Prokaryotes

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 within the cyanobacterial line of descent, as do the green chloroplasts, but it is not a specific relative of green chloroplasts . Thus, similar pigment compositions do not necessarily reflect close evolutionary relationships.

Nature, 1989 Jan 26, 337(6205), 382 - 5
psbA genes indicate common ancestry of prochlorophytes and chloroplasts; Morden CW et al.; It has long been suspected that chloroplasts evolved after an endosymbiotic event involving a photosynthetic prokaryote, presumably a cyanobacterium, and a eukaryotic organism . Recent studies have provided strong evidence about the cyanobacterial nature of chloroplasts . Since the discovery of prochlorophytes, oxygen-evolving photosynthetic prokaryotes containing chlorophyll a and chlorophyll b and lacking phycobiliproteins, there has been speculation that these represent evolutionary intermediates between cyanobacteria and chloroplasts . Prochloron sp., the first described prochlorophyte, proved difficult to work with because it is an obligate symbiont of marine ascidians . Prochlorothrix hollandica, a recently isolated, freshwater filamentous prochlorophyte, is easily maintained in the laboratory . Overall pigment composition and thylakoid membrane structure of P . hollandica suggest it has intermediate characteristics between cyanobacteria and the chloroplasts of higher plants . The P . hollandica psbA genes, which encode the photosystem II thylakoid protein D1, were cloned and sequenced and the sequences compared to those reported for cyanobacteria, a green alga, a liverwort, and several higher plants . The two psbA genes present in P . hollandica encode an identical amino-acid sequence . As in all chloroplast psbA genes, there is a seven amino-acid gap near the C terminus of the derived protein relative to the protein predicted by cyanobacterial genes, suggesting that P . hollandica is part of the lineage that led to chloroplasts after a divergence from cyanobacteria . This hypothesis is also supported by phylogenetic analysis of derived D1 amino-acid sequences from psbA genes of thirteen taxa on the basis of parsimony.

J Mol Biol, 1989 Jan 20, 205(2), 315 - 30
Sequences linked to prokaryotic promoters can affect the efficiency of downstream termination sites; Telesnitsky AP et al.; The efficiency of transcription termination at certain well-defined prokaryotic rho-independent terminators depends on the promoter unit from which transcription is initiated . Some promoter units allow substantial readthrough of strong termination signals, a phenomenon we term "factor-independent antitermination" . This observation is not easily explained by current models for prokaryotic terminator function that consider the terminator to be a "cassette" involving only sequences and RNA transcript structures in the immediate region of the terminator or directly upstream . When transcription is carried out in vitro employing only purified Escherichia coli RNA polymerase, up to 20 times as many RNA polymerase molecules pass through a particular terminator when transcription is initiated from the E . coli tac promoter unit, as compared to transcription initiated from the T7A1 or rrnB P2 promoters . This effect cannot be attributed to antitermination factors separate from the core RNA polymerase . Similar differences in termination efficiency are found for the same promoters in vivo . These termination differences are affected by sequences just downstream from the start site for transcription, including those in the +25 region of the nascent transcript . These early transcribed sequences can confer factor-independent antitermination onto a heterologous promoter, but only when the sequences are precisely positioned relative to the start site for transcription . We have considered several possible models to explain how early transcribed sequences might affect termination, including those in which the 5' end of the transcript interacts with either the terminator RNA or the polymerase . We favor an alternative model in which these sequences interact with the core RNA polymerase to convert the enzyme from a termination-proficient state (T-state) to a conformation resistant to termination (R-state) . Such enzyme conformations may be an important component of factor-dependent antitermination systems.

Biochem Biophys Res Commun, 1989 Jan 16, 158(1), 45 - 51
Amino acid residues essential for catalysis by peptidyl dipeptidase-4 from Pseudomonas maltophilia; Lanzillo JJ et al.; To assess residues essential for catalysis by prokaryotic peptidyl dipeptidase-4, the enzyme was subjected to chemical modification by a series of reagents . Treatment with either tetranitromethane or N-acetylimidazole abolished catalytic activity . Hydroxylamine reversed inactivation by acetylimidazole only . Thus, an essential tyrosine is indicated . Enzymatic activity also was quenched by either trinitrobenzenesulfonic acid or diethyl pyrocarbonate . Inactivation by these reagents was not reversed by hydroxylamine . These data suggest an essential lysine . The competitive inhibitor Phe-Arg protected partially against inactivation by tetranitromethane, and fully against inactivation by N-acetylimidazole . The substrate Hip-Phe-Arg protected against inactivation by trinitrobenzenesulfonic acid and diethyl pyrocarbonate . Thus, both tyrosine and lysine are located at the catalytic site.

Eur J Biochem, 1989 Jan 15, 179(1), 39 - 45
Structural features of lipoprotein lipase . Lipase family relationships, binding interactions, non-equivalence of lipase cofactors, vitellogenin similarities and functional subdivision of lipoprotein lipase; Persson B et al.; A structural homology between lipoprotein lipase, pancreatic lipase and hepatic lipase is known and indicates that all three lipases are members of a common protein family . Lipoprotein lipase and pancreatic lipase utilize small protein co-factors, apolipoprotein C-II and co-lipase, respectively, but comparisons reveal no homology between the co-factor molecules . Hence, they do not show the same relationship as their target enzymes . Neither do screenings detect any extensive similarities between lipoprotein lipase, serine hydrolases, or apolipoproteins . Scannings against data bank proteins show that a 105-residue segment of lipoprotein lipases exhibits a 35-40% residue identity with a sub-region of Drosophila vitellogenins . One fifth of the conserved amino acid residues (8 of 40) are glycine, a pattern which is typical of distantly related forms of protein families . This supports a true relationship between large segments of Drosophila vitellogenins and lipases . Physiological and functional aspects of the vitellogenin/lipoprotein lipase similarities are given . The region concerned is entirely within the N-terminal domain of lipoprotein lipase and constitutes the segment where the similarity to hepatic and pancreatic lipases is most pronounced . Within this lipase region a 10-residue putative lipid-binding site exists for which further similarities have been found to the otherwise not closely related lingual/gastric lipases, prokaryotic lipases and lecithin-cholesterol acyltransferase . Another segment in lipoprotein lipase, where the heparin-binding site has been mapped, exhibits a correlation between strength of heparin binding and extent of basic residues among members of the lipase family . It further exhibits weak similarities with the 'Zn-finger' DNA-binding segment of steroid hormone receptors and may indicate convergence in a binding interaction . Thus, a functional subdivision of lipoprotein lipase into different segments can be distinguished.

Biochem J, 1989 Jan 15, 257(2), 529 - 34
Cloning, sequence analysis and over-expression of the gene for the class II fructose 1,6-bisphosphate aldolase of Escherichia coli; Alefounder PR et al.; Nucleotide sequence analysis of the Escherichia coli chromosomal DNA inserted in the plasmid pLC33-5 of the Clarke and Carbon library {Clarke & Carbon (1976) Cell 9, 91-99} revealed the existence of the gene, fda, encoding the Class II (metal-dependent) fructose 1,6-bisphosphate aldolase of E . coli . The primary structure of the polypeptide chain inferred from the DNA sequence of the fda gene comprises 359 amino acids, including the initiating methionine residue, from which an Mr of 39,146 could be calculated . This value is in good agreement with that of 40,000 estimated from sodium dodecyl sulphate-polyacrylamide gel electrophoresis of the purified dimeric enzyme . The amino acid sequence of the Class II aldolase from E . coli showed no homology with the known amino acid sequences of Class I (imine-forming) fructose 1,6-bisphosphate aldolases from a wide variety of sources . On the other hand, there was obvious homology with the N-terminal sequence of 40 residues already established for the Class II fructose 1,6-bisphosphate aldolase of Saccharomyces cerevisiae . These Class II aldolases, one from a prokaryote and one from a eukaryote, evidently are structurally and evolutionarily related . A 1029 bp-fragment of DNA incorporating the fda gene was excised from plasmid pLC33-5 by digestion with restriction endonuclease HaeIII and subcloned into the expression plasmid pKK223-3, where the gene came under the control of the tac promoter . When grown in the presence of the inducer isopropyl-beta-D-thiogalactopyranoside, E . coli JM101 cells transformed with this recombinant expression plasmid generated the Class II fructose 1,6-bisphosphate aldolase as approx . 70% of their soluble protein . This unusually high expression of an E . coli gene should greatly facilitate purification of the enzyme for any future structural or mechanistic studies.

Cell, 1989 Jan 13, 56(1), 111 - 8
Transcription-driven supercoiling of DNA: direct biochemical evidence from in vitro studies; Tsao YP et al.; The translocation of an RNA polymerase elongation complex along double helical DNA has been proposed to generate positive supercoiling waves ahead of and negative supercoiling waves behind the transcription ensemble . This twin supercoiled domain model has been tested in vitro . In the presence of prokaryotic DNA topoisomerase I, which selectively removes negative supercoils, transcription from a single promoter results in rapid and extensive positive supercoiling of the DNA template . The accumulation of positive supercoils in the DNA template requires continued movement of the elongation complex as well as sizable nascent RNA chains . These in vitro results provide direct biochemical evidence supporting the twin supercoiled domain model of transcription . Furthermore, the magnitute of DNA supercoiling (torsional) waves generated by transcription is much greater than previously expected, suggesting that transcription is one of the principal factors affecting intracellular DNA supercoiling.

Nature, 1989 Jan 5, 337(6202), 44 - 7
GroE heat-shock proteins promote assembly of foreign prokaryotic ribulose bisphosphate carboxylase oligomers in Escherichia coli; Goloubinoff P et al.; Assembly of foreign prokaryotic ribulose bisphosphate carboxylases (Rubiscos) in Escherichia coli requires both heat-shock proteins groEL and groES . GroEL is related to a chloroplast protein implicated in Rubisco assembly . Bacteria and chloroplasts therefore have a conserved mechanism that uses auxiliary proteins to assist in the assembly of Rubisco.

Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 247 - 51
Cloning, analysis, and expression of murine perforin 1 cDNA, a component of cytolytic T-cell granules with homology to complement component C9; Lowrey DM et al.; The nucleotide sequence coding for the cytotoxic T-lymphocyte (CTL) protein perforin 1 (P1) has been determined and the corresponding protein sequence has been derived . Murine CTL cDNA libraries contained in the vector lambda gt11 were screened by using a monospecific antiserum to purified P1 . Three recombinant phages were isolated and their cDNA inserts were sequenced . The derived protein sequence contains 554 amino acids and displays, as expected, considerable homology with certain functional domains in the complement components C9, C8 alpha, C8 beta, and C7 . The identity of P1 cDNA clones was verified by prokaryotic expression and the reactivities of antisera produced to the expressed proteins . In addition, antisera were produced to two synthetic peptides located in the center and C-terminal portions of P1 . All antisera reacted with purified P1 . In Northern blot analyses, P1 cDNA probes recognized a 2.9-kilobase mRNA only in CTL . Perforin mRNA was found in all cloned CTL and in all mixed lymphocyte reactions that gave rise to cytotoxic cells . Perforin mRNA was also detected in virus-specific CTL that had been generated in vivo and isolated from liver tissue of mice infected with lymphocytic choriomeningitis virus . The cell-specific expression of perforin is consistent with its postulated role in cytolysis.

Boll Soc Ital Biol Sper, 1989 Jan, 65(1), 13 - 8
{Construction of an apparatus for pulsed field electrophoresis for the analysis of high molecular weight DNA}; Gennarelli M et al.; An inexpensive apparatus for Pulsed Field Gel Electrophoresis constructed by the Authors, is described . According to our experimental condition it was found that this apparatus is capable to resolve DNA fragments in order of 50-1500 Kb . This range of analysis is adequate for the molecular studies adjacent regions of prokaryotes, primitive eukaryotes and mammals.

Genome, 1989, 31(2), 668 - 70
125 years of experimental heat shock research: historical roots of a discipline; Nover L; The history of experimental heat shock research over the last 125 years is briefly, outlined . Starting with reports on the upper temperature limits of plant survival (1864) and on the spontaneous regression of skin cancer after a severe local inflammation (1866), studies on the heat shock response today are a major field of modern cell biology and include all types of prokaryotic and eukaryotic organisms.

Biol Cell, 1989, 66(3), 215 - 23
Localization of functional centers on the prokaryotic ribosome: immuno-electron microscopy approach; Spirin AS et al.; Immuno-electron microscopy studies on the morphology of the Escherichia coli ribosome and the topography of its functional centers are summarized . A three-dimensional model of the ribosome with indications of the mRNA-binding site, the peptidyl-transferase center, the EF-Tu and EF-G-binding sites and the tRNA-binding sites in given . Thus, the mutual arrangement of the centers corresponding to the main functional activities of the ribosome during the process of translation is presented.

Int J Biochem, 1989, 21(5), 455 - 61
A proposal for a possible role of nucleosome positioning in the evolutionary adjustment of introns; Csordas A; 1 . Prokaryotes and yeast have mostly intronless genes, whereas the presence of a large number of extended introns are characteristic of the genes of of multicellular eukaryotic organisms which, however, as an exception also have a few intronless genes . 2 . According to the current view, the lack of introns in prokaryotic organisms and yeast is due to the selective pressure of a short cell division time . On the other hand, the presence of introns in multicellular eukaryotic organisms is explained by the lack of selective forces against them . 3 . In the present hypothesis it is proposed that introns were used as tools in the course of evolution for the organization of eukaryotic genes within the repeating units of nucleosomes, since the distinct DNA conformations of the nucleosome core particle and of the linker region, respectively, represent a constraint for the positioning of genes . 4 . Recently it was shown that initiation of transcription is inhibited when the promoter sequence is within a nucleosome . 5 . Since the nucleosomal organization of DNA leads to a severely deformed DNA helix and recognition of sequences by regulatory proteins is likely to depend on the conformation of the double helix, it is postulated that for the different sizes of eukaryotic genes which have to be organized within repeating units of nucleosomes, introns provided the flexibility of adjustment for the positioning of regulatory sequences, by drifting in length, sequence and position.

Ann Ist Super Sanita, 1989, 25(1), 145 - 8
The pR plasmid: a tool for studying DNA repair and mutagenesis in prokaryotic and eukaryotic cells; Battaglia PA et al.; The pR plasmid, a derivative of R46 plasmid, offers the possibility to have an experimental approach to three important problems related to UV repair and mutagenesis . By using this plasmid we were able to show: a) the pR mucAB genes need the cooperation of uvpl gene product to carry out their UV repair function; b) the expression of mucAB genes is regulated not only by lexA gene, but by a gene localized in the rep region of pR itself . This gene acts as an antirepressor of lexA; c) mammalian cells show an enhanced resistance to UV light when transformed by pR plasmid carrying the mucAB genes.

Mol Biol (Mosk), 1989 Jan-Feb, 23(1), 184 - 92
{Theoretical analysis of mechanisms of occurrence of DNA deletions in prokaryotic genomes based on direct repeats}; Kel' AE et al.; In the present work a mechanism of deletions emergence on the basis of complementary DNA regions mispairing of direct repeats has been investigated theoretically . A quantitative dependence of the rates of deletions emergence on such parameters of the flanking repeats as the nucleotide composition of repeats, the number of homology damages and the distance between repeated regions has been constructed . It has been proved, that using this relationship one can reliably evaluate the total rates of deletions emergence in the lacI gene sequence of E . coli according to the repeats arrangement in this gene.

Mol Biol (Mosk), 1989 Jan-Feb, 23(1), 175 - 83
{Statistical evidence for the correlation of DNA deletions in prokaryotic genomes with direct repeats}; Kolchanov NA et al.; In the present work a computer analysis of deletion localization in the sequence of the E . coli lacI gene has been carried out by the statistical weight method . Reliable statistical correlation of the deletions location sites with the arrangement of the most perfect direct repeats revealing the shortest distance between repeated fragments has been shown . At the same time statistical analysis did not reveal reliable connection of deletions localization regions with the expected sites of gyrase recognition, sites and other recombination sites . A conclusion has been drawn, that the mechanism of deletions emergence on the basis of repeats appears to be predominant.

Anal Biochem, 1989 Jan, 176(1), 78 - 81
A colony-blot technique for the detection of specific transcripts in eukaryotes; Maniak M et al.; We present a simple and rapid technique for the detection of specific transcripts in eukaryotic cells . The method allows the screening of large numbers of clones for the expression of a gene of interest, similar to the colony blotting techniques described for prokaryotes . We have used this method to monitor developmentally regulated transcription of endogenous genes and the expression of foreign genes in transformed Dictyostelium discoideum cells . The same procedure can be applied to detect specific transcripts in yeast and should thus provide a useful molecular tool for most biological systems.

Mol Gen Genet, 1989 Jan, 215(2), 209 - 16
Structural and functional organization of ColE2 and ColE3 replicons; Yasueda H et al.; The complete nucleotide sequences of the 1.5 kb regions of ColE2 and ColE3 plasmids containing the segments sufficient for autonomous replication have been determined . They are quite homologous (greater than 90%), indicating that these two plasmids share common mechanisms of initiation of replication and its regulation . An open reading frame with a coding capacity for a protein of about 300 amino acids is present in both ColE2 and ColE3 and it actually specifies the Rep (for replication) protein, which is the plasmid specific trans-acting factor required for autonomous replication . The amino acid sequences of the Rep proteins of ColE2 and ColE3 are quite homologous (greater than 90%) . The cis-acting sites (origins) where replication initiates in the presence of the trans-acting factors consist of 32 bp for ColE2 and 33bp for ColE3 . They are the smallest of all the prokaryotic replication origins so far reported . They are nonhomologous only at two positions, one of which, a deletion of a single nucleotide in ColE2 (or an insertion in ColE3), determines the plasmid specificity in interaction of the origins with the Rep proteins . Both plasmids carry a region with an identical nucleotide sequence and the one in ColE2, the IncA region, has been shown to express incompatibility against both ColE2 and ColE3 . These results indicate that these plasmids share a common IncA determinant . A possibility that a small anti-sense RNA is involved in copy number control and incompatibility (IncA function) was suggested.

DNA, 1989 Jan-Feb, 8(1), 1 - 13
The P450 superfamily: updated listing of all genes and recommended nomenclature for the chromosomal loci; Nebert DW et al.; In this update we provide a list of the 71 P450 genes and the four P450 pseudogenes that have been characterized as of September 30, 1988 . The chromosomal locations of many of these genes are also summarized . A modest revision of the initially proposed nomenclature of the P450 superfamily (Nebert et al., DNA 6, 1-11, 1987) is described specifically for the human and mouse chromosomal loci . The motivation for this revision is to conform to the rules of nomenclature for human and mouse genes . Recommendations for the naming of chromosomal loci include the root symbol "CYP" for human ("Cyp" for mouse), denoting "cytochrome P450." We recommend that this root also be used for other organisms . For a chromosomal locus, the root symbol is followed by an Arabic numeral designating the P450 family, a letter indicating the subfamily, and an Arabic numeral representing the individual gene within the family or subfamily . Numbers of the individual genes usually will be assigned in the order the genes are identified . This system is consistent with our earlier proposed nomenclature for P450 families and gene products from all eukaryotes and prokaryotes.

Proc Natl Acad Sci U S A, 1989 Jan, 86(2), 409 - 13
Presence of the hypermodified nucleotide N6-(delta 2-isopentenyl)-2-methylthioadenosine prevents codon misreading by Escherichia coli phenylalanyl-transfer RNA; Wilson RK et al.; The overall structure of transfer RNA is optimized for its various functions by a series of unique post-transcriptional nucleotide modifications . Since many of these modifications are conserved from prokaryotes through higher eukaryotes, it has been proposed that most modified nucleotides serve to optimize the ability of the tRNA to accurately interact with other components of the protein synthesizing machinery . When a cloned synthetic Escherichia coli tRNAPhe gene was transfected into a bacterial host that carried a defective phenylalanine tRNA-synthetase gene, tRNAPhe was overexpressed by 11-fold . As a result of this overexpression, an undermodified tRNAPhe species was produced that lacked only N6-(delta 2-isopentenyl)-2-methylthioadenosine (ms2i6A), a hypermodified nucleotide found immediately 3' to the anticodon of all major E . coli tRNAs that read UNN codons . To investigate the role of ms2i6A in E . coli tRNA, we compared the aminoacylation kinetics and in vitro codon-reading properties of the ms2i6A-lacking and normal fully modified tRNAPhe species . The results of these experiments indicate that while ms2i6A is not required for normal aminoacylation of tRNAPhe, its presence stabilizes codon-anticodon interaction and thereby prevents misreading of the genetic code.

Proc Natl Acad Sci U S A, 1989 Jan, 86(1), 2 - 6
Esperamicins, a class of potent antitumor antibiotics: mechanism of action; Long BH et al.; The esperamicins represent a class of antitumor antibiotics characterized by an unusual chemical core structure and extremely potent cytotoxicity . The mechanism by which these drugs produce cytotoxicity was investigated and found to be related to the formation of single- and double-strand DNA breaks . Using five structurally related analogs, we defined a structure-activity relationship for cytotoxicity in various eukaryotic and DNA-repair-deficient prokaryotic cell lines, for DNA breakage in a human colon carcinoma cell line, and for DNA breakage in vitro in pBR322 DNA . Mild reducing agents such as dithiothreitol greatly increased the DNA breakage potency of these analogs in vitro . Results suggest that the pendant aromatic chromophore of esperamicin A1 may contribute to the uptake of the drug into cells but may also hinder double-strand DNA break formation . Little DNA breakage specificity was observed for the drug in a 139-base-pair fragment of pBR322 DNA . Evidence supports a previously proposed mechanism whereby esperamicins may produce the observed DNA breaks through reduction of the methyl trisulfide group to a thiolate anion followed by a Michael addition of the anion across the alpha,beta-unsaturated ketone . This addition may result in the saturation of the bridgehead double bond, thus allowing the two triple bonds to approach each other, causing cyclization of the diyn-ene to form a phenylene diradical . It is likely that this diradical is the active form of the drug responsible for single- and double-strand DNA breakage produced by this class of antitumor agents.

FASEB J, 1989 Jan, 3(1), 14 - 21
Human DNA polymerase alpha: predicted functional domains and relationships with viral DNA polymerases; Wang TS et al.; The primary sequence of human DNA polymerase alpha deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage phi 19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus . The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene . Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction . Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction . One region toward the carboxyl-terminus has the potential to be the DNA interacting domain, whereas a potential DNA primase interaction domain is predicted toward the amino-terminus . The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DNA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.

Eksp Med Morfol, 1989, 28(4), 49 - 53
{An in vitro micromethod for studying phagocytic and bacteriocidal activity of granulocytes from peripheral blood}; Divanian Kh et al.; A microcultivation method convenient for clinical objectives was prepared for simultaneous examination of phagocytic and bactericidal capability of human granulocytes, based on vital staining with acridine-orange . Slides, on which nonheparinized blood from the examined individuals was poured out drop by drop, were assembled on the microcultivation camera, constructed by us . After one hour incubation the clot was removed and a suspension from Staphylococcus aureus was poured out on the cellular film . After proper cultivation the adherent cells were stained with acridine-orange as the live bacteria and nuclei of intact granulocytes were stained green under the action of the dye, but the dead prokaryotes and degenerated nuclei-red . The phagocytic index, phagocytic number, bacteriocidal index were followed-up in dynamics and it was established that a 45-minute incubation appeared to be the most suitable for simultaneous examination of these parameters.

Cytogenet Cell Genet, 1989, 52(1-2), 93 - 4
In situ methylation of human chromosomes with methylase-AluI; Fernandez-Piqueras J et al.; A method for in situ DNA methylation with the prokaryotic methylase AluI has been developed for use on fixed human chromosomes . Incorporation of methyl groups into the chromosomal DNA has been shown by autoradiography using a labeled substrate . The methylation prevents the digestion of chromosomal DNA by AluI, allowing direct visualization of clusters of nonmethylated AGCT targets along the human complement.

Genome, 1989, 31(2), 520 - 7
Homologous recombination in prokaryotes: enzymes and controlling sites; Smith GR; A common step in prokaryotic recombination appears to be the synapsis of the 3'-end of single-stranded DNA with duplex DNA to form a D-loop . The enzymatic mechanisms by which 3'-ends are produced and by which D-loops are converted into recombinant molecules are illustrated by proposed mechanisms of recombination by the Escherichia coli RecBCD pathway and the phage lambda Red pathway . The enzymes promoting recombination and the special DNA sites at which they act are emphasized . Recombination by other E . coli pathways and in other prokaryotes is compared with these mechanisms.

Proteins, 1989, 5(4), 281 - 8
A protein structural motif that bends DNA; White SW et al.; The prokaryotic protein HU, integration host factor (IHF) from Escherichia coli, and transcription factor 1 (TF1) from bacteriophage SPO1 are closely related molecules . Biochemical results suggest that the role of these proteins is to bind and bend DNA . From the high-resolution structure of HU, we propose a model for this interaction with DNA . Crucial amino acid differences between the proteins can be rationalized in terms of their different specific functions.

Arch Microbiol, 1989, 152(3), 258 - 62
Unusually strong immunological cross-reaction between elongation factor Tu of Escherichia coli and Bacillus subtilis; Wenzig P et al.; Polyclonal antibodies were prepared against the purified elongation factor Tu (EF-Tu) of Escherichia coli and Bacillus subtilis . Using the methods of Western blotting and microcomplement fixation the cross-reactivities of EF-Tu of 19 different prokaryotes were determined . The immunological distance were compared with the results of 16S rRNA oligonucleotide analysis . An unexpectedly high cross-reactivity was revealed between the EF-Tu of B . subtilis and the antiserum against the EF-Tu of E . coli . A comparison of the predicted amino acid sequences from the tufgenes of E . coli and B . subtilis yielded two identical peptide fragments that are likely candidates for antibody binding sites.

Dev Biol Stand, 1989, 70, 257 - 69
Biologicals production by recombinant DNA technology in Cuba; Herrera L et al.; During 1981 we started an intensive programme for the development of biotechnology in Cuba . The first project was related to the production of leucocyte interferon and the cloning an expression of these genes in prokaryotic and yeast cells . These products are currently obtained in large amounts and their physical and chemical characterization are presented . The use of mammalian cells in culture for the production of recombinant proteins has also been carried out . The results obtained with the expression and amplification of the hepatitis B surface antigen (HBsAg) in CHO cells are discussed and compared with that obtained in yeast . The construction of a recombinant vaccinia virus carrying the HBsAg was also achieved . The specific transcripts were characterized and the Ab levels tested in animals . The results presented here indicate that the host system is a very important aspect to be taken into consideration and in some cases mammalian cells appear to be the best choice.

Ann N Y Acad Sci, 1989, 573, 113 - 29
Molecular biology of the human pyruvate dehydrogenase complex: structural aspects of the E2 and E3 components; Thekkumkara TJ et al.; The availability of the primary amino acid sequences of the E2 of PDC, alpha-KGDC and BCKADC from several prokaryotic and eukaryotic species has allowed us to compare the structural aspects of human PDC-E2 with those of the E2 components from the other complexes . The PDC-E2 components from all the species examined so far contain three structurally identifiable regions: the lipoyl-bearing domain, the E3-binding site, and the catalytic domain . The primary structure of the lipoyl-bearing domain shows considerable variation in its size, ranging from one to three repeating units of approximately 110 amino acids, but essentially preserving its function in the E2 components . In contrast, the sizes of the E3-binding site and the catalytic domain of PDC-E2 from several species are essentially similar and show considerable conservation of specific amino acid residues . Obviously, additional studies are warranted to better understand the structure-function relationships of these domains and the evolutionary conservation of PDC-E2 in different species . Similarly, the availability of the primary amino acid sequences of E3 from several prokaryotes and eukaryotes has also permitted comparison of the structural domains of these proteins with that of the known structure of human GR, a flavoprotein member of the pyridine nucleotide-disulfide oxidoreductase family . Four structural domains (FAD, NAD+, central, and interface domains) have been identified in the E3 components . On the basis of the comparison of the secondary structural elements of GR and E3, the core structure of these two proteins are shown to be similar . It is hoped that further analysis of E3 using site-directed mutagenesis and determination of its crystal structure will provide better insight into its structure-function relationships.

Scanning Microsc Suppl, 1989, 3, 109 - 15
Structural features of Borrelia burgdorferi--the Lyme disease spirochete: silver staining for nucleic acids; Garon CF et al.; Borrelia burgdorferi--the Lyme disease spirochete--was grown in modified Kelly medium and characterized by transmission and by scanning electron microscopy . Using silver staining procedures which preferentially bind to nuclear components of eukaryotic cells, signal could be detected by backscattered electron imaging throughout the length of the prokaryotic spirochete . Interestingly, however, the highest levels of backscattered signal were observed in naturally elaborated membrane blebs that were visible attached to cell surfaces and free in the medium . These membrane vesicles could be enriched by filtration through nitrocellulose or Anopore membranes and by differential centrifugation . The possibility of contaminating cellular DNA coating the membrane vesicles was ruled out by exhaustive digestion with pancreatic DNAse I . Intact DNA was demonstrated both by lysing blebs directly on the surface of microscope grids and by extracting molecules from purified bleb preparation with detergents and solvents . Both linear and circular DNA molecules could be identified in purified membrane blebs . A simple, one-step, alternative silver staining procedure is described which appears to effectively label the protein-nucleic acid complexes contained in the membrane vesicles of the human pathogen B . burgdorferi, and may provide an important method to track and to define the biological function of these structures.

Acta Leprol, 1989, 7 Suppl 1, 231 - 3
Studies on ribosomal RNA genes of mycobacteria including M . leprae; Katoch VM et al.; Information about specific genes specially of pathogenic mycobacteria could be used to unequivocally identify isolates of mycobacteria which are of clinical interest . Both eukaryotic and prokaryotic ribosomal RNA (rRNA) genes have been shown to comprise sequences which are conserved and others which are divergent . In the present study, rRNA genes from several cultivable mycobacteria including M . tuberculosis and armadillo derived M . leprae have been investigated . rRNA was isolated, made radioactive in vitro and then used to identify restriction fragments of DNA containing rRNA gene sequences . It was observed that restriction endonuclease patterns of rRNA genes are characteristic . By probing with homologous and heterologous rRNA probes, fragments hybridizing maximum with homologous probes could be identified and it appears that sequences flanking the rRNA genes are not identical . These fragments need to be further sequenced to identify the nucleotide sequences specific to rRNA gene cluster . It would also be necessary to analyse several isolates of each species including armadillo derived M . leprae before reaching any conclusions.

J Protozool, 1989 Jan-Feb, 36(1), 12S - 14S
A method for isolation of RNA from Pneumocystis carinii; Cushion MT et al.; Total RNA from Pneumocystis carinii obtained directly from the rat lung and from short term culture on A549 cells was evaluated for size and purity . An isolation procedure using guanidine isothiocyanate and lithium chloride was preferable to a hot phenol method . Host cells were eliminated by hypotonic lysis and a series of microfiltrations . Pneumocystis carinii were pretreated with Zymolyase for increased susceptibility to chaotropic agents . The major ribosomal species of P . carinii RNA migrated similarly to Saccharomyces cerevisiae rRNA . The 28s-like species migrated well ahead of rat and A549 cell rRNA and well behind the prokaryotic large rRNA species.

Gene, 1988 Dec 30, 74(2), 365 - 73
An Escherichia coli vector to express and purify foreign proteins by fusion to and separation from maltose-binding protein; Maina CV et al.; A plasmid vector has been constructed that directs the synthesis of high levels (approximately 2% of total cellular protein) of fusions between a target protein and maltose-binding protein (MBP) in Escherichia coli . The MBP domain is used to purify the fusion protein in a one step procedure by affinity chromatography to crosslinked amylose resin . The fusion protein contains the recognition sequence (Ile-Glu-Gly-Arg) for blood coagulation factor Xa protease between the two domains . Cleavage by factor Xa separates the two domains and the target protein domain can then be purified away from the MBP domain by repeating the affinity chromatography step . A prokaryotic (beta-galactosidase) and a eukaryotic (paramyosin) protein have been successfully purified by this method.

Gene, 1988 Dec 25, 74(1), 9 - 12
Cloning of a mammalian DNA methyltransferase; Bestor TH; Cloning and sequencing of cDNA clones has shown that mammalian DNA (cytosine-5)-methyltransferase comprises a 1000-amino acid (aa) N-terminal region of unknown function and a 570-aa C-terminal region that is clearly related to bacterial type-II cytosine restriction methyltransferases . These findings indicate that the mammalian enzyme contains at least two structural domains and suggest a common evolutionary origin for mammalian and prokaryotic DNA (cytosine-5)-methyltransferases.

Gene, 1988 Dec 25, 74(1), 253 - 9
The amino acid sequence of the eukaryotic DNA {N6-adenine}methyltransferase, M.CviBIII, has regions of similarity with the prokaryotic isoschizomer M.TaqI and other DNA {N6-adenine} methyltransferases; Narva KE et al.; The sequences of the genes coding for M.CviBIII (from virus NC-1A which infects a eukaryotic alga) {Narva et al., Nucleic Acids Res . 15 (1987) 9807-9823} and M.TaqI (from the bacterium Thermus aquaticus) {Slatko et al., Nucleic Acids Res . 15 (1987) 9781-9796} have been determined recently . Both enzymes methylate adenine in the sequence TCGA . We have compared the predicted amino acid sequences of these two methyltransferases (MTases), with each other and with ten other N6 A-MTases and find regions of similarity . M.CviBIII and M.TaqI were most closely related followed by M.PaeR7, whose recognition sequence (CTCGAG) contains the M.TaqI/M.CviBIII recognition sequence TCGA, and M.PstI, whose recognition sequence is CTGCAG . All of the N6-MTases contain the sequence Asp/Asn-Pro-Pro-Tyr (B-P-P-Y) referred to by Hattman et al . {J . Bacteriol . 164 (1985) 932-937} as region IV . The predicted secondary structure of this region forms a finger-like structure ('beta finger') containing a beta-pleated sheet (...XXXB), two beta-turns (P-P) followed by another beta-pleated sheet {Y/FXXX...}.

J Chromatogr, 1988 Dec 23, 458, 117 - 28
High-performance liquid chromatographic analysis of DNA composition and DNA modification by chloroacetaldehyde; Singhal RP et al.; The separation of common and modified deoxyribonucleosides derived from DNA hydrolyzates was examined under different chromatographic conditions on silica-based octadecyl (C18) columns, involving hydrophobic interactions with the matrix . A novel method for the analysis of the DNA composition is described . It involves the removal of RNA contaminants and enzymatic hydrolysis of DNA, first to deoxyribonucleoside monophosphates and then dephosphorylation of the latter to deoxyribonucleosides . Hydrolysis conditions were sought to avoid deamination of dA and dC residues to dI and dU contaminants, respectively . Elution of these contaminants and the artifacts (ribonucleosides derived from RNA) is described in relation to the elution of deoxyribonucleosides . Chromatographic separation of the hydrolyzate derived from a 15-micrograms sample of DNA under selected separation conditions and on one high-performance liquid chromatographic column is achieved in 18 min at room temperature . Detection of modified components (and contaminants) present in minute amounts is enhanced with the use of a diode-array detector . The power of this technique lies in its ability to characterize and quantitate accurately the amount of modified species present in the DNA structure (less than 2% of all the other residues) . Examples of the composition analysis of DNA derived from a prokaryote (Escherichia coli B) and a eukaryote (salmon sperm) are described . Details of quantitation (calibration graphs) of different nucleosides are furnished for peak-area integration by commercially available software, and spectral properties of the nucleoside in the elution buffer are described for quantitation by other means . Application of the composition analysis is shown here for probing the DNA conformation in solution by chemical means, while using chloroacetaldehyde as the modifying agent.

EMBO J, 1988 Dec 20, 7(13), 4305 - 9
Secondary structure determination for the Antennapedia homeodomain by nuclear magnetic resonance and evidence for a helix-turn-helix motif; Otting G et al.; The homeodomain encoded by the Antennapedia (Antp) gene of Drosophila was studied in aqueous solution by nuclear magnetic resonance (NMR) . Sequence-specific resonance assignments have been obtained for the complete polypeptide chain of 68 amino acid residues . The secondary structure determined from nuclear Overhauser effects (NOE) and information about slowly exchanging amide protons includes three helical segments consisting of the residues 10-21, 28-38 and 42-52, respectively . Combination of the presently available NMR data with computer modeling provided preliminary evidence for the presence of a helix-turn-helix motif in the homeodomain . Near the turn, this supersecondary structure appears to be very similar to the DNA binding site in the 434 and P22 c2 repressors, but both helices in the homeodomain include 2-3 additional residues when compared with these prokaryotic DNA-binding proteins.

Eur J Biochem, 1988 Dec 15, 178(2), 381 - 6
Fidelity of aminoacylation by rat-liver tyrosyl-tRNA synthetase . Effect of age; Takahashi R et al.; The possibility of change in the rate of misrecognition of amino acids by rat liver tyrosyl-tRNA synthetase during aging was investigated . Frequency of misrecognition of phenylalanine vs tyrosine was determined at two temperatures by competitive assay using partially purified enzymes . At 25 degrees C, the error frequencies were 5.17 x 10(-8) and 8.24 x 10(-8) in young and old animals, respectively . These values are much below the reported error frequencies for the prokaryotic enzymes: i.e . approximately 5 x 10(-6) . Although the fidelity of tyrosyl-tRNA synthetase from old animals appeared to be slightly lower, the difference was not statistically significant . At 37 degrees C, the error frequencies were increased 1.3-1.5-fold, but again the difference between young and old animals was not significant . To our knowledge, this is the first report in which fidelity of aminoacyl-tRNA synthetase of animals of various ages has been compared using natural amino acids.

Gene, 1988 Dec 15, 73(1), 227 - 35
The T7 phage gene 10 leader RNA, a ribosome-binding site that dramatically enhances the expression of foreign genes in Escherichia coli; Olins PO et al.; Expression of foreign genes in Escherichia coli requires the juxtaposition of prokaryotic transcription and translation elements with a coding region for the foreign gene . Commonly, this results in only modest expression of the foreign gene product . Here we describe a novel ribosome-binding site (RBS; phage T7 'gene 10 leader') which is able to drive the translation of several foreign genes . This RBS dramatically enhanced the translation efficiency of all the genes we have tested to date, and was particularly effective for foreign genes . The enhanced expression was often more than 40-fold greater than that obtained using a 'consensus' RBS . A general plasmid vector has been constructed, incorporating the T7 gene 10 leader sequence, which allows the facile expression of important gene products . In this report we demonstrate the application of this system for the high-level expression of plant, mammalian and bacterial proteins in E . coli.

Gene, 1988 Dec 10, 72(1-2), 3 - 14
Repetitive extragenic palindromic sequences, mRNA stability and gene expression: evolution by gene conversion? A review; Higgins CF et al.; Repetitive extragenic palindromic (REP) sequences are highly conserved inverted repeats present in up to 1000 copies on the Escherichia coli chromosome . We have shown both in vivo and in vitro that REP sequences can stabilize upstream mRNA by blocking the processive action of 3'----5' exonucleases . In a number of operons, mRNA stabilization by REP sequences plays an important role in the control of gene expression . Furthermore, differential mRNA stability mediated by the REP sequences can be responsible for differential gene expression within polycistronic operons . Despite the key role of REP sequences in mRNA stability and gene expression in a number of operons, several lines of evidence suggest that this is unlikely to be the primary reason for the exceptionally high degree of sequence conservation between REP sequences . Other possible functions for REP sequences are discussed . We propose that REP sequences may be a prokaryotic equivalent of 'selfish DNA' and that gene conversion may play a role in the evolution and maintenance of REP sequences.

Gene, 1988 Dec 10, 72(1-2), 15 - 23
Mechanisms of mRNA decay in bacteria: a perspective; Belasco JG et al.; Messenger RNA decay plays an important role in prokaryotic gene expression . The disparate stabilities of bacterial messages in vivo are a consequence of their differential susceptibility to degradation by cellular endoribonucleases and 3' -exoribonucleases, which in turn results from differences in mRNA sequence and structure . RNase II and polynucleotide phosphorylase, the major bacterial exonucleases involved in mRNA turnover, rapidly degrade single-stranded RNA from the 3' end, but are impeded by 3' stem-loop structures . At present, the identify and substrate specificity of the endonucleases that control mRNA decay rates are relatively poorly defined . Ribosomes and antisense RNA also can influence the stability of transcripts with which they associate . Differences in mRNA stability can contribute to differential expression of genes within polycistronic operons and to modulation of gene expression in response to changes in bacterial growth conditions.

Gene, 1988 Dec 10, 72(1-2), 35 - 44
Naturally occurring antisense RNA control--a brief review; Simons RW; Biological control by naturally occurring anti-sense RNAs has been documented in a number of prokaryotic cases, and strongly suggested in several eukaryotic systems . The biological activities controlled are diverse, including transposition, phage development, chromosomal gene expression, and plasmid replication, compatibility and conjugation . Control is exerted at many different levels, by both direct and long-range effects . The stem/loop structures common to all anti-sense RNAs are important functional domains: loops are the sites of critical interactions in the initiation of pairing to the target RNA; stems determine anti-sense RNA stability in vivo . These features need to be considered in the design of artificial anti-sense RNA control . Details of RNA/RNA pairing have emerged; pairing initiates at single-stranded regions in anti-sense RNA loops, and stable complex formation involves the nearby end of one or both molecules.

J Mol Biol, 1988 Dec 5, 204(3), 569 - 80
Structure and regulation of controlling sequences for the Streptomyces coelicolor glycerol operon; Smith CP et al.; The pathway for glycerol catabolism in Streptomyces coelicolor is determined by the gylABX operon . The sequence of about 1500 base-pairs (bp) preceding the structural genes of the operon has been determined, and related to a detailed transcriptional analysis of this region . The gylABX operon contains two major promoters, gylP1 and gylP2, separated by 50 bp . Both promoters are glycerol-inducible and glucose-repressible . A 900-base transcription unit, gylR, is situated immediately upstream of the gylABX promoter region and contains an open reading frame for a 27,600 Mr protein . The predicted sequence of this protein contains a region that is similar to the helix-turn-helix domains of certain DNA-binding proteins . Transcription of gylR is also glycerol-inducible, but is only weakly glucose-repressible, and initiates predominantly from a single promoter, gylRp . The three promoters, gylRP, gylP1 and gylP2, each resemble the "typical" prokaryotic consensus promoter sequence . The DNA sequence of the gylR and gylABX promoter regions share some striking features . These include almost identical operator-like elements (segments of which are tandemly repeated around gylRP) and tracts of alternating purine-pyrimidine residues.

Biochim Biophys Acta, 1988 Dec 2, 957(3), 323 - 34
Assembly of Rubisco from native subunits; Roy H et al.; Large subunits of ribulosebisphosphate carboxylase/oxygenase (Rubisco) (3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39) from prokaryotic sources can assemble into intact enzyme either in vitro or in Escherichia coli cells . Large subunits of higher plant Rubisco do not assemble into Rubisco in E . coli cells, nor is it possible to reconstitute higher plant Rubisco from its dissociated subunits in vitro . This behavior represents an obstacle to any practical attempts at engineering the higher plant enzyme, and it suggests that the in vivo assembly mechanism of higher plant Rubisco must be more complex than is commonly expected for oligomeric proteins of organelles . In pea chloroplasts, a binding protein interacts with newly synthesized large subunits, in quantities expected for an intermediate in the assembly process, as judged by Western blotting . Radiotracer-labeled large subunits which interact with this binding protein can be shown to assemble into Rubisco in reactions which lead to changes in the aggregation state of the binding protein . Antibody to this binding protein specifically inhibits the assembly of these subunits into Rubisco . Rubisco synthesis appears to be subject to many types of control: gene dosage, transcription rate, selective translation of message, post-translational degradation and threshold concentration effects have been observed in various organisms' synthesis of Rubisco . The biochemical mechanisms underlying most of these effects have not been elucidated . The post-translational assembly mechanism in particular appears to require further study.

J Cell Sci, 1988 Dec, 91 ( Pt 4), 577 - 86
Supramolecular structure of the thylakoid membrane of Prochlorothrix hollandica: a chlorophyll b-containing prokaryote; Miller KR et al.; Prochlorothrix hollandica is a newly described photosynthetic prokaryote, which contains chlorophylls a and b . In this paper we report the results of freeze fracture and freeze etch studies of the organization of the photosynthetic thylakoid membranes of Prochlorothrix . These membranes exhibit four distinct fracture faces in freeze fractured preparations, two of which are derived from membrane splitting in stacked regions of the thylakoid membrane, and two of which are derived from nonstacked regions . The existence of these four faces confirms that the thylakoid membranes of Prochlorothrix, like those of green plants, display true membrane stacking and have different internal composition in stacked and non-stacked regions, a phenomenon that has been given the name lateral heterogeneity . The general details of these fracture faces are similar to those of green plants, although the intramembrane particles of Prochlorothrix are generally smaller than those of green plants by as much as 30% . Freeze etched membrane surfaces have also been studied, and the results of these studies confirm freeze fracture observations . The outer surface of the thylakoid membrane displays both small (less than 8.0 nm) and large (greater than 10.0 nm) particles . The inner surface of the thylakoid membrane is covered with tetrameric particles, which are concentrated into stacked membrane regions, a situation that is similar to the inner surfaces of the thylakoid membranes of green plants . These tetramers have never before been reported in a prokaryote . The photosynthetic membranes of Prochlorothrix therefore represent a prokaryotic system that is remarkably similar, in structural terms, to the photosynthetic membranes found in chloroplasts of green plants.

Biokhimiia, 1988 Dec, 53(12), 1980 - 6
{Local homology of amino acid sequences of histones H3, H4 and the protein repressor lambda Cro . Possible conformation of homologous histone sites in DNP}; Mil' EM et al.; A mathematical analysis of amino acid sequences was carried out with a view of detecting possible homology between histones H3 and H4 and repressor-activator proteins of prokaryotes according to the A . I . criterion which reflects the similarity of their primary structure . It was found that the sites of eukaryotic histones H3 (102-123) and H4 (68-85) and site alpha 3 (24-25) of the prokaryotic repressor protein lambda Cro, i . e., the site of protein interaction with DNA, reveal a statistically significant homology . The A . I . value for the H3 site of lambda Cro is 3.37, that for the H4 site of calf thymus and sea horse is 3.28 . The amino acid sequences of these proteins in the alpha 2-alpha 3 site, i . e., the site in which the homology between amino acid sequences of histones and DNA-binding proteins had been established previously, with regard to similarity of their secondary structure of the helix-turn-helix type, were analyzed . A pairwise comparison of H3 and protein lambda Cro showed that the A . I . value for histones H3 from various sources is approximately 2.7; however, the homology of the alpha 2 site is lower than that of site alpha 3 . It is concluded that there exists an evolutionary relationship between homologous segments of histones H3 and H4 and protein lambda Cro, which can be preserved in order to maintain a definite secondary structure, presumably for binding to DNA.

Genes Dev, 1988 Dec, 2(12B), 1800 - 11
On the role of homologous sequences in chromosomal rearrangements; Singer BS; Deletions and other chromosomal rearrangements can be generated by recombination between repeated sequences . It has been shown in a number of systems that the probability of exchange or gene conversion decreases with increasing distance between repeats . This paper examines the question of how repeats find each other, using deletion formation in bacteriophage T4 as a model system . Homologous sequences adjacent to the repeats can either stimulate or inhibit recombination, depending on their orientation . I present evidence that the spatial separation between repeats is the key determinant of the distance dependence and conclude that adjacent homologous sequences affect recombination by aligning chromosomes so as to position the recombining sites either closer together or farther apart . Analogous examples of apparent 'targeting' by homologous sequences in eukaryotes and other prokaryotes are noted.

FEMS Microbiol Rev, 1988 Dec, 4(4), 271 - 97
Thioredoxin and related proteins in procaryotes; Gleason FK et al.; Thioredoxin is a small (Mr 12,000) ubiquitous redox protein with the conserved active site structure: -Trp-Cys-Gly-Pro-Cys- . The oxidized form (Trx-S2) contains a disulfide bridge which is reduced by NADPH and thioredoxin reductase; the reduced form {Trx(SH)2} is a powerful protein disulfide oxidoreductase . Thioredoxins have been characterized in a wide variety of prokaryotic cells, and generally show about 50% amino acid homology to Escherichia coli thioredoxin with a known three-dimensional structure . In vitro Trx-(SH)2 serves as a hydrogen donor for ribonucleotide reductase, an essential enzyme in DNA synthesis, and for enzymes reducing sulfate or methionine sulfoxide . E . coli Trx-(SH)2 is essential for phage T7 DNA replication as a subunit of T7 DNA polymerase and also for assembly of the filamentous phages f1 and M13 perhaps through its localization at the cellular plasma membrane . Some photosynthetic organisms reduce Trx-S2 by light and ferredoxin; Trx-(SH)2 is used as a disulfide reductase to regulate the activity of enzymes by thiol redox control . Thioredoxin-negative mutants (trxA) of E . coli are viable making the precise cellular physiological functions of thioredoxin unknown . Another small E . coli protein, glutaredoxin, enables GSH to be hydrogen donor for ribonucleotide reductase or PAPS reductase . Further experiments with molecular genetic techniques are required to define the relative roles of the thioredoxin and glutaredoxin systems in intracellular redox reactions.

Biochimie, 1988 Dec, 70(12), 1743 - 8
Bacterial proteins with N-terminal leader sequences resembling mitochondrial targeting sequences of eukaryotes; Saier MH Jr et al.; Amphipathic, alpha-helical, leader sequences, analogous to those that direct nuclear-encoded eukaryotic proteins into mitochondria, have been found in one and only one class of bacterial integral membrane proteins . These bacterial proteins are the sugar permeases of the phosphoenolpyruvate-dependent phosphotransferase system . The amphipathic leader sequence in each of these proteins is terminated by a helix breaker, either a prolyl residue or 2 adjacent glycyl residues . Preliminary evidence suggests that these leader sequences function to target the proteins to the envelope fraction of the prokaryotic cell during their biosynthesis.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8830 - 4
Evidence for a trans-acting factor that regulates the transcription of class II major histocompatibility complex genes: genetic and functional analysis; Calman AF et al.; The study of specific trans-acting transcription factors in prokaryotes and lower eukaryotes has been greatly facilitated by genetic analysis of mutant strains deficient in such factors . We have developed such a system to study mammalian trans-acting factors that regulate the transcription of class II major histocompatibility complex genes, using the mutant cell lines RM2 and RM3 . These cells, derived from the human B-cell line Raji, specifically fail to transcribe their class II major histocompatibility complex genes . Here we show that a transfected HLA-DR alpha class II major histocompatibility complex gene, like the endogenous HLA-DR alpha genes, is efficiently transcribed in Raji cells but not in RM2 or RM3 cells, demonstrating that the mutant cells are deficient in a specific trans-acting factor required for transcription of these genes . HLA-DR expression in RM2 and RM3 cells is rescued by fusion to another B-cell line but not by fusion to each other . Thus, the defects in the two cell lines are recessive and noncomplementing and define a locus whose wild-type product we designate TF-X1 . We show that TF-X1 influences the activity of a 24-base-pair B-cell-specific cis-acting transcription element in the HLA-DR alpha promoter . However, in three different biochemical assays, we detect no difference between wild-type and mutant cells in the DNA-binding proteins that interact with these DNA sequences . Thus, the defective version of TF-X1 may be a DNA-binding protein that binds to the HLA-DR alpha promoter but fails to activate transcription . Alternatively, TF-X1 may not be a DNA-binding protein at all.

Proc Natl Acad Sci U S A, 1988 Dec, 85(24), 9396 - 400
Charge configurations in viral proteins; Karlin S et al.; The spatial distribution of the charged residues of a protein is of interest with respect to potential electrostatic interactions . We have examined the proteins of a large number of representative eukaryotic and prokaryotic viruses for the occurrence of significant clusters, runs, and periodic patterns of charge . Clusters and runs of positive charge are prominent in many capsid and core proteins, whereas surface (glyco)proteins frequently contain a negative charge cluster . Significant charge configurations are abundant in regulatory proteins implicated in transcriptional transactivation and cellular transformation . Proteins with charge structures are much more predominant in animal DNA viruses as compared to animal RNA viruses and prokaryotic viruses . This contrast might reflect the role of protein charge structures in facilitating competitive virus-host interactions involving the cellular transcription, translation, protein sorting, and transport apparatus.

Proc Natl Acad Sci U S A, 1988 Dec, 85(23), 8850 - 4
DNA gyrase binds to the family of prokaryotic repetitive extragenic palindromic sequences; Yang Y et al.; A family of repetitive extragenic palindromic (REP) sequences is composed of hundreds of copies distributed throughout the chromosome . Their palindromic nature and conservation suggested that they are specifically recognized by a protein(s) . We have identified DNA gyrase {DNA topoisomerase (ATP-hydrolysing), EC 5.99.1.3} as one of the REP-binding proteins . Gyrase has at least a 10-fold higher affinity for DNA containing REP sequences than for DNA not containing REP sequences . Binding effectiveness correlates directly with the number of REP sequences in the DNA . DNase I footprinting shows that gyrase protects 205 base pairs on a REP-containing DNA fragment enclosing the REP sequences . In agreement with the above results, a comparison of the REP consensus sequence with the sequence of previously identified pBR322 "strong" gyrase cleavage sites reveals a high degree of homology . Because REP sequences are numerous and found throughout the genome, we suggest they have physiological functions mediated through their interaction with gyrase, such as being sites of action for the maintenance of DNA supercoiling . In addition, we speculate that these interactions may be of a structural nature, such as involvement in the higher-order structure of the bacterial chromosome.

Science, 1988 Nov 25, 242(4882), 1164 - 6
Intron existence predated the divergence of eukaryotes and prokaryotes; Shih MC et al.; Nucleotide sequences for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDHs) from Arabidopsis thaliana were determined . Comparison of nucleotide sequences indicates that the divergence of chloroplast and cytosolic GAPDH genes preceded the divergence of prokaryotes and eukaryotes . In addition, some intron-exon junctions are conserved among GapB, GapC, and chicken GAPDH genes . These results provide evidence at the molecular level to support the idea that introns existed before the divergence of prokaryotes and eukaryotes.

Science, 1988 Nov 25, 242(4882), 1162 - 4
A continuous cell-free translation system capable of producing polypeptides in high yield; Spirin AS et al.; A cell-free translation system has been constructed that uses a continuous flow of the feeding buffer {including amino acids, adenosine triphosphate (ATP), and guanosine triphosphate (GTP)} through the reaction mixture and a continuous removal of a polypeptide product . Both prokaryotic (Escherichia coli) and eukaryotic (wheat embryos, Triticum sp.) versions of the system have been tested . In both cases the system has proven active for long times, synthesizing polypeptides at a high constant rate for tens of hours . With the use of MS2 phage RNA or brome mosaic virus RNA 4 as templates, 100 copies of viral coat proteins per RNA were synthesized for 20 hours in the prokaryotic or eukaryotic system, respectively . With synthetic calcitonin messenger RNA, 150 to 300 copies of calcitonin polypeptide were produced per messenger RNA in both types of continuous translation systems for 40 hours.

FEBS Lett, 1988 Nov 21, 240(1-2), 45 - 8
Nucleotide sequence of a cDNA for the dihydrolipoamide acetyltransferase component of human pyruvate dehydrogenase complex; Thekkumkara TJ et al.; Deoxynucleotide sequencing of a cDNA for the dihydrolipoamide acetyltransferase (PDC-E2) component of human pyruvate dehydrogenase complex (PDC) revealed an open reading frame of 1848 base pairs corresponding to a leader sequence of 54 amino acids and a mature protein of 561 amino acids (59,551 Da) . Both an amino-terminal lipoyl-bearing domain and a carboxy-terminal catalytic domain are present in the deduced amino acid sequence . The lipoyl-bearing domain contains two repeating units of 127 amino acids, each harboring one lipoic acid-binding lysine . Thus, mammalian PDC-E2 differs as to the number of lipoic acid-binding sites from other dihydrolipoamide acyltransferases in both prokaryotic and eukaryotic organisms.

Biochim Biophys Acta, 1988 Nov 10, 951(1), 61 - 70
Isolation and characterization of a cDNA for mitochondrial manganese superoxide dismutase (SOD-3) of maize and its relation to other manganese superoxide dismutases; White JA et al.; We describe the isolation of a cDNA clone for the nuclear-encoded manganese superoxide dismutase (SOD-3) of maize mitochondria . The cDNA, pSod3.1c, selects by hybridization an RNA which produces the SOD-3 precursor upon in vitro translation . The DNA sequence of pSod3.1c was determined from fragments subcloned in M13 . The amino-acid sequence deduced from the nucleotide sequence displays significant homology with the amino-acid sequences of prokaryotic and eukaryotic Mn-SODs, but displays greater homology with mammalian Mn-SOD than it does with yeast or bacterial Mn-SOD . A 31 amino-acid transit peptide also is encoded by the pSod3.1c clone . Analysis of poly(A)+ RNA indicates that Sod3 mRNA is approx . 1250 nucleotides in length . The amount of Sod3 transcript in seedling leaves is increased by light.

J Biol Chem, 1988 Nov 5, 263(31), 16401 - 7
Characterization of vaccinia virus DNA topoisomerase I expressed in Escherichia coli; Shuman S et al.; The putative structural gene encoding the vaccinia virus type I DNA topoisomerase (EC 5.99.1.2) was expressed in Escherichia coli under the control of a bacteriophage T7 promoter . Provision of T7 RNA polymerase resulted in the accumulation to high level of a Mr = 33,000 type I topoisomerase with the properties of the vaccinia enzyme . A simple purification scheme yielded approximately 8 mg of recombinant vaccinia topoisomerase from 400 ml of bacteria . DNA unwinding by the enzyme was stimulated by magnesium, manganese, calcium, cobalt, and spermidine, but inhibited by copper and zinc . Like eukaryotic cellular type I topoisomerases, but unlike the prokaryotic counterpart, the recombinant topoisomerase relaxed positively and negatively supercoiled DNA . The viral topoisomerase I was, however, resistant to the effects of camptothecin, a drug that specifically inhibits cellular type I topoisomerases.

Biochimie, 1988 Nov, 70(11), 1493 - 504
Structure and biosynthesis of prokaryotic glycoproteins; Wieland F; Glycoproteins as components of cell surfaces are not restricted to eukaryotes . The prokaryotic glycoprotein studied in greatest detail so far is the cell surface glycoprotein of the archaebacterium Halobacterium halobium . This bacterial glycoprotein contains 3 different types of glycoconjugates, and each type of glycoconjugate involves a different carbohydrate-protein linkage unit: 1) One glycosaminoglycan chain, constructed from a repeating sulfated pentasaccharide block, is linked to one protein molecule via the novel N-glycosyl linkage unit asparaginyl-N-acetylgalactosamine . 2) Ten sulfated oligosaccharides that contain glucose, glucuronic acid and iduronic acid are bound to the protein via the hitherto unknown N-glycosyl linkage unit asparaginylglucose . 3) About 15 disaccharides, glucosylgalactose, are O-glycosyl-linked to a cluster of threonine residues close to the C-terminus of the core protein . The overall structure of the cell surface glycoprotein of halobacteria is thus reminiscent of animal proteoglycans and a functional role of the glycosaminoglycan chain in maintaining the rod shape of halobacteria is discussed . Biosynthesis of the two N-glycosyl linkage units involves dolichol monophosphate and dolicholdiphosphate-linked saccharide precursors . Sulfation and epimerization of the glycoconjugates occur at the lipid-linked level and the mature saccharides are transferred to the protein core on the cell surface . The sulfated oligosaccharides that finally become bound to asparagine via glucose are transiently methylated at their lipid-linked stage and this transient chemical modification seems to be required for the biosynthesis of the corresponding N-glycosyl bond.

J Neurochem, 1988 Nov, 51(5), 1394 - 9
Studies on DNA damage and repair in the mammalian brain; Walker AP et al.; Methods for studying breaks in DNA strands and their repair, originally developed for prokaryotes and cultured cell lines, have been applied to preparations from rat brain . The relative sensitivities of these methods, which include alkaline sucrose density gradient sedimentation, nucleoid sedimentation, and ADP-ribosyltransferase assay, are compared.

Arthritis Rheum, 1988 Nov, 31(11), 1337 - 45
Autoantibodies to intracellular proteins in human systemic lupus erythematosus are not due to random polyclonal B cell activation; Gharavi AE et al.; Antibody binding to total protein extracted from a mammalian source (HeLa cells) and from a prokaryotic source (Escherichia coli) was compared in sera from patients with systemic lupus erythematosus (SLE) and sera from normal subjects . When the average numbers of peptides or proteins recognized by IgG antibodies were compared on immunoblots, SLE sera bound to a significantly greater number of proteins from the HeLa cell extract than did sera from normal individuals (P less than 0.001) . In contrast, SLE sera actually bound to fewer E coli proteins than did the sera obtained from normal controls, although the difference was not statistically significant . There was no correlation between the number of E coli proteins and HeLa proteins recognized by individual SLE sera, and there was no trend toward reactivity with a larger number of antigens in sera containing higher levels of IgG . IgG from SLE sera did not bind to 6 purified eukaryotic protein standards (selected solely on the basis of differences in size and charge) either in their denatured state or in their native state . These findings indicate that the high levels of IgG antibodies against selected eukaryotic intracellular proteins in patients with SLE cannot be explained by a random polyclonal B cell activation.

Gene, 1988 Oct 30, 70(2), 411 - 3
Versatile plasmid vectors for use in studies of eukaryotic gene expression; Cab-Barrera EL et al.; A new pair of plasmid vectors useful in transient expression experiments of genes from higher eukaryotes was constructed . The vectors have been developed as derivatives of pSEG, a cloning and expression vector that has been used in studies of gene promoter structure and function {Barrera-Saldana et al., EMBO J . 4 (1985) 3839-3849} . pUANL1 and pUANL2 include in their configuration a complete rabbit beta-globin transcriptional unit as internal control for gene expression the simian virus 40 (SV40) enhancer sequences, conveniently located unique restriction sites, the gene for resistance to ampicillin as a selectable marker and a prokaryotic ori for propagation in Escherichia coli . The new pair of pUANL vectors facilitates the cloning of foreign genes, placing two copies of the enhancer at either the 3' or the 5' side of gene . Our vectors completely lack SV40 ori, promoter and upstream sequences, which renders them ideal for gene expression studies where enhancer sequences are required but promoter and upstream sequences may interfere . Finally, by carrying an internal beta-globin reference gene, they are of special value for the standardization of quantitative S1 nuclease mapping studies of gene promoters.

Science, 1988 Oct 21, 242(4877), 433 - 6
Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70; Riabowol KT et al.; Synthesis of a small group of highly conserved proteins in response to elevated temperature and other agents that induce stress is a universal feature of prokaryotic and eukaryotic cells . Although correlative evidence suggests that these proteins play a role in enhancing survival during and after stress, there is no direct evidence to support this in mammalian cells . To assess the role of the most highly conserved heat shock protein (hsp) family during heat shock, affinity-purified monoclonal antibodies to hsp70 were introduced into fibroblasts by needle microinjection . In addition to impairing the heat-induced translocation of hsp70 proteins into the nucleus after mild heat shock treatment, injected cells were unable to survive a brief incubation at 45 degrees C . Cells injected with control antibodies survived a similar heat shock . These results indicate that functional hsp70 is required for survival of these cells during and after thermal stress.

Biochim Biophys Acta, 1988 Oct 11, 947(3), 445 - 64
The membrane channel-forming colicin A: synthesis, secretion, structure, action and immunity; Lazdunski CJ et al.; The study of colicin release from producing cells has revealed a novel mechanism of secretion . Instead of a built-in 'tag', such as a signal peptide containing information for secretion, the mechanism employs coordinate expression of a small protein which causes an increase in the envelope permeability, resulting in the release of the colicin as well as other proteins . On the other hand, the mechanism of entry of colicins into sensitive cells involves the same three stages of protein translocation that have been demonstrated for various cellular organelles . They first interact with receptors located at the surface of the outer membrane and are then transferred across the cell envelope in a process that requires energy and depends upon accessory proteins (TolA, TolB, TolC, TolQ, TolR) which might play a role similar to that of the secretory apparatus of eukaryotic and prokaryotic cells . At this point, the type of colicin described in this review interacts specifically with the inner membrane to form an ion channel . The pore-forming colicins are isolated as soluble proteins and yet insert spontaneously into lipid bilayers . The three-dimensional structures of some of these colicins should soon become available and site-directed mutagenesis studies have now provided a large number of modified polypeptides . Their use in model systems, particularly those in which the role of transmembrane potential can be tested for polypeptide insertion and ionic channel gating, constitutes a powerful handle with which to improve our understanding of the dynamics of protein insertion into and across membranes and the molecular basis of membrane excitability . In addition, their immunity proteins, which exist only in one state (membrane-inserted) will also contribute to such an understanding.

Nucleic Acids Res, 1988 Oct 11, 16(19), 9233 - 51
The left end of rat L1 (L1Rn, long interspersed repeated) DNA which is a CpG island can function as a promoter; Nur I et al.; Here we report that the 600 bp promoter-like region at the left end of a newly isolated and characterized rat L1 DNA element can activate the prokaryotic chloramphenicol acyltransferase gene in a rat cell line . Activation only occurs when the promoter region is oriented to the transferase gene as it is to the L1 protein encoding sequences and is 75% inhibited by methylation of just 5 of the 22 CpGs present in the promoter . The G + C rich promoter contains enough CpGs to qualify it as a CpG island, but in contrast to other CpG islands, genomic L1 promoters are fully methylated in both somatic cell and sperm DNA as judged by restriction enzyme analysis . Partial demethylation of the genomic promoters by treatment with 5-azacytidine failed to produce discrete L1 transcripts . The relationship of methylation to the evolutionary history and fate of the rat L1 promoter is discussed.

Proc Natl Acad Sci U S A, 1988 Oct, 85(19), 7174 - 6
Nonenzymatic synthesis of 5-aminoimidazole ribonucleoside and recognition of its facile rearrangement; Groziak MP et al.; 5-Amino-1-beta-D-ribofuranosylimidazole 5'-monophosphate (AIR, 1) is the ubiquitous precursor to the purine ribonucleotides in vivo, and it serves as the biochemical precursor to the pyrimidine portion of thiamin (vitamin B1) in certain prokaryotic organisms . The corresponding ribonucleoside (AIRs, 5b) was prepared via chemical (nonenzymatic) synthesis from 5-amino-1-beta-D-ribofuranosylimidazole-4-carboxamide . The tri-O-acetylated derivative of AIRs (5a) was also prepared, and it was shown to undergo a facile ring transformation in aqueous pH 7 buffer to afford N-(imidazol-4-yl)-2,3,5-tri-O-acetyl-D-ribofuranosylamine as a 1:2 mixture of alpha and beta anomers (6a) . Under similar conditions, compound 5b affords the corresponding unprotected beta-ribonucleosides 6b . This Dimroth-type ring transformation reaction of 5 to 6, which occurs primarily in neutral aqueous solution, may be responsible for the previously reported lability of AIRs and its derivatives . It may also have relevance to the postulated early biotic pathway to the 9- and 3-substituted purine nucleotide components of an all-purine biopolymer.

Med Hypotheses, 1988 Oct, 27(2), 115 - 26
RISH . VI: General evolutionary aspects of the immune system; Daunter B; Both the prokaryotic and eukaryotic cells are considered to have arisen from a common progenitor cell . The plasma membrane of the prokaryotic cell became specialized to carry out functions that the eukaryotic cell delegated to cellular organelles . Thus the plasma membrane of the eukaryotic cell remained flexible to evolutionary influences . Thus, provided the structural integrity of the plasma membrane was maintained, alterations within this infrastructure could be tolerated . This gave rise to basic speciation at the cellular level . Such differences in the plasma membrane of these primitive eukaryotic cells were of no importance until the dawn of sexual reproduction, then only like-cells could associate to exchange genetic information . Thus in the protozoa cell-surface, antigens are demonstrable in mating, whereas alloincompatability is intracellular . With the evolution of the Metazoa, in order for like-cells to identify each other, alloincompatability changed from intracellular expression to become expressed on the plasma membrane . Like-cell identification was derived and evolved from the basic feeding mechanism of primitive eukaryotic cells, which involved the induction of lectins that were expressed at the cell-surface . These lectins were induced by the RNA that was complementary to, and complexed with cell surface components of the organisms upon which the eukaryotic cells fed . This RNA was also inserted along with the lectin in the eukaryotic cell plasma membrane, and acted as a template for DNA synthesis . This DNA was then incorporated into the genome of the eukaryotic cell and it became an inheritable characteristic . Thus these lectins could be expressed intracellularly as well as on the plasma membrane . The intracellular expression of these inheritable lectins may have constituted intracellular-alloincompatibility, as well as being used for feeding by agglutination of the organism on the plasma membrane of the eukaryotic cell . With the development of colony formation and the true metazoa, the cell-surface lectins became incorporated into cell-surface components for the identification of like-cells . This represents, in part, the histocompatability antigens of the organisms . At the same time, the lectins were also being increasingly used for the regulation of differentiation, and for what we would classify as immunological reactions.(ABSTRACT TRUNCATED AT 400 WORDS)

Biofactors, 1988 Oct, 1(3), 245 - 50
Selenocysteine, a highly specific component of certain enzymes, is incorporated by a UGA-directed co-translational mechanism; Bock A et al.; The opal termination codon UGA is used in both prokaryotic and eukaryotic species to direct the specific insertion of selenocysteine into certain selenium-dependent enzymes . So far a formate dehydrogenase (hydrogenase-linked) of Escherichia coli and glutathione peroxidases of murine, human and rat origin have been identified as enzymes containing selenocysteine residues encoded by UGA . A novel seryl-tRNA, anticodon UCA, that specifically recognizes the UGA codon is required for selenocysteine incorporation into formate dehydrogenase . A eukaryotic UGA suppressor tRNA with UCA anticodon that accepts serine and is phosphorylated to O-phosphoseryl-tRNA may have a corresponding function in glutathione peroxidase synthesis . Other factors required for the unusual usage of the in-frame UGA codons to specify selenocysteine incorporation and the biochemical mechanism involved in distinguishing these from normal UGA termination codons are discussed.

Microbiol Sci, 1988 Oct, 5(10), 316 - 9
Evolution and phylogenetic distribution of the specialized isozymes of 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase in superfamily-B prokaryotes; Jensen RA et al.; Purple sulphur (Superfamily B) bacteria vary greatly in the isozyme number and regulatory properties corresponding to the initial step of aromatic amino acid biosynthesis, 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAHP) synthase . The signal evolutionary events that are responsible for generation of the three regulatory isozymes of DAHP synthase found in the contemporary Escherichia coli and other enteric bacteria have been deduced, starting from a common ancestor of all Superfamily-B organisms . This analysis of one prokaryotic grouping is a model for analogous studies of the evolutionary history of other biochemical pathways . It is also the beginning of an analysis that can be extended to include the entire assemblage of prokaryotic organisms.

Cell, 1988 Sep 23, 54(7), 915 - 8
A model for initiation at origins of DNA replication; Bramhill D et al.; Many prokaryotic origins resemble E . coli oriC in possessing essential AT-rich sequences, tandemly repeated . The role of these repeats may be in the initial opening of the duplex by the initiator protein, as has been found for the 13-mers in oriC and is implied for the 11-mers of the lambda origin . Regulatory influences on the effective action of the initiator protein of E . coli (dnaA protein) include transcriptional activation of the origin, nucleotide binding and membrane attachment of the protein, and interactions leading to the introduction of helicases to start replication forks.

Nucleic Acids Res, 1988 Sep 12, 16(17), 8525 - 39
Oligonucleotide site-directed mutagenesis in Xenopus egg extracts; Almouzni G et al.; Addition of M13mp18 single-stranded DNA annealed with an oligonucleotide to a Xenopus egg extract results in a rapid and efficient incorporation of the oligonucleotide in a complete double-stranded supercoiled molecule . Both the efficiency of DNA synthesis and the recovery of complete double-stranded molecules are increased relative to the reaction carried out by the classical technique using the E . coli Klenow DNA polymerase, DNA ligase, dNTPs, ATP and ions . Site specific mutagenesis was assayed by reverting a point mutation in the lacz region of M13mp18 . The color assay described by Messing and sequencing of the DNA extracted from isolated plaques was used to check for the reversion . A 2 hr incubation of the heteroduplex carrying the mutagenic oligonucleotide in the Klenow-ligase-dNTP mixture allows a recovery of 6% mutant phage after transformation of competent cells with the reaction products . Using the Xenopus egg extract, 83% mutant phage were recovered after the same incubation time, in reactions entirely performed in parallel . The Xenopus extract is stable and contains all components required for the assay, including all ionic and protein factors; thus the only addition is the annealed DNA . Such an eukaryotic system is therefore an attractive alternative to the reconstituted prokaryotic DNA polymerase-DNA ligase system for site specific mutagenesis.

Nucleic Acids Res, 1988 Sep 12, 16(17), 8411 - 31
Recognition of prokaryotic transcription terminators by spinach chloroplast RNA polymerase; Chen LJ et al.; To determine whether chloroplast RNA polymerase will accurately terminate transcription in vitro, we have fused the spinach chloroplast rbcL promoter to the 3' end of the rbcL gene as well as to various factor independent transcription terminators from E . coli . Transcription of the rbcL minigene did not result in production of the expected 265 nucleotide RNA . However, the spinach chloroplast RNA polymerase did terminate transcription with varying efficiency at the thra, rrnB, rrnC and gene 32 terminators . The most efficient transcription termination was observed for the threonine attenuator . For each of the prokaryotic terminators, the chloroplast enzyme ceased transcription at essentially the same position as the E . coli RNA polymerase . These data indicate that the transcription termination process in chloroplasts has some features in common with the mechanism used in prokaryotes.

Biochimie, 1988 Sep, 70(9), 1269 - 76
Molecular sorting of proteins into the cisternal secretory pathway; Scheele GA; Cotranslational translocation of exportable proteins across the RER membrane prior to their release into the extracellular space has been essentially described by use of canine pancreatic microsomal membranes . Intracisternal segregation of nascent secretory proteins was observed to be irreversible and proteolytic removal of signal sequences resulted in conformationally mature and stable proteins . Structural studies on various translocation peptides from both eukaryotic and prokaryotic preparations showed that many of them have a comparable three-domain organization . A hydrophilic amino-terminal domain is followed by a core region of hydrophobic amino acids and by the region in which the proteolytic cleavage occurs . Membrane components involved in the translocation process namely the signal recognition particle and the SRP receptor as well as the way the vectorial transport mechanism of nascent secretory proteins occurs are also discussed.

Mutagenesis, 1988 Sep, 3(5), 373 - 80
In vitro complementation of xeroderma pigmentosum; Kaufmann WK; In vitro complementation is a powerful strategy for isolation and characterization of individual components of multienzyme biochemical pathways . Application of the method to elucidate DNA metabolic pathways in prokaryotes enabled the successful identification of pathways of DNA replication and repair . In practice the technique requires the availability of genetic mutants that display defective operation of a selected biochemical pathway, and an in vitro assay system that allows detection of effective operation of the pathway . Xeroderma pigmentosum is a human disease syndrome characterized by partial or severe deficit in the operations of the nucleotidyl DNA excision repair pathway . This pathway of DNA repair appears to respond to DNA lesions which produce substantial distortion of helical structure, the best characterized of which are the UV radiation-induced pyrimidine dimers . This review summarizes a variety of approaches to analysis of the reparative deficiencies in xeroderma pigmentosum by in vitro complementation.

Proc Natl Acad Sci U S A, 1988 Sep, 85(18), 6632 - 6
Role of the Escherichia coli DnaK and DnaJ heat shock proteins in the initiation of bacteriophage lambda DNA replication; Liberek K et al.; We examined the role of two Escherichia coli heat shock proteins, the dnaK and dnaJ gene products, during the initiation of lambda dv DNA replication in vitro . Using 14C-labeled lambda P protein we showed that the DnaK and DnaJ heat shock proteins function together to release lambda P protein from the preprimosomal complex consisting of lambda origin of replication-lambda O-lambda P-DnaB protein . Hydrolysis of ATP, catalyzed presumably by DnaK, is required during this reaction . Substitution of DnaK protein with that of the mutant DnaK756 protein blocks lambda P release . After DnaK and DnaJ action, the preprimosomal complex, isolated on Sepharose 4B, can support lambda dv DNA replication without any additional prepriming proteins . Using DnaK-affinity chromatography we showed that both lambda O and lambda P proteins bind to DnaK protein . The lambda P protein interacts with DnaK protein in a salt-resistant, hydrophobic manner, and ATP hydrolysis is necessary to elute at least part of lambda P protein from the DnaK-affinity column . The proposed mechanism of action of the prokaryotic DnaK and DnaJ heat shock proteins agrees with the hypothesis that Hsp70, the DnaK analogue of eukaryotes, uses ATP to disrupt hydrophobic aggregates {Pelham, H . R . B . (1986) Cell 46, 959-961}.

Virology, 1988 Sep, 166(1), 166 - 74
Reactivation of the methylation-inhibited late E2A promoter of adenovirus type 2 by a strong enhancer of human cytomegalovirus; Knebel-Morsdorf D et al.; Promoter inactivation by sequence-specific methylation was demonstrated by using a construct which contained the late E2A promoter of adenovirus type 2 (Ad2) DNA and the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as indicator . After the in vitro methylation of 5'-CCGG-3' sequences at positions -215, +6, and +24 relative to the cap site of the promoter, the construct was inactive upon transfection into mammalian cells . The same pAd2E2AL-CAT construct was active in the unmethylated form . Promoter inactivation could be overcome when the strong immediate early enhancer of human cytomegalovirus DNA, which lacked 5'-CCGG-3' sites, was inserted into the construct either in a position immediately antecedent to the promoter or in a location several thousand nucleotides remote from it . Reactivation of the 5'-CCGG-3' methylated pAd2E2AL-CAT construct entailed initiation of transcription at the authentic cap site of the late E2A promoter and maintenance of methylation at least during the duration of the transient expression experiment . Reactivation of the methylated late E2A promoter had also been demonstrated by the trans-activating 289 amino acid protein which was encoded in the E1A region of adenoviruses (B . Weisshaar et al., 1988, J . Mol . Biol . 202, 255-270) . Thus there are several ways in which a methylated and silenced promoter can be reactivated in mammalian cells.

Biokhimiia, 1988 Aug, 53(8), 1278 - 87
{The effect of SSB-protein from Ehrlich ascites carcinoma cells on activity of various enzymes of DNA replication and repair}; Koterov AN et al.; The effect of a single-stranded DNA-binding protein (SSB-protein) form Ehrlich ascites tumour cells (EAT) on the activity of homologous purified DNA-polymerases alpha and beta, DNA-replicase, primase and DNA-polymerases from phage T4 and Bacillus stearothermophillus was studied . It was shown that the SSB-protein caused a 1.5-2.5-fold stimulation of the DNA-polymerase alpha activity on different templates (e.g., denaturated and activated DNA, poly(dA) . The degree of stimulation depended on the template type, protein/template ratio and purity of DNA-polymerase alpha . The activity of DNA-polymerase was inhibited by the SSB-protein, when the activated DNA was used as a matrix and was unchanged on the denaturated DNA . The activity of some prokaryotic DNA-polymerases was increased under the influence of the SSB-protein . The protein enhanced the processivity of T4 DNA-polymerase and strongly inhibited the activity of replicase and primase . A conclusion about the complex effect of the SSB-protein on the activity of replicative and repair enzymes is drawn.

Eur J Biochem, 1988 Aug 1, 175(2), 405 - 11
Prokaryotic hopanoids: the biosynthesis of the bacteriohopane skeleton . Formation of isoprenic units from two distinct acetate pools and a novel type of carbon/carbon linkage between a triterpene and D-ribose; Flesch G et al.; Incorporation of 13C-labelled acetate into the hopanoids of the purple non-sulfur bacteria Rhodopseudomonas palustris and Rhodopseudomonas acidophila and the facultative methylotroph Methylobacterium organophilum showed that the bacteriohopane skeleton is built from an unique carbon/carbon linkage between the triterpenic hopane moiety and the C-5 carbon of a D-ribose derivative arising from the non-oxidative pentose phosphate pathway . Furthermore a probable compartmentation of the acetate metabolism could be observed in these bacteria . Whereas exogenous acetate was directly incorporated into the glucose derivatives and poly-(3-hydroxybutyrate), the isoprenic units were apparently solely synthesized from two acetate units arising from the glyoxylate cycle and a third one issued either from the glyoxylate cycle or from the Entner-Doudoroff pathway of glucose catabolism . Although an unknown biosynthetic pathway different from that usually proposed for isoprenoid biosynthesis can not be excluded, the former hypothesis explained all labelling patterns observed on the triterpenic skeleton.

Int J Radiat Biol, 1988 Aug, 54(2), 131 - 50
The repairability of oxidative free radical mediated damage to DNA: a review; Teebor GW et al.; Many DNA repair enzyme activities are present in both prokaryotic and eukaryotic organisms . Among these are DNA exo- and endonucleases and DNA glycosylases which remove oxidatively damaged portions of the DNA molecule, thereby initiating excision-repair . The existence of these enzymes may be taken as evidence that cellular DNA is continuously subject to endogenous oxidative stress . Many of the lesions introduced by ionizing and ultraviolet radiation are identical to those introduced into DNA by reactive oxygen species generated by activated white cells, and are substrates for the repair enzymes . The chemical nature of the lesions, their biologic effects, and the mechanism of their repairability are described.

Proc Natl Acad Sci U S A, 1988 Aug, 85(16), 5794 - 8
Primary structure of cotranscribed genes encoding the Rieske Fe-S and cytochrome f proteins of the cyanobacterium Nostoc PCC 7906; Kallas T et al.; The thylakoid membrane cytochrome b6-f complex (plastoquinol:oxidized-plastocyanin oxidoreductase, EC 1.10.99.1) catalyzes electron-transfer and proton-translocation reactions essential for oxygenic photosynthesis . We have isolated and determined the nucleotide sequences of the petC and petA genes encoding the Rieske Fe-S and cytochrome f polypeptides from the filamentous cyanobacterium Nostoc PCC 7906 . These genes occur as single genomic copies, are tightly linked, and, as indicated by hybridization of gene-specific probes to Nostoc RNA, are cotranscribed as a 2.0-kilobase message . The Rieske Fe-S/cytochrome f gene pair thus represents an example of clustering and cotranscription in cyanobacteria of functionally related genes that, in photosynthetic eukaryotes, reside on separate nuclear and plastid genomes . These data are consistent with the progressive degeneration of the modern chloroplast genome from the ancestral, cyanobacterial-like genome of an endosymbiont . The Rieske Fe-S and the mature cytochrome f apoproteins are encoded by 537 and 867 nucleotides and have molecular masses of 19.2 and 31.2 kDa, respectively . They show 59% and 60% protein sequence identity, respectively, relative to spinach . Forty-four amino acids (4.7 kDa) resembling a prokaryotic signal sequence precede apocytochrome f . In contrast, the Rieske Fe-S protein appears to be translated without a presequence . The 183 bases separating the Rieske Fe-S and preapocytochrome f genes contain two families of 7- to 9-base tandem repeats, and some part of this sequence is highly reiterated in the genome . The C terminus of the Rieske Fe-S protein contains cysteine and histidine residues (probable ligands for the Fe2S2 center) in two peptides, Cys-Thr-His-Leu-Gly-Cys-Val and Cys-Pro-Cys-His-Gly-Ser, which have been conserved in spinach and in the five available Rieske Fe-S sequences from the mitochondrial-type cytochrome b-c1 complexes . Cytochrome f shows the heme binding residues Cys-Xaa-Xaa-Cys-His near its N terminus . Single, long hydrophobic stretches occur near the N and C termini, respectively, of the Rieske Fe-S and cytochrome f proteins and may form membrane-spanning helices.

Proc Natl Acad Sci U S A, 1988 Aug, 85(15), 5463 - 7
Expression of the human erythrocyte glucose transporter in Escherichia coli; Sarkar HK et al.; The gene encoding the human erythrocyte glucose transporter, cloned from HepG2 hepatoma cells, was expressed in Escherichia coli by introducing a prokaryote-type ribosome binding site, subcloning the gene into the T7 promoter/T7 polymerase expression system, and transforming a strain that is defective in glucose transport . Cells bearing plasmids with the transporter gene take up 2-deoxy-D-glucose and D-glucose, unlike cells bearing plasmids without the transporter gene . Moreover, 2-deoxy-D-glucose uptake is inhibited by unlabeled D-glucose, cytochalasin B, or mercuric chloride but not by L-glucose . The glucose transport protein is inserted into the membrane of E . coli, as evidenced by immunoblotting experiments with two site-directed polyclonal antibodies, one directed against the COOH terminus of the glucose transporter and the other directed against a synthetic peptide containing amino acid residues 225-238 . As detected with both antibodies, the protein migrates with apparent molecular mass of 34 kDa in sodium dodecyl sulfate/12% polyacrylamide, a size similar to that of the unglycosylated glucose-transport protein synthesized in vitro.

Virology, 1988 Aug, 165(2), 406 - 18
Common epitopes of glycoprotein B map within the major DNA-binding proteins of bovine herpesvirus type 2 (BHV-2) and herpes simplex virus type 1 (HSV-1); Hammerschmidt W et al.; Bovine herpesvirus 2 (BHV-2) specifies a glycoprotein of 130 kDa (gB BHV-2) which shows extensive homology to glycoprotein B (gB-1) of herpes simplex virus 1 (HSV-1) . The BHV-2-specific 130-kDa glycoprotein is able to induce cross-reacting antibodies, some of which even cross-neutralize HSV-1 . In order to determine the genome localization of gB BHV-2 and in order to identify conserved antigenic domains in both glycoproteins, we established libraries of subgenic fragments of BHV-2 and HSV-1 DNA in the prokaryotic expression vector lambda gt11 and screened them with cross-reacting monoclonal antibodies which allowed us to identify recombinant lambda gt11 clones expressing gB fusion protein . Nucleotide sequencing of inserted DNA fragments within these recombinant lambda gt11 clones revealed that they originated from the carboxy-terminal part of the major DNA-binding proteins (dbp) of BHV-2 (dbp BHV-2) and its counterpart ICP8 in HSV-1 . Antisera raised against the beta-galactosidase fusion protein of recombinant phage lambda-113/2 coding for an 84 amino acid (aa) polypeptide originating from dbp BHV-2 neutralized infectivity of BHV-2 and HSV-1 in the presence of complement and precipitated {3H} glucosamine-labeled gB BHV-2 and gB-1 . This antiserum also reacts with ICP8 and presumably with dbp BHV-2 . Two hypotheses are discussed to explain this unexpected result: (i) epitopes in the carboxy-terminal part of gB BHV-2 and gB-1 are similar to antigenic determinants in the amino-terminal region of the gBs, thus providing cross-reacting antibody-binding sites; (iii) during gene expression a carboxy-terminal part of dbp BHV-2 and ICP8 genes might be spliced to the amino-terminal region of the glycoproteins gB BHV-2 and gB-1.

Nucleic Acids Res, 1988 Jul 25, 16(14A), 6477 - 92
Bent DNA structures associated with several origins of replication are recognized by a unique enzyme from trypanosomatids; Linial M et al.; Sequence-directed bending of the DNA double helix is a conformational variation found in both prokaryotic and eukaryotic organisms . The utilization of bent DNA structures from various sources as specific signals recognized by an enzyme is demonstrated here using a unique endonuclease purified from trypanosomatid cells . Crithidia fasciculata nicking enzyme was previously shown to recognize specifically the bent structure found in kinetoplast DNA minicircles . The binding constant measured for this specific interaction is of two orders of magnitude higher than that measured for the binding of the enzyme to a non-curved sequence . As determined by binding competition and mobility shift electrophoresis analyses, this enzyme recognizes the sequence-directed bends associated with the origins of replication of bacteriophage lambda and simian virus 40 (SV40), as well as that located within the autonomously replicating sequence (ARS1) region of the yeast S . cerevisiae.

J Biol Chem, 1988 Jul 25, 263(21), 10295 - 9
An N-terminally fused Xenopus transcription factor IIIA synthesized in Escherichia coli is biologically active; Pao CI et al.; A 1.5-kilobase DNA fragment containing the Xenopus transcription factor IIIA (TFIIIA) gene was inserted into the prokaryotic expression vector pIN-III(A) containing the lpp/lac promoter . The recombinant DNA was introduced into Escherichia coli K-12 strain SB221 . The expression TFIIIA gene was induced by isopropyl-beta-D-thiogalactopyranoside, which resulted in the synthesis of a recombinant TFIIIA with an extra 17 amino acids fused to its N terminus as predicted from the nucleotide sequence . The engineered gene product, purified to at least 90% homogeneity, retained its binding affinity to the intragenic control region of the 5 S RNA gene, as well as its activity to stimulate 5 S RNA gene transcription in vitro.

J Biol Chem, 1988 Jul 15, 263(20), 9578 - 81
The 5'-terminal guanylate of chloroplast histidine tRNA is encoded in its gene; Burkard U et al.; Among tRNA species histidine tRNAs possess the unique feature of having an additional nucleotide, a guanylate, at their 5'-end . In prokaryotes this G is encoded in the gene sequence and retained in the mature tRNA as result of an unusual cleavage by RNase P (Orellana, O., Cooley, L., and Soll, D . (1986) Mol . Cell . Biol . 6, 525-529), while in eukaryotes it is added post-transcriptionally by a special tRNA guanylyl transferase (Cooley, L., Appel, B., and Soll, D . (1982) Proc . Natl . Acad . Sci . U . S . A . 79, 6475-6479) . Here we show that the additional G of chloroplast tRNAHis species is gene-encoded and retained in the tRNA by a processing step analogous to the prokaryotic one . However, the structural requirements for recognition by the different RNase P activities are not the same.

Gene, 1988 Jul 15, 67(1), 141 - 5
The instability of a recombinant plasmid, caused by a prokaryotic-like promoter within the eukaryotic insert, can be alleviated by expression of antisense RNA; Futterer J et al.; A region of the cauliflower mosaic virus genome was found to direct the expression of a nucleic-acid-binding protein in Escherichia coli . This protein is apparently toxic for the bacteria and leads to a destabilization of plasmids containing that region . Antisense RNA was used to diminish the unwanted expression and to stabilize the respective recombinant plasmids . The approach described may prove useful in other cases where problems with cloning of eukaryotic DNA arise.

Biochim Biophys Acta, 1988 Jul 14, 966(1), 30 - 5
Escherichia coli isocitrate lyase: properties and comparisons; Hoyt JC et al.; The glyoxylate cycle was first discovered during studies on bacteria and fungi with the ability to grow on acetate or ethanol as the sole carbon source . Isocitrate lyase, the first enzyme unique to the glyoxylate cycle, has been studied in numerous prokaryotic and eukaryotic organisms . However, information on this enzyme from Escherichia coli is limited . We have recently reported the purification and in vitro phosphorylation of this enzyme . In the present study we have examined and characterized a variety of inhibitors, the divalent cation requirement and the amino acid composition of E . coli isocitrate lyase and compared these results to those obtained with other organisms.

Biochim Biophys Acta, 1988 Jul 13, 950(2), 234 - 7
Induction of a light-inducible gene in Arthrobacter sp . by exposure of cells to chelating agents and pH 5; Hoober JK et al.; Transcription of a light-inducible gene in the prokaryote Arthrobacter sp . is induced in the dark when cells are incubated with chelating agents or in medium at pH 5 . However, repletion of metal ions such as Ca2+, Mn2+ or Zn2+ or an increase in pH is required for accumulation of the gene product, an Mr 21,000 polypeptide . But such changes in condition restore repression of the gene, and the decay in the rate of synthesis of the polypeptide follows the same time-course as when photodynamically induced cells are transferred to the dark . These results are consistent with regulation of expression of this gene at transcriptional and posttranscriptional steps by mechanisms that involve metal-protein complexes.

Biochemistry, 1988 Jul 12, 27(14), 5179 - 88
Helix stability in prokaryotic promoter regions; Margalit H et al.; Prokaryotic promoters have been extensively studied to relate sequence features to promoter function . Here we examine the relationship between double-helix stability and promoter activity . The double-helix stability is evaluated from sequence data by free energy computation, based on reported values of dinucleotide free energies for strand separation . For a collection of 168 promoters, we find that within a 500-nucleotide span around the transcription initiation site the -10 region is the least stable . There is no correlation between the free energies and the rates of RNA polymerase-promoter open complex formation measured for 25 promoters . We also compare the free energies of 121 promoter mutations across the -35 and -10 consensus regions with the free energies of the corresponding wild-type sequences . These pairwise mutant-wild-type comparisons provide a particularly good test since the examined sequences differ only in one nucleotide so that all other sequence-dependent effects remain the same . About 80% of the mutations in the -10 region that show increased/reduced promoter activity are less/more stable than the wild types . The observed high free energy peak and the mutation data strongly support the conjecture that the instability, or melting properties, of the -10 region plays a significant role in promoter function.

J Biol Chem, 1988 Jul 5, 263(19), 9271 - 5
Cloning and nucleotide sequence of the isoamylase gene from Pseudomonas amyloderamosa SB-15; Amemura A et al.; The gene (iam) coding for isoamylase (glycogen 6-glucanohydrolase) of Pseudomonas amyloderamosa SB-15 was cloned . Its nucleotide sequence contained an open reading frame of 2313 nucleotides (771 amino acids) encoding a precursor of secreted isoamylase . The precursor contained a signal peptide of 26 amino acid residues at its amino terminus and three regions homologous with those conserved in alpha-amylases (1,4-alpha-D-glucan 4-glucanohydrolase) of species ranging from prokaryotes to eukaryotes . These homologous regions were also found in another debranching enzyme, pullulanase (pullulan 6-glucanohydrolase) from Klebsiella aerogenes . Sequences of the isoamylase also showed significant homology with those between positions 300 and the carboxyl terminus of pullulanase . The regions required for the specificity of isoamylase were discussed on the basis of a comparison of its amino acid sequence with those of alpha-amylases, cyclomaltodextrin glucanotransferases, and pullulanase.

J Biol Chem, 1988 Jul 5, 263(19), 9443 - 8
Extended polysialic acid chains (n greater than 55) in glycoproteins from human neuroblastoma cells; Livingston BD et al.; Polysialic acid-containing glycoproteins consisting of extended chains of at least 55 sialyl residues (DP55, where DP represents degree of polymerization) are expressed on human neuroblastoma cells, CHP-134 . The strategy used for detecting these unique carbohydrate structures was based on the use of two highly specific prokaryotic-derived enzyme systems and an anti-polysialosyl antibody (H.46) . These probes were developed for the detection of polysialic acid on neural cell adhesion molecules (Troy, F . A., Hallenbeck, P . C., McCoy, R . D., and Vimr, E . R . (1987) Methods Enzymol . 138, 169-185) . Proof for the presence of long chain multimers of sialic acid was based on two types of experiments which utilized: 1) a glycopeptide fraction of CHP-134 cells, labeled metabolically with D-{3H}GlcN and 2) a membrane fraction from CHP-134 cells which served as an exogenous acceptor of {14C} NeuNAc residues in an Escherichia coli K1 sialyltransferase assay . In vitro, this enzyme CMP-NeuNAc:poly-alpha-2,8-sialosyl sialyltransferase catalyzes the transfer of {14C}NeuNAc from CMP-{14C}NeuNAc to exogenous acceptors containing at least 3 sialyl residues . In the first series of experiments, endo-N-acetylneuraminidase (Endo-N), a bacteriophage-derived enzyme specific for hydrolyzing poly-alpha-2,8-sialosyl chains containing a minimum of 5 sialyl residues was used . Limit Endo-N digestion of the 3H-glycopeptides from the {3H} GlcN-labeled cells released short {3H}sialyl oligomers {( 3H}DP1-6) which were degraded to {3H}NeuNAc by exosialidase . Partial Endo-N digestion released a series of {3H}sialyl oligomers extending up to DP55 . The longer (DP20-55) and intermediate sized (DP10-20) oligomers were isolated and converted to short oligomers ((3H}DP1-6) by retreating with Endo-N, thus confirming their identity as homo-oligomers of alpha-2,8-linked {3H}NeuNAc residues . In the second series of experiments, a membrane fraction of CHP-134 cells was radiolabeled in vitro with {14C}NeuNAc by E . coli K1 sialyltransferase . The membrane fraction had a major portion of radioactivity that was high Mr and polydisperse (Mr 100,000-250,000) as demonstrated in sodium dodecyl sulfate-polyacrylamide gels . Using Western blotting, pre-existing material of similar size was shown to react with antibody H.46.(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Biol Evol, 1988 Jul, 5(4), 377 - 91
Molecular coevolution among cryptically simple expansion segments of eukaryotic 26S/28S rRNAs; Hancock JM et al.; The set of "expansion segments" of any eukaryotic 26S/28S ribosomal RNA (rRNA) gene is responsible for the bulk of the difference in length between the prokaryotic 23S rRNA gene and the eukaryotic 26S/28S rRNA gene . The expansion segments are also responsible for interspecific fluctuations in length during eukaryotic evolution . They show a consistent bias in base composition in any species; for example, they are AT rich in Drosophila melanogaster and GC rich in vertebrate species . Dot-matrix comparisons of sets of expansion segments reveal high similarities between members of a set within any 28S rRNA gene of a species, in contrast to the little or spurious similarity that exists between sets of expansion segments from distantly related species . Similarities among members of a set of expansion segments within any 28S rRNA gene cannot be accounted for by their base-compositional bias alone . In contrast, no significant similarity exists within a set of "core" segments (regions between expansion segments) of any 28S rRNA gene, although core segments are conserved between species . The set of expansion segments of a 26S/28S gene is coevolving as a unit in each species, at the same time as the family of 28S rRNA genes, as a whole, is undergoing continual homogenization, making all sets of expansion segments from all ribosomal DNA (rDNA) arrays in a species similar in sequence . Analysis of DNA simplicity of 26S/28S rRNA genes shows a direct correlation between significantly high relative simplicity factors (RSFs) and sequence similarity among a set of expansion segments . A similar correlation exists between RSF values, overall rDNA lengths, and the lengths of individual expansion segments . Such correlations suggest that most length fluctuations reflect the gain and loss of simple sequence motifs by slippage-like mechanisms . We discuss the molecular coevolution of expansion segments, which takes place against a background of slippage-like and unequal crossing-over mechanisms of turnover that are responsible for the accumulation of interspecific differences in rDNA sequences.

Mutat Res, 1988 Jul-Aug, 200(1-2), 99 - 116
The role of blood platelets in nucleoside metabolism: assay, cellular location and significance of thymidine phosphorylase in human blood; Shaw T et al.; The enzyme thymidine phosphorylase (thymidine: orthophosphate deoxyribosyltransferase, EC 2.4.2.4), which plays a crucial role in nucleic acid metabolism in both prokaryotic and eukaryotic cells by regulating the availability of thymidine, is present in mammalian blood . Here we describe a simple, rapid HPLC-based micromethod for the assay of blood thymidine phosphorylase . We have arbitrarily defined 1 unit of blood thymidine phosphorylase activity as the activity required to produce a 1-nM increment in the plasma concentration of thymine after incubation for 1 h at 37 degrees C with a saturating concentration of exogenous thymidine . In normal adults, whole (peripheral venous) blood thymidine phosphorylase activity with blood cells intact was 64 +/- 11 units (mean +/- S.D., n = 20, range 45-89) . The apparent Michaelis constant for thymidine was of the order of 10(-4) M but varied nearly 5-fold between different individuals . Activity increased when blood cells were permeabilised or lysed with non-ionic detergents, implying that thymidine phosphorylase is an intracellular enzyme which may be influenced by exogenous as well as intracellular factors . When blood from normal donors was fractionated, thymidine phosphorylase activity consistently co-isolated with platelets . Whole-blood thymidine phosphorylase activity correlated well with platelet parameters . Although thymidine phosphorylase activity was also detected in plasma and serum, the small size and notorious fragility of platelets suggest its platelet origin . Blood from leukaemic donors showed significantly increased thymidine phosphorylase activity compared to normal controls (mean activity +/- S.D . was 96 +/- 27 units; range 58-140, n = 8) . Thymine formation from thymidine was temperature- and pH-dependent in whole blood . 2'-Deoxyuridine and 3 of its 5-halogenated analogues (but not 3'-azido-3'-deoxythymidine (AZT), were catabolised by blood thymidine phosphorylase, even during blood clotting at room temperature . Assumptions about in vivo concentrations of these compounds should therefore be interpreted cautiously . In the presence of high concentrations of thymine and suitable deoxyribose donors, small amounts of thymidine were formed in some blood samples, so it is conceivable that thymidine catabolism may be reversible in vivo under some circumstances.

Proc Natl Acad Sci U S A, 1988 Jul, 85(14), 4981 - 5
Transmitter and receiver modules in bacterial signaling proteins; Kofoid EC et al.; Prokaryotes are capable of sophisticated sensory behaviors . We have detected sequence motifs in bacterial signaling proteins that may act as transmitter or receiver modules in mediating protein-protein communication . These modules appear to retain their functional identities in many protein hosts, implying that they are structurally independent elements . We propose that the fundamental activity characterizing these domains is specific recognition and association of matched modules, accompanied by conformational changes in one or both of the interacting elements . Signal propagation is a natural consequence of this behavior . The versatility of this information-processing strategy is evident in the chemotaxis machinery of Escherichia coli, where proteins containing transmitters or receivers are linked in "dyadic relays" to form complex signaling networks.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1988 Jul, 269(1), 26 - 33
An electron microscopy study of Legionella pneumophila after in vitro and in vivo culture; Surgot M et al.; The ultrastructure of Legionella pneumophila serogroup 1 (Philadelphia Strain) was studied by Scanning Transmission Electron Microscopy (STEM) following in vitro and in vivo culture . The results obtained were identical for all the samples tested; the structure belonged to the prokaryotic type, with features consistent with the known structure of Gram-negative rods . A characteristic feature is the loose undulated outer membrane, a peptidoglycan layer which was difficult to visualize and was better seen in lysed bacteria . The outermost layer was stained by ruthenium red for polysaccharides . In guinea-pig lung tissue and in chick embryo yolk sac membranes, the bacteria were more often intracellular in intracytoplasmic ribosome-studded vacuoles.

Biochem Pharmacol, 1988 Jul 1, 37(13), 2551 - 8
Pethidine analogues, a novel class of potent inhibitors of mitochondrial NADH: ubiquinone reductase; Filser M et al.; Analogues of the analgetic drug pethidine were synthesized . Two N-aralkylen derivatives displayed a superior inhibitory effect on the activity of NADH:ubiquinone reductase in beef heart mitochondrial membranes . Dose-response curves revealed that the potency of these compounds is very comparable to that of the standard probe rotenone . The inhibitors were characterized by (a) their action on the reductase activity in various (eukaryotic and prokaryotic) organisms, (b) their influence on the enzyme kinetics, (c) their effects on the NADH dependent reduction of different electron acceptors, (d) their interference with the activities of other mitochondrial oxido-reductases . With regard to many of these aspects a close analogy between pethidine derivatives and rotenone was observed . A computer simulation of the steric structures of these molecules indicates that both classes of the chemically rather unrelated inhibitors may imitate very similar conformations . The potential advantages of the pethidine derivatives for the investigation of structure - function relationships within complex I of the respiratory chain is discussed.

Ann Inst Pasteur Virol, 1988 Jul-Sep, 139(3), 263 - 76
Expression of CaMV ORF IV in Escherichia coli; Albrecht H et al.; A CaMV DNA fragment corresponding to nucleotides 2200-3992 and including the coding sequence (2200-3670) of open reading frame IV was inserted in the pTG908 prokaryotic expression vector . In the recombinant pTG-IV plasmid, ORF IV, which codes for the coat protein precursor, was fused to the N-terminal coding sequence of the lambda CII gene, which is under transcriptional control of the lambda PL promoter . The expected fusion protein CII-ORF IV had a calculated molecular weight of 58.4 Kd . Nevertheless, temperature induction of the PL promoter resulted in synthesis of a major 76-Kd fusion protein: the coat protein precursor migrated abnormally on SDS polyacrylamide gel.

Horm Metab Res, 1988 Jul, 20(7), 411 - 20
Insulin-related materials in the nervous system of vertebrates and non-vertebrates: possible extrapancreatic production; LeRoith D et al.; Studies from multiple laboratories with a range of methods raised the possibility that insulin production occurs naturally at extrapancreatic sites . Part A covers the presence of insulin-related materials in organisms that do not have an endocrine pancreas, including unicellular prokaryotes and eukaryotes as well as multicellular non-vertebrate animals (insects et al.) and plants . Part B covers possible production of insulin by extrapancreatic tissues of vertebrates that are remote from a source of pancreatic insulin e.g . early chick embryos and mammalian cells in culture . Part C covers possible extrapancreatic insulin production in mammals in vivo . Each section ends with an outline summary with evidence in favor of and against the hypothesis.

Proc Natl Acad Sci U S A, 1988 Jul, 85(14), 5166 - 70
Site-specific DNA recombination in mammalian cells by the Cre recombinase of bacteriophage P1; Sauer B et al.; The Cre protein encoded by the coliphage P1 is a 38-kDa protein that efficiently promotes both intra- and intermolecular synapsis and recombination of DNA both in Escherichia coli and in vitro . Recombination occurs at a specific site, called lox, and does not require any other protein factors . The Cre protein is shown here also to be able to cause synapsis of DNA and site-specific recombination in a mammalian cell line . A stable mouse cell line was established that expresses the Cre protein under the control of the Cd2+-inducible metallothionein I gene promoter . DNA recombination was monitored with DNA substrates containing two directly repeated lox sites . One such substrate is a circular plasmid with two directly repeated lox sites (lox2) flanking a marker gene and was introduced into cells by Ca3(PO4)2 transformation . As a second substrate we used a pseudorabies virus (a herpesvirus) containing a lox2 insertion designed to provide a sensitive detection system for recombination . In both cases, site-specific recombination in vivo is dependent on the presence of the Cre protein and occurs specifically at the 34-base-pair lox sites . These results demonstrate the controlled site-specific synapsis of DNA and recombination by a prokaryotic protein in mammalian cells and suggest that Cre-mediated site-specific recombination may be a useful tool for understanding and modulating genome rearrangements in eukaryotes.

Gene, 1988 Jun 30, 66(2), 215 - 22
Identification of functional open reading frames in chloroplast genomes; Wolfe KH et al.; We have used a rapid computer dot-matrix comparison method to identify all DNA regions which have been evolutionarily conserved between the completely sequenced chloroplast genomes of tobacco and a liverwort . Analysis of these regions reveals 74 homologous open reading frames (ORFs) which have been conserved as to length and amino acid sequence; these ORFs also have an excess of nucleotide substitutions at silent sites of codons . Since the nonfunctional parts of these genomes have become saturated with mutations and show no sequence similarity whatsoever, the homologous ORFs are almost certainly functional . A further four pairs of ORFs show homology limited to only a short part of their putative gene products . Amino acid sequence identities range between 50 and 99%; some chloroplast proteins are seen to be among the most slowly evolving of all known proteins . A search of the nucleotide and amino acid sequence databanks has revealed several previously unidentified genes in chloroplast sequences from other species, but no new homologies to prokaryotic genes.

J Biol Chem, 1988 Jun 25, 263(18), 8864 - 71
Transformation of glutamate to delta-aminolevulinic acid by soluble extracts of Synechocystis sp . PCC 6803 and other oxygenic prokaryotes; Rieble S et al.; delta-Aminolevulinic acid is the first committed precursor in the biosynthesis of hemes, phycobilins, and chlorophylls . Plants and algae synthesize delta-aminolevulinic acid from glutamate via an RNA-dependent 5-carbon pathway . Previous reports demonstrated that cyanobacteria form delta-aminolevulinic acid from glutamate in vivo . We now report the direct measurement of this activity in vitro . Three oxygenic prokaryotes were examined, the unicellular cyanobacteria Synechocystis sp . PCC 6803 and Synechococcus sp . PCC 7002 (Agmenellum quadruplicatum PR-6) and the chlorophyll a- and b-containing filamentous prochlorophyte Prochlorothrix hollandica . delta-Aminolevulinic acid-forming activity was detected in soluble extracts of all three species . delta-Aminolevulinic acid formation by Synechocystis extracts was further characterized . Activity depended upon addition of reduced pyridine nucleotide, ATP, and Mg2+ to the incubation mixture . NADPH was a more effective pyridine nucleotide than NADH at low concentrations, but NADPH inhibited delta-amino-levulinic acid formation above 1 mM, whereas NADH did not . The pH optimum was about 7.6, and the ATP concentration optimum was 0.1 mM . Activity was stimulated by addition of RNA derived from Synechocystis or Chlorella, and abolished by preincubation with RNase A . After RNase inactivation, activity was restored by addition of RNasin to block further RNase action, followed by supplementation with Synechocystis RNA . Activity was inhibited by micromolar concentrations of hemin, as was previously found with plant and algal extracts . Complete dependence on added glutamate could not be achieved . Radioactivity was incorporated into delta-aminolevulinic acid when the incubation mixture contained 1-{14C}glutamate . Activity in the Synechocystis enzyme extract was stimulated by the addition of a partially purified enzyme fraction from Chlorella . It thus appears that prokaryotic oxygenic organisms share with chloroplasts the capacity for biosynthesis of photosynthetic pigments from glutamate via the RNA-dependent 5-carbon pathway.

Biochem Biophys Res Commun, 1988 Jun 16, 153(2), 498 - 501
Assembly of M13 and M13am8H1R1 procoat protein into microsomes is stimulated by rabbit reticulocyte lysate and ATP; Sagstetter M et al.; Processing of M13 procoat protein to transmembrane coat protein by dog pancreas microsomes is stimulated by a component of rabbit reticulocyte lysate and ATP . We asked whether this ATP-dependent reaction, involved in membrane assembly of procoat protein in the eukaryotic system, is related to the membrane potential dependent reaction observed for the membrane assembly of procoat protein in E . coli . Specifically, we asked if a mutant procoat protein which had been previously shown to be independent of the membrane potential with respect to its assembly in E . coli (M13am8H1R1 procoat protein) shows a stimulation by reticulocyte lysate and ATP in its assembly into microsomes . Since the mutant procoat protein behaved exactly as the wild type procoat protein in the eukaryotic in vitro system, we propose that the ATP-dependent reaction observed for the eukaryotic system does not substitute for the membrane potential dependent reaction in the prokaryotic system.

Biochem J, 1988 Jun 15, 252(3), 825 - 31
Escherichia coli promoter -10 and -35 region homologies correlate with binding and isomerization kinetics; Studnicka GM; The DNA promoter sequence at which gene transcription is initiated in Escherichia coli contains two distinct regions of conserved nucleotides . Coefficients of homology to the -10 region and the -35 region were computed for many different prokaryotic promoters . Linear equations were derived that relate the degree of homology for each promoter region to the two kinetic constants that describe the interaction of RNA polymerase with the promoter site on DNA: the strength KB of binding to form closed complex, and the rate kf of isomerization to form open complex . A graphical plot of -35 versus -10 promoter region homologies for many promoters suggest that certain classes of prokaryotic operons might utilize differential degrees of consensus homology in these two regions to achieve specific control over kf and KB, in addition to modulating overall promoter strength.

J Biol Chem, 1988 Jun 15, 263(17), 8288 - 93
Characterization of a chloroplast sequence-specific DNA binding factor; Lam E et al.; The large subunit of ribulose 1,5-bisphosphate carboxylase (rbcL) and the beta subunit of chloroplast ATP synthase (atpB) are encoded by divergently transcribed genes on the plastid genome . We have identified DNA binding factors specific for sequences located in the intergenic region between these two genes . Soluble plastid extracts from pea or whole cell extracts from maize protected a maize chloroplast DNA probe containing the 160-base pair region between the 5' ends of rbcL and atpB genes from exonuclease III digestion between positions -16 and -101 relative to the rbcL gene transcription start site . Competition assay with partial sequences from this intergenic region demonstrated that specific sequence(s) are required for the protection . The borders of the binding domain are conserved among the homologous regions of maize, tobacco, spinach, and pea chloroplast genomes . Gel filtration chromatography revealed a molecular weight of about 115,000 for the active complex involved in DNA binding . Using the exonuclease III protection assay, we have also shown that purified Escherichia coli RNA polymerase protects from +25 to -20 of the rbcL gene and from +21 to -23 of the atpB gene relative to their respective transcription start sites . These regions are analogous to open complexes found when E . coli RNA polymerase interacts with the prokaryotic promoters and are consistent with the ability of E . coli RNA polymerase to initiate transcription correctly on linear templates containing these chloroplast promoters . Possible role(s) for the chloroplast DNA binding factor in chloroplast gene expression and its regulation are discussed.

Science, 1988 Jun 10, 240(4858), 1435 - 9
Research on bacteria in the mainstream of biology; Magasanik B; The study of the genetics, biochemistry, and physiology of bacteria during the last 40 years has provided the concepts and methods for the study of cells of all types at the molecular level . Although much is already known about the mechanisms bacteria use to regulate the expression of their genes, a great deal more remains to be discovered that will have relevance to both prokaryotic and eukaryotic cells . Similarly, the study in bacteria of the transactions of DNA, of the synthesis and function of the cell membrane, of differentiation, and of the interaction with eukaryotic cells will undoubtedly produce results of general importance . The advantages of using bacteria for these studies include their simple noncompartmented structure, the accessibility of their genetic material, and the possibility of correlating the expression of a gene in the intact cell with its expression in a system composed of highly purified components . Finally, the comparative study of a wide variety of microorganisms may result in a better understanding of the evolution of prokaryotes and eukaryotes and lead to a comprehensive theory of cell biology.

J Biol Chem, 1988 Jun 5, 263(16), 7760 - 6
Synthesis and secretion of multiple forms of beta 2-interferon/B-cell differentiation factor 2/hepatocyte-stimulating factor by human fibroblasts and monocytes; May LT et al.; The cDNA for human beta 2-interferon (IFN-beta 2)/B-cell differentiation factor 2/hepatocyte-stimulating factor was expressed in Escherichia coli to yield a fusion protein which contains the 182 carboxyl-terminal amino acids of IFN-beta 2 fused to a 34-amino acid prokaryotic leader peptide (rIFN-beta 2) . When added to cultures of human hepatoma cell line Hep3B2, rIFN-beta 2 as well as preparations of natural IFN-beta 2 enhance secretion of positive acute phase reactants such as alpha 1-antichymotrypsin, complement C3, fibrinogen, and alpha 1-acid glycoprotein and inhibit secretion of albumin, confirming that a protein derived from the IFN-beta 2 gene can have hepatocyte-stimulating factor activity . We have prepared a rabbit polyclonal antiserum to the E . coli-derived human IFN-beta 2 fusion protein . This polyclonal antiserum inhibits the hepatocyte-stimulating and B-cell differentiation activities of appropriate IFN-beta 2 preparations . The anti-rIFN-beta 2 antiserum has been used in immunoprecipitation experiments and in Western blots to help define the secretory proteins derived from the IFN-beta 2 gene in fibroblasts and monocytes . "Uninduced" human FS-4 fibroblasts as well as those induced with interleukin-1 alpha, tumor necrosis factor, or bacterial lipopolysaccharide secrete at least five forms of IFN-beta 2 of apparent molecular mass in the range from 23 to 30 kDa which can be resolved by polyacrylamide gel electrophoresis under denaturing and reducing conditions . The three higher molecular mass forms are not observed when FS-4 cells are induced in the presence of tunicamycin, suggesting that these forms are N-glycosylated (gp28, gp29, and gp30) . Although secretion of the two lower molecular mass forms is resistant to tunicamycin, they are labeled by {3H}glucosamine (gp23-gp25) . The inclusion of cycloheximide during the {35S}methionine labeling of induced FS-4 cells results in the preferential synthesis and secretion of the 29-kDa triplet . Human monocytes induced with bacterial lipopolysaccharide also secrete several distinct forms of IFN-beta 2 in the size range from 23 to 30 kDa which co-migrate in polyacrylamide gels with those obtained from FS-4 cells . Our observations help relate previous descriptions of multiple forms of hepatocyte-stimulating factor to specific proteins derived from the IFN-beta 2 gene.

Scanning Microsc, 1988 Jun, 2(2), 925 - 35
Preparation of biological samples for transmission X-ray microanalysis: a review of alternative procedures to the use of sectioned material; Sigee DC; Although transmission X-ray microanalysis of biological material has traditionally been carried out mainly on sectioned preparations, a number of alternative procedures exist . These are considered under three major headings - whole cell preparations, analysis of cell homogenates and biological fluids, and applications of the technique to microsamples of purified biochemicals . These three aspects provide a continuous range of investigative level - from the cellular to the molecular . The use of X-ray microanalysis with whole cell preparations is considered in reference to eukaryote (animal) cells and prokaryotes - where it has particular potential in environmental studies on bacteria . In the case of cell homogenates and biological fluids, the technique has been used mainly with microdroplets of animal material . The use of X-ray microanalysis with purified biochemicals is considered in relation to both particulate and non-particulate samples . In the latter category, the application of this technique for analysis of thin films of metalloprotein is particularly emphasised . It is concluded that wider use could be made of the range of preparative techniques available - both within a particular investigation, and in diverse fields of study . Transmission X-ray microanalysis has implications for environmental, physiological and molecular biology as well as cell biology.

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 3772 - 6
DNA polymerase I gene of Saccharomyces cerevisiae: nucleotide sequence, mapping of a temperature-sensitive mutation, and protein homology with other DNA polymerases; Pizzagalli A et al.; A 5600-base pair segment spanning the coding region of the Saccharomyces cerevisiae DNA polymerase I gene was sequenced and found to contain an open reading frame of 1468 codons, corresponding to a polypeptide of Mr 166,794 . A pol1 temperature-sensitive mutation, encoding a DNA-polymerase-primase complex with altered stability, has a single base-pair substitution that changes the glycine at position 493 to a positively charged arginine . Protein sequence comparison with other prokaryotic and eukaryotic DNA polymerases reveals three major regions of homology . This observation suggests that certain DNA polymerases might require the conservation of critical amino acid residues for activity.

EMBO J, 1988 Jun, 7(6), 1821 - 9
The nucleocapsid of bacteriophage phi 6 penetrates the host cytoplasmic membrane; Romantschuk M et al.; Bacteriophage phi 6 infects its host, the Gram-negative bacterium Pseudomonas syringae, by a protein-targeted fusion of the virus envelope with the host outer membrane . In this investigation we present results suggesting that the phage nucleocapsid penetrates the host cytoplasmic membrane via a membrane invagination and an intracellular vesicle . This indicates that the prokaryotic plasma membrane might be more dynamic and have more common features with eukaryotic membrane systems than previously expected . Most of the nucleocapsid surface lattice protein is degraded in the cell, and the nucleocapsid core particle containing the viral dsRNA segments and the proteins necessary for the viral RNA polymerase activity can be isolated from the infected cells . The penetration is dependent on the energized state of the host cytoplasmic membrane . About 25% of the entering core particles are re-used in the progeny viruses.

Mol Gen Genet, 1988 Jun, 212(3), 418 - 25
Cloning, nucleotide sequence and mutational analysis of the gene encoding the Photosystem II manganese-stabilizing polypeptide of Synechocystis 6803; Philbrick JB et al.; Affinity purified, polyclonal antibodies raised against the Photosystem II 33 kDa manganese-stabilizing polypeptide of the spinach oxygen-evolving complex were used to isolate the gene encoding the homologous protein from Synechocystis 6803 . Comparison of the amino acid sequence deduced from the Synechocystis psb1 nucleotide sequence with recently published sequences of spinach and pea confirms the homology indicated by antigenic cross-reactivity and shows that the cyanobacterial and higher plant sequences are 43% identical and 63% conserved . Regions of identity, varying in length from 1 to 10 consecutive residues, are distributed throughout the protein . The 28 residues at the amino terminus of the psb1 gene product, characteristic of prokaryotic signal peptides, show homology with the carboxyl-terminal third of the transit sequences of pea and spinach and are most likely needed for the transport of the manganese-stabilizing protein across the thylakoid membrane to its destination of the lumen . Synechocystis mutants which contain a kanamycin resistance gene cassette inserted into the coding region for the 32 kDa polypeptide were constructed . These mutants contain no detectable 32 kDa polypeptide, do not evolve oxygen, and are incapable of photoautotrophic growth.

Mutat Res, 1988 Jun, 208(2), 109 - 13
Suppressive effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in the G1 phase of human lymphocytes; Kishi K; 1-beta-D-Arabinofuranosylcytosine (ara C) induces chromosome-type aberrations in mammalian cells by inhibiting repair replication in the G1 phase . The effect of novobiocin, an inhibitor of prokaryotic gyrases, on G1 repair in human cells was studied cytogenetically using this characteristic of ara C . The experiment was based on the assumption that if novobiocin inhibits the relaxation of chromatin required prior to repair replication, it would reduce the frequency of chromosome-type aberrations in cells treated with a mutagen followed by posttreatment with ara C . It has also been shown that in lymphocytes ara C induces chromosome-type aberrations which were not caused by any induced DNA lesion, and that the frequency of these aberrations changes with the age of the blood donor . The effect of novobiocin on the frequency of chromosome-type aberrations induced by ara C in lymphocytes without mutagen pretreatment was also investigated for blood samples from donors of different ages . Human peripheral blood lymphocytes, which were either untreated of treated with 4-nitroquinoline-N-oxide (4NQO) or methyl methanesulfonate (MMS), were posttreated in their early G1 phase with ara C only or ara C and novobiocin . The resulting chromosome-type aberrations were observed in cells in their first mitoses, and a comparison was made between the frequency of aberrations occurring in the presence of novobiocin and in its absence . The results showed that novobiocin reduced the frequency of chromosome-type aberrations induced by ara C in both mutagen-pretreated and -non-pretreated cells, and that lymphocytes from younger donors were less sensitive to novobiocin . The present study demonstrated cytogenetically the existence of a novobiocin-sensitive process to induce chromosome recombination in G1 lymphocytes.

Biochem Cell Biol, 1988 Jun, 66(6), 584 - 93
Transcriptional activation of heat-shock genes in eukaryotes; Tanguay RM; Prokaryotes and eukaryotes respond to thermal or various chemical stresses by the rapid induction of a group of genes collectively referred to as the heat shock genes . In eucaryotes, the expression of these genes is primarily regulated at the transcriptional level . The early observations that transfected heat shock genes were inducible in heterologous systems suggested the existence of common regulatory elements in these ubiquitous genes . Sequence analysis of cloned Drosophila heat shock genes revealed a conserved 14 base pair (bp) inverted repeat, which is essential for heat induction . This regulatory sequence, referred to as the heat shock element (HSE), is found in multiple imperfect copies upstream of the TATA box of all heat shock genes . While studies in heterologous systems indicated that a single copy of HSE was sufficient for inducibility, further analysis in homologous assays suggests that multiple HSE can act in a cooperative way and that the efficiency of transcriptional activation is related, within limits, to the number of HSE . Comparative analysis of heat shock genes reveals that HSE can be positioned at different distances from the TATA box in either orientation, a behavior reminiscent of enhancer elements . However, the presence of HSE does not necessarily confer heat inducibility, as shown by their presence in the constitutively expressed but non-heat-inducible homologous cognate genes . Footprinting and nuclease mapping have been used to show that a protein factor (HSTF: heat shock transcription factor) binds to the HSE element, activating heat shock gene transcription in a dose-dependent manner . The recent progress in the isolation and characterization of HSTF in Drosophila, yeast, and human cells is reviewed . Finally, different models suggested to account for the positive regulation of heat shock genes by the HSTF are presented.

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 4104 - 8
Cis-acting sequences that modulate atrial natriuretic factor gene expression; Seidman CE et al.; Nucleotide sequences necessary to direct transcription of the gene encoding atrial natriuretic factor (ANF) in neonatal and fetal hearts have been defined by using expression of the prokaryotic marker gene chloramphenicol acetyltransferase (CAT) as a functional assay . Hybrid ANF-CAT genes were introduced into primary cultured cardiocytes by electroporation . A 3.4-kilobase (kb) fragment containing sequences on the 5' side of the ANF gene promoted significant CAT activity in atrial but not ventricular cardiocytes derived from 1-day-old rats . Deletion analysis of putative regulatory regions demonstrated that 2.4 kb of 5' ANF sequences were sufficient for high-level atrial transcription, whereas hybrid genes containing less than 700 base pairs of ANF sequences promoted less CAT activity . Cardiocytes derived from embryonic ventricles expressed the 3.4-kb ANF-CAT hybrid gene at levels comparable to atrial cells, suggesting that the nucleotide sequences controlling developmental regulation of ANF expression are contained in this 5' region . Nucleotide sequence analysis of this 3.6-kb region identified segments that may contribute to the regulated expression of the ANF gene.

EMBO J, 1988 Jun, 7(6), 1907 - 11
Gamma delta transposase and integration host factor bind cooperatively at both ends of gamma delta; Wiater LA et al.; gamma delta, a prokaryotic transposon, encodes a transposase that is essential for its transposition . We show here, by DNase I protection experiments, that purified gamma delta transposase binds at the transposon's inverted repeats (IRs) . Immediately adjacent to each transposase binding site (and within gamma delta DNA) we have identified a binding site for an additional protein factor, the Escherichia coli-encoded integration host factor (IHF) . The binding of transposase and IHF to these adjacent sites is mutually cooperative . An IHF binding-site was also found in the original target DNA, just outside one of the ends of gamma delta . The affinity of IHF for this flanking site is reduced by transposase . These results demonstrate that gamma delta transposase binds at the IRs of gamma delta, and suggest that IHF may be involved in forming a transposase-DNA complex and/or influencing the target site selection during the transposition of gamma delta.

DNA, 1988 Jun, 7(5), 307 - 16
Expression of coxsackievirus B3 capsid proteins in Escherichia coli and generation of virus-specific antisera; Werner S et al.; Subgenomic fragments of cloned infectious coxsackievirus B3 (CVB3) cDNA up to the size of the complete coding sequence of the viral polyprotein were inserted into the prokaryotic expression vector pPLc24 and expressed in Escherichia coli . Fusion proteins, containing 54 amino acids of MS2 replicase at their amino terminus followed by different parts of the CVB3 structural proteins, were expressed from several constructs . The expression product of a plasmid encoding the capsid proteins VP4, VP2, and the amino-terminal part of VP3 was obtained in high amounts . However, primary expression products containing the complete viral capsid precursor VP4-VP1 were completely degraded, indicating the presence of domains downstream from VP3 that are accessible to E . coli proteases . This finding is consistent with the observation that the structural intact expression product of the separately subcloned VP1 gene is also extremely unstable and consequently obtained only in low amounts . Two fusion proteins of non-overlapping parts of the viral structural proteins containing VP4, VP2, and VP3 or VP1, respectively, were isolated and used for the generation of antisera in rabbits . The antisera obtained recognize distinct CVB3 structural proteins in infected cell cultures as well as from purified CVB3 preparations . In addition, significant cross-reactivity of the described antisera with the corresponding structural proteins of other enteroviruses was observed, indicating that these antisera provide a valuable tool for an improved broad spectrum diagnosis of enteroviral infections.

J Gen Virol, 1988 Jun, 69 ( Pt 6), 1195 - 204
Prokaryotic expression of immunogenic polypeptides of the large phosphoprotein (pp150) of human cytomegalovirus; Scholl BC et al.; The large phosphorylated matrix protein pp150 of human cytomegalovirus (HCMV) is the polypeptide most frequently reactive in immunoblotting analyses with human antisera when compared with other viral proteins . Several defined regions of pp150 were expressed as beta-galactosidase fusion proteins and these were tested for their immunoreactivity with human sera and their immunogenicity . One antigenic region could be expressed in large amounts and was found to carry immunodominant epitopes, as shown by immunoblotting and ELISA . A rabbit antiserum raised against recombinant pp150 antigens produced in bacteria proved to be useful for immunofluorescence and immunohistochemistry studies of HCMV-infected cells and tissues . The results suggest that this anti-pp150 serum will help to elucidate the process of virus assembly and antigen detection in infected cells.

Proc Natl Acad Sci U S A, 1988 Jun, 85(11), 4000 - 4
Mapping antigenic domains expressed by Chlamydia trachomatis major outer membrane protein genes; Baehr W et al.; Chlamydia trachomatis is an obligate prokaryotic intracellular pathogen of humans that infects mucosal epithelial cells . Exposed domains of its major outer membrane protein (MOMP) are both serotyping and protective antigenic determinants . To identify these domains, we have cloned and epitope-mapped the genes of serovars A, C (C serogroup) and L2, B (B serogroup) with a panel of monoclonal antibodies (mAbs) . Predominantly conserved regions of the genes of both serogroups are interspersed with four short variable domains (I-IV) . Recombinant phage clones expressing specific MOMP antigenic determinants revealed that protective serotype-specific recognized epitopes in variable domains I and II . Protective subspecies and serogroup-specific mAbs recognized overlapping determinants in variable domain IV near the C terminus . A nonprotective species-specific mAb mapped to an invariant peptide of nine residues contained within variable domain IV . In the intact chlamydial organism of serovar B, variable domains II and IV were susceptible to proteolytic digestion, whereas both N and C termini were protected . These results suggest an arrangement of MOMP in the outer membrane in which three of the four variable domains are exposed to the outside and in which both N and C termini are presumably oriented toward the periplasmic space . This molecular analysis of MOMP antigenic determinants and their surface topology on intact chlamydiae will be useful toward the development of a recombinant subunit or synthetic chlamydial vaccine.

Gene, 1988 May 30, 65(2), 305 - 14
Expression of cloned genes by in vivo insertion of tac promoter using a mini-Mu bacteriophage; Gramajo HC et al.; The strong trp-lac(tac) promoter has been incorporated into the mini-Mu bacteriophage genome to form a mini-Mu-tac (mMu-tac) expression transposon . This mMu-tac element can transpose efficiently in Escherichia coli cells when derepressed and occasionally insert into a recombinant plasmid . When mMu-tac integration occurs upstream of a cloned gene in the orientation of its transcription, the tac promoter can direct the expression of the gene insert . mMu-tac contains translation stop codons downstream of the tac promoter . Thus, mMu-tac should be useful to express only those genes containing their own translational information . We report here the successful expression in E . coli of several prokaryotic genes using the transposon expression system.

Nature, 1988 May 26, 333(6171), 330 - 4
Homologous plant and bacterial proteins chaperone oligomeric protein assembly; Hemmingsen SM et al.; An abundant chloroplast protein is implicated in the assembly of the oligomeric enzyme ribulose bisphosphate carboxylase-oxygenase, which catalyses photosynthetic CO2-fixation in higher plants . The product of the Escherichia coli groEL gene is essential for cell viability and is required for the assembly of bacteriophage capsids . Sequencing of the groEL gene and the complementary cDNA encoding the chloroplast protein has revealed that these proteins are evolutionary homologues which we term 'chaperonins' . Chaperonins comprise a class of molecular chaperones that are found in chloroplasts, mitochondria and prokaryotes . Assisted post-translational assembly of oligomeric protein structures is emerging as a general cellular phenomenon.

Nucleic Acids Res, 1988 May 25, 16(10), 4407 - 18
Sites of termination of in vitro DNA synthesis on cis-diamminedichloroplatinum(II) treated single-stranded DNA: a comparison between E . coli DNA polymerase I and eucaryotic DNA polymerases alpha; Villani G et al.; We have compared the capacity of the large fragment of E . coli DNA polymerase I and highly purified DNA polymerases alpha from either Drosophila melanogaster embryos or calf thymus to replicate single-stranded M13 mp10 DNA treated with the antitumoral drug cis-diamminedichloroplatinum(II) (cis-DDP) . We report that: a) although prokaryotic and eukaryotic enzymes have different structural complexity and dissimilar in vivo functions, their synthesis was blocked in vitro at similar sites on cis-DDP treated DNA; b) this inhibition occurred not only at d(G)n sequences, as previously reported for E . coli DNA polymerase I, (Pinto & Lippard (1985) Proc . Natl . Acad . Sci . USA, 82, 4616-4619) but also at other sequences which may represent putative cis-DDP-DNA adducts.

Biochim Biophys Acta, 1988 May 22, 960(2), 190 - 9
Polyterpenoids as cholesterol and tetrahymanol surrogates in the ciliate Tetrahymena pyriformis; Raederstorff D et al.; The tetracyclic sterol precursors, cyclolaudenol, cycloartenol and lanosterol, inhibit efficiently the tetrahymanol biosynthesis in the ciliate Tetrahymena pyriformis, as reported earlier for cholesterol and other sterols . The prokaryotic bacteriohopanetetrols have little effect, and diplopterol, another hopanoid, as well as the carotenoid, canthaxanthin, have no effect . In the presence of triparanol, a hypocholesterolemic drug inhibiting the squalene cyclase of T . pyriformis and modifying the fatty acid metabolism, the cells do not grow further, but growth can be restored by the addition to the culture medium of suitable polyterpenoids . Thus, growth in presence of triparanol (13 microM) is almost normal after addition of a sterol such as sitosterol and cyclolaudenol, and longer lag times and lower absorbances than those of untreated cultures are observed in presence of cyclartenol, lanosterol, euphenol (a lanosterol isomer), bacteriohopanetetrols and three carotenoids . No growth at all is observed in the presence of tetrahymanol and diplopterol, although these triterpenoids are the normal reinforcers of the ciliate, probably because of a poor bioavailability . Thus, structurally different polyterpenoids are (at least partially) functionally equivalent and capable of replacing tetrahymanol or sterols and might act as membrane reinforcers in T . pyriformis cells.

J Biol Chem, 1988 May 15, 263(14), 6854 - 6
Enzymatic removal of O6-ethylguanine from mitochondrial DNA in rat tissues exposed to N-ethyl-N-nitrosourea in vivo; Satoh MS et al.; DNA repair is essential for maintaining the integrity of the genetic material, and a number of DNA repair mechanisms have been fairly well characterized for the nuclear DNA of eukaryotic cells as well as prokaryotes . However, little is known about DNA repair in mitochondria . Using highly sensitive immunoanalytical methods to detect specific DNA alkylation products, we found active removal of O6-ethyl-2'-deoxyguanosine (O6-EtdGuo) from rat liver mitochondrial DNA after pulse-exposure to N-ethyl-N-nitrosourea in vivo . In the kidney, O6-EtdGuo was removed from mitochondrial DNA with moderate efficiency, but nearly no removal was observed from the DNA of brain mitochondria . Among the rat tissues examined, the kinetics of O6-EtdGuo elimination from mitochondrial DNA was very similar to the kinetics of removal from nuclear DNA . O4-Ethyl-2'-deoxythymidine, another premutagenic DNA ethylation product, was stable in both mitochondrial and nuclear DNA of rat liver.

J Biol Chem, 1988 May 15, 263(14), 6518 - 24
Specificity of proofreading by the 3'----5' exonuclease of the DNA polymerase-primase of Drosophila melanogaster; Reyland ME et al.; The DNA polymerase-primase from Drosophila melanogaster contains a cryptic 3'----5' exonuclease that can be detected after separation of the 182-kDa polymerase subunit from the four-subunit enzyme . To determine the specificity of excision of mispaired nucleotides by the exonuclease, we have utilized primed phi X174am3 single-stranded DNA containing a noncomplementary nucleotide at the 3'-primer terminus, opposite deoxyadenosine at position 587 in the amber3 codon of the template strand . In the absence of polymerization, the preference for excision of the mispaired nucleotide from the primer is C greater than A much greater than G . Excision under these conditions is inhibited by the addition of deoxyguanosine monophosphate . Under conditions of concomitant DNA synthesis, the preference for excision at this site becomes A = G much greater than C, and excision is insensitive to deoxyguanosine monophosphate . The high fidelity of DNA synthesis exhibited by the isolated 182-kDa polymerase subunit is not reduced by concentrations of deoxyguanosine monophosphate or adenosine monophosphate that inhibit proofreading by prokaryotic DNA polymerases . Thus, the 3'----5' exonuclease of the Drosophila DNA polymerase-primase participates in exonucleolytic proofreading by excising noncomplementary nucleotides prior to extension of the primer by polymerase action . The deoxynucleoside triphosphate analogs N2-(p-butylphenyl)deoxyguanosine triphosphate and N2-(p-butylphenyl)deoxyadenosine triphosphate are potent inhibitors of DNA polymerase alpha . Like calf thymus DNA polymerase delta, recently determined to have proofreading capability, DNA synthesis by the isolated Drosophila 182-kDa polymerase subunit was not inhibited by the two analogs . In contrast, DNA synthesis by the intact Drosophila polymerase-primase complex was inhibited greater than 95% by these analogs.

FEBS Lett, 1988 May 9, 232(1), 115 - 20
Primary and secondary structure of the 18 S ribosomal RNA of the insect species Tenebrio molitor; Hendriks L et al.; The sequence of the 18 S rRNA of Tenebrio molitor is reported . A detailed secondary structure model for eukaryotic small subunit rRNAs is proposed . The model comprises 48 universal helices that eukaryotic and prokaryotic small subunit rRNAs have in common, plus a number of helices in areas of variable secondary structure . For the central area of the model, an alternative structure is possible, applicable only to eukaryotic small subunit rRNAs . Possibly, small subunit rRNA switched to this alternative conformation after the eukaryotic branch had been established in evolution . Another possibility is that the two conformers represent a dynamic structural switch functioning during the translational activity of the eukaryotic ribosome.

J Biol Chem, 1988 May 5, 263(13), 6209 - 14
Prepro-alpha-factor has a cleavable signal sequence; Waters MG et al.; MAT alpha Saccharomyces cerevisiae secrete a small peptide mating pheromone termed alpha-factor . Its precursor, prepro-alpha-factor, is translocated into the endoplasmic reticulum and glycosylated at three sites . The glycosylated form is the major product in a yeast in vitro translation/translocation system . However, there is another translocated, nonglycosylated product that contains a previously unidentified modification . Contrary to previous results suggesting that the signal sequence of prepro-alpha-factor is not cleaved, amino-terminal radiosequencing has identified this product as prepro-alpha-factor without its signal sequence, that is, pro-alpha-factor . The translocated, glycosylated proteins are also processed by signal peptidase . Moreover, we have found that both purified eukaryotic and prokaryotic signal peptidase can process prepro-alpha-factor . Experiments using a yeast secretory mutant (sec 18) blocked in transport from the endoplasmic reticulum to the Golgi indicate that the protein is also cleaved in vivo . Finally, characterization of the Asn-linked oligosaccharide present on pro-alpha-factor in the yeast in vitro system by use of specific glucosidase and mannosidase inhibitors indicates that they have had the three terminal glucoses and probably one mannose removed . Therefore they most likely consist of Man8GlcNAc2 structures, identical to those found in the endoplasmic reticulum in vivo.

J Mol Biol, 1988 May 5, 201(1), 81 - 90
Redundancy of information in enhancers as a principle of mammalian transcription control; Schaffner G et al.; In contrast to prokaryotes, in which strong transcriptional signals can be located within very short DNA segments, typical mammalian enhancers are about 200 base-pairs long . We reasoned that a minimal length of enhancer-active DNA is required for a high transcription rate in higher eukaryotes, and that segments from a single enhancer or from different enhancers might be multimerized or combined to satisfy such a requirement . To test this, enhancer fragments from different viruses were joined in a recombinant simian virus 40 (SV40) and screened for efficiency of viral growth . The 48 combinations tested show that the hypothesis is basically correct . For example, two subfunctional heterologous enhancer fragments can together form a functional enhancer . No enhancer shorter than 84 base-pairs could promote SV40 growth, i.e . in no case did we find a short "superstrong" enhancer segment . To test whether multimerization of a short fragment would result in a strong enhancer, we have synthesized a 50 base-pair enhancer segment derived from Herpesvirus saimiri . One to six copies of this oligonucleotide gave an incremental increase in enhancer activity . We propose, therefore, that mammalian gene regulation is based on a redundancy of information that can be provided either by a combination of different DNA sequence elements, or by multiple copies of the same element . Also, the finding of strong and weak enhancers suggests that in most cases an enhancer is permanently required for transcription of a gene, rather than acting in an all-or-none fashion to establish a transcription complex, after which it becomes dispensable.

DNA, 1988 May, 7(4), 287 - 95
Direct detection of HIV-1 RNA from AIDS and ARC patient samples; Murakawa GJ et al.; Human immunodeficiency virus (HIV), formerly termed human T-lymphotropic virus (HTLVIII/LAV), is the etiological agent of acquired immune deficiency syndrome (AIDS) . Direct detection of HIV-1 nucleic acid sequences in patient tissue or blood samples is possible in only a minor fraction of cases due to the low percentage of infected cells (Shaw et al., 1984) . We report a modification of the polymerase chain reaction method (PCR) (Saiki et al., 1985), in which we amplify sequences from HIV-1 RNA templates, for the identification of HIV-1 in peripheral blood and tissue samples obtained from AIDS and AIDS-related complex (ARC) patients . This method of HIV-1 detection is at least six orders of magnitude more sensitive than standard nucleic acid detection methods and has direct clinical applications . In vitro tissue culturing of the virus is not required for HIV-1 detection . Using this technique, the sequence in the orfB region of HIV-1 has been amplified and detected from less than 1 microgram of total RNA prepared from a few milliliters of peripheral blood samples . This technique enables the rapid and unambiguous clinical detection of potential HIV-infected individuals and can be used to assay the efficacy of anti-HIV-1 drugs . To enhance the efficiency of this technique, we have appended the prokaryotic T7 RNA polymerase promoter sequence to one of the priming oligonucleotides . After several cycles of PCR with the promoter-containing oligo, a small aliquot of the reaction can be utilized to direct specific and efficient T7 RNA polymerase-mediated transcription of the amplified sequences, thus enhancing the sensitivity and simplifying the labor of the experiment.

Radiat Res, 1988 May, 114(2), 307 - 18
Comparative ultraviolet action spectra (254-320 nm) of five "wild-type" eukaryotic microorganisms and Escherichia coli; Calkins J et al.; The action spectra of five eukaryotic organisms and the prokaryote, Escherichia coli, were examined over the wavelength range, 254-320 nm . Both the repair competent and three repair defective strains (E . coli, Caenorhabditis elegans, Saccharomyces) were examined . Tetrahymena pyriformis action spectra were performed with and without the excision repair inhibitor caffeine present . Others have observed that lethality, mutation, and the production of pyrimidine dimers show much the same wavelength dependence as DNA absorption . The results presented here demonstrate several action spectra which deviate from the DNA absorption spectra . Ultraviolet sensitization ratios (repair competent/repair defective) were also examined and were shown to change over the wavelength range . These findings suggest that DNA may not be the only important chromophore leading to cell death in the uv wavelength range studied . Since uv-B is of major importance in solar uv damage, these findings may also yield important implications for solar uv studies.

Biochem J, 1988 May 1, 251(3), 691 - 9
Primary- and secondary-structural analysis of a unique prokaryotic metallothionein from a Synechococcus sp . cyanobacterium; Olafson RW et al.; Biochemical and physiological studies of Synechococcus cyanobacteria have indicated the presence of a low-Mr heavy-metal-binding protein with marked similarity to eukaryotic metallothioneins (MTs) . We report here the characterization of a Synechococcus prokaryotic MT isolated by gel-permeation and reverse-phase chromatography . The large number of variants of this molecule found during chromatographic separation could not be attributed to the presence of major isoproteins as assessed by amino acid analysis and amino acid sequencing of isoforms . Two of the latter were shown to have identical primary structures that differed substantially from the well-described eukaryotic MTs . In addition to six long-chain aliphatic residues, two aromatic residues were found adjacent to one another near the centre of the molecule, making this the most hydrophobic MT to be described . Other unusual features included a pair of histidine residues located in repeating Gly-His-Thr-Gly sequences near the C-terminus and a complete lack of association of hydroxylated residues with cysteine residues, as is commonly found in eukaryotes . Similarly, aside from a single lysine residue, no basic amino acid residues were found adjacent to cysteine residues in the sequence . Most importantly, sequence alignment analyses with mammalian, invertebrate and fungal MT sequences showed no statistically significant homology aside from the presence of Cys-Xaa-Cys structures common to all MTs . On the other hand, like other MTs, the prokaryotic molecule appears to be free of alpha-helical structure but has a considerable amount of beta-structure, as predicted by both c.d . measurements and the Chou & Fasman empirical relations . Considered together, these data suggested that some similarity between the metal-thiolate clusters of the prokaryote and eukaryote MTs may exist.

Mol Microbiol, 1988 May, 2(3), 323 - 9
Proteins from the prokaryotic nucleoid: primary and quaternary structure of the 15-kD Escherichia coli DNA binding protein H-NS; Falconi M et al.; The primary sequence of H-NS (136 amino acid residues, Mr = 15,402), an abundant Escherichia coli DNA-binding protein, has been elucidated and its quaternary structure has been investigated by protein-protein cross-linking reactions . It was found that H-NS exists predominantly as a dimer, even at very low concentrations, but may form tetramers at higher concentrations and that the protein-protein interaction responsible for the dimerization is chiefly hydrophobic.

J Gen Microbiol, 1988 May, 134 ( Pt 5), 1131 - 9
The nucleotide sequence of the LPD1 gene encoding lipoamide dehydrogenase in Saccharomyces cerevisiae: comparison between eukaryotic and prokaryotic sequences for related enzymes and identification of potential upstream control sites; Ross J et al.; The complete nucleotide sequence of the LPD1 gene, which encodes the lipoamide dehydrogenase component (E3) of the pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase multienzyme complexes of Saccharomyces cerevisiae, has been established . The flanking region 5' to the LPD1 gene contains DNA sequences which show homology to known control sites found upstream of other yeast genes . The primary structure of the protein, determined from the DNA sequence, shows strong homology to a group of flavoproteins including Escherichia coli lipoamide dehydrogenase and pig heart lipoamide dehydrogenase . The amino acid sequence also reveals the presence of a potential targeting sequence at its N-terminus which may facilitate transport to and entry into mitochondria.

Tsitologiia, 1988 May, 30(5), 516 - 23
{Possible mechanisms regulating the entry of normal and neoplastic cells into the DNA synthesis phase}; Kazhdan IIa et al.; Literature data concerning some aspects in the regulation of the entry of normal and neoplastic cells into the phase of DNA synthesis are reviewed . Basing on the analysis of data reported by different authors, a hypothetical model is suggested for regulation of cell passing from G- to S-period . The main chain in the regulation is the constitutive synthesis of protein-repressor which blocks the expression of genes whose products are necessary for replication ("replication genes") . The repressor inactivation is achieved by growth factors through protein kinases . A comparison is made between the transcriptional mechanisms in eukaryotic and prokaryotic cells . Four hypothetic types of genes have been isolated responsible for regulation of progression of the cell to phase S . The role of quantitative and/or qualitative changes in the products of these genes in neoplastic transformation is discussed.

Mol Gen Genet, 1988 May, 212(2), 225 - 31
Replication of ColE2 and ColE3 plasmids: the regions sufficient for autonomous replication; Horii T et al.; We have localized the regions sufficient for autonomous replication on the genomes of the colicin E2 (ColE2) and colicin E3 (ColE3) plasmids and analyzed the replication functions carried by these regions . A 1.3 kb segment of each plasmid is sufficient for autonomous replication . Plasmids carrying this segment retain the replication properties of the original plasmid . The 1.3 kb segment consists of three functional portions . Firstly, a 0.9 kb region which specifies at least one trans-acting factor required for replication of each plasmid . Secondly, a 0.4 kb region located adjacent to one end of the 0.9 kb region, which is required for expression of the trans-acting factor(s) and probably contains the promoter . The region across the border of these two portions of ColE2 is involved in copy number control of the plasmid . The third portion is a 50 bp region adjacent to the other end of the 0.9 kb region, which contains a cis-acting site (origin) where replication initiates in the presence of the trans-acting factor(s) . The action of the trans-acting factor(s) on the origin is plasmid specific . The 50 bp regions functioning as the origins of replication of ColE2 and ColE3 are the smallest among those in prokaryotic replicons so far identified and analyzed.

Biochem Biophys Res Commun, 1988 Apr 29, 152(2), 808 - 17
Anion-dependent modulations of DNA topoisomerase II-mediated reactions in potassium-containing solutions; Zwelling LA et al.; DNA binding proteins operate in an intracellular environment of low chloride concentration, yet in vitro assays of the activities of these proteins are often performed in isotonic chloride-containing solutions . Previously, the activity of bacterial DNA-binding proteins was found to be enhanced in potassium-containing solutions in which the anion glutamate (Glu) was substituted for chloride . We have extended this observation to include eukaryotic topoisomerase I and II activities . The concentration ranges over which DNA strand passing activities of these enzymes were observed was broader in KGlu than in KCl . This was also true for the topoisomerase II-mediated DNA strand passage and antineoplastic drug-dependent DNA cleavage produced by nuclear extracts from HL-60 human leukemia cells . The rate of topoisomerase II-mediated DNA strand passage was also dependent on anion moiety and concentration in potassium-containing buffers . Drug-dependent topoisomerase II-mediated DNA cleavage in intact HL-60 cell nuclei was also anion-dependent, suggesting that anion type and concentration may influence topoisomerase II-mediated events in mammalian cells as had been described for other DNA binding proteins in prokaryotic systems . This should be considered in developing biochemical assays of topoisomerase activities to reproduce intracellular conditions.

J Theor Biol, 1988 Apr 21, 131(4), 477 - 85
Stop is not the end: physiological implications of translational readthrough; Engelberg-Kulka H et al.; The translational readthrough mechanism permits the occasional misreading of termination codons by normal charged tRNAs causing extended translation beyond the stop signal . In both prokaryotes and eukaryotes translational readthrough is involved in the regulation of gene expression, as for example in the synthesis of the enzyme reverse transcriptase of the Murine Leukemia Virus (MuLV) (Yoshinaka et al., 1985) . Here we particularly deal with the sensitivity of the translational readthrough process to two parameters which are affected by changes in physiological conditions: (1) fluctuations in the concentration of readthrough tRNAs and (b) The affinity of the tRNAs to termination codons . We also discuss the possible role of translational readthrough during major changes in cell physiology.

Gene, 1988 Apr 15, 64(1), 165 - 72
Cloning and analysis of a yeast genomic DNA sequence capable of directing gene transcription in Escherichia coli as well as in yeast; Kwak JW et al.; A DNA fragment was isolated from yeast genomic sequences by its ability to direct the transcription of promoterless CmR (cat) gene in Escherichia coli and in yeast . Nucleotide sequencing and primer extension analysis showed that yeast DNA contains sets of consensus sequences pertaining to prokaryotic and yeast-type promoter elements . It was designated as yeast- and E . coli-type promoter (YEP1) . Typical E . coli-type promoter elements are found at appropriate positions: TATTTT from -12 to -7 and TTGTCC from -35 to -30 with their spacing of 17 bp from the single mRNA start point determined by the primer extension . Analysis of cat transcripts from yeast cells showed that the YEP1 caused multiple transcription initiations at more than 20 different points that are spaced over a 100-bp region . The DNA is composed of A + T-rich sequences and putative TATA-like sequences are found at several places upstream from the transcription start points . Deletion analysis showed that a 276-bp sequence between -872 and -596 from the initiating ATG codon was required for the maximal promoter activity in yeast but not in E . coli.

Am J Physiol, 1988 Apr, 254(4 Pt 1), E459 - 67
Arginine metabolism in wounds; Albina JE et al.; Arginine metabolism in wounds was investigated in the rat in 1) lambda-carrageenan-wounded skeletal muscle, 2) Schilling chambers, and 3) subcutaneous polyvinyl alcohol sponges . All showed decreased arginine and elevated ornithine contents and high arginase activity . Arginase could be brought to the wound by macrophages, which were found to contain arginase activity . However, arginase was expressed by macrophages only after cell lysis and no arginase was released by viable macrophages in vitro . Thus the extracellular arginase of wounds may derive from dead macrophages within the injured tissue . Wound and peritoneal macrophages exhibited arginase deiminase activity as demonstrated by the conversion of {guanido-14C}arginine to radiolabeled citrulline during culture, the inhibition of this reaction by formamidinium acetate, and the lack of prokaryotic contamination of the cultures . These findings and the known metabolic fates of the products of arginase and arginine deiminase in the cellular populations of the wound suggest the possibility of cooperativity among cells for the production of substrates for collagen synthesis.

Nucleic Acids Res, 1988 Apr 11, 16(7), 2765 - 85
Terminase host factor: a histone-like E . coli protein which can bind to the cos region of bacteriophage lambda DNA; Shinder G et al.; Terminase Host Factor (THF), an E . coli protein capable of fulfilling the host factor requirement for in vitro bacteriophage lambda terminase activity, displays properties characteristic of the prokaryotic type II DNA-binding or "histone-like" proteins . It is a 22 K basic, heat- and acid-stable protein which binds non-specifically to various DNAs . Conditions can be established, however, where THF binds preferentially to the cohesive end site (cos) of lambda DNA forming several distinct complexes as visualized by band retardation in polyacrylamide gels . DNase I footprinting reveals that THF can protect several regions of the top strand on the right side (+) of cos but does not bind as well to the left side (-) . The binding regions are separated either by unprotected or by DNase I- hypersensitive bases . Under the conditions used in these experiments, DNA which does not contain cos lambda sequences does not show this pattern of protection . Several repeated motifs in the cos lambda nucleotide sequence may represent a consensus sequence for THF interaction . THF may be similar to other "histone-like" proteins which display both non-specific and selective DNA-binding capacities.

J Biol Chem, 1988 Apr 5, 263(10), 4932 - 8
Wheat germ cytoplasmic ribosomes . Structure of ribosomal subunits and localization of N6,N6-dimethyladenosine by immunoelectron microscopy; Montesano L et al.; Cytoplasmic ribosomes have been isolated from wheat germ, and the structure of ribosomal subunits has been examined by electron microscopy of negatively stained preparations . Small (40 S) subunits show structural features generally regarded as characteristic of eukaryotic particles, while large (60 S) subunits show shapes that are equally well described by models of prokaryotic 50 S particles . Small subunit 18 S RNA contains 2 residues of N6,N6-dimethyladenosine 19 and 20 residues from the 3'-end (Hagenbuchle, O., Santer, M., Steitz, J . A., and Mans, R . J . (1978) Cell 13, 551-563) . Nucleoside analysis by high performance liquid chromatography shows no other residues of this component in the RNA . Anti-dimethyladenosine immunoglobulins were reacted with wheat germ 40 S subunits, and the resulting complexes were studied by electron microscopy in order to localize the nucleoside . In about 90% of the complexes observed, antibody-subunit contact was consistent with a single binding site . We place the dimethyladenosine residues at or near the end of the platform of the 40 S particle in a position nearly equivalent to that previously identified in prokaryotic and chloroplast subunits (Trempe, M . R., and Glitz, D . G . (1981) J . Biol . Chem . 256, 11873-11879).

Proc Natl Acad Sci U S A, 1988 Apr, 85(8), 2672 - 6
Intron conservation across the prokaryote-eukaryote boundary: structure of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize; Quigley F et al.; The nuclear gene encoding chloroplast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from maize has been cloned and sequenced . The gene is G + C rich in its coding sequences and, in addition, contains a CpG-rich region surrounding the promoter . Further upstream several enhancer-like repetitions have been identified that may control the light- and phytochrome-mediated expression of this gene . The gene is interrupted by three introns . Introns 1 and 2 are located within the sequence encoding the transit peptide, dividing it into three parts, each containing one of the three major homology blocks typical for transit peptides of nucleus-encoded chloroplast proteins . Intron 3 is located at codon 166 (glycine) at the same nucleotide position as intron 1 in the GAPDH gene from the nematode Caenorhabditis elegans, suggesting that this intron was present in the parental GAPDH gene from which these two modern descendants originated . Intron 3 divides the GAPDH protein into its two constituent domains, the NAD-binding and the catalytic domain, immediately after helix alpha 1 at a position homologous to that of intron 9 in the gene for maize alcohol dehydrogenase, thereby confirming the prediction of Branden et al . on the basis of gene-protein structure correlations in maize alcohol dehydrogenase for the placement of introns in the GAPDH gene {Branden, C.I., Eklund, H., Cambillau, C . & Pryor, A.J . (1984) EMBO J . 3, 1307-1310} . These results suggest that intron 3 is an archetypical relic of early GAPDH and alcohol dehydrogenase evolution, whereas introns 1 and 2 were implicated in the evolution of chloroplast transit peptides.

Protein Eng, 1988 Apr, 2(1), 15 - 20
Distinctive properties of signal sequences from bacterial lipoproteins; Klein P et al.; We have compared a number of attributes (hydrophobicity, amino acid size, charge and secondary structure propensities) of signal sequences from bacterial lipoproteins with the same attributes of signal peptides from other prokaryotic proteins (non-lipoproteins) . Lipoprotein leader sequences tend to be shorter, more hydrophobic and bulky, and they have stronger conformational preferences, the most conspicuous being a predicted beta-turn comprising positions 2 or 3 of the mature protein . Another distinctive feature is a maximum in the local energy profile between positions -1 and +2 . With one exception (beta-lactamase III), the lipoproteins do not have Pro in their signal peptides, and they tend to have fewer Ser and Thr but more Gly than non-lipoproteins . Lipoproteins also lack a net negative charge in the N-terminal regions of the mature proteins . The signal peptides of the bacteriocin plasmid-coded lysis proteins appear to be unique in that they have all the ascribed features of lipoprotein signals; these characteristics can be used to guide signal peptide mutagenesis experiments and to construct new secretion vehicles.

J Gen Microbiol, 1988 Apr, 134 ( Pt 4), 997 - 1004
Developmental regulation of the cysteine-rich outer-membrane proteins of murine Chlamydia trachomatis; Sardinia LM et al.; The developmental cycle of the obligate intracellular prokaryote Chlamydia trachomatis involves the serial alternation of two distinct morphological forms of the organism . To examine the basis of chlamydial differentiation we have searched for developmentally regulated gene products in this species . Chlamydia-infected cells were pulse-labelled with {35S} cysteine at various stages of development and the products of synthesis examined by SDS-PAGE . Our results indicate that the synthesis of the cysteine-rich outer-membrane proteins is developmentally regulated, occurring only late in the cycle during the conversion of reticulate bodies to elementary bodies . Both hydroxyurea and ampicillin block this conversion; as a result of this blockade the cysteine-rich outer-membrane proteins are not produced in the presence of either drug.

Microbiol Sci, 1988 Apr, 5(4), 121 - 3
The metabolism of sporulation in yeast; Dickinson JR; An holistic view of the metabolism of sporulation in Saccharomyces cerevisiae is emerging . Furthermore, there may be a general biochemical signal for the initiation of differentiation that is conserved in both prokaryotes and eukaryotes.

FEMS Microbiol Rev, 1988 Apr-Jun, 4(2), 85 - 110
Microbial metabolism of mandelate: a microcosm of diversity; Fewson CA; This review highlights the diversity of prokaryotic and eukaryotic microorganisms that can metabolise mandelate and it describes how a wide range of compounds related to mandelate is formed in many environments . The chief aspects that are summarised include the various pathways whereby mandelate and its structural analogues are converted into catechol or protocatechuate, the properties of the enzymes that are involved in the pathways, and the regulation and genetics of the pathways . The review incorporates the idea that the study of peripheral metabolic pathways is particularly useful for illuminating evolutionary speculations and it concludes with a list of questions that need to be answered.

Can J Microbiol, 1988 Apr, 34(4), 547 - 51
Bacterial evolution; Doolittle WF; The deliberate application of the methodology of contemporary molecular genetics to problems in bacterial systematics has led to a broad new understanding of the evolutionary history of both prokaryotes and eukaryotes . In this review, I discuss some of the major conclusions of this endeavour and try to predict future directions.

Antiviral Res, 1988 Apr, 9(3), 205 - 18
Suppression of BK virus replication and cytopathic effect by inhibitors of prokaryotic DNA gyrase; Portolani M et al.; Nalidixic acid and oxolinic acid, two antibacterial agents known to inhibit bacterial DNA gyrase, are shown to suppress the replication, as well as the cytopathic effect, of BK virus in Vero cell cultures . The inhibition of virus replication was detectable at day 4 post infection in cultures which had been continuously exposed to drugs at concentrations as low as 0.02 to 0.04 mM of nalidixic acid and 0.2 mM of oxolinic acid . These active concentrations are inferior to plasma levels attained in the course of clinical use of the drugs for antibacterial chemotherapy . Also, under these circumstances, no cytotoxicity occurred . The inhibition of development of cytopathology and of virus-induced cell death was demonstrable in cultures treated for 12 days with the drugs . Under these circumstances of prolonged action, oxolinic acid proved to be slightly cytotoxic in that virus inhibitory doses reduced the viability of normal cells . No alterations in the topological conformation of the viral genome or accumulation of end products of viral DNA replication were detected . However, accumulation of viral DNA form I at 48 h post infection suggests that the drugs act through a mechanism involving DNA topoisomerase.

Proc Natl Acad Sci U S A, 1988 Apr, 85(7), 2076 - 80
Illegitimate recombination mediated by calf thymus DNA topoisomerase II in vitro; Bae YS et al.; We have found that purified calf thymus DNA topoisomerase II mediates recombination between two phage lambda DNA molecules in an in vitro system . The enzyme mainly produced a linear monomer recombinant DNA that can be packaged in vitro . Novobiocin and anti-calf thymus DNA topoisomerase II antibody inhibit this ATP-dependent recombination . The recombinant molecules contain duplications or deletions, and most crossovers take place between nonhomologous sequences of lambda DNA, as judged by the sequences of recombination junctions . Therefore, the recombination mediated by the calf thymus DNA topoisomerase II is an illegitimate recombination that is similar to recombination mediated by Escherichia coli DNA gyrase or phage T4 DNA topoisomerase . The subunit exchange model, which has been suggested for the DNA gyrase-mediated recombination, is now generalized as follows: DNA topoisomerase II molecules bind to DNAs, associate with each other, and lead to the exchange of DNA strands through the exchange of topoisomerase II subunits . Illegitimate recombination might be carried out by a general mechanism in organisms ranging from prokaryotes to higher eukaryotes.

Can J Microbiol, 1988 Apr, 34(4), 421 - 6
Why can't a cell grow infinitely fast?
Koch AL.
Living cells are esoteric physiochemical systems that have evolved to survive and reproduce in their naturl environment . Under balanced conditions of growth, bacteria are probably systems as simple as any kind of free-living organism . Evolutionary forces, seemingly, should have driven prokaryotes to be very efficient . In part that is so; they make effective use of the machinery most expensive for the cell, i.e., the ribosomes and associated factors . But the evidence is that the efficiency with which they use the ribosomal machinery increases as the environment provides more favorable conditions for balanced growth . This article emphasizes the limitation to growth under optimal conditions . The role of fluctuations in the environment and the cost of accurate protein synthesis are discussed as reasons for the upper limit in obtainable specific growth rate.

Nucleic Acids Res, 1988 Mar 25, 16(5), 2235 - 46
Expression of the plasmid-encoded type I dihydrofolate reductase gene in cultured mammalian cells: a novel selectable marker; Simonsen CS et al.; A recombinant plasmid has been designed to express the gene encoding a type I methotrexate-resistant dihydrofolate reductase, derived from the bacterial plasmid R483, in DHFR- Chinese hamster ovary cells . Vectors containing the wild type gene, whose coding sequence initiates with a GTG codon, fail to direct the synthesis of detectable levels of protein . Substitution of the GTG codon by an AG codon using in vitro mutagenesis overcomes this block; cells transfected with the modified vector synthesize a functional prokaryotic protein that sustains the growth of these cells in the presence of dihydrofolic acid in the culture media . This property is consistent with the inability of the type I enzyme to reduce folate to dihydrofolate, and enabled the development of a selection strategy whereby prokaryotic and mammalian DHFRs genes could be used sequentially as independently selectable markers.

J Biol Chem, 1988 Mar 25, 263(9), 4450 - 9
Exonucleolytic proofreading enhances the fidelity of DNA synthesis by chick embryo DNA polymerase-gamma; Kunkel TA et al.; The high fidelity of chick embryo DNA polymerase-gamma (pol-gamma) observed during in vitro DNA synthesis (Kunkel, T . A . (1985) J . Biol . Chem . 260, 12866-12874) has led us to examine this DNA polymerase for the presence of an exonuclease activity capable of proofreading errors . Highly purified chick embryo pol-gamma preparations do contain exonuclease activity capable of digesting radiolabeled DNA in a 3'----5' direction, releasing deoxynucleoside 5'-monophosphates . The polymerase and exonuclease activities cosediment during centrifugation in a glycerol gradient containing 0.5 M KCl . In the absence of dNTP substrates, this exonuclease excises both matched and mismatched primer termini, with a preference for mismatched bases . Excision is inhibited by the addition of nucleoside 5'-monophosphates to the digestion reaction . In the presence of dNTP substrates to permit competition between excision and polymerization from the mismatched primer, the exonuclease excises mismatched bases from preformed terminal mispairs with greater than 98% efficiency . The preference for excision over polymerization can be diminished by addition of either high concentrations of dNTP substrates or nucleoside 5'-monophosphates to the exonuclease/polymerase reaction . To determine if this exonuclease is capable of proofreading misinsertions produced during a normal polymerization reaction, a sensitive base substitution fidelity assay was developed based on reversion of an M13mp2 lacZ alpha nonsense codon . In this assay using reaction conditions that permit highly active exonucleolytic proofreading, pol-gamma exhibits a fidelity of less than one error for every 260,000 bases polymerized . As for terminal mismatch excision, fidelity is reduced by the addition to the synthesis reaction of high concentrations of dNTP substrates or nucleoside 5'-monophosphates, both hallmarks of exonucleolytic proofreading by prokaryotic enzymes . Taken together, these observations suggest that the 3'----5' exonuclease present in highly purified chick embryo pol-gamma preparations proofreads base substitution errors during DNA synthesis . It remains to be determined if the polymerase and exonuclease activities reside in the same or different polypeptides.

FEBS Lett, 1988 Mar 14, 229(2), 283 - 8
The bacteriophage Mu transposase protein can form high-affinity protein-DNA complexes with the ends of transposable elements of the Tn 3 family; Cameron RK et al.; The 37 kb transposable bacteriophage Mu genome encodes a transposase protein which can recognize and bind to a consensus sequence repeated three times at each extremity of its genome . A subset of this consensus sequence (5'-PuCGAAA(A)-3') is found in the ends of many class II prokaryotic transposable elements . These elements, like phage Mu, cause 5 bp duplications at the site of element insertion, and transpose by a cointegrate mechanism . Using the band retardation assay, we have found that crude protein extracts containing overexpressed Mu transposase can form high-affinity protein-DNA complexes with Mu att R and the ends of the class II elements Tn 3 (right) and IS101 . No significant protein-DNA complex formation was observed with DNA fragments containing the right end of the element IS102, or a non-specific pBR322 fragment of similar size . These results suggest that the Mu transposase protein can specifically recognize the ends of other class II transposable elements and that these elements may be evolutionarily related.

Science, 1988 Mar 4, 239(4844), 1145 - 7
A gene for dihydrofolate reductase in a herpesvirus; Trimble JJ et al.; The enzyme dihydrofolate reductase (DHFR) is found ubiquitously in both prokaryotes and eukaryotes . It is essential for de novo synthesis of purines and of deoxythymidine monophosphate for DNA synthesis . Among viruses, however, only the T-even and T5 bacteriophage have been found to encode their own DHFR . In this study a gene for DHFR was found in a specific subgroup of the gamma or lymphotropic class of herpesviruses . DNA sequences for DHFR were found in herpesvirus saimiri and herpesvirus ateles but not in Epstein-Barr virus, Marek's disease virus, herpes simplex virus, varicella-zoster virus, herpesvirus tamarinus, or human cytomegalovirus . The predicted sequence of herpesvirus saimiri DHFR is 186 amino acids in length, the same length as human, murine, and bovine DHFR . The human and herpesvirus saimiri DHFRs share 83 percent positional identity in amino acid sequence . The herpesvirus saimiri DHFR gene is devoid of intron sequences, suggesting that it was acquired by some process involving reverse transcription . This is to our knowledge the first example of a mammalian virus with a gene for DHFR.

Mol Biol (Mosk), 1988 Mar-Apr, 22(2), 338 - 56
{Characteristics of nucleotide blocks in coding and non-coding DNA sequences from different organisms}; Sprizhitskii IuA et al.; A statistical analysis of occurrence of particular nucleotide runs (1 divided by 10 nucleotides long) in DNA sequences of different species has been carried out . There are considerable differences in run distributions in DNA sequences of prokaryotes, invertebrates and vertebrates . Distribution of various types of runs has been found to be different in coding and non-coding sequences . There is an abundance of short runs 1 divided by 2 nucleotides long in coding sequences, and there is a deficiency of such runs in the non-coding regions . However, some interesting exceptions from this rule exist: for run distribution of adenine in prokaryotes and for distribution of purine-pyrimidine runs in eukaryotes . This may be stipulated by the fact that the distribution of runs are predetermined by structural peculiarities of the entire DNA molecule . Runs of guanine or cytosine of three to six nucleotides long occur predominantly in the non-coding DNA regions in eukaryotes, especially in vertebrates.

Microbiol Sci, 1988 Mar, 5(3), 68 - 73
Electron microscopic approaches to the study of bacterial DNA organization; Meyer J et al.; Electron microscopic techniques have been applied to the study of all aspects of structure, function and organization of prokaryotic and eukaryotic genomes . Today these powerful methods are mainly used in determining sequence relationships of large DNA molecules and in structural studies of nucleic acid-protein complexes . Some recent applications in the analysis of prokaryotic genomes are discussed.

Mutat Res, 1988 Mar, 198(1), 115 - 29
Analysis of metal-induced mutations altering the expression or structure of a retroviral gene in a mammalian cell line; Biggart NW et al.; Carcinogenic metal compounds, with the exception of chromium(VI), have been found to be poorly mutagenic in both prokaryotic and mammalian cell mutagenesis assays, yet they are clearly clastogenic (Hansen and Stern, 1984) . Thus, the role of metals as initiators in carcinogenesis has been difficult to delineate . In an effort to develop a model system capable of assaying DNA damage caused by carcinogenic metals, we have investigated the role of NiCl2, CdCl2, Na2CrO4, and NMU in a murine sarcoma virus-infected mammalian cell line in which expression of the retroviral v-mos gene is growth-temperature regulated . This cell line, designated 6m2, contains a single-copy, stably integrated, mutant Moloney murine sarcoma virus DNA (designated MuSVts110) and is temperature sensitive for morphological transformation due to a conditionally defective viral RNA-splicing event that in turn regulates expression of the viral transforming gene . Mutations affecting the viral DNA in 6m2 cells can be detected if these alterations lead to changes in the structure or expression of the transforming protein encoded by the MuSVts110 v-mos gene . Analysis of the viral proteins from 6m2 'revertant' cell lines (as defined by reversion to the transformed phenotype at all growth temperatures) selected after treatment with the above agents showed that NiCl2, NMU, and Na2CrO4 each induced a different yet specific type of mutation . NiCl2 and NMU each altered the temperature sensitivity of viral RNA splicing, possibly due to base substitution mutations, but did so to distinctly different extents . Na2CrO4 affected the structure of the viral proteins by inducing what appear to be short frameshift mutations that resulted in the temperature-dependent translation of a novel virus-encoded transforming protein, P100gag-mos . CdCl2 also induced frameshift mutations but, in one case, induced a mutation which may result from a deletion of about 300 bases within the MuSVts110 DNA.

Proc Natl Acad Sci U S A, 1988 Mar, 85(5), 1394 - 7
Tn10-encoded tet repressor can regulate an operator-containing plant promoter; Gatz C et al.; The Tn10-encoded tet repressor-operator system was used to regulate transcription from the cauliflower mosaic virus (CaMV) 35S promoter . Expression was monitored in a transient assay system by using electric field-mediated gene transfer ("electroporation") into tobacco protoplasts . The tet repressor, being expressed in the plant cells under the control of eukaryotic transcription signals, blocks transcription of a CaMV 35S promoter chloramphenicol acetyltransferase (cat) fusion gene when the two tet operators flank the "TATA" box . In the presence of the inducer tetracycline, expression is restored to full activity . Location of the operators 21 base pairs downstream of the transcription start site does not significantly affect transcription in the presence of the repressor . These experiments show that a prokaryotic regulatory protein can function in plant cells . The tet repressor-operator complex may be useful for specifically inducing transferred genes at different stages of plant development.

FEBS Lett, 1988 Feb 29, 229(1), 197 - 202
Proteins from the prokaryotic nucleoid . Interaction of nucleic acids with the 15 kDa Escherichia coli histone-like protein H-NS; Friedrich K et al.; The interaction between nucleic acids and Escherichia coli H-NS, an abundant 15 kDa histone-like protein, has been studied by affinity chromatography, nitrocellulose filtration and fluorescence spectroscopy . Intrinsic fluorescence studies showed that the single Trp residue of H-NS (position 108) has a restricted mobility and is located within an hydrophobic region inaccessible to both anionic and cationic quenchers . Binding of H-NS to nucleic acids, however, results in a change of the microenvironment of the Trp residue and fluorescence quenching; from the titration curves obtained with addition of increasing amounts of poly(dA)-poly(dT) and poly(dC)-poly(dG) it can be estimated that an H-NS dimer in 1.5 x SSC binds DNA with an apparent Ka approximately equal to 1.1 x 10(4) M-1.bp-1 . H-NS binds to double-stranded DNA with a higher affinity than the more abundant histone-like protein NS(HU) and, unlike NS, prefers double-stranded to single-stranded DNA and DNA to RNA; both monovalent and divalent cations are required for optimal binding.

Nature, 1988 Feb 25, 331(6158), 723 - 5
Gene for a novel tRNA species that accepts L-serine and cotranslationally inserts selenocysteine; Leinfelder W et al.; The biological requirement of the trace element selenium was recognized 40 years ago . Selenium is incorporated into several enzymes and transfer RNA species of both prokaryotic and eukaryotic origin . In enzymes which contain a selenopolypeptide, selenium is present as covalently bound selenocysteine which participates in the catalytic reaction . Sequence analysis of the genes coding for two selenoproteins, formate dehydrogenase H from Escherichia coli and glutathione peroxidase from mouse and man, demonstrated that an in-frame UGA opal nonsense codon directs the incorporation of selenocysteine . In the case of formate dehydrogenase incorporation occurs cotranslationally . Recently, we identified four genes whose products are required for selenocysteine incorporation in E . coli . We report here that one of these genes codes for a tRNA species with unique properties . It possesses an anticodon complementary to UGA and deviates in several positions from sequences, until now, considered invariant in all tRNA species . This tRNA is aminoacylated with L-serine by the seryl-tRNA ligase which also charges cognate tRNASer . Selenocysteine, therefore, is synthesized from a serine residue bound to a natural suppressor tRNA which recognizes UGA.

J Biol Chem, 1988 Feb 25, 263(6), 2817 - 23
Glutamate biosynthesis in Bacillus azotofixans . 15N NMR and enzymatic studies; Kanamori K et al.; Pathways of ammonia assimilation into glutamic acid in Bacillus azotofixans, a recently characterized nitrogen-fixing species of Bacillus, were investigated through observation by NMR spectroscopy of in vivo incorporation of 15N into glutamine and glutamic acid in the absence and presence of inhibitors of ammonia-assimilating enzymes, in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and alanine dehydrogenase . In ammonia-grown cells, both the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways contribute to the assimilation of ammonia into glutamic acid . In nitrate-grown and nitrogen-fixing cells, the glutamine synthetase/glutamate synthase pathway was found to be predominant . NADPH-dependent glutamate dehydrogenase activity was detectable at low levels only in ammonia-grown and glutamate-grown cells . Thus, B . azotofixans differs from Bacillus polymyxa and Bacillus macerans, but resembles other N2-fixing prokaryotes studied previously, as to the pathway of ammonia assimilation during ammonia limitation . Implications of the results for an emerging pattern of ammonia assimilation by alternative pathways among nitrogen-fixing prokaryotes are discussed, as well as the utility of 15N NMR for measuring in vivo glutamate synthase activity in the cell.

J Biol Chem, 1988 Feb 15, 263(5), 2447 - 51
Processing of histidine transfer RNA precursors . Abnormal cleavage site for RNase P; Burkard U et al.; The 5'-terminal guanylate residue (G-1) of mature Escherichia coli tRNA(His) is generated as a result of an unusual cleavage by RNase P (Orellana, O., Cooley, L., and Soll, D . (1986) Mol . Cell . Biol . 6, 525-529) . We have examined the importance of the unique acceptor stem structure of E . coli tRNA(His) in determining the specificity of RNase P cleavage . Mutant tRNA(His) precursors bearing substitutions of the normal base G-1 or the opposing, potentially paired base, C73, can be cleaved at the +1 position, in contrast to wild-type precursors which are cut exclusively at the -1 position . These data indicate that the nature of the base at position -1 is of greater importance in determining the site of RNase P cleavage than potential base pairing between nucleotides -1 and 73 . In addition, processing of the mutant precursors by M1-RNA or P RNA under conditions of ribozyme catalysis yields a higher proportion of +1-cleaved products in comparison to the reaction catalyzed by the RNase P holoenzyme . This lower sensitivity of the holoenzyme to alterations in acceptor stem structure suggests that the protein moiety of RNase P may play a role in determining the specificity of the reaction and implies that recognition of the substrate involves additional regions of the tRNA . We have also shown that the RNase P holoenzyme and tRNA(His) precursor of Saccharomyces cerevisiae, unlike their prokaryotic counterparts, do not possess these abilities to carry out this unusual reaction.

J Mol Biol, 1988 Feb 5, 199(3), 447 - 65
Molecular characterization and evolution of sequences encoding light-harvesting components in the chromatically adapting cyanobacterium Fremyella diplosiphon; Conley PB et al.; The major light-harvesting complex in eukaryotic red algae and prokaryotic cyanobacteria is the phycobilisome, a water-soluble complex located on the outer surface of the photosynthetic membranes and composed of both pigmented phycobiliproteins (85%) and non-pigmented linker (15%) polypeptides . The phycobiliproteins are encoded by a gene family and exhibit varying degrees of sequence homology (25 to 55%) . Some cyanobacteria can maximize the absorption of prevalent wavelengths of light by adjusting the phycobiliprotein composition of the phycobilisome, a process called complementary chromatic adaptation . In the chromatically adapting species Fremyella displosiphon, there are at least two sets of phycocyanin genes; one is transcribed as two red light-induced transcripts and the other is encoded on a single transcript present in both red and green light . We have determined the complete nucleotide sequences of both sets of phycocyanin subunit genes and their associated 5' and 3' regulatory regions . Based on S1 nuclease protection experiments, the transcripts (1600 and 3800 bases) encoding the inducible phycocyanin subunits have the same 5' end, and possible mechanisms for their synthesis are presented . The 5' end of the 1500-base transcript encoding the constitutive phycocyanin subunits was determined and revealed an Escherichia coli-like "-10" and "-35" region, and sequences near the transcription initiation site homologous to the analogous region of the phycocyanin gene set of Anabaena sp . 7120 . Determination of the 3' ends of the transcripts encoding both F . diplosiphon phycocyanin gene sets revealed regions of potential secondary structure that may be important for transcription termination and/or transcript stability . In addition, the sequence of an open reading frame (encoding a 30 kDa polypeptide), located 3' to the constitutive phycocyanin gene set in F . diplosiphon and highly conserved in at least three cyanobacterial species, is presented . The same high degree of sequence homology between the two F . diplosiphon PC alpha and PC beta sequences (85 and 77%, respectively) was found at both the nucleotide and amino acid levels, and similar results were obtained for interspecies comparisons . Implications of these homologies with regard to the evolution of phycobiliprotein subunits are discussed.

Proc Natl Acad Sci U S A, 1988 Feb, 85(3), 846 - 50
rbcS genes in Solanum tuberosum: conservation of transit peptide and exon shuffling during evolution; Wolter FP et al.; Five genes of the rbcS gene family of Solanum tuberosum (potato) were studied . One of these is a cDNA clone; the other four are located on two genomic clones representing two different chromosomal loci containing one (locus 1) and three genes (locus 2), respectively . The intron/exon structure of the three genes in locus 2 is highly conserved with respect to size and position . These genes contain two introns, whereas the gene from locus 1 contains three introns . Although in most cases the amino acid sequences in the transit peptide part of different rbcS genes from the same species varied considerably more than the corresponding mature amino acid sequences, one exception found in tomato and potato indicates that the transit peptide of rbcS could have a special function . A comparison of the rbcS genes of higher plants with those of prokaryotes offers suggestive evidence that introns first served as spacer material in the process of exon shuffling and then were removed stepwise during the evolution of higher plants.

Mutat Res, 1988 Feb, 197(2), 243 - 60
The Allium test--an alternative in environmental studies: the relative toxicity of metal ions; Fiskesjo G; Among the test systems suitable for toxicity monitoring, the Allium test (A . cepa) is well known and commonly used in many laboratories . The onions are easy to store and to handle, and the root tip cells constitute a convenient system for macroscopic (growth, EC50 values) as well as for microscopic parameters (c-mitosis, stickiness, chromosome breaks) . Since the cells possess important plant activation enzymes, the Allium test has a wide area of application . Furthermore, results from the Allium test have shown good agreement with results from other test systems, eukaryotic as well as prokaryotic . A modified version of the test method, comprising series of onions for each concentration of the test liquids, was applied to salts of eight metals: Hg (as methyl mercury chloride (MMC) and as HgCl2), Cu, Ni, Cd, Be, Al (diluted in tap water and distilled water), Mn and Li . The highest toxicity, in EC50 values, was caused by Hg (for MMC 9.0 X 10(-7) M, for HgCl2 3.3 X 10(-6) M), Cu (2.7 X 10(-6) M), Ni (1.7 X 10(-5) M) and Cd (3.1 X 10(-5) M); medium toxicity was caused by Be (4.8 X 10(-4) M) and Al (in tap water 8.0 X 10(-4) M, in distilled water 2.8 X 10(-5) M), and low toxicity by Mn (5.2 X 10(-3) M) and Li (2.0 X 10(-2) M) . Some of the metals induced specific microscopic effects, requiring particular mention: thus, Ni treatment induced an unusual form of c-mitosis with the c-mitotic chromosomes remaining on the equatorial plate, Be treatment induced a type of 'banded' or 'fragmented' chromosomes . Treatment with Al led to the development, in the cytoplasm of certain root tip cells, of two oblong hyaline structures formed by material extruded from the nucleus . Chromosome breaks were mainly observed as fragments at mitotic anaphase . The metal ions tested here caused only low amounts of fragments, usually in less than 1% of the cells; only Be caused a higher frequency (4.1%) . It was not possible to group the few metals tested here according to their cytological effects . The standard parameters, such as the most commonly occurring c-mitosis or stickiness, showed no correlation to atomic weight or to ion charge of the metals . Still, they gave valuable information in relation to environmental screening; thus, the finding of c-mitosis may indicate risks of aneuploidy . Generally speaking, the Allium test is a very useful tool for evaluating and ranking environmental chemicals with reference to their toxicity.

What Is Bioengineering?, What Is Biofilter?, What Is Molecular Biology?, What Is Biotechnology?, What Is Genetic Engineering?, n, Bacteria, o, Microbiology, r, Microorganisms, r, Microbe, c, Bacterium, s, Klebsiella, c, Minimum inhibiting concentration, c, Escherichia coli, a, S. cerevisiae, i, Rhizobacter, s, Thermophile, i, Escherichia coli, i, Staphylococcus, e, Enterococci, r, S. cerevisiae, i, Staphylococcus, a, Yeasts, s, Penicillin, e, Bacillus, i, Staphylococcus, n, Microbiological, r, Escherichia coli, o, Acinetobacter, n, Culture medium, s, Yeasts, n, Mueller hinton broth

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

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

Last modified: May 25, 2005