J Biol Chem, 1988 Dec 15, 263(35), 18810 - 5 The ATP binding site on rho protein . Affinity labeling of Lys181 by pyridoxal 5'-diphospho-5'-adenosine; Dombroski AJ et al.; We have labeled the nucleoside triphosphate-binding domain of Escherichia coli rho factor with the ATP affinity analog {3H}pyridoxal 5'-diphospho-5'-adenosine (PLP-AMP) . PLP-AMP completely inactivates the RNA-dependent ATPase activity of rho upon incorporation of 3 mol of reagent/mol of hexameric rho protein . Although the potency of PLP-AMP is enhanced when an RNA substrate such as poly(C) is present, the stoichiometry for inhibition remains the same as in the absence of poly(C) . The nucleotide substrate ATP competes very effectively for the binding site and protects against PLP-AMP inactivation . A domain of rho called N2, which comprises the distal two-thirds of the molecule (residues 152-419) and encompasses the region proposed to bind ATP, is labeled specifically in the presence of poly(C) . Amino acid sequence analysis of the single {3H}PLP-AMP labeled proteolytic fragment showed Lys181 to be the site of modification, suggesting that this residue normally interacts with the gamma-phosphoryl of bound ATP . These results agree with our proposed tertiary structure for the ATP-binding domain of rho that places this lysine residue in a flexible loop above a hydrophobic nucleotide-binding pocket comprised of several parallel beta-strands, similar to adenylate kinase, F1-ATPase, and related ATP-binding proteins . Parallel studies of rho structure and function by site-directed mutagenesis and chemical modification support this interpretation. Gene, 1988 Dec 15, 73(1), 141 - 52 Structure of yeast glucokinase, a strongly diverged specific aldo-hexose-phosphorylating isoenzyme; Albig W et al.; Saccharomyces cerevisiae glucokinase (GLK) is the only described hexose-phosphorylating enzyme specific for aldo-hexoses . The gene was cloned by complementation of a triple mutant lacking all hexose-phosphorylating isoenzymes . Restriction sites were confirmed by genomic hybridization and GLK1 was mapped on chromosome III by ROFAGE, a method derived from the orthogonal field alteration gel electrophoresis . The mapping data were in agreement with previous genetic data . The open reading frame was established by two transcription start points in front of the initial ATG codon and by C-terminal beta-galactosidase fusions . The mRNA is 1.75 kb long and codes for 500 amino acid (aa) residues . Diversity of GLK from hexokinases PI and PII is very marked, with only 26 and 28% overall aa homology . A central core of about 350 aa shows 39% homology . No cross-hybridization could be observed by Southern hybridization . However, strong homologies were found over a range of 11 aa between glucokinase, yeast hexokinases (PI, PII) and rat hexokinase with 8 aa in common . These strongly conserved homologies give support to the view that this aa region corresponds to the binding site for glucose . Unlike all other hexose-phosphorylating enzymes, there is no proline residue indicating a conformational turn next to this glucokinase region . This finding may explain the failure of fructose phosphorylation . In both GLK and the hexokinases, a lysine residue is also conserved at aa position 110 which probably corresponds to the ATP-binding site . Additionally, a consensus sequence of 8 aa residues which is common for ATP-binding enzymes is conserved within the C-terminal part of GLK . The codon bias index for GLK1 is 0.25, which is very low compared with other glycolytic enzymes described so far . The gene is moderately expressed and constitutive on different carbon sources investigated . GLK1 null alleles had no detectable effects on sporulation and growth . Hence, a physiological role for GLK, which might explain its preservation, could not be detected under our laboratory test conditions. Biochem J, 1988 Dec 15, 256(3), 741 - 9 Overexpression and mutagenesis of the lipoamide dehydrogenase of Escherichia coli; Allison N et al.; A 'split-gene' technique for the overexpression and mutagenesis of the gene encoding the lipoamide dehydrogenase of Escherichia coli was developed in order to overcome the instability problems encountered when attempting to mutate the intact gene . The lipoamide dehydrogenase gene, lpd, was dissected into two fragments which were separately subcloned into M13 vectors for mutagenesis in vitro followed by reconstitution in the pJLA504 expression vector under the transcriptional control of the lambda PR and lambda PL promoters and a temperature-sensitive lambda repressor . After thermo-induction, E . coli cells transformed with the plasmid carrying the reconstituted lpd gene contained 4-5 times more lipoamide dehydrogenase activity than is normally found in the wild-type organism . The strategy was used to engineer a Glu-188----Asp replacement in lipoamide dehydrogenase, and this generated an enzyme with markedly different kinetic properties. Eur J Biochem, 1988 Dec 15, 178(2), 445 - 50 Catalytic-site mapping of pyruvate formate lyase . Hypophosphite reaction on the acetyl-enzyme intermediate affords carbon-phosphorus bond synthesis (1-hydroxyethylphosphonate); Plaga W et al.; Pyruvate formate-lyase of Escherichia coli cells, a homodimeric protein of 2 x 85 kDa, is distinguished by the property of containing a stable organic free radical (g = 2.0037) in its resting state . The enzyme (E-SH) achieves pyruvate conversion to acetyl-CoA via two distinct half-reactions (E-SH + pyruvate in equilibrium E-S-acetyl + formate; E-S-acetyl + CoA in equilibrium E-SH + acetyl-CoA), the first of which has been proposed to involve reversible homolytic carbon-carbon bond cleavage {J . Knappe et al . (1984) Proc . Natl Acad . Sci . USA 81, 1332-1335} . Present studies identified Cys-419 as the covalent-catalytic cysteinyl residue via CNBr fragmentation of E-S-{14C}acetyl and radio-sequencing of the isolated peptide CB-Ac (amino acid residues 406-423) . Reaction of the formate analogue hypophosphite with E-S-acetyl was investigated and found to produce 1-hydroxyethylphosphonate with a thioester linkage to the adjacent Cys-418 . The structure was determined from the chymotryptic peptide CH-P (amino acid residues 415-425), using 31P-NMR spectroscopy (delta = 44 ppm) and by chemical characterisation through degradation into 1-hydroxyethylphosphonate with phosphodiesterase or bromine . This novel P-C-bond synthesis involves the enzyme-based free radical and is proposed to resemble the physiological C-C-bond synthesis (pyruvate production) from formate and E-S-acetyl . These findings are interpreted as proof of a radical mechanism for the action of pyruvate formate-lyase . The central Cys-418/Cys-419 pair of the active site shows a distinctive thiolate property even in the inactive (nonradical) form of the enzyme, as determined using an iodoacetate probe. Eur J Biochem, 1988 Dec 15, 178(2), 351 - 5 Interaction of Escherichia coli translation-initiation factor IF-1 with ribosomes; Celano B et al.; The interaction between Escherichia coli translation-initiation factor IF-1 and ribosomes was studied in binding experiments by Airfuge centrifugation . IF-1 binds to the 30S, but not to the 50S, ribosomal subunit and its binding is strongly stimulated by IF-3 and IF-2, either alone or in combination . From the dependence of the Kd of the 30S-subunit--IF-1 complex on ionic strength, it can be concluded that IF-1 binds primarily via an ionic interaction, most likely with the 16S rRNA, with the minimum number of ion pairs involved being 2.7-3.6 . The 30S-subunit--IF-1 interaction is unaffected by temperature changes between 11 degrees C and 44 degrees C and is thus accompanied by a negligible enthalpy change . It is concluded that the interaction is an entropy-driven process triggered mainly by the release of counter ions from the RNA phosphates . Titration of 30S-subunit--IF-1 complexes with 50S subunits causes the ejection of the factor indicating that IF-1 is released from the ribosomes during the subunit association step which marks the transition from a 30S-initiation-complex to a 70S initiation complex. J Biol Chem, 1988 Dec 15, 263(35), 19174 - 80 Proteolytic cleavage of Ada protein that carries methyltransferase and transcriptional regulator activities; Yoshikai T et al.; The 39-kDa Ada protein that carries two distinct methyltransferase activities and an activity to promote transcription of the ada and alkA genes is cleaved to smaller polypeptides in the case of incubation with a crude extract of Escherichia coli . A protease that specifically cleaves the Ada protein was partially purified and characterized . The enzyme showed maximal activity at pH 7.8-8.0 and did not require specific ions or ATP for the reaction . The activity was inhibited by p-chloromercuribenzoic acid, thereby suggesting that it is a thiol protease . When a purified preparation of Ada protein was incubated with the protease preparation, definite sizes of cleavage products were formed; at the initial stage of proteolysis, the 20- and 19-kDa proteins were formed as the major products . When each of these products was purified by successive column chromatography, the 20-kDa protein was found to catalyze transfer of a methyl group from methylphosphotriester of methylated polynucleotides, whereas the 19-kDa protein transfers a methyl group from O6-methylguanine of methylated DNA to each of the molecules . Neither the 20- nor 19-kDa protein, even after methyl acceptance, exhibited an activity to promote transcription of the ada gene . However, alkA transcription was promoted by the 20-kDa protein provided that it was methylated. J Biol Chem, 1988 Dec 15, 263(35), 19147 - 53 Nucleotide sequence and analysis of the purA gene encoding adenylosuccinate synthetase of Escherichia coli K12; Wolfe SA et al.; Adenylosuccinate synthetase (EC 6.3.4.4), encoded by the purA gene of Escherichia coli K12, catalyzes the synthesis of adenylosuccinate (SAMP) from IMP, the first committed step in AMP biosynthesis . The E . coli K12 purA gene and flanking DNA was cloned by miniMu-mediated transduction, and the nucleotide sequence was determined . The mature SAMP synthetase subunit, as deduced from the DNA sequence, contains 427 amino acid residues and has a calculated Mr of 47,277 . The size of the purA mRNA was determined by Northern blotting to be approximately 1.5 kilobase pairs . The 5'-end of the purA mRNA was identified by primer extension and is located 23 nucleotides upstream of the ATG translational initiation codon . Comparison of the purA control region with the guaBA control region revealed a common region of dyad symmetry which may suggest mutual elements of regulation . The purA control region did not resemble the control regions of the other known pur loci. J Biol Chem, 1988 Dec 15, 263(35), 19077 - 82 Inhibition and resumption of processing of the staphylokinase in some Escherichia coli prlA suppressor mutants; Iino T et al.; Escherichia coli strains carrying certain prlA mutations (prlA4 and prlA401) could not support the processing and export of staphylokinase, resulting in the accumulation of the precursor form under high-level synthesis conditions . In order to clarify the cause of the defect in the structure of staphylokinase, we constructed signal peptide mutations of sak which suppressed the processing defect in the prlA4 cells by site-directed mutagenesis . The processing defect was suppressed when glycine or asparagine was introduced in place of the serine residue at position 17 from the amino terminus of the signal peptide . Substitutions of glycine for the leucine residue at position 15 and for the serine residue at position 19 were also effective . Other mutations we constructed had no suppression activity . Taking account of the correlation between the suppression activity and the parameter value of each substituted amino acid for the beta-turn probability, we predict that the staphylokinase signal peptide requires a more bending structure at the end of the hydrophobic core to act efficiently in the prlA4 cells than in the prl+ cells and that a function of the PrlA protein necessary to recognize the staphylokinase signal peptide has become deficient through the prlA4 mutation. J Biol Chem, 1988 Dec 15, 263(35), 19053 - 9 An artificial hydrophobic sequence functions as either an anchor or a signal sequence at only one of two positions within the Escherichia coli outer membrane protein OmpA; MacIntyre S et al.; The 325-residue outer membrane protein, OmpA, of Escherichia coli, like most other outer membrane proteins with known sequence, contains no long stretch of hydrophobic amino acids . A synthetic oligonucleotide, encoding the sequence Leu-Ala-Leu-Val, was inserted four times between the codons for amino acid residues 153 and 154 and two, three, or four times between the codons for residues 228 and 229, resulting in the OmpA153-4, OmpA-228-2, -3, and -4 proteins, respectively . In the first case, the lipophilic sequence anchored the protein in the plasma membrane . In the OmpA228 proteins, 16 but not 12 or 8 lipophilic residues most likely also acted as an anchor . By removal of the NH2-terminal signal peptide, the function of the insert in OmpA153-4 was converted to that of a signal-anchor sequence . Possibly due to differences in amino acid sequences surrounding the insert, no signal function was observed with the insert in OmpA228-4 . Production of the OmpA153-4 protein, with or without the NH2-terminal signal sequence, resulted in a block of export of chromosomally encoded OmpA . Clearly, long hydrophobic regions are not permitted within proteins destined for the bacterial outer membrane, and these proteins, therefore, have had to evolve another mechanism of membrane assembly. J Biol Chem, 1988 Dec 15, 263(35), 18946 - 52 Purification and characterization of an inducible Escherichia coli DNA polymerase capable of insertion and bypass at abasic lesions in DNA; Bonner CA et al.; We have investigated the ability of DNA polymerases from SOS-induced and uninduced Escherichia coli to incorporate nucleotides at a well-defined abasic (apurinic/apyrimidinic) DNA template site and to extend these chains from this unpaired 3' terminus . A DNA polymerase activity has been purified from E . coli, deleted for DNA polymerase I, that appears to be induced 7-fold in cells following treatment with nalidixic acid . Induction of this polymerase (designated DNA polymerase X) appears to be part of the SOS response of E . coli since it cannot be induced in strains containing a noncleavable form of the LexA repressor (Ind-) . The enzyme is able to incorporate nucleotides efficiently opposite the abasic template lesion and to continue DNA synthesis . Although we observe an approximate 2-fold induction of DNA polymerase III in cells treated with nalidixic acid, several lines of evidence argue that DNA polymerase X is unrelated to DNA polymerase III (pol III) . In contrast to pol X, pol III shows almost no detectable ability to incorporate at or extend beyond the abasic site; incorporation efficiency at the abasic lesion is at least 100-fold larger for pol X compared to pol III holoenzyme, pol III core, or pol III* (the polymerase III holoenzyme subassembly lacking the beta subunit) . Pol X does not cross-react with polyclonal antibody directed against pol III holoenzyme complex or with monoclonal antibody prepared to the alpha subunit of pol III . Despite these structural and biochemical differences, pol X appears to interact specifically with the beta subunit of the pol III holoenzyme in the presence of single-stranded binding protein . Pol X has a molecular mass of 84 kDa . Our results indicate that this novel activity is likely to be identical to DNA polymerase II of E . coli. Gene, 1988 Dec 15, 73(1), 11 - 20 Structural analysis of mouse S-antigen; Tsuda M et al.; Mouse S-antigen clones were isolated from a mouse retinal cDNA library using a bovine S-antigen cDNA probe . The largest clone (MSC-242) comprised 1532 bp and contained the entire coding sequence . The nucleotide sequence homology between the mouse and bovine coding regions was 84%, while non-coding regions appeared to be more divergent . The deduced amino acid sequence indicated that the mouse S-antigen had 403 residues and its molecular ratio was 44,930 . An overall amino acid sequence similarity of 84% was observed between the mouse and bovine proteins . This degree of similarity dropped to 60% and 47% at the N and the C termini, respectively . The local homology with alpha-transducin observed in the bovine proteins, including the putative phosphoryl and rhodopsin binding sites, was conserved in the mouse as well . There was no overall sequence similarity with other proteins listed in the National Biomedical Research Foundation (NBRF) protein sequence database . Among the uveitopathogenic sites for experimental autoimmune uveitis (EAU), peptides N and M were identical to their bovine counterparts . Peptides 3 and K, however, were more divergent . The short repeats within these peptides were conserved. J Biol Chem, 1988 Dec 15, 263(35), 18857 - 63 Transcriptional mapping and nucleotide sequence of the Escherichia coli fepA-fes enterobactin region . Identification of a unique iron-regulated bidirectional promoter; Pettis GS et al.; The iron-controlled fepA and fes-entF transcripts from the Escherichia coli enterobactin gene complex are expressed divergently from a limited genetic region, thereby suggesting the existence of a single, possibly overlapping promoter junction for these genes . The nucleotide sequence of a 1,997-base pair HpaI fragment specific for this genetic region allowed for the identification of an 1,122-base pair open reading frame as the previously uncharacterized fes gene . Its product, Fes (approximately Mr 42,573) plays an essential but as yet ambiguous role in the release of ferric iron from the ligand . An additional small open reading frame of 216 nucleotides (encoding a potential product of calculated Mr 8,271) was also identified between fes and entF . A portion of the remaining nucleotide sequence defined a 320-base pair control region for both the fepA and fes-entF messages . Primer extension analyses placed the major in vivo transcription initiation sites to within 18 nucleotides of one another, thereby revealing a novel, extensively overlapping bidirectional promoter as well as long dual leader transcripts . This promoter region contains multiple overlapping nucleotide stretches which show strong homology to the consensus Fur repressor-binding sequence, forms of which are found in all E . coli iron-regulated promoters characterized to date. Cancer Lett, 1988 Dec 15, 43(3), 191 - 5 Human renal carcinoma: asparagine independence with asparaginase susceptibility in culture; Prager MD et al.; A human renal carcinoma cell line (Caki-1) was examined for asparagine (Asn) dependence and susceptibility to Escherichia coli asparaginase . Because this enzyme hydrolyzes glutamine (Gln) as well as Asn, even though at only 2-3% the rate, Asn- Gln+ and Asn- Gln- media were prepared . Only the former supported Caki growth . The Asn- Gln- medium was then repleted with Asn, Gln, or both . Although Asn repletion failed to promote growth, addition of Gln alone or the combination supported growth as well as complete medium . With {3H}leucine and {3H}mannose incorporation to indicate protein and glycoprotein synthesis, respectively, the Gln repleted medium supported these processes as well as complete medium . Asparaginase added to complete medium was highly toxic to the Caki cells, but this is a reflection of Gln depletion rather than Asn depletion. Biochem Biophys Res Commun, 1988 Dec 15, 157(2), 816 - 20 3-Deoxy-D-manno-octulosonate-8-phosphate synthase catalyzes the C-O bond cleavage of phosphoenolpyruvate; Hedstrom L et al.; The mechanism of 3-deoxy-D-manno-octulosonate-8-phosphate (KDO8P) synthase was investigated . When {18O}-PEP specifically labeled in the enolic oxygen is a substrate for KDO8P synthase, the 18O is recovered in Pi . This indicates that the KDO8P synthase reaction proceeds with C-O bond cleavage of PEP similar to that observed in the 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase catalyzed condensation of PEP and erythrose-4-phosphate (1) . No evidence for a covalent enzyme-PEP intermediate could be obtained . No {32P}-Pi exchange into PEP nor scrambling of bridge 18O to non-bridging positions in {18O}-PEP was observed in the presence or absence of arabinose-5-phosphate or its analog ribose-5-phosphate . Bromopyruvate inactivated KDO8P synthase in a time dependent process . It is likely that bromopyruvate reacts with a functional group at the PEP binding site since PEP, but not arabinose-5-phosphate, protects against inactivation. Gene, 1988 Dec 15, 73(1), 245 - 50 Jekyll, a family of phage-plasmid shuttle vectors; Burmeister M et al.; A series of shuttle vectors has been constructed, which consist of a plasmid carrying a polylinker sequence and an M13 origin integrated into a lambda vector . A short direct repeat flanking the plasmid allows plasmid excision by homologous recombination . Sequences are cloned into unique restriction sites within the plasmid, and can be recovered either in phage or plasmid form, or can be packaged further as single-stranded DNA phage . These vectors therefore combine the efficiency of phage lambda cloning and screening with the ease of handling or analysing plasmid or M13 clones. J Biol Chem, 1988 Dec 15, 263(35), 18802 - 9 Site-directed alterations in the ATP-binding domain of rho protein affect its activities as a termination factor; Dombroski AJ et al.; We have utilized oligonucleotide site-directed mutagenesis to test our prediction that Escherichia coli rho factor has an ATP-binding domain separate from its RNA-binding domain and similar to that of adenylate kinase . Single amino acid substitutions were generated in regions thought to be within the active site and catalytically important for the ATPase activity, changing lysine 181 and/or lysine 184 to glutamine, and aspartate 265 to valine and asparagine . The altered proteins were purified and characterized in vitro for RNA- and ATP-binding ability, ATPase activity, helicase activity, and ability to catalyze transcription termination . Our results indicate that 1) these amino acid alterations in the proposed ATP-binding domain do not interfere with RNA binding; 2) substitution of lysine 184 by glutamine actually improves the ATPase and related activities while the same substitution at lysine 181 reduces but does not eliminate activity; 3) the double mutation changing both lysine 181 and lysine 184 to glutamine eliminates ATPase activity; and 4) the aspartate at 265 is also required for ATP hydrolysis but not for ATP binding . These results are consistent with our proposal that the general tertiary structure of rho's ATP-binding domain is similar to that of adenylate kinase. Biochemistry, 1988 Dec 13, 27(25), 9020 - 30 Investigation into the nature of substrate binding to the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase; Warren MJ et al.; The formation of the dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was shown to depend on the presence of 5-aminolevulinic acid . A hemA- mutant formed inactive deaminase when grown in the absence of 5-aminolevulinic acid since this strain was unable to biosynthesize the dipyrromethane cofactor . The mutant formed normal levels of deaminase, however, when grown in the presence of 5-aminolevulinic acid . Porphobilinogen, the substrate, interacts with the free alpha-position of the dipyrromethane cofactor to give stable enzyme-intermediate complexes . Experiments with regiospecifically labeled intermediate complexes have shown that, in the absence of further substrate molecules, the complexes are interconvertible by the exchange of the terminal pyrrole ring of each complex . The formation of enzyme-intermediate complexes is accompanied by the exposure of a cysteine residue, suggesting that substantial conformational changes occur on binding substrate . Specific labeling of the dipyrromethane cofactor by growth of the E . coli in the presence of 5-amino{5-14C}levulinic acid has confirmed that the cofactor is not subject to catalytic turnover . Experiments with the alpha-substituted substrate analogue alpha-bromoporphobilinogen have provided further evidence that the cofactor is responsible for the covalent binding of the substrate at the catalytic site . On the basis of these cumulative findings, it has been possible to construct a mechanistic scheme for the deaminase reaction involving a single catalytic site which is able to catalyze the addition or removal of either NH3 or H2O . The role of the cofactor both as a primer and as a means for regulating the number of substrates bound in each catalytic cycle is discussed. Biochemistry, 1988 Dec 13, 27(25), 8915 - 23 Evidence for a singlet intermediate in catalysis by Escherichia coli DNA photolyase and evaluation of substrate binding determinants; Jordan SP et al.; Escherichia coli DNA photolyase contains 1,5-dihydro-FAD (FADH2) plus 5,10-methenyl-tetrahydrofolate (5,10-CH+-H4folate) . Both chromophores are fluorescent, and either can function as a sensitizer in catalysis . At 77 K separate fluorescence emission bands are observed for FADH2 (lambda max = 505 nm, shoulder at 540 nm) and 5,10-CH+-H4folate (lambda max = 465, 440 nm) whereas at 5 degrees C only a shoulder at 505 nm is attributable to FADH2 . Formation of an enzyme-substrate complex with various dimer-containing oligothymidylates {UV-oligo(dT)n} quenches the fluorescence due to FADH2 at 5 degrees C or 77 K and also stabilizes FADH2 against air oxidation . The fluorescence of 5,10-CH+-H4folate is unaffected by substrate . Reduction of the pterin chromophore eliminates the chromophore's fluorescence but does not affect catalytic activity or the ability of substrate to quench FADH2 fluorescence . Quenching of FADH2 fluorescence is fully reversible upon dimer repair . The results are consistent with the proposal that the singlet state of FADH2 functions as an intermediate in catalysis . Fluorometric titrations indicate that the enzyme has a similar affinity for dimers in UV-oligo(dT)4 (KD = 2.5 X 10(-7) M, delta G = 8.4 kcal/mol at 5 degrees C) or UV-oligo(dT)6, except for dimers located at the unphosphorylated 3' end of the oligomers where binding is considerably weaker.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1988 Dec 13, 27(25), 9062 - 70 Dihydrofolate synthetase and folylpolyglutamate synthetase: direct evidence for intervention of acyl phosphate intermediates; Banerjee RV et al.; The transfer of 17O and/or 18O from (COOH-17O or -18O) enriched substrates to inorganic phosphate (Pi) has been demonstrated for two enzyme-catalyzed reactions involved in folate biosynthesis and glutamylation . COOH-18O-labeled folate, methotrexate, and dihydropteroate, in addition to {17O}-glutamate, were synthesized and used as substrates for folylpolyglutamate synthetase (FPGS) isolated from Escherichia coli, hog liver, and rat liver and for dihydrofolate synthetase (DHFS) isolated from E . coli . Pi was purified from the reaction mixtures and converted to trimethyl phosphate (TMP), which was then analyzed for 17O and 18O enrichment by nuclear magnetic resonance (NMR) spectroscopy and/or mass spectroscopy . In the reactions catalyzed by the E . coli enzymes, both NMR and quantitative mass spectral analyses established that transfer of the oxygen isotope from the substrate 18O-enriched carboxyl group to Pi occurred, thereby providing strong evidence for an acyl phosphate intermediate in both the FPGS- and DHFS-catalyzed reactions . Similar oxygen-transfer experiments were carried out by use of two mammalian enzymes . The small amounts of Pi obtained from reactions catalyzed by these less abundant FPGS proteins precluded the use of NMR techniques . However, mass spectral analysis of the TMP derived from the mammalian FPGS-catalyzed reactions showed clearly that 18O transfer had occurred. Gene, 1988 Dec 10, 72(1-2), 141 - 9 The differential stability of the Escherichia coli ompA and bla mRNA at various growth rates is not correlated to the efficiency of translation; Lundberg U et al.; Using two monocistronic gene transcripts, bla and ompA, we have studied the relationship between mRNA stability and translational efficiency . It was found that changes in the ompA mRNA stability are not correlated with an alteration in translational efficiency . In addition, at slow bacterial growth rates, the ompA transcript is translated ten times more efficiently than the bla messenger although the stability of the two transcripts is about equal . At rapid bacterial growth rate, chloramphenicol slightly stabilises both the bla and ompA transcripts without affecting their characteristic difference in half-life . Thus, control of mRNA stability seems not necessarily to be mediated either by the efficiency of loading ribosomes on a transcript, or by the arrest or slowing down of translating ribosomes. Gene, 1988 Dec 10, 72(1-2), 131 - 9 Post-transcriptional control in Escherichia coli: translation and degradation of the atp operon mRNA; McCarthy JE et al.; An attractive subject for investigations of post-transcriptional control is the atp operon, whose nine genes are differentially expressed . The primary mode of control of atp gene expression is exercised at the translational level . It has been clearly demonstrated for almost all of the atp genes that the primary and secondary structures of their respective translational initiation regions direct translational initiation rates that correspond well to the requirements for these subunits in the cell . The relationship between the structure of the translational initiation region, including bases upstream from the Shine-Dalgarno region and downstream from the start codon, and the rates of initiation that it determines, has been investigated in more detail using various polycistronic and monocistronic systems . No evidence could be found for a role of codon usage bias in controlling overall translation rates . The functional half-lives of atpE and of the other six cistrons downstream from it are similar . The chemical stabilities of the first two cistrons of the polycistronic atp mRNA may, however, be lower, and we are investigating the possibility that there may also be control of atp gene expression exercised at the level of mRNA stability . The effects of manipulations of the intercistronic regions of at least the plasmid borne atp operon are consistent with a model of mRNA decay in which rate control is associated with endonucleolytic cleavages within individual cistrons . The experimental data are discussed in relation to the possible ways in which primary and secondary structures of the mRNA might control translational efficiency and stability. Gene, 1988 Dec 10, 72(1-2), 349 - 60 Alpha-anomeric DNA: beta-RNA hybrids as new synthetic inhibitors of Escherichia coli RNase H, Drosophila embryo RNase H and M-MLV reverse transcriptase; Bloch E et al.; Nuclease-resistant alpha-anomeric DNA:beta-RNA hybrids are inhibitors of Escherichia coli RNase H, and Drosophila embryo RNase H . RNase H activities were measured by polyacrylamide gel electrophoresis, employing a short substrate, (A)12:d{G-G-(T)12-G-G}, or by acid-solubility techniques, using a long substrate, poly(A):poly(dT) . Strand exchanges which could be responsible for the observed inhibition have been ruled out by S1 nuclease experiments and by using inhibitors which do not allow strand exchange . Our results suggest that RNase H, for which DNA:RNA duplexes are the natural substrates, binds to non-physiological alpha-DNA:RNA hybrids and is consequently inhibited . These hybrids also inhibit the RNA-dependent DNA polymerase activity of M-MLV reverse transcriptase, therefore appearing as potential inhibitors of at least two reverse transcriptase activities . However, the inhibitory effect of these hybrids with respect to M-MLV reverse transcriptase is also observed with the single-stranded alpha-DNA itself . Unexpectedly, polymerase activity is highly stimulated by alpha-oligos, analogous in their sequence to the beta primer used at a concentration unable to generate a detectable synthesis . These results suggest that the inhibition of reverse transcriptase activity with the alpha:beta may occur at different levels. Gene, 1988 Dec 10, 72(1-2), 247 - 52 Antisense RNA does not significantly affect expression of the galK gene of Escherichia coli or the N gene of coliphage lambda; Hasan N et al.; The effect of antisense RNA on the expression of genes galK and N was studied in vivo . These two genes were either present in the Escherichia coli chromosome, as single copies, or were cloned on plasmid vectors . Antisense RNA was supplied from multicopy vectors where the entire galK or N gene, or only their N-proximal portions, were cloned in the antisense orientation downstream from the strong PL, PR or lacZp promoters . In all of the experiments there was no significant inhibition of the galK or N expression by up to a 50-fold excess of the specific antisense RNAs, for both the in cis and in trans experimental designs . The excess of the antisense RNA was calculated as based on respective copy numbers, but was not experimentally measured . The apparent five-fold regulatory effect observed in one of the experiments was found to be artifactually caused by unexpected creation of a terminator in one of our constructs . To avoid such artifacts, all our constructs were equipped with the nut-N antitermination system . We conclude that the reported antimessenger-mediated inhibition of gene expression is not a general phenomenon, but must require some special features which are not present in the galK and N systems. Gene, 1988 Dec 10, 72(1-2), 219 - 36 Analysis of the promoters and transcripts involved in IS10 anti-sense RNA control; Case CC et al.; Genetic analysis of eleven mutations affecting the IS10 promoters, pIN and pOUT, involved in anti-sense RNA control of transposase gene expression, and characterization of the transcripts, reveal that: (i) The transposase message (RNA-IN) and the anti-sense RNA (RNA-OUT) have been unambiguously identified in vivo . (ii) Five mutations affect pIN activity, and establish that pIN is the only IS10 promoter transcribing the tnp gene, and the only such IS10 promoter that responds to DNA-adenine methylation . (iii) Six mutations alter pOUT activity, and establish that pOUT is the only IS10 promoter specifying the anti-sense RNA-OUT . (iv) The latter, however, need not be so: heterologous promoters, if properly positioned, can also specify active anti-sense RNAs . (v) These heterologously promoted anti-sense RNAs are processed to species closely resembling native RNA-OUT. Gene, 1988 Dec 10, 72(1-2), 179 - 86 Unexpected translation initiation within the coding region of eukaryotic genes expressed in Escherichia coli; Preibisch G et al.; When expressing several eukaryotic genes in Escherichia coli, we observed N-terminally truncated proteins which were attributed to translation initiation at downstream AUG codons . These AUG codons are located between 4 and 20 nucleotides 3' from sequences resembling bacterial SD elements . Although the presence of such downstream SD sequences is not sufficient for downstream initiation to occur, in two cases their removal abolishes synthesis of the truncated proteins . In one construct, a potential hairpin-loop structure is likely to inhibit translation initiation at the correct site and favor downstream initiation. Nucleic Acids Res, 1988 Dec 9, 16(23), 11339 - 54 Dihydrothymidine and thymidine glycol triphosphates as substrates for DNA polymerases: differential recognition of thymine C5-C6 bond saturation and sequence specificity of incorporation; Ide H et al.; The ability of dihydrothymidine (DHdTTP) and thymidine glycol (dTTP-GLY) 5'-triphosphates to serve as substrates for different DNA polymerases was investigated . DHdTTP but not dTTP-GLY was used as a substrate by E . coli DNA polymerase I (Pol I) . Within the detection limit of the assay used, neither T4 DNA polymerase nor avian myeloblastosis virus (AMV) reverse transcriptase used DHdTTP or dTTP-GLY as substrates . The ability of DHdTTP and dTTP-GLY to undergo enzyme-catalyzed turnover to the monophosphate paralleled their ability to serve as substrates for polymerization . These results, along with kinetic parameters for the incorporation of DHdTTP with Pol I, strongly suggest that the saturation of thymine C5-C6 bond and the substituent groups at C5 and C6 differentially exert effects on binding to DNA polymerases . DNA sequencing gel analysis of the polymerization products revealed that most single adenine sites were capable of templating DHdTTP, however, DNA synthesis was partially arrested at multiple adenine sites, suggesting that sequential incorporation of DHdTTP produced significant disorder in the primer terminus. Biochim Biophys Acta, 1988 Dec 9, 972(3), 249 - 56 Increase of histidine decarboxylase activity in murine myelomonocytic leukemia cells (WEHI-3B) in parallel to their differentiation into macrophages; Kawaguchi-Nagata K et al.; When cells of mouse myelomonocytic leukemia cell line, WEHI-3B, were cultured in the presence of actinomycin D plus the serum which was obtained from mice injected with bacterial endotoxin, i.e., lipopolysaccharide, their histidine decarboxylase (L-histidine carboxy-lyase, EC 4.1.1.22) (HDC) activity increased about 100-fold with a peak at 48 h . According to the increase in HDC activity, the expression of surface antigens associated with macrophages, such as Mac II, Mac III and Iad, increased markedly on WEHI-3B cells as well as their morphological changes to macrophages . Histamine levels in the culture medium increased concomitantly with the increase in the HDC activity in WEHI-3B cells, whereas the histamine contents inside the cells did not increase remarkably . Furthermore, the addition of lipopolysaccharide to the culture medium caused an additional 2-fold increase in the HDC activity of WEHI-3B cells . These results indicate that the increase in HDC activity in WEHI-3B cells may represent an event in the process of the differentiation to macrophages. J Biol Chem, 1988 Dec 5, 263(34), 18086 - 92 Mechanism of ferritin iron uptake: activity of the H-chain and deletion mapping of the ferro-oxidase site . A study of iron uptake and ferro-oxidase activity of human liver, recombinant H-chain ferritins, and of two H-chain deletion mutants; Levi S et al.; To study the functional differences between human ferritin H- and L-chains and the role of the protein shell in the formation and growth of the ferritin iron core, we have compared the kinetics of iron oxidation and uptake of ferritin purified from human liver (90% L) and of the H-chain homopolymer overproduced in Escherichia coli (100% H) . As a control for iron autocatalytic activity, we analyzed the effect of Fe(III) on the iron uptake reaction . The results show that the H-chain homopolymer has faster rates of iron uptake and iron oxidation than liver ferritin in all the conditions analyzed and that the difference is reduced in the conditions in which iron autocatalysis in high: i.e . at pH 7 and in presence of iron core . We have also analyzed the properties of two engineered H-chains, one lacking the last 22 amino acids at the carboxyl terminus and the other missing the first 13 residues at the amino terminus . These mutant proteins assemble in ferritin-like proteins and maintain the ability to catalyze iron oxidation . The deletion at the carboxyl terminus, however, prevents the formation of a stable iron core . It is concluded that the ferritin H-chain has an iron oxidation site which is separated from the sites of iron transfer and hydrolysis and that either the integrity of the molecule or the presence of the amino acid sequences forming the hydrophobic channel is necessary for iron core formation. J Biol Chem, 1988 Dec 5, 263(34), 18043 - 51 Genomic structure and amino acid sequence domains of the human La autoantigen; Chambers JC et al.; La is an autoimmune RNA-binding protein of 47 kDa that plays a role in the transcription of RNA polymerase III . Both genomic and complementary DNAs were isolated that encompass the coding sequence of the human La molecule . The genomic clones encompass 11 exons and a putative G/C-rich promoter upstream of the mRNA start site . The cDNA sequence encodes a protein of 408 amino acids and can be divided into two structural domains based upon amino acid content and protease sensitivity . An unusually long stretch of 130 amino acids, much of which was predicted to form a stable alpha-helix, was found near the middle of the protein between the two domains . A ribonucleoprotein (RNP) consensus sequence was found just NH2-terminal to the long alpha-helix . The RNP consensus sequence is split into two exons by the fifth intron . Expression of three separate fragments of the La protein in Escherichia coli showed that a strongly autoimmune-reactive portion resides in the fragment containing the RNP consensus sequence and most of the long alpha-helical core . Autoantibodies from La patients also reacted with the terminal regions of the protein, but the extent of reactivity varied among patients . Differences in reactivity of autoantibodies to each portion of La protein may reflect an evolution of recognition of different epitopes during the development of the autoimmune response . These findings support an antigen-driven mechanism for autoimmune reactivity. J Biol Chem, 1988 Dec 5, 263(34), 18452 - 8 Production, biological activity, and structure of recombinant basic fibroblast growth factor and an analog with cysteine replaced by serine; Fox GM et al.; We have chemically synthesized the gene encoding bovine basic fibroblast growth factor (bFGF) and cloned it into a plasmid vector . This gene was then used as a template for site-directed mutagenesis to produce the human bFGF gene and a gene coding for an analog in which serine residues were substituted for the cysteine residues at positions 70 and 88 . All three constructs were cloned and expressed in Escherichia coli and the proteins purified . The recombinant human and bovine bFGFs exhibited the potent mitogenic activity toward both fibroblasts and endothelial cells, which characterizes natural bFGF . The serine-70,88 analog and natural sequence bovine and human forms were equally active in all assays . Sulfhydryl titration of the purified recombinant bovine bFGF in 4.8 M guanidine hydrochloride indicated the presence of approximately two free sulfhydryl groups . This was consistent with the sequence analysis of peptides derived from trypsin digestion, which suggests that cysteines 70 and 88 exist in free sulfhydryl form while cysteines 26 and 93 form a disulfide bond . Circular dichroism shows that the protein has little ordered structure but is folded into a rigid tertiary configuration . Carboxymethylation of the free sulfhydryl groups resulted in no change in the mitogenic activity or conformation . These results are consistent with previous suggestions that, for tissue-derived bFGF, at least 2 of the 4 cysteines in the molecule are not involved in a disulfide bond. J Biol Chem, 1988 Dec 5, 263(34), 18343 - 9 In vitro attachment of bilins to apophycocyanin . I . Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C-phycocyanin; Arciero DM et al.; Expression of cloned alpha and beta subunit genes of Synechococcus sp . PCC7002 C-phycocyanin in Escherichia coli led to the production of large amounts of apophycocyanin . The apophycocyanin was purified to homogeneity and shown to be an alpha beta monomer . The reactivity of the apoprotein toward a number of open chain and cyclic tetrapyrroles was examined . Phycocyanobilin (PCB), phycoerythrobilin, and biliverdin all formed covalent adducts with apophycocyanin in 50 mM sodium phosphate buffer at pH 7.0 . Mesobiliverdin, bilirubin, PCB dimethyl ester, protoporphyrin IX, and hemin did not react with the apoprotein . None of these tetrapyrroles reacted with 2 mM 2-mercaptoethanol, cysteine, or reduced glutathione under the same conditions . The adduct with PCB was investigated in greater detail . Its visible absorption spectrum, with a maximum at 646 nm, is more similar to that of allophycocyanin than phycocyanin . Two PCBs are bound per alpha beta monomer when the reaction is performed with excess bilin . While tryptic digestion of the adduct generates numerous bilin peptides, amino acid analysis of these chromopeptides revealed that PCB reacted specifically at alpha-Cys-84 and beta-Cys-82, two of the three cysteinyl residues that serve as the attachment sites for PCB in native phycocyanin . The major bilin peptides arising from in vitro adduct formation at each of these sites differed both in chromatographic behavior and in spectroscopic properties from the corresponding PCB peptides isolated from tryptic digests of native C-phycocyanin. J Mol Biol, 1988 Dec 5, 204(3), 725 - 47 Complex of N-phosphonacetyl-L-aspartate with aspartate carbamoyltransferase . X-ray refinement, analysis of conformational changes and catalytic and allosteric mechanisms; Ke HM et al.; The allosteric enzyme aspartate carbamoyltransferase of Escherichia coli consists of six regulatory chains (R) and six catalytic chains (C) in D3 symmetry . The less active T conformation, complexed to the allosteric inhibitor CTP has been refined to 2.6 A (R-factor of 0.155) . We now report refinement of the more active R conformation, complexed to the bisubstrate analog N-phosphonacetyl-L-aspartate (PALA) to 2.4 A (R-factor of 0.165, root-mean-square deviations from ideal bond distances and angles of 0.013 A and 2.2 degrees, respectively) . The antiparallel beta-sheet in the revised segment 8-65 of the regulatory chain of the T conformation is confirmed in the R conformation, as is also the interchange of alanine 1 with the side-chain of asparagine 2 in the catalytic chain . The crystallographic asymmetric unit containing one-third of the molecule (C2R2) includes 925 sites for water molecules, and seven side-chains in alternative conformations . The gross conformational changes of the T to R transition are confirmed, including the elongation of the molecule along its threefold axis by 12 A, the relative reorientation of the catalytic trimers C3 by 10 degrees, and the rotation of the regulatory dimers R2 about the molecular twofold axis by 15 degrees . No changes occur in secondary structure . Essentially rigid-body transformations account for the movement of the four domains of each catalytic-regulatory unit; these include the allosteric effector domain, the equatorial (aspartate) domain, and the combination of the polar (carbamyl phosphate) and zinc domain, which moves as a rigid unit . However, interfaces change, for example the interface between the zinc domain of the R chain and the equatorial domain of the C chain, is nearly absent in the T state, but becomes extensive in the R state of the enzyme; also one catalytic-regulatory interface (C1-R4) of the T state disappears in the more active R state of the enzyme . Segments 50-55, 77-86 and 231-246 of the catalytic chain and segments 51-55, 67-72 and 150-153 of the regulatory chain show conformational changes that go beyond the rigid-body movement of their corresponding domains . The localized conformational changes in the catalytic chain all derive from the interactions of the enzyme with the inhibitor PALA; these changes may be important for the catalytic mechanism . The conformation changes in segments 67-72 and 150-153 of the regulatory chain may be important for the allosteric control of substrate binding . On the basis of the conformational differences of the T and R states of the enzyme, we present a plausible scheme for catalysis that assumes the ordered binding of substrates and the ordered release o J Mol Biol, 1988 Dec 5, 204(3), 581 - 91 Construction and characterization of the deletion mutant of hupA and hupB genes in Escherichia coli; Wada M et al.; Insertion and deletion mutations of the hupB and hupA genes, which encode the HU-1 and HU-2 proteins, respectively, of Escherichia coli, have been constructed in vitro and transferred to the hup loci on the bacterial chromosome . The mutations were constructed by inserting a gene encoding chloramphenicol resistance or kanamycin resistance into the coding region of the hupB or hupA gene, respectively . A complete deletion of the hupA gene was constructed by replacing the entire hupA coding region with the kanamycin resistance gene . Cells in which either the hupB or the hupA gene is defective grow normally, but cells in which both of the hup genes are defective exhibit phenotypes different from the wildtype strain . The hupA-hupB double mutants are cold-sensitive, although their growth rate is normal at 37 degrees C . Furthermore, the viability of the hupA-hupB double mutants is severely reduced when the cells are subjected to either cold shock or heat shock, indicating that the hup genes are essential for cell survival under some conditions of stress . The double mutants also exhibit filamentation when grown in the lower range of permissive growth temperature. FEBS Lett, 1988 Dec 5, 241(1-2), 46 - 50 Engineering of an intersubunit disulphide bridge in glutathione reductase from Escherichia coli; Scrutton NS et al.; By site-directed mutagenesis, Thr-75 was converted to Cys-75 in the glutathione reductase (EC 1.6.4.2) of Escherichia coli . This led to the spontaneous formation of an intersubunit disulphide bridge across the 2-fold axis of the dimeric enzyme . The disulphide bridge had no deleterious effect on the catalytic activity, but nor did it increase the thermal stability of the enzyme, possibly because of local conformational flexibility on the dimer interface . The T75C mutant, like the wild-type enzyme, was inactivated by NADPH, proving that this inactivation cannot be due to simple dissociation of the dimer. FEBS Lett, 1988 Dec 5, 241(1-2), 33 - 7 Spectroscopic studies on the mode of binding of ATP, UTP and alpha-amanitin with yeast RNA polymerase II; Bhargava P et al.; The binding affinity between the substrates ATP and UTP with the purified yeast RNA polymerase II have been studied here in the presence and absence of Mn2+ . In the absence of template DNA, both ATP and UTP showed tight binding with the enzyme without preference for any specific nucleotide, unlike Escherichia coli RNA polymerase . Fluorescence titration of the tryptophan emission of the enzyme by nucleoside triphosphate substrates gave an estimated Kd value around 65 microM in the absence of Mn2+ whereas in the presence of Mn2+, the Kd was 20 microM . The effect of substrates on the longitudinal relaxation of the HDO proton in enzyme-substrate complex also yielded a similar Kd value. FEBS Lett, 1988 Dec 5, 241(1-2), 141 - 4 Flagellin parts acquiring a regular structure during polymerization are disposed on the molecule ends; Kostyukova AS et al.; Flagellins of two Escherichia coli strains have been investigated by limited proteolysis and scanning microcalorimetry . It has been shown that a monomer flagellin consists of two parts: a central one cooperatively melting, rather resistant to proteases, and the other without a stable tertiary structure and thus easily degrading terminals . Just these terminals acquire a regular structure during polymerization . Core fragments of the two strains have been isolated and characterized. J Biol Chem, 1988 Dec 5, 263(34), 18437 - 42 Reconstitution of mitochondrial protein transport with purified ornithine carbamoyltransferase precursor expressed in Escherichia coli; Murakami K et al.; Ornithine carbamoyltransferase (OTC; subunit, 36,000 Da) is initially synthesized as a precursor (pOTC) with a transient NH2-terminal presequence of 32 amino acid residues and imported posttranslationally into the mitochondrial matrix . The rat pOTC was synthesized in Escherichia coli using an expression vector containing a thermoinducible lambda pL promoter . The recombinant pOTC represented 5-10% of the total bacterial protein and was present in the precipitate of the disrupted bacteria . The precipitate was washed and pOTC was extracted with 8 M urea or 0.1% cetyltrimethylammonium bromide . The extracted pOTC was essentially homogeneous, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The purified pOTC was cleaved to the intermediate-sized product of 37,000 Da by a processing protease partially purified from the matrix fraction of rat liver mitochondria . The purified recombinant pOTC, but not the mature form of OTC synthesized in E . coli and purified, competed with the in vitro-synthesized, radiolabeled pOTC for uptake and processing by the isolated rat liver mitochondria . The radiolabeled and purified recombinant pOTC could be imported into the isolated mitochondria and processed to the mature form in an energy- and rabbit reticulocyte lysate-dependent manner . When the purified pOTC was subjected to sucrose gradient centrifugation, it sedimented as a large aggregate of greater than 60 S in the absence of reticulocyte lysate, whereas it sedimented as a complex of about 5 S in the presence of the lysate . These observations together with our previous results indicate that a protein factor(s) present in the lysate interacts with pOTC and holds it in an import-competent form. J Biol Chem, 1988 Dec 5, 263(34), 18277 - 85 The role of exonucleolytic processing and polymerase-DNA association in bypass of lesions during replication in vitro . Significance for SOS-targeted mutagenesis; Shwartz H et al.; The role of exonuclease activity in trans-lesion DNA replication with Escherichia coli DNA polymerase III holoenzyme was investigated . RecA protein inhibited the 3'----5' exonuclease activity of the polymerase 2-fold when assayed in the absence of replication and had no effect on turnover of dNTPs into dNMPs . In contrast, single-stranded DNA-binding protein, which had no effect on the exonuclease activity in the absence of replication, showed a pronounced 7-fold suppression of the 3'----5' exonuclease activity during replication . The excision of incorporated dNMP alpha S residues from DNA by the 3'----5' exonuclease activity of DNA polymerase III holoenzyme was inhibited 10-20-fold; still no increase in bypass of pyrimidine photodimers was observed . Thus, in agreement with our previous results in which the exonuclease activity was inhibited at the protein level (Livneh, Z . (1986) J . Biol . Chem . 261, 9526-9533), inhibition at the DNA level also did not increase bypass of photodimers . Fractionation of the replication mixture after termination of DNA synthesis on a Bio-Gel A-5m column under conditions which favor polymerase-DNA binding yielded a termination complex which could perform turnover of dNTPs into dNMPs . Adding challenge-primed single-stranded DNA to the complex yielded a burst of DNA synthesis which was promoted most likely by DNA polymerase III holoenzyme molecules transferred from the termination complex to the challenge DNA thus demonstrating the instability of the polymerase-DNA association . Addition of a fresh sample of DNA polymerase III holoenzyme to purified termination products, which consist primarily of partially replicated molecules with nascent chains terminated at UV lesions, did not result in any net DNA synthesis as expected . However, reactivation of lesion-terminated primers was achieved by pretreatment with a 3'----5' exonuclease which excised 200 nucleotides or more, generating new 3'-OH termini located away from the UV lesions . When these exonuclease-treated products were subjected to a second round of replication, an increased level of DNA synthesis was observed including additional bypass of photodimers . These results suggest the possibility that 3'----5' exonuclease processing might be required at least transiently during one of the stages of trans-lesion DNA replication, which is believed to be the mechanism of SOS-targeted mutagenesis. J Biol Chem, 1988 Dec 5, 263(34), 18017 - 22 Yeast DNA 3'-repair diesterase is the major cellular apurinic/apyrimidinic endonuclease: substrate specificity and kinetics; Johnson AW et al.; DNA strand breaks with damaged 3' termini are potentially toxic lesions caused by free radicals . The purified yeast diesterase that removes small nucleotide fragments from such 3' termini in oxidized DNA has been further characterized with respect to its substrate specificity . In addition to the 3'-phosphoglycolaldehyde esters used to monitor the activity during purification, the enzyme efficiently hydrolyzed a variety of other 3'-esters in DNA . These included 3'-phosphates, 3'-(2,3-didehydro-2,3-dideoxyribose phosphates), and the 3'-blocking damages formed in vivo in Escherichia coli by H2O2 or in vitro by DNA treatment with bleomycin . This same transition metal-dependent enzyme also constitutes the major yeast endonuclease for apurinic/apyrimidinic sites in DNA, hydrolyzing these damages to yield normal 3'-hydroxyl nucleotides and 5'-phosphoryl base-free sugar termini (a Type II apurinic/apyrimidinic endonuclease) . Yeast 3'-phosphoglycolaldehyde diesterase therefore appears to be involved in two distinct pathways of DNA repair: initiation of the repair of oxidative strand breaks in DNA and the restoration of sites of base loss caused by many types of DNA-damaging agents. J Biol Chem, 1988 Dec 5, 263(34), 17933 - 41 Trypanosoma brucei ornithine decarboxylase: enzyme purification, characterization, and expression in Escherichia coli; Phillips MA et al.; Ornithine decarboxylase from the African trypanosome is an important target for antitrypanosomal chemotherapy . Despite this, the enzyme had not been previously purified or extensively characterized as it is a very low level protein . In this paper we describe the purification of Trypanosoma brucei brucei ornithine decarboxylase from bloodstream form trypomastigotes by 107,000-fold to a specific activity of 2.7 x 10(6) nmol CO2/h/mg of protein in the parasite . T . brucei ornithine decarboxylase had a native molecular weight of 90,000 and a subunit molecular weight of 45,000 . The isoelectric point of the protein was 5.0 . The Km for ornithine was 280 microM and the Ki for the irreversible inhibitor alpha-difluoromethylornithine (DFMO) was 220 microM with a half-time of inactivation at saturating DFMO concentration of 2.7 min . T . brucei ornithine decarboxylase appears similar to mouse ornithine decarboxylase, further supporting our previous suggestion that the selective toxicity of DFMO to the parasite is not due to catalytic differences between the two proteins . Although a small quantity of T . brucei ornithine decarboxylase was purified from T . brucei, extensive structural and kinetic studies will require a more ample source of the enzyme . We therefore expressed our previously cloned T . brucei ornithine decarboxylase gene in Escherichia coli using a vector that contains an inducible lambda promoter . T . brucei ornithine decarboxylase activity was induced in E . coli to levels that were 50 to 200 fold of that present in the long-slender bloodstream form of T . brucei . Ornithine decarboxylase activity in the crude E . coli lysate was 1500-6000 nmol of CO2/h/mg of protein and represented 0.05-0.2% of the total cell protein . The recombinant T . brucei ornithine decarboxylase was purified to apparent homogeneity from the transformed E . coli . The purified recombinant enzyme had kinetic and physical properties essentially identical to those of the native enzyme. J Biol Chem, 1988 Dec 5, 263(34), 17909 - 12 Transfer RNA is a substrate for RNase D in vivo; Zhang JR et al.; RNase D is a 3'-exonuclease whose in vitro specificity has suggested a role in tRNA processing . However, since mutant Escherichia coli strains devoid of RNase D display a normal phenotype, it has not been possible to ascertain the enzyme's function or even to determine which RNA is its substrate in vivo . Here we show that transformation of strains devoid of tRNA nucleotidyltransferase with a multicopy plasmid carrying the rnd+ gene leads to extremely slow growth due to elevated levels of RNase D activity . Analysis of such a slow-growing strain revealed that less tRNA is present in the cell and that the tRNA that could be recovered is substantially damaged . These studies demonstrate that RNase D can act at the 3' terminus of tRNA in vivo, and they support the conclusion that RNase D participates in tRNA metabolism. J Biol Chem, 1988 Dec 5, 263(34), 17905 - 8 Synthetic peptides as substrates and inhibitors of human immune deficiency virus-1 protease; Billich S et al.; Retroviruses code for a virus-specific protease which is essential for polyprotein processing and viral infectivity . The human immune deficiency virus-1 protease is an aspartic protease of 9 kDa which was synthesized by recombinant DNA technology and arises by autocatalytic processing from a polyprotein precursor which has recently been demonstrated by use of a protease-specific monoclonal antibody . The protease was shown to form dimers . Here we demonstrate that synthetic peptides can be used as both model substrates as well as inhibitors for investigation of the protease . 14 synthetic peptides, 7-18 amino acids in length, containing putative protease cleavage sites of the viral polyprotein gag and pol precursors, have been analyzed with the partially purified protease by the use of high performance liquid chromatography . In seven cases, where cleavage was observed, the length of the peptides did not significantly influence the cleavage efficiencies, heptapeptides being large enough as model substrates . No cleavage was observed with a protein preparation purified in parallel from control bacteria not expressing the human immune deficiency virus-1 protease . The protease was not only able to cut next to a proline but also between other peptides indicating that the proline is not a prerequisite . Three peptides with either reduced bonds at the cleavage site or a substitution by statin were inhibitory while another uncleaved substrate was not . The usefulness of small model substrates for characterization of the protease is further demonstrated by determination of a kinetic optimum pH (3.5-5.5) and incubation temperature (37 degrees C). FEBS Lett, 1988 Dec 5, 241(1-2), 251 - 6 Renaturation of guanidine-unfolded tryptophan synthase by multi-mixing stopped-flow dilution in D2O; Blond-Elguindi S et al.; Guanidine hydrochloride (GdnHCl) at high concentrations, e.g . 4 to 8 M, has been used extensively to promote reversible denaturation of several proteins . The refolding is induced by removal of the denaturing agent by diluting the denatured protein at least 50-100-fold in a 'renaturation buffer' . Fast kinetic studies, using a stopped-flow apparatus to achieve such dilutions, are difficult for two reasons: firstly, injecting widely different volumes of the two reagents does not afford a proper mixing . Secondly, the density differences existing between the concentrated GdnHCl solution and the renaturation buffer often causes important mixing and redistribution artefacts particularly in vertical stopped-flows . Here, it is shown that these difficulties can be overcome by using a multi-mixing stopped-flow to achieve 2 successive 7-fold dilutions and by using heavy water (D2O) to adjust the density of the renaturation buffer . This enabled us to study the appearance of a short-lived intermediate during the folding of the beta 2-subunit of Escherichia coli tryptophan synthase. J Biol Chem, 1988 Dec 5, 263(34), 18099 - 103 Mutants of translational components that alter reading frame by two steps forward or one step back; Falahee MB et al.; External suppressors, sufS, of a -1 frameshift mutant cause ribosomes to shift into the -1 frame when reading the sequence CAG GGA GUG . The resulting product is not Gln-Gly-Val but Gln-Gly-Ser with Ser being encoded by the underlined AGU . The alleles investigated are approximately 2% efficient in causing frameshifting . Two other suppressors, hopR and hopE of the same -1 frameshift mutant, cause some ribosomes reading the sequence GUG UG to decode a single amino acid, Val, from the five nucleotides . The possibility is considered that peptidyl-tRNA(Val) dissociates from the mRNA, but re-pairs in a triplet manner after the mRNA slips forward by two bases. J Mol Biol, 1988 Dec 5, 204(3), 507 - 22 Domain VI of Escherichia coli 23 S ribosomal RNA . Structure, assembly and function; Leffers H et al.; Domain VI at the 3' end of the 23 S ribosomal RNA from Escherichia coli was prepared using the in vitro T7 RNA polymerase system . Its structure was examined by probing with ribonucleases and chemical reagents, including a psoralen derivative, of various nucleotide specificities, using a reverse transcriptase procedure for analysis . The data provided support for the most recent secondary structure derived from phylogenetic sequence comparisons and for additional structuring that was inferred from earlier experimental data . Moreover, the structure was essentially the same in the free domain, in renatured 23 S RNA and in 50 S subunits . Protein L3 bound to the isolated domain and its binding site was located at a long-range double helix containing a large internal loop . This structure is unusual for a protein-RNA binding site and it may characterize a new (third) class of site . Protein L3 has been implicated, together with L24, in initiating assembly of the 50 S subunit and it shares the exceptional property with L24 that it binds adjacent to the junction of two RNA domains from where it can maximally influence RNA folding . Protein L6 also assembled to domain VI and, in a control experiment, protein L2 bound to isolated domain IV . Domain VI was largely inaccessible in the 50 S subunit and the few accessible RNA sites occurred mainly within conserved sequence regions that constitute potential functional sites . alpha-Sarcin inactivates ribosomes by cutting at one of these sites in 50 S subunits; it also recognized the same site in the free 23 S RNA and in the free domain . Both the EF-Tu ternary complex, and the EF-G ternary complex stabilized by fusidic acid or by a non-hydrolyzable GTP derivative, inhibited alpha-sarcin attack while non-enzymatically bound tRNA did not, thus providing evidence, more direct than before, for the involvement of the RNA region in a common elongation factor binding site. Science, 1988 Dec 2, 242(4883), 1290 - 5 Sugar and signal-transducer binding sites of the Escherichia coli galactose chemoreceptor protein; Vyas NK et al.; D-galactose-binding (or chemoreceptor) protein of Escherichia coli serves as an initial component for both chemotaxis towards galactose and glucose and high-affinity active transport of the two sugars . Well-refined x-ray structures of the liganded forms of the wild-type and a mutant protein isolated from a strain defective in chemotaxis but fully competent in transport have provided a molecular view of the sugar-binding site and of a site for interacting with the Trg transmembrane signal transducer . The geometry of the sugar-binding site, located in the cleft between the two lobes of the bilobate protein, is novel in that it is designed for tight binding and sequestering of either the alpha or beta anomer of the D-stereoisomer of the 4-epimers galactose and glucose . Binding specificity and affinity are conferred primarily by polar planar side-chain residues that form intricate networks of cooperative and bidentate hydrogen bonds with the sugar substrates, and secondarily by aromatic residues that sandwich the pyranose ring . Each of the pairs of anomeric hydroxyls and epimeric hydroxyls is recognized by a distinct Asp residue . The site for interaction with the transducer is about 18 A from the sugar-binding site . Mutation of Gly74 to Asp at this site, concomitant with considerable changes in the local ordered water structures, contributes to the lack of productive interaction with the transmembrane signal transducer. Cell, 1988 Dec 2, 55(5), 849 - 56 Supercoiling of intracellular DNA can occur in eukaryotic cells; Giaever GN et al.; The supercoiling of 2 micron DNA in yeast by a process or processes that generate positively and negatively supercoiled domains was shown by the use of yeast DNA topoisomerase mutants expressing Escherichia coli DNA topoisomerase I, an enzyme that relaxes negative supercoils specifically . Intracellular 2 micron DNA becomes positively supercoiled in yeast top1 top2 ts strains expressing the E . coli enzyme when neither one of the yeast DNA topoisomerases I and II is functional . Examination of the linking number distributions of plasmids bearing the inducible promoters of GAL1 and GAL10 genes indicates that the generation of supercoiled domains of opposite signs is related to transcription. J Clin Invest, 1988 Dec, 82(6), 1934 - 8 Interleukin 1 administration in mice produces hypoferremia despite neutropenia; Gordeuk VR et al.; To determine whether the hypoferremic response to inflammation requires neutrophils, we administered human recombinant IL-1 to mice made neutropenic with cyclophosphamide . With single intraperitoneal injections of IL-1 the plasma iron concentrations decreased significantly in mice with either normal neutrophil counts or neutropenia . After single injections transferrin concentrations were not significantly changed, but the decrease in serum iron lowered mean transferrin saturations from a baseline of 45 to 24-30% in nonneutropenic mice, and from 99 to 70-77% in neutropenic mice . Similar changes were observed after intraperitoneal injections of Escherichia coli . 4-d continuous infusions of IL-1 also led to reductions in serum iron concentrations, but transferrin concentrations doubled . The combination of a decrease in serum iron and an increase in transferrin concentration after chronic infusion in neutropenic mice led to a greater decline in mean transferrin saturations, from a baseline of 110 to 25% . In mice not given cyclophosphamide, chronic IL-1 infusion was associated with a reduction in mean hemoglobin concentrations from 14.7 to 13.5 g/dl, consistent with restricted availability of iron for erythropoiesis associated with low saturation of transferrin . We conclude that IL-1 can decrease the serum iron despite profound peripheral neutropenia and that transferrin in a positive acute phase reactant in the mouse. Genes Dev, 1988 Dec, 2(12B), 1812 - 23 Gene activities and segmental patterning in Drosophila: analysis of odd-skipped and pair-rule double mutants; Coulter DE et al.; The odd-skipped (odd) gene is required to generate anterior regions of the odd-numbered segments in Drosophila; homozygous embryos show pattern deletions that are always less than a segment in width and are associated with mirror-image duplications of adjacent regions . To define further the role of odd and determine how it interacts with other segmentation genes, we have described the effects of combining odd with mutations at other pair-rule loci . We have observed phenotypic suppression in double-mutant combinations with even-skipped (eve), paired, sloppy paired, and engrailed (en) . In the most thoroughly characterized combination (odd eve), both naked cuticle and specific denticle rows are restored that would normally have been deleted by one of the two mutants alone . In the odd en double mutant, we observe nearly complete suppression of the odd phenotype, such that the mirror image duplications are eliminated and the odd-numbered denticle bands are restored . We conclude that the requirements of pattern elements for specific gene activities are not absolute, and propose mechanisms by which these genes interact to specify cell fates. Biophys J, 1988 Dec, 54(6), 983 - 93 Particle counting by fluorescence correlation spectroscopy . Simultaneous measurement of aggregation and diffusion of molecules in solutions and in membranes; Meyer T et al.; A method for simultaneous determination of molar weights (M) and lateral diffusion constants (D) of particles in three- and two-dimensional systems is described . Spontaneous concentration fluctuations in space and time are analyzed, by monitoring fluctuations in the fluorescence from fluorescein-labeled molecules (1 dye/molecule is sufficient), excited by a rotating laser spot . For particles in solution, M values are determined over the range of 3 x 10(2) to 3 x 10(11) daltons, and D values can be determined from approximately 10(-7) to 10(-10) cm2/s . The time for a determination is approximately 1 min . Aggregation can be followed by changes of either M or D . This method is used to study the calcium dependence of vesicle aggregation or fusion, and the time course of aggregate formation of porin (an Escherichia Coli outer membrane protein) in lipid monolayers . Essential parameters for the development of the method are described . Equations to estimate the signal-to-noise ratios and to find the optimal free parameters for a specific application are derived . The theoretical predictions for the correlation function of the signal and for the signal-to-noise ratio are compared with observed values. Epidemiol Infect, 1988 Dec, 101(3), 611 - 21 A study of intranasally administered interferon A (rIFN-alpha 2A) for the seasonal prophylaxis of natural viral infections of the upper respiratory tract in healthy volunteers; Tannock GA et al.; The efficacy of interferon A (rIFN-alpha 2A), an Escherichia coli-derived interferon, in the prophylaxis of acute upper respiratory tract infection, was evaluated in a community-based double-blind placebo-controlled study in the Australian winter of 1985 . The trial population of 412 healthy volunteers (190 males and 222 females, aged 18-65 years) self-administered 1.5, 3.0 and 6.0 megaunits (MU) of interferon A per day or a placebo, intranasally for 28 days . The period of study coincided with an outbreak of H3N2 influenza A (detected in 35 of the 107 acute specimens) as well as substantial numbers of respiratory syncytial virus and adenovirus infections . Rhinoviruses were isolated from only three specimens . In many cases, subjects had laboratory and clinical evidence of having had more than one respiratory tract infection during the period of the study . Viruses were detected in 54 or 107 acute specimens (49%) . No statistically significant differences were noted between the various treatment groups in the incidence of laboratory-proven viral infection (virus isolation and/or antibody response) . Analysis of reported symptoms indicated that blood-tinged mucus and nasal stuffiness occurred more frequently with higher doses of interferon . There appeared to be no clinical benefit from the use of interferon A in the amelioration of symptoms. Am J Med Sci, 1988 Dec, 296(6), 381 - 6 Endotoxin-induced neutrophilic alveolitis and macrophage chemotaxin production in sheep; Duke SS et al.; The mechanism(s) responsible for endotoxin-induced pulmonary leukostasis has not yet been elucidated . We studied ten unanesthetized sheep by performing serial bronchoalveolar lavages (BAL) before and after infusing Escherichia coli endotoxin to determine whether or not neutrophils appeared in the airways and whether or not endotoxemia stimulated alveolar macrophages to produce neutrophil chemotactic activity . The absolute number and percentage of neutrophils recoverable by BAL increased significantly at 24 hours after infusion of endotoxin . Alveolar macrophages isolated from the BAL of sheep after endotoxemia produced a substance that is chemotactic for neutrophils, a response that is also maximal 24 hours after endotoxin infusion . We conclude that endotoxemia causes migration of neutrophils into lung air spaces and that this response may result from in vivo production of a chemotactic factor(s) by activated alveolar macrophages. EMBO J, 1988 Dec 1, 7(12), 3975 - 82 The Escherichia coli LexA repressor-operator system works in mammalian cells; Smith GM et al.; We have demonstrated the use of the Escherichia coli LexA repressor-operator system to down-regulate gene expression in mouse cells . The LexA gene was placed downstream of the RSVLTR promoter with polyadenylation and splice signals from SV40 . This expression unit was introduced into mouse Ltk- cells by calcium phosphate transfection and stable transfectants selected which express LexA protein . We have used the bacterial chloramphenicol acetyltransferase gene (CAT) as our reporter gene . Transcription of this gene was driven by the HSV tk promoter, into which we have introduced one or two synthetic LexA operator sequences in various positions throughout the promoter . Necessary 3' signals were from the HSV tk gene . Repression by LexA was assessed by comparing the transient expression of tkCAT target constructs, containing LexA operator sequences in the promoter, in cells expressing LexA protein with that in control cells not expressing the repressor . We have observed up to 10-fold repression of CAT expression in LexA+ cells from promoters containing LexA operator sequences. Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8924 - 8 Cocrystal structure of an editing complex of Klenow fragment with DNA; Freemont PS et al.; High-resolution crystal structures of editing complexes of both duplex and single-stranded DNA bound to Escherichia coli DNA polymerase I large fragment (Klenow fragment) show four nucleotides of single-stranded DNA bound to the 3'-5' exonuclease active site and extending toward the polymerase active site . Melting of the duplex DNA by the protein is stabilized by hydrophobic interactions between Phe-473, Leu-361, and His-666 and the last three bases at the 3' terminus . Two divalent metal ions interacting with the phosphodiester to be hydrolyzed are proposed to catalyze the exonuclease reaction by a mechanism that may be related to mechanisms of other enzymes that catalyze phospho-group transfer including RNA enzymes . We suggest that the editing active site competes with the polymerase active site some 30 A away for the newly formed 3' terminus . Since a 3' terminal mismatched base pair favors the melting of duplex DNA, its binding and excision at the editing exonuclease site that binds single-stranded DNA is enhanced. Mol Gen Mikrobiol Virusol, 1988 Dec, (12), 33 - 8 {Mapping of "nonessential" regions in the genome of vaccinia virus}; Loparev VN et al.; The special molecular probe for mapping the "nonessential" regions in the genome of vaccinia virus has been obtained by the genetic engineering methods . The probe included the gene for beta-galactosidase of E . coli under the control of vaccinia virus 7.5 K protein promoter as well as the gene for kanamycin resistance . In its final version the probe is obtainable from the plasmid pUCZ beta using the restriction endonucleases SalI, BamHI, EcoRI . The probe included by the BamHI fragment of DNA was inserted into the HindIII-E-fragment of the vaccinia virus (cloned into a plasmid) in 8 of the existing 9 BglII cleavage sites . The latter plasmids were introduced into the chicken embryo cells infected by the vaccinia virus . The plasmid having the probe inserted into the 5th BglII site (from the left end) of the HindIII-E fragment permitted to obtain the live vaccinia strain expressing the beta-galactosidase . Thus, the "nonessential" region of vaccinia virus, that was not described previously, is mapped. Biochimie, 1988 Dec, 70(12), 1807 - 14 Allosteric transition of aspartokinase I-homoserine dehydrogenase I studied by time-resolved fluorescence; Jullien M et al.; The allosteric transition of threonine-sensitive aspartokinase I-homoserine dehydrogenase I from Escherichia coli has been studied by time-resolved fluorescence spectroscopy . Fluorescence decay can be resolved into 2 distinct classes of tryptophan emitters: a fast component, with a lifetime of about 1.5 ns; and a slow component, with a lifetime of about 4.5 ns . The fluorescence properties of the slow component are modified by the allosteric transition . In the T-form of the enzyme stabilized by threonine, the lifetime of the slow component is longer, with a red-shifted spectrum; its accessibility to quenching by acrylamide becomes slightly higher without any decrease of fluorescence anisotropy . These results indicate a change in polarity of the slow component environment . The quaternary structure change associated with the allosteric transition probably involves global movements of structural domains without leading to any local mobility on the nanosecond time-scale . We suggest that the slow component corresponds to the unique tryptophan of the buried kinase domain. J Photochem Photobiol B, 1988 Dec, 2(4), 491 - 501 Effect of UV irradiation at defined wavelengths on the tertiary structure of double-stranded covalently closed circular DNA; Boullard A et al.; Double-stranded, covalently closed, supercoiled circular DNA from phage fd (replicative form) was irradiated with increasing doses of UV light at 254 nm, 290 nm, 313 nm and 365 nm, and subjected to electrophoresis on agarose slab gels . Increasing the doses of UV light at 254 and 290 nm promotes a smooth reduction in the electrophoretic mobility of the sample, as would be expected if the major effect of light at these two wavelengths were to induce the formation of photoproducts leading to the unwinding of the double strand . At high doses, UV light at 290 nm introduces single-strand breaks (1.2 kJ m-2 per nick per million phosphodiester bonds) . UV light at 313 nm promotes an abrupt change in the electrophoretic mobility, as would be expected if the effect of this wavelength were to induce single-strand breaks, leading to the transformation of the supercoiled molecules in their relaxed form (23 kJ m-2 in order to introduce one nick per million phosphodiester bonds) . UV light at 365 nm also promotes single-strand breaks in DNA (140 kJ m-2 per nick per million phosphodiester bonds). AIDS, 1988 Dec, 2(6), 477 - 80 A new second-generation anti-HIV-1 enzyme immunoassay using recombinant envelope and core proteins; Backer U et al.; In a multicenter collaborative study a new second-generation HIV-1 antibody enzyme immunoassay (Abbott recombinant HIV-1 EIA) using Escherichia coli-expressed recombinant p24 and p41 proteins as solid-phase antigens was compared with the first-generation H9 cell-line-based Abbott HIV-1 EIA . The results of the confirmatory assays (Western blot, immunofluorescence), combined with clinical information, were used as the reference standard for the detection of HIV-1 antibodies in 10,676 random blood donor serum specimens, in a panel of 840 specimens from symptomatic and asymptomatic patients and a total of 63 serial blood specimens from 23 people at risk . With fresh blood donor sera, the specificity of the first-generation assay ranged between 99.54 and 99.76% (95% confidence limits, CL) compared with 99.81-99.95% (95% CL) for the second-generation EIA . With panel specimens the recombinant HIV-1 EIA achieved an overall sensitivity of 100% and a specificity range of 98.3-99.7% (95% CL); the corresponding sensitivity and specificity ranges observed for the first-generation EIA were 98.0-99.5% (95% CL) and 94.3-96.8% (95% CL), respectively . The improved sensitivity for the second-generation assay was confirmed by testing serial samples from seroconverting patients . The use of recombinant proteins eliminated non-specific reactions due to class II human leukocyte antigen (HLA)-directed antibodies. Mol Gen Genet, 1988 Dec, 215(1), 161 - 4 The isolation and preliminary characterisation of a novel Escherichia coli mutant rorB with enhanced sensitivity to ionising radiation; Debenham PG et al.; Escherichia coli K803 cells were mutagenized and screened for the presence of clones sensitive to gamma-rays but not to ultraviolet light . One new mutant of this type, named rorB, was isolated . This mutant is both cross-sensitive to mitomycin C and shows reduced conjugal recombination frequencies, but to a lesser extent than the phenotypically similar mutant recN . Unlike previously reported mutants of E . coli or yeast with an enhanced sensitivity to ionising radiations, rorB appears to be near wild type in ability to rejoin DNA double-strand breaks . The rorB gene maps close to ilvGEDAC at 84.5 min of the E . coli chromosome. Mol Gen Genet, 1988 Dec, 215(1), 156 - 60 The cloning of the rorB gene of Escherichia coli; Debenham PG et al.; A segment of the Escherichia coli genome which complements the ionising radiation sensitivity of the rorB mutation was cloned into pBR322 . This DNA segment also complements the mitomycin C sensitivity of the rorB mutation . The gene was subcloned until defined in a fragment of 1.05 kb . Only one gene product, a protein of approximately 16.5 kDa, was found on maxicell analysis of the various subclones . Iso-electric focusing of this gene product suggests it may function in a complex. Genes Dev, 1988 Dec, 2(12B), 1851 - 8 Escherichia coli heat shock gene mutants are defective in proteolysis; Straus DB et al.; Heat shock proteins in Escherichia coli are relatively abundant and some are essential for growth, but the function that they provide is unknown . The observation that heat shock proteins are induced by some abnormal, rapidly degraded polypeptides, and that strains with mutations in the rpoH gene, the positive regulator of heat shock gene expression, are defective in proteolysis, has led to the proposal that heat shock proteins are required for normal degradation of polypeptides . We have investigated this hypothesis by examining the degradation of polypeptide fragments generated by puromycin and the degradation of a nonsense fragment of beta-galactosidase . Mutations in the dnaK, dnaJ, grpE, and groEL heat shock genes result in defective proteolysis . Furthermore, overproduction of heat shock proteins results in enhanced rates of puromycyl fragment decay . The proteolysis defect of the heat shock gene mutants primarily affects energy-dependent protein degradation . These results indicate that at least one general function of heat shock proteins is to contribute to the ability of the cell to degrade abnormal polypeptides. Vet Microbiol, 1988 Dec, 18(3-4), 313 - 25 Conservation of antigenicity in a 31-kDa Brucella protein; Bricker BJ et al.; A 31-kilodalton (kDa) protein extracted from Brucella abortus was previously cloned into Escherichia coli and expressed at high levels . The E . coli-derived protein can be purified by a simple 2-step procedure entailing detergent extraction followed by ion-exchange chromatography . Subsequent analyses show that the E . coli-derived protein is identical to the Brucella-derived protein in molecular weight and isoelectric point . A partial amino acid sequence of the N-terminus of the protein of E . coli origin matches the predicted sequence, based on DNA sequence data . Using specific antiserum raised against the E . coli-derived protein, 34 strains of Brucella, representing all 6 recognized species, were examined for expression of the 31-kDa protein by Western blotting . This protein was detectable in all, but one Brucella species (B . ovis), including all 8 biovars of B . abortus tested . This degree of conservation supports further study of the 31-kDa protein for potential exploitation as a vaccine or diagnostic component. Genetics, 1988 Dec, 120(4), 863 - 73 Mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position; Sambamurti K et al.; In order to examine the mechanisms of mutagenesis by a bulky DNA lesion at the guanine N7 position, the replicative form DNA of phage M13AB28 (mp8 without the amber codons in phage genes) was modified in vitro with aflatoxin B1-2,3-dichloride and transfected into appropriate Escherichia coli cells . Forward mutations in the lacZ alpha-complementing gene segment were identified as light blue or colorless plaques on appropriate indicator plates, isolated, and defined by DNA sequencing . Transfection of modified DNA into uvrA-/mucAB+ cells without prior UV (SOS) induction increased mutation frequency eight-fold over untreated DNA, whereas this increase was 12-fold upon SOS induction . Transfection of modified DNA after conversion of the primary guanine-aflatoxin lesions to the stable imidazole ring-opened formamidopyrimidine-aflatoxin suggested that these lesions were nearly equally mutagenic . A majority of point mutations under all conditions affected G:C bp . Base substitutions were in the majority, but significant frameshift mutagenesis was also detected in SOS-induced cells . Both G-to-T transversions and G-to-A transitions were produced at equal efficiency and together accounted for virtually all of the base substitutions induced by the primary lesions . Point mutations occurred predominantly at predicted damage hotspots . The characteristics of base substitution and frameshift mutations, together with available information point to multiple mechanisms of mutagenesis by this class of mutagens . The data indicate that primary lesions have the properties of both a noninstructional and pseudo-instructional lesion . In addition, the sequence context appears to play a role in determining whether a frameshift or a base substitution is induced by this bulky lesion. Eur J Immunol, 1988 Dec, 18(12), 1881 - 7 Specificity of proliferative response of human CD8 clones to mycobacterial antigens; Rees A et al.; Human CD8 T lymphocyte clones (TLC) were generated from the pleural effusion of patients with tuberculosis using a protocol that required, in addition to antigen, coculture of purified CD8+ T cells, accessory cells, interleukin 2 (IL2) and anti-CD3-Sepharose . The TLC obtained were stimulated by mycobacterial soluble extracts in an IL2-dependent and MHC class I-restricted manner . When antigen-responsive TLC were screened with extracts from the recombinant mycobacterial library they were found to respond to either the Y3125 (100-kDa) or the Y3111 (71-kDa) lambda gt11 clones . Polyacrylamide gel immunoblot analysis demonstrated that the CD8 TLC responded to fractions with the molecular mass range 27-45 kDa in the Y3125 lysogen and 60-90 kDa in the mycobacterial soluble extract . The specificity of TLC reactive with the Y3111 clone was confirmed using the 71-kDa antigen purified from the same lysogen . These TLC recognized sequences common to the 71-kDa protein derived from mycobacteria, E . coli or a human cell line . Studies of three TLC using antigen-presenting cells of known genetic haplotype indicated that stimulation with both the Y3125 and the 71-kDa antigens were restricted by determinants encoded by HLA-B8. Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8968 - 72 Dual mechanism of repression at a distance in the lac operon; Flashner Y et al.; The mechanism by which the internal lacZ gene sequence O2 influences lac repression was investigated by using in vivo footprinting of operon mutants . Quantitative in vivo binding curves show that O2 strengthens by approximately 3-fold repressor binding to O1 that is located 400 base pairs upstream at the transcription start site . The internal O2 sequence also contributes to repression by a second mechanism: repressor bound internally blocks elongation of beta-galactosidase gene expression . This secondary mechanism of repression is facilitated by the remote O1 operator that strengthens binding to O2 12-fold . Thus, lac repression involves two mechanisms, both of which involve cooperation between remote operator elements . During mild repression only the initiation mechanism applies, but more severe repression favors formation of the presumptive O1-O2 repression loop that allows both mechanisms to act simultaneously. J Bacteriol, 1988 Dec, 170(12), 5949 - 52 Analysis of the diphtheria tox promoter by site-directed mutagenesis; Boyd J et al.; By oligonucleotide-directed mutagenesis, we introduced alterations in the two putative -10 regions of the diphtheria tox promoter which are positioned at -50 and -56 from the GUG tox initiation signal . The -10 region positioned at -50 is favored in the expression of ADP-ribosyltransferase activity from the wild-type tox promoter in recombinant Escherichia coli; however, the promoter down mutation at position -50 is compensated for by increased activity of the -10 region positioned at -56. J Bacteriol, 1988 Dec, 170(12), 5890 - 4 Molecular cloning and high-level expression of a bromoperoxidase gene from Streptomyces aureofaciens Tü24; van Pee KH; A bromoperoxidase gene was cloned from Streptomyces aureofaciens Tu24 into Streptomyces lividans TK64 by using the promoter-probe vector pIJ486 . Subcloning of DNA from the original, unstable clone allowed the gene to be localized to a 1.7-kilobase (kb) fragment of DNA . Southern blotting showed that the cloned 1.7-kb insert hybridized to a 4.3-kb fragment in an SstI digest of S . aureofaciens Tu24 total DNA . The 1.7-kb insert was shown to code for a protein with the electrophoretic properties of the subunits of the nonheme bromoperoxidase isolated from S . aureofaciens Tu24 . The protein produced by S . lividans TK64 transformed with pHM621, which contained an 8.0-kb insert, was shown to be identical to the S . aureofaciens Tu24 bromoperoxidase in terms of its electrophoretic mobility on denaturing and nondenaturing polyacrylamide gels and its NH2-terminal amino acid sequence . The bromoperoxidase was overproduced (up to 180 times) by S . lividans TK64 containing pHM621 . Based on the heat stability of the S . aureofaciens Tu24 bromoperoxidase, a new and simple purification procedure with very high yields was developed. J Bacteriol, 1988 Dec, 170(12), 5460 - 7 Regulation of gene expression in plasmid ColE1: delayed expression of the kil gene; Zhang SP et al.; cea, imm, and kil are a cluster of three functionally related genes of the plasmid ColE1 . The cea and kil genes are in the same inducible operon, with transcription being initiated from a promoter adjacent to the cea gene . The imm gene is located between the cea and kil genes, but it is transcribed in the opposite direction . Complementary interaction between the imm mRNA and the anti-imm sequences in the middle of the cea-kil transcript causes a pronounced delay in expression of the kil gene when the cea-kil operon is induced . A segment in the overlapping region between the cea and imm genes causes delayed expression of the kil gene in the absence of imm gene transcription . This delay effect increases the yields of colicin synthesized in induced cells. FEMS Microbiol Immunol, 1988 Dec, 1(3), 117 - 25 Non-replicating oral whole cell vaccine protective against enterotoxigenic Escherichia coli (ETEC) diarrhea: stimulation of anti-CFA (CFA/I) and anti-enterotoxin (anti-LT) intestinal IgA and protection against challenge with ETEC belonging to heterologous serotypes; Evans DG et al.; An oral killed (non-replicating) whole-cell anti-ETEC vaccine was prepared by treating enterotoxigenic Escherichia coli strain H-10407 (ST + LT +; 078: H11: CFA/I) with a 100%-lethal amount of colicin E2 . Colicin E2 is a potent DNA endonuclease which enters the target bacterial cells without disrupting cellular integrity . Thus the vaccine consists of intact cells lacking chromosomal and plasmid DNA but possessing a normal complement of antigens, including CFA/I and enterotoxin(s), unaltered by chemical- or heat-treatment . Young healthy volunteers were administered two oral doses, one month apart, of approximately 3 x 10(10) vaccine cells . Of 22 vaccinees, 17 (77.3%) showed an intestinal anti-CFA/I IgA response and 19 (86.4%) showed an increase in intestinal anti-LT IgA . Twenty of 22 (90.9%) vaccinees had antibody responses to either CFA/I, LT, or both antigens, demonstrating that colicin E2-treated CFA-positive E . coli cells are an efficient vehicle in terms of delivery of antigens to the gut immune system . We previously demonstrated protection of vaccinees against challenge with the living homologous ETEC (strain H-10407) . In this study, two groups of 8 vaccinees were challenged with a diarrheagenic dose of virulent ST + LT + ETEC of heterologous serotype; one group was challenged with a CFA/I-positive 063: H- strain and the other group was challenged with a CFA/II-positive 06: H16 strain . Approximately 75% efficacy was achieved in both challenge groups . None of the 16 vaccinees who had responded to both CFA/I and LT became ill upon challenge while both of the vaccinees who had not responded to either antigen did.(ABSTRACT TRUNCATED AT 250 WORDS) J Gen Microbiol, 1988 Dec, 134 ( Pt 12), 3129 - 39 Mutants of Escherichia coli specifically deficient in respiratory formate dehydrogenase activity; Mandrand-Berthelot MA et al.; Escherichia coli K12 mutants lacking phenazine-methosulphate-linked formate dehydrogenase (FDH-PMS) activity, but still capable of producing normal levels of benzyl-viologen-linked formate dehydrogenase (FDH-BV) and nitrate reductase activities, have been isolated following P1 localized mutagenesis . The relevant mutations mapped with the same cotransduction frequency close to the rhaD gene, at 88 min on the E . coli chromosome . They were further subdivided into two classes . Class I consisted of six fdhD mutants which synthesized an inactive FDH-PMS protein with the same subunit composition as the wild-type enzyme . In contrast, class II contained four fdhE mutants totally devoid of this antigen . Construction of merodiploid strains harbouring various combinations of the mutated alleles, fdhE on the episome and fdhD on the chromosome, led to the restoration of FDH-PMS activity by complementation of the products encoded by the respective wild-type alleles . Difference spectroscopy suggested that both fdhD and fdhE mutants contained normal amounts of the cytochrome b559 associated with FDH-PMS although the cytochrome had lost its capacity for formate-dependent reduction. Tsitologiia, 1988 Dec, 30(12), 1463 - 6 {Heterogeneous packing of the Escherichia coli chromosome and its decompaction in in vitro experiments}; Likhoshvai EV et al.; The chromatin organization of E . coli cells, taken on various growth stages of the culture (active, stationary, grown with heightened density), displays different characters when examined by the Miller method . In the active phase of growth, the cell chromatin is released as threads and loops of DNA, threads of nucleosome-like particles and granules 25-38 nm in size . The chromatin from cells in the stationary phase of growth, grown in heightened density conditions, contains not only granules of average size 30 and 100 nm, but also larger conglomerates consisting of several 100 nm particles . The chromatin decompaction of cells grown under heightened density, in conditions of weak alkali medium and low salt buffer, was in general of two types: creation of diffusion cloud with no clear-cut outlines, and spherical structure of 1.5-2 microns in diameter with electron dense centre . In one chromosome both the types of chromatin decompaction can be found at the same time with regions of compact chromatin, which undoubtedly shows different functional activity of some regions of the chromosome. Mol Cell Probes, 1988 Dec, 2(4), 255 - 60 Modification of the DNA colony hybridization technique for multiple filter analysis; Kaysner CA et al.; Inexpensive fiberglass mesh window screens were used as spacers between colony blot filters to increase the number of bacterial isolates that could be tested by DNA colony hybridization . Sixty filters with up to 48 isolates per filter were tested at one time . This modified technique reduced the amount of radiolabelled probe and other materials needed for simultaneous analysis of up to 2880 test isolates. Mol Cell Biol, 1988 Dec, 8(12), 5425 - 31 Determination of the spectrum of mutations induced by defined-wavelength solar UVB (313-nm) radiation in mammalian cells by use of a shuttle vector; Keyse SM et al.; Mutations induced by UVB (313-nm) radiation, a wavelength in the region of peak effectiveness for sunlight-induced skin cancer in humans, have been analyzed at the sequence level in simian cells by using a plasmid shuttle vector (pZ189) . We find that significant differences exist between the types of mutations induced by this solar wavelength and those induced by nonsolar UVC (254-nm) radiation . Compared with 254-nm radiation, 313-nm radiation induces more deletions and insertions in the region sequenced . In addition, although the types of base substitutions induced by the two wavelengths are broadly similar (in both cases, the majority of changes occur at G-C base pairs and the G-C to A-T transition is predominant), an analysis of the distribution of these base changes within the supF gene following irradiation at 313 nm reveals additional hot spots for mutation not seen after irradiation at 254 nm . These hot spots are shown to arise predominantly at sites of mutations involving multiple base changes, a class of mutations which arises more frequently at the longer solar wavelength . Lastly, we observed that most of the sites at which mutational hot spots arise after both UVC and UVB irradiation of the shuttle vector are also sites at which mutations arise spontaneously . Thus, a common mechanism may be involved in determining the site specificity of mutations, in which the DNA structure may be a more important determinant than the positions of DNA photoproducts. Microb Pathog, 1988 Dec, 5(6), 419 - 26 Cloning and nucleotide sequence analysis of the genes determining verocytotoxin production in a porcine edema disease isolate of Escherichia coli; Gyles CL et al.; The structural genes determining the edema disease principle were cloned from the total cellular DNA of Escherichia coli strain 412 (O139:K82) isolated from a case of porcine edema disease . An assay for cytotoxicity in Vero cells was used to detect the edema disease principle . A 7.5 kb EcoRI-SalI fragment specifying cytotoxin production was subcloned in pUC18 . Sequences which specified production of cytotoxin were localized to a 0.9 kb region by transposon Tn5 mutagenesis . A 2.4 kb EcoRI-BglII fragment encompassing this region was subcloned into pUC18 . Using nucleotide sequence analysis, two open reading frames separated by 12 bp were identified . They encoded proteins of 319 (A subunit) and 87 (B subunit) amino acids which both had N-terminal sequences typical of E . coli signal peptides . Comparison of these with the published sequence for the Shiga-like toxin II (SLT-II) showed 91% overall nucleotide sequence similarity . The nucleotide sequence similarity extended to 200 base pairs upstream of the putative A subunit translational start site suggesting a common regulatory mechanism . The deduced amino acid sequences of the processed A and B subunits had 94% and 84% similarity, respectively . These findings confirm the close genetic relationship between SLT-II and edema disease principle. Mol Gen Genet, 1988 Dec, 215(1), 38 - 45 Targeting a foreign protein to chloroplasts using fusions to the transit peptide of a chlorophyll a/b protein; Kavanagh TA et al.; We have constructed chimaeric genes consisting of sequences encoding the transit peptide and 4, 16, 24, 53 or 126 amino-terminal residues of the mature chlorophyll a/b binding (Cab) apoprotein fused to the Escherichia coli gene encoding beta-glucuronidase (GUS) . These genes were introduced into tobacco plants and the fate of the fusion proteins they encode was analysed . Less than 1% of the total activity of fusion proteins containing the transit peptide and 4 (FP4) or 16 (FP16) amino-terminal amino acids of the mature Cab protein was associated with chloroplasts . Moreover, FP4 appears to be unprocessed . This is in striking contrast to fusion proteins containing the transit peptide and 24 (FP24), 53 (FP53) or 126 (FP126) amino-terminal residues of the mature Cab polypeptide . Approximately 98%, 96% or 75%, respectively, of the total activity of these fusion proteins was associated with purified intact chloroplasts, and protease protection experiments showed that of this, approximately 98%, 87% or 50%, respectively, was located within this organelle . Furthermore, both FP24 and FP53 appear to be processed . However, less than 10% of the activity of those fusion proteins translocated into chloroplasts was associated with thylakoid membranes. Mol Gen Genet, 1988 Dec, 215(1), 146 - 51 Killing of Escherichia coli cells modulated by components of the stability system ParD of plasmid R1; Bravo A et al.; The proteins P10 and P12 have been shown to be gene products of a new stability system, ParD, of plasmid R1 . It is now shown that an R1 miniplasmid, pAB112, carrying a trans-complementable amber mutation in the gene of the P10 protein, is lethal for the host in the absence of suppression . This lethal effect is suppressed in a supF background and also by deletions in pAB112 that affect the gene of the P12 protein . These data indicate that the P12 protein has a lethal effect on the host and that this effect is neutralized by the P10 protein . The possibility that the stabilization conferred by the ParD system could be due to a counterselection, mediated by P12, of cells that lose the plasmid at cell division, is discussed. Am J Vet Res, 1988 Dec, 49(12), 2030 - 3 Effects of weaning on diarrhea caused by enterotoxigenic Escherichia coli in three-week-old pigs; Sarmiento JI et al.; We attempted to determine whether weaning is required for induction of diarrhea in pigs with postweaning enterotoxigenic Escherichia coli infection . Three-week-old newly weaned pigs and their suckling littermates were inoculated with the K88+ enterotoxigenic E coli strain M1823B . Fourteen of 21 weaned and 12 of 20 suckling pigs were genetically resistant to intestinal adhesion by the K88+ strain of E coli; they remained healthy, and gained weight at similar rates . Both groups of K88-resistant pigs gained weight faster, and shed fewer bacteria of strain M1823B in their feces, than did their K88-susceptible counterparts . Diarrhea developed in K88-susceptible pigs in the weaned (6 of 7 pigs) and suckling (4 of 8 pigs) groups, and 1 of the 4 affected suckling pigs died from complications resulting from diarrhea . The incidences of diarrhea, weight gain rates, and the numbers of strain M1823B shed in feces of susceptible weaned and suckling pigs were not significantly (P greater than 0.05) different . Diarrhea scores of susceptible weaned pigs were significantly (P less than 0.02) higher than those of susceptible suckling pigs on the second day after inoculation . In this experimental model, it was concluded that weaning is not required for induction of diarrhea, but may modestly increase its severity. Anal Biochem, 1988 Dec, 175(2), 373 - 85 An efficiently mutagenizable recombinant plasmid for in vitro transcription of the Escherichia coli 16 S RNA gene; Krzyzosiak WJ et al.; The portion of the rrnB operon coding for 16 S RNA was modified to permit efficient in vitro transcription by T7 RNA polymerase of full-length, correctly terminated, biologically active 16 S RNA (W . Krzyzosiak et al., 1987, Biochemistry 26, 2353-2364) . The 5'-end of the gene was fused to the class III T7 promoter and the 3'-end was modified so that cleavage with MstII would generate correctly terminated RNA upon runoff transcription . The modified gene was placed in pUC19 by a four-way ligation reaction involving linearized pUC19, a 1490-bp fragment of 16 S rDNA, and two synthetic oligodeoxynucleotides . Because of the cohesive end design, phosphorylation of the synthetic oligomers was not necessary . Single and tandem cassette insertions were used to generate single base changes in the C-1400 region of 16 S RNA . Three examples are described . This method is generally applicable to the 16 S RNA molecule as suitable singlecleavage restriction sites allow all regions to be mutated by this approach. Mol Immunol, 1988 Dec, 25(12), 1291 - 8 Purification and characterization of radiolabelled biosynthetic human insulin from Escherichia coli . Kinetics of processing by antigen presenting cells; Semple JW et al.; An Escherichia coli strain transfected with a plasmid containing four linked human proinsulin genes was grown in the presence of 35S and 3H labelled amino acids to gain access to human insulin that was radiolabelled at 19 evenly distributed sites throughout the amino acid sequence . The multi-proinsulin precursor was cleaved at methionine residues with cyanogen bromide, then the individual proinsulin units were folded via their S-cysteine sulfonate derivative and converted to insulin by enzymatic digestion . Purification steps were carried out by ion-exchange and reverse-phase HPLC techniques . The final radiolabelled biosynthetic human insulin was produced at a specific activity of up to 300 Ci/mmol, and was shown to be indistinguishable from commercially available human insulin according to HPLC behavior, amino acid analysis, immunoreactivity and biological activity . A comparison of the kinetics of processing of 35S/3H-labelled biosynthetic human insulin and 125I-labelled commercial human insulin by murine TA3 hybridoma antigen presenting cells demonstrated that radiolabelled biosynthetic insulin was processed approximately 16 times slower than its iodinated counterpart . Measurable 125I TCA soluble radioactivity was detected extracellularly within 15 min whereas the same amount of extracellular TCA soluble 3H/35S radioactivity was not seen until 240 min . These results begin to address the importance of using a biosynthetically labelled protein as opposed to an iodinated protein to study how an APC handles antigen in a physiological manner. DNA, 1988 Dec, 7(10), 729 - 34 Gene manipulation by homologous recombination in Escherichia coli; Crouse GF et al.; The use of homologous recombination in Escherichia coli is described as a tool for DNA manipulation . The utility of the method is illustrated by the addition of 3'-flanking sequences to a dhfr minigene by plasmid-phage recombination involving a supF-containing dhfr minigene plasmid and a lambda Charon4A phage containing the 3' end of the dhfr gene . In addition, other uses of both plasmid-phage and phage-phage recombination in gene manipulation are described. Scand J Immunol, 1988 Dec, 28(6), 783 - 9 Avidity of IgA antibody to Escherichia coli polysaccharide and diphtheria toxin in breast milk from Swedish and Pakistani mothers; Roberton DM et al.; The avidity of breast milk IgA antibody was studied with the aid of thiocyanate elution of antibody from solid-phase bound E.coli polysaccharides and diphtheria toxoid . The relative avidity index for each sample was determined by the molarity of thiocyanate required to elute 50% of the bound IgA antibody under conditions of antigen excess . Milk samples collected from Pakistani mothers during early lactation (2-4 weeks after delivery; n = 12) had a significantly lower median relative avidity index of IgA antibody to E.coli antigens than did early lactation samples from Swedish mothers (n = 11; avidity indices 1.78 M and 2.65 M; P less than 0.02) . Samples collected from Pakistani mothers in mid-lactation showed a significant rise in the relative avidity index to a median of 2.50 M (P less than 0.01), with a subsequent fall in late lactation (28-36 weeks after delivery) to 1.75 M (P less than 0.01) . Milk samples from Pakistani mothers in mid-lactation (n = 12) also had a lower median relative avidity index of IgA antibody to diphtheria toxoid than did samples from Swedish mothers (n = 14; avidity indices 2.35 M and 4.30 M; P less than 0.002) . The lower avidity of breast milk IgA in Pakistani mothers in comparison with Swedish mothers may arise from differences in antigen exposure or nutritional status or could possibly be genetically determined. Pathol Res Pract, 1988 Dec, 184(1), 60 - 8 Induction of lymph follicle formation with endotoxin lipopolysaccharide in the draining lymph node of athymic nude mice; Hoshi H et al.; Formation of lymph follicles in draining popliteal lymph nodes in athymic nude mice and their phenotypically normal littermates (PN mice) was investigated after footpad injection with either endotoxin lipopolysaccharide (LPS) or phytohemagglutinin in soluble and precipitated forms (PHA) . The dose of LPS injected ranged from 10 to 100 micrograms, and that of PHA was 50 micrograms . After any dose of LPS, draining nodes of nude mice produced new lymph follicles in the peripheral cortex outside pre-existing follicles, though the number of follicles induced was somewhat less than in the case of PN mice treated with LPS . After injection of PHA in soluble or precipitated form, PN mice developed a significant number of new follicles in the draining nodes, but nude mice failed to do so .