic properties and stabilization with substrates}; Bezirdzhian KhO et al.; Some properties of hexameric purine nucleoside phosphorylase II (EC 2.4.2.1) from Escherichia coli K-12 were studied . The enzyme obeys the Michaelis-Menten kinetics with respect to purine substrates (Km for inosine, deoxyinosine and hypoxanthine are equal to 492, 106 and 26.6 microM, respectively) and exhibits negative kinetic cooperativity towards phosphate and ribose-1-phosphate . The Hill coefficient is equal to approximately 0.5 for both substrates . Hexameric purine nucleoside phosphorylase II is not a metal-dependent enzyme; its activity is inhibited by Cu2+, Zn2+, Ni2+ and SO4(2-) . The enzyme is the most stable at pH 6.0; it contains essential thiol groups . All substrates partly protect the enzyme against inactivation by 5.5'-dithiobis(2-nitrobenzoic acid) and heat-inactivation and, with the exception of phosphate-against inactivation by p-chloromercuribenzoate . Hypoxanthine, especially in combination with phosphate, afford the best protection against inactivation.

EMBO J, 1987 Oct, 6(10), 3155 - 61
Singlet oxygen-induced mutations in M13 lacZ phage DNA; Decuyper-Debergh D et al.; The mutagenic consequences of damages to M13 mp19 RF DNA produced by singlet oxygen have been determined in a forward mutational system capable of detecting all classes of mutagenic events . When the damaged M13 mp19 RF DNA is used to transfect competent E . coli JM105 cells, a 16.6-fold increase in mutation frequency is observed at 5% survivors when measured as a loss of alpha-complementation . The enhanced mutagenicity is largely due to single-nucleotide substitutions, frameshift events and double-mutations . The single-nucleotide substitutions occur in the regulatory and in the structural part of the lacZ gene under the predominant form of a G:C to T:A transversion . The spectrum of mutations detected among the M13 lacZ phages surviving the singlet oxygen treatment is totally different from those appearing spontaneously . SOS induction mediated through u.v.-irradiation of bacteria leads to an increase of the mutation frequency in the M13 surviving to the singlet oxygen treatment . The mutation spectrum in this case is a mixture between those observed with the spontaneous mutants and the mutants induced by singlet oxygen . Lesions introduced in the M13 mp19 RF DNA can be partly repaired by the enzymatic machinery of the bacteria . It turns out that excision-repair and SOS repair are probably involved in the removal of these lesions by singlet oxygen.

Tohoku J Exp Med, 1987 Oct, 153(2), 151 - 60
Antitumor activity of cyclophosphamide and lipopolysaccharide in tumor-bearing mice pretreated with BCG; Sato H et al.; Antitumor effects of cyclophosphamide (CY) and lipopolysaccharide (LPS) were investigated in BCG-treated mice . C3H/He mice and CDF1 mice were injected with BCG and were inoculated subcutaneously with syngeneic mouse hepatoma and mastocytoma P815 respectively . A subsequent injection of LPS caused hemorrhagic necrosis and retarded growth of tumor . When mice were treated with LPS plus suboptimal dose of CY, tumor growth was retarded and survival time was prolonged . The antitumor effects were more remarkable when mice were treated with CY prior to the injection of LPS . Without BCG pretreatment, LPS showed no antitumor activity in mice . Sera from mice treated with BCG plus LPS was cytotoxic for cultured tumor cells . However treatment of mice with CY did not increase the in vitro cytotoxicity . In this experimental condition, CY had no effect on delayed type hypersensitivity when evaluated by the footpad reaction to purified protein derivative (PPD) . These results seem to indicate that the antitumor effects of the treatment with CY and LPS in BCG-treated mice are mediated by the reduction of tumor burden by CY and a serum factor induced by LPS.

Mol Cell Biol, 1987 Oct, 7(10), 3620 - 8
Expression of the Aplysia californica rho gene in Escherichia coli: purification and characterization of its encoded p21 product; Anderson PS et al.; A new family of highly conserved genes, designated rho, has recently been isolated and characterized (P . Madaule and R . Axel, Cell 41:31-40, 1985) . These genes have been found in Saccharomyces cerevisiae, Drosophila melanogaster, rats, and humans, and their 21,000-dalton products are highly homologous . The rho p21 protein shares 35% amino acid homology with the Harvey ras p21 protein and on this basis has been proposed to be a G protein . We expressed the Aplysia californica rho gene in Escherichia coli and purified its p21 protein to more than 90% purity . The availability of the rho protein in high quantities made it possible to establish its high affinity for guanine nucleotides . The rho p21 protein had nucleotide-binding properties similar to those of the ras p21 protein . However, a comparison of these proteins revealed some important differences regarding their specificities and affinities . Finally, the rho p21 protein had GTPase activity almost identical to that of a normal ras p21 protein, the rates being 0.106 and 0.105 mol/min per mol of p21, respectively . Thus, the results suggest that the degree of homology found between the ras and rho genes products most likely is related to the conservation of sequences relevant to their ability to bind and hydrolyze guanine nucleotides . The fact that the rho p21 protein binds and hydrolyzes GTP strongly suggests that it is a G protein with a potential regulatory function conserved in evolution.

Eur J Biochem, 1987 Oct 1, 168(1), 239 - 43
Production and purification of human growth-hormone-releasing factor from continuous cultures of recombinant-plasmid-containing Escherichia coli; Pages JM et al.; A recombinant gene comprising phoS (the gene for the phosphate-binding protein PhoS) fused to a synthetic gene for a modified human growth-hormone-releasing factor (mhGRF) has been constructed . This gene was highly expressed in cells growing under conditions of phosphate starvation . Various conditions of continuous culture, varying in phosphate concentrations and dilution rates, have been tested to optimize the expression of the hybrid gene product (PhoS-mhGRF) . Conditions were obtained such that a large amount of the hybrid protein was no longer exported as a result of saturation of export sites, which also induce the inhibition of processing of pre-PhoE and pre-OmpA . The pre-PhoS-mhGRF, accumulated in the cell, was recovered mainly in the particulate fraction after cell fractionation . This protein was purified . Besides the methionine residues located within the signal sequence, the only other one is located in the fusion joint of the hybrid protein . Thus cyanogen bromide treatment allowed the isolation of pure mhGRF . The yield obtained is about of 1 mg/l culture.

Proc Natl Acad Sci U S A, 1987 Oct, 84(19), 6795 - 9
A beta-galactosidase hybrid protein targeted to nuclei as a marker for developmental studies; Bonnerot C et al.; The Escherichia coli lacZ gene has been used as an indicator gene for the study of cell lineage in vivo . To adapt this marker for gene expression studies, a sequence encoding a modified beta-galactosidase and including the simian virus 40 large tumor nuclear location signal (nls-beta-Gal) has been introduced into vectors . In differentiated cells, multipotential cells, and embryos, the constructs led to the expression of an enzymatically active protein . Its location was examined by its beta-galactosidase activity or by using antibodies and electron microscopy . The results show that the nls-beta-Gal protein remains mainly located at the nuclear periphery (probably at the nuclear pores) but does not reach the nucleoplasm . It suggests that an interaction with the nuclear membrane is necessary but not sufficient for protein uptake into the nucleus . In multipotential cells, the expression of nuclear location signal LacZ (nls-LacZ) interferes neither with cell growth nor with differentiation . Using various lacZ constructs, the transcriptional activity of embryos was studied . At the two-cell stage, the promoters of the Rous sarcoma virus, simian virus 40, and the beta-actin gene are functional but the Moloney murine leukemia virus long terminal repeat is not . Thus, transcriptional specificity must already be present at the stage of activation of the embryonic genome.

Proc Natl Acad Sci U S A, 1987 Oct, 84(19), 6697 - 701
Nucleotide sequence of the argR gene of Escherichia coli K-12 and isolation of its product, the arginine repressor; Lim DB et al.; In Escherichia coli, the arginine repressor, the product of the argR gene, in conjunction with L-arginine controls the synthesis of the enzymes of arginine biosynthesis . We describe the nucleotide sequence of the argR gene, including its control region, and show that formation of the repressor is autoregulated . The argR control region contains two promoters, one of which overlaps the operator site and, as with other arg genes, consists of two adjacent palindromic sequences ("ARG boxes") . The arginine repressor protein and an arginine repressor-beta-galactosidase fusion protein were purified, and the amino acid sequence of the N-terminal end of the repressor protein portion of the fusion protein was determined . Antibodies prepared against the fusion protein react with the repressor . The repressor is precipitable by L-arginine, which facilitates its purification . The native repressor is a hexamer with a molecular weight of 98,000; its monomeric subunit has a molecular weight of 16,500 . To verify its properties postulated from genetic studies, we show that in the presence of L-arginine, repressor inhibits transcription of argF and binds to the ARG boxes of argF and argR.

Mutat Res, 1987 Oct, 180(2), 163 - 9
Potent induction of the adaptive response by a weak mutagen, methyl iodide, in Escherichia coli; Takahashi K et al.; Methyl iodide (MeI), a weakly mutagenic and highly chemoselective chemical, was tested for its abilities to induce the adaptive and SOS responses in E . coli CSH26/pMCP1000 (alkA'-lacZ') and CSH26/pSK1002 (umuC'-lacZ') . MeI induced the adaptive response effectively but gave a very weak SOS response . Its potent ability in inducing the adaptive response was also demonstrated by adaptation to both the mutagenic and killing effects of N-methyl-N-nitrosourea (MNU) in E . coli WP2 cells . Simultaneous treatment with MeI in a non-growth medium slightly increased the mutagenicity of MNU, probably as a result of depletion of the repair enzyme, O6-methylguanine-DNA methyltransferase, which is constitutively present in the cells . As MeI itself proved to be only weakly mutagenic, a small part of the adaptive response which we have observed may involve indirect methylation of the repair enzyme by methyl transfer from MeI-induced O6-methylguanine residues in DNA . But the extent of the induced adaptive response seems to be much higher than would be expected from the observed weak mutagenicity of MeI . It is therefore suggested that the mechanism of induction of the adaptive response may involve direct methylation of the O6-methylguanine-DNA methyltransferase itself.

J Bacteriol, 1987 Oct, 169(10), 4668 - 73
Genetic analysis of amino acid transport in the facultatively heterotrophic cyanobacterium Synechocystis sp . strain 6803; Labarre J et al.; The existence of active transport systems (permeases) operating on amino acids in the photoautotrophic cyanobacterium Synechocystis sp . strain 6803 was demonstrated by following the initial rates of uptake with 14C-labeled amino acids, measuring the intracellular pools of amino acids, and isolating mutants resistant to toxic amino acids . One class of mutants (Pfa1) corresponds to a regulatory defect in the biosynthesis of the aromatic amino acids, but two other classes (Can1 and Aza1) are defective in amino acid transport . The Can1 mutants are defective in the active transport of three basic amino acids (arginine, histidine, and lysine) and in one of two transport systems operating on glutamine . The Aza1 mutants are not affected in the transport of the basic amino acids but have lost the capacity to transport all other amino acids except glutamate . The latter amino acid is probably transported by a third permease which could be identical to the Can1-independent transport operating on glutamine . Thus, genetic evidence suggests that strain 6803 has only a small number of amino acid transport systems with fairly broad specificity and that, with the exception of glutamine, each amino acid is accumulated by only one major transport system . Compared with heterotrophic bacteria such as Escherichia coli, these permeases are rather inefficient in terms of affinity (apparent Km ranging from 6 to 60 microM) and of Vmax.

J Biomol Struct Dyn, 1987 Oct, 5(2), 435 - 46
The ribosomal E site at low Mg2+: coordinate inactivation of ribosomal functions at Mg2+ concentrations below 10 mM and its prevention by polyamines; Rheinberger HJ et al.; Under standard conditions (Mg2+/150 mM NH4+) ribosomes can quantitatively participate in tRNA binding at Mg2+ concentrations of 12 to 15 mM . The overall poly(U)-directed Phe incorporation and the extent of tRNA binding to either P, E or A sites decrease in a parallel manner when the Mg2+ concentration is lowered below 10 mM . At 4 mM the inactivation amounts to about 80% . The coordinate inactivation of all three binding sites is accompanied by an increasing impairment of the ability to translocate A-site bound AcPhe-tRNA to the P site . The translocation efficiency is already reduced at 10 mM Mg2+, and is completely blocked at 6-8 mM . The severe inactivation seen at 6 mM Mg2+ vanishes when the polyamines spermine (0.6 mM) and spermidine (0.4 mM) are present in the assay; tRNA binding again becomes quantitative, the total Phe synthesis even exceeds that observed in the absence of polyamines by a factor of 4 . In the presence of polyamines and low Mg2+ (3 and 6 mM) two essential features of the allosteric three-site model (Rheinberger and Nierhaus, J . Biol . Chem . 261, 9133 (1986} are demonstrated . 1) Deacylated tRNA is not released from the P site, but moves to the E site during the course of translocation . 2) Occupation of the E site reduces the A site affinity and vice versa (allosteric interactions between E and A sites) . The quality of an in vitro system for protein synthesis can be assessed by two criteria . First, the incubation conditions must allow a near quantitative tRNA binding . Secondly, protein synthesis should proceed with near in vivo rate and accuracy . The 3 mM Mg2+/NH4+/polyamine-system seems to be the best compromise at present between these two requirements.

Cancer Res, 1987 Oct 1, 47(19), 5092 - 6
Formation of blocking lesions at identical DNA sequences by the nitrosourea and platinum classes of anticancer drugs; Gralla JD et al.; cis-Diamminedichloroplatinum (II) (cisplatin) compounds and the chloroethylnitrosoureas are two different classes of anticancer drugs that work by modifying DNA covalently . We have compared the platinating drug cisplatin with the alkylating drug bischloroethylnitrosourea and other chloroethylnitrosoureas by modifying double stranded DNA in vitro and identifying blocking lesions that impede the progress of Escherichia coli DNA polymerase . Despite their very different structures and reactivities, cisplatin and the chloroethylnitrosoureas from primary blocking lesions at identical sequences, those containing adjacent guanosines on the same DNA strand . In tumor virus SV 40 DNA, a very strong target for both types of drugs is the regulatory sequence GGGCGG, which is repeated six times and is an important sequence for viral replication and an essential sequence for expression of the viral transforming gene . Sequences related to these GC box elements are known to be present in the flanking regions of many retroviruses and oncogenes, thus raising the possibility that the targeting of these sequences in tumor cells contributes to drug activity.

Can J Vet Res, 1987 Oct, 51(4), 528 - 30
Ocular lesions in chickens inoculated with Escherichia coli; Nakamura K et al.; Specific-pathogen-free chickens (two, four and ten weeks of age) which were inoculated via the air sac with Escherichia coli developed ocular lesions . Histologically, the main ocular lesions consisted of hyphema, hemorrhages of the iris, hypopyon, keratitis and uveitis . Hyphema was associated with hemorrhages of the iris, and hypopyon with keratitis and uveitis . Cyclophosphamide treatment enhanced the incidence and severity of hyphema and hemorrhages of the iris in the chickens.

J Gen Microbiol, 1987 Oct, 133 ( Pt 10), 2707 - 17
Lambda transducing phage and clones carrying genes of the cysJIHDC gene cluster of Escherichia coli K12; Hunt CL et al.; DNA from each of two specialized transducing lambda phage, lambda dcysJIHD and lambda cysJ, has been analysed by heteroduplex mapping . The segment of the Escherichia coli chromosome carried by lambda dcysJIHD was shown to be large, approximately 18 kb in length, and to replace a large length of lambda DNA, approximately 11 kb, which includes the genes for integration and recombination . Thus lambda dcysJIHD is a bio-type transducing phage . lambda cysJ was shown to have lost very little lambda DNA and to carry about 8 kb of bacterial DNA . Sites for several restriction endonucleases were mapped in the DNA from each phage and cloning experiments located some of the genes of the cluster in relation to the restriction map . Cysteine regulation of the cloned cysJ and cysD genes was shown as well as cysteine regulation of beta-galactosidase in some constructs . The direction of transcription of the cysD gene was established, and from physical evidence the size of the 'silent section' between the cysH and cysD genes was estimated to be at least 11 kb.

Mol Gen Mikrobiol Virusol, 1987 Oct, (10), 37 - 40
{Phenomenon of restriction alleviation in Escherichia coli strains}; Torosian MV et al.; The alleviation of foreign DNA restriction after treatment of cells by UV and gamma-rays or ocr+-gene product of T3, T7 phages has been studied . Both UV and gamma-radiation were shown to induce in restriction alleviation of unmodified phage . The results of restriction alleviation caused by T3 and T7 ocr+-gene products have been evaluated by F-lac+ transfer efficiencies in heterologic crosses and plating of unmodified phage lambda . The phenomenon of restriction alleviation was established to depend on the strain used and to be expressed mostly in AB157 and its derivatives.

EMBO J, 1987 Oct, 6(10), 3139 - 44
Promoter recognition and promoter strength in the Escherichia coli system; Brunner M et al.; The strength of Escherichia coli promoters in vivo as well as the rates of association between RNA polymerase and promoter sequences differ by more than an order of magnitude . Since efficient promoter recognition and rapid binding of the enzyme might be a prerequisite for exceptional promoter strength we have determined the forward rate constants kon (as well as koff) for nine promoters including PL, PA1, and PN25 from phages lambda, T7, and T5, respectively as well as Pbla and PlacUV5 from E . coli . The second order forward rate constants span a 30-fold range from 1 X 10(7) M-1 s-1 for Pbla and PL up to 2.9 X 10(8) M-1 S-1 for PN25 . Little correlation between 'promoter recognition' as defined by the rate of complex formation of a promoter sequence with RNA polymerase and its strength in vivo as defined by the rate of RNA synthesis has been found . This adds to the evidence that the complex functional pathway encoded in a promoter sequence can be limited at various levels and that promoter strength in vivo is the result of an optimization process involving more than just one functional parameter.

Proc Natl Acad Sci U S A, 1987 Oct, 84(19), 6673 - 6
Missing contact probing of DNA-protein interactions; Brunelle A et al.; We have examined the positions of contact between lambda phage repressor protein and operator OR1 DNA by scanning populations of lightly depurinated or depyrimidated DNA for bases essential to or irrelevant to repressor binding . This global scanning technique delineates the apparent contact region between lambda repressor and operator and shows bases previously demonstrated or predicted to be contacted plus some additional bases . A mutant repressor, previously shown to contact DNA as wild-type repressor does with the exception of a missing contact to guanosine G4' {Hochschild, A . & Ptashne, M . (1986) Cell 44, 925-933}, similarly failed to contact G4' when assayed by this method . Coupled with altering a test residue of a DNA-contacting protein to glycine or alanine so as to eliminate a specific contact, the method appears to provide an efficient means of scanning for specific residue-base contacts.

What Is Water Purification?, What Is Growth Medium?, What Is Yeast?, What Is MIC?, What Is Nitrification?, i, Microbiology, e, Microbe, r, Bacterium, n, Microorganism, s, Bacteria, e, Rhizobacter, r, Micrococci, s, Shigella, n, Escherichia coli, n, Waste treatment, r, Escherichia coli, e, Petri dish, o, Shigella, o, Escherichia coli, o, Microorganisms, a, Bactericidal, r, S. cerevisiae, o, Gram negative, s, Escherichia coli, e, Staphylococcus, n, Cell cultures, e, Bacillus, s, Haemophilus, e, Brevibacteria, c, Biodegradation, o, Biodegradation

Scientific Publications - Work Done by Microbiology Reader Bioscreen C
Agricultural Microbiology Anaerobic Microbiology Antimicrobial Susceptibility Artificial Atmosphere Bioassay of Antibiotics Biofilm Microbiology Bioreactor Technology Biotechnology Cell Biology Clinical Microbiology Environmental Microbiology Experiments with Yeast	Fermentation Food Microbiology Functional Genomics Gene Technology Growth Media Development Growth Rate and Lag Time Industrial Microbiology Medical/Pharmaceutical Field Microbiological Assay Microbiological Research Microbiology of Cosmetics go to a specific theme...	Military Microbiology Molecular Microbiology Mutagenicity and Genotoxicity Oral Microbiology Patents Postantibiotic Studies Soil Microbiology Spore Microbiology Veterinary Microbiology Waste/Wastewater Treatment Water Microbiology Wine Microbiology

� 2005 Transgalactic Ltd (manufacturer of Bioscreen C software) | Privacy Statement | P.O. Box 1393, 00101 Helsinki, Finland, phone: +358 9 85172920, fax: +358 9 8749481, e-mail: microbiology@bionewsonline.com

Last modified: May 25, 2005