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 .