J Mol Evol, 1989 Aug, 29(2), 157 - 69 Evolutionary conservation of protein regions in the protonmotive cytochrome b and their possible roles in redox catalysis; Howell N; The amino acid sequences of the protonmotive cytochrome b from seven representative and phylogenetically diverse species have been compared to identify protein regions or segments that are conserved during evolution . The sequences analyzed included both prokaryotic and eukaryotic examples as well as mitochondrial cytochrome b and chloroplast b6 proteins . The principal conclusion from these analyses is that there are five protein regions--each comprising about 20 amino acid residues--that are consistently conserved during evolution . These domains are evident despite the low density of invariant residues . The two most highly conserved regions, spanning approximately consensus residues 130-150 and 270-290, are located in extramembrane loops and are hypothesized to constitute part of the Qo reaction center . The intramembrane, hydrophobic protein regions containing the heme-ligating histidines are also conserved during evolution . It was found, however, that the conservation of the protein segments extramembrane to the histidine residues ligating the low potential b566 heme group showed a higher degree of sequence conservation . The location of these conserved regions suggests that these extramembrane segments are also involved in forming the Qo reaction center . A protein segment putatively constituting a portion of the Qi reaction center, located approximately in the region spanned by consensus residues 20-40, is conserved in species as divergent as mouse and Rhodobacter . This region of the protein shows substantially less sequence conservation in the chloroplast cytochrome b6 . The catalytic role of these conserved regions is strongly supported by locations of residues that are altered in mutants resistant to inhibitors of cytochrome b electron transport. Gene, 1989 Aug 1, 80(1), 29 - 38 Nucleotide sequence of a Bacillus subtilis arginine regulatory gene and homology of its product to the Escherichia coli arginine repressor; North AK et al.; In Bacillus subtilis, arginine represses its biosynthetic enzymes and activates its catabolic ones via a regulator gene ahrC . A 6.2-kb EcoRI fragment of B . subtilis chromosomal DNA that includes the ahrC gene has previously been cloned . Gene ahrC was localised in a 0.8-kb HindIII sub-fragment whose nucleotide sequence was determined . An open reading frame (ORF) was present whose translated amino acid sequence showed homology to that of the Escherichia coli arginine repressor encoded by that organism's argR gene . That this ORF corresponded to ahrC was confirmed by (i) the location of the transposon in an ahrC::Tn917 insertion mutant within the ORF; and (ii) by the appearance of an AhrC- phenotype when plasmids carrying restriction fragments lying wholly within this ORF were permitted to integrate by Campbell-type recombination into the B . subtilis chromosome . This represents the first description of a repressor in a 'housekeeping' biosynthetic system in a Bacillus, and indeed of homology between regulatory proteins for any 'housekeeping' system across such a wide taxonomic barrier among prokaryotes. Proc Natl Acad Sci U S A, 1989 Aug, 86(15), 5748 - 52 Homology of the human intestinal Na+/glucose and Escherichia coli Na+/proline cotransporters; Hediger MA et al.; Cotransport proteins are responsible for the active accumulation of organic substrates in cells . Na+ gradients provide the driving force for uptake of most substrates into eukaryotes and for a few substrates in some prokaryotes . We report here the cloning and sequencing of the human intestinal Na+/glucose cotransporter (SGLT1) and compare its structure with other cloned transporters . At the DNA level and the predicted amino acid and secondary structure levels, close homology is evident between the human and rabbit intestinal Na+/glucose cotransporters, and a significant homology is found between these and the Escherichia coli Na+/proline cotransporter (putP) . No homology is detectible with other known proteins . We infer from these results that the mammalian Na+/glucose and prokaryote Na+/proline cotransporters share a common ancestral gene. Surgery, 1989 Aug, 106(2), 283 - 90; discussion 290-1 Change in hepatic gene expression after shock/resuscitation; Buchman TG et al.; In response to specific stresses, such as a heat pulse or the sequence of hypoxia-reoxygenation, each isolated cell (prokaryotic and eukaryotic) that has been studied characteristically alters gene expression to synthesize a set of proteins (heat-shock proteins) that are important for intracellular homeostasis . To determine whether a corresponding response occurs within parenchymal cells in vivo when subjected to the complex stress of circulatory shock, liver biopsy specimens were obtained from a swine model of cardiogenic shock before and after shock/resuscitation . With use of complementary DNA prepared from post-shock/resuscitation messenger RNA, a library was constructed and screened for differential gene expression . Of 32/4000 clones initially screened as positive for induction after shock/resuscitation, six were confirmed positive by Northern blot analysis . The nucleotide sequences of two of these six have been determined, and one has been unambiguously identified as metallothionein. J Mol Biol, 1989 Jul 20, 208(2), 225 - 32 Characterization of a new prokaryotic transcriptional activator and its DNA recognition site; Barthelemy I et al.; The expression of the Bacillus subtilis phage phi 29 DNA is controlled by the viral gene 4 product, which is required for the initiation of transcription at the unique late promoter A3 . Protein p4 binds specifically to a phi 29 DNA fragment containing the A3 promoter . DNase I footprinting analysis has shown that the DNA binding region for protein p4 is located between nucleotides -50 and -100 relative to the transcription start site . Methylation interference assays suggest that two eight base-pair long inverted repeats located within this binding region are the protein p4 recognition sequence . These results, together with the fact that the protein p4-dependent in vitro transcription requires the B . subtilis sigma 43-RNA polymerase, indicate that protein p4 is a transcriptional activator . The protein p4 DNA recognition region is statically bent as suggested by gel retardation and chemical cleavage assays . A model of protein p4 binding to its DNA target site is proposed. Gene, 1989 Jul 15, 79(2), 289 - 98 The Drosophila melanogaster RPS17 gene encoding ribosomal protein S17; Maki C et al.; A human ribosomal protein S17 cDNA {Chen et al., Proc . Natl . Acad . Sci . USA 83 (1986) 6907-6911} was used as heterologous probe to isolate S17 clones from Drosophila genomic and cDNA recombinant libraries . Five S17 genomic clones were recognized; all contained overlapping regions of a single chromosomal site . Subsequently the Drosophila RPS17 gene was mapped by in situ hybridization to chromosome 3L, band 67B1-5 . The locus spans approximately 1000 bp of DNA and includes four exons . It is preceded by conventional CAAT and TATA RNA polymerase II promoter motifs . The 131 amino acid protein encoded within Drosophila RPS17 is similar to ribosomal proteins from several other eukaryotes . Comparison of eukaryotic S17 proteins' primary structures as well as the number and location of their genes' intervening sequences suggest that S17 is a relatively recent addition to the ribosomal protein family, probably post-dating divergence of eukaryotes and prokaryotes. FEBS Lett, 1989 Jul 3, 250(2), 317 - 22 Localisation of the binding site for the initiating substrate on the RNA polymerase from Sulfolobus acidocaldarius; Grachev MA et al.; RNA polymerase from the archaebacterium Sulfolobus acidocaldarius was chemically modified with AMP o-formylphenyl ester followed by reduction with borohydride . The modified protein catalyzes the labeling of its own largest subunit when incubated with {alpha-33P}UTP in the presence of poly{d(A-T)} . On cleaving of the labeled protein using cyanogen bromide, hydroxylamine or amino acid-specific endoproteinases for a very brief period, the pattern and size of the radioactive fragments formed are best explained by attachment of the label between Gly843 and Met895 of the largest subunit . In this region there exists a highly conserved sequence which is also found in other archaebacterial, eukaryotic and prokaryotic RNA polymerase . This suggests that the binding site for the initiating substrate of RNA polymerases has been conserved during evolution. Biol Chem Hoppe Seyler, 1989 Jul, 370(7), 763 - 8 Purification and N-terminal amino-acid sequences of bacterial malate dehydrogenases from six actinomycetales strains and from Phenylobacterium immobile, strain E; Rommel TO et al.; Malate dehydrogenases from Streptosporangium roseum (DSM 43021), Planomonospora venezuelensis (DSM 43178), Microtetraspora glauca (ATCC 23057), Actinoplanes missouriensis (DSM 43046), Streptomyces atratus (ATCC 14046), Kibdelosporangium aridum (ATCC 39323), and from Phenylobacterium immobile, strain E (DSM 1986) were purified to homogeneity . The N-terminal amino-acid sequences were determined and compared with known prokaryotic and eukaryotic sequence data . The partial sequences from Actinomycetales enzymes include a string of amino acids which is also present in the N-terminal region of malate dehydrogenases from Thermus flavus and from mammalian cytoplasm. J Cell Biochem, 1989 Jul, 40(3), 309 - 20 Phospholipase A2 engineering: design, synthesis, and expression of a gene for bovine (pro)phospholipase A2; Noel JP et al.; A gene coding for the (pro)phospholipase A2 (PLA2) from bovine pancreas has been designed, synthesized, and expressed in Escherichia coli . The gene was designed with a variety of restriction sites that will facilitate future mutagenesis studies . Codons occurring frequently in prokaryotic systems were chosen whenever possible . The total gene spans 404 base pairs and was divided into 33 oligonucleotides . The gene was constructed in two halves of 224 and 180 base pairs from the oligonucleotides by the shotgun ligation technique using pBSM13- as the cloning vehicle . The two fragments were then ligated and cloned into pBSM13- to complete the gene . The (pro)PLA2 gene was then verified by restriction site mapping and dideoxy sequencing . The gene was expressed to high levels from a high copy number vector, designated as pJPN, derived from the E . coli secretion vector pIN-III-ompA3 . Although the protein failed to be excreted and was in the form of insoluble inclusion body, active PLA2 could be obtained by renaturation of the inclusion body pellet followed by tryptic activation, which removes the signal sequence and the pro-peptide of proPLA2 . The PLA2 thus obtained reacted with the antisera raised against the natural PLA2 purified from bovine pancreas, and the specific activity of the expressed PLA2 was identical to that of the natural PLA2 . The shotgun ligation and synthetic gene approaches are simple and inexpensive and can be adapted to express most of the enzymes in the phospholipase A2 family. Rev Infect Dis, 1989 Jul-Aug, 11 Suppl 5, S898 - 901 The new quinolones: back to the future; Davies J; Few agents developed for the treatment of infectious diseases have been heralded to the same extent as the new quinolone derivatives . While these compounds are extremely promising, we still have much to learn about their biologic properties . Detailed analyses of, for example, the mode of action at the target level could have implications for the study and design of both prokaryotic and eukaryotic topoisomerase inhibitors . It is significant that, in spite of the fact that these potent new antimicrobial agents have been in use for only a few years, resistant strains have appeared and have begun to affect the therapeutic potential of the quinolones . Prudent use of these agents will be an important measure of guaranteeing their maximal benefit in future clinical practice. Mutat Res, 1989 Jul, 226(3), 211 - 4 Reduction of the translation fidelity by kanamycin: effects on growth and mutant frequency in S . typhimurium TA102; Gocke E; Kanamycin reduces the translation fidelity in prokaryotes . By read-through of the ochre stop codon (hisG428) of S . typhimurium TA102 enough functional enzyme is produced to allow the his- cells to form a dense background growth on the minimal agar plates . The influence on the revertant colony numbers is similar to the effect of histidine supplementation. Mol Microbiol, 1989 Jul, 3(7), 957 - 67 A Mycoplasma genetic element resembling prokaryotic insertion sequences; Ferrell RV et al.; Nucleotide sequence analysis of two Mycoplasma hyopneumoniae-derived copies of a repetitive genetic element revealed structural similarities to typical prokaryotic insertion sequences . This is the first such sequence identified in the class Mollicutes . The element spans approximately 1550bp, with 28bp inverted terminal repeats . Two open reading frames occur within the sequence, one potentially encoding a protein with a size-variant alpha-helical domain containing heptameric leucine periodicity . Hybridization data with several strains from each of two mycoplasma species showed that the repetitive sequence is variably distributed within the M . hyopneumoniae and Mycoplasma hyorhinis chromosomes and indicated that in some cases the repeated sequence is contained within a larger genetic element which may be the result of phage or plasmid insertion. Int J Pept Protein Res, 1989 Jul, 34(1), 2 - 5 Site-directed mutagenesis at amino terminus of recombinant ricin A chain; Bradley JL et al.; Successful immunotoxin therapy may depend upon reduction of the size of the components in order to decrease antigenicity and rate of clearance . In initial attempts to modify the A chain of ricin by deletion analyses within a prokaryotic expression system, coding sequences were modified by the insertion of unique restriction endonuclease sites and by the removal of 22 codons near the 5' terminus . The work presented here examines the expression, solubility, and activity of these mutant proteins and demonstrates that while amino acid residues may be altered in this region, the deletion of residues 19 through 40 yields an insoluble and inactive toxin molecule. Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5497 - 501 Dysfunction of chromosomal loop attachment sites: illegitimate recombination linked to matrix association regions and topoisomerase II; Sperry AO et al.; A family of A + T-rich sequences termed MARs ("matrix association regions") mediate chromosomal loop attachment . Here we demonstrate that several MARs both specifically bind and contain multiple sites of cleavage by topoisomerase II, a major protein of the mitotic chromosomal scaffold . Interestingly, "hotspots" of enzyme cutting occur within the MAR of the mouse immunoglobulin kappa-chain gene at the breakpoint of a previously described chromosomal translocation . Since topoisomerase II can mediate illegitimate recombination in prokaryotes, we explored further the possibility that MARs might be targets for this process in eukaryotes . We found that a MAR had been deleted from one of the two rabbit immunoglobulin kappa-chain genes and that MARs reside next to a long interspersed repetitive element within the recombination junction of a human ring chromosome 21 . These results, taken together with other accounts of nonhomologous recombination, lead to the proposal that a dysfunction of MARs is illegitimate recombination. Bioorg Khim, 1989 Jul, 15(7), 997 - 1000 {Structure of the recombination zone during abnormal excision of the transducing bacteriophage lambda plac9}; Shpakovskii GV et al.; Investigating molecular mechanism of illegitimate recombinations in prokaryote we study transducing bacteriophages of the lambda lac series . We have carried out physical mapping of bacteriophage lambda plac9 DNA and, by comparing the obtained results with the data on the structure of lambda DNA and lac operon of E . coli, located the phage-bacterial junction corresponding to the lambda-lac9 abnormal excision and elucidated the nucleotide sequence around the junction . It led to the primary structure of phage and bacterial segments in the lysogenic bacterium which took part in the recombinational act leading to the abnormal excision and lambda lac9 formation . Structural homology of the partners in the lambda plac9 excision proved to be lower than in case of the earlier studied lambda plac5 and lambda plac10 whose excision proceeded regioselectively . Various aspects of the crossover area, including the crossover point's probable position and enzymic systems participating in the abnormal excision, are discussed. Sex Transm Dis, 1989 Jul-Sep, 16(3), 141 - 7 The heat shock response of type 1 and type 4 gonococci; Klimpel KW et al.; A heat shock response, characterized by the elevated expression of certain proteins in response to a shift to a higher temperature, has been observed in both eukaryotic and prokaryotic organisms . We have characterized the heat shock response of the pathogenic organism N . gonorrhoeae, colony types 1 and 4 . Following a shift up in temperature from 37 C to 43 C, two-dimensional gel electrophoretic analysis was used to identify 19 heat shock proteins in Type 1 and 37 heat shock proteins in Type 4 gonococci . One heat shock protein was found only in Type 1, 19 were common to both Type 1 and Type 4, and 19 were found only in Type 4 gonococci . Most heat shock proteins were found in the cytoplasm, some in the cytoplasmic membrane, and none could be detected in the outer membrane . Pulse-chase experiments revealed that heat shock proteins were very stable. Biochem J, 1989 Jul 1, 261(1), 113 - 8 Demonstration and partial characterization of ADP-ribosylation in Pseudomonas maltophilia; Edmonds C et al.; ADP-ribosylation of proteins occurs in many eukaryotes, and it is also the mechanism of action of a growing number of important bacterial toxins . To date, however, there is only one well-characterized ADP-ribosylation system where the ADP-ribosyltransferase and the substrate protein are both bacterial in origin, namely within the nitrogen-fixing bacterium Rhodospirillum rubrum . The present paper demonstrates the endogenous ADP-ribosylation of two proteins of Mr 32,000 and 20,000 within Pseudomonas maltophilia, a Gram-negative aerobe . The proteins have been partially purified: two apparently separate species of modified protein can be separated by ion-exchange chromatography and gel filtration (V0 and Mr 158,000 - Vi) . The substrate protein(s) either has, or is co-eluted with, NAD+ glycohydrolase activity . The modification is mono-ADP-ribosyl in nature . The linkage between the acceptor amino acid and the ADP-ribose moiety is alkali-labile and stable to hydroxylamine, possibly indicating an S-glycosidic bond . The activity appears to be a true ADP-ribosylation reaction and not an NAD+ glycohydrolase activity followed by non-enzymic addition of ADP-ribose to protein . The results presented here indicate that ADP-ribosylation may have a wider significance within prokaryotic systems than previously thought. J Mol Evol, 1989 Jul, 29(1), 95 - 7 The border residues of the dihydrofolate reductase domain in Escherichia coli beta-galactosidase correspond to the positions of introns 1 and 5 of dihydrofolate reductase of chicken; Kuchinke W; In beta-galactosidase of Escherichia coli residues 820-934 are similar to residues in dihydrofolate reductase of E . coli . Dihydrofolate reductase of E . coli and chicken are also similar and have identical tertiary structures . I used the similarity of the three-dimensional structure of prokaryotic and eukaryotic dihydrofolate reductases to align the chicken dihydrofolate reductase and the similar residues of beta-galactosidase . The positions of introns 1 and 5 of the chicken dihydrofolate reductase gene correspond exactly to the start and the end of the dihydrofolate reductase-like domain in the beta-galactosidase polypeptide chain . This equivalence of intron positions in a eukaryotic gene and domain structure in a prokaryotic protein was interpreted as evidence for a common origin of both genes. J Mol Evol, 1989 Jul, 29(1), 68 - 88 Electrophoretic and immunological comparisons of chloroplast and prokaryotic ribosomal proteins reveal that certain families of large subunit proteins are evolutionarily conserved; Randolph-Anderson BL et al.; Antibodies to individual chloroplast ribosomal (r-)proteins of Chlamydomonas reinhardtii synthesized in either the chloroplast or the cytoplasm were used to examine the relatedness of Chlamydomonas r-proteins to r-proteins from the spinach (Spinacia oleracea) chloroplast, Escherichia coli, and the cyanobacterium Anabaena 7120 . In addition, 35S-labeled chloroplast r-proteins from large and small subunits of C . reinhardtii were co-electrophoresed on 2-D gels with unlabeled r-proteins from similar subunits of spinach chloroplasts, E . coli, and Anabaena to compare their size and net charge . Comigrating protein pairs were not always immunologically related, whereas immunologically related r-protein pairs often did not comigrate but differed only slightly in charge and molecular weight . In contrast, when 35S-labeled chloroplast r-proteins from large and small subunits of a closely related species C . smithii were coelectrophoresed with unlabeled C . reinhardtii chloroplast r-proteins, only one pair of proteins from each subunit showed a net displacement in mobility . Analysis of immunoblots of one-dimensional SDS and two-dimensional urea/SDS gels of large and small subunit r-proteins from these species revealed more antigenic conservation among the four species of large subunit r-proteins than small subunit r-proteins . Anabaena r-proteins showed the greatest immunological similarity to C . reinhardtii chloroplast r-proteins . In general, antisera made against chloroplast-synthesized r-proteins in C . reinhardtii showed much higher levels of cross-reactivity with r-proteins from Anabaena, spinach, and E . coli than did antisera to cytoplasmically synthesized r-proteins . All spinach r-proteins that cross-reacted with antisera to chloroplast-synthesized r-proteins of C . reinhardtii are known to be made in the chloroplast (Dorne et al . 1984b) . Four E . coli r-proteins encoded by the S10 operon (L2, S3, L16, and L23) were found to be conserved immunologically among the four species . Two of the large subunit r-proteins, L2 and L16, are essential for peptidyltransferase activity . The third (L23) and two other E . coli large subunit r-proteins (L5 and L27) that have immunological equivalents among the four species are functionally related to but not essential for peptidyltransferase activity. Biochim Biophys Acta, 1989 Jun 28, 1003(3), 321 - 6 3-Hydroxy-3-methylglutaryl coenzyme A lyase from Pseudomonas mevalonii; Scher DS et al.; HMG-CoA lyase, the putative second intracellular enzyme of mevalonate catabolism in Pseudomonas mevalonii (which we previously referred to as Pseudomonas sp . M (Gill et al . (1984) J . Bacteriol . 160, 294-298, Gill et al . (1985) J . Biol . Chem . 250, 9393-9398 and Sherban, M.S., Thesis, Purdue University), was purified 650-fold from cell extracts to a specific activity of 22 mumol acetyl-CoA formed per min per mg protein . This represents the first published report of the partial purification and characterization of an HMG-CoA lyase from a prokaryotic source . Cleavage of HMG-CoA produced acetyl-CoA and acetoacetate . Activity was optimal at pH 8.8 and was undetectable at or below pH 6.5 . The estimated Km for S-HMG-CoA was 100 microM . Both a reduced thiol and Mg2+ or Mn2+ were required for activity . While Mn2+ was preferred at low concentrations, 1 mM or higher concentrations of either cation supported the same maximum velocity . An apparent native Mr of 40,400 +/- 3100 was estimated from gel filtration and sucrose density gradient ultracentrifugation data. Nucleic Acids Res, 1989 Jun 26, 17(12), 4713 - 30 Two related superfamilies of putative helicases involved in replication, recombination, repair and expression of DNA and RNA genomes; Gorbalenya AE et al.; In the course of systematic analysis of protein sequences containing the purine NTP-binding motif, a new superfamily was delineated which included 25 established or putative helicases of Escherichia coli, yeast, insects, mammals, pox- and herpesviruses, a yeast mitochondrial plasmid and three groups of positive strand RNA viruses . These proteins contained 7 distinct highly conserved segments two of which corresponded to the "A" and "B" sites of the NTP-binding motif . Typical of the new superfamily was an abridged consensus for the "A" site, GxGKS/T, instead of the classical G/AxxxxGKS/T . Secondary structure predictions indicated that each of the conserved segments might constitute a separate structural unit centering at a beta-turn . All previously characterized mutations impairing the function of the yeast helicase RAD3 in DNA repair mapped to one of the conserved segments . A degree of similarity was revealed between the consensus pattern of conserved amino acid residues derived for the new superfamily and that of another recently described protein superfamily including a different set of prokaryotic, eukaryotic and viral (putative) helicases. J Biol Chem, 1989 Jun 25, 264(18), 10888 - 96 Expression and initial characterization of a recombinant human thrombospondin heparin binding domain; Yabkowitz R et al.; Thrombospondin (TSP) is a trimeric glycoprotein of Mr 420,000 . It was originally described as a major component of human platelet alpha granules and is essential for the secondary phase of platelet aggregation . TSP is also synthesized and secreted by a variety of nucleated cells where it functions in processes involving growth and adhesion of cells to the extracellular matrix . Many of these processes are heparin-inhibitable and are mediated by a proteolytic fragment of TSP called the heparin binding domain (HBD) . In order to facilitate the analysis of the structure and function(s) of this domain, we have expressed this molecule in Escherichia coli . A fragment of a TSP cDNA that encodes the heparin binding domain was inserted into the prokaryotic expression vector pJBL6 . In bacterial cells grown at 42 degrees C, this vector directs the synthesis of a 24,000-Da polypeptide . Milligram quantities of this protein were purified to homogeneity from E . coli lysates . The structure of the recombinant HBD was confirmed by protein sequencing . The protein was further characterized by analysis of its conformation and function . The recombinant HBD binds {3H}heparin with a Kd of 71 nM, almost identical to that of TSP-derived HBD (80 nM) . Additionally, the recombinant HBD is able to compete for TSP binding to 11B carcinoma cells . These studies indicate that the recombinant HBD is synthesized and purified in a native configuration and is functionally equivalent to thrombospondin-derived HBD . They further indicate that glycosylation of the thrombospondin HBD is not necessary for its interaction with heparin and that sequences essential to this interaction reside within the first 229 amino acids of secreted thrombospondin. Science, 1989 Jun 23, 244(4911), 1493 - 6 Transfer RNA genes: landmarks for integration of mobile genetic elements in Dictyostelium discoideum; Marschalek R et al.; In prokaryotes and eukaryotes mobile genetic elements frequently disrupt the highly conservative structures of chromosomes, which are responsible for storage of genetic information . The factors determining the site for integration of such elements are still unknown . Transfer RNA (tRNA) genes are associated in a highly significant manner with different putative mobile genetic elements in the cellular slime mold Dictyostelium discoideum . These results suggest that tRNA genes in D . discoideum, and probably tRNA genes generally in lower eukaryotes, may function as genomic landmarks for the integration of different transposable elements in a strictly position-specific manner. J Biol Chem, 1989 Jun 15, 264(17), 9953 - 9 RNA secondary structure is an integral part of the in vitro mechanism of attenuation in simian virus 40; Resnekov O et al.; At late times after infection with SV40, a prematurely terminated transcript that initiates at the major late promoter (MLP) and has a 3'-end about 95 nucleotides downstream has been identified and termed an attenuated RNA (Hay, N., Skolnik-David, H., and Aloni, Y . (1982) Cell 29, 183-193) . The DNA template of the attenuated RNA has two regions of dyad symmetry, and the attenuated RNA can therefore fold into two hairpin elements . The hairpin element at the 3'-end of the attenuated RNA is followed by a stretch of Us and resembles a rho-independent terminator in prokaryotes . We have suggested that folding of the RNA into two hairpin elements will lead to a block of transcription elongation . Using site-directed mutagenesis, we created two templates that either strengthened or weakened the proposed hairpin structures . The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared between the templates in a cell-free extract . We have shown that RNA polymerase II recognizes the SV40 sequence that leads to a block of transcription elongation, even when it is under the control of the MLP of adenovirus 2 . The extent of the block of transcription elongation is directly dependent on the stability of the hairpin structure of the RNA as assessed by a comparison of transcription of the wild-type and mutated templates . The addition of Sarkosyl and transcription at an elevated temperature during the elongation reaction enhanced the production of the attenuated RNA from all templates. Experientia, 1989 Jun 15, 45(6), 535 - 41 Interferons and indoleamine 2,3-dioxygenase: role in antimicrobial and antitumor effects; Carlin JM et al.; Indoleamine 2,3-dioxygenase (IDO) is an interferon (IFN)-induced protein that initiates the metabolism of tryptophan along the kynurenine pathway . Although IDO can be induced by IFN-gamma in many cell types, only mononuclear phagocytes have been shown to be induced to decyclize tryptophan by all three IFN classes . Since tryptophan is an essential amino acid necessary for a variety of metabolic processes, depletion of available tryptophan may be an important mechanism for control of rapidly-dividing microbial pathogens and tumors . The purpose of this review is to present evidence that documents the effects of IFN-induced IDO on prokaryotic and eukaryotic pathogens, as well as on a variety of tumor cell lines. EMBO J, 1989 Jun, 8(6), 1841 - 8 Co-operative interactions between NFI and the adenovirus DNA binding protein at the adenovirus origin of replication; Cleat PH et al.; The DNA-protein and protein-protein interactions proposed for the stability of nucleoprotein complexes at the origin of replication in prokaryotes are also thought to impart regulatory precision in eukaryotic DNA replication . This type of specificity can be observed, for example, during adenovirus DNA replication where efficient initiation requires that nuclear factor I (NFI) binds to the origin of DNA replication . Addition of purified NFI stimulates the initiation of adenovirus DNA replication in vitro in a reaction that is dependent on the concentration of the adenovirus DNA binding protein (DBP) . However, the molecular basis for the synergistic action of NFI and DBP during replication is at present unknown . We report here that DBP increases the affinity of NFI for its binding site in the replication origin . DBP did not, however, increase the affinity of another eukaryotic sequence-specific DNA binding protein, EBP1, for its recognition site . Other single-stranded DNA binding proteins could not substitute for DBP in increasing NFI affinity for its binding site . In addition, DBP was found to alter the binding kinetics of NFI, both by increasing the rate of association and decreasing the rate of dissociation of NFI with the DNA template . The co-operativity between NFI and DBP was also demonstrated on another DNA template, a human NFI site (FIB2), suggesting that this interaction is of general occurrence and not restricted to the adenovirus origin of replication. Proc Natl Acad Sci U S A, 1989 Jun, 86(11), 3997 - 4001 An element downstream of the cap site is required for transcription of the gene encoding mouse ribosomal protein L32; Moura-Neto R et al.; To identify the elements that regulate transcription of the mouse gene encoding ribosomal protein L32 (rpL32), we transfected monkey kidney (COS or CV-1) cells with mutants bearing progressive 5' deletions or an internal deletion in exon I and measured their transient expression by S1 nuclease protection analysis . When the mutant genes were tested in the vector pi SVHSplac, which contains a short segment of the oriregion of simian virus 40, maximum expression was observed with as little as 36 base pairs of 5' flanking sequence, and the mutant bearing the exon I deletion was expressed very efficiently . However, when the genes were tested in a simple prokaryotic (pUC) vector, the expression was increased 3- to 4-fold by sequences between -36 and -159, and the exon I segment was absolutely required for expression . Gel mobility-shift and methylation interference analyses revealed that a nuclear factor specifically binds to a GGCTGCCATC sequence within this exon I segment . These results, taken together with other recent findings, indicate that the elements involved in transcriptional regulation of the rpL32 gene are distributed over a 200-base-pair region that spans the cap site . The contributions of some of these elements are apparently masked in the presence of simian virus 40 ori-region elements. Risk Anal, 1989 Jun, 9(2), 157 - 68 Transgenic mice as future tools in risk assessment; Cordaro JC; Historically, mice have served a routine and useful purpose in the research, development, and testing of biologicals, chemicals, and drugs for efficacy, toxicity, and carcinogenic risk . The literature is replete with examples using mice to study organic compounds both in short-term tests involving tumor initiation and promotion and in long-term experiments dealing with fertility, reproduction, and teratology . During the past two decades, a virtual explosion of advances has occurred in modern biology that includes the discoveries of retroviruses, oncogenes, DNA restriction enzymes, nucleotide sequence analyses, and microinjection techniques . Fusion of these milestones in genetic, molecular, and cell biology with recent developments in mouse embryology has opened novel avenues and methods of experimentation as significant additions to the risk assessment armamentarium that currently uses both prokaryotes and eukaryotes . Some promising directions afforded by transgenic mice as powerful future tools in risk assessment will be summarized below. Proc Natl Acad Sci U S A, 1989 Jun, 86(12), 4382 - 6 Mammalian aspartate transcarbamylase (ATCase): sequence of the ATCase domain and interdomain linker in the CAD multifunctional polypeptide and properties of the isolated domain; Simmer JP et al.; Mammalian aspartate transcarbamylase (ATCase; carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2) is part of a 240-kDa multifunctional polypeptide called CAD, which also has carbamoyl-phosphate synthetase and dihydroorotase activities . We have sequenced selected restriction fragments of a Syrian hamster CAD cDNA that are clearly homologous to three prokaryotic ATCases . These studies, combined with previous sequence data, showed that the ATCase domain of CAD is encoded by 924 base pairs and has a mass of 34,323 Da and a pI of 9.8 . While the bacterial pyrimidine biosynthetic enzymes are separate proteins, in mammals the ATCase domain is fused to the carboxyl end of the CAD chimera via a 133-amino acid (14-kDa) linker with an unusual amino acid composition, a pI of 10.2, and pronounced hydrophilic character . The fully active domain isolated from proteolytic digests was characterized by partial amino acid sequencing and amino acid analysis . Trypsin cleavage produced the ATCase domain with a 20-residue amino-terminal extension . Hydrodynamic studies showed that the isolated domain is a 110-kDa trimer with a Stokes radius of 41 A . The mammalian ATCase domain and the prokaryotic enzymes have virtually identical active-site residues and are likely to have the same tertiary fold. FEMS Microbiol Immunol, 1989 Jun, 1(6-7), 331 - 40 Carbohydrates as recognition molecules in infection and immunity; Weir DM; Consideration of host-parasite interactions encompasses a wide range of phenomena from adhesion to epithelial surfaces to interactions with cells of the immune system . This review focuses on the role of carbohydrates as recognition molecules in these complex interactions . The abundant glycoproteins and glycolipids of cell surfaces of both prokaryotic and eukaryotic cells have the ability to exist in a variety of spatial configurations through alpha- and beta-linkages and the formation of branched structures . This ability carries with it the opportunity of acting as informational molecules greater than that possible for proteins or nucleic acids . The blood group substances are probably the best characterized of these carbohydrate containing molecules . Whilst at present a detailed understanding of the importance of these molecules in host-parasite interaction is lacking, the material covered in this discussion emphasizes the way in which carbohydrate based recognition has been shown to be involved and may provide the basis for further understanding. Gene, 1989 May 30, 78(2), 309 - 21 The phosphofructokinase genes of yeast evolved from two duplication events; Heinisch J et al.; Yeast phosphofructokinase (PFK) is an octameric enzyme composed of four alpha-subunits and four beta-subunits, encoded by the genes PFK1 and PFK2, respectively . PFK1 was mapped 23 cM distal to ADE3 on chromosome VII, and PFK2 30 cM proximal to RNA1 on chromosome XIII . The entire nucleotide sequences for the two genes were obtained by sequencing both DNA strands . Only one major open reading frame was found for each gene . They encode 987 aa for PFK1 (Mr 107,984) and 959 aa for PFK2 (Mr 104,589) . Both genes show a biased codon usage . The deduced amino acid sequences showed: (i) 20% homology between the N- and the C-terminal halves of each subunit, (ii) 55% homology between the two subunits, and (iii) significant homologies to the PFK sequences from human and rabbit muscle (42%), Escherichia coli (34%), and Bacillus (36%) . These data support the view that two gene duplication events occurred in the evolution of the yeast PFK genes . The first duplication event took place soon after the separation of prokaryotic and eukaryotic lineage and the second in Saccharomyces later in the phylogeny . Functional domains in the yeast subunits were deduced by comparison to the rabbit muscle enzyme. Gene, 1989 May 15, 78(1), 73 - 84 A self-inducing runaway-replication plasmid expression system utilizing the Rop protein; Giza PE et al.; A highly efficient prokaryotic expression system has been developed that produces proteins at levels exceeding 150 micrograms/ml of culture medium . The system consists of a temperature-sensitive-copy-number plasmid that carries the rop gene and promoter downstream from the trp promoter . Any sequence cloned into the PvuII site of the rop gene alters Rop protein activity and causes lethal runaway plasmid DNA replication . This plasmid replication can be suppressed in trans by complementation with a similar wild-type plasmid . Cells harboring both plasmids are quite stable, and induction of plasmid DNA synthesis occurs only after cells are grown for several generations under conditions that lead to the loss of the trans-acting repressor . Large amounts of Rop fusion proteins accumulate in the cell as the trp operon is gradually induced via repressor titration . All chimeric proteins accumulate as insoluble aggregates, and are therefore easily purified . They can be solubilized using relatively mild conditions, and the partially purified proteins are highly amenable to cleavage by chemical methods . Using this system we have made Rop fusions with the HIV Tat protein, the herpes simplex virus type-2 38K protein, and Chinese hamster metallothionin. Biokhimiia, 1989 May, 54(5), 726 - 9 {Pyridoxal phosphate-dependent enzymes of sulfur amino acid metabolism}; Gabibov AG et al.; Cystathionine beta-synthase and gamma-cystathionase, the two major enzymes of the transsulfuration pathway of methionine metabolism, are described . These enzymes are responsible for inborn errors, e.g., homocystinuria and cystathioninuria . The interaction of gamma-cystathionase with the cofactor, substrates and inhibitors of the general formula RONH2 containing structural fragments of substrates has been studied . A non-radioactive avidin-biotin system for the microdetection of gamma-cystathionase in dot blots has been developed . This system was applied for immunoscreening of a rat liver cDNA library in the prokaryotic expression vector lambda gt 11. J Electron Microsc Tech, 1989 May, 12(1), 1 - 10 Ultrastructural localization of DNA on ultrathin sections of resin-embedded tissues by the lactoferrin-gold complex; Benhamou N; Lactoferrin, a DNA-binding protein, was complexed to colloidal gold and applied on ultrathin sections of resin-embedded plant tissues and bacterial cultures . Optimal results were obtained when lactoferrin was tagged to colloidal gold particles at pH 9.2 . Postfixation with osmium tetroxide and embedding with Epon did not prevent the accessibility of the protein towards its corresponding binding sites . In plant nuclei, labeling was observed over the dense chromatin and to a lesser extent over the dispersed chromatin . Nucleolar labeling was preferentially located over the dense fibrillar component . Gold particles were also found to be associated with chloroplasts and mitochondria . In prokaryotic cells, a dispersed labeling was noted over the cytoplasm and, in some cases, the aggregation of few gold particles suggested the presence of packed DNA fibrils . Various control experiments confirmed the specificity of the labeling pattern obtained . Lactoferrin-gold complex appears to be a valuable probe for the intracellular demonstration of DNA molecules in double-fixed and Epon-embedded tissues. Proc Natl Acad Sci U S A, 1989 May, 86(9), 3296 - 300 Prediction of major histocompatibility complex binding regions of protein antigens by sequence pattern analysis; Sette A et al.; We have previously experimentally analyzed the structural requirements for interaction between peptide antigens and mouse major histocompatibility complex (MHC) molecules of the d haplotype . We describe here two procedures devised to predict specifically the capacity of peptide molecules to interact with these MHC class II molecules (IAd and IEd) . The accuracy of these procedures has been tested on a large panel of synthetic peptides of eukaryotic, prokaryotic, and viral origin, and also on a set of overlapping peptides encompassing the entire staphylococcal nuclease molecule . For both sets of peptides, IAd and IEd binding was successfully predicted in approximately 75% of the cases . This suggests that definition of such sequence "motifs" could be of general use in predicting potentially immunogenic peptide regions within proteins. Mol Biol (Mosk), 1989 May-Jun, 23(3), 629 - 38 {Antisense polynucleotides and prospects for their use in fighting viruses}; Tikhonenko TI; Natural or synthetic anti-sense (as) polynucleotides complementary to distinct functional regions of mRNA (asRNA or asDNA) are able to inhibit the expression of any target gene . If certain viral mRNAs important for virus replication are targeted the inhibition of viral infection by asRNA or asDNA takes place . Inhibitory effects of complementary polynucleotides on gene activity in eukaryotic cells is due to the disturbance of translation of corresponding mRNAs as well as to the impairment of their splicing or transportation from the nuclei to cytoplasm . In prokaryotic cells, obviously, only the first factor is operating . The recombinant genes programming anti-viral asRNA can confer the resistance to the infection by other virus to the transformed cells . The resistance to viral infection observed in transgenic animals, expressing asRNA genes, may be considered as a new unnatural form of informational immunity. Genes Dev, 1989 May, 3(5), 598 - 605 A bacterial environmental sensor that functions as a protein kinase and stimulates transcriptional activation; Igo MM et al.; Transcription of the genes that encode the major outer membrane porin proteins OmpF and OmpC of Escherichia coli is regulated in response to changes in medium osmolarity by EnvZ and OmpR . EnvZ functions to sense environmental conditions and to relay this information to the DNA-binding protein OmpR . We have used a truncated EnvZ protein (EnvZ115), which is defective in sensory function but able to communicate with OmpR, to study the biochemical interactions between these two proteins and their effects on transcription from the ompF promoter . We show that purified EnvZ115 can phosphorylate OmpR in the presence of ATP . In addition, EnvZ115 stimulates the ability of OmpR to activate ompF transcription in vitro . Using antibodies specific for EnvZ, we have purified the wild-type protein and have shown that it is also an OmpR kinase . These results provide a prokaryotic example of a transmembrane sensory protein that functions as a protein kinase and suggest a mechanism by which EnvZ communicates with OmpR in signal transduction. Mol Biochem Parasitol, 1989 May 1, 34(2), 155 - 66 Structure, genomic organization and transcription of the bifunctional dihydrofolate reductase-thymidylate synthase gene from Crithidia fasciculata; Hughes DE et al.; The bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) gene from the monogenetic kinetoplastid protozoan, Crithidia fasciculata, was isolated and characterized . The gene is located on a single chromosome of approximately one megabase, and shows significant sequence similarity to other eukaryotic and prokaryotic DHFR and TS genes . There is a single low-abundance polyadenylated DHFR-TS transcript of approximately 3100 nt . One major miniexon splice site was identified by primer extension analysis . The 5' flanking region of the gene is divergently transcribed and shows strong similarities to a consensus DHFR promoter as well as to other eukaryotic 'housekeeping' gene promoter regions . A sequence downstream of the DHFR promoter consensus region is complementary to the 3' end of the C . fasciculata miniexon-derived RNA . This suggests a means by which the two separately transcribed RNAs may be juxtaposed for trans-splicing . In the 3' flanking region of the DHFR-TS gene, there is a sequence that is present in all of the chromosomes from this species and also from Leishmania tarentolae. Biokhimiia, 1989 May, 54(5), 757 - 64 {Protein-nucleic acid interactions in reactions catalyzed by eukaryotic and prokaryotic DNA-polymerases}; Lavrik OI et al.; A new method of estimation of dissociation constants for ligands and free energies of its binding based on the affinity modification of active centers in the presence of competitive ligands was developed . This method is designed for the analysis of protein-nucleic acid interactions in template systems . Deoxyoligoribonucleotides containing the reactive residue of cis-aquadihydroxydiaminoplatinum (II) and oligonucleotides ethylated at phosphate groups were used for the study of interactions of human placental DNA-polymerase alpha and the Klenow fragment of DNA-polymerase I from E . coli with templates and primers . A model was constructed which postulates the formation of a single Me2+-dependent electrostatic bond and of a hydrogen bond by one of template phosphates with the enzyme active center . Similar bonds form the basis for the enzyme interaction with the 3'-terminal phosphate group of the primer . Other monomeric units of the template are likely to interact with the enzyme by forming hydrophobic bonds . Other mononucleotide units of the primer are involved in complementary interactions with the template . The primer activity of dNMP and NMP in these systems has been demonstrated for the first time . The efficiency of dNMP, dNDP and dNTP interaction with DNA-polymerase was estimated from the affinity modification of the enzymes by dNTP and dNMP imidazolides . The key role of the template-primer interaction in the formation of the dNTP-binding site of DNA-polymerases was demonstrated . A significant contribution of dNTP gamma-phosphate to the template--dependent specific tuning of substrate dNTP was revealed. J Mol Evol, 1989 May, 28(5), 403 - 17 Sequence and secondary structure of the central domain of Drosophila 26S rRNA: a universal model for the central domain of the large rRNA containing the region in which the central break may happen; de Lanversin G et al.; An 890-bp sequence from the central region of Drosophila melanogaster 26S ribosomal DNA (rDNA) has been determined and used in an extensive comparative analysis of the central domain of the large subunit ribosomal RNA (lrRNA) from prokaryotes, organelles, and eukaryotes . An alignment of these different sequences has allowed us to precisely map the regions of the central domain that have highly diverged during evolution . Using this sequence comparison, we have derived a secondary structure model of the central domain of Drosophila 26S ribosomal RNA (rRNA) . We show that a large part of this model can be applied to the central domain of lrRNA from prokaryotes, eukaryotes, and organelles, therefore defining a universal common structural core . Likewise, a comparative study of the secondary structure of the divergent regions has been performed in several organisms . The results show that, despite a nearly complete divergence in their length and sequence, a common structural core is also present in divergent regions . In some organisms, one or two of the divergent regions of the central domain are removed by processing events . The sequence and structure of these regions (fragmentation spacers) have been compared to those of the corresponding divergent regions that remain part of the mature rRNA in other species. J Gen Virol, 1989 May, 70 ( Pt 5), 1217 - 29 The type-specific epitopes of the Epstein-Barr virus nuclear antigen 2 are near the carboxy terminus of the protein; Rowe DT et al.; The Epstein-Barr virus nuclear antigen 2 (EBNA 2) shows serotype variation and two serologically distinct groups of viruses have been identified . These correspond to the two hybridization groups of viruses (A and B) that are distinguished by a highly substituted nucleic acid sequence in the middle of the open reading frame of the EBNA 2 gene . An epitope survey of the EBNA 2-coding region was carried out using a new prokaryotic expression vector tailored to express DNA fragments from the M13 sequencing libraries of the B95-8 (type A) and Jijoye (type B) prototype virus strains . Short overlapping stretches of EBNA 2 sequence were expressed as fusion proteins and used in Western blotting with human sera that contained serotype-specific antibodies . The type A-specific epitope was located between residues 378 and 435 of the B95-8 EBNA 2 polypeptide and the type B-specific epitope mapped between residues 390 and 454, at the carboxy terminus of the Jijoye polypeptide chain . All of the type-specific anti-EBNA 2 sera tested reacted with fusion proteins containing one or other of these epitopes . Despite the direct correlation between the hybridization and serological phenotypes, the type-specific epitopes appear to lie in the relatively conserved carboxy-terminal region of EBNA 2 . There was no indication that the residues of the non-homologous region contributed to the formation of antibody-combining sites. Nucleic Acids Res, 1989 Apr 25, 17(8), 2947 - 57 The narX and narL genes encoding the nitrate-sensing regulators of Escherichia coli are homologous to a family of prokaryotic two-component regulatory genes; Nohno T et al.; The nucleotide sequence of a 4.4-kilobase SacII-SspI fragment encoding the narXL operon and a part of the narK gene of Escherichia coli has been determined . The narX and narL genes encode proteins of molecular weight 67,275 and 23,927, respectively, and are transcribed from a common promoter, narXp, locating within 429 bases upstream of narX . Transcription from narXp is not significantly induced by nitrate under anaerobiosis, whereas transcription from narK promoter, which overlaps narXp region and is transcribed divergently, is fully induced by nitrate . The N-terminal two-thirds of the NarL protein has extensive homology with those of a diverse set of prokaryotic regulatory proteins, including OmpR, PhoB, SfrA, UhpA, CheY, CheB, NtrC, DctD, FixJ, VirG, SpoOF, and SpoOA . A segment locating in the C-terminal half of the NarL protein seems to have potential most likely to form the helix-turn-helix structure characteristic of a class of DNA-binding protein . The protein is considered to play a role as a transcriptional activator of the nitrate reductase operon, narCHJI, and the narK gene . The C-terminal region of the NarX protein also has homology with other regulatory proteins known as counterparts of two-component regulatory systems, such as EnvZ, PhoR, PhoM, CpxA, NtrB, DctB, FixL, and VirA . Presence of two copies of hydrophobic segments in the N-terminal half of the NarX protein suggests the role as a transmembrane receptor sensing nitrate. J Chromatogr, 1989 Apr 19, 466, 331 - 7 High-performance liquid chromatographic separation of variants of chromosomal proteins from prokaryotes; Chartier F et al.; The separation of variants of chromosomal proteins exhibiting closely related amino acid compositions has been achieved using weak cation-exchange or reversed-phase high-performance liquid chromatography . The purity of the isolated proteins has been ascertained by polyacrylamide gel electrophoresis and in several cases by micro-sequencing. J Biol Chem, 1989 Apr 15, 264(11), 6504 - 8 Suppression of amber codons in vivo as evidence that mutants derived from Escherichia coli initiator tRNA can act at the step of elongation in protein synthesis; Seong BL et al.; The absence of a Watson-Crick base pair at the end of the amino acid acceptor stem is one of the features which distinguishes prokaryotic initiator tRNAs as a class from all other tRNAs . We show that this structural feature prevents Escherichia coli initiator tRNA from acting as an elongator in protein synthesis in vivo . We generated a mutant of E . coli initiator tRNA in which the anticodon sequence is changed from CAU to CUA (the T35A36 mutant) . This mutant tRNA has the potential to read the amber termination codon UAG . We then coupled this mutation to others which change the C1.A72 mismatch at the end of the acceptor stem to either a U1:A72 base pair (T1 mutant) or a C1:G72 base pair (G72 mutant) . Transformation of E . coli CA274 (HfrC Su- lacZ125am trpEam) with multicopy plasmids carrying the mutant initiator tRNA genes show that mutant tRNAs carrying changes in both the anticodon sequence and the acceptor stem suppress amber codons in vivo, whereas mutant tRNA with changes in the anticodon sequence alone does not . Mutant tRNAs with the above anticodon sequence change are aminoacylated with glutamine in vitro . Measurement of kinetic parameters for aminoacylation by E . coli glutaminyl-tRNA synthetase show that both the nature of the base pair at the end of the acceptor stem and the presence or absence of a base pair at this position can affect aminoacylation kinetics . We discuss the implications of this result on recognition of tRNAs by E . coli glutaminyl-tRNA synthetase. Gene, 1989 Apr 15, 77(1), 79 - 86 Helpers for efficient encapsidation of SV40 pseudovirions; Oppenheim A et al.; Plasmid DNA that carries the simian virus 40 (SV40) ori can be packaged as SV40 pseudovirions . The pseudovirions are very efficient in gene transmission into a variety of cell types, including human hemopoietic cells . They are routinely prepared with wild-type (wt) SV40 as a helper . In the present study, several parameters required for the helper function were investigated . Plasmids that carry pBR322 sequences in addition to the late genes of SV40 were inefficient in providing helper functions, presumably because the prokaryotic sequences interfered with expression of the SV40 late genes . Efficient helpers were plasmid pSVPiC {Villarreal and Soo, Mol . Appl . Genet . 3 (1985) 62-71} and an SV40 defective virus SLT3 (presently constructed) . Plasmid pSVPiC carries a duplication of the SV40 ori and enhancer regions, and pi AN7 sequences . Because of its large size it was not packaged into virion particles . However, it underwent extensive recombination generating infective SV40 particles . Almost no prokaryotic sequences are included in SLT3, that carries the SV40 late gene . In spite of its small size (3.5 kb) it was packaged efficiently, creating defective (T-antigen-negative) SV40 virions . The availability of T-antigen positive and negative pseudovirion mixtures enabled us to suggest that T-antigen drives gene amplification in the target human hemopoietic cells. Mol Microbiol, 1989 Apr, 3(4), 479 - 86 Molecular analysis of linear plasmid-encoded major surface proteins, OspA and OspB, of the Lyme disease spirochaete Borrelia burgdorferi; Bergstrom S et al.; The ospA and ospB genes encode the major outer membrane proteins of the Lyme disease spirochaete Borrelia burgdorferi . The deduced translation products from the ospA and ospB genes were: (OspA) 273 amino acids long with a molecular weight of 29,334, and (OspB) 296 amino acids long with a molecular weight of 31,739 . The two Osp proteins showed a great degree of sequence similarity indicating a recent evolutionary event . Molecular analysis and sequence comparison of OspA and OspB with other proteins revealed a sequence similarity to the signal peptides of prokaryotic lipoproteins . These are the first sequences from Borrelia and provide interesting data on the evolutionary relationship between spirochaetes and other species as well as providing potential for spirochaete diagnostics and vaccines. Mol Gen Genet, 1989 Apr, 216(2-3), 254 - 68 Nucleotide sequence, organization, and nature of the protein products of the carotenoid biosynthesis gene cluster of Rhodobacter capsulatus; Armstrong GA et al.; Carotenoid pigments are essential for the protection of both photosynthetic and non-photosynthetic tissues from photooxidative damage . Although carotenoid biosynthesis has been studied in many organisms from bacteria to higher plants, little is known about carotenoid biosynthetic enzymes, or the nature and regulation of the genes encoding them . We report here the first DNA sequence of carotenoid genes from any organism . We have determined the complete nucleotide sequence (11,039 bp) of a gene cluster encoding seven of the eight previously known carotenoid genes (crtA, B, C, D, E, F, I) and a new gene, designated crtK, from Rhodobacter capsulatus, a purple non-sulfur photosynthetic bacterium . The 5' flanking regions of crtA, I, D and E contain a highly conserved palindromic sequence homologous to the consensus binding site for a variety of prokaryotic DNA-binding regulatory proteins . This putative regulatory palindrome is also found 5' to the puc operon, encoding the light-harvesting II antenna polypeptides . Escherichia coli-like sigma 70 promoter sequences are located 5' to crtI and crtD, suggesting for the first time that such promoters may exist in purple photosynthetic bacteria . The crt genes form a minimum of four distinct operons, crtA, crtIBK, crtDC and crtEF, based on inversions of transcriptional orientation within the gene cluster . Possible rho-independent transcription terminators are located 3' to crtI, B, K, C and F . The 3' end of crtA may overlap transcription initiation signals for a downstream gene required for bacteriochlorophyll biosynthesis . We have also observed two regions of exceptional amino acid homology between CrtI and CrtD, both of which are dehydrogenases. Bioessays, 1989 Apr, 10(4), 127 - 30 Metabolic compartmentation; Spivey HO et al.; Evidence for the association of 'soluble' enzymes in vivo is extensive and compelling . These associations occur in all compartments of the cell of both prokaryotes and eukaryotes . Several factors present in vivo promote these associations among enzymes whose association in vitro is often too weak to detect . Several physiological advantages of the associated enzyme complexes can be identified, most (but not all) of which are the consequence of microcompartmentation of metabolites (substrate channeling) . Substrate channeling of intermediates by either a 'direct transfer' process or 'proximity effects' can occur . The latter mechanism does not require the special molecular features needed for the direct transfer mechanism and may, therefore, exist in more general situations in the cell . Criticisms of these views are discussed . We argue that these criticisms have been largely answered by experiment and theory in recent years . Studies on simple systems in vitro, nevertheless, contribute important insights concerning the more complex phenomena in vivo. Biochem J, 1989 Apr 1, 259(1), 307 - 10 Effect of the antibiotic purpuromycin on cell-free protein-synthesizing systems; Rambelli F et al.; Purpuromycin, an antibiotic isolated from the culture broth of Actinoplanes ianthinogenes, which is very active against Gram-positive bacteria and fungi, inhibits protein synthesis in both prokaryotic and eukaryotic cell-free systems . The ID50 was 9 microM with the endogenous mRNA-directed rabbit reticulocyte lysate, 17 microM with a poly(U)-directed system from Escherichia coli and 69 microM with a poly(U)-directed system from Artemia salina cysts . Of the three steps of elongation, purpuromycin does not affect the peptidyl-transferase reaction, inhibits the elongation factor 1 (EF-1) dependent binding of phenylalanyl-tRNA and stimulates the GTP-dependent binding of EF-2 . When protein synthesis is stopped by the addition of purpuromycin, the nascent peptide chains are found in the puromycin-reactive P site . The results suggest that the mechanism of action of purpuromycin is similar to that of fusidic acid . Both antibiotics would seem to produce a stable guanine nucleotide-ribosome-EF-2 complex which allows one round of translocation but prevents, because of a common or overlapping ribosomal binding site for the two elongation factors, the subsequent EF-1-dependent binding of aminoacyl-tRNA. J Mol Recognit, 1989 Apr, 1(4), 172 - 8 A subsequence-specific DNA-binding domain resides in the 13 kDa amino terminus of the bacteriophage Mu transposase protein; Tolias PP et al.; We have previously reported that the 13 kDa amino terminus of the 70 kDa bacteriophage D108 transposase protein (A gene product) contains a two-component, sequence-specific DNA-binding domain which specifically binds to the related bacteriophage Mu's right end (attR) in vitro . To extend these studies, we examined the ability of the 13 kDa amino terminus of the Mu transposase protein to bind specifically to Mu attR in crude extracts . Here we report that the Mu transposase protein also contains a Mu attR specific DNA-binding domain, located in a putative alpha-helix-turn-alpha-helix region, in the amino terminal 13 kDa portion of the 70 kDa transposase protein as part of a 23 kDa fusion protein with beta-lactamase . We purified for this attR-specific DNA-binding activity and ultimately obtained a single polypeptide of the predicted molecular weight for the A'--'bla fusion protein . We found that the pure protein bound to the Mu attR site in a different manner compared with the entire Mu transposase protein as determined by DNase I-footprinting . Our results may suggest the presence of a potential primordial DNA-binding site (5'-PuCGAAA-3') located several times within attR, at the ends of Mu and D108 DNA, and at the extremities of other prokaryotic class II elements that catalyze 5 base pair duplications at the site of element insertion . The dissection of the functional domains of the related phage Mu and D108 transposase proteins will provide clues to the mechanisms and evolution of DNA transposition as a mode of mobile genetic element propagation. Mol Gen Genet, 1989 Apr, 216(2-3), 287 - 92 Cloning and DNA sequence of plasmid determinant iss, coding for increased serum survival and surface exclusion, which has homology with lambda DNA; Chuba PJ et al.; Escherichia coli K12 cells carrying a cloned 1.4 kb HindIII fragment from plasmid ColV2-K94, showed increased survival in guinea pig serum . The recombinant plasmid also conferred group II surface exclusion, i.e . the cells were reduced in recipient ability towards the incoming plasmid R538drd in conjugation experiments . Southern blotting suggested homology with bacteriophage lambda DNA and to the insertion element IS2 . Determination of the DNA sequence of the fragment demonstrated the presence of a truncated IS2 (165 bp), separated by 250 bp from a 900 bp stretch of homology with lambda DNA, beginning within the Rz gene and continuing in the rightward direction on the lambda map . A 97 amino acid open reading frame (ORF) adjacent to Rz and on the opposite strand, remained intact in iss, with several amino acid changes . The ORF in iss is preceded by sequences resembling prokaryotic ribosome binding sites and promoters. Genes Dev, 1989 Apr, 3(4), 510 - 26 Specific recognition nucleotides and their DNA context determine the affinity of E2 protein for 17 binding sites in the BPV-1 genome; Li R et al.; The DNA context of nucleotides that a protein recognizes can influence the strength of the protein-DNA interaction . Moreover, in prokaryotes, understanding the quantitative differences in binding affinities that result in part from the DNA context is often important in describing regulatory mechanisms . Nevertheless, these issues have not been a major focus yet for the investigation of protein-DNA interactions in eukaryotes . In this study, we explored the binding specificity and the range of affinities that the BPV-1 E2 transcriptional activator has for DNA . Because E2 binding sites are positioned near several different BPV-1 promoters, such quantitative information may be important to understand transcriptional regulatory mechanisms in BPV-1 . Gel retardation assays and DNA footprinting were used to quantitate the affinities of the E2 binding sites in the viral genome . In the process, five sites were discovered, which, on the basis of sequence, had not been predicted previously to interact with the E2 protein . Equilibrium and kinetic studies show that the range of E2 affinities of the 17 sites varied over 300-fold . The sequence elements responsible for E2 recognition of DNA were determined by missing contact analysis of several sites and a point mutation analysis of one site . The results presented show that the affinity of an E2 binding site is to a large extent determined by the availability of specific contacts, but the data also strongly suggest that DNA structure plays an important role. Proc Natl Acad Sci U S A, 1989 Apr, 86(8), 2540 - 4 Ubiquitin fusion augments the yield of cloned gene products in Escherichia coli; Butt TR et al.; Despite the availability of efficient transcription and translation signals, some heterologous gene products are not adequately expressed when introduced into prokaryotes and eukaryotes . An expression system has been established in Escherichia coli to increase the yield of cloned gene products, where the C terminus of ubiquitin was fused to the N terminus of unstable or poorly expressed proteins . Fusion of ubiquitin to yeast metallothionein or to the alpha subunit of the adenylate cyclase-stimulatory GTP-binding protein increased the yield from undetectable to 20% of the total cellular protein . A ubiquitin-N alpha-protein hydrolase has been partially purified from rabbit reticulocytes; this enzyme faithfully cleaves the junction peptide bound between the C-terminal Gly-76 of ubiquitin and the fusion protein . The increased yield of cloned gene products is very likely due to increased stability and/or more efficient translation of the fusion proteins . Possible mechanisms for the augmentation of ubiquitin fusion-protein expression in prokaryotes and eukaryotes are discussed. Med Hypotheses, 1989 Apr, 28(4), 219 - 23 Prokaryotic mechanisms in eukaryotes: experimental data and speculations; Grossgebauer K; Surprising experimental data recently provided could indicate ancestral genes common to mammals and bacteria . The detection of prokaryotic structures and/or mechanisms even in higher eukaryotes shows the extreme conservation of gene structures throughout evolution . The occurrence of common structures in pro- and eukaryotes is in good agreement with the new view concerning the early steps of cellular evolution . Because of the relative paucity of research in this area, further prokaryotic features of transcription and translation process may also be evolutionarily conserved in eukaryotes. J Vet Diagn Invest, 1989 Apr, 1(2), 160 - 4 Etiologic studies on late-term swine abortions; Nielsen JN et al.; One hundred thirty-eight swine abortions were studied in detail in an effort to identify an etiologic agent . A viral agent was implicated in 7 cases . Bacteria were isolated in less than half of the examined cases: however, in 61% of the cases, motile, filamentous organisms were observed in tissues and fluids . Although swine sera from farms experiencing reproductive problems had a high reactor rate to Leptospira bratislava antigen, electron microscopy of the observed organism revealed a wall-free prokaryote morphologically typical of the class Mollicutes. J Gen Microbiol, 1989 Apr, 135 ( Pt 4), 931 - 9 Cloning and sequence analysis of the 10 kDa antigen gene of Mycobacterium tuberculosis; Baird PN et al.; The gene encoding a major protein antigen of Mycobacterium tuberculosis has been cloned and sequenced using oligonucleotide probes derived from the N-terminal sequence of the analogous protein from Mycobacterium bovis BCG . The gene analysis revealed a sequence encoding a protein of 99 amino acid residues, with a molecular mass of 10.7 kDa . Computer prediction suggests that the protein contains three T-cell-determined epitopes (of which one has been demonstrated experimentally) and three B-cell-determined epitopes . The protein sequence was homologous to two prokaryote heat-shock proteins and the gene possessed heat-shock-like promoter sequences upstream of the initiation codon . A hairpin loop identified in the upstream sequence may also be important in regulation of the gene. J Mol Biol, 1989 Mar 20, 206(2), 305 - 12 Cytosine-specific type II DNA methyltransferases . A conserved enzyme core with variable target-recognizing domains; Lauster R et al.; Comparisons of the amino acid sequences of m5C DNA methyltransferases (Mtases) from 11 prokaryotes and one eukaryote reveal a very similar organization . Among all the enzymes one can distinguish highly conserved "core" sequences and "variable" regions . The core sequences apparently mediate steps of the methylation reaction that are common to all the enzymes . The major variable region has been shown in our previous studies on multispecific phage Mtases to contain the target-recognizing domains (TRDs) of these enzymes . Here we have compared the amino acid sequences of various TRDs from phage Mtases . This has revealed the presence of both highly conserved and variable amino acids . We postulate that the conserved residues represent a "consensus" sequence defining a TRD, whereas the specificity of the TRD is determined by the variable residues . We have observed similarity between this consensus sequence and sequences in the variable region of the monospecific Mtases . We predict that the regions thus identified represent part of the TRDs of monospecific Mtases. J Biol Chem, 1989 Mar 15, 264(8), 4367 - 73 Expression of human parathyroid hormone in Escherichia coli; Wingender E et al.; Human parathyroid hormone (PTH) has been expressed in Escherichia coli as a cro-beta-galactosidase-hPTH fusion protein under temperature-sensitive control of the lambda phage PR promoter . The lacZ gene has been truncated to a different extent revealing an optimal length of the prokaryotic peptide portion between 199 and 407 amino acid residues . Up to 250 mg of pure fusion protein have been obtained from 1-liter E . coli culture by stepwise solubilization with urea . The linkage between the prokaryotic and the eukaryotic protein moiety consists of an Asp-Pro peptide bond and therefore is easily cleavable by acid treatment . A simple procedure for the purification of the hormone is described . The resulting recombinant hormone reacts with anti-PTH antibodies and stimulates renal adenylate cyclase identically to bovine or human PTH. Nucleic Acids Res, 1989 Mar 11, 17(5), 2057 - 80 Translation of chloroplast-encoded mRNA: potential initiation and termination signals; Bonham-Smith PC et al.; A survey of 196 protein-coding chloroplast DNA sequences demonstrated the preference for AUG and UAA codons for initiation and termination of translation, respectively . As in prokaryotes at every nucleotide position from -25 to +25 (AUG is +1 to +3) and for 25 nucleotides 5' and 3' to the termination codon an A or U is predominant, except for C at +5 and G at +22 . A Shine-Dalgarno (SD) sequence (GGAGG or tri- or tetranucleotide variant) was found within 100 bp 5' to the AUG codon in 92% of the genes . In 40% of these cases, the location of the SD sequence was similar to that of the consensus for prokaryotes (-12 to -7 5' to AUG), presumed to be optimal for translation initiation . A SD sequence could not be located in 6% of the chloroplast sequences . We propose that mRNA secondary structures may be required for the relocation of a distal SD sequences to within the optimal region (-12 to -7) for initiation of translation . We further suggest that termination at UGA codons in chloroplast genes may occur by a mechanism, involving 16S rRNA secondary structure, which has been proposed for UGA termination in E . coli. J Biol Chem, 1989 Mar 5, 264(7), 4093 - 103 Gene synthesis, expression, and processing of human ubiquitin carboxyl extension proteins; Monia BP et al.; In order to study 1) the mechanisms responsible for generating free ubiquitin monomer and 2) the function of ubiquitin carboxyl extension proteins in eukaryotes, we have developed a system for expression of human ubiquitin carboxyl extension proteins in prokaryotic and eukaryotic hosts . When expressed in Saccharomyces cerevisiae, the intact ubiquitin carboxyl extension proteins were rapidly processed to free ubiquitin monomer and extension protein . Furthermore, expression in this host conferred a slow growth phenotype mediated by the extension protein . Expression in Escherichia coli did not result in processing of the fusion proteins . However, when the expressed fusion proteins were purified from E . coli and incubated with a rabbit reticulocyte extract, the proteins were rapidly processed to free ubiquitin monomer and extension protein . These results show that human ubiquitin carboxyl extension proteins are processed to ubiquitin and extension protein when expressed in eukaryotic but not prokaryotic cells and that pre- and co-translational events are not necessary for their processing . Establishment of this system will allow for large scale purification of these proteins which will aid future studies on the function and structure of ubiquitin carboxyl extension proteins, as well as the mechanisms responsible for their processing. J Biol Chem, 1989 Mar 5, 264(7), 3840 - 8 Nucleotide sequence of the Neurospora crassa trp-3 gene encoding tryptophan synthetase and comparison of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae and Escherichia coli; Burns DM et al.; The complete nucleotide sequence of the Neurospora crassa trp-3 gene-encoding tryptophan synthetase has been determined; we present an analysis of its structure . A comparison of the deduced amino acid sequence of the trp-3 polypeptide with its homologs in Saccharomyces cerevisiae (encoded by the TRP5 gene) and Escherichia coli (encoded by the trpA and trpB genes) shows that the A and B domains (amino acid segments homologous to the trpA and trpB polypeptides, respectively) of the N . crassa and yeast polypeptides are in the same order (NH2-A-B-COOH) . This arrangement is the reverse of the gene order characteristic of all prokaryotes that have been examined . N . crassa tryptophan synthetase has strong homology to the yeast TRP5 polypeptide (A domains have 54% identity; B domains have 75% identity), and somewhat weaker homology to the E . coli trpA and trpB polypeptides (A domains have 31% identity; B domains have 50% identity) . The two domains of the N . crassa polypeptide are linked by a connector of 54-amino acid residues that has less than 25% identity to the 45-residue connector of the yeast polypeptide, although secondary structure analysis predicts both connectors would be alpha-helical . In contrast to the yeast TRP5 gene, which has no introns, the trp-3 coding region is interrupted by two introns 77 and 71 nucleotides in length . Both introns are located near the 5'-end of the gene and therefore not near the segment encoding the connector. Mutat Res, 1989 Mar, 211(1), 13 - 7 Variability of the adaptive response to ionizing radiations in humans; Bosi A et al.; Human lymphocytes exposed to low doses of ionizing radiations from incorporated tritiated thymidine ({3H}dThd) or from X-rays become less susceptible to the induction of chromatid aberrations by high doses of X-rays . This indicates that low doses of ionizing radiation can produce an effect similar to the adaptive response observed with alkylating agents in prokaryotes, animal and plant cells . To determine whether there is individual variability in the adaptive response to ionizing radiations we exposed human lymphocytes from 18 different healthy donors to 'adapting' doses of {3H}dThd (0.01 microCi/ml) or X-rays (0.01 Gy) and subsequently to a 'challenge' treatment of 0.75 Gy of X-rays delivered 2 h before fixation . Four of the 18 donors did not show an adaptive response; in some cases in these individuals a synergistic response of increased, rather than decreased, damage was found . Two of these 4 donors showed no adaptive response in 3 subsequent experiments separated by 4-month intervals . This suggests that the human population exhibits a heterogeneity in the adaptive response to ionizing radiations which might be, at least in part, genetically determined. Mutat Res, 1989 Mar, 217(2), 153 - 61 Homologous recombination is not enhanced in UV-irradiated normal and repair-deficient human fibroblasts; van der Lubbe JL et al.; Many mammalian cells exhibit damage-inducible phenomena that resemble the bacterial SOS functions . However, whereas RecA plays a prominent role in the prokaryotic SOS response, in mammalian cells so far no enhanced recombination as a result of treatment with DNA-damaging agents of the cells, rather than of infecting viruses, has been found . In order to study recombination as a UV-inducible cellular phenomenon we infected UV-irradiated normal and repair-deficient human fibroblasts with a mixed population of adenovirus 5 (Ad5) mutants that carried a deletion in the E1A or the E2A gene . Wild-type recombinant progeny viruses were readily obtained, but no enhanced recombination was observed at any UV dose given to the cells, nor at any time point between -6 h and +4 days between irradiation and infection . Control experiments, in which we infected unirradiated cells with UV-irradiated Ad5 deletion mutants (a test for recombination targeted at UV-damaged DNA) showed a strong increase in wild-type recombinant viruses when both deletion mutants had been irradiated compared to the additive effect of irradiation of either one of the mutants alone . Therefore, this study shows that UV irradiation results in an enhanced recombination activity in cells that is specifically targeted to damaged DNA, but it does not cause a general (untargeted) recombinational response (enhanced recombination) in the cell. Mol Biol (Mosk), 1989 Mar-Apr, 23(2), 537 - 44 {Theoretical analysis of the DNA duplication mechanisms in the prokaryotic genomes on the basis of repeats}; Kolchanov NA et al.; In the present work a theoretical analysis of the molecular mechanisms on duplications emergence in the genomes of prokaryotes on the basis of direct repeats has been carried out . The correlations obtained have shown, that the duplication rate depends on such parameters as the distance between repeated regions, repeats nucleotide composition and the number of homology damages in them . It has been revealed that the rate of duplications decreases more readily than the deletion rate upon the growth of the distance between the repeats . Such prevalence of deletions over duplications must lead to the elimination of various types of direct repeats from the prokaryotic genomes in the course of their evolution. Curr Genet, 1989 Mar, 15(3), 221 - 9 Chloroplast ribosomal DNA organization in the chromophytic alga Olisthodiscus luteus; Delaney TP et al.; There are almost no data describing chloroplast genome organization in chromophytic (chlorophyll a/c) plants . In this study chloroplast ribosomal operon placement and gene organization has been determined for the golden-brown alga Olisthodiscus luteus . Ribosomal RNA genes are located on the chloroplast DNA inverted repeat structure . Nucleotide sequence analysis, demonstrated that in contrast to the larger spacer regions in land plants, the 16S-23S rDNA spacer of O . luteus is only 265 bp in length . This spacer contains tRNA(Ile) and tRNA(Ala) genes which lack introns and are separated by only 3 bp . The sequences of the tRNA genes and 16S and 23S rDNA termini flanking the spacer were examined to determine homology between O . luteus, chlorophytic plant chloroplast DNA, and prokaryotes. Antonie Van Leeuwenhoek, 1989 Mar, 55(3), 291 - 6 Rhodospirillum centenum, sp . nov., a thermotolerant cyst-forming anoxygenic photosynthetic bacterium; Favinger J et al.; A novel non-sulfur purple photosynthetic bacterium, designated Rhodospirillum centenum, was isolated from an enrichment culture designed to favor growth of anoxygenic photosynthetic N2-fixing bacteria . R . centenum grows optimally at 40-42 degrees C and has the capacity to produce cytoplasmic 'R bodies', refractile structures not observed hitherto in photosynthetic prokaryotes . The bacterium is also unusual among photosynthetic bacteria in that it forms desiccation-resistant cysts when grown aerobically in darkness with butyrate as the sole carbon source. Anticancer Res, 1989 Mar-Apr, 9(2), 373 - 81 The interaction between fibronectin and DNA; Siddiqa A et al.; An examination of the DNA binding domain structure of bovine plasma fibronectin (Fn) was undertaken by a combination of limited proteolytic cleavage and Western blotting . A time course digestion of fibronectin with cathepsin D produced a number of proteolytic fragments possessing DNA binding activity . After two min digestion, two DNA binding fibronectin fragments of Mr approximately 180kd and 120kd were detected . Upon further digestion, a fibronectin fragment of Mr 18kd was detected . Thus it would appear that under physiological ionic strength only a single DNA binding domain exists in the fibronectin molecule . It was also demonstrated that the interaction of fibronectin with DNA is not ionic in nature, as heat denaturation of protein totally abolishes the DNA binding activity . An examination of possible sequence specificity of DNA binding activity of fibronectin was also undertaken by dot blotting the bovine plasma fibronectin and using {32P} labelled lambda FC 40 DNA containing approximately 16 kd of 5' end of the chicken fibronectin gene . Its binding to fibronectin was approximately twice the binding of {32P} labelled wild type lambda, where as binding to control of equimolar concentration of calf thymus histone H 2 A was approximately equal . The use of a smaller subcloned region, a approximately 1.9kb fragment of DNA from the 5' end of chicken fibronectin gene and wild type lambda DNA showed approximately 2 fold increase in histone binding and approximately 7 fold increase in fibronectin binding, indicating preferential fibronectin binding with eukaryotic DNA as compared to prokaryotic DNA . Further investigation of sequence specificity showed that a 0.45kb DNA fragment from the 5' end of chicken fibronectin gene, containing a number of elements characteristic of promoter, demonstrated approximately 2 fold higher level of binding with fibronectin and approximately 3.5 fold less binding activity with equimolar concentration of histone H2A when compared with a 1.4kb fragment of chicken fibronectin gene from the 1st exon . These results suggest fibronectin binding may be preferential to the promoter region of its own gene which could have possibly a regulatory function. Shi Yan Sheng Wu Xue Bao, 1989 Mar, 22(1), 99 - 110 {Whole nucleotide sequence of penicillin G acylase gene and its flanking region from E . coli}; Guo LH et al.; Some of microorganisms have been known to possess penicillin G acylase activity . The E . coli derived penicillin G acylase (PGA) can catalyze the conversion of penicillin G into phenylacetic acid and 6-amino-penicillanic acid, the latter is used as the starting compound for the industrial formation of semi-synthetic penicillins . Apart from its industrial importance, the enzyme PGA displays a number of interesting properties . Catalytically active enzyme is localized in the periplasmic space of E . coli cells and composed of two dissimilar subunits . The two subunits are apparently produced from a precursor protein, via a processing pathway hitherto unique in its features for a prokaryotic enzyme . The studies on processing of the precursor and on the relationship between structure and function of the mature enzyme are important theoretically . Previously we cloned a 3.5 kb DNA fragment from a strain (E . coli AS 1.76), which displays PGA activity . In this paper, we report a nucleotide sequence of the 3.5 kb DNA fragment containing PGA gene . After insertion of the DNA fragment into EcoR I and Hind III sites in pWR 13, pPGA 20 had been obtained . We subcloned the Hind III and Bg1 II treated fragment of 1.6 kb in length from pPGA 20 into Hind III and BamH I sites of pWR 13 to get a pPGA 1.6, and Bg1 II and EcoR I treated fragment of 1.9 kb in length into BamH I and EcoR I sites of pWR 13 to get a pPGA 1.9 . The linearized pPGA 1.9 which were digested with appropriate restriction enzymes were progressively shortened from both ends respectively by digestion with Bal 31 nuclease, followed by cleavage of shortened target DNA off vector DNA molecules with appropriate restriction enzymes . The series of the DNA fragments shortened from EcoR I end were then cloned into plasmid pWR 13 which had previously digested with Hind III and Sma I enzymes (Fig . 1) . The DNA fragment cloned in pWR 13 were directly sequenced on the resulted plasmids by using primer I and primer II . Thus we have obtained the complete nucleotide sequence of the 3.5 kb DNA fragment . The 3.5 kb fragment contains an intact PGA gene which is 2.6 kb.(ABSTRACT TRUNCATED AT 400 WORDS) Mikrobiol Zh, 1989 Mar-Apr, 51(2), 107 - 17 {A mechanism of transcription in prokaryotes: stages and place in the realization of genetic information}; Babichev VV; Transcription, being the first stage on the way of genetic information realization, is the most important to regulate expression of genes . It is a complex process in procaryotes . At different stages it is controlled by definite systems of a cell, that in the end, provides a regulated information flow initiating all processes which proceed in the organisms . At present the problem of the gene activity regulation plays a key role in the molecular biology and intensively develops both in theoretical and applied aspects. Mutat Res, 1989 Mar-May, 220(2-3), 269 - 78 Use of data from bacteria to interpret data on DNA damage processing in mammalian cells; Hutchinson F; The most important reason for determining the changes in base sequence in the processing of DNA damage is to determine mechanisms . Currently, much more is known about these mechanisms in prokaryotes, partly because the experiments are easier and quicker to do in bacteria, and partly because of the wealth of well characterized bacterial mutants deficient in various DNA repair pathways . This paper summarizes some information on the mechanisms in bacteria that are involved in the induction by various agents of base change mutations, 1- and 2-base deletions or additions that cause frameshifts, and more complicated insertions and deletions that involve up to tens of base pairs . For gross DNA rearrangements such as large deletions involving hundreds or thousands of base pairs, there is actually more information available in mammalian cells than in bacterial cells . It is suggested that deletions of several kilobases or more in bacteria are not easy to detect because they have a high probability of deleting both the gene under study and an adjacent essential gene, forming a nonviable cell . In mammalian cells, the large size (30-40-kb pairs) of the average gene, including both introns and exons, means that a large deletion is more likely to be confined to a single gene and less likely to lead to a nonviable cell. Biotechniques, 1989 Mar, 7(3), 276 - 80 A DNA cassette containing a trimerized SV40 polyadenylation signal which efficiently blocks spurious plasmid-initiated transcription; Maxwell IH et al.; A head-to-tail trimer of the SV40 Bcl I-Bam H1 DNA fragment, specifying polyadenylation of RNA transcripts, was cloned as a cassette flanked by multiple restriction sites . Insertion of the trimer into several expression vectors efficiently prevented spurious expression of reporter genes resulting from transcriptional initiation in prokaryotic plasmid sequences in transfected mammalian cells. Plasmid, 1989 Mar, 21(2), 113 - 9 In vivo assembly of chromatin on pBR322 sequences cloned into yeast plasmids; Estruch F et al.; In order to study the in vivo assembly of chromatin on prokaryotic DNA templates, we have transformed yeast cells with plasmids pAJ50 and pRB58, which contain pBR322 sequences . In both cases nucleosomes are assembled in vivo on pBR322 DNA, although the nucleosomes are not homogeneous in size . To explore whether there is any preference for nucleosome assembly along pBR322 sequences, we have used an indirect end labeling method . The results indicate that most nucleosomes are placed at random on pBR322, although the probability for histone octamers to interact with some short regions is somewhat reduced . These regions coincide with sequences in which the frequency distribution of nucleosomes reconstituted in vitro (E . Caffarelli et al . (1988) Eur . J . Biochem . 171, 497-501) is low . Sequence determinants that direct chromatin assembly in vitro seem thereby to act to some extent in vivo. Rev Infect Dis, 1989 Mar-Apr, 11 Suppl 2, S431 - 5 Serology of mycobacteria: characterization of antigens recognized by monoclonal antibodies; Young DB et al.; Analysis of the antibody response to mycobacterial extracts has identified a limited set of proteins that are recognized as immunodominant in the BALB/c strain of mice . Detailed characterization has revealed that several of these antigens are homologues of proteins known to be induced in response to environmental stress stimuli in other prokaryotic and eukaryotic cell types . It is proposed that differential gene expression may play a role in determining which antigens are recognized during infection and that highly conserved stress proteins could be involved in generation of autoimmune responses. Mol Microbiol, 1989 Mar, 3(3), 405 - 10 Functional characterization of a putative internal promoter sequence between the 16S and the 23S RNA genes within the Escherichia coli rrnB operon; Zacharias M et al.; Transcription of ribosomal RNAs in Escherichia coli is started from two strong tandem promoters, P1 and P2 . It is known, however, that internal promoter-like structures occur and in a recent report (Mankin et al., 1987) a promoter sequence Pi within the 16S and 23S RNA spacer region showing good homology to the prokaryotic consensus promoter structure was identified . It was proposed that this putative promoter has a possible function in the transcription of ribosomal RNAs in E . coli . Fusion of various DNA fragments containing the putative promoter sequence and different parts of the 16S/23S spacer region as well as the 23S RNA to the galactokinase gene allowed us to assess the functional activity of the promoter in vivo . To determine any growth rate dependent function of the putative promoter, the measurements were performed under different growth conditions . The promoter activity did not exceed 7% of the lac promoter under in vivo assay conditions . In addition, transcription starting at the promoter Pi did not proceed through the entire 23S RNA gene . We conclude, therefore, that transcription from Pi does not contribute significantly to the synthesis of ribosomal RNAs . Thus its functional significance, if any, remains elusive. FEBS Lett, 1989 Feb 27, 244(2), 439 - 46 Species-specific variation in signal peptide design . Implications for protein secretion in foreign hosts; von Heijne G et al.; Secretory signal peptides from individual prokaryotic and eukaryotic species have been analyzed, and the lengths and amino acid compositions of the positively charged amino-terminal region, the central hydrophobic region, and the carboxy-terminal cleavage-region have been compared . We find distinct differences between species in all three regions . Implications for protein secretion in foreign hosts are discussed. Nucleic Acids Res, 1989 Feb 25, 17(4), 1459 - 74 Related sites in human and herpesvirus DNA recognized by methylated DNA-binding protein from human placenta; Zhang XY et al.; Methylated DNA-binding protein (MDBP) from mammalian cells binds specifically to six pBR322 and M13mp8 DNA sequences but only when they are methylated at their CpG dinucleotide pairs . We cloned three high-affinity MDBP recognition sites from the human genome on the basis of their binding to MDBP . These showed much homology to the previously characterized prokaryotic sites . However, the human sites exhibited methylation-independent binding apparently because of the replacement of m5C residues with T residues . We also identified three other MDBP sites in the herpes simplex virus type 1 genome, two of which require in vitro CpG methylation for binding and are in the upstream regions of viral genes . A comparison of MDBP sites leads to the following partially symmetrical consensus sequence for MDBP recognition sites: 5'-R T m5Y R Y Y A m5Y R G m5Y R A Y-3'; m5Y (m5C or T), R (A or G), Y (C or T) . This consensus sequence displays an unusually high degree of degeneracy . Also, interesting deviations from this consensus sequence, including a one base-pair deletion in the middle, are sometimes observed in high-affinity MDBP sites. J Mol Biol, 1989 Feb 20, 205(4), 771 - 5 A lower size limit exists for export of fragments of an outer membrane protein (OmpA) of Escherichia coli K-12; Freudl R et al.; The ompA gene codes for a 346 residue precursor of a 325 residue protein of the outer membrane of Escherichia coli K-12 . Internally and/or COOH-terminally deleted genes were constructed that encode 123, 116, 88, 72 or 68 residue precursors . The former three were processed and localized to the periplasmic space; the latter two were not processed and remained cytosolic . These data suggest that the signal sequence has to interact with a component of the export apparatus (the Sec pathway) before translation is finished . Comparison of these results with others obtained for prokaryotic and eukaryotic systems shows that: (1) a very similar lower size limit exists for membrane translocation of the 147 residue chicken prelysozyme or the 229 residue bovine preprolactin; (2) precursors smaller than those reported here can be translocated in both systems; (3) the latter translocation, in contrast to, for example, the ompA gene products, does not depend on the cellular export machinery but most likely requires folding of the precursors into an export-competent conformation . In general, at least two quite different, not necessarily mutually exclusive, mechanisms for translocation of a protein across or assembly into a membrane appear to exist. J Biol Chem, 1989 Feb 15, 264(5), 2672 - 7 One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556; Kita K et al.; Complex II (succinate:ubiquinone oxidoreductase) is an important component of both the tricarboxylic acid cycle and of the aerobic respiratory chains of eukaryotic and prokaryotic organisms . The enzyme has been purified from numerous sources and appears to be highly conserved from considerations of both the amino acid sequences of the catalytic subunits and from the prosthetic groups associated with the enzyme . The sdh operon has been cloned and sequenced from Escherichia coli, but the enzyme from this source has, so far, resisted attempts at biochemical purification . In this work, a one-step purification of the enzyme is described which yields a stable four-subunit enzyme which has a high specific activity . This purification takes advantage of a strain which overproduces the enzyme by 10-fold due to the presence of a multicopy plasmid containing the cloned sdh operon . The purified complex II has one FAD, eight non-heme irons, seven acid-labile sulfides, and one protoheme IX per molecule . The enzyme has been reconstituted in phospholipid vesicles and demonstrated to reduce ubiquinone-8, the natural electron acceptor, at a high rate. Arch Biochem Biophys, 1989 Feb 15, 269(1), 1 - 10 Functional reconstitution of prokaryote and eukaryote membrane proteins; Maloney PC et al.; A new strategy for the functional reconstitution of membrane proteins is described . This approach introduces a new class of protein stabilizing agents--osmolytes--whose presence at high concentration (10-20%) during detergent solubilization prevents the inactivations that normally occur when proteins are extracted from natural membranes . Osmolytes that act in this way include compounds such as glycerol and higher polyols (erythritol, xylitol, sorbitol), sugars (glucose, trehalose), and certain amino acids (glycine, proline, betaine) . The beneficial effects of osmolytes are documented by reconstitution of a variety of prokaryote and eukaryote membrane proteins, including several proton- and calcium-motive ATPases, cation- and anion-linked solute carriers (symport and antiport), and a membrane-bound hydrolase from endoplasmic reticulum . In all cases, the presence of 20% glycerol or other osmolyte during detergent solubilization led to 10-fold or more increased specific activity in proteoliposomes . These positive effects did not depend on use of any specific detergent for protein solubilization, nor on any particular method of reconstitution, but for convenience most of the work reported here has used octylglucoside as the solubilizing agent, followed by detergent-dilution to form proteoliposomes . The overall approach outlined by these experiments is simple and flexible . It is now feasible to use reconstitution as an analytical tool to study the biochemical and physiological properties of membrane proteins. Biochim Biophys Acta, 1989 Feb 13, 979(1), 69 - 76 The role of the positively charged N-terminus of the signal sequence of E . coli outer membrane protein PhoE in export; Bosch D et al.; Signal sequences of prokaryotic exported proteins have a dipolar character due to positively charged amino-acid residues at the N-terminus and to a preferentially negatively charged region around the cleavage site . The role of the two lysine residues at the N-terminus of the signal sequence of outer membrane protein PhoE of E . coli-K12 was investigated . Replacement of both of these residues by aspartic acid slightly affected the kinetics of protein translocation in vivo . This export defect, which was observed only when PhoE was overproduced, could not be suppressed by the prlA4 mutation, which has been shown to restore export defects caused by alterations in the hydrophobic core of the signal sequences of various exported proteins . In an in vitro translocation assay, the export defect was more pronounced . Replacement of both lysines by uncharged residues did not disturb the kinetics of protein export in vivo . In the in vitro assay, an extraordinarily efficient processing was detected upon incubation of this precursor with inverted cytoplasmic membrane vesicles . However, this efficient processing was not accompanied by more efficient translocation of the protein . We conclude that the positively charged residues at the N-terminus of the signal sequence are not essential for protein export, but contribute to the efficiency of the process. Cell, 1989 Feb 10, 56(3), 455 - 65 Intron mobility in the T-even phages: high frequency inheritance of group I introns promoted by intron open reading frames; Quirk SM et al.; Intron mobility in the T-even phages has been demonstrated . Efficient nonreciprocal conversion of intron minus (In-) alleles to intron plus (In+) occurred for the td and sunY genes, but not for nrdB . Conversion to In+ was absolutely dependent on expression of the respective intron open reading frame (ORF) . Introns were inserted at their cognate sites in an intronless phage genome via an RNA-independent, DNA-based, duplicative recombination event that was stimulated by exon homology . The td intron ORF product directs the endonucleolytic cleavage of DNA, targeting the site of intron integration . A 21 nucleotide deletion of the integration site abolished high frequency intron inheritance . These experiments provide a novel example of gene conversion in prokaryotes, while suggesting a molecular rationale for the inconsistent distribution of introns within highly conserved exon contexts of the T-even phage genomes. J Biol Chem, 1989 Feb 5, 264(4), 1968 - 71 Chloroplast ribosomal protein L13 is encoded in the nucleus and is considerably larger than its bacterial homologue . Construction, immunoisolation, and nucleotide sequence (including transit peptide) its cDNA clone from an angiosperm; Phua SH et al.; Chloroplast ribosomes of higher plants are of the prokaryotic ribosome motif but, unlike in bacteria, their ribosomal protein (r-protein) genes are distributed between the organelle and the nucleus . In order to isolate some of the nuclear-encoded r-protein genes, we have raised antibodies to several spinach chloroplast r-proteins and constructed spinach cDNA expression libraries in lambdagt11 . Screening the libraries with one of the antisera yielded three cDNA clones for r-protein L13, an early 50 S subunit assembly protein essential for RI50 formation . The cDNA clone encodes, beginning with a Met codon in the consensus plant initiator context, a polypeptide of 250 amino acid residues . The NH2-terminal 60 residues bear the characteristic features of a chloroplast transit peptide . The putative mature L13 protein, which has common immunoepitopes with Escherichia coli L13, is 34% longer than the E . coli homologue . It has 56% sequence identity with E . coli L13 in the homologous region, but no identity to any known protein in the extra stretch . There are two neighboring ATG codons in the 5' region and two putative plant polyadenylation signals in the 3'-untranslated region of the cDNA . Their possible effect to increase translational efficiency is discussed, and the importance of encoding a RI50 protein in the nuclear genome for possible nuclear control of chloroplast protein synthesis is noted. J Biol Chem, 1989 Feb 5, 264(4), 1915 - 8 Fused bacterial luciferase subunits catalyze light emission in eukaryotes and prokaryotes; Boylan M et al.; A monocistronic luxAB gene containing the luxA and luxB genes coding for bacterial luciferase has been generated by site-specific mutagenesis so that it is now possible to express luciferase under control of a single promoter in eukaryotes as well as in prokaryotes . The fused luciferase subunits (alpha and beta), linked by a decapeptide, were synthesized in yeast under the Gal-4 promoter, in Escherichia coli under the T7-phage promoter, and in vitro in a rabbit reticulocyte lysate after transcription and capping of the mRNA under the SP6 phage promoter . Replacement of the ATG codon initiating the luxB sequence in the fused luxAB gene with CAG prevented internal initiation and allowed purification of a highly active fused luciferase in the absence of the beta-luciferase subunit . Consequently luciferase activity can be directly attributed to the fused luciferase alone and does not require complementation with free beta subunit of luciferase . Light emission could be measured in yeast and bacterial cells without the need for cell lysis providing the basis for measuring gene expression directly in vivo . These results demonstrate the potential applicability of the fused bacterial luciferase genes as a reporter of gene expression both in prokaryotic and eukaryotic systems. J Endocrinol Invest, 1989 Feb, 12(2), 77 - 86 Binding of thyrotropin to selected Mycoplasma species: detection of serum antibodies against a specific Mycoplasma membrane antigen in patients with autoimmune thyroid disease; Sack J et al.; Radiolabeled human (hTSH) and bovine (bTSH) thyroid stimulating hormone was shown to bind to five species of Mycoplasma, the wall-less prokaryotes . The maximum binding capacity of 125I-bTSH to these five species was about 7.9 x 10(-13) moles-1.4 x 10(-12) moles for 50-100 micrograms protein with dissociation constants of approximately 1.7 to 2.2 x 10(-7)M . Approximately 50% of the 125I-bTSH binding was displaced by excess, unlabeled bTSH or hTSH, but labeled bTSH was not effectively displaced by growth hormone, LH, FSH, prolactin, or the beta subunit of hTSH, FSH and LH . Antisera prepared against Mycoplasma gallisepticum and Mycoplasma pneumoniae bound to human thyroid membranes and guinea pig fat cells, suggesting that receptors on human thyroid tissues and on Mycoplasma cells may have similarities in antigenicity . These findings were substantiated by the occurrence of TSH binding to Mycoplasma antisera . Further, sera from three of six patients with Graves' disease containing antibodies to thyroid tissues also reacted to a 108 Kd polypeptide of Mycoplasma gallisepticum. Electrophoresis, 1989 Feb, 10(2), 73 - 5 Report of workshop on cellular protein databases derived from two-dimensional polyacrylamide gel electrophoresis; Neidhardt FC; A workshop entitled Cellular Protein Databases from Two-Dimensional Gel Electrophoresis was held in Atlanta, Georgia, 28 February-1 March 1987 . Its purpose was to assess the status of two-dimensional gel electrophoresis of proteins as a research methodology in biological systems, particularly in the generation of cellular protein databases . The workshop participants summarized current studies on a variety of biological systems, both prokaryotic and eukaryotic . Analysis of the progress being made led to the conclusion that electrophoretic techniques, supported by automatic scanning of gel images and computer-assisted processing, analysis and matching of gel images, are now capable of generating databases of great potential value . Factors affecting the reproducibility of protein spot patterns on gels were identified, and the extent to which gel pattern variability causes difficulties in communicating results and in integrating information from different laboratories was assessed . Measures were suggested to help overcome obstacles to the generation of comprehensive cellular protein databases from the electrophoretic resolution of total cellular proteins. EMBO J, 1989 Feb, 8(2), 577 - 85 Mutational analysis of a prokaryotic recombinational enhancer element with two functions; Hubner P et al.; The site-specific DNA inversion system Cin encoded by the bacteriophage P1 consists of a recombinase, two inverted crossing-over sites and a recombinational enhancer . The latter approximately 75 bp long genetic element is bifunctional due to its location within the 5' part of the cin gene encoding the recombinase . In order to determine the essential nucleotides for each of its two biological functions we randomly mutated the recombinational enhancer sequence sis(P1) and analysed both functions of the mutants obtained . Three distinct regions of this sequence were found to be important for the enhancer activity . One of them occupies the middle third of the enhancer sequence and it can suffer a number of functionally neutral base substitutions, while others are detrimental . The other two regions occupy the two flanking thirds of the enhancer . They coincide with binding sites of the host-coded protein FIS (Factor for Inversion Stimulation) needed for efficient DNA inversion in vitro . These sequences appear to be highly evolved allowing only a few mutations without affecting either of the biological functions . Taking the effect of mutations within these FIS binding sites into account a consensus sequence for the interaction with FIS was compiled . This FIS consensus implies a palindromic structure for the recombinational enhancer . This is in line with the orientation independence of enhancer action with respect to the crossing-over sites. Proc Natl Acad Sci U S A, 1989 Feb, 86(4), 1234 - 8 Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana; Harper JF et al.; In plants, the transport of solutes across the plasma membrane is driven by a proton pump (H+-ATPase) that produces an electric potential and pH gradient . We have isolated and sequenced a full-length cDNA clone that encodes this enzyme in Arabidopsis thaliana . The protein predicted from its nucleotide sequence encodes 959 amino acids and has a molecular mass of 104,207 Da . The plant protein shows structural features common to a family of cation-translocating ATPases found in the plasma membrane of prokaryotic and eukaryotic cells, with the greatest overall identity in amino acid sequence (36%) to the H+-ATPase observed in the plasma membrane of fungi . The structure predicted from a hydropathy plot contains at least eight transmembrane segments, with most of the protein (73%) extending into the cytoplasm and only 5% of the residues exposed on the external surface . Unique features of the plant enzyme include diverged sequences at the amino and carboxyl termini as well as greater hydrophilic character in three extracellular loops. Biochem Biophys Res Commun, 1989 Jan 31, 158(2), 562 - 8 Evidence for a membrane-associated GTP-binding protein in Stigmatella aurantiaca, a prokaryotic cell; Derijard B et al.; Signal transducing G proteins are present in all eukaryotic cells, but they have not been found in prokaryotes so far . Myxobacteria, especially Stigmatella aurantiaca, are prokaryotic organisms able to exchange signals . Moreover, they exhibit an active phosphoinositide metabolism, whose intensity is dependent on the physiological state of the cell . Therefore G proteins potentially involved in the activation of phospholipid metabolism or any other event stimulated by external signals were looked for in S . aurantiaca membranes . Using a photoaffinity technique based on cross-linking of radioactive GTP to membrane-associated proteins under UV irradiation, only one major band in the range of 54 kDa was detected . This GTP-binding protein present specifically in membrane preparations binds also GDP, whereas it does not react with other nucleotides, such as ATP, UTP and CTP . The membrane-bound G protein of S . aurantiaca needs further characterization but could be homologous to G alpha subunits found in cytoplasmic membranes of eukaryotes. Nature, 1989 Jan 26, 337(6205), 380 - 2 The relationship of a prochlorophyte Prochlorothrix hollandica to green chloroplasts; Turner S et al.; It is generally accepted that chloroplasts arose from one or more endosymbiotic events between an ancestral cyanobacterium and a eukaryote . Such an origin fits well in the case of the chloroplasts of rhodophytes that, like cyanobacteria, contain chlorophyll a and phycobilin pigments . The green chloroplasts from higher plants, green algae, and euglenoids however, contain chlorophyll b as well as chlorophyll a, and lack phycobilins . Consequently, it has been suggested that they arose independently of the rhodophyte chloroplasts, from an ancestral prokaryote containing that complement of pigments . The 'prochlorophytes' Prochloron didemni (an exosymbiont on didemnid ascidians) and Prochlorothrix hollandica (a recently discovered, free-living, filamentous form) have been suggested to be modern counterparts of the ancestor of the green chloroplasts because they are prokaryotes that also contain both chlorophylls a and b, and lack phycobilins . We report here a 16S rRNA-based phylogenetic analysis of P . hollandica . The organism is found to fall wi