Prokaryotes

Biophys Chem, 1988 Feb, 29(1-2), 51 - 62
About the organisation of condensed and decondensed non-eukaryotic DNA and the concept of vegetative DNA (a critical review); Kellenberger E; Experiments are reviewed that allow one to assign naturally occurring DNA-containing plasmas to either of two classes by virtue of their sensitivity to aggregation upon dehydration in organic solvents . The interphase nuclei of higher cells are relatively insensitive, while the DNA plasmas represented by bacterial nucleoids, vegetative bacteriophage and the chromosomes of dinoflagellates are sensitive . In higher cells the bulk of DNA is organised with histones in the form of nucleosomes . In prokaryotes and in the pool of vegetative phage DNA the most abundant histone-like protein HU is not associated with the bulk DNA, but localised in the border region with ribosomes where transcription and translation occur . These experimental results strongly suggest that the two classes of DNA plasmas are distinguishable by a low (1:10) or high (1:1) protein-to-DNA ratio . The hypothesis is formulated that the vegetative DNA (replicating and transcribing), throughout the living world, is nucleosome-free; during evolution, nucleosomes would have been introduced as a simple and adequate means for compacting the resting DNA . Condensation of DNA does not occur with prokaryotic nucleoids, but does take place when DNA is withdrawn from the vegetative phage pool to become packaged into phage heads . Dinoflagellate chromosomes are rather condensed although structurally different from eukaryotic chromosomes (e.g., those from Euglena) and are much more aggregation-sensitive.

J Oral Maxillofac Surg, 1988 Feb, 46(2), 128 - 33
In vitro effects of low intensity direct current generated silver on eukaryotic cells; Hall RE et al.; An examination of the effects of low intensity direct current generated silver, (LIDC Ag) on several types of eukaryotic microorganisms and cell culture lines suggests that LIDC Ag has an appropriate selective toxicity for prokaryotic cells . It appears that levels of this agent could be obtained clinically that would have marked bacteriostatic activity and yet have little or no effect on mammalian cells.

Mutat Res, 1988 Feb, 207(2), 53 - 6
Molecular analysis of Drosophila melanogaster AdhnLA405 confirms reliability of DNA-sequencing methodology; LoMonaco MB et al.; An alcohol dehydrogenase (ADH) null mutant of Drosophila melanogaster (AdhnLA405) originally recovered following X-ray irradiation of mature sperm (Aaron, 979) is analyzed by Southern blotting, Western blotting, and DNA sequencing . The genetic, immunologic, and nucleic acid sequence data are consistent with the hypothesis that a cross-over event, independent of X-irradiation, between parental chromosomes is responsible for the ADH null phenotype of AdhnLA405 . By DNA-sequence analysis we show that molecular cloning of this locus (i.e., propagation in prokaryotic hosts) apparently does not introduce any spurious changes (substitutions, additions, deletions, or rearrangements) within the DNA.

Biotechnol Appl Biochem, 1988 Feb, 10(1), 6 - 12
Transfer and expression of plasmids containing human cytomegalovirus immediate-early gene 1 promoter-enhancer sequences in eukaryotic and prokaryotic cells; Davis MG et al.; The human cytomegalovirus immediate-early gene region 1 promoter-enhancer is active in bacteria and in many mammalian cells . Recombinant plasmids containing portions of this DNA can be used to promote the expression of foreign proteins in many cells . In this communication, we report the optimal conditions for transfer of plasmid DNA to cells by electroporation and the transient expression assays which document the activity of different promoter constructions . The observed activity of the human cytomegalovirus promoter is more than 100-fold higher than the activity of the early promoter of SV40.

Curr Genet, 1988 Feb, 13(2), 137 - 44
Nucleotide sequence of the Aspergillus niger trpC gene: structural relationship with analogous genes of other organisms; Kos T et al.; The nucleotide sequence of the Aspergillus niger tryptophan C (trpC) gene was determined . Northern hybridization and S1-mapping experiments showed the presence of a 2.6 kb trpC poly(A)+ RNA with two very short (5 and 6 nucleotides) noncoding 5'-regions . Comparison of the predicted amino acid sequence with that of trp gene proteins of pro- and eukaryotic organisms revealed three functional domains (G, C, F) in the A . niger TrpC protein which catalyse the glutamine amidotransferase reaction (GAT), the indole-3-glycerol phosphate synthase reaction (IGPS) and the N-(5'-phosphoribosyl) anthranilate isomerase reaction (PRAI), respectively . These domains are highly conserved and bordered by short areas showing less homology . Within the F domain of the trpC gene in A . niger, A . nidulans and Neurospora crassa, a region encoding 30 amino acids was found which is absent in the analogous genes of Saccharomyces cerevisiae and prokaryotic organisms . This region has features of a mutated in-phase intron.

J Bioenerg Biomembr, 1988 Feb, 20(1), 59 - 83
Organization and structure of the genes for the cytochrome b/c1 complex in purple photosynthetic bacteria . A phylogenetic study describing the homology of the b/c1 subunits between prokaryotes, mitochondria, and chloroplasts; Gabellini N; The cytochrome b/c1 complex is an ubiquitous energy transducing enzyme, part of the electron transport chain of prokaryotes, mitochondria, and chloroplasts (b6/f) . In the ancient purple photosynthetic bacteria, the b/c1 complex occupies a central metabolic role, being part of their photosynthetic and respiratory electron transport chain . In Rhodobacter the three subunits of the b/c1 complex are FeS protein, cytochrome b, and cytochrome c1, and they are encoded by a constitutively expressed operon named fbc . The organization of the genes for the cytochrome b/c1 complex, the modality of transcription, and the biogenesis of the encoded polypeptides will be described . The Rhodobacter species used to isolate the fbc genes, previously reported as R . sphaeroides was identified as R . capsulatus . Further biochemical characterization of the prokaryotic b/c1 complex indicated that the three polypeptides encoded by the fbc operon comprise the entire catalytic structure: ubiquinol-cytochrome-c reductase . The amino acid sequences of the three b/c1 subunits from the photosynthetic bacterium Rhodobacter capsulatus were compared with the corresponding sequences from yeast mitochondria and spinach chloroplasts . The high homology found between the sequences of all three redox polypeptides from R . capsulatus and yeast mitochondria (cytochrome b 41%, FeS protein 46%, cytochrome c1 31%) provided further evidence that mitochondria arose from the phylogenetic line of purple bacteria . The structure of cytochrome b also exhibited considerable homology to chloroplast cytochrome b6 plus subunit IV (26%) . The amino acid sequence of the Rieske FeS protein from R . capsulatus and chloroplasts were found to be conserved only in the C-terminal part (14% total identity), whereas the homology between cytochrome c1 and cytochrome f is very weak (12%), despite similar topology of the two polypeptides . Analysis of the homology suggested that the catalytic sites quinol oxidase (Q0) and quinone reductase (Qi) arose monophonetically, whereas cytochrome c and plastocyanin reductase sites are not homologous and could derive from diverse ancestral genes by convergent evolution.

Eur J Biochem, 1988 Feb 1, 171(3), 551 - 64
Complex RNA maturation in chloroplasts . The psbB operon from spinach; Westhoff P et al.; The psbB operon of the spinach plastid chromosome encodes the genes for the 51-kDa chlorophyll a apoprotein (psbB), the 10-kDa phosphoprotein (psbH), both associated with photosystem II, as well as cytochrome b6 (petB) and subunit IV (petD) of the cytochrome b/f complex in the order given . These genes are not expressed coordinately . The RNA pattern of this DNA region is complex and resolves into eighteen major RNA species . Using northern and S1 protection analysis we demonstrate (a) that all RNA species derive from one DNA strand and hybridize in an overlapping fashion; and (b) that they arise by processing rather than by multiple transcription initiation/termination . (c) The operon is bordered by a single prokaryote-like promotor in front of psbB, and by a putative factor-independent terminator with characteristic sequence elements following petD . The terminator appears to function bidirectionally . (d) At least four distinct modification activities operate on the putative primary transcript of 5650 nucleotides and on the processing intermediates, including a novel endonucleolytic activity cleaving within a characteristic hexanucleotide motif, 3'-exonucleolytic activity at discrete RNA ends, 5' shortage of mRNA (psbB), and excision of class II intervening sequences (petB and petD) . (e) Kinetically, maturation of the primary transcript is largely a stochastic process . (f) Processing results ultimately in the formation of monocistronic mRNAs for each of the two photosystem II polypeptides and a bicistronic mRNA encoding both subunits of the cytochrome b/f complex . We postulate that these RNA species represent the translationally active components in the non-coordinate dark/light expression of these genes . (g) Light is without any noticeable effect on posttranscriptional modification . Under our conditions it appears to operate at a translational rather than a transcriptional or posttranscriptional level indicating that the biogenesis of thylakoid membranes is regulated at various levels.

Virology, 1988 Feb, 162(2), 300 - 10
The glycoprotein C gene of herpes simplex virus 1 resident in clonal L cell lines manifests two regulatory domains conferring a dominant B and a subordinate gamma 2 regulation; Arsenakis M et al.; Earlier studies have shown that late, gamma 2, genes of herpes simplex virus 1 stably incorporated into the environment of the cell are regulated as beta genes . For example, the induction of the intact gene specifying glycoprotein C (gC) resident in a clonal L cell line superinfected with a gC- virus was enhanced in the presence of inhibitory concentrations of phosphonoacetate (PAA), a viral DNA synthesis inhibitor . Moreover, the gene was induced by superinfection at the nonpermissive temperature with tsHA1, a temperature-sensitive (ts) DNA- virus mutant . In the viral genome, the gC gene is not expressed by the tsHA1 mutant at the nonpermissive temperature or by the mutant or wild-type virus at the permissive temperature in the presence of inhibitory concentrations of PAA . We report that expression of a truncated gC gene introduced in the same fashion and resident in a clonal L cell line was at least partially sensitive to PAA and was not induced by tsHA1 at the nonpermissive temperature . The gene was transcribed from a prokaryotic transcription initiation site in the plasmid . Induction of gene expression by superinfecting virus did not result in appreciable amplification of the gene . We conclude that gC, a gamma 2 gene, contains two regulatory domains . The gene domain upstream from nucleotide +22 confers beta-like regulation, whereas the gene domain downstream from +22 confers gamma 2 regulation . In the gene resident in cells in culture the upstream regulatory domain is dominant, whereas the converse is true for the gene resident in the viral genome.

Biochemistry, 1988 Jan 26, 27(2), 582 - 92
Chemical probing of adenine residues within the secondary structure of rabbit 18S ribosomal RNA; Rairkar A et al.; The location of unpaired adenine residues within the secondary structure of rabbit 18S ribosomal RNA was determined by chemical probing . Naked 18S rRNA was first prepared by digestion of purified 40S subunits with matrix-bound proteinase K in sodium dodecyl sulfate, thereby omitting the use of nucleic acid denaturants . Adenines within naked 18S rRNA were chemically probed by using either diethyl pyrocarbonate or dimethyl sulfate, which specifically react with unpaired nucleotides {Peattie, D . A., & Gilbert, W . (1980) Proc . Natl . Acad . Sci . U.S.A . 77, 4679-4682} . Adenine modification sites were identified by polyacrylamide sequencing gel electrophoresis either upon aniline-induced strand scission of 32P-end-labeled intact and fragmented rRNA or by primer extension using sequence-specific DNA oligomers with reverse transcriptase . The data indicate good agreement between the general pattern of adenine reactivity and the location of unpaired regions in 18S rRNA determined by comparative sequence analysis {Chan, Y.-L., Gutell, R., Noller, H . F., & Wool, I . G . (1984) J . Biol . Chem . 259, 224-230} . The overall reactivity of adenine residues toward single-strand-specific chemical probes was, also, similar for both rabbit and Escherichia coli small rRNA . The number of strongly reactive adenines appearing within phylogenetically determined helical segments, however, was greater in rabbit 18S rRNA than for E . coli 16S rRNA . Some of these adenines were found clustered in specific helices . Such differences suggest a greater irregularity of many of the helical elements within mammalian 18S rRNA, as compared with prokaryotic 16S rRNA . These helical irregularities could be important for protein association and also may represent biologically relevant flexible regions of the molecule.

Biochemistry, 1988 Jan 26, 27(2), 536 - 9
Immunological relatedness between Bordetella pertussis and rat brain adenylyl cyclases; Monneron A et al.; A prokaryotic adenylyl cyclase, secreted by Bordetella pertussis, shares a common functional property with eukaryotic adenylyl cyclases, i.e., regulation by the eukaryotic protein calmodulin . Making use of polyclonal antibodies raised against the bacterial adenylyl cyclase and the rat brain adenylyl cyclase catalytic component, respectively, we showed an immunological cross-reactivity between the two enzymes . Furthermore, B . pertussis adenylyl cyclase was inhibited and immunoprecipitated by the homologous and one of the heterologous immune sera . These results suggest an evolutionary relationship between the B . pertussis enzyme and its eukaryotic counterpart.

J Biol Chem, 1988 Jan 15, 263(2), 609 - 12
Transcription attenuation; Yanofsky C; Prokaryotic transcription attenuation mechanisms are described in which different metabolic signals and sensing events are used to regulate transcription termination at sites preceding structural genes . Suggestive eukaryotic examples also are mentioned.

Eur J Biochem, 1988 Jan 15, 171(1-2), 131 - 6
Immunochemical properties of elongation factors 1 of plant origin; Pulikowska J et al.; Elongation factors 1 (EF-1) have been isolated from different plants: wheat, yellow lupine, blue lupine, Chinese cabbage and Norway maple . Antibodies for EF-1 from yellow lupine have been obtained in rabbits; antibodies for wheat EF-1 were elicited in mice . The immunological properties of EF-1 were assayed by the following methods: western blotting, double immunodiffusion and rocket immunoelectrophoresis . Our results suggest that one antigenic site is similar for all plant elongation binding factors tested . This epitope probably overlaps the centre of biological activity of EF-1, as was shown for wheat EF-1 . The hypothesis concerning the potential presence of plant EF-1 as a subunit of turnip yellow mosaic virus RNA replicase (similar to prokaryotic EF-Tu in the Q beta RNA replicase system) has also been tested using immunotechniques as well as tests of biological activity, but has not been confirmed.

J Biol Chem, 1988 Jan 15, 263(2), 833 - 8
Sequence of peptides from Saccharomyces cerevisiae glutamine synthetase . N-terminal peptide and ATP-binding domain; Kim KH et al.; Sequences of seven tryptic peptides derived from Saccharomyces cerevisiae glutamine synthetase have been determined . The amino terminus of yeast enzyme is acetylated and has the following sequence: acetyl-Ala-Glu-Ala-Ser-Ile-Glu-Lys . Neither higher eukaryotic nor bacterial glutamine synthetase contain sequences homologous to this yeast amino terminus . 8-Azidoadenosine 5'-triphosphate {( alpha-32P}8-N3ATP) has been used to photolabel the ATP-binding site in yeast glutamine synthetase . Only one 32P-labeled tryptic peptide was obtained as a major fraction and its sequence is Glu-Gly-Tyr-Gly-X-Phe-Glu-Asp-Arg . Similar photolabeling experiments with bovine glutamine synthetase yielded a tryptic peptide whose sequence is Gly-X-Phe-Glu-Asp-Arg, where X is likely Tyr covalently attached by nitrene derived from {alpha-32P}8-N3ADP . Sequences very homologous to this nucleotide-binding site can be found in other eukaryotic enzymes but not in prokaryotic enzymes . In addition, the sequences of two cysteine-containing peptides and three other tryptic peptides were established . Sequences homologous to all these five peptides can be found in mammalian and plant enzymes . The homology between yeast and higher eukaryotic glutamine synthetases was sufficiently strong to suggest that the overall tertiary and quaternary structures of these enzymes must be similar . The sequences presented here, particularly the amino terminus sequence will be valuable in identifying the structural gene of yeast glutamine synthetase, thereby making it possible to study its transcriptional regulation . In addition, the sequences of the cysteine-containing peptides will be useful in determining whether or not the covalent modification of a sulfhydryl group(s) is responsible for the modulation of glutamine synthetase activity.

Nucleic Acids Res, 1988 Jan 11, 16(1), 165 - 78
Crosslinking of the anticodon of P site bound tRNA to C-1400 of E.coli 16S RNA does not require the participation of the 50S subunit; Denman R et al.; Crosslinking of the 5'-anticodon base of ribosomal P site bound AcVal-tRNA to residue C-1400 of 16S RNA or to its equivalent in 18S RNA has been shown to occur on 70S or 80S ribosomes of both prokaryotes and eukaryotes {Ciesiolka, J., Nurse, K., Klein, J . and Ofengand, J . (1985) Biochemistry 24, 3233-3239} . In the present work, we show that the crosslinking rate, crosslinking yield, and site of crosslinking are all unchanged when the 50S subunit is omitted . Therefore, all of the positional features of tRNA-ribosome complexes which allow crosslinking to occur are entirely contained in the 30S subunit . Blockage of reverse transcription by crosslink formation was used to determine the site of crosslinking . This analysis revealed that RNA modifications which do not directly block base-pairing ligands sometimes allow the modified base to be transcribed, leading to doublet band formation even when there is only a single crosslink site.

J Biol Chem, 1988 Jan 5, 263(1), 461 - 7
Animal viruses are able to fuse with prokaryotic cells . Fusion between Sendai or influenza virions and Mycoplasma; Citovsky V et al.; Sendai and influenza virions are able to fuse with mycoplasmata . Virus-Mycoplasma fusion was demonstrated by the use of fluorescently labeled intact virions and fluorescence dequenching, as well as by electron microscopy . A high degree of fusion was observed upon incubation of both virions with Mycoplasma gallisepticum or Mycoplasma capricolum . Significantly less virus-cell fusion was observed with Acholeplasma laidlawii, whose membrane contains relatively low amounts of cholesterol . The requirement of cholesterol for allowing virus-Mycoplasma fusion was also demonstrated by showing that a low degree of fusion was obtained with M . capricolum, whose cholesterol content was decreased by modifying its growth medium . Fluorescence dequenching was not observed by incubating unfusogenic virions with mycoplasmata . Sendai virions were rendered nonfusogenic by treatment with trypsin, phenylmethylsulfonyl fluoride, or dithiothreitol, whereas influenza virions were made nonfusogenic by treatment with glutaraldehyde, ammonium hydroxide, high temperatures, or incubation at low pH . Practically no fusion was observed using influenza virions bearing uncleaved hemagglutinin . Trypsinization of influenza virions bearing uncleaved hemagglutinin greatly stimulated their ability to fuse with Mycoplasma cells . Similarly to intact virus particles, also reconstituted virus envelopes, bearing the two viral glycoproteins, fused with M . capricolum . However, membrane vesicles, bearing only the viral binding (HN) or fusion (F) glycoproteins, failed to fuse with mycoplasmata . Fusion between animal enveloped virions and prokaryotic cells was thus demonstrated.

FEBS Lett, 1988 Jan 4, 226(2), 247 - 9
Vestiges of lost introns in the thermal stability map of DNA; Hanai R et al.; The absence of introns in prokaryotic genes has been explained by intron loss on various bases . Here we report another piece of evidence on intron loss, which was found in the thermal stability map of DNA . We calculated the local melting temperature of cDNA sequences and found that (i) gaps in thermal stability tend to occur near intron positions with a statistical significance, and (ii) one-third of the gaps far from intron positions can be assigned to lost introns . From these results we conclude that the gaps of thermal stability in protein coding regions are the vestiges of lost introns.

Ann N Y Acad Sci, 1988, 534, 283 - 98
Carcinogenic and mutagenic potential of several fluorocarbons; Longstaff E; A series of chlorofluorocarbons (CFC) have been evaluated for carcinogenic potential in two comprehensive toxicity studies . The first of these studies involved an assessment of their potential carcinogenicity using in vitro short-term predictive tests followed by a limited gavage validation assay in rats . The second study was a more conventional inhalation study of CFC22 employing rats and mice coupled with an assessment of in vivo genotoxicity of the material . The current paper briefly reviews these two studies and assesses the overall genotoxicity profile of CFC22 in terms of risk to man . It is concluded that CFCs are not biologically inert, but that the series contains bacterial mutagens, cell-transforming agents, and rodent carcinogens, and for this series of compounds at least, prokaryotic mutation does not accurately predict carcinogenic potential . In addition it is concluded that CFC22 does not represent a carcinogenic or mutagenic threat to man.

EMBO J, 1988 Jan, 7(1), 37 - 47
Human DNA polymerase alpha gene expression is cell proliferation dependent and its primary structure is similar to both prokaryotic and eukaryotic replicative DNA polymerases; Wong SW et al.; We have isolated cDNA clones encoding the human DNA polymerase alpha catalytic polypeptide . Studies of the human DNA polymerase alpha steady-state mRNA levels in quiescent cells stimulated to proliferate, or normal cells compared to transformed cells, demonstrate that the polymerase alpha mRNA, like its enzymatic activity and de novo protein synthesis, positively correlates with cell proliferation and transformation . Analysis of the deduced 1462-amino-acid sequence reveals six regions of striking similarity to yeast DNA polymerase I and DNA polymerases of bacteriophages T4 and phi 29, herpes family viruses, vaccinia virus and adenovirus . Three of these conserved regions appear to comprise the functional active site required for deoxynucleotide interaction . Two putative DNA interacting domains are also identified.

Agents Actions Suppl, 1988, 24, 178 - 83
Mechanisms of gold resistance; Wollheim FA; The efficacy of gold treatment in rheumatoid arthritis is hampered by toxicity, most prevalent in early phases, and unresponsiveness which can only be assessed after more than 6 months on therapy . There is some evidence that more severe disease increases the likelihood of drug resistance . No data indicate an altered pharmacokinetics in resistant individuals, although increased dosage of parenteral gold has been claimed effective in uncontrolled studies . Exposure to metals induces cell adaption and resistance in both prokaryotic and eukaryotic cell lines . In particular this has been shown for gold chloride, sodium aurothiomalate and auranofin . Auranofin induces increased synthesis of metallothionein, a low molecular weight cystein-rich peptide with metal-binding and -homeostatic properties . Thus there is experimental evidence suggesting cellular adaptation as a potential mechanism for gold resistance . However, with the possible exception of auranofin, the nature of the processes remains unknown.

Prep Biochem, 1988, 18(3), 303 - 20
Large scale purification of factor X by hydrophobic chromatography; Friedberg RC et al.; Factor X is a critical enzyme in the blood coagulation cascade, however, in recent years the coagulation zymogen factor X has received additional interest as a selective proteinase to allow production of functional eukaryotic proteins in a prokaryotic expression system . Traditional factor X purification schemes suffer from low yields, low capacity, lengthy dialysis steps, and contamination by the autoproteolytic activated enzyme factor Xa . By incorporating a reversible inhibitor of factor X activation, we were able to recover 67% of the factor X present without any detectable activated enzyme . Six liters of plasma could be processed onto a 50 mL phenylalanine-Sepharose hydrophobic chromatography column without saturating the matrix . The final product is devoid of detectable proteolytic activity . At time of use, the zymogen is specifically activated with a Sepharose-bound activating enzyme isolated from Russell's Viper Venom, resulting in factor Xa free of other detectable proteinases.

Toxicon, 1988, 26(8), 733 - 46
Isolation of a hemolysin from a spore-crystal mixture of Bacillus thuringiensis israelensis (serotype H-14); Weinstein SA et al.; A hemolytic toxin from Bacillus thuringiensis israelensis was obtained by alkaline extraction and fast protein liquid chromatography (chromatofocusing followed by gel filtration) . The toxin displayed a pI of 4.6-4.8 and an Mr of 26,000 . Amino acid analysis demonstrated large amounts of serine, glycine and glutamic acid . The toxin was strongly lytic for rabbit, human and non-human primate erythrocytes, and was weakly lytic toward equine, feline, canine, bovine, amphibian and reptilian erythrocytes . It was weakly entomocidal as indicated by a lethal potency (LC50) of 25 micrograms/ml for second instar larvae of Anopheles stephansi . Direct micro-ELISA indicated that the toxin lacked antigenic identity with numerous eukaryotic and prokaryotic toxins and venoms.

Chem Biol Interact, 1988, 68(3-4), 241 - 57
Interaction of lysinoalanine with the protein synthesizing apparatus; Lifsey BJ Jr et al.; Lysinoalanine {N epsilon-(DL-2-amino-2-carboxyethyl)-L-lysine; LAL}, a nephrotoxic lysine analog, inhibits the lysyl-tRNA-synthetase (EC 6.1.1.6) of prokaryotic and eukaryotic cells competitively at micromolar concentrations . Incorporation of {14C}lysine into protein by a cell-free eukaryotic protein-synthesizing system was inhibited by LAL . Inhibition was 69.7% and 18.4% at LAL concentrations of 1.0 mM and 0.1 mM, respectively . LAL was incorporated into protein as well as being an inhibitor as indicated by the incorporation of {14C}LAL into protein by the cell-free eukaryote protein-synthesizing system . The proteins labeled with {14C}LAL co-electrophoresed with those labeled with {14C}lysine . These results indicate that LAL is an inhibitor of both prokaryote and eukaryote lysyl-tRNA-synthetase . Furthermore, it is incorporated into protein . Both of these actions can be factors in the nephrotoxicity of this common food contaminant . Possible mechanisms for the toxicity of lysinoalanine are discussed.

J Mol Evol, 1988, 27(3), 222 - 7
Unusual sequences, homologous to 5S RNA, in ribosomal DNA repeats of the nematode Meloidogyne arenaria; Vahidi H et al.; There are sequences homologous to 5S ribosomal RNA in the ribosomal DNA (rDNA) repeats of the plant-parasitic nematode Meloidogyne arenaria . This is surprising, because in all other higher eukaryotes studied to date, the genes for 5S RNA are unlinked to and distinct from a tandem rDNA repeat containing the genes for 18S, 5.8S, and 28S ribosomal RNA . Previously, only prokaryotes and certain "lower eukaryotes" (protozoa and fungi) had been found to have both the larger rRNAs and 5S rRNA represented within a single DNA repeat . This has raised questions on the organization of these repeats in the earliest cell (progenote), and on subsequent evolutionary relationships between pro- and eukaryotes . Evidence is presented for rearrangements and deletions within Meloidogyne rDNA . The unusual life cycles (different levels of ploidy, reproduction by meiotic and mitotic parthenogenesis) of members of this genus might allow rapid fixation of any variants with introduced 5S RNA sequences . The 5S RNA sequences in Meloidogyne rDNA may not be expressed, but their presence raises important questions as to the evolutionary origins and stability of repeat gene families.

Biosystems, 1988, 21(3-4), 333 - 40
Nuclear division and chromosome cycle in microsporidia; Canning EU; Nuclear division and chromosome cycle of microsporidia are reviewed in the light of recent speculation that the group diverged as an early branch in the eukaryotic line of descent . Microsporidia are primitive eukaryotes with simple cytoplasmic organisation, lacking mitochondria, peroxisomes and a classical Golgi apparatus . The ribosomes resemble prokaryotic ribosomes in size and in having sequences complementary to the eukaryotic 5.8S rRNA contained within their large (23S) ribosomal subunit, rather than a separate 5.8S molecule . Nuclei may be isolated or closely appressed as a diplokaryotic pair which divide synchronously . Mitosis is intranuclear: there are no centrioles but spindle termini are electron dense plaques in pores in the nuclear envelope . Some genera have isolated nuclei throughout the life cycle, while others have diplokaryotic nuclei throughout: meiosis is not known in either of these categories of genera . In contrast, certain polymorphic species, which are transmitted horizontally between copepods and mosquitoes and vertically between generations of mosquitoes, alternate between stages with isolated nuclei and stages with diplokaryotic nuclei . Haploid spores in copepods are infective to mosquito larvae, in which gametogenesis and plasmogamy occur to give diplokaryotic (diploid) stages . Stages remain diplokaryotic through transovarial transmission to the next generation, when an unusual form of meiosis is initiated in both nuclei of the diplokaryon (as indicated by synaptonemal complexes), and mingling of chromosomes occurs when the two nuclei fuse during pachytene.

Prep Biochem, 1988, 18(4), 431 - 42
Purification of isocitrate lyase from Escherichia coli and watermelon using fast protein liquid chromatography; Conder MJ et al.; The enzyme isocitrate lyase has been purified to gel electrophoretic homogeneity from Escherichia coli and watermelon . From cotyledons of the latter source, the enzyme is obtained in less than 8 hours after precipitation with (NH4)2 SO4 followed by fractionation on cationic Mono S microbeads and anionic Mono Q microbeads using Fast Protein Liquid Chromatography (FPLC) . From a genetically engineered E . coli strain, in which high-level expression of isocitrate lyase occurs, the enzyme has been purified in one step from the high-speed supernatant using a Mono Q column with FPLC . These purifications, both of which give satisfactory yields, potentiate rapid access to isocitrate lyase from both prokaryotic and eukaryotic sources.

Cancer Surv, 1988, 7(2), 303 - 15
DNA repair in human cells: from genetic complementation to isolation of genes; Bootsma D et al.; The genetic disease xeroderma pigmentosum (XP) demonstrates the association between defective repair of DNA lesions and cancer . Complementation analysis performed on XP cell strains and on repair deficient rodent cell lines has revealed that at least nine and possibly more than 13 genes are involved in early steps of the excision of ultraviolet light-induced DNA lesions in mammalian cells . Two of these genes have been cloned and others are in an advanced stage of cloning . One cloned gene, ERCC-1, has been characterized at the molecular level . This human gene is homologous with excision repair genes in yeast and in Escherichia coli . These results indicate that the excision repair system is conserved during evolution . It is expected that the cloning and characterization of prokaryotic and eukaryotic repair genes will pave the way to a deeper understanding of mammalian repair systems and their association with cancer.

Proteins, 1988, 3(4), 243 - 51
Structural comparison of the prokaryotic ribosomal proteins L7/L12 and L30; Leijonmarck M et al.; The structures of two prokaryotic ribosomal proteins, the carboxyterminal half of L7/L12 from Escherichia coli (L12CTF) and L30 from Bacilus stearothermophilus display a remarkably similar fold in which alpha-helices pack onto one side of an antiparallel, three-stranded, beta-pleated sheet . A detailed comparison of the structures by least-squares methods reveals that more than two-thirds of the alpha carbons can be superimposed with a root mean square distance of 2.33 A . The principal difference is an extra alpha-helix in L12CTF . The sequences of the proteins display a distinct conservation in regions which are crucial to the common fold, in particular the hydrophobic core . It is proposed that the similarity is a result of divergent evolution.

Comp Biochem Physiol A, 1988, 90(2), 209 - 23
The evolutionary origin of eukaryotic transmembrane signal transduction; Janssens PM; 1 . A comparison was made of transmembrane signal transduction mechanisms in different eukaryotes and prokaryotes . 2 . Much attention was given to eukaryotic microbes and their signal transduction mechanisms, since these organisms are intermediate in complexity between animals, plants and bacteria . 3 . Signal transduction mechanisms in eukaryotic microbes, however, do not appear to be intermediate between those in animals, plants and bacteria, but show features characteristic of the higher eukaryotes . 4 . These similarities include the regulation of receptor function, adenylate cyclase activity, the presence of a phosphatidylinositol cycle and of GTP-binding regulatory proteins . 5 . It is proposed that the signal transduction systems known to operate in present-day eukaryotes evolved in the earliest eukaryotic cells.

Mol Microbiol, 1988 Jan, 2(1), 19 - 30
The calmodulin-sensitive adenylate cyclase of Bordetella pertussis: cloning and expression in Escherichia coli; Glaser P et al.; The adenylate cyclase toxin of the prokaryote Bordetella pertussis is stimulated by the eukaryotic regulatory protein, calmodulin . A general strategy, using the adenylate-cyclase-calmodulin interaction as a tool, has permitted cloning and expression of the toxin in Escherichia coli in the absence of any B . pertussis trans-activating factor . We show that the protein is synthesized in a large precursor form composed of 1706 amino acids . The calmodulin-stimulated catalytic activity resides in the amino-terminal 450 amino acids of the adenylate cyclase . The enzyme expressed in E . coli is recognized in Western blots by antibodies directed against purified B . pertussis adenylate cyclase, and its activity is inhibited by these antibodies.

Mol Reprod Dev, 1988, 1(1), 57 - 62
Identification of specific gene sequences in preimplantation embryos by genomic amplification: detection of a transgene; King D et al.; Endogenous and foreign DNA sequences can be detected in an extremely small number of cells via sequence amplification in vitro . The polymerase chain reaction (PCR) technique applied in multiple cycles allows the amplification of specific short regions of the genome to levels that can be detected by DNA blotting techniques . Cow and mouse blastocysts were analyzed by PCR for the presence of an endogenous singlecopy gene or an integrated foreign gene . The endogenous single-copy gene encoding the beta chain of bovine luteinizing hormone was detectable in cow blastocysts and in purified bovine genomic DNA representing as few as 25 cells . To determine whether exogenous genes (transgenes) can be detected in preimplantation embryos, transgenic male mice hemizygous for the prokaryotic gene encoding neomycin resistance were bred to nontransgenic females, and the resulting blastocysts were analyzed . The neo gene was detected in approximately half of the embryos . The capability to identify specific gene sequences in a limited number of embryonic cells affords investigators the opportunity to study genetics in early development.

Free Radic Biol Med, 1988, 5(5-6), 377 - 85
Biosynthesis and regulation of superoxide dismutases; Hassan HM; The past two decades have witnessed an explosion in our understanding of oxygen toxicity . The discovery of superoxide dismutases (SODs) (EC.1.15.1.1), which specifically catalyze the dismutation of superoxide radicals (O2-) to hydrogen peroxide (H2O2) and oxygen, has indicated that O2- is a normal and common byproduct of oxygen metabolism . There is an increasing evidence to support the conclusion that superoxide radicals play a major role in cellular injury, mutagenesis, and many diseases . In all cases SODs have been shown to protect the cells against these deleterious effects . Recent advances in molecular biology and the isolation of different SOD genes and SOD c-DNAs have been useful in proving beyond doubt the physiological function of the enzyme . The biosynthesis of SODs, in most biological systems, is under rigorous controls . In general, exposure to increased pO2, increased intracellular fluxes of O2-, metal ions perturbation, and exposures to several environmental oxidants have been shown to influence the rate of SOD synthesis in both prokaryotic and eukaryotic organisms . Recent developments in the mechanism of regulation of the manganese-containing superoxide dismutase of Escherichia coli will certainly open new research avenues to better understand the regulation of SODs in other organisms.

Braz J Med Biol Res, 1988, 21(1), 1 - 7
Biology of metastasis; Brentani RR et al.; 1 . Metastasis, i.e., dissemination of tumor cells throughout the organism, is the cause of death for most cancer patients . We discuss here the role of the interaction between basement membrane laminin and specific cell receptors in the process . We further show that such interaction is mediated by receptors not only in cancer cells but also in normal granulocytes, in a parasitic protozoan and even in an invasive prokaryote . 2 . Metastatic potential has been correlated with the number of receptors/cell and preliminary results with a monoclonal antibody against the S . aureus laminin receptor suggest that the binding site at the receptor level might be conserved . This conclusion is supported by the facts that this monoclonal antibody can recognize the eukaryotic receptor and inhibit laminin binding.

Gan To Kagaku Ryoho, 1988 Jan, 15(1), 1 - 14
{Biological function of DNA topoisomerases and its implication in cancer chemotherapy}; Andoh T et al.; It has been generally recognized that in a variety of biological systems the structural changes such as circularization, looping and supercoiling of DNA play important roles in carrying out genetic processes such as replication, transcription, recombination etc . Since the discovery of DNA topoisomerase I in 1971 (then named omega protein) in E . coli type I and type II topoisomerases have been found in a wide variety of organisms . In addition the finding that these enzymes are the only ones which can cope with the torsional strain accumulating within the DNA molecules carrying out the genetic processes by swiveling the strands through breakage and rejoining of phosphodiester bonds strongly suggests that they are involved in various aspects of DNA metabolism such as replication, transcription, recombination, differentiation etc . In this article the function of DNA topoisomerases elucidated so far in prokaryotic and eukaryotic cells with special emphasis on their roles in gene expression has been briefly reviewed.

EMBO J, 1987 Dec 20, 6(13), 4219 - 25
Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases; Bernad A et al.; The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation . Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase . This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases . Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology . Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains . These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases.

Philos Trans R Soc Lond B Biol Sci, 1987 Dec 15, 317(1187), 395 - 420
Prokaryotic DNA replication mechanisms; Alberts BM; The three different prokaryotic replication systems that have been most extensively studied use the same basic components for moving a DNA replication fork, even though the individual proteins are different and lack extensive amino acid sequence homology . In the T4 bacteriophage system, the components of the DNA replication complex can be grouped into functional classes as follows: DNA polymerase (gene 43 protein), helix-destabilizing protein (gene 32 protein), polymerase accessory proteins (gene 44/62 and 45 proteins), and primosome proteins (gene 41 DNA helicase and gene 61 RNA primase) . DNA synthesis in the in vitro system starts by covalent addition onto the 3'OH end at a random nick on a double-stranded DNA template and proceeds to generate a replication fork that moves at about the in vivo rate, and with approximately the in vivo base-pairing fidelity . DNA is synthesized at the fork in a continuous fashion on the leading strand and in a discontinuous fashion on the lagging strand (generating short Okazaki fragments with 5'-linked pppApCpXpYpZ pentaribonucleotide primers) . Kinetic studies reveal that the DNA polymerase molecule on the lagging strand stays associated with the fork as it moves . Therefore the DNA template on the lagging strand must be folded so that the stop site for the synthesis of one Okazaki fragment is adjacent to the start site for the next such fragment, allowing the polymerase and other replication proteins on the lagging strand to recycle.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8287 - 91
Bacteriophage PRD1 DNA polymerase: evolution of DNA polymerases; Jung GH et al.; A small lipid-containing bacteriophage PRD1 specifies its own DNA polymerase that utilizes terminal protein as a primer for DNA synthesis . The PRD1 DNA polymerase gene has been sequenced, and its amino acid sequence has been deduced . This protein-primed DNA polymerase consists of 553 amino acid residues with a calculated molecular weight of 63,300 . Thus, it appears to be the smallest DNA polymerase ever isolated from prokaryotic cells . Comparison of the PRD1 DNA polymerase sequence with other DNA polymerase sequences that have been published yielded segmental but significant homologies . These results strongly suggest that many prokaryotic and eukaryotic DNA polymerase genes, regardless of size, have evolved from a common ancestral gene . The results further indicate that those DNA polymerases that use either an RNA or protein primer are related . We propose to classify DNA polymerases on the basis of their evolutionary relatedness.

Arch Biochem Biophys, 1987 Dec, 259(2), 481 - 96
Metabolism of exogenous long-chain fatty acids by spinach leaves; Roughan PG et al.; When applied in liquid paraffin to the upper surface of expanding spinach leaves, {1-14C}palmitic acid was efficiently and exclusively incorporated into the sn-1 position of cellular glycerolipids, principally phosphatidylcholine and triacylglycerol . A slow transfer of fatty acids from phosphatidylcholine to chloroplast glycolipids subsequently occurred with the positional specificity of the label remaining intact . Labeled palmitate at the sn-1 position of monogalactosyldiacylglycerol was desaturated to hexadecatrienoate so that 1-{14C}hexadecatrienoyl-2-linolenoyl-3-galactosoylglycerol became the major labeled species of the lipid between 8 and 24 h . There was no evidence of deacylation/reacylation reactions modifying the acyl compositions of spinach leaf glycerolipids for at least 48 h after labeling with {1-14C}palmitic acid; even the partially prokaryotic glycerolipids remained firmly labeled at the sn-1 position . Exogenous {1-14C}stearic acid was also incorporated into the sn-1 position of MGD, presumably by the same mechanism, and was there desaturated to {14C}linolenate . Exogenous {1-14C}oleic acid was initially incorporated equally into both sn-1 and sn-2 positions of phosphatidylcholine, and was desaturated to linoleate at both positions before the label was rapidly transferred to monogalactosyldiacylglycerol . There, desaturation of linoleate to linolenate took place . Galactolipids remained equally labeled at both positions throughout the 6 days of the experiment, but label was concentrated in the 1-saturated-2-{14C}linolenoyl molecular species of phosphatidylcholine as those species with two {14C}linoleoyl residues were drawn off for monogalactolipid synthesis . Glycerolipids synthesised from exogenous {1-14C}acetate by spinach leaves were labeled equally at both the sn-1 and the sn-2 positions . These results are interpreted as providing strong support for the two-pathway scheme of glycerolipid synthesis in plants.

Proc Natl Acad Sci U S A, 1987 Dec, 84(24), 8859 - 63
Mutants of Escherichia coli formylmethionine tRNA: a single base change enables initiator tRNA to act as an elongator in vitro; Seong BL et al.; We show that the absence of a Watson-Crick base pair at the end of the amino acid acceptor stem, which is a hallmark of all prokaryotic initiator tRNAs, is one of the key features that prevents them from acting as an elongator in protein synthesis . We generated mutants of Escherichia coli formylmethionine tRNA that have a base pair at the end of the acceptor stem . The mutants generated were C1----T1, which had a U.A base pair, A72----G72, which had a C.G base pair, and the C1A72----T1G72 double mutant, which lacked the base pair . After aminoacylation, the activity of these and other mutant initiator methionyl-tRNAs (Met-tRNAs) in elongation were assayed in a MS2 RNA-directed E . coli protein-synthesizing system and in binding to the elongation factor Tu (EF-Tu) . Unlike wild-type initiator tRNA or the T1G72 double mutant, the T1 and G72 mutant Met-tRNAs were active in elongation, the G72 mutant being more active than the T1 mutant . The T1 and G72 mutant Met-tRNAs also formed a ternary complex with elongation factor EF-Tu.GTP, and their relative affinities for EF-Tu.GTP paralleled their activities in elongation . Combination of the T1 or G72 mutation with mutations in the GGG.CCC sequence conserved in the anticodon stem of initiator tRNAs led to a further increase in the activities of these mutant tRNAs in elongation such that one of these mutants was now almost as good an elongator as E . coli elongator methionine tRNA.

Vet Immunol Immunopathol, 1987 Dec, 17(1-4), 279 - 89
Expression of rabbit IgA heavy chain genes in E . coli and in murine myeloma cells; Knight KL et al.; Rabbit IgA-heavy chain cDNA and germline genes were cloned into prokaryotic and eukaryotic expression vectors, respectively . The Fc alpha encoding portion of six C alpha cDNA clones were cloned into pUC8 and E . coli were transformed . Radioimmunoassay of the molecules synthesized by these clones showed that molecules with Fc alpha antigenic determinants were produced at the level of approximately 0.1 to 1.0 microgram per ml culture . Radiobinding analysis showed that each of the clones encoded heavy chains of the IgA-g subclass . Southern blot analysis of rabbit germline DNA revealed 10 germline C alpha genes . Five of these, isolated from recombinant cosmid libraries, were cloned into a eukaryotic expression vector containing a rearranged murine VDJ gene, the CH enhancer region and the Eco-gpt gene . Murine myeloma cells, J558L, were transfected with each of the heavy chain constructs and stable transfectants was selected with mycophenolic acid . The immunoglobulins produced by each transfectant were analyzed by radiobinding and by SDS-PAGE . Each transfectant were shown to synthesize IgA molecules and thus all five C alpha genes are expressible . The heavy chains from the transfectants ranged in size from 55,000 to 60,000 daltons . Radiobinding analyses indicated that four of the five genes encode molecules of the IgA-f subclass; the serological identity of the fifth gene is not yet established.

Zh Mikrobiol Epidemiol Immunobiol, 1987 Dec, (12), 23 - 8
{Ultracytochemistry of phosphohydrolase of the microorganisms and phagocytes in gonococcal infection}; Dmitriev GA; The complex study of phosphohydrolases (Mg2+ATPase, Na+,K+ATPase) and adenylate cyclase in gonococci, both in vivo and in vitro, and in phagocytes in gonococcal infection has been carried out with the use of ultracytochemical techniques . The enzymatic activity in the cellular structures of micro- and macroorganisms under study has been visualized . The prospects of the ultracytochemical approach to the study of the enzymes of nucleotide metabolism, their role in the life of eu- and prokaryotes, as well as the ways for affecting the enzymatic systems of cells are discussed.

Antimicrob Agents Chemother, 1987 Dec, 31(12), 1925 - 8
Effect of DNA gyrase inhibitors pefloxacin, five other quinolones, novobiocin, and clorobiocin on Escherichia coli topoisomerase I; Tabary X et al.; Two coumarins, inhibitors of the B subunit of DNA gyrase, and six quinolones, inhibitors of the A subunit, were tested against Escherichia coli topoisomerase I-catalyzed DNA relaxation . Coumarins had no effect, whereas quinolones were inhibitors of the enzyme . This inhibition was compared with that of DNA gyrase and calf thymus topoisomerase I . The 50% inhibitory concentrations for E . coli topoisomerase I were about one order of magnitude higher than the corresponding values for E . coli DNA gyrase but were far lower than the known values for calf thymus topoisomerase I . There was a good relationship between inhibition of the two prokaryotic topoisomerases and MICs for E . coli, and the quinolones could be ranked in the same order in the three cases.

Proc Natl Acad Sci U S A, 1987 Dec, 84(23), 8360 - 4
Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene; Bzik DJ et al.; Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced . The deduced DHFR-TS protein contained 608 amino acids (71,682 Da) . The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%) . The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein . The TS domain was more conserved than the DHFR domain and both P . falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes . Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes.

Biochem Biophys Res Commun, 1987 Nov 30, 149(1), 27 - 33
Evidence for nucleoside channeling in vivo: deoxythymidine incorporation into rat liver dTTP and nuclear matrix DNA; Panzeter PL et al.; Previous studies in prokaryotes and in eukaryotic cell lines have indicated the possible existence of more than one dTTP pool accessible to DNA synthesis . To investigate this possibility in eukaryotes in vivo, the incorporation of {3H} deoxythymidine into nuclear matrix-attached DNA and intracellular dTTP was examined in regenerating rat liver . The labeling of matrix DNA reached a maximum after a 5 min pulse and then began to rapidly decrease . Conversely, {3H} deoxythymidine incorporation into dTTP began to increase after 5 min and peaked 10 min after injection . Since the peak specific activity for {3H} deoxythymidine incorporation into matrix DNA precedes that into dTTP, there seems to be channeling of exogenous thymidine directly to sites of DNA replication, bypassing existing nucleotide pools.

FEBS Lett, 1987 Nov 30, 224(2), 257 - 60
Two separable functional domains in the sigma-subunit of RNA polymerase in Bacillus subtilis?
Errington J.
The sigma-subunit of RNA polymerase is responsible for promoter recognition in prokaryotes {(1969) Nature 221, 43-46} . Alterations in the sigma-subunit are thought to be involved in controlling 'global' changes in gene expression, such as those involved in differentiation in the spore-forming bacterium Bacillus subtilis {(1981) Cell 25, 582-584} . Stragier et al . {(1985) FEBS Lett . 195, 3-11} have proposed that sigma-factors are composed of two domains: a C-terminal domain involved in promoter recognition and an N-terminal domain involved in interactions with RNA polymerase . We have sequenced another developmental gene from B . subtilis, spoIIIC, and the strong homology of its predicted product suggests that it too may be a sigma-factor . However, the spoIIIC product is small and lacks completely the conserved N-terminal domain of the sigma-subunits . I propose that the product of the spoIIIC gene may carry out the DNA-recognition functions of a sigma-factor but that it probably requires an auxiliary factor to interact with core RNA polymerase.

J Biol Chem, 1987 Nov 15, 262(32), 15756 - 8
Crystallization of rat cellular retinol binding protein II . Preliminary X-ray data obtained from the apoprotein expressed in Escherichia coli; Sacchettini JC et al.; Rat cellular retinol-binding protein II (CRBP II) is a member of a family of cytoplasmic proteins which bind hydrophobic ligands . CRBP II is thought to participate in the intestinal absorption and intracellular metabolism of retinoids . We have previously described the crystallization of a homologous rat intestinal fatty acid-binding protein (I-FABP) isolated from Escherichia coli containing a suitably constructed prokaryotic expression vector (Sacchettini, J . C., Meininger, T . A., Lowe, J . B., Gordon, J . I., and Banaszak, L . J., J . Biol . Chem . 262, 5428-5430) . We have now efficiently expressed rat CRBP II in E . coli . The E . coli-derived protein, which does not contain any bound retinoid, has been purified and crystals grown from solutions of polyethylene glycol 4000 . Crystals of apo-CRBP II are triclinic, space group P1, a = 36.8 A, b = 64.0 A, c = 30.4 A; alpha = 92.8 degrees, beta = 113.5 degrees, gamma = 90.1 degrees . Each unit cell contains two molecules of the 134-residue apoprotein . X-ray diffraction data suggest that the unit cell parameters of crystalline apo-CRBP II resemble those of I-FABP . Comparison of the tertiary structures of E . coli-derived rat I-FABP and CRBP II should provide insights about how these proteins evolved to bind different hydrophobic ligands.

Science, 1987 Nov 13, 238(4829), 964 - 7
Unwinding of duplex DNA from the SV40 origin of replication by T antigen; Dodson M et al.; The T antigen specified by SV40 virus is the only viral-encoded protein required for replication of SV40 DNA . T antigen has two activities that appear to be essential for viral DNA replication: specific binding to duplex DNA at the origin of replication and helicase activity that unwinds the two DNA strands . As judged by electron microscopy, DNA unwinding is initiated at the origin of replication and proceeds bidirectionally . Either linear or circular DNA molecules containing the origin of replication are effective substrates; with closed circular DNA, a topoisomerase capable of removing positive superhelical turns is required for an efficient reaction . Presence of an origin sequence on duplex DNA and a single-strand DNA-binding protein appear to be the only requirements for T antigen to catalyze unwinding . This reaction mediated by T antigen defines a likely pathway to precise initiation of DNA replication: (i) the sequence-specific binding activity locates the origin sequence, (ii) the duplex DNA is unwound at this site, and (iii) the DNA polymerase and primase begin DNA replication . A similar pathway has been inferred for the localized initiation of DNA replication by bacteriophage lambda and by Escherichia coli in which a sequence-specific binding protein locates the origin and directs the DnaB helicase to this site . Observations with the SV40 system indicate that localized initiation of duplex DNA replication may be similar for prokaryotes and eukaryotes.

Nucleic Acids Res, 1987 Nov 11, 15(21), 8693 - 711
A comparison of eukaryotic viral 5'-leader sequences as enhancers of mRNA expression in vivo; Gallie DR et al.; The 5'-untranslated leader sequences of several plant RNA viruses, and a portion of the 5'-leader of an animal retrovirus, were tested for their ability to enhance expression of contiguous open reading frames for chloramphenicol acetyltransferase (CAT) or beta-glucuronidase (GUS) in tobacco mesophyll protoplasts, Escherichia coli and oocytes of Xenopus laevis . Translation of capped or uncapped transcripts was substantially enhanced in almost all systems by the leader sequence of either the U1 or SPS strain of TMV . All leader sequences, except that of TYMV, stimulated expression of 5'-capped GUS mRNA with the native prokaryotic initiation codon context, in electroporated protoplasts . Only the TMV leaders enhanced translation of uncapped GUS mRNAs in protoplasts and increased expression of uncapped CAT mRNA in microinjected X . laevis oocytes . In oocytes, the TYMV leader sequence was inhibitory . In transformed E . coli, the TMV-U1 leader enhanced expression of both the native and eukaryotic context forms of GUS mRNA about 7.5-fold, despite the absence of a Shine-Dalgarno region in any of the transcripts . The absolute levels of GUS activity were all about 6-fold higher with mRNAs containing the native initiation codon context . In E . coli, the leaders of AlMV RNA4 and TYMV were moderately stimulatory whereas those of BMV RNA3, RSV and the SPS strain of TMV enhanced GUS expression by only 2- to 3-fold.

J Biol Chem, 1987 Nov 5, 262(31), 15256 - 61
Rho-dependent transcriptional polarity in the ilvGMEDA operon of wild-type Escherichia coli K12; Wek RC et al.; It has been generally accepted that transcriptional polarity in prokaryotic systems is due to an uncoupling of translation and transcription which unmasks latent rho-dependent termination sites in a polycistronic messenger RNA . In this report, we identify and characterize rho-dependent termination sites responsible for transcriptional polarity in the ilvGMEDA operon of wild-type Escherichia coli K12 . The ilvG gene in the wild-type E . coli K12 ilvGMEDA operon contains a frameshift site which results in termination of translation in the middle of the gene . Mutations have been characterized which restore the reading frame of this gene . In addition to allowing full-length expression of the ilvG product, these mutations cause a 3-4-fold elevation in the expression of the operon distal genes . This transcriptional polarity effect on operon distal genes also has been shown to be relieved by rho suppressor mutations . We have used in vitro transcription experiments to identify rho-dependent transcriptional termination sites downstream of the frameshift site in the ilvG gene . Three tandem rho-dependent sites have been located in the ilv'GM' gene region using transcription reactions containing linear or supercoiled plasmid DNA templates . Accumulatively, these rho-dependent termination sites account for about 80% in vitro transcription termination, which is in agreement with the in vivo measurements of transcriptional polarity on operon distal gene expression . These transcriptional experiments provide in vitro confirmation for the latent rho-dependent termination site model of transcriptional polarity.

Biochemistry, 1987 Nov 3, 26(22), 7113 - 21
Covalent coupling of 4-thiouridine in the initiator methionine tRNA to specific lysine residues in Escherichia coli methionyl-tRNA synthetase; Leon O et al.; A new method has been developed to couple a lysine-reactive cross-linker to the 4-thiouridine residue at position 8 in the primary structure of the Escherichia coli initiator methionine tRNA (tRNAfMet) . Incubation of the affinity-labeling tRNAfMet derivative with E . coli methionyl-tRNA synthetase (MetRS) yielded a covalent complex of the protein and nucleic acid and resulted in loss of amino acid acceptor activity of the enzyme . A stoichiometric relationship (1:1) was observed between the amount of cross-linked tRNA and the amount of enzyme inactivated . Cross-linking was effectively inhibited by unmodified tRNAfMet, but not by noncognate tRNAPhe . The covalent complex was digested with trypsin, and the resulting tRNA-bound peptides were purified from excess free peptides by anion-exchange chromatography . The tRNA was then degraded with T1 ribonuclease, and the peptides bound to the 4-thiouridine-containing dinucleotide were purified by high-pressure liquid chromatography . Two major peptide products were isolated plus several minor peptides . N-Terminal sequencing of the peptides obtained in highest yield revealed that the 4-thiouridine was cross-linked to lysine residues 402 and 439 in the primary sequence of MetRS . Since many prokaryotic tRNAs contain 4-thiouridine, the procedures described here should prove useful for identification of peptide sequences near this modified base when a variety of tRNAs are bound to specific proteins.

Virus Genes, 1987 Nov, 1(1), 97 - 116
Human RNA polymerase II can prematurely terminate transcription of the adenovirus type 2 late transcription unit at a precise site that resembles a prokaryotic termination signal; Seiberg M et al.; Premature termination of transcription has been demonstrated by eukaryotic RNA polymerase II at specific sites in the major late transcriptional unit of SV40 and in one of the transcriptional units of the parvovirus, minute virus of mice (MVM) (Y . Aloni and N . Hay, CRC Critical Reviews of Biochem., 18:327-383, 1985) . In both cases the prematurely terminated (attenuated) RNA can be folded into a hairpin structure followed by U-residues that resemble a termination signal in prokaryotes . The experiments presented herein demonstrate premature termination of transcription 185 nucleotides (nt) downstream from the major late promoter of adenovirus type 2 (Ad2) in vivo, and in vitro in isolated nuclei and in HeLa whole cell extract . As in SV40 and MVM the attenuated RNA of Ad2 can be folded into a hairpin structure followed by U-residues . Transcription-termination was significantly reduced when ITP replaced GTP and when Br-UTP replaced UTP in the transcription reaction mixture, indicating that RNA secondary structure and the rU-dA interactions, respectively, are parts of the termination signal . Moreover, in isolated nuclei transcription-termination at the attenuation site occurred when the reaction mixture contained between 50-150 mM NaCl but not when it contained 300 mM NaCl . These results indicate that, at least in isolated nuclei, attenuation can be regulated . The possible involvement of termination factor(s) in the regulation of attenuation is discussed.

Cell Tissue Res, 1987 Nov, 250(2), 475 - 7
Prokaryotic-eukaryotic cell junctions between spiral-shaped bacteria and cecal epithelium of the guinea pig; Mora-Galindo J; Scanning electron microscopy demonstrated that the cecum of the guinea-pig is colonized by numerous spiral-shaped bacteria; these microorganisms, which adhere to mucosa at one end, were found exclusively on the brush border of the surface epithelium . The membranes of sectioned bacteria have a set of electron-dense bands girdling the tip adhered to epithelium . Freeze-fracture replicas of the bacteria revealed the prokaryote-eukaryote junction as a set of ridges on the P-face of outer membrane; the numerous particles of E-face were arranged in parallel rows; on the other hand, the apical plasma membrane and subjacent cytoplasm of epithelium occupied by the spiral-shaped bacteria did not show a structural counterpart . Observations suggest that one end of the spiral-shaped bacteria possesses specialized membrane components that permit specific attachment to the apical surface of epithelial cells.

Ann Inst Pasteur Microbiol, 1987 Nov-Dec, 138(6), 639 - 50
Monoclonal antibodies against the bacterial-like organism associated with citrus greening disease; Garnier M et al.; Hybridoma clones secreting specific monoclonal antibodies (mAb) against the bacterium-like organism associated with citrus greening disease were produced from homogenates of infected phloem tissues used as immunogens in mice immunizations . Differential ELISA and immunofluorescence were used to screen for hybridomas secreting mAb against the greening organism (GO) . Two such hybridoma clones were obtained . The mAb were specific for the GO . No cross-reactions were seen with any of the other phloem-limited prokaryotes tested nor with healthy plant material.

Ann Inst Pasteur Microbiol, 1987 Nov-Dec, 138(6), 625 - 37
Production of monoclonal antibodies against phloem-limited prokaryotes of plants: a general procedure using extracts from infected periwinkles as immunogen; Martin-Gros G et al.; Using Spiroplasma citri-infected periwinkle extracts as the source of antigens, together with monoclonal antibody technology, we determined the conditions for mouse immunization and developed a screening assay to detect those hybridomas which produce immunoglobulins specifically directed against the pathogen.

Comput Appl Biosci, 1987 Nov, 3(4), 287 - 95
Statistical method for predicting protein coding regions in nucleic acid sequences; Fichant G et al.; Protein coding regions of a genome fragment can be mathematically predicted by studying variations in the statistical properties or by searching the signals characteristic of the junctions between the coding and non-coding regions . We propose here a new statistical method using correspondence analysis . This method does not use any reference codon set but takes into account the codon usage homogeneity along the studied genome fragment . Comparison with previously published methods especially the 'codon usage method' of Staden has been made, and two examples are presented here . Applications to analysis of prokaryotic operon and eukaryotic split genes are also discussed . Use of the method has also shown two structures not previously described: i) in the human prt gene, a strong triplet structure exists in a non-coding region; ii) in the human tp-a codon usage is not uniform between the different exons.

Virus Res, 1987 Nov, 8(4), 317 - 26
Activation of an insect baculovirus promoter in mammalian cells by adenovirus functions; Knebel D et al.; The insect baculovirus Autographa californica nuclear polyhedrosis virus (AcNPV) replicates in insect cell lines in culture . In mammalian cells, however, the virus cannot be propagated . AcNPV DNA does not replicate or persist and is not transcribed in mammalian cells (Tjia et al., 1983) . In insect cells productively infected with AcNPV, at least two major late viral gene products have been recognized, the polyhedrin, which makes up the bulk of the polyhedral inclusion bodies in infected cell nuclei, and a 10,000 Da protein (p10) of unknown function . The p10 promoter has been fused to the prokaryotic gene for chloramphenicol acetyltransferase (CAT) as a reporter gene (Knebel et al., 1985) . Activity of this construct can be elicited in AcNPV-infected insect cells but not in uninfected insect cells or in mammalian cells . Presumably, the late p10 promoter requires other AcNPV gene products for activity . When the pAcp10-CAT construct is transfected into BHK21 hamster cells at about 18 h after infection with human adenovirus type 5 (Ad5), the insect AcNPV promoter is transactivated in cells of the heterologous mammalian species . The results of S1 protection analyses on the RNA from Ad5-infected and pAcp10-CAT transfected cells reveal that the p10 promoter is used for initiation of transcription . Similarly, the p10 insect virus promoter is activated in BHK21 hamster cells cotransfected with the HindIII-G fragment of adenovirus type 2 (Ad2) DNA which contains the E1A and parts of the E1B region in the left terminal 7.8% of the Ad2 genome . Moreover, in human 293 cells or in BHK297-C131 hamster cells, which both carry and constitutively express the E1 region of Ad5 DNA, the pAcp10-CAT construct is also expressed, and similarly in HE7 hamster cells which carry appreciable portions of the Ad2 genome (Klimkait and Doerfler, 1985) . It is concluded that adenovirus functions are capable of transactivating a heterologous insect virus promoter in mammalian cells.

Virology, 1987 Nov, 161(1), 18 - 28
Characterization of adeno-associated virus rep proteins in human cells by antibodies raised against rep expressed in Escherichia coli; Trempe JP et al.; The rep gene of the defective human parvovirus, adeno-associated virus, (AAV) mediates several trans-acting functions important to virus replication, transcription, and gene expression . At least four overlapping polypeptides are expressed from the rep gene . We have constructed a prokaryotic vector which expressed in Escherichia coli a region of AAV comprising 93% of the largest AAV rep protein . The protein expressed in E . coli, rep 78.93, was used to raise specific antibodies in rabbits . These antibodies were capable of detecting all four AAV rep proteins in human cells transfected with AAV-containing plasmids as well as new species of 47 and 35 kDa in molecular weight . These new rep proteins originate from the transcription promoter at map unit 19 in the AAV genome and may indicate use of alternate AUG codons or protein modification . The antibodies also recognized novel forms of the rep proteins expressed from mutant AAV genomes . Immunofluorescence analysis of AAV-infected human cells revealed that the rep proteins are localized primarily in the nucleus of the infected cell and have a distribution different from that of AAV capsid protein . These results demonstrate that antisera raised against an AAV rep protein synthesized in E . coli are capable of detecting wild-type AAV rep proteins in virus-infected mammalian cells . These specific antibodies should facilitate further characterization of the functionally pleiotropic viral rep proteins.

FASEB J, 1987 Nov, 1(5), 375 - 9
Specific occurrence of selenium in enzymes and amino acid tRNAs; Stadtman TC; In contrast to the widespread ability of bacteria, plants, and animals to incorporate selenium nonspecifically into proteins in the form of selenomethionine residues, the selenoamino acid selenocysteine occurs as a highly specific component of a few selenium-dependent enzymes . Selenocysteine has been identified in glycine reductase, formate dehydrogenase, and hydrogenase of bacterial origin and glutathione peroxidase from mammalian and avian sources . In these enzymes there is evidence that the selenol group, which is largely ionized at physiological pH, functions as a redox center . It now seems clear, from studies with both prokaryotes and eukaryotes, that the UGA opal stop codon is used to specify the cotranslational insertion of selenocysteine into proteins . The factors that allow this unusual use of the stop codon are, however, unknown . The occurrence of selenium as a normal constituent of several bacterial tRNA species has been established . The presence of a selenonucleoside, 5-methylaminomethyl-2-selenouridine, in the first or wobble position of the anticodons of certain glutamate and lysine iso-acceptor species influences codon-anticodon interaction and thus may serve to regulate translational processes . The biosynthesis of the selenonucleoside appears to involve the ATP-dependent activation of the sulfur in a preformed 5-methylaminomethyl-2-thiouridine residue in tRNA and replacement of the sulfur with selenium.

J Biol Chem, 1987 Oct 25, 262(30), 14633 - 9
Purification and characterization of RNA polymerase from the cyanobacterium Anabaena 7120; Schneider GJ et al.; A procedure for the purification of RNA polymerase from vegetative cells of the filamentous cyanobacterium Anabaena 7120 is described . Polyethyleneimine precipitation followed by gel filtration and affinity chromatography steps results in greater than 99% purification with 46% yield . The enzyme has a novel core component of Mr = 66,000, designated gamma, in addition to the typical prokaryotic beta'beta alpha 2 core enzyme . The sigma subunit has been identified by reconstitution of specific transcriptional activity from core enzyme and gel-purified sigma . In transcription assays, this RNA polymerase initiates at a number of Anabaena vegetative cell promoters, as well as from a bacteriophage T4 early promoter, but does not initiate at nitrogen fixation (nif) promoters used in heterocysts . The promoter specificity of Anabaena RNA polymerase is compared with that of Escherichia coli RNA polymerase.

Cell, 1987 Oct 23, 51(2), 165 - 73
The tko locus, site of a behavioral mutation in D . melanogaster, codes for a protein homologous to prokaryotic ribosomal protein S12; Royden CS et al.; The tko (technical knockout) mutation is one of a family of behavioral mutations that cause "bang sensitivity" in D . melanogaster . Using P-element-mediated transformation, we show that a 3.1 kb piece of genomic DNA complements tko . This fragment contains only one complete transcript, 0.68 kb in length . This transcript is abundantly expressed through all stages of the life cycle, and we have isolated cDNAs corresponding to this transcript . Their sequence implies a protein product composed of 140 amino acids, which exhibits considerable sequence similarity to ribosomal protein S12 from both Euglena gracilis chloroplasts and E . coli . We suggest that tko codes for a mitochondrial ribosomal protein and that the tko phenotype results from defective mitochondria.

Science, 1987 Oct 23, 238(4826), 530 - 3
Recombinant fragment of protein kinase inhibitor blocks cyclic AMP-dependent gene transcription; Grove JR et al.; Transcriptional regulation by cyclic adenosine monophosphate (cAMP) in mammalian cells could be mediated by a phosphoprotein substrate of the cAMP-dependent protein kinase or, as in prokaryotes, by a cAMP-binding protein . Two synthetic genes that code for an active fragment of the protein inhibitor of this kinase and a mutant inactive fragment were constructed and used to distinguish these alternatives . Transient expression of the active peptide product specifically inhibited the cAMP-stimulated expression of a cotransfected reporter gene by more than 90 percent, whereas the expression of the inactive peptide did not alter cAMP-stimulated gene expression . The results indicate that an active kinase catalytic subunit is a necessary intermediate in the cAMP stimulation of gene transcription.

J Theor Biol, 1987 Oct 21, 128(4), 457 - 61
A purine-pyrimidine motif verifying an identical presence in almost all gene taxonomic groups; Arques DG et al.; A statistical parameter identifies, with a high degree of significance, a motif which is present in protein-coding sequences of eukaryotes, prokaryotes, chloroplasts, mitochondria, viral introns, ribosomal RNA genes, and transfer RNA genes . The random probability of occurrence of such a situation is 10(-12) . This motif has the following properties: (i) its significant presence in almost all present-day genes explains why it can be considered as primitive oligonucleotide, (ii) its nucleotide order is: YRY (N)6YRY, R being a purine base, Y a pyrimidine one and N any base, (iii) its length and its terminal trinucleotides YRY suggest a primordial function related to the spatial structure of the DNA sequences . This motif is found in some viral protein-coding genes, but not in eukaryotic introns.

Eur J Biochem, 1987 Oct 15, 168(2), 295 - 300
Chlorophyll-protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b; Bullerjahn GS et al.; The chlorophyll-protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions . Five complexes, CP1-CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods . CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm . CP4 contains the major chlorophyll-a-binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross-reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6 . Furthermore, fluorescence excitation studies at 77 K indicated that only a Chl a is bound to CP4 . Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20 degrees C and fluorescence excitation spectra at 77 K . CP2, CP3 and CP5 all contain polypeptides of 30-33 kDa which are immunologically distinct from the LHC-II complex of higher plant thylakoids.

Cancer Res, 1987 Oct 15, 47(20), 5249 - 55
Heat shock proteins in thermotolerance and other cellular processes; Carper SW et al.; Heat shock proteins appear to be causatively involved in the acquisition of thermotolerance in prokaryotes but not in eukaryotes . Further, the enhanced synthesis of hsps may be necessary for some cellular responses to stress but not others . In prokaryotic cells the development of thermotolerance, as measured by cell survival, is dependent upon protein synthesis . However, in eukaryotes, enhanced hsp synthesis following an inducing stress and prior to a subsequent heat shock is neither necessary nor sufficient for the development of thermotolerance as measured by colony-forming assays . The enhanced expression of hsps may be required for some mammalian cellular stress responses, such as the ability to reform both actin microfilament bundles and nucleolar morphology . These latter two thermotolerant responses have not been correlated with colony-forming ability . Future work should address the relationships between these various physiological responses to stress and determine if hsps function in some repair mode with regard to colony formation responses . Evidence is accumulating that hsps or their cognates may function in growth and differentiation in some manner as yet to be fully explained . Recent studies indicate that genes controlling cell division in E . coli may be linked to those of several stress regulons, and it would not be surprising to find a similar relationship in eukaryotes . At this time, it is important that studies investigating the role of hsps in stress and other cellular responses such as growth and differentiation define the specific gene (including its regulatory sequences) that encodes the protein being investigated, in order to avoid apparently contradictory and confusing reports of hsps expression.

J Biol Chem, 1987 Oct 15, 262(29), 14105 - 11
DNA ligase from Drosophila melanogaster embryos . Substrate specificity and mechanism of action; Rabin BA et al.; DNA ligase has been purified to homogeneity from 6-12 h Drosophila melanogaster embryos (Rabin, B . A., Hawley, R . S., and Chase, J . W . (1986) J . Biol . Chem . 261, 10637-10645) . This enzyme had an apparent Km for ATP of 1.6 microM . Of a variety of nucleotides tested, only adenosine 5'-O-(3-thio)triphosphate could substitute for ATP in the joining reaction . The enzyme was competitively inhibited by dATP, with an apparent Ki of 2.3 microM . The apparent Km for DNA using p(dT)20 annealed with poly(dA) as substrate was 1.0 microM . Studies utilizing synthetic homopolymers showed that in addition to joining DNA to DNA, this enzyme could join the 5'-phosphoryl termini of RNA to the 3'-hydroxyl termini of DNA or RNA, when they were annealed with DNA . In addition, p(dT)7U could be joined when annealed with poly(dA) . No joining was detected when RNA served as the template . Drosophila DNA ligase also catalyzed the joining of oligonucleotides containing a single mismatched nucleotide at their 3'-hydroxyl termini, as well as DNA containing short, complementary 5'-protruding ends, and in the presence of polyethylene glycol 6000, blunt-ended duplex DNA . The overall reaction mechanism was shown to be identical to that of the homologous prokaryotic DNA ligases . The joining reactions catalyzed by the Drosophila and T4 DNA ligases were shown to be reversible . Incubation of superhelical closed circular DNA molecules with the purified enzymes and AMP resulted in the production of a population of DNA molecules which had lost most, if not all, of their superhelical density.

Nucleic Acids Res, 1987 Oct 12, 15(19), 7831 - 48
Mismatch and blunt to protruding-end joining by DNA ligases; Wiaderkiewicz R et al.; A nuclear DNA ligase activity from immature chicken erythrocytes, and to a lesser extent T4-induced DNA ligase, can join cohesive-ends (3 and 5-nucleotides long) having one of the mismatches, A/A, T/T, C/C, G/G, at the middle position . The rate of ligation depends on the length and stability of the mispaired intermediate (G/G, T/T greater than A/A, C/C) . When the non-complementary overhanging-ends are short (i.e . 1-nucleotide) both ligases catalyze the joining of the single-stranded protruding-end with a blunt-end . This reaction occurs at low but significant rates compared to blunt-end ligation . The chicken ligase has lower flush-end joining activity than T4 DNA ligase, but it is more permissive since it joins C/C or A/A mismatched-ends, whereas the prokaryotic ligase does not . Possible biological implications of the reactions are discussed . We have also found that BstEII easily cleaves at sites harboring a C/C or a G/G mismatch at the center of its recognition sequence, whereas AvaII (T/T or A/A), HinfI (G/G) and DdeI (G/G) do not.

EMBO J, 1987 Oct, 6(10), 3125 - 31
Restoration of u.v.-induced excision repair in Xeroderma D cells transfected with the denV gene of bacteriophage T4; Arrand JE et al.; The heritable DNA repair defect in human Xeroderma D cells, which results in failure to incise at u.v . light-induced pyrimidine dimers, has been partially but stably corrected by transfection of immortalised cells with the denV pyrimidine dimer glycosylase gene of bacteriophage T4 . Transfectants selected either for a dominant marker on the mammalian vector carrying the prokaryotic gene or for the dominant marker plus resistance to killing by u.v . light, have been shown to express the denV gene to varying degrees . denV expression results in significant phenotypic change in the initially repair-deficient, u.v.-hypersensitive cells . Increased resistance to u.v . light and more rapid recovery of replicative DNA synthesis following u.v . irradiation have been correlated both with improved repair DNA synthesis and with a novel dimer incision capability present in denV transfected Xeroderma cells but not as evident in transfected normal cells . Most of the transfectants contain a single integrated copy of the denV gene; increase in denV copy number does not result in either increased gene expression or enhanced survival to u.v . light . These results show that expression of a heterologous prokaryotic repair gene can partially compensate for the genetic defect in a human Xeroderma D cell.

Blood, 1987 Oct, 70(4), 1124 - 30
Immunohistochemical characterization of a 183 KD myeloid-specific-DNA-binding protein in B5 fixed, paraffin-embedded tissues, and bone marrow aspirates by monoclonal antibody BM-1; Epstein AL et al.; A monoclonal antibody, designated BM-1, which is reactive in B5 formalin-fixed, paraffin-embedded tissues, has been generated against a cytoplasmic and nuclear antigen expressed in human myeloid precursor cells and derived leukemias . Using the avidin-biotin-complex immunoperoxidase procedure, BM-1 was found to stain selectively myeloid precursor cells in normal bone marrow and mature granulocytes in the blood . In a screen of 26 normal adult and fetal human organs fixed in B5 formalin, BM-1 was negative in all nonhematopoietic tissues with the exception of tissue granulocytes and scattered cells in the peripheral cortex of the thymus . Likewise a screen of 30 solid tumor cell lines including a spectrum of carcinomas, sarcomas, and neural-derived tumors was negative . BM-1 was also negative with 21 T and B cell lymphomas and 11 Hodgkin's disease tumors . A preliminary study of tumors of the hematopoietic system revealed that BM-1 was reactive with M2 and M3 acute myelogenous leukemias (AML), chronic myelogenous leukemias (CML) and myelomonocytic leukemias, and granulocytic sarcomas . M1, M4, M5, and M6 AML clot preparations were negative in this study, indicating that BM-1 may have a role in the histopathologic diagnosis of myelogenous leukemia . Myeloid leukemic cell lines HL-60, ML-2, KG1, and TPH-1-O showed BM-1 nuclear and/or cytoplasmic reactivity in a subpopulation of cells, but erythroid and lymphoid leukemias and all lymphoma cell lines were negative . Immunoperoxidase studies of a panel of fetal tissues showed BM-1 positive cells in the peripheral cortex of the thymus and portal myelopoietic regions of the liver at 18 weeks gestation . Finally, DNA-cellulose and solid phase radioimmunoassay (RIA) techniques developed in our laboratory demonstrate that the BM-1 antigenic domain is reactive only after binding to eukaryotic but not prokaryotic single- or double-stranded DNA . Immunoblot techniques using a DNA-cellulose purified protein sample revealed that BM-1 recognizes a 183 kD protein . These studies indicate that BM-1 is recognizing a myeloid-specific antigen that, because of its DNA binding characteristics, may have an important role in the differentiation of myeloid cells at the molecular level.

EMBO J, 1987 Oct, 6(10), 3027 - 34
The bidirectional upstream element of the adenovirus-2 major late promoter binds a single monomeric molecule of the upstream factor; Lennard AC et al.; The adenovirus-2 major late promoter (Ad2MLP) upstream element (Ad2MLP-UE) contains a sequence of interrupted dyad symmetry . By inverting this element we have found that it functions in a bidirectional manner both in vivo and in vitro . Footprinting and binding kinetics studies have demonstrated that both orientations of the upstream element bind the sequence-specific upstream factor (UEF) in a similar fashion . These data strongly suggest that the dyad symmetric sequence is sufficient for fully functional binding of the UEF . Binding studies of the UEF to the Ad2MLP-UE indicate that, contrary to prokaryotic palindromic promoter elements which bind multimers of specific factors, the entire Ad2MLP dyad symmetric upstream element binds a single monomeric UEF molecule.

Proc Natl Acad Sci U S A, 1987 Oct, 84(20), 7024 - 7
Supercoiling of the DNA template during transcription; Liu LF et al.; Transcription of a right-handed double-helical DNA requires a relative rotation of the RNA polymerase and its nascent RNA around the DNA . We describe conditions under which the resistance to the rotational motion of the transcription ensemble around the DNA can be large . In such cases, the advancing polymerase generates positive supercoils in the DNA template ahead of it and negative supercoils behind it . Mutual annihilation of the positively and negatively supercoiled regions may be prevented by anchoring points on the DNA to a large structure, or, in the case of an unanchored plasmid, by the presence of two oppositely oriented transcription units . In prokaryotes, DNA topoisomerase I preferentially removes negative supercoils and DNA gyrase (topoisomerase II) removes positive ones . Our model thus provides an explanation for the experimentally observed high degree of negative or positive supercoiling of intracellular pBR322 DNA when DNA topoisomerase I or gyrase is respectively inhibited . We discuss the implications of our model in terms of supercoiling regulation, DNA conformational transitions, and gene regulation in both prokaryotes and eukaryotes.

EMBO J, 1987 Oct, 6(10), 3133 - 7
AIDS virus reverse transcriptase defined by high level expression in Escherichia coli; Larder B et al.; The causative agent of AIDS the human immunodeficiency virus (HIV) encodes as part of its pol gene a reverse transcriptase (RT) which has a key role in the replication of the virus and thus constitutes an ideal target for antiviral chemotherapy . The purified HIV RT from virus particles consists of two related polypeptides of 66 and 51 kd mol . wt and similar polypeptides are found on expression of the complete HIV pol gene using prokaryotic systems . Here we describe the expression of the 66-kd protein in Escherichia coli and demonstrate that this polypeptide alone has authentic RT activity . Thus, a central HIV pol gene segment encodes and is sufficient for high levels of RT activity . The RT has been purified from E . coli extracts using a purification procedure involving two chromotography steps resulting in an enzyme preparation near homogeneity . Deletion of the C-terminal region of the RT thought to encode the RNase H domain resulted in loss of polymerase activity.

J Biol Chem, 1987 Sep 15, 262(26), 12879 - 86
The primary structure of rat ribosomal protein L5 . A comparison of the sequence of amino acids in the proteins that interact with 5 S rRNA; Chan YL et al.; The covalent structure of rat ribosomal protein L5, which associates with 5 S rRNA in the organelle, was deduced from the sequence of nucleotides in a recombinant cDNA (pL5-6-4) and confirmed from the sequences of amino acids in portions of the protein . Ribosomal protein L5, encoded by pL5-6-4, contains 296 amino acids and has a molecular weight of 34,298 . However, a second recombinant cDNA, pL5-8-5, encodes a protein with an additional methionyl residue at position 236 and may be the product of a second active L5 gene . Rat L5 is homologous to yeast YL3 and to Halobacterium cutirubrum HL13, proteins that also bind to 5 S rRNA . No significant structural similarity, however, was found between rat L5 and other 5 S rRNA-binding proteins; not with a second H . cutirubrum protein HL19, nor the Escherichia coli ribosomal proteins, L5, L18, or L25, nor the Xenopus laevis transcription factor IIIA . H . cutirubrum HL19, however, has structural identity with E . coli L5 and seems to be related to yeast YL3 and, hence, may be an evolutionary link between the prokaryotic and eukaryotic 5 S rRNA-binding proteins . A group of ribosomal proteins not known to be associated with 5 S rRNA are also related to rat L5 . They include: rat L39, Euglina gracilis chloroplast S7, Saccharomyces cerevisiae L31 and L46, Homo sapiens L32 and, perhaps, several others as well . There is an especially close interrelationship between rat L5, rat L39, yeast L46, human L32, and mouse L32 . These results, and others, suggest that ribosomal proteins form an extended family and that L5 may contain in its structure traces of this affinity.

J Biol Chem, 1987 Sep 15, 262(26), 12570 - 4
Maintenance of intracellular calcium in Escherichia coli; Gangola P et al.; Recently a series of fluorescent calcium indicator dyes have been developed for measurement of free intracellular calcium in eukaryotic cells . Here we report the use of one such dye, fura-2, for the study of intracellular calcium levels in the prokaryote Escherichia coli . Cells of E . coli were loaded with the membrane-permeable acetoxymethyl ester of fura-2, which was cleaved intracellularly to give the free pentaacid . The concentration of free {Ca2+}i in unstarved cells was maintained at 90 +/- 10 nM, irrespective of the Ca2+ concentration in the extracellular medium . Cells of a strain lacking the H+-translocating ATPase were depleted of endogenous energy reserves and loaded with calcium . In this strain oxidative phosphorylation is uncoupled, so ATP is not produced by respiration . In starved cells {Ca2+}i varied from 0.2 to 0.7 microM when the loading Ca2+ concentration varied from 10 microM to 10 mM . Addition of glucose lowered the Ca2+ levels to 90 nM . Addition of respiratory substrates as energy donors produced cyanide-sensitive efflux . Total cell Ca2+ increased in parallel to the extracellular calcium, but the pool of free calcium did not equilibrate with the total cellular pool . These results demonstrate that 1) the pool of total Ca2+ in the bacterial cell is large and responds to extracellular calcium, 2) the free {Ca2+}i is independent of extracellular calcium, and 3) energy in the form of a proton motive force is required for maintenance of the free intracellular pool of calcium.

Nature, 1987 Sep 3-9, 329(6134), 84 - 5
A bacterial calcium-binding protein homologous to calmodulin; Swan DG et al.; Many of the effects of calcium ions in eukaryotic cells are mediated by calcium-binding regulatory proteins such as calmodulin, in which each calcium-binding site has a distinctive helix-loop-helix conformation termed the EF hand . Protein S from the spore coat of the Gram-negative bacterium Myxococcus xanthus has been shown to resemble calmodulin in its internally-duplicated structure and ability to bind calcium . However, it has a beta-sheet secondary structure rather than the helix-loop-helix arrangement of the eukaryotic proteins . We have determined the complete amino-acid sequence of a calcium-binding protein from the Gram-positive bacterium "Streptomyces erythraeus" by cloning and sequencing the corresponding gene . It contains four EF-hand motifs bearing remarkable sequence similarity to the calcium-binding sites in calmodulin . This implies that the EF-hand super-family may have evolved from ancient proteins present in prokaryotes.

Nature, 1987 Sep 3-9, 329(6134), 79 - 81
Association of DNA-bound progesterone receptors; Theveny B et al.; Steroid hormone-receptor complexes regulate the transcription of specific genes . Recent studies of high-affinity interactions between the receptors and discrete regions of DNA, together with gene-transfer experiments, have led to the precise mapping of hormone regulatory elements . Nothing is known, however, about the mechanisms whereby DNA-bound receptors modulate gene transcription . At the start of transcription in prokaryotes two oligomeric molecules of several regulatory proteins must bind to two specific DNA sites and interact with one another to regulate the binding of RNA polymerase to DNA . Using electron microscopy to observe progesterone receptor binding to regulatory regions of uteroglobin and mouse mammary tumour virus genes, we demonstrate a similar binding between receptor oligomers at two DNA sites . DNA loops are formed when the hormone regulatory elements are at a distance from one another . Thus, in common with certain prokaryotic systems, protein-protein interactions may be important in steroid hormone regulation of gene transcription.

Carcinogenesis, 1987 Sep, 8(9), 1333 - 6
Enhanced repair of pyrimidine dimers in coding and non-coding genomic sequences in CHO cells expressing a prokaryotic DNA repair gene; Bohr VA et al.; We have previously demonstrated that the active dihydrofolate reductase (DHFR) gene is efficiently repaired in Chinese hamster ovary (CHO) cells which remove only a small fraction of u.v.-induced pyrimidine dimers from the overall genome . Preferential DNA repair of essential genes may explain why the u.v . resistance of normal CHO cells is as high as that of fully repair-proficient normal human cells . In this report, we have studied the removal of pyrimidine dimers in a CHO cell line expressing the cloned denV gene from bacteriophage T4 which codes for the pyrimidine dimer specific enzyme T4 endonuclease V (T4 endo V) . This cell line was derived from a u.v.-sensitive excision deficient mutant of a CHO wild type line by transformation with the denV gene, and partial restoration of u.v . resistance was achieved . We have examined an important aspect of the u.v . excision repair in these denV+ cells by studying the repair efficiencies in the active DHFR gene and in a non-coding sequence located downstream from it . In the u.v.-sensitive CHO mutant cell line from which the denV+ was derived, we detected no pyrimidine dimer removal from the gene or from the downstream sequence after irradiation of the cells with 20 J/m2 u.v . (254 nm) light . In the wild type CHO cells, approximately 50% of the pyrimidine dimers were removed from a sequence in the DHFR gene within 8 h, whereas none were removed from the downstream sequence in that period . This represents the normal pattern of preferential DNA repair of active genes, which we have described in previous communications . In the denV+ cells, approximately 70% of the pyrimidine dimers were removed from both the DHFR gene and from the downstream sequence; these cells thus repair both coding and non-coding regions of the genome and show no pattern of preferential repair . The endogenous activity that initiates excision repair in normal CHO cells is evidently much more restricted in its accessibility to DNA lesions in chromatin than is the activity in cells containing substantial amounts of the small T4 endo V enzyme.

Mutat Res, 1987 Sep, 180(1), 43 - 53
Chemical and biological characterization of hazardous industrial waste . II . Eukaryotic bioassay of a wood-preserving bottom sediment; Donnelly KC et al.; The eukaryotic haploid and diploid forms of Aspergillus nidulans were used to detect gene mutations and various types of chromosome damage, respectively, in the acid, base and neutral fractions of a wood-preserving bottom sediment . The corresponding response to prokaryotic mutagenicity assays and major chemical constituents of the 3 waste fractions were described by Donnelly et al . (1987) . The haploid methionine system detected genotoxic compounds in all 3 primary waste fractions without metabolic activation . With metabolic activation, the maximum response observed in the gene mutation assay was induced by the base fraction . In the diploid assay without metabolic activation, the acid fraction induced the maximum number of major chromosome abnormalities, while the base fraction induced the maximum number of minor deletions or insertions . These results appear to reflect the different composition of the waste fractions since each fraction induced a different type of genetic damage in the two bioassays employed . Alternately, because exposure in the diploid assay was during a growth stage, the results may reflect a varying response at different points of the cell division cycle . The results obtained using eukaryotic bioassays indicate that the wood preserving waste contains compound(s) capable of inducing point mutations, chromosome damage, recombination, and compound(s) acting as spindle poisons.

Mutat Res, 1987 Sep, 180(1), 31 - 42
Chemical and biological characterization of hazardous industrial waste . I . Prokaryotic bioassays and chemical analysis of a wood-preserving bottom-sediment waste; Donnelly KC et al.; Prokaryotic bioassays, capable of detecting point mutations and lethal damage to DNA, and a GC/MS/Data System analysis were employed to evaluate the genotoxic characteristics of wood-preserving bottom sediment . Organic compounds in the waste were initially extracted with dichloromethane and then fractionated by liquid-liquid extraction into acid, base and neutral fractions . The crude extract and each of 3 subfractions were tested in 4 strains of S . typhimurium to detect point mutations and 6 strains of B subtilis to detect lethal damage to DNA . The assay using S . typhimurium responded to indirect-acting mutagens in the crude extract and all 3 primary fractions, with the maximum mutagenic response of 181 net revertants induced by the base fraction at a dose of 500 micrograms/plate . In the DNA-repair assay, the survival ratio for the repair-deficient strain recE4 when compared to the repair-proficient strain 168 wt was 0.17 and 0.09 in the acid and base fractions, respectively, at a dose of 100 micrograms/plate . Potentially genotoxic compounds identified in the waste fractions by GG/MS/DS analysis include acenaphthylene, pentachlorophenol, methyl phenanthrene, fluoranthene and pyrene . However, it appears that these identified chemicals did not contribute significantly to the observed mutagenic activity of the sample extracts.

EMBO J, 1987 Sep, 6(9), 2849 - 53
Construction of hybrid Tn501/Tn21 transposases in vivo: identification of a region of transposase conferring specificity of recognition of the 38-bp terminal inverted repeats; Evans LR et al.; In order to study the transposase enzymes of Class II prokaryotic transposable elements, we have constructed genes encoding hybrid transposase proteins . This was done by recombination in vivo between the tnpA genes of transposons Tn501 and Tn21 . These hybrid genes can complement in trans a transposition-defective mutant of Tn501 . The structures of the products of this complementation indicate whether the specificity of the hybrid transposase in recognising the 38 bp terminal inverted repeats is that of Tn501 or that of Tn21 . The determinant of this specificity is in the N-terminal region of the transposase protein, between amino acids 28 and 216 . The predicted amino acid sequences so far determined of transposases from the Class II family reveal an area of homology in this region.

Br J Cancer, 1987 Sep, 56(3), 245 - 50
Detection of human papillomavirus DNA in biopsies of human oral tissue; Maitland NJ et al.; We have employed molecular probes produced from DNA fragments of human papillomavirus, cloned into prokaryotic vectors, to detect virus nucleic acid sequences in extracts of human oral tissues . The study was conducted with duplicate coded snap-frozen tissue biopsies from which frozen sections had been taken to accurately assess the pathology of each particular sample . The results show that a large proportion of the oral biopsies contained DNA which hybridized to the viral DNA probes, even under conditions of high stringency . The presence of virus did not correlate with neoplasia in the tissues examined, but HPV like sequences were found in a high proportion (80%) of biopsies taken from areas of keratosis and lichen planus and also in 41 to 46% of normal and tumour tissues.

J Cell Physiol, 1987 Sep, 132(3), 552 - 8
Identification of an enhancer-like element upstream from a cell cycle dependent human H4 histone gene; Helms SR et al.; We have identified a segment of DNA in the region 6,500 nucleotides upstream from a cell-cycle-dependent human H4 histone gene (pF0108A) which exhibits properties of an enhancer element . This distal element is not required for cap site initiation from the F0108A H4 histone gene . When the enhancer element is present in the genome as a stable integrated sequence, either in its natural upstream location or in a construct where the element is moved just upstream from the proximal promoter sequences, a 25-fold increase in the level of human H4 histone RNAs is observed . This increased level of mRNA reflects an increase in the rate of transcription . The enhancer effect is also observed when the distal element is inserted in inverse orientation with respect to this gene . In addition, the far upstream element can increase expression of a prokaryotic chloramphenicol acetyl transferase (CAT) gene under control of the simian virus 40 (SV40) early promotor, indicating that the ability to influence transcription is not confined to the gene with which it is normally associated . The ability of the histone gene distal enhancer element to function in both mouse and human cells indicates that transacting regulatory factors encoded by either the human or murine genome are capable of mediating the functional properties of this element, further supporting the cross-species compatibility of regulatory sequences and molecules that influence transcription of human histone genes.

Virology, 1987 Sep, 160(1), 151 - 61
Identification of an Epstein-Barr virus early gene encoding a second component of the restricted early antigen complex; Pearson GR et al.; When the latent Epstein-Barr virus (EBV) genome in B95-8 cells is induced into a replicative phase, two abundant early RNAs are transcribed rightward from the EBV BamHI H DNA fragment into BamHI F . Analysis of cDNA clones prepared from the RNA of cells replicating EBV revealed that both RNAs contain the BHRF1 open reading frame . Part of BHRF1, cloned into a prokaryotic fusion protein expression vector, expressed a fusion protein in Escherichia coli and the purified fusion protein was used to generate a monoclonal antibody against BHRF1 . This antibody was then employed to characterize the protein encoded by BHRF1 in cells replicating EBV . The monoclonal antibody reacted with a 17-kDa protein component of the restricted early antigen (EA) complex . The distribution of the protein in cells was similar to that noted when sera from patients with African Burkitt's lymphoma were used to stain these cells . The protein was synthesized before the major 47-56 kDa protein associated with the diffuse component of EA in superinfected Raji cells . All human sera containing antibodies to EA as determined by immunofluorescence (IF) reacted with the protein as did some sera determined to be anti-VCA positive and anti-EA negative by IF . The predicted amino acid sequence of the protein has characteristics which suggest that it is a membrane protein . It also has significant homology with both the anchor region of polyoma middle T antigen and with the predicted protein product of the bcl-2 mRNA activated by the 14/18 chromosome translocation characteristic of follicular lymphomas . This latter homology is extensive and colinear, suggesting common evolution and function . However, neither a mRNA which could efficiently translate the BHRF1 protein nor the BHRF1 protein could be detected in latently infected cells . Thus, the bcl-2 predicted protein is similar to an EBV protein synthesized in the early phase of virus infection.

Proc Natl Acad Sci U S A, 1987 Sep, 84(18), 6486 - 90
Early genes that were oligomeric repeats generated a number of divergent domains on their own; Ohno S; One of the more popular concepts to emerge in recent years is that new proteins evolved by domain exchanges between preexisting proteins . The presence of introns within eukaryotic genes is thought to enhance such exchanges . Yet domain exchanges must necessarily be the secondarily developed process in evolution, for they would have been effective only after multitudes of domains came into being . Many of the proteins with functionally divergent domains were established before the division of prokaryotes from eukaryotes; i.e., soon after the creation of life on this earth . I attribute the extreme innovativeness of early coding sequences to their construction; i.e., being repeats of oligomeric units . The rhodopsin family of proteins--with seven hydrophobic, alpha-helical transmembrane domains, four extracellular domains, and four intracytoplasmic domains--indeed arose before the division of prokaryotes from eukaryotes and later gave rise to muscarinic acetylcholine receptor and beta-adrenergic receptor among others . In this paper, I show that the entire coding sequence for porcine muscarinic acetylcholine receptor is still replete with copies of three heptameric units that are very closely related to each other . Original heptameric units are more stringently conserved in parts encoding the seven transmembrane domains, whereas new repeating units are comingled with the old in parts encoding extracellular and intracytoplasmic domains.

Biochemistry, 1987 Aug 25, 26(17), 5471 - 7
Ferric uptake regulation protein acts as a repressor, employing iron (II) as a cofactor to bind the operator of an iron transport operon in Escherichia coli; Bagg A et al.; The Fur (ferric uptake regulation) protein is a negative regulator of the aerobactin operon and of several other siderophore-mediated, high-affinity iron transport systems in Escherichia coli . The purified Fur protein and a plasmid containing a lacZ fusion to the aerobactin operon were used in conjunction with an in vitro coupled transcription/translation system to demonstrate that the Fur protein requires Fe(II) or certain other divalent metals as a cofactor to negatively regulate expression of the aerobactin operon . In a second set of experiments, using a restriction site protection assay, Fur was shown to bind to and block the aerobactin promoter in a metal-dependent fashion . It is concluded that Fur acts as a classical negative repressor that, under in vivo conditions, uses ionic Fe(II) as a corepressor . Our results support the hypothesis {Williams, R.J.P . (1982) FEBS Lett . 140, 3-10} that prokaryotic cells may contain a standing pool of free or loosely bound Fe(II) that is capable of acting in a regulatory capacity.

Experientia, 1987 Aug 15, 43(8), 920 - 2
Fusion polypeptides in gene cloning: potential problems due to conformational alterations at the junction; Querol E et al.; Many eukaryotic genes are cloned in bacterial hosts as fusion polypeptides . Prediction of the secondary structures for some common prokaryotic fusion polypeptides shows that many junction sites correspond to important secondary structures . It is suggested that such structures could affect (hinder, etc.) the conformation or drive the folding of the neighboring eukaryotic counterparts . Thus the prokaryotic junction should be better performed in random coil regions, or short fusion prokaryotic polypeptides should be used.

Biochem J, 1987 Aug 15, 246(1), 115 - 20
The amino acid sequence of the cytochrome c2 from the phototrophic bacterium Rhodopseudomonas globiformis; Ambler RP et al.; The amino acid sequence of the principal soluble cytochrome c from the phototrophic acidophilic bacterium Rhodopseudomonas (or Rhodopila) globiformis was determined . By the criteria of percentage sequence identity and fewness of internal insertions and deletions it is more similar in sequence to some mitochondrial cytochromes c than to any known bacterial cytochrome . The organism does not have any properties that commend it as being particularly similar to postulated prokaryotic precursors of the mitochondrion . We consider that the relatively high degree of sequence similarity is an instance of convergence, and is an example of the limitations that are imposed on attempts to deduce distant evolutionary relationships from sequence information . Detailed evidence for the amino acid sequence of the protein has been deposited as Supplementary Publication SUP 50136 (12 pages) at the British Library Lending Division, Boston Spa, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment {see Biochem . J . (1987) 241, 5}.

Nucleic Acids Res, 1987 Aug 11, 15(15), 5985 - 6005
Restriction endonucleases for pulsed field mapping of bacterial genomes; McClelland M et al.; Fundamental to many bacterial genome mapping strategies currently under development is the need to cleave the genome into a few large DNA fragments that can be resolved by pulsed field gel electrophoresis . Identification of endonucleases that infrequently cut a genome is of key importance in this process . We show that the tetranucleotide CTAG is extremely rare in most bacterial genomes with G+C contents above 45% . As a consequence, most of the sixteen bacterial genomes we have tested are cleaved less than once every 100,000 base pairs by one or more endonucleases that have CTAG in their recognition sequences: Xba I (TCTAGA), Spe I (ACTAGT), Avr II (CCTAGG) and Nhe I (GCTAGC) . Similarly, CCG and CGG are the rarest trinucleotides in many genomes with G+C content of less than 45% . Thus, Sma I (CCCGGG), Rsr II (CGGWCCG), Nae I (GCCGGC) and Sac II (CCGCGG) are often suitable endonucleases for producing fragments that average over 100,000 base pairs from such genomes . Pulsed field gel electrophoresis of the fragments that result from cleavage with endonucleases that cleave only a few times per genome should assist in the physical mapping of many prokaryotic genomes.

J Theor Biol, 1987 Aug 7, 127(3), 301 - 14
Structure and evolution of prokaryotic and eukaryotic RNA polymerases: a model; Armaleo D; A comparative overview of the subunit taxonomy and sequences of eukaryotic and prokaryotic RNA polymerases indicates the presence of a core structure conserved between both sets of enzymes . The differentiation between prokaryotic and eukaryotic polymerases is ascribed to domains and subunits peripheral to the largely conserved central structure . Possible subunit and domain functions are outlined . The core's flexible shape is largely determined by the elongated architecture of the two largest subunits, which can be oriented along the DNA axis with their bulkier amino-terminal head regions looking towards the 3' end of the gene to be transcribed and their more slender carboxyl-terminal domains at the tail end of the enzyme . The two largest prokaryotic subunits appear originally derived from a single gene.

Anal Biochem, 1987 Aug 1, 164(2), 554 - 8
Methods for in situ visualization and assay of sulfurtransferases; Aird BA et al.; The dansyl derivative 5-dimethylamino-1-naphthalene thiosulfonate (DANTS) can serve as a sulfane sulfur-donor substrate for several of the sulfurtransferases, the reaction being dependent on the acceptor substrates supplied . Enzymatic cleavage of the sulfur-sulfur bond of DANTS releases the intrinsic fluorescence of the molecule, with an emission maximum of 500-510 nm (excitation at 325 nm) . This process permits selective visualization of active sulfurtransferase enzymes separated in nondenaturing polyacrylamide gels, even from impure preparations . This technique was used to locate rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1), thiosulfate reductase (EC unassigned), and a recently isolated prokaryotic enzyme that has been called sulfane sulfurtransferase . In addition, a refinement of the thiosulfate reductase assay technique is reported.

Tsitologiia, 1987 Aug, 29(8), 934 - 41
{Detection and identification of Mycoplasma infections by DNA hybridization}; Borkhsenius SN et al.; Infection of cell cultures by mycoplasmas can be detected by hybridization of the DNA of suspected cell cultures with recombinant plasmids containing fragments of the mycoplasma DNA . The test is very sensitive and allows detection of as little as 1 ng of mycoplasmal DNA, roughly equivalent to the DNA amount of 10(6) mycoplasmas . This approach turns out to be effective for detection and identification of mycoplasmas in clinical material, plant and insect tissues . A set of DNA probes for detection of mycoplasmas infecting cell cultures by dot hybridization has been constructed . This set consists of specific DNA probes and universal DNA probe . Recombinant plasmids, pAl32, pMa13, pMh9, containing specific DNA fragments of Acholeplasma-laidlawii, Mycoplasma arginini, Mycoplasma hominis (the prevalent mycoplasma contaminants of home cell cultures) are species-specific DNA probes . Recombinant plasmid pMg16 containing rRNA genes of Mycoplasma gallisepticum is the universal DNA probe for detection of any mycoplasma (or any prokaryote) contaminations . These two classes of DNA probes may be considered as complementing each other . These 32P labeled probes do not hybridize with eukaryotic DNA . The set of DNA probes allows not only to detect infection of cell cultures by mycoplasmas but also to identify the species of mycoplasmas and to evaluate the multiplicity of mycoplasma infection.

Radiat Res, 1987 Aug, 111(2), 254 - 66
Expression of prokaryotic genes after transfection of X-irradiated plasmids into primate cells; Piazza L et al.; Expression of the prokaryotic gene for chloramphenicol acetyltransferase (EC 2.3.1.28) (CAT) in primate cells transfected with X-irradiated plasmid pSV2CAT was determined in transient expression assays . CAT expression did not depend upon the presence of supercoiled plasmids, but relaxed circular forms were essential . X-ray conversion of relaxed circles to linear forms paralleled the loss of CAT expression, with identical D0's in the first part of dose-response curves . X-ray-induced loss of supercoiled forms was complete at much lower doses . The D0 for inactivation of CAT expression by X irradiation of the plasmids in 1 mM Tris buffer was 270 Gy; it was 13 Gy for plasmids irradiated in water . The D0's for conversion of pSV2CAT to relaxed circle forms were only one-seventh as large as the D0's for CAT inactivation after X-ray in water or in 1 mM Tris buffer . Expression of the CAT gene in some representative repair-deficient human fibroblasts transfected with X-irradiated pSV2CAT was less than in monkey CV-1 cells or cell lines from normal human subjects . These results demonstrate a novel means to study low levels of X-ray damage in DNA correlating specific X-ray damage in the DNA with expression of the gene in unirradiated primate cells.

Biochemistry, 1987 Jul 28, 26(15), 4646 - 52
Interaction between adenovirus DNA-binding protein and single-stranded polynucleotides studied by circular dichroism and ultraviolet absorption; van Amerongen H et al.; The adenovirus DNA-binding protein (AdDBP) is a multifunctional protein required for viral DNA replication and control of transcription . We have studied the binding of AdDBP to single-stranded M13 DNA and to the homopolynucleotides poly(rA), poly(dA), and poly(dT) by means of circular dichroism (CD) and optical density (OD) measurements . The binding to all these polynucleotides was strong and nearly stoichiometric . Titration experiments showed that the size of the binding site is 9-11 nucleotides long for M13 DNA, poly(dA), and poly(rA) . A higher value (15.0 +/- 0.8) was found for poly(dT) . Pronounced changes in the circular dichroism and optical density spectra were observed upon binding of AdDBP . In general, both the positive peak around 260-270 nm and the negative peak around 240-250 nm in the CD spectra decreased in intensity, and a shift of the crossover point to longer wavelengths was found . The OD spectra observed upon binding of AdDBP are remarkably similar to those obtained with prokaryotic helix-destabilizing proteins like bacteriophage T4 gene 32 protein and fd gene 5 protein . The data can best be interpreted by assuming that the AdDBP-polynucleotide complex has a regular, rigid, and extended configuration that satifies two criteria: (1) a considerable tilt of the bases in combination with (2) a small rotation per base and/or a shift of the bases closer to the helix axis.

J Mol Biol, 1987 Jul 20, 196(2), 363 - 77
Isolation and structural analysis of the Escherichia coli trp leader paused transcription complex; Landick R et al.; Transcription pausing is a key step in many prokaryotic transcription attenuation mechanisms . Pausing is thought to occur when an RNA hairpin forms near the 3' end of a growing transcript . We report here the isolation of the trp leader paused transcription complex containing a defined 92-nucleotide nascent transcript . Digestion of isolated paused complexes with RNase T1 suggests that the trp leader RNA hairpin designated 1:2 forms in the paused transcription complex . The transcription factor NusA alters the RNase T1 digestion pattern of the 92-nucleotide pause transcript in the complex but not the cleavage patterns of purified pause RNA, suggesting that NusA specifically affects the 1:2 hairpin in the paused transcription complex . The isolated paused transcription complex retains the ability to resume transcription . Kinetic studies on the resumption of elongation suggest that NusA is a non-competitive inhibitor of paused complex release and that the Ks for GTP is around 300 microM . RNA polymerase in the paused transcription complex protects approximately 30 base-pairs on both DNA strands from exonuclease digestion.

Science, 1987 Jul 10, 237(4811), 182 - 4
Variable occurrence of the nrdB intron in the T-even phages suggests intron mobility; Pedersen-Lane J et al.; The bacteriophage T4 nrdB gene, encoding nucleoside diphosphate reductase subunit B, contains a self-splicing group I intervening sequence . The nrdB intron was shown to be absent from the genomes of the closely related T-even phages T2 and T6 . Evidence for variable intron distribution was provided by autocatalytic 32P-guanosine 5'-triphosphate labeling of T-even RNAs, DNA and RNA hybridization analyses, and DNA sequencing studies . The results indicate the nonessential nature of the intron in nrdB expression and phage viability . Furthermore, they suggest that either precise intron loss from T2 and T6 or lateral intron acquisition by T4 occurred since the evolution of these phages from a common ancestor . Intron movement in the course of T-even phage divergence raises provocative questions about the origin of these self-splicing elements in prokaryotes.

Nucleic Acids Res, 1987 Jul 10, 15(13), 5105 - 24
Supercoiling in prokaryotic and eukaryotic DNA: changes in response to topological perturbation of plasmids in E . coli and SV40 in vitro, in nuclei and in CV-1 cells; Esposito F et al.; Changes in DNA linking number have been observed in plasmid DNA purified from E . coli cells after the cells were treated with chloroquine . Chloroquine, a DNA intercalating drug, unwinds the DNA, decreasing the levels of negative supercoiling . Following this in vivo topological perturbation, within minutes DNA gyrase decreases DNA linking number producing more negatively supercoiled DNA topoisomers . Following the removal of the drug from cells, within minutes topoisomerase 1 or DNA gyrase increases the linking number restoring the original level of supercoiling . Analogous changes in DNA linking number after addition of chloroquine are observed in purified plasmid DNA, and in purified SV40 minichromosomes in the presence of exogenous topoisomerase . Changes in linking number are also observed in SV40 chromosomes in isolated nuclei and in SV40 DNA purified from CV-1 cells following topological perturbation with chloroquine . These results suggest that eukaryotic cells may have mechanisms to maintain a defined level of DNA supercoiling.

Nature, 1987 Jul 30-Aug 5, 328(6129), 454 - 6
Giant linear plasmids in Streptomyces which code for antibiotic biosynthesis genes; Kinashi H et al.; A number of examples of circular plasmids with specific functions are known in both prokaryotes and eukaryotes . Several linear plasmids have also been identified, but these are all relatively small: large linear plasmids cannot be separated from chromosomal DNA by conventional techniques . There are several cases where the genetic evidence suggests that a character is encoded by a plasmid but no plasmid can be physically detected . This has been the case for antibiotic synthesis genes in Streptomyces; in particular a plasmid SCP1 in Streptomyces coelicolor has been shown to be involved in methylenomycin production by genetic evidence . We report here the application of orthogonal-field-alternation gel electrophoresis to the isolation of linear plasmids from Streptomyces . We have discovered a large linear plasmid of around 520 kilobases in Streptomyces lasaliensis and subsequently similar giant linear plasmids in other Streptomyces strains . We have confirmed that genes for methylenomycin biosynthesis are located on a series of giant linear plasmids in S . coelicolor . These observations may bear on the genetic variability and unstable genetic character of Streptomyces species.

J Biol Chem, 1987 Jul 5, 262(19), 9098 - 108
Purified RNA polymerase II recognizes specific termination sites during transcription in vitro; Dedrick RL et al.; We have studied the ability of certain well-defined prokaryotic DNA sequences to act as specific termination signals for highly purified calf thymus RNA polymerase II . We used duplex DNA fragments modified to direct efficient and specific transcription of defined DNA templates to follow transcription with RNA polymerase alone in the absence of additional protein factors . Elongation of RNA chains by RNA polymerase II is processive through most DNA sequences . However, certain DNA sequences serve as effective "intrinsic" terminators for RNA polymerase II; in this they resemble the "rho-independent" terminators for the bacterial RNA polymerase . Several rho-independent bacterial terminators are also able to act as termination signals for RNA polymerase II . However, there is no apparent correlation between the efficiency of termination for the bacterial enzyme and that found for the calf thymus enzyme . One very efficient bacterial terminator (phage T7 early terminator) gives no termination with RNA polymerase II, and we have identified at least two sites that cause the eukaryotic enzyme to terminate but have no effect on transcription by the bacterial enzyme . Hence, the signals recognized as intrinsic termination sites for the two enzymes are substantially different . All of the sites that act as intrinsic terminators for RNA polymerase II contain a series of consecutive thymidine residues in the nontranscribed DNA strand (T-run), and the 3' end of the completed RNA normally lies within this sequence . It is plausible that the T-run is part of the signal for an RNA polymerase II termination site; however, there is no apparent correlation between the number of T residues and the efficiency of the terminator, suggesting that other sequence elements are required for, or modulate, termination . Several lines of evidence suggest that the formation of RNA secondary structures in the nascent transcript is not an essential element of the intrinsic RNA polymerase II termination signal.

Mutat Res, 1987 Jul, 186(1), 1 - 34
The development of ideas about the effect of DNA repair on the induction of gene mutations and chromosomal aberrations by radiation and by chemicals; Kimball RF; An historical overview is given of the development of ideas about chromosomal and DNA repair as they relate to the induction of mutations, chromosomal aberrations, and sister-chromatid exchanges by radiations and chemicals . The genetic and molecular bases of the various repair pathways are reviewed whenever possible . Work on both prokaryotes and eukaryotes is included . Mention is made, when deemed appropriate, of major developments in other areas that served as essential background for the repair work, but no attempt is made to cover these background developments in any detail . Near the end, a brief review is given of factors affecting polymerase fidelity . The history is subdivided into approximately 10-year intervals . For the most part, references are to reviews and symposia in which the ideas of the time were brought together . The implications of these findings for some practical problems in genetic toxicology and for our understanding of the maintenance of the genome are discussed at the end.

J Mol Biol, 1987 Jun 20, 195(4), 909 - 17
Ribosomal proteins EL11 from Escherichia coli and L15 from Saccharomyces cerevisiae bind to the same site in both yeast 26 S and mouse 28 S rRNA; el-Baradi TT et al.; The heterologous interaction of Escherichia coli ribosomal protein EL11 with yeast 26 S and mouse 28 S rRNA was studied by analysing the ability of this protein to form a specific complex with various synthetic rRNA fragments that span the structural equivalent of the EL11 binding site present in these eukaryotic rRNAs . The fragments were obtained by SP6 polymerase-directed in-vitro run-off transcription of parts of the yeast or mouse large rRNA gene cloned behind the SP6 promoter . EL11 was found to protect an oligonucleotide fragment of 63 nucleotides from both the yeast and mouse transcripts against digestion by RNase T1 . In both cases, the position of this fragment in the L-rRNA sequence coincides almost exactly with that of the fragment previously found to be protected by EL11 in E . coli 23 S rRNA . Moreover, the protected yeast fragment was shown to be able to re-bind to EL11 by a nitrocellulose filter binding assay . A ribosomal protein preparation from Saccharomyces cerevisiae containing L15 (YL23) as well as the acidic proteins L44', L44 and L45 protects exactly the same oligonucleotide fragment as does EL11 in both the yeast and mouse transcripts . Evidence is provided that L15, which is known to be structurally and functionally equivalent to EL11, is the rRNA-binding protein in this preparation . Thus the structural equivalent of the EL11 binding site present in yeast 26 S rRNA constitutes the second example of functional conservation of a ribosomal protein-binding site on rRNA between prokaryotes and eukaryotes.

Nature, 1987 Jun 18-24, 327(6123), 638 - 40
Preferential relaxation of supercoiled DNA containing a hexadecameric recognition sequence for topoisomerase I; Busk H et al.; In prokaryotes, the degree of supercoiling of DNA can profoundly influence the use of specific promoters . In eukaryotes, a variety of indirect observations suggest that DNA topology has a similar importance in proper gene expression . Much attention has therefore been focused on the cellular proteins that control DNA supercoiling, among which are the enzymes topoisomerase I and II . A hexadecameric sequence functions as a strong attraction site for topoisomerase I . Here we report that the interaction of topoisomerase I with this sequence motif is highly specific, because a single base-pair substitution prevents strand cleavage and thereby catalytic activity at the sequence . Thus, supercoiled DNA containing the recognition sequence is relaxed preferentially by topoisomerase I compared to a control, but no difference in the relaxation rate is observed for supercoiled DNA carrying the mutated sequence . The preference for the recognition sequence seems to be an intrinsic property of all eukaryotic type I topoisomerases, suggesting that the interaction might be important in a fundamental biological process.

J Biol Chem, 1987 Jun 5, 262(16), 7586 - 93
On the metabolic relationships between monogalactosyldiacylglycerol and digalactosyldiacylglycerol molecular species in Dunaliella salina; Cho SH et al.; Dunaliella salina cells were pulse-labeled for 2 min with {14C}palmitic acid, {14C}oleic acid, or {14C}lauric acid in order to trace the pathway of galactolipid biosynthesis and desaturation . Through the use of high performance liquid chromatography it was possible to follow the movement of radioactivity through many individual molecular species of monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) for periods of 24 h and, in some cases, as much as 120 h . Analysis of the fatty acid fluxes permitted us to refine current views regarding biosynthesis of the predominantly "prokaryotic" galactolipids . The initial D . salina MGDG molecular species, containing paired oleate and palmitate (18:1/16:0), can follow two metabolic routes . If the palmitoyl chain is desaturated to 16:1, the resulting 18:1/16:1 MGDG is subject to rapid further desaturation to varying degrees, and a part of these products is subsequently galactosylated to DGDG . Contrary to widely held opinions, these DGDG molecular species can themselves be further desaturated toward a 18:3/16:4 final product . In a separate series of reactions, a smaller portion of the nascent 18:1/16:0 MGDG is directly galactosylated to 18:1/16:0 DGDG . This molecular species can then be sequentially desaturated to 18:2/16:0 DGDG and 18:3/16:0 DGDG . However, there is only very limited desaturation of the palmitoyl group attached to these molecular species.

Zh Mikrobiol Epidemiol Immunobiol, 1987 Jun, (6), 22 - 5
{Role of the lipid peroxidation system of Escherichia coli cells in maintaining their viability in air}; Bogoslovskaia OA et al.; The study of 6 E . coli strains differing in their capacity for survival in the air has revealed that the physicochemical characteristics of lipids in microbial cells, such as antioxidizing activity, the concentration of peroxidation products, the content of lipids and their capacity for oxidation, are interrelated, which confirms the existence of the system regulating the peroxidation of lipids in prokaryotic cells, similar to the system regulating lipid peroxidation in eukaryotic cells . The capacity of cells for survival in the air has been shown to depend on the physicochemical state of lipids in cellular membranes.

Genetics, 1987 Jun, 116(2), 191 - 9
A Tn10-lacZ-kanR-URA3 gene fusion transposon for insertion mutagenesis and fusion analysis of yeast and bacterial genes; Huisman O et al.; We describe here a new variant of transposon Tn10 especially adapted for transposon analysis of cloned yeast genes; it can equally well be used for analysis of prokaryotic genes . We have applied this element to analysis of the LEU2, RAD50, and CDC48 genes of Saccharomyces cerevisiae . This transposon, nicknamed mini-Tn10-LUK, contains a lacZ gene without efficient transcription or translation start signals, an intact URA3 gene, and a kanR determinant . The lacZ gene can be activated by appropriate insertion of the element into an actively expressed gene . Other yeast genes can easily be substituted for URA3 in the available constructs . The mini-Tn10-LUK system has several important advantages . Transposition events occur in Escherichia coli at high frequency and into many different sites in yeast DNA . It is easy to obtain enough insertions to sensitively define the functional limits of a gene . Transposon insertions can be obtained in a single step by standard transposon procedures and can be screened immediately for phenotype either in yeast or in E . coli . The LacZ phenotypes of the insertion mutations provide a good circumstantial indication of the orientation of the target gene . Under favorable circumstances, usable lacZ protein fusions are created . Transposon insertion mutations obtained by this method directly facilitate additional genetic, functional, physical and DNA sequence analysis of the gene or region of interest.

Sci Sin {B}, 1987 Jun, 30(6), 591 - 8
DNA fragments of Bombyx mori nuclear polyhedrosis virus containing the promoters active in prokaryotes; Xin JH et al.; Using a promoter probe plasmid in E . coli called pHE5, eight different HindIII and one SalI DNA fragments of Bombyx mori nuclear polyhedrosis virus, directing the expression of the tetracycline resistance gene, have been cloned and isolated . The tetracycline resistance levels of the strains containing the recombinant plasmids were measured . Among them, the highest level of the resistance to tetracycline was 30 micrograms/ml . Part of the nucleotide sequence of a DNA fragment was determined . A sequence similar to the E . coli promoter was found.

J Biol Chem, 1987 May 15, 262(14), 6564 - 71
Isolation and characterization of a fibronectin receptor from Staphylococcus aureus; Froman G et al.; Attachment of bacteria to the host tissue is considered a first step in the development of many infections . Previous studies have shown that fibronectin, a protein shown to mediate substrate adhesion of eukaryotic cells, also binds to some pathogenic bacteria and mediates the tissue adherence of these prokaryotes . In the present communication, we report on the isolation and characterization of a fibronectin receptor from Staphylococcus aureus strain Newman . A 210-kDa fibronectin binding protein was isolated from a bacterial lysate by affinity chromatography followed by gel chromatography . Additional smaller peptides with fibronectin binding properties were also obtained . These peptides seem to represent degradation products of the large receptor protein since the former dominated when the purification was carried out in the absence of protease inhibitors . Furthermore, degradation of the purified receptor protein by staphylococcal V8 protease generated a large number of peptides that retained fibronectin binding activity . This observation also suggests that the large receptor protein contains several binding sites for fibronectin, and analysis of the binding of the 29-kDa amino-terminal fibronectin fragment to the 210-kDa receptor adsorbed in microtiter wells suggests that one receptor molecule can bind six to nine fibronectin molecules.

J Biol Chem, 1987 May 5, 262(13), 6238 - 47
Partial purification and characterization of an insulin-like material from spinach and Lemna gibba G3; Collier E et al.; The existence in invertebrates, unicellular eukaryotes, and prokaryotes of materials that resemble several vertebrate peptide hormones led to the suggestion that these peptide messengers may have arisen earlier in evolution than had previously been thought . Consistent with this hypothesis, we describe here material in two plants, spinach and Lemma gibba G3, that is very similar to mammalian insulin, yet distinctive . In each of the early purification steps, which consisted of acidic methanol chloroform extraction and sequential chromatography on C-18 hydrophobic resin, Sephadex G-50, CM-Sepharose, and a short C-3 high performance liquid chromatography column, the immunoactive material from plants resembled the common vertebrate insulins . The protein nature of the material was suggested by its destruction by Pronase but not by the inactivated enzyme . In addition, on TSK chromatography it eluted in a position similar to that of insulin, i.e . equivalent to a protein of 6000 daltons . Using an isocratic high performance liquid chromatography system, the plant immunoactivity eluted earlier, and thus was more hydrophilic, than most of the common mammalian insulins, including pork insulin . The interaction of the plant material with anti-insulin antibodies in a radioimmunoassay was confirmed by using an affinity column of anti-insulin antibodies which adsorbed the plant immunoactivity at neutral pH, and released the material with acid elution . Using a quantitative double radioimmunoassay, the plant insulin-like material was distinguished immunologically from chicken insulin . Although the plant insulin-like material is clearly distinct from pork insulin chromatographically, and from chicken insulin immunologically, it resembles vertebrate insulins in its overall configuration . The plant insulin-like material bound to insulin receptors on IM-9 lymphocytes and stimulated glucose oxidation and lipogenesis in isolated adipocytes from young rats . The bioactivity was neutralized in the presence of anti-insulin antibodies, but not in the presence of normal guinea pig IgG . The role of this insulin-like material in plants is unknown but its existence is consistent with an early evolutionary origin of the insulin messenger peptide family . Alternatively we cannot exclude a later convergent development of this family or introduction of vertebrate DNA into plants.

EMBO J, 1987 May, 6(5), 1467 - 73
Membrane fusion in prokaryotes: bacteriophage phi 6 membrane fuses with the Pseudomonas syringae outer membrane; Bamford DH et al.; Protein-triggered membrane fusion in the prokaryotic system is described using the lipid-containing enveloped bacterial virus phi 6 and its host, the Gram-negative bacterium Pseudomonas syringae . Bacteriophage particles can be fused to form multiple particles where two or more nucleocapsids are surrounded by a single membrane vesicle with a volume proportional to the number of fused particles . For fusion to occur, a fusogenic protein is required in the membrane of the participating phage particles . Upon infection of the host cell, fusion of the viral membrane with the bacterial membrane takes place without leakage of the periplasmic enzyme alkaline phosphatase to the extracellular supernatant . There is a time-dependent mixing of fluorescent phage phospholipids with the bacterial membrane lipids between 5 and 20 min post-infection . The phage membrane proteins and phospholipids co-purify with the bacterial outer membrane of infected cells . The fusion is independent of divalent cations and pH, resembling Sendai virus fusion with the plasma membrane . This is the first targeted, protein-dependent fusion event described in prokaryotes.

Virology, 1987 May, 158(1), 206 - 10
Determination of the promoter region of an early vaccinia virus gene encoding thymidine kinase; Weir JP et al.; Nine recombinant vaccinia viruses that contain overlapping segments of the putative promoter region of the vaccinia virus thymidine kinase (TK) gene linked to DNA coding for the prokaryotic enzyme chloramphenicol acetyltransferase (CAT) were constructed . In each case, the RNA start site and 5 bp of DNA downstream were retained . No significant difference in CAT expression occurred as the deletion was extended from 352 to 32 bp before the RNA start site . Deletion of a further 10 bp, however, led to complete cessation of early promoter activity . Primer extension analysis of the 5' ends of the transcripts verified that the natural TK RNA start site was still used when only 32 bp of upstream DNA remained . Loss of early promoter activity was previously found when deletions were extended from 31 to 24 bp before the RNA start site of another vaccinia gene that is expressed constitutively throughout infection (M.A . Cochran, C . Puckett, and B . Moss, 1985, Proc . Natl . Acad . Sci . USA 82, 19-23) . Sequence similarities in the promoter regions of these two genes were noted.

Zh Evol Biokhim Fiziol, 1987 May-Jun, 23(3), 361 - 72
{Origin and evolution of peptide-protein bioregulators}; Chipens GI et al.; Possible evolutionary pathways of cellular regulatory systems are discussed . Analysis of animal evolution suggests that peptide and protein bioregulators emerged at an early stage during formation of biochemical systems in prokaryotic cells involving protein synthesis on ribosomes, the processes of exo- and endocytosis and limited proteolysis reactions . Primary autocrine bioregulators are compared with growth factors . Models for cellular bioregulation are discussed in which both cell receptors and peptide/protein ligands, primarily immunoglobins, act as prehormones . Their internalization and limited proteolysis can lead to formation of low-molecular peptides (tetines) acting as autocrine or paracrine bioregulators . Basing on the concept of biochemical universality, it is suggested that the effects of many growth factors, hormones, immunoglobulins, mono- and lymphokins are mediated by identical or similar (carrying the same signatures) fragments which are produced in cells due to limited proteolysis reactions and which are directly involved in activation of biochemical systems in these cells.

Mol Gen Genet, 1987 May, 207(2-3), 413 - 20
Novel rearrangements of IS30 carrying plasmids leading to the reactivation of gene expression; Dalrymple B; Unusual DNA rearrangements involving the prokaryote mobile genetic element IS30 have been identified . In order to study the potential mechanisms for the reactivation of a gene after IS element insertion, IS30 was introduced between the lacUV5 promoter and the galK gene of the multicopy plasmid pFR100 . In this plasmid terminators of RNA transcription in the sequence of IS30 prevent expression of the galK gene from the lacUV5 promoter . A number of independently isolated mutant plasmids re-expressing the galK gene were studied and shown to be tandemly repeated dimers of the original plasmid . However, the two copies of IS30 were tandemly repeated at one of the original sites of insertion, while at the other site IS30 and three base pairs were missing . The repeated copies of IS30 were separated by two base pairs, the same as those originally flanking the elements . An apparently identical mechanism generated cointegrates between a derivative of plasmid pFD51 carrying IS30 upstream of a promoterless galK gene and a derivative of plasmid pACYC177 carrying IS30 inserted into the beta-lactamase gene . This arrangement brought the galK gene under the control of the beta-lactamase promoter of pACYC177 . A mechanism involving aborted conservative transposition of IS30 is discussed as a possible route for the generation of these novel cointegrates . In a third experiment we isolated an insert of IS30 which was also two base pairs away from an already resident IS30 element . This insertion of IS30 created a strong promoter of RNA transcription, which has the potential to increase the expression of the transposase in the downstream copy of the element.

Proc Natl Acad Sci U S A, 1987 May, 84(10), 3117 - 21
Sequences from a prokaryotic genome or the mouse dihydrofolate reductase gene can restore the import of a truncated precursor protein into yeast mitochondria; Baker A et al.; Sequences that are capable of restoring mitochondrial targeting to a truncated yeast cytochrome c oxidase subunit IV presequence are encoded within the genome of Escherichia coli and within the gene for a higher eukaryotic cytosolic protein, mouse dihydrofolate reductase . These sequences, which resemble authentic presequences in their overall amino acid composition and degree of hydrophobicity, are rather frequent; greater than 2.7% of clones generated from E . coli DNA and greater than 5% of clones from the dihydrofolate reductase gene were functional in our screening system . These results suggest that, during evolution, mitochondrial precursor proteins could arise as a result of DNA rearrangements that place potential mitochondrial presequences at the amino terminus of existing open reading frames . Primitive eukaryotic cells may have used this mechanism to target proteins to their endosymbiotic protomitochondria.

J Gen Virol, 1987 May, 68 ( Pt 5), 1449 - 55
Varicella-zoster virus specifies a thymidylate synthetase; Thompson R et al.; A homology search of proteins predicted from the recently reported complete DNA sequence of varicella-zoster virus (VZV) revealed that the product of gene 13 was highly homologous to eukaryotic and prokaryotic thymidylate synthetases (TSs) . The VZV protein was shown to be a TS by three functional tests . Firstly, a plasmid designed to express the native protein was able to complement a strain of Escherichia coli in which the natural TS gene is deleted . Secondly, in an enzyme assay for TS, extracts of the complemented strain were capable of releasing tritiated water from 2'-deoxy{5-3H}uridylate . Thirdly, these extracts contained a protein that bound isotopically labelled 5-fluoro-2'-deoxyuridylate, a ligand specific for the active site of TS . In addition, a novel ligand-binding protein was detected in human cells infected with VZV.

J Gen Virol, 1987 May, 68 ( Pt 5), 1429 - 33
The herpes simplex virus type 1 DNA polymerase gene: site of phosphonoacetic acid resistance mutation in strain Angelotti is highly conserved; Knopf CW; By comparative sequence analysis of the herpes simplex virus type 1 DNA polymerase gene of strain Angelotti and a phosphonoacetic acid-resistant (PAAr) derivative, the site of the PAAr mutation was identified as a single nucleotide (C----T) conversion within the mapping limits of the known PAAr mutations of strains KOS and 17 . The conservative amino acid change at residue 719 from alanine to valine results in a radical change in the properties of the polymerase, rendering the mutant enzyme resistant to PAA and various antiviral compounds . Amino acid homologies as well as secondary structure analysis reveal that the PAAr mutation is contained in a 14 amino acid sequence which is highly conserved, and detected in the central domain of prokaryotic and eukaryotic DNA polymerases.

Mol Biol (Mosk), 1987 May-Jun, 21(3), 831 - 6
{Construction and use of phasmid vectors of large capacity}; Iankovskii NK et al.; lambda vector phages--pUC19 phasmid hybrids were constructed . The hybrids, phasmids lambda pMYF131 and lambda pSL51, were used as vector molecules for making genomic libraries . Vector phasmids exist as plasmids in vivo . They contain all the genes and specific sites necessary for lytic development, but the DNA molecules are not long enough to be packaged in lambda capsid . Elongation of the molecule due to insertion of a foreign DNA fragment renders phasmid all features of non-defective phage . Output of recombinants is up to 3 X 10(6) per 1 microgram of phasmid vector DNA . The fraction of non-recombinants in libraries is less than 1-0.1% . The capacity of the vectors is 19.6 and 22 kb for lambda pMYE131 and lambda pSL51 accordingly . It is possible to clone DNA fragments with blunt ends and various sticky-end fragments obtained by digestion with 14 prototype restrictases, into nine unique restriction sites of vector lambda pMYF131 . The created vectors allowed to construct more than 30 representative genomic libraries of eukaryotic and prokaryotic organisms . 13 individual genes were identified within the libraries.

J Biol Chem, 1987 Apr 25, 262(12), 5546 - 53
Imbalanced deoxyribonucleoside triphosphate pools and spontaneous mutation rates determined during dCMP deaminase-defective bacteriophage T4 infections; Sargent RG et al.; DNA precursor imbalances are known to be mutagenic in both eukaryotic and prokaryotic systems . Almost certainly, such mutagenesis involves competition between correctly and incorrectly base-paired precursors at replication sites . Since other factors may be involved, it is important to identify specific mutations induced by specific pool imbalances . Using bacteriophage T4, we have developed a system for such analysis . We prepare double mutants of T4; one mutation affects a phage-coded enzyme of deoxyribonucleoside triphosphate (dNTP) metabolism, while the second is an rII mutation known to revert along a specific pathway . We determine dNTP pools in infection by such a mutant and measure both the spontaneous reversion rate of the rII mutation and, in some cases, the nucleotide sequence at the mutant site . In this paper we analyze mutations induced by a deficiency of T4-encoded deoxycytidylate deaminase . This causes pools of 5-hydroxymethyl-dCTP to expand some 30-fold, while dTTP pools contract . This specifically stimulates AT-to-GC reversion . One of the four AT-to-GC reverters tested, rIIUV215, increases its reversion rate at least 1000-fold under these pool-imbalance conditions, while the other mutants tested show increases of only about 10-fold . Therefore, factors other than dNTP competition, including local DNA sequence environment, must be invoked to fully explain mechanisms of dNTP pool imbalance-induced mutagenesis . We discuss models for this, and we also report unexpected effects of the dCMP deaminase deficiency upon pools of ribonucleoside triphosphates.

Nucleic Acids Res, 1987 Apr 10, 15(7), 3155 - 75
DNA synthesis arrest sites at the right terminus of rat long interspersed repeated (LINE or L1Rn) DNA family members; d'Ambrosio E et al.; An approximately equal to 150-bp GC-rich (approximately equal to 60%) region is at the right end of rat long interspersed repeated DNA (LINE or L1Rn) family members . We report here that one of the DNA strands from this region contains several non-palindromic sites that strongly arrest DNA synthesis in vitro by the prokaryotic Klenow and T4 DNA polymerases, the eukaryotic alpha polymerase, and AMV reverse transcriptase . The strongest arrest sites are G-rich (approximately equal to 70%) homopurine stretches of 18 or more residues . Shorter homopurine stretches (12 residues or fewer) did not arrest DNA synthesis even if the stretch contains 11/12 G residues . Arrest of the prokaryotic polymerases was not affected by their respective single strand binding proteins or polymerase accessory proteins . The region of duplex DNA which contains DNA synthesis arrest sites reacts with bromoacetaldehyde when present in negatively supercoiled molecules . By contrast, homopurine stretches that do not arrest DNA synthesis do not react with bromoacetaldehyde . The presence of bromoacetaldehyde-reactive bases in a G-rich homopurine-containing duplex under torsional stress is thought to be caused by base stacking in the homopurine strand . Therefore, we suggest that base-stacked regions of the template arrest DNA synthesis.

FEBS Lett, 1987 Apr 6, 214(1), 1 - 7
Intron-dependent evolution: preferred types of exons and introns; Patthy L; Exon insertions and exon duplications, two major mechanisms of exon shuffling, are shown to involve modules that have introns of the same phase class at both their 5'- and 3'-ends . At the sites of intronic recombinations exon insertions and duplications create new introns which belong to the same phase class as the recipient introns . As a consequence of repeated exon insertions and exon duplications introns of a single phase class predominate in the resulting genes, i.e . gene assembly by exon shuffling is reflected both by this nonrandom intron phase usage and by the correlation between the domain organization of the proteins and exon-intron organization of their genes . Genes that appeared before the eukaryote-prokaryote split do not show these diagnostic signs of exon shuffling . Since ancestral introns (e.g . self-splicing introns) did not favour intronic recombination, exon shuffling may not have been significant in the early part of protein evolution.

J Mol Biol, 1987 Apr 5, 194(3), 557 - 64
Systematic method for the detection of potential lambda Cro-like DNA-binding regions in proteins; Dodd IB et al.; We have developed and tested a systematic method for the location and statistical evaluation of potential DNA-binding regions of the lambda Cro type in protein sequences . Using this approach to examine proteins expected to contain such regions, we have been able to compile a statistically homogeneous master set of 37 lambda Cro-like DNA-binding domains . Examination of a protein database revealed other prokaryotic proteins that are similar to this lambda Cro-like group . There are also many DNA-binding proteins that are not found to be significantly similar to the lambda Cro group, consistent with previous suggestions that different types of protein sequence may be able to achieve a similar mode of binding and that there exist other modes of sequence-specific DNA-binding . A useful feature of the method is that it can be applied without a computer.

Mol Endocrinol, 1987 Apr, 1(4), 312 - 20
Comparison of human mouse P1450 upstream regulatory sequences in liver- and nonliver-derived cell lines; Jaiswal AK et al.; The foreign chemical tetrachlorodibenzo-p-dioxin (TCDD) is known to interact with the aromatic hydrocarbon receptor and, in turn, activate transcription of the mouse P1450 and P3450 genes . Various lengths of DNA upstream from the human P1450 gene were inserted into the promoterless pSVO-cat prokaryotic expression vector and compared with mouse P1450 upstream sequences similarly treated . The constructs were cotransfected with pSV2-neo into human, mouse, and monkey liver- and nonliver-derived cell lines . After selection in G418, the transformed colonies were treated with control medium, TCDD, or, in some cases, cycloheximide . Pooled transformants were then assayed for chloramphenicol acetyltransferase activity . The data are consistent with the presence of several functional regulatory regions within the upstream DNA: a promoter region, a region that is negatively autoregulated, and a region further upstream that activates transcription and is dependent upon a functional aromatic hydrocarbon receptor . Compared with 1604 base pairs of human P1450 upstream sequences, 1646 base pairs of mouse P1450 upstream sequences exhibit an increased sensitivity to TCDD; this effect was found to require both trans-acting protein factors and cis-acting DNA elements . Our results demonstrate the successful interaction of mouse trans-acting factors with human P1450 upstream sequences and human trans-acting factors with mouse P1450 upstream sequences.

Antibiot Med Biotekhnol, 1987 Apr, 32(4), 248 - 54
{Optimization of the gene expression for human alpha F- and beta 1-interferons in Escherichia coli cells}; Mashko SV et al.; The basic results of the studies on expression of the genes of human alpha F- and beta 1-interferons in E . coli cells are presented . To synthesize the fibroblast interferon, the respective fragment of the human chromosome was cloned, the complete nucleotide sequence of the structural moiety of mature beta-interferon was determined and the genes of "hybrid (interferon-like) proteins" and "hybrid sites of ribosome binding" were constructed with control of the beta-interferon gene by the prokaryotic regulatory areas . Synthesis of beta-interferon was achieved (1.10(7)-5.10(7) IU per 1 l of the bacterial culture) with the use of the tryptophan operon promoter . A new procedure for optimization of allogenic genetic information in E . coli cells: constructing of "hybrid operons with partially overlapping genes" or artificial "overlappons" was developed following the example of the alpha F-interferon gene cloned in the Laboratory headed by E . D . Sverdlov at the Institute of Bioorganic Chemistry of the USSR Academy of Sciences . The use of this procedure enabled production of up to 5.10(7) IU/l of alpha F-interferon under the control of the lacUV5-promoter . On the basis of the newly constructed vector molecules expression of the genes of alpha F- and beta 1-interferons was amplified with the "overlappon" procedure.

Microbiol Sci, 1987 Apr, 4(4), 100 - 5
Bacterial membrane proteins; Salton MR; Bacterial membranes have diverse functions, depending on whether they are specialized membranes or cytoplasmic membranes possessing transport, mitochondrial activities and biosynthetic functions for assembly of membranes, walls and capsules . In contrast to plasma membranes which serve as major biochemical organelles of both Gram-positive and Gram-negative bacteria, the outer membranes of the latter group confer barrier functions on the cells, providing a variety of selective channels . Although prokaryotic cells lack the array of membranous organelles characteristic of eukaryotic cells, bacteria with specific physiological and genetic capabilities form specialized membrane systems such as the bacteriorhodopsin purple membrane, chromatophore membranes of phototrophs, and forespore membranes essential to bacterial endospore formation . Unravelling the structure, function and proteins of these membranes presents a formidable biochemical, immunochemical and structural challenge.

Proc Natl Acad Sci U S A, 1987 Apr, 84(8), 2185 - 8
In vivo aminoacylation of human and Xenopus suppressor tRNAs constructed by site-specific mutagenesis; Ho YS et al.; Amber suppressor tRNA genes were constructed by site-specific mutagenesis of the anticodons of human lysine-inserting tRNA (tRNA(Lys)) and glutamine-inserting tRNA (tRNA(Gln)) genes, and a Xenopus laevis tyrosine-inserting tRNA (tRNA(Tyr)) gene . As previous in vitro studies in prokaryotes have shown that substitution of nucleotides in the anticodon region can profoundly affect tRNA aminoacylation, it is important to determine whether the mutation affects aminoacylation of these eukaryotic tRNAs . We present a method for quantitating the tRNA aminoacylation in vivo in mammalian cells, and we have determined that the suppressor tRNA(Tyr) is fully aminoacylated and suppressor tRNA(Lys) and tRNA(Gln) are aminoacylated 40-50% and 80%, respectively . This in vivo method of estimating aminoacylation may be applied to other mutations in the tRNA genes.

Virology, 1987 Apr, 157(2), 560 - 4
Expression of the poliovirus genome from infectious cDNA is dependent upon arrangements of eukaryotic and prokaryotic sequences in recombinant plasmids; Kuhn RJ et al.; The introduction of a cDNA copy of poliovirus type 1 (Mahoney) into cultured primate cells results in the production of infectious virus . The level of infectious virus can be increased by incorporation of eukaryotic signals of transcription and replication . We have utilized the SV40 DNA sequence coding for the early and late promoters, the SV40 origin of replication, and the enhancer elements, along with the cDNA of poliovirus, to determine the important parameters for the level of infectious virus produced following transfection . Although plasmid replication increases the level of infectivity, the major determinant of infectivity is promoter activity.

Genetics, 1987 Apr, 115(4), 591 - 5
Mutations that improve the pRE promoter of coliphage lambda; Mahoney ME et al.; The dya5 mutation, a C----T change at position -43 of the lambda pRE promoter, results in a twofold increase in pRE activity in vivo . Smaller increases in pRE activity are found for the dya2 mutation, a T----C change at position -1 of pRE, and the dya3 mutation, an A----G change at +5 of pRE . The mutant pRE promoters retain complete dependence on cII protein for activity . These observations argue, at least for pRE-like promoters, that promoter activities are influenced by nucleotide sequences at least eight nucleotides to the 5'-side of the conventional -35 region consensus sequence, and by nucleotide sequences near the start-site of transcription . Although Hawley and McClure (1983) found A-T pairs more frequently than G-TC pairs in the region of -40 to -45 of prokaryotic promoters, other mutations that change a G-TC pair to an A-T pair at positions -41, -44 and -45 of pRE do not result in increased promoter activity . We also found that a T----C change at positions -42 results in a mild decrease in promoter activity . These observations argue that Ts at positions -42 and -43 of pRE are required for maximum promoter activity, but do not support the hypothesis that As and Ts in the -40 and -45 region generally lead to higher promoter activities.

Tsitologiia, 1987 Apr, 29(4), 379 - 90
{Mycoplasmas}; Borkhsenius SN et al.; Structural peculiarities of the mycoplasmas--the smallest prokaryotic organisms--are reviewed, in addition to their complicated relationships with the eukaryotic cells and with the whole organisms of plants and animals.

J Biol Chem, 1987 Mar 25, 262(9), 4195 - 9
The rabbit muscle phosphofructokinase gene . Implications for protein structure, function, and tissue specificity; Lee CP et al.; Sequence homologies between bacterial and rabbit muscle phosphofructokinases and between the amino- and carboxyl-terminal halves of the latter suggest that the mammalian enzyme evolved from a prokaryotic progenitor by gene duplication and divergence (Poorman, R . A., Randolph, A., Kemp, R . G., and Heinrikson, R . L . (1984) Nature 309, 467-469) . We have isolated the gene for the rabbit enzyme and determined the nucleotide sequence for all the exons and most of the introns . This represents the first eukaryotic phosphofructokinase gene ever sequenced . The cloned gene is 17 kilobase pairs long . The coding sequence for 780 amino acids is split into 22 exons ranging in size from 15 to 63 codons . Sequence analysis shows that 75% of the bases at the third position of the codons in these exons are either G or C . Exons XV and XVI code for the 30 amino acid residues which were left unidentified in the published primary structure for this enzyme . When overlaid on the structure of the protein, most of the introns are located between or near the ends of the secondary structural elements but not at analogous positions in the two protein-coding halves of the gene.

J Biol Chem, 1987 Mar 5, 262(7), 3388 - 97
Assembly of the mitochondrial membrane system . MRP1 and MRP2, two yeast nuclear genes coding for mitochondrial ribosomal proteins; Myers AM et al.; Nuclear respiratory deficient mutants of Saccharomyces cerevisiae impaired in mitochondrial protein synthesis have been screened for lesions in ribosomal protein constituents . Two mutants, each representative of a separate pet complementation group, have been analyzed . One of the mutants, E795, was found to have altered mitochondrial ribosomes as evidenced by the absence of some ribosomal proteins . The second mutant studied, C167, appeared to have more grossly altered ribosomes that could not be isolated by standard preparative procedures . In addition to being defective in mitochondrial protein synthesis, the mutants exhibit an absence of "a" and "b" type cytochromes, are partially blocked in processing of intron bI4 of the apocytochrome b gene, have reduced levels of mitochondrial 15 S rRNA, and convert to rho- and rho 0 mutants at a high frequency . The wild type genes MRP1 and MRP2 were cloned by transformation of the pet mutations in E795 and C167, respectively, with a recombinant plasmid library of wild type yeast genomic DNA . MRP1 codes for a basic protein of 37 kDa with no significant homology to any known prokaryotic or eukaryotic ribosomal protein . MRP2 codes for a 14-kDa polypeptide homologous to protein S14 of the Escherichia coli small ribosomal subunit and to a chloroplast-encoded component of chloroplast ribosomes . The levels of MRP1 and MRP2 mRNAs were examined in glucose-repressed cells and in cells undergoing adaptation to aerobic metabolism of ethanol . The steady state concentrations of the mRNAs increased during the first 3 h of derepression, indicating that expression of these mitochondrial ribosomal protein genes is transcriptionally regulated by glucose in a fashion analogous to respiratory carriers such as cytochrome c.

Nature, 1987 Mar 26-Apr 1, 326(6111), 411 - 4
Ribosomal RNA sequence suggests microsporidia are extremely ancient eukaryotes; Vossbrinck CR et al.; The microsporidia are a group of unusual, obligately parasitic protists that infect a great variety of other eukaryotes, including vertebrates, arthropods, molluscs, annelids, nematodes, cnidaria and even various ciliates, myxosporidia and gregarines . They possess a number of unusual cytological and molecular characteristics . Their nuclear division is considered to be primitive, they have no mitochondria, their ribosomes and ribosomal RNAs are reported to be of prokaryotic size and their large ribosomal subunit contains no 5.8S rRNA . The uniqueness of the microsporidia may reflect their phylogenetic position, because comparative sequence analysis shows that the small subunit rRNA of the microsporidium Vairimorpha necatrix is more unlike those of other eukaryotes than any known eukaryote 18S rRNA sequence . We conclude that the lineage leading to microsporidia branched very early from that leading to other eukaryotes.

Proc Natl Acad Sci U S A, 1987 Mar, 84(5), 1192 - 6
Prokaryotic and eukaryotic RNA polymerases have homologous core subunits; Sweetser D et al.; Eukaryotic RNA polymerases are complex aggregates whose component subunits are functionally ill-defined . The gene that encodes the 140,000-dalton subunit of Saccharomyces cerevisiae RNA polymerase II was isolated and studied in detail to obtain clues to the protein's function . This gene, RPB2, exists in a single copy in the haploid genome . Disruption of the gene is lethal to the yeast cell . RPB2 encodes a protein of 138,750 daltons, which contains sequences implicated in binding purine nucleotides and zinc ions and exhibits striking sequence homology with the beta subunit of Escherichia coli RNA polymerase . These observations suggest that the yeast and the E . coli subunit have similar roles in RNA synthesis, as the beta subunit contains binding sites for nucleotide substrates and a portion of the catalytic site for RNA synthesis . The subunit homologies reported here, and those observed previously with the largest RNA polymerase subunit, indicate that components of the prokaryotic RNA polymerase "core" enzyme have counterparts in eukaryotic RNA polymerases.

Mol Gen Genet, 1987 Mar, 206(3), 485 - 90
A transcriptional terminator sequence in the prokaryotic transposable element IS1; Hubner P et al.; The prokaryotic transposable element IS1 is known to exert a strong polar effect upon integration into an operon . To elucidate this polar effect, we constructed a plasmid which has an IS1 integrated between the 5' half of the tet gene for tetracycline resistance and the cat structural gene for chloramphenicol resistance . The cat gene is expressed by the tet promoter and the presence of IS1 in orientation I, in which the IS1 transposase genes insA and insB are in the same orientation as the cat gene, reduced the cat expression . By introducing deletions or insertions within the IS1 sequence, we were able to map a rho-dependent terminator TIS1A between the insA and insB genes . Translational interruption between these ins genes is important for TIS1A to be an active terminator.

J Biol Chem, 1987 Feb 15, 262(5), 2228 - 33
Biochemical characterization of recombinant human phenylalanine hydroxylase produced in Escherichia coli; Ledley FD et al.; A full-length human phenylalanine hydroxylase cDNA has been recombined with a prokaryotic expression vector and introduced into Escherichia coli . Transformed bacteria express phenylalanine hydroxylase immunoreactive protein and pterin-dependent conversion of phenylalanine to tyrosine . Recombinant human phenylalanine hydroxylase produced in E . coli has been partially purified, and biochemical studies have been performed comparing the activity and kinetics of the recombinant enzyme with native phenylalanine hydroxylase from human liver . The optimal reaction conditions, kinetic constants, and sensitivity to inhibition by aromatic amino acids are the same for recombinant phenylalanine hydroxylase and native phenylalanine hydroxylase . These data indicate that the recombinant human phenylalanine hydroxylase is an authentic and complete phenylalanine hydroxylase enzyme and that the characteristic aspects of phenylalanine hydroxylase enzymatic activity are determined by a single gene product and can be constituted in the absence of any specific accessory functions of the eukaryotic cell . The availability of recombinant human phenylalanine hydroxylase produced in E . coli will expedite physical and chemical characterization of human phenylalanine hydroxylase which has been hindered in the past by inavailability of the native enzyme for study.

J Mol Biol, 1987 Feb 5, 193(3), 507 - 15
Messenger ribonucleic acid for the lipoprotein of the Escherichia coli outer membrane is polyadenylated; Taljanidisz J et al.; Earlier studies had shown that a large portion of bacterial messenger RNA carries 3'-terminal polyadenylate sequences, albeit of somewhat shorter length than those associated with eukaryotic mRNA . In this paper, we show for the first time that a specific prokaryotic mRNA is polyadenylated . Three independent lines of evidence demonstrate that a 3'-terminal polyadenylate sequence 10 to 15 nucleotides in length is associated with about 40% of the mRNA of the outer membrane lipoprotein of Escherichia coli: 40% of lipoprotein mRNA binds to oligodeoxythymidylate-substituted cellulose at high ionic strength and is eluted by water; treatment of lipoprotein mRNA with oligodeoxythymidylate and ribonuclease H destroys its ability to bind to oligodeoxythymidylate-cellulose; and in the presence of oligodeoxythymidylate, lipoprotein mRNA can serve as template for the synthesis of DNA complementary to lipoprotein mRNA by reverse transcriptase . In view of the fact that the lpp gene and its downstream-flanking region contain no continuous deoxyadenylate sequences longer than five nucleotides, the polyadenylate moiety must be added post-transcriptionally . It was possible to demonstrate the synthesis of polyadenylated lipoprotein mRNA in toluene-permeabilized cells of E . coli, opening the way for the study of its biosynthesis.

Nature, 1987 Feb 26-Mar 4, 325(6107), 823 - 6
Long-range cooperativity between gene regulatory sequences in a prokaryote; Dandanell G et al.; Regulation of transcription initiation by proteins binding at DNA sequences some distance from the promoter region itself seems to be a general phenomenon in both eukaryotes and prokaryotes . Proteins bound to an enhancer site in eukaryotes can turn on a distant gene, whereas efficient repression of some prokaryotic genes such as the gal, ara and deo operons of Escherichia coli, requires the presence of two operator sites, separated by 110, 200 and 600 base pairs (bp) respectively . In the deo operon, which encodes nucleoside catabolizing enzymes, we have shown that efficient and cooperative repression can be obtained when the distance between the two sites ranges from 224 to 997 bp . Here, we report that transcription initiation can be regulated from an operator site placed 1 to 5 kilobases (kb) downstream of the deoP2 promoter (and downstream of the transcribed gene), and present the first experimental data for prokaryotic regulation at distances greater than 1 kb . Our results support the model of DNA loop formation as a common regulatory mechanism explaining both some prokaryotic regulation and the action of eukaryotic enhancers.

Tohoku J Exp Med, 1987 Feb, 151(2), 241 - 4
Intratumoral doxycycline for skin metastases of human malignancies; Okuyama S et al.; As soon as its inducibility of lethality via prompt deprivation of heavy metals and HDL-cholesterol was born out, an intratumoral administration of doxycycline, an anti-prokaryotic agent, was undertaken in 7 patients presenting with skin metastases from a variety of malignancies . Complete remission of these lesions was attained in 6 . The histological observation of elongated areas of acellularity along with central vessels appeared to suggest doxycycline-mediated cellular disintegration, resulting in empty tumor cords . Specific tumor selectivity of the treatment can be sustained by the intra- and/or peri-tumoral confinement of the agent . The technique may constitute a novel mode of cancer cell elimination.

Cell, 1987 Jan 30, 48(2), 297 - 310
Stabilization of translationally active mRNA by prokaryotic REP sequences; Newbury SF et al.; The REP sequence is a highly conserved inverted repeat that is present in about 25% of all E . coli transcription units . We show that the REP sequence can stabilize upstream RNA, independently of any other sequences, by protection from 3'-5' exonuclease attack . The REP sequence is frequently responsible for the differential stability of different segments of mRNA within an operon . We demonstrate that REP-stabilized mRNA can be translated in vivo and that cloning the REP sequence downstream of a gene can increase protein synthesis . This provides direct evidence that alterations in mRNA stability can play a role in determining bacterial gene expression . The implications of these findings for the mechanisms of mRNA degradation and for the role of RNA stability in the regulation of gene expression are discussed.

J Biol Chem, 1987 Jan 25, 262(3), 962 - 5
Reductive trapping of substrate to bovine plasma amine oxidase; Hartmann C et al.; Plasma amine oxidases catalyze the oxidative deamination of amines to aldehydes, followed by a 2e- reduction of O2 to H2O2 . Pyrroloquinoline quinone (PQQ), previously believed to be restricted to prokaryotes, has recently been proposed to be the cofactor undergoing reduction in the first half-reaction of bovine plasma amine oxidase (Ameyama, M., Hayashi, U., Matsushita, K., Shinagawa, E., and Adachi, O . (1984) Agric . Biol . Chem . 48, 561-565; Lobenstein-Verbeek, C . L., Jongejan, J . A., Frank, J., and Duine, J . A . (1984) FEBS Lett . 170, 305-309) . This result is unexpected, since model studies with PQQ implicate Schiff's base formation between a reactive carbonyl and substrates, whereas experiments with bovine plasma amine oxidase have failed to provide evidence for a carbonyl cofactor . We have, therefore, re-examined putative adducts between substrate and enzyme-bound cofactor, employing a combination of {14C}benzylamine and {3H}NaCNBH3 . The use of the relatively weak reductant, NaCNBH3, affords Schiff's base specificity and permits the study of enzyme below pH 7.0 . As we show, enzyme can only be inactivated by NaCNBH3 in the presence of substrate, leading to the incorporation of 1 mol of {14C}benzylamine/mol of enzyme subunit at complete inactivation . By contrast, we are unable to detect any labeling with {3H}NaCNBH3, analogous to an earlier study with {3H}NaCNBH4 (Suva, R . H., and Abeles, R . H . (1978) Biochemistry 17, 3538-3545) . We conclude, first, that our inability to obtain adducts containing both carbon 14 and tritium rules out the reductive trapping either of amine substrate with pyridoxal phosphate or of aldehyde product with a lysyl side chain and, second, that the observed pattern of labeling is fully consistent with the presence of PQQ at the active site of bovine plasma amine oxidase.

Cell, 1987 Jan 16, 48(1), 63 - 71
Two domains for splicing in the intron of the phage T4 thymidylate synthase (td) gene established by nondirected mutagenesis; Hall DH et al.; Of 97 nondirected T4 thymidylate synthase-defective (td) mutations, 27 were mapped to the intron of the split td gene . Clustering of these intron mutations defined two domains that are functional in splicing, each within approximately 220 residues of the respective splice sites . Two selected mutations, tdN57 and tdN47, fell within phylogenetically conserved pairings, with tdN57 disrupting the exon I-internal guide pairing (P1) in the 5' domain and tdN47 destabilizing the P9 helix in the 3' domain . A splicing assay with synthetic oligonucleotides complementary to RNA junction sequences revealed processing defects for T4tdN57 and T4tdN47, both of which are impaired in cleavage at the 5' and 3' splice sites . Thus prokaryotic genetics facilitates association of specific residue changes with their consequences to splicing.

Nucleic Acids Res, 1987 Jan 12, 15(1), 345 - 60
Potential secondary structure at translation-initiation sites; Ganoza MC et al.; Since translational start codons also occur internally, more-complex features within mRNA must determine initiation . We compare the potential secondary structure of 123 prokaryotic mRNA start regions to that of regions coding for internal methionines . The latter display an unexpectedly-uniform, almost-periodic pattern of pairing potential . In contrast, sequences 5' to start codons have little self-pairing, and do not pair extensively with the proximal coding region . Pairing potential surrounding start codons was found to be less than half of that found near internal AUGs . In groups of random sequences where the distribution of nucleotides at each position, or of trinucleotides at each in-frame codon position, matched the observed natural distribution, there was no periodicity in the pairing potential of the internal sequences . Randomized internal sequences had less pairing: the ratio of pairing intensity between internals and starts was reduced from 2.0 to 1.6 by randomization . We propose that the transition from the relatively-unstructured start domains to the highly-structured internal sequences may be an important determinant of translational start-site recognition.

Science, 1987 Jan 9, 235(4785), 211 - 4
Metalloregulatory DNA-binding protein encoded by the merR gene: isolation and characterization; O'Halloran T et al.; The MerR protein mediates the induction of the mercury resistance phenotype in bacteria; it has been isolated in order to study the effects of metal-ion induced changes in the metabolism of prokaryotic cells at the molecular level . After DNA sequences responsible for negative autoregulation were removed, the 16-kilodalton protein was overproduced and purified to more than 90 percent homogeneity by a salt extraction procedure that yields about 5 milligrams of protein per gram of cells . Complementation data, amino terminal analysis, gel filtration, and deoxyribonuclease I protection studies demonstrate that the purified merR gene product is a dimer under nondenaturing conditions and that it binds specifically to DNA, in the presence and absence of mercury, at a palindromic site which is directly between the -10 and -35 regions of the structural genes and adjacent to its own promoter . These initial results indicate that MerR is a DNA-binding metalloregulatory protein that plays a central role in this heavy metal responsive system and they delineate an operator site in the mer operon.

J Biol Chem, 1987 Jan 5, 262(1), 239 - 44
Crucial role of the connecting region joining the two functional domains of yeast tryptophan synthetase; Crawford IP et al.; We constructed a hybrid plasmid expressing yeast tryptophan synthetase in Escherichia coli . Several deletion variants lacking the A or B domains of this polypeptide (recognized by their homology to the alpha and beta subunits of prokaryotic tryptophan synthetase) showed no enzymatic activity and failed to substitute for the corresponding E . coli subunits . To examine the role of a presumed interdomain connecting region in the yeast enzyme, we constructed a variant lacking 18 amino acids in that region . The variant polypeptide was completely inactive . Replacing 14 of the 18 missing amino acids with a segment having a different sequence partially restored activity . A spontaneous revertant was characterized and shown to have a duplication of 16 amino acid residues in this region; the activity of the duplication polypeptide was better than that of the 14-residue replacement . If confirmed by additional studies, our finding that the length of the connecting region is more critical than its sequence has implications for understanding the origin of gene fusions during evolution as well as for designing artificial fusions.

J Mol Biol, 1987 Jan 5, 193(1), 1 - 13
The rudimentary gene of Drosophila melanogaster encodes four enzymic functions; Freund JN et al.; We have determined the 7168 nucleotide DNA sequence corresponding to the messenger RNA of the rudimentary gene of Drosophila melanogaster . By sequence comparison with genes involved in the pyrimidine pathway of prokaryotes and lower eukaryotes, we conclude that the rudimentary gene encodes four enzymically different functions . Each function is restricted to a specific coding domain but in an order different from that previously defined by genetic data . We have found that the corresponding mammalian gene, the CAD gene, exhibits a similar functional organization, and we propose schemes for the evolution of the corresponding coding sequences.

Arch Biol Med Exp (Santiago), 1987, 20(3-4), 343 - 57
The evolution of hexokinases; Ureta T et al.; Recent advances in the knowledge of the structural and functional aspects of the enzymes catalyzing sugar phosphorylation by ATP are reviewed . Hexokinases may exist, mainly in prokaryotes, as sugar-specific kinases (glucokinase, fructokinase, mannokinase) or as ubiquitous hexose-kinases which are relatively unspecific for the natural hexoses . Enzymes presenting intermediate specificity (e.g . mannofructokinases) have been also described . With a few exceptions, the molecular mass of a variety of hexokinases may be either 25 kDa, 50 kDa or 100 kDa . The smaller hexokinases have been found in some microorganisms whereas the 50 kDa enzymes are found (with only one exception) in most invertebrates and in a particular isozyme from vertebrates (hexokinase D) . The 100 kDa enzymes are restricted to vertebrates (hexokinases A, B and C) . These facts have led to the speculation that gene duplication events have played an important role in the evolutionary development of the hexokinases from present day organisms . The fact that the 100 kDa hexokinases are allosterically inhibited by the product, glucose 6-P, may indicate that a duplicated active site has evolved to a regulatory binding site . Comparisons of the amino acid sequence of a few peptides from hexokinase C are presented to support the gene duplication hypothesis . Also, partial sequence comparisons of vertebrate hexokinases with the sequences of two hexokinase isozymes from yeast show strong similarities suggesting a rather slow amino acid substitution rate of homologous genes.

Mutat Res, 1987 Jan, 183(1), 53 - 60
Pyrimidine dimers in Drosophila chromatin become increasingly accessible after irradiation; Harris PV et al.; A prokaryotic DNA-repair enzyme has been utilized as a probe for changes in the accessibility of pyrimidine dimers in Drosophila chromatin following UV irradiation . The results demonstrate a rapid cellular response to physiologically relevant doses of radiation which results in at least a 40% increase in accessible dimers . This increase occurs in two incision-deficient mutants which indicates that the excision-repair process, at or beyond the incision step, is not required or responsible for the increase . In the absence of excision the increase in accessibility persists for at least 2 days following irradiation . The observed increase in accessibility is inhibited by both novobiocin and coumermycin . These inhibitors do not inhibit the initial rate of incision, but do reduce dimer excision measured over more extended periods . A pre-incision process is proposed which actively exposes DNA lesions to excision repair . A fraction of the genome is postulated to be accessible without the intervention of that process.

Bioorg Khim, 1987 Jan, 13(1), 58 - 68
{Eukaryotic and prokaryotic DNA-polymerase . II . The role of internucleotide phosphate groups of a template in its binding with the enzyme}; Nevinskii GA et al.; The affinity of different ligands (phosphate, nucleoside monophosphates, oligonucleotides) to the template binding site of DNA polymerase alpha from human placenta was estimated . To this goal, dependences of rate of the enzyme inactivation by the affinity reagent d(pT)2pC{Pt2+(NH3)2OH}(pT)7 on the concentration of these ligands as competitive inhibitors were determined . Minimal ligands capable to bind with the template site of DNA polymerase alpha were shown to be triethylphosphate (Kd 600 microM) and phosphate (Kd 53 microM) . Ligand affinity increases by the factor 1.71 per added monomer unit from phosphate to d(pT) and then for oligothymidylates d(Tp)nT (n 1 to 14) . The partial ethylation of phosphodiester groups does not change the efficiency of the oligothymidylate binding with the enzyme . However, the complete ethylation of these groups lowers affinity of the oligothymidylates to the enzyme by 7-9 times . The decrease is comparable with the change of Pt2+-decathymidylate affinity to the enzyme caused by Mn2+-ions . The data obtained led to suggestion that an electrostatic contact (most likely, Me2+-dependent) of phosphodiester group with the enzyme takes place . The type of contact is confirmed by Gibbs' energy change 1.1-1.4 kcal/mole . Formation of a hydrogen bond with the oxygen atom of P = O group of the same phosphate is also assumed (delta G =--4.4 . . .--4.5 kcal/mole) . The other internucleotide phosphates and all bases of oligonucleotides form neither hydrogen bonds nor electrostatic contacts with the template binding site . Gibbs' energy changes by 0.32 kcal/mole when the template is lengthened by one unit . We suppose that this value characterizes the energy gain in the transition of oligonucleotide template from aquous medium to the hydrophobic environement of the enzyme active site . Comparison of Km values of oligothymidylates and their partially or completely ethylated analogues as templates in the reaction of DNA polymerization catalysed by DNA polymerase alpha from human placenta and Klenow's fragment of E . coli DNA polymerase I suggests a similar mechanism of template recognition by both enzymes.

Bioorg Khim, 1987 Jan, 13(1), 45 - 57
{Prokaryotic and eukaryotic DNA-polymerase . I . The role of internucleotide phosphate groups in the binding of a primer with the enzyme}; Nevinskii GA et al.; The mechanism of binding and elongation of the oligothymidylate primers in the systems of the DNA polymerase alpha from human placenta and DNA polymerase I from E . coli with the poly(dA) as a template was investigated . Both dTMP and dTTP were shown to be the minimal primers of DNA polymerase alpha, the affinity and V increasing 1.8- and 1.4-fold respectively upon lengthening the primer by each unit from dTMP to d(Tp)9T . Further elongation is accompanied by 1.3-fold affinity enhancement and a decrease in V . For the E . coli enzyme, a similar dependence of affinity of primer d(Tp)4T-d(Tp)14T was observed with the inflexion point corresponding to d(Tp)8T . The individual diastereomers of oligothymidylate ethyl esters (with p' and p'' corresponding to enantiomeric configuration) such as d{Tp'(Et)Tp}3Tp'(Et)T, d{Tp''(Et)Tp}3Tp''(Et)T, d(Tp)8Tp'(Et)T, d(Tp)8Tp''(Et)T, d(Tp)8Tp'(Et)TpT, d(Tp)8 X X Tp''(Et)TpT and completely esterified analogues d{Tp(Et)}7T, d{Tp(Et)}14T were shown to initiate the poly (dA)-dependent polymerization catalyzed by both enzymes . A sum of the obtained results provided the basis for a number of conjectures on the mode of primer and template binding to the enzyme, possible role of their preformed complex, as well as electrostatic interactions and hydrogen bonding.

Prikl Biokhim Mikrobiol, 1987 Jan-Feb, 23(1), 3 - 13
{The role of free radical oxidation in the regulation of growth and lipid formation in eukaryotic and prokaryotic organisms}; Feofilova EP et al.; The review considers the effect of free-radical oxidation on the growth of microorganisms, intensity of their metabolism, and composition of the membrane lipids . Using both own research evidence and this presented in the literature, the authors put forward a hypothesis about a certain correlation between physico-chemical properties of the membrane lipids and the intensity of the growth of eucaryotic and procaryotic microorganisms.

J Cell Sci Suppl, 1987, 7, 51 - 65
The role of sequence-specific DNA-binding proteins in adenovirus DNA replication; Hay RT et al.; In prokaryotes it is well established that proteins which recognise defined DNA sequences are involved in the control of gene expression and replication . Cellular proteins in eukaryotes which may perform a similar function have been identified by their interactions with control regions of the human adenovirus genome . Immediately after infection a small region (E1a) at the left end of the adenovirus genome is expressed . Proteins coded by the E1a region transcriptionally activate the viral early genes . The products of a number of these early genes are directly involved in replication of the viral DNA . DNA sequences which are required for efficient E1a transcription and for the initiation of DNA replication have been identified by mutational analysis . Cellular proteins which recognise these sequences were detected using a sensitive gel retention assay . The basis of this assay is that during electrophoresis DNA-protein complexes migrate more slowly through a polyacrylamide gel than free DNA . In this way a cellular protein which binds to a conserved sequence in the adenovirus enhancer has been identified and partially purified . Cellular proteins which bind to adenovirus type 2 and 4 origins of replication have also been fractionated from nuclear extracts of uninfected HeLa cells . The roles of these proteins in adenovirus replication will be discussed.

Haematol Blood Transfus, 1987, 31, 511 - 8
Repetition as the essence of life on this earth: music and genes; Ohno S; In prebiotic nucleic acid replication, templates appear to have been in short supply . A single round of tandem duplication of existing oligomers assured progressive extension of templates to the length adequate for encoding of polypeptide chains . Thus, the first set of coding sequences had to be repeats of base oligomers encoding polypeptide chains of various periodicities . On one hand, the readiness of these periodical polypeptide chains to assume alpha-helical and/or beta-sheet secondary structures contributed to the extremely rapid initial functional diversification of these polypeptide chains . It would be recalled that most, if not all, of the sugar-metabolizing enzymes had already achieved the inviolable functional competence before the division of prokaryotes from eukaryotes . On the other hand, a certain (dipeptidic?) of the peptidic periodicities was apparently chosen as the timekeeping unit by the biological clock . Musical compositions too apparently evolved originally as a timekeeping device . Accordingly, repetitiousness is evident in all musical compositions . Evolution of musical compositions from the early Baroque to the late Romantic parallels that of coding sequences from rather exact repeats of base oligomers to more complex modern coding sequences in which repetitious elements are less conspicuous and more varied . Inasmuch as the earth is governed by the hierarchy of periodicities (days, months and years), such reliance on periodicities is rather expected.

Haematol Blood Transfus, 1987, 31, 493 - 5
Atavistic mutations reflect the long life span of dispensable genes; Ohno S; Most of the major innovations in evolution occurred at the very beginning of life on this earth some 3.5 billion years ago before the division of eukaryotes from prokaryotes . This initial innovativeness was due, in no small part, to the peculiar construction of primordial coding sequences that were repeats of base oligomers, the number of bases in oligomeric units not being a multiple of three . Such coding sequences are conferred with a measure of immortality . Because of this initial immortality and of long life span of genes after becoming dispensable, the ancient gene may remain silenced in particular phylogenetic trees for a very long time, only to be resurrected later . Hemoglobin genes expressed in exceptional bacteria, plants, worms, insects, as well as in all vertebrates are a good example of this . Atavistic mutations are more dramatic visible examples of such resurrection of long dormant genes . A few interesting examples are given.

Basic Appl Histochem, 1987, 31(4), 465 - 73
Histochemical demonstration of oxidoreductase activities in the fat body and symbionts of Blattella germanica (Blattodea) following chlortetracycline-treatment; De Piceis Polver P et al.; The metabolic apport of prokaryotic symbionts in the fat body of Blattella germanica was investigated by histoenzymatic methods, using chlortetracycline-treated and normal strains . In the experimental insects, bacteriocytes showed a decreased oxidoreductase activity, whereas the staining intensity of the other cell types was generally unchanged . Electron microscopic observations showed that some bacteria were still present in the bacteriocytes of the treated insects, but exhibited degeneration patterns to a different extent; therefore, they are not likely to carry on any enzymatic activity . Hence, chlortetracycline, an antibiotic that blocks the transovaric transmission of the symbionts, is active also on the endocellular symbionts of the fat body.

Biochem Soc Symp, 1987, 54, 33 - 43
Patterns of diversity of citric acid cycle enzymes; Weitzman PD; The citric acid cycle performs a dual role in cell metabolism, acting as a source of both 'energy' and biosynthetic starting materials . The widespread occurrence of the cycle throughout Nature is an excellent example of the unity of biochemistry, but closer examination reveals that there is considerable diversity in the citric acid cycle of different organisms with respect to metabolic role, molecular enzymology and mode of regulation . Two enzymes of the cycle--citrate synthase and succinate thiokinase--have been found to exhibit particularly striking patterns of diversity in structure and catalytic and regulatory function . Some of these patterns show a correlation with the taxonomic groupings of the organisms and with their physiological characteristics . Comparative enzyme studies have a contribution to make to an ultimate understanding of the cycle and its cellular operation, and there are substantial benefits to be gained from interactive studies on both prokaryotic and eukaryotic systems.

Biochem Soc Symp, 1987, 54, 3 - 16
Evolutionary roots of the citric acid cycle in prokaryotes; Gest H; Advances in biochemistry, microbiology, and molecular biology suggest new approaches for exploring the early evolution of bioenergetic systems . These approaches, still in their infancy, are necessarily directed to detection of 'molecular fossils' in diverse extant prokaryotes . Since the Earth was devoid of atmospheric oxygen during early cellular evolution, it is likely that 'precursor fragments' of the classical citric acid cycle are to be found in contemporary anaerobic bacteria . Accumulating evidence indicates that such fragments originally served biosynthetic roles of one kind or another, before they were recruited for assembly of the energy-yielding aerobic cycle . The extraordinary versatility of citric acid cycle intermediates and reactions for multiple uses raises the possibility that origination of the aerobic cycle, viewed as an evolutionary event, occurred more than once.

Biochem Soc Symp, 1987, 53, 39 - 49
The development of novel hepatitis B vaccines; Zuckerman AJ; The development of vaccines against hepatitis B has proceeded along four main lines . (i) Human plasma-derived vaccines are safe, effective, and in general use . (ii) Subunit polypeptide vaccines formulated in micelles have reached the stage of clinical trials . (iii) Recombinant DNA vaccines have been produced in prokaryotic and eukaryotic cells, notably in yeast . The yeast-derived recombinant vaccine has proved safe and effective in extensive clinical trials, eliciting antibodies of equal quantity and quality of specificity to those elicited by plasma-derived vaccine . DNA recombination has also been applied to the development of hybrid vaccinia virus vaccines which are capable of immunological 'priming' . (iv) Finally, chemical synthesis has succeeded in producing small peptides which include specific epitopes eliciting antibody responses in experimental animals.

Gene, 1987, 59(2-3), 223 - 30
Chemical synthesis and expression of the human calcitonin gene; Ivanov I et al.; A gene coding for the peptide hormone, human calcitonin (hCT), has been constructed and expressed in bacteria using a prokaryotic vector containing the strong T5P25 promoter and a strong ribosome-binding site . The bacterial hCT is different from the natural hCT by having an additional Met residue at the N terminus and a non-amidated C terminus . Despite these differences, the bacterial hCT is biologically active in rat cells . The results are important for future structure-function relationship studies using mutants constructed by genetic engineering.

Gene, 1987, 59(2-3), 191 - 200
T7 RNA polymerase can direct expression of influenza virus cap-binding protein (PB2) in Escherichia coli; Rosenberg AH et al.; Influenza virus cap-binding protein (PB2; Mr 85,000) is made in Escherichia coli when the cloned cDNA is transcribed by T7 RNA polymerase . Translation begins at the probable natural start codon and also from at least five internal sites in the same reading frame . The eukaryotic initiation site is not typical of protein initiation sites of E . coli, in that the closest potential Shine-Dalgarno sequence is far (15 nucleotides) from the start codon . Nevertheless, protein synthesis initiates efficiently at this site even in competition with a strong upstream prokaryotic initiation site . PB2 is somewhat unstable in the cell, but accumulates to a level where it is easily detectable in electrophoresis patterns of total cell protein . The full-length protein and various subfragments of it are insoluble in crude extracts, but have been useful for producing antibodies.

Ann N Y Acad Sci, 1987, 510, 9 - 15
Sensory transduction in flagellate bacteria; Armitage JP et al.; Flagellate bacteria can respond to a wide range of environmental chemicals and a variety of physical parameters, and integrate those responses . The most important thing for a cell is to maintain its energy level; bacteria therefore respond directly to any changes in their PMF . This has been likened to higher organisms responding to a physiological change, for example, a fall in blood glucose . In addition, if the PMF is high, the cell is free to respond to a limited range of metabolites and possibly move to an area that will allow an increased growth rate . Bacteria do not sense all amino acids, as the space available on the cytoplasmic membrane is limited, and a change in a few important metabolites is probably a good measure of the general environment around the cell . The sensory response does not require either transport into the cell or metabolism of the chemical, only the binding to the specific MCP . The cell could have a mutation in the pathway metabolizing the chemoeffector, but it would still respond to changes in the concentration of that compound . This taken with the ability of the cells to adapt to the stimulus has been considered to be the prokaryotic equivalent of smell and taste.

Gene, 1987, 57(1), 121 - 30
The complete nucleotide sequence of the left very early region of Escherichia coli bacteriophage PRD1 coding for the terminal protein and the DNA polymerase; Savilahti H et al.; DNA molecules replicating in a linear form have been found in certain viruses and plasmids of both prokaryotic and eukaryotic origin . Characteristic of this type of molecules are the proteins covalently linked to their 5' ends and inverted terminal nucleotide sequences . The molecules replicate via a protein-priming mechanism, where participants include terminal protein and a specific polymerase . We report here the nucleotide sequence of the left very early region of Escherichia coli bacteriophage PRD1 . This region codes for the terminal protein and the phage DNA polymerase . The predicted amino acid sequence of the terminal protein does not share homology with those of other known terminal proteins . The PRD1 DNA polymerase shows four regions of extensive homology to that of Bacillus subtilis phage phi 29 . One of these conserved regions is also found in several animal virus DNA polymerases.

Gene, 1987, 57(2-3), 171 - 81
Cloning and characterization of the isopenicillin N synthetase gene mediating the formation of the beta-lactam ring in Aspergillus nidulans; Ramon D et al.; Genomic clones containing an Aspergillus nidulans isopenicillin N synthetase (IPNS) gene have been identified by heterologous hybridization with a Cephalosporium acremonium DNA probe . The open reading frame encodes a 331 amino acid polypeptide with extensive homology with the genes of other beta-lactam-producing fungi . The gene product has been overexpressed in Escherichia coli and shown to have activity of IPNS . This represents the first evidence at the molecular level that the biosynthesis of penicillins in A . nidulans occurs by the same pathway as in other beta-lactam-producing microorganisms . Comparison of available nucleotide sequences from IPNS genes suggests a horizontal transmission of the gene between the prokaryotic beta-lactam producers of the genus Streptomyces and the filamentous fungi.

J Immunoassay, 1987, 8(4), 283 - 95
Slide immunoenzymatic assay (SIA): improving sensitivity to measure antibodies when samples are very small and dilute, and antigen is scarce; Conway de Macario E et al.; Modifications to the slide immunoenzymatic assay (SIA) using the PAP reagent (SIA-PAP) were developed which increase sensitivity considerably . These SIA modifications are particularly useful for measuring speedily antibodies in dilute samples available only in microliter-volumes when antigen is scarce, whether a molecule, or a prokaryotic or eukaryotic cell.

CRC Crit Rev Biochem, 1987, 22(2), 111 - 80
Aspects of the structure, function, and applications of superoxide dismutase; Bannister JV et al.; The current status of superoxide dismutase (SOD) is that it is an enzyme with diverse ramifications . This review attempts an understanding of SOD as a structural, functional, and biological entity . Accordingly, the review is in three parts . The first part discusses SOD in terms of protein structure, proceeding from primary to secondary and three-dimensional structure for the three forms of SOD: copper/zinc SOD, manganese SOD, and iron SOD . This is the order of structural knowledge of the enzyme . Iron SOD is an enzyme of prokaryotes and some higher plants . Manganese SOD is an enzyme of prokaryotes and eukaryotes . Copper/zinc SOD is an enzyme of eukaryotes and certain prokaryotes . The evolutionary relationships of the three forms of SOD, the status of the copper/zinc SOD gene in prokaryotes, and the cloning and sequencing of SOD genes are discussed . The second part of the review deals with the catalytic mechanism of SOD in the three forms of the enzyme . Structural and mechanistic conclusions from various spectroscopic studies are critically considered . A detailed picture is given of the active site of copper/zinc SOD . The third part is a review of SOD in the general context of oxygen toxicity . After consideration of the question of superoxide toxicity and superoxide pathology, several areas in which SOD has been investigated or used as a tool in a biochemical, pharmacological, or clinical context are discussed, including population genetics; trisomy 21; development and senescence; the nutritional copper, zinc, and manganese status; hemolysis and anemia; oxygen toxicity in the lung and nervous system; inflammation, autoimmune disease and chromosome breakage, ischemia and degenerative changes; radiation damage; and malignancy . A comprehensive picture is given of measurements of SOD activity in disease states, and the question of superoxide-related disease is considered at several points.

Gene, 1987, 55(2-3), 189 - 96
High-level secretion of human growth hormone by Escherichia coli; Chang CN et al.; A gene encoding the mature form of human growth hormone (hGH) was fused to the secretion signal coding sequence of the Escherichia coli heat-stable enterotoxin II (STII) . This hybrid gene was preceded by two Shine-Dalgarno sequences derived from the trp and STII-coding genes and was expressed in E . coli under the transcriptional control of the E . coli alkaline phosphatase (phoA) promoter . In low-phosphate growth media, cells synthesized about 15 to 25 micrograms of hGH/ml/1 A550 unit of cells . This represents 6 to 10% of total cellular protein . The majority of the hGH produced (more than 90%) was processed precisely and secreted into the periplasmic space . These results demonstrate that E . coli cells are able to synthesize and secrete high levels of this human protein using a prokaryotic signal sequence.

J Mol Evol, 1987, 26(4), 320 - 8
Endosymbiotic origin and codon bias of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize; Brinkmann H et al.; The nuclei of plant cells harbor genes for two types of glyceraldehyde-3-phosphate dehydrogenases (GAPDH) displaying a sequence divergence corresponding to the prokaryote/eukaryote separation . This strongly supports the endosymbiotic theory of chloroplast evolution and in particular the gene transfer hypothesis suggesting that the gene for the chloroplast enzyme, initially located in the genome of the endosymbiotic chloroplast progenitor, was transferred during the course of evolution into the nuclear genome of the endosymbiotic host . Codon usage in the gene for chloroplast GAPDH of maize is radically different from that employed by present-day chloroplasts and from that of the cytosolic (glycolytic) enzyme from the same cell . This reveals the presence of subcellular selective pressures which appear to be involved in the optimization of gene expression in the economically important graminaceous monocots.

J Mol Evol, 1987, 26(3), 252 - 6
Enzyme-coding genes as molecular clocks: the molecular evolution of animal alpha-amylases; Hickey DA et al.; We constructed a cDNA library for the beetle, Tribolium castaneum . This library was screened using a cloned amylase gene from Drosophila melanogaster as a molecular probe . Beetle amylase cDNA clones were isolated from this bank, and the nucleotide sequence was obtained for a cDNA clone with a coding capacity for 228 amino acids . Both the nucleotide sequence and predicted amino acid sequence were compared to our recent results for D . melanogaster alpha-amylases, along with published sequences for other alpha-amylases . The results show that animal alpha-amylases are highly conserved over their entire length . A broader comparison, which includes plant and microbial alpha-amylase sequences, indicates that parts of the gene are conserved between prokaryotes, plants, and animals . We discuss the potential importance of this and other enzyme-coding genes for the construction of molecular phylogenies and for the study of the general question of molecular clocks in evolution.

J Mol Evol, 1987, 25(4), 325 - 9
Evolution from primordial oligomeric repeats to modern coding sequences; Ohno S; It seems as though nature was most innovative at the very beginning of life on this Earth a few billion years ago . For example, the functional competence of most, if not all, of the sugar-metabolizing enzymes was clearly established before the division of eukaryotes from prokaryotes eons ago, each critical active-site amino acid sequence being conserved ever since by bacteria as well as by mammals . I contend that this initial innovativeness was due to the first set of coding sequences being repeats of base oligomers, thus encoding polypeptide chains of various periodicities; such periodical polypeptide chains can easily acquire alpha-helical and beta-sheet-forming segments . In fact, the entire length of sugar-metabolizing enzymes is comprised of alternating alpha-helical and beta-sheet-forming segments . In the prebiotic (therefore nonenzymatic) replication of nucleic acids, what was in short supply was long templates, for there apparently was no inherent obstacle in copying of long templates, if such existed, in the presence of Zn2+ . I submit that in this prebiotic condition, only those nucleotide oligomers that were internal doubles were automatically assured of progressive elongation to become long templates . For example, a decamer that was a pentameric repeat and its complementary sequence may pair unequally to initiate the next round of replication: first unit pairing with second, and a paired segment serving as a primer . As a consequence of this unequal pairing, decameric templates managed to become pentadecameric templates only after one round of replication, and this elongation process had no inherent limit.

Biosystems, 1987, 20(3), 275 - 88
Development of structural organization of protein-synthesizing machinery from prokaryotes to eukaryotes; Ryazanov AG et al.; Though the mechanisms of protein biosynthesis are similar in the cells of prokaryotes and eukaryotes, the eukaryotic translational machinery in the cell is arranged more intricately . One of the most striking characteristic features of the eukaryotic translational machinery is that the eukaryotic proteins involved in the translational process, such as initiation factors, elongation factors and aminoacyl-tRNA synthetases, in contrast to their prokaryotic analogs, possess a non-specific affinity for RNA . Due to the RNA-binding ability, these eukaryotic proteins can be compartmentalized on polyribosomes . In addition to the proteins of the translational apparatus, several other eukaryotic RNA-binding proteins can be also compartmentalized on polyribosomes; these proteins include glycolytic enzymes, steroid hormone receptors and intermediate filament proteins . Thus, the eukaryotic polyribosome is an element of the cytoplasmic labile structure on which various proteins can be compartmentalized and, consequently, different biochemical pathways can be integrated.

J Mol Evol, 1987, 24(3), 236 - 51
The secondary structure of human 28S rRNA: the structure and evolution of a mosaic rRNA gene; Gorski JL et al.; We have determined the secondary structure of the human 28S rRNA molecule based on comparative analysis of available eukaryotic cytoplasmic and prokaryotic large-rRNA gene sequences . Examination of large-rRNA sequences of both distantly and closely related species has enabled us to derive a structure that accounts both for highly conserved sequence tracts and for previously unanalyzed variable-sequence tracts that account for the evolutionary differences in size among the large rRNAs . Human 28S rRNA is composed of two different types of sequence tracts: conserved and variable . They differ in composition, degree of conservation, and evolution . The conserved regions demonstrate a striking constancy of size and sequence . We have confirmed that the conserved regions of large-rRNA molecules are capable of forming structures that are superimposable on one another . The variable regions contain the sequences responsible for the 83% increase in size of the human large-rRNA molecule over that of Escherichia coli . Their locations in the gene are maintained during evolution . They are G + C rich and largely nonhomologous, contain simple repetitive sequences, appear to evolve by frequent recombinational events, and are capable of forming large, stable hairpins . The secondary-structure model presented here is in close agreement with existing prokaryotic 23S rRNA secondary-structure models . The introduction of this model helps resolve differences between previously proposed prokaryotic and eukaryotic large-rRNA secondary-structure models.

Crit Rev Microbiol, 1987, 14(1), 1 - 48
Molecular aspects of nitrogen fixation by photosynthetic prokaryotes; Hallenbeck PC; The photosynthetic prokaryotes possess diverse metabolic capabilities, both in carrying out different types of photosynthesis and in their other growth modes . The nature of the coupling of these energy-generating processes with the basic metabolic demands of the cell, such as nitrogen fixation, has stimulated research for many years . In addition, nitrogen fixation by photosynthetic prokaryotes exhibits several unique features; the oxygen-evolving cyanobacteria have developed various strategies for protection of the oxygen-labile nitrogenase proteins, and some photosynthetic bacteria have been found to regulate their nitrogenase (N2ase) activity in a rapid response to fixed nitrogen, thus saving substantial amounts of energy . Recent advances in the biochemistry, physiology, and genetics of nitrogen fixation by cyanobacteria and photosynthetic bacteria are reviewed, with special emphasis on the unique features found in these organisms . Several major topics in cyanobacterial nitrogen fixation are reviewed . The isolation and characterization of N2ase and the isolation and sequence of N2ase structural genes have shown a great deal of similarity with other organisms . The possible pathways of electron flow to N2ase, the mechanisms of oxygen protection, and the control of nif expression and heterocyst differentiation will be discussed . Several recent advances in the physiology and biochemistry of nitrogen fixation by the photosynthetic bacteria are reviewed . Photosynthetic bacteria have been found to fix nitrogen microaerobically in darkness . The regulation of nif expression and possible pathways of electron flow to N2ase are discussed . The isolation of N2ase proteins, particularly the covalent modification of the Fe protein, the nature of the modifying group, properties of the activating enzyme, and regulating factors of the inactivation/activation process are reviewed.

EMBO J, 1987 Jan, 6(1), 139 - 44
Identification of early proteins of the human papilloma viruses type 16 (HPV 16) and type 18 (HPV 18) in cervical carcinoma cells; Seedorf K et al.; We have sequenced 1730 bp of human papilloma virus type 18 (HPV 18) DNA containing the open reading frames (ORF) E6, E7, the N-terminal part of E1 and, additionally, 120 bp of the N-terminal part of L1 . Based on these sequencing data, together with the human papilloma virus type 16 (HPV 16) DNA sequence published recently, we identified and cloned the ORF E6, E7, E1 and L1 of HPV 18 and the ORF E6, E7, E1, E4, E5, L2 and L1 of HPV 16 into prokaryotic expression vectors . The expression system used provides fusions to the N-terminal part of the MS2 polymerase gene controlled by the heat-inducible lambda PL promoter . Using the purified fusion proteins as immunogens we raised antisera against the proteins encoded by the ORF E6, E7 and E1 of HPV 18 as well as those encoded by the ORF E6, E7, E4 and L1 of HPV 16 . By Western blot analysis we could show that the E7 gene product is the most abundant protein in cell lines containing HPV 16 or HPV 18 DNA . It is a cytoplasmic protein of 15 kd in the SiHa and the CaSki cell lines which contain HPV 16 DNA, and 12 kd in the HeLa, the C4-1 and the SW756 cell lines which contain HPV 18 DNA . These results were confirmed by in vitro translation of hybrid-selected HPV 16 and HPV 18 specific poly(A)+ RNA from SiHa, CaSki and HeLa cells . Additionally, these experiments led to the identification of an 11-kd E6 and a 10-kd E4 protein in the CaSki cell line as well as a 70-kd E1 protein in HeLa cells.

Toxicol Pathol, 1987, 15(1), 82 - 7
Alteration of DNA tertiary structure by physical and chemical carcinogens: involvement in DNA repair processes; Spadari S et al.; Parameters defining the topological state of DNA seem extremely important for describing the reactive state of the same DNA molecules . We have shown that physical and chemical DNA modifying agents alter the tertiary structure of DNA molecules . Variations in the tertiary structure of DNA were studied by one dimensional electrophoresis on an agarose gel of partially relaxed plasmid DNA topoisomers, a technique allowing the measurement of alterations in the degree of supercoiling equivalent to fractions of superhelical turns . Unwinding angles of -10.1 degrees or -8.7 degrees per pyrimidine or thymine dimer respectively, of -12 degrees per apurinic site, and of -3.4 degrees per methylated purine were obtained by titrating the number of damaged sites necessary to reduce the number of superhelical turns by one in each topoisomer . On the contrary, enzymatic methylation of the C-5 position of cytosine (a modified base present in prokaryotic and eukaryotic DNAs) did not alter the DNA tertiary structure . We have also shown that local alterations in DNA structure caused by UV-irradiation inhibit bacterial DNA topoisomerase I and DNA methylase, and that the topological state of DNA substrate influences the mode of methylation of Hpa II DNA methylase . These findings suggest that the natural topological state of DNA substrate (linear, relaxed, or covalently closed duplex DNA with varying degrees of supercoiling) influences the mode of action of enzymes possibly involved in DNA repair processes, while DNA structural alterations caused by DNA modifying agents might influence DNA repair processes in two ways: either by driving the interaction between repair enzymes and the modified sites of DNA, or by inhibiting or changing the mode of action of enzymes normally acting on unmodified DNA.

Gene, 1987, 61(3), 373 - 83
Evidence for prokaryotic transcription and translation control regions in the human factor IX gene; Simpson PJ et al.; A human factor IX cDNA clone isolated from a liver cDNA library constructed in phage lambda gt11 vector was shown to express factor IX protein in Escherichia coli . A factor IX immunospecific protein of 46.8 kDa was expressed, but was not a beta-galactosidase-factor IX fusion protein . Expression was seen when the factor IX cDNA was cloned into two different vector systems, lambda gt11 and pUC9, in both orientations with respect to the vector lacZ promoter . The expression of factor IX was not under control of the lacZ promoter of either vector system . In addition, when the factor IX cDNA fragment was subcloned in both orientations into a promoterless cloning vector (p CPP3), the factor IX cDNA fragment demonstrated promoter activity when inserted in only one orientation resulting in expression of chloramphenicol acetyl transferase in E . coli and Bacillus subtilis . A DNA computer search of the N-terminal sequences of the factor IX gene revealed prokaryotic-like promoter and ribosome-binding site (RBS) sequences with strong homology to the E . coli consensus sequences . The predicted sites homologous with prokaryotic promoter and RBS consensus sequences are followed by an in-frame methionine which could correspond to the translation start codon of the expressed factor IX . This report provides the first evidence that a eukaryotic gene encodes the information necessary for both transcription and translation to control gene expression in a prokaryotic host.

Gene, 1987, 53(2-3), 173 - 80
Expression of adenovirus type 12 E1b 58-kDa protein in Escherichia coli and production of antibodies raised against a 58-kDa::beta-galactosidase fusion protein; Schughart K et al.; DNA fragments coding for the N-terminal 185 amino acids (aa) and for the entire coding region of the adenovirus (Ad)12 E1b 58-kDa protein have been cloned in a prokaryotic expression vector . The N-terminal region of the 58-kDa viral protein (aa 21-205) is expressed as a beta-galactosidase (beta Gal) fusion protein encoded by plasmid pB58Ngal . Escherichia coli strains transformed with this plasmid synthesize a full-length fusion protein of 150-kDa and two truncated proteins: a 140-kDa protein containing aa 64-205 and a 120-kDa polypeptide containing aa 158-205 of the E1b 58-kDa protein . Antibodies raised against purified fusion proteins specifically immunoprecipitate the E1b 58-kDa protein from Ad12-infected and transformed cells . Bacteria transformed with plasmid pB58 carrying the entire E1b 58-kDa coding region (minus the first N-terminal 20 aa which are replaced by 4 aa of beta Gal) showed dramatically reduced growth properties after induction of 58K gene expression . We have not been able to detect substantial amounts of the 58-kDa protein in these cells . However, the viral 58-kDa polypeptide could be synthesized in vitro from plasmid pB58 in a DNA-dependent translation system from E . coli.

Hepatology, 1987 Jan-Feb, 7(1 Suppl), 30S - 35S
The pAS vector system and its application to heterologous gene expression in Escherichia coli; Shatzman AR et al.; There are numerous proteins of biological interest which cannot be obtained from natural sources in quantities sufficient for detailed biochemical and physical analysis . The limited bioavailability of these molecules has made it impossible to consider their potential utilization as either pharmacological agents and/or targets . One solution to this problem has been the development of recombinant vector systems which are designed to achieve efficient expression of cloned genes in a variety of biological systems . This paper will describe the development and application of a particular set of vectors which have been designed to achieve efficient expression of essentially any gene coding sequence in Escherichia coli . The system utilizes efficient phage-derived transcriptional and translational regulatory signals and provides a strong regulatable promoter, an antitermination mechanism to ensure efficient transcription across any gene insert, high stability and, when appropriate, efficient translation initiation information . In addition, a wide variety of host strains have been developed in order to help control, stabilize and maximize expression of various cloned genes . The system has now been used to express efficiently more than 75 different prokaryotic and eukaryotic gene products . The application of this system to the expression and characterization of several oncogene products will be described.

Curr Genet, 1987, 12(4), 263 - 9
Structure and transcription of the 5S rRNA gene from spinach chloroplasts; Audren H et al.; The nucleotide sequence of the spinach chloroplast 5S rRNA gene and its flanking regions has been determined . A prokaryotic type promoter is to be found upstream of the 5S rRNA gene . Northern blot experiments with selected gene probes show that the 5S gene is co-transcribed with the other ribosomal genes of the operon . This result is confirmed by 5' S1 mapping of in vivo RNAs synthesised in chloroplasts or in an E . coli strain harboring a multicopy plasmid containing the 5S rRNA gene and its flanking regions . In vitro transcription experiments show that initiation of transcription does not occur at the level of the putative 5S rRNA gene promoter . Therefore, we conclude that the 5S rRNA is synthesized only be co-transcription of its gene with the other ribosomal genes of the operon . 3' S1 nuclease mapping in the spacer region between the 4.5S and the 5S rRNA genes reveals a set of protected fragments located in an A.T rich region downstream of a very stable hairpin and immediately upstream of the putative 5S promoter . This result is interpreted by the presence of preterminated transcripts or processing sites in this region.

Curr Genet, 1987, 12(3), 225 - 9
Neurospora crassa nuclear genome contains analogy of Saccharomyces cerevisiae genes for ribosomal RNA processing; Dutta SK et al.; Neurospora crassa wild type genome shows DNA sequences which are homologous to the sequences present in the rRNA processing genes of the yeast Saccharomyces cerevisiae . Five such processing genes from yeast, viz., RNA1 through RNA5, cloned in plasmid pBR322 were transformed in Escherichia coli strain LE392 . Southern blots containing DNAs from these clones were restricted with several restriction endonucleases along with DNAs from lambda phage, rice (plant) and neuroblastoma (animal), were hybridized with 32P-labelled nick-translated N . crassa nuclear DNA under very stringent conditions . Autoradiograms of these blots revealed that four yeast rRNA processing genes (RNA1, RNA2, RNA3, and RNA4) showed homology with N . crassa nuclear DNA but such analogs were not found in DNAs representing prokaryotes, phages, higher plants and animals.

Gene, 1987, 60(2-3), 217 - 25
Characterisation of the 5'-leader sequence of tobacco mosaic virus RNA as a general enhancer of translation in vitro; Sleat DE et al.; Uncapped messenger RNAs (mRNAs) encoding calf preprochymosin, chicken prelysozyme, or Escherichia coli beta-glucuronidase (GUS) were synthesized in vitro, with or without a 5'-terminal 67-nucleotide sequence (omega') derived from the untranslated 5'-leader (omega) of tobacco mosaic virus (TMV) RNA . Messenger RNAs were translated in vitro, in messenger-dependent systems derived from rabbit reticulocytes (MDL), wheat-germ (WG) or E . coli (EC) . The omega' sequence enhanced expression of each mRNA in almost every translation system . While MDL was the least responsive to omega', this sequence proved particularly efficient in permitting translation of the eukaryotic mRNAs in EC, despite the absence of a consensus Shine-Dalgarno sequence in either the mRNAs or omega' . The local context of the initiation codon (AUG) in two GUS mRNA constructs did not influence the relative enhancement caused by the omega' sequence . These findings extend the utility of omega' as a general enhancer of translation for both prokaryotic and eukaryotic mRNAs in either 80S- or 70S-ribosome-based systems.

Gene, 1987, 58(1), 45 - 57
Nucleotide sequence homologies in control regions of prokaryotic genomes; Studnicka GM; Functional recognition sites for several regulatory factors, including RNA polymerase, cyclic adenosine monophosphate receptor protein and ribosomes, do not always have strong consensus nucleotide sequence homology, yet they are capable of biological activity . Using the computer, other nucleotide sequences can be found that have equal or significantly greater consensus homology, but whose biological function has not been characterized . This analysis shows that no arbitrary 'cutoff score' can successfully distinguish active recognition sites from uncharacterized homologies, due to the great natural diversity in the strength and conservation of functional sites . It also predicts that the strong 'cryptic' homologies presented here are of two types: some might already have a biological function which has so far not been detected, whereas certain single-point mutations might be able to confer activity upon the others by correcting a key structural defect.

Gene, 1987, 57(2-3), 159 - 69
Expression of the human alpha-galactosidase A in Escherichia coli K-12; Hantzopoulos PA et al.; We used the prokaryotic expression vector, ptrpL1, for the expression in Escherichia coli K-12 of a cDNA clone specific for the human lysosomal hydrolase, alpha-galactosidase A . The 5' terminus of the cDNA clone was engineered so that an ATG codon precedes the first codon of the mature form of the enzyme . A clone with elevated expression of this human enzyme was constructed by increasing the distance between the Shine-Dalgarno site and the ATG start codon from 6 to 8 bp . Clones with alpha-galactosidase A specific cDNA encoding the proenzyme produce a protein of 45 kDa, the size expected for the intact proenzyme . The 45-kDa protein is specifically precipitated by antibody to alpha-galactosidase A, and its expression is repressed by tryptophan and induced by 3-beta-indoleacrylic acid as expected for this expression vector . The human enzyme is produced in E . coli in a catalytically active form at levels sufficient to support the growth of cells using alpha-galactosides as sole sources of carbon and energy . In addition, bacterial colonies that produce the human enzyme turn blue in the presence of 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside.

Gene, 1987, 60(1), 29 - 37
Transcriptional analysis of the cyanobacterial gvpABC operon in differentiated cells: occurrence of an antisense RNA complementary to three overlapping transcripts; Csiszar K et al.; Cyanobacteria are photosynthetic prokaryotes able to colonize almost all kinds of ecosystems . Some of them exhibit differentiation processes and/or may establish tight symbiotic associations . Upon changes in the environmental conditions, the cyanobacterium Calothrix 7601 differentiates hormogonia which are short filaments of small cells resulting from cellular division and fragmentation of the long filaments of vegetative cells . In Calothrix, hormogonia are characterized by their gliding motility and by a massive production of gas vesicles which confer buoyancy . At least four genes are involved in the formation of gas vesicles, three of which are organized in one operon (gvpABC) . Four different RNA species, only present after induction of hormogonia differentiation, result from transcription of this operon . Mapping of the 5' and 3' ends of these transcripts demonstrates the presence of gvpA, gvpAB and gvpABC transcripts, all three having the same 5' end . Each of the three transcripts terminates a few bases downstream from stem-and-loop structures . Most interestingly, the fourth transcript is an antisense RNA starting from the 3' end of the gvpB gene and ending within the gvpA gene . This antisense RNA can thus form an homologous duplex with the three other transcripts, thereby being able to impair translation and/or modify mRNA stability.

Biosystems, 1987, 21(1), 51 - 68
Evolution of the mitochondrial protein synthetic machinery; Benne R et al.; Comparative analysis of the components of the mitochondrial translational apparatus reveals a remarkable variability . For example the mitochondrial ribosomal rRNAs, display a three-fold difference in size in different organisms as a result of insertions or deletions, which affect specific areas of the rRNA molecule . This suggests that such areas are either not essential for mitoribosome function or that they can be replaced by proteins . Also mitochondrial tRNAs and mitoribosomal proteins are much less conserved than their cytoplasmic counterparts . Not only do the mitochondrial translational molecules vary in properties, also the location of the genes from which they are derived is not the same in all cases: mitochondrial tRNA genes which usually are found in the mtDNA, may have a nuclear location in protozoa and, conversely, only in fungi one finds a mitoribosomal protein gene in the organellar genome . The high rate of change of the components of the mitochondrial protein synthesizing machinery is accompanied by a number of unique features of the translation process: (i) the mitochondrial genetic code differs substantially from the standard code in a species-specific manner; (ii) special codon-anticodon recognition rules are followed; (iii) unusual mechanisms of translational initiation may exist . These observations suggest that the evolutionary pressures that have shaped the present day mitochondrial translational apparatus have been different in different organisms and also distinct from those acting on the cytoplasmic machinery . In spite of the interspecies variability, however, many features of the mitochondrial and bacterial protein synthetic apparatus show a clear resemblance, providing support for the hypothesis of a prokaryotic endosymbiont ancestry of mitochondria.

Development, 1987 Jan, 99(1), 15 - 23
Distribution, expression and germ line transmission of exogenous DNA sequences following microinjection into Xenopus laevis eggs; Etkin LD et al.; We analysed the fate, expression and germ line transmission of exogenous DNA which was microinjected into fertilized eggs of Xenopus laevis . DNA was injected into fertilized eggs within 1 h following fertilization . The injected DNA was dispersed around the site of injection and became localized to cleavage nuclei by stage 6 . Injected DNA persisted in the tissues of 6- to 8-month-old frogs and exhibited a mosaic pattern of distribution with regard to the presence or absence and copy number between different tissues . We detected the exogenous DNA sequences in 60% of injected frogs . Restriction digestion analysis of this DNA suggested that it is not rearranged and was organized as head-to-tail multimers . The copy number varied from 2 to 30 copies/cell in various tissues of the same frog . Plasmid pSV2CAT which contains the prokaryotic gene coding for chloramphenicol acetyl transferase (CAT) enzyme linked to the SV40 early gene promoter was expressed in 50% of the animals containing the gene . The pattern of expression, however, varied between different animals and could not be correlated with copy number . We also showed that the exogenous DNA sequences were transmitted through the male germ line and that each offspring contained the gene integrated into a different region of the genome.

Nucleic Acids Res, 1986 Dec 22, 14(24), 9843 - 60
Methylated DNA-binding protein from human placenta recognizes specific methylated sites on several prokaryotic DNAs; Wang RY et al.; Methylated DNA-binding protein (MDBP) from human placenta recognizes specific DNA sequences containing 5-methylcytosine (m5C) residues . Comparisons of binding of various prokaryotic DNAs to MDBP indicate that m5CpG is present in the recognition sites for this protein but is only part of the recognition sequence . Specific binding to MDBP was observed for bacteriophage XP12 DNA, which naturally contains approximately 1/3 of its residues as m5C, and for Micrococcus luteus DNA, M13mp8 replicative form (RF) DNA, and pBR322 when these three DNAs were methylated at CpG sites by human DNA methyltransferase . Five DNA regions binding to MDBP have been localized by DNase I footprinting or restriction mapping in methylated pBR322 and M13mp8 RF DNAs . A comparison of their sequences reveals a common 5'-m5CGRm5CG-3' element or closely related sequence in which one of the m5C residues may be replaced by a T . In addition to this motif, one upstream and one downstream m5CpG as well as other common residues over an approximately 20-bp long region may be recognized by MDBP.

Eur J Biochem, 1986 Dec 15, 161(3), 635 - 45
Characterization of the elongation factors from calf brain . 1 . Purification, molecular and immunological properties; Crechet JB et al.; This work describes a method for the purification of the elongation factors (EF) from calf brain . The elongation factor responsible for the binding of aminoacyl-tRNA to the ribosome is found in this organ as a light form (EF-1 alpha) and as a component of heavy, polydispersed aggregates (EF-1H) . EF-1 beta, the factor enhancing the EF-1 alpha GDP/GTP exchange, is part of EF-1H and of smaller aggregates . The fraction of EF-1 alpha and EF-1 beta not associated with EF-1H, and EF-2 have been purified to homogeneity after several chromatographic steps . EF-1H consists of many proteins; among them, EF-1 alpha, EF-1 beta and an EF-1 gamma-like protein represent three of the major components . This conclusively shows that EF-1H from calf brain is not a polydispersed aggregate of only EF-1 alpha . EF-1 beta has also been purified to homogeneity from EF-1H . The property of EF-1 beta to aggregate with other proteins suggests that this factor plays an important role in the organization of EF-1H . The relative molecular mass of the purified factors have been determined as: EF-1 alpha, 50,000; EF-1 beta, 30,000; the EF-1 gamma-like component, 49,000; EF-2, 85,000 . Some cross-reactivity with the antibodies against the prokaryotic counterparts has been shown for EF-1 alpha, EF-1 beta and EF-2 by functional and immuno-precipitation methods, suggesting the existence of structural homologies.

Brain Res, 1986 Dec, 395(2), 262 - 7
The 5B4 antigen expressed on sprouting neurons contains alpha-2,8-linked polysialic acid; Rosenberg J et al.; Monoclonal antibody 5B4 recognizes a large (approximately 185,000-255,000 Da) developmentally regulated membrane glycoprotein, whose expression on fetal rat neurons is coincident with neuronal sprouting . By the use of two prokaryotic-derived probes specific for recognizing alpha-2,8-linked polysialosyl units, we demonstrate the presence of this unusual carbohydrate moiety (a characteristic of neural cell adhesion molecules, NCAMs) on the fetal form of the 5B4 antigen . The 5B4 antigens expressed in adult rat brain (approximately 140,000 and 180,000 Da) do not contain polysialic acid.

Proc Natl Acad Sci U S A, 1986 Dec, 83(24), 9551 - 5
Direct introduction of genes into rats and expression of the genes; Benvenisty N et al.; A method of introducing actively expressed genes into intact mammals is described . DNA precipitated with calcium phosphate has been injected intraperitoneally into newborn rats . The injected genes have been taken up and expressed by the animal tissues . To examine the generality of the method we have injected newborn rats with the chloramphenicol acetyltransferase prokaryotic gene fused with various viral and cellular gene promoters and the gene for hepatitis B surface antigen, and we observed appearance of chloramphenicol acetyltransferase activity and hepatitis B surface antigen in liver and spleen . In addition, administration of genes coding for hormones (insulin or growth hormone) resulted in their expression.

Comput Appl Biosci, 1986 Dec, 2(4), 269 - 75
Quantitative computer analysis of signal sequence homologies in DNA; Studnicka GM; Homologies to prokaryotic recognition sites for RNA polymerase, ribosomes, and cyclic-AMP receptor protein (CRP), are analyzed by a new computer program using weighting factors to account for the statistical variation at each position of the consensus . Known signal sequence sites are easily detected by this algorithm, and other sites with equally strong homology are found whose biological function is still unknown . Some sites are biologically active even though they have very weak homology . No arbitrary 'cutoff score' can distinguish active recognition sites from inactive homologies; experiments must determine why certain weak homologies are able to function while others are not.

Proc Natl Acad Sci U S A, 1986 Dec, 83(23), 8849 - 53
A GTP-binding protein of Escherichia coli has homology to yeast RAS proteins; Ahnn J et al.; The DNA sequence of a gene (era) located immediately downstream of the gene (rnc) encoding ribonuclease III of Escherichia coli was determined and found to encode a protein of 316 amino acid residues . The amino acid sequence of this protein, Era, has significant similarity to the yeast RAS proteins . Overexpression of the Era protein was achieved and GTP cross-linking experiments demonstrated that the protein was indeed capable of binding GTP, as are the yeast and mammalian ras gene products . These data indicate that ras-related sequences occur not only in eukaryotes but also in prokaryotes.

EMBO J, 1986 Dec 1, 5(12), 3305 - 11
Novobiocin inhibits passive chromatin assembly in vitro; Sealy L et al.; Novobiocin, an inhibitor of prokaryotic DNA gyrase and eukaryotic type II topoisomerase enzymes, interferes with in vitro chromatin assembly using purified histones, DNA and nucleoplasmin . The target of inhibition is not topoisomerase II; this energy-independent assembly system lacks any ATP and Mg2+-dependent type II topoisomerase or gyrase activities . Rather, novobiocin interacts with histones, disrupting histone-histone associations required for octamer formation, and causing histones to precipitate from both nucleoplasmin-histone and histone-DNA complexes . Thus, novobiocin is able to generate 'dynamic' chromatin in vitro in the absence of ATP and Mg2+ by removing histones from previously assembled static chromatin, so that the DNA supercoils, previously constrained by conventional nucleosomes, become susceptible to removal by topoisomerase I.

J Cell Biol, 1986 Dec, 103(6 Pt 1), 2263 - 72
The NH2 terminus of preproinsulin directs the translocation and glycosylation of a bacterial cytoplasmic protein by mammalian microsomal membranes; Eskridge EM et al.; To investigate putative sorting domains in precursors to polypeptide hormones, we have constructed fusion proteins between the amino terminus of preproinsulin (ppI) and the bacterial cytoplasmic enzyme chloramphenicol acetyltransferase (CAT) . Our aim is to identify sequences in ppI, other than the signal peptide, that are necessary to mediate the intracellular sorting and secretion of the bacterial enzyme . Here we describe the in vitro translation of mRNAs encoding two chimeric molecules containing 71 and 38 residues, respectively, of the ppI NH2 terminus fused to the complete CAT sequence . The ppI signal peptide and 14 residues of the B-chain were sufficient to direct the translocation and segregation of CAT into microsomal membrane vesicles . Furthermore, the CAT enzyme underwent N-linked glycosylation, presumably at a single cryptic site, with an efficiency that was comparable to that of native glycoproteins synthesized in vitro . Partial amino-terminal sequencing demonstrated that the downstream sequences in the fusion proteins did not alter the specificity of signal peptidase, hence cleavage of the ppI signal peptide occurred at precisely the same site as in the native precursor . This is in contrast to results found in prokaryotic systems . These data demonstrate that the first 38 residues of ppI encode all the information necessary for binding to the endoplasmic reticulum membrane, translocation, and proteolytic (signal sequence) processing.

Nucleic Acids Res, 1986 Nov 25, 14(22), 8829 - 44
Upstream regulatory sequences of immunoglobulin genes are recognized by nuclear proteins which also bind to other gene regions; Mocikat R et al.; The decanucleotide sequence (dc) TNATTTGCAT is an upstream regulatory sequence of immunoglobulin genes and occurs also upstream of certain other eukaryotic and prokaryotic genes (compiled in the accompanying paper) . We now investigated the binding of proteins from nuclear extracts of a number of cell types and organisms to the dc sequence using a sensitive gel electrophoretic DNA binding assay . Binding studies with specifically designed oligonucleotides led to the following conclusions: the central T of the dc sequence can be altered with only a slight decrease of protein binding activity: the sequences in the neighborhood of dc have a positive or negative effect on the efficiency of protein binding; C-rich sequences which occur in many K chain promoters have a protein binding activity independent of dc; the dc binding protein(s) of human lymphoid cells elute from a Sephadex column in the 30.000-60.000 molecular weight range; dc binding proteins were found in nuclear extracts of lymphoid as well as non-lymphoid human and murine cell lines, of Xenopus oocytes, and of yeast cells . The finding of dc binding proteins in a wide variety of different organisms and the occurrence of dc-related sequences in the regulatory regions of several gene families point to a general role in the transcriptional regulation of the respective genes.

Nucleic Acids Res, 1986 Nov 25, 14(22), 8819 - 27
Sequences closely related to an immunoglobulin gene promoter/enhancer element occur also upstream of other eukaryotic and of prokaryotic genes; Falkner FG et al.; Decanucleotide sequences closely related to the TNATTTGCAT element which occurs upstream of the immunoglobulin genes and in the immunoglobulin gene enhancer were found also upstream of other eukaryotic and of prokaryotic genes . The possibility of evolutionary and functional relationships between the various transcriptional systems is discussed.

J Mol Biol, 1986 Nov 20, 192(2), 287 - 90
Net N-C charge imbalance may be important for signal sequence function in bacteria; von Heijne G; The net charge distribution in a region around the signal sequence cleavage site has been calculated for samples of 41 prokaryotic and 165 eukaryotic exported proteins . The results show that prokaryotic proteins in particular have a markedly higher incidence of acidic than of basic residues in this region . The possibility that a "dipolar" structure with a positive net charge difference between the N and C-terminal regions is important for signal sequence function in bacteria is suggested, and invoked to rationalize a number of known export-defective signal sequence mutations.

Biochem Biophys Res Commun, 1986 Nov 14, 140(3), 916 - 23
Structural determination of a new naturally occurring cyclic vitamin K; Howarth OW et al.; The respiratory quinone composition of 5 members of the genus Nocardia was examined . All species contained a hitherto unknown cyclic vitamin K-like molecule as their predominant lipoquinone . On the basis of mass spectrometry, 1H and 13C nuclear magnetic resonance spectrometry, the novel quinone was shown to correspond to (2E, 14E, 18E, 22E)-2-{3,7, 11,15,19,23-hexamethyl-25-(2,6,6-trimethylcyclohex-2- enyl)pentacosa-2, 14,18,22-tetraenyl}-3-methyl-1,4-naphthoquinone . Results indicate this molecule may represent a valuable phylogenetic marker for the prokaryote genus Nocardia.

Nature, 1986 Nov 6-12, 324(6092), 87 - 9
Bent DNA at a yeast autonomously replicating sequence; Snyder M et al.; DNA fragments that show retarded electrophoretic mobility through polyacrylamide gels have been found in both prokaryotes and eukaryotes . In the case of kinetoplast DNA, evidence has been presented that the DNA is curved or 'bent' . Bent DNA has previously been found at the lambda and simian virus 40 (SV40) DNA replication origins . Here we show the existence of bent DNA at a yeast autonomously replicating sequence (ARS1), a putative replication origin . The bent DNA has been localized to a 40-55 base pair (bp) segment and contains six (A)3-5 stretches (that is, six poly(A) stretches, three to five nucleotides in length) phased approximately every 10.5 bp . This region contains a DNA binding site for a yeast protein factor . This site lies at the 3' end of the TRP1 gene, in a region devoid of nucleosomes, and is positioned 80 bp away from the ARS consensus sequence; removal of this region impairs ARS function in vivo . The bent DNA may be involved in transcription termination or the prevention of nucleosome assembly in this region.

J Med Chem, 1986 Nov, 29(11), 2400 - 3
Carbocyclic puromycin: synthesis and inhibition of protein biosynthesis; Vince R et al.; The carbocyclic analogue of puromycin was prepared by the coupling of N-(benzyloxycarbonyl)-p-methoxy-L-phenylalanine to the racemic aminonucleoside (+/-)-9-{3 beta-amino-2 beta-hydroxy-4 alpha-(hydroxymethyl)cyclopent-1 alpha-yl}-6-(dimethylamino)purine, followed by separation of the diastereomers and subsequent removal of the Cbz blocking group . Kinetic studies indicate that carbocyclic puromycin is an excellent substrate for the peptidyltransferase reaction with both prokaryotic and eukaryotic ribosomes . A comparison of carbocyclic puromycin with previously synthesized analogues indicate that the furanosyl ring oxygen and the hydroxymethyl group of puromycin do not contribute to ribosomal binding, but both moieties contribute to the rate of product formation from the enzyme-substrate complex . Carbocyclic puromycin was equal to puromycin when evaluated for cytotoxicity using P-388 mouse lymphoid leukemia cells in culture.

FEBS Lett, 1986 Oct 27, 207(2), 198 - 204
Mechanism of translational initiation in prokaryotes . Evidence for a direct effect of IF2 on the activity of the 30 S ribosomal subunit; Canonaco MA et al.; Initiation factor IF2 from either Escherichia coli or Bacillus stearothermophilus was found to possess the previously undetected property of stimulating the template-dependent ribosomal binding of aminoacyl-tRNAs with free alpha-NH2 groups . IF1, which had no detectable activity alone, was found to stimulate the activity of E . coli IF2 and, to a lesser extent, that of B . stearothermophilus IF2 . Since in the absence of ribosomes not even a weak interaction between the two IF2 molecules and the aminoacyl-tRNAs was detected, the present findings indicate that IF2 can act at the ribosomal level stimulating aminoacyl-tRNA binding without prior formation of a binary complex with the aminoacyl-tRNA . IF2 does not appear to open or strengthen a weak A-site binding, but rather to enhance aminoacyl-tRNA binding to a 30 S site equivalent to the P-site by slowing down the rate of aminoacyl-tRNA dissociation from ribosomes.

Nucleic Acids Res, 1986 Oct 24, 14(20), 7915 - 27
Tn7 transposition: a multigene process . Identification of a regulatory gene product; Smith GM et al.; Tn7 transposition is abolished by the deletion of a 2.2kb HindIII fragment from the central region of the transposon . Transposition is restored when the fragment is present in trans . When this fragment is present in trans with wild-type Tn7, transposition frequencies are stimulated 10-100-fold . The DNA sequence of this fragment has been determined and found to contain one long open reading frame coding for a protein of molecular weight 61 187 . We have visualised this protein using a DNA-directed prokaryotic transcription-translation system . This gene may fill a regulatory role in the mechanism of Tn7 transposition . When present in trans the 2.2kb HindIII fragment alleviates a transcriptional block of promoter activity detected in Tn7.

J Biol Chem, 1986 Oct 15, 261(29), 13389 - 92
A cDNA encoding protein kinase C identifies two species of mRNA in brain and GH3 cells; Makowske M et al.; Antiserum raised against purified protein kinase C (the Ca2+/phospholipid-dependent enzyme) (Ballester, R., and rosen, O . M . (1985) J . Biol . Chem . 260, 15194-15199) was used to screen a rat brain cDNA library in the prokaryotic expression vector lambda gt11 . Three positive clones were isolated and shown to have overlapping restriction endonuclease maps . The positive recombinant phage with the longest cDNA insert (1.4 kilobases (kb)) was used for production of a beta-galactosidase fusion protein . Rabbit antiserum raised against the fusion protein recognized a single rat brain polypeptide of Mr 80,000 which was identified as protein kinase C by the following criteria: electrophoretic co-migration with purified protein kinase C, partial co-purification with protein kinase C, and disappearance from the cytosol of phorbol 12-myristate 13-acetate-treated GH3 cells . The nick-translated cDNA hybridized with two mRNAs, 8 kb and 3.5 kb, whose tissue distribution was in agreement with that reported for protein kinase C activity . Hybrid selection with immobilized cDNA identified mRNA encoding a protein of Mr 80,000 that could be precipitated by antibody to purified protein kinase C . Treatment of GH3 cells with phorbol 12-myristate 13-acetate, which promotes translocation and subsequent degradation of protein kinase C, did not alter the level of either message.

Philos Trans R Soc Lond B Biol Sci, 1986 Oct 14, 313(1162), 347 - 58
Interaction, functional relations and evolution of large and small subunits in Rubisco from prokaryota and eukaryota; McFadden BA et al.; In early biological evolution anoxygenic photosynthetic bacteria may have been established through the acquisition of ribulose bisphosphate carboxylase-oxygenase (Rubisco) . The establishment of cyanobacteria may have followed and led to the production of atmospheric oxygen . It has been postulated that a unicellular cyanobacterium evolved to cyanelles which were evolutionary precursors of chloroplasts of both green and non-green algae . The latter probably diverged from ancestors of green algae as evidenced by the occurrence of large (L) and small (S) subunit genes for Rubisco in the chloroplast genome of the chromophytic algae Olisthodiscus luteus . In contrast, the gene for the S subunit was integrated into the nucleus in the evolution of green algae and higher plants . The evolutionary advantages of this integration are uncertain because the function of S subunits is unknown . Recently, two forms of Rubisco (L8 and L8S8) of almost equivalent carboxylase and oxygenase activity have been isolated from the photosynthetic bacterium Chromatium vinosum . This observation perpetuates the enigma of S subunit function . Current breakthroughs are imminent, however, in our understanding of the function of catalytic L subunits because of the application of deoxyoligonucleotide-directed mutagenesis . Especially interesting mutated Rubisco molecules may have either enhanced carboxylase activity or higher carboxylase:oxygenase ratios . Tests of expression, however, must await the insertion of modified genes into the nucleus and chloroplasts . Methodology to accomplish chloroplast transformation is as yet unavailable . Recently, we have obtained the first transformation of cyanobacteria by a colE1 plasmid . We regard this transformation as an appropriate model for chloroplast transformation.

Science, 1986 Oct 10, 234(4773), 200 - 3
Brominating oxidants generated by human eosinophils; Weiss SJ et al.; Eosinophils are white blood cells that in humans are found in association with helminthic infections and various inflammatory disease processes . These cells contain a unique lysosomal peroxidase that oxidizes halides to generate highly reactive and toxic hypohalous acids . Although chloride is found in vivo at concentrations at least 1000-fold greater than those of other halides, human eosinophils did not preferentially oxidize chloride under physiologic conditions . Instead, eosinophils used bromide, a halide with a hitherto unknown function in humans, to generate a halogenating oxidant with characteristics similar, if not identical, to those of hypobromous acid . These results indicate that physiological concentrations of bromide arm human eosinophils with the ability to generate and release an unusual oxidant capable of destroying a wide range of prokaryotic and eukaryotic targets.

Science, 1986 Oct 10, 234(4773), 179 - 86
In vivo half-life of a protein is a function of its amino-terminal residue; Bachmair A et al.; When a chimeric gene encoding a ubiquitin-beta-galactosidase fusion protein is expressed in the yeast Saccharomyces cerevisiae, ubiquitin is cleaved off the nascent fusion protein, yielding a deubiquitinated beta-galactosidase (beta gal) . With one exception, this cleavage takes place regardless of the nature of the amino acid residue of beta gal at the ubiquitin-beta gal junction, thereby making it possible to expose different residues at the amino-termini of the otherwise identical beta gal proteins . The beta gal proteins thus designed have strikingly different half-lives in vivo, from more than 20 hours to less than 3 minutes, depending on the nature of the amino acid at the amino-terminus of beta gal . The set of individual amino acids can thus be ordered with respect to the half-lives that they confer on beta gal when present at its amino-terminus (the "N-end rule") . The currently known amino-terminal residues in long-lived, noncompartmentalized intracellular proteins from both prokaryotes and eukaryotes belong exclusively to the stabilizing class as predicted by the N-end rule . The function of the previously described posttranslational addition of single amino acids to protein amino-termini may also be accounted for by the N-end rule . Thus the recognition of an amino-terminal residue in a protein may mediate both the metabolic stability of the protein and the potential for regulation of its stability.

Nature, 1986 Oct 2-8, 323(6087), 451 - 3
Domainal evolution of a prokaryotic DNA repair protein and its relationship to active-transport proteins; Doolittle RF et al.; The ABC excision nuclease of Escherichia coli is an ATP-dependent DNA repair enzyme composed of three protein subunits, UvrA, UvrB and UvrC . The DNA sequences of all three genes have been reported . UvrA, the component that binds directly to the DNA, and UvrB, which attaches itself to the UvrA-DNA complex, both contain consensus sequences though to be diagnostic of ATP-binding sites, although the UvrC sequence does not . We now report that a computer analysis of the UvrA sequence has revealed an unusual series of internal duplications centering around putative metal-binding sites which may be involved in the interaction with DNA . We also find a strong evolutionary relationship to a family of prokaryotic membrane-associated active-transport proteins.

Arch Microbiol, 1986 Oct, 146(1), 19 - 24
Structural and chemical characterization of S-layers of selected strains of Bacillus stearothermophilus and Desulfotomaculum nigrificans; Sleytr UB et al.; The structures, amino acid- and neutral sugar compositions of the crystalline surface layers (S-layers) of four selected strains each of Bacillus stearothermophilus and Desulfotomaculum nigrificans were compared . Among the four strains of each species a remarkable diversity in the molecular weights of the S-layer subunits and in the geometry and constants of the S-layer lattices was apparent . The crystalline arrays included hexagonal (p6), square (p4) and oblique (p2) lattices . In vitro self-assembly of isolated S-layer subunits (or S-layer fragments) led to the formation of flat sheets or open-ended cylindrical assembly products . The amino acid composition of the S-layers exhibited great similarities and was predominantly acidic . With the exception of the S-layers of two strains of B . stearothermophilus (where only traces of neutral sugars could be detected), all other S-layer proteins seemed to be glycosylated . Among these strains significant differences in the amount and composition of the glycan portions were found . Based on this diversity interesting questions may be asked about the biological significance of the carbohydrate units of glycoproteins in prokaryotic organisms.

Proc Natl Acad Sci U S A, 1986 Oct, 83(19), 7251 - 5
Crystal structure of a prokaryotic ribosomal protein; Wilson KS et al.; The structure of ribosomal protein L30 from Bacillus stearothermophilus has been solved to a resolution of 2.5 A . The molecule is somewhat elongated and contains two helices and a three-stranded, anti-parallel beta-pleated sheet . The protein fold, in which helices pack on the same side of the sheet, generates a simple helix-sheet, two-layered motif . It is possible to distinguish three hydrophobic patches on the molecular surface, and one end has six isolated arginine and lysine residues . It is proposed that these reflect sites of protein-protein and protein-RNA interaction, respectively . The protein fold is very similar to that of the only other known ribosomal protein structure, L7/L12 from Escherichia coli, and, based on this similarity, an attempt is made to align the amino acid sequences of the two proteins.

Mol Gen Genet, 1986 Oct, 205(1), 122 - 6
Repeated sequences with unusual properties from the divergent region of Crithidia oncopelti maxicircle kinetoplast DNA; Tarassoff IA et al.; The occurrence of bacterial promoter-like sequences was shown in the divergent region of Crithidia oncopelti maxicircular kinetoplast DNA . A promoter-cloning vector based on the neomycin phosphotransferase II (NPT II) gene was used to localize promoters in three homologous blocks of repeated sequences . The elements found displayed greater promoter strength in Escherichia coli than the normal promoter of the NPT II gene . Sequences of three promoter-containing inserts from different blocks were determined and typical prokaryotic promoter sequences were localized.

J Mol Biol, 1986 Sep 20, 191(2), 221 - 9
Yeast HIS3 expression in Escherichia coli depends upon fortuitous homology between eukaryotic and prokaryotic promoter elements; Struhl K; The yeast imidazoleglycerolphosphate dehydratase gene HIS3, when introduced into Escherichia coli, is transcribed and translated with sufficient fidelity to produce functional enzyme . The following lines of evidence indicate that E . coli RNA polymerase recognizes a particular region of HIS3 DNA as a promoter sequence . First, this promoter contains nucleotide sequences that resemble the canonical prokaryotic promoter elements, the -10 and -35 regions . Second, HIS3 transcription in vitro by E . coli RNA polymerase is initiated at the predicted site downstream from the conserved sequences . Third, deletion mutations that successively encroach upon the 5' end of the HIS3 gene indicate that the promoter is necessary and sufficient for expression in E . coli . Fourth, a single base-pair change that behaves as an "up-promoter" mutation alters the -35 region such that it becomes identical with the consensus sequence . Because the -10 region of this promoter coincides with the TATA promoter element that is necessary for expression in yeast cells, it is possible directly to compare prokaryotic and eukaryotic promoter function . Analysis of 51 deletion and substitution mutations indicates that the patterns of mutant phenotypes are quite different for each organism . Therefore, although prokaryotic -10 regions are similar in sequence to eukaryotic TATA elements and although the same his3 region serves both functions, it appears that this represents an evolutionary coincidence whose current functional basis is minimal . The evolutionary significance of the homology between prokaryotic and eukaryotic promoter elements is discussed.

J Biol Chem, 1986 Sep 15, 261(26), 12317 - 23
Functional assembly in vitro of phycobilisomes with isolated photosystem II particles of eukaryotic chloroplasts; Kirilovsky D et al.; Phycobiliproteins obtained by dissociation of phycobilisomes were reassociated in vitro with intact thylakoids or isolated photosystems I and II preparations obtained from cyanophytes (prokaryotes) or green algae (eukaryotes) to form bound phycobilisome complexes . Energy transfer from Fremyella diplosiphon phycobiliproteins to chlorophyll a of reaction centers I and II was measured in: complexes containing intact thylakoids of the cyanophytes F . diplosiphon or Anacystis nidulans and the eukaryotic algae Euglena gracilis and mutants of Chlamydomonas reinhardtii; complexes containing isolated photosystem II particles of A . nidulans or C . reinhardtii; and complexes containing reaction center I of F . diplosiphon or C . reinhardtii . Energy transfer from phycoerythrin to chlorophyll a of photosystem II could be demonstrated in complexes containing phycobilisomes bound to cyanophyte thylakoids or isolated photosystem II particles of A . nidulans or C . reinhardtii . Bound phycobilisomes did not transfer energy to photosystem II within green algae thylakoids containing altered forms of light-harvesting chlorophyll a/b-protein complex (LHC) II antenna, reduced amounts of LHC II, or chlorophyll b, or chlorophyll b-less mutants, nor to chlorophyll a of photosystem I of intact thylakoids or isolated reaction centers . We conclude that phycobilisomes can form a specific and functional association with photosystem II particles of both cyanophytes and eukaryotic thylakoids . This interaction appears to be hindered by the presence of LHC II antenna in the eukaryotic thylakoids.

J Biol Chem, 1986 Sep 15, 261(26), 12109 - 13
Reconstitution of the H+-ATPase complex of Rhodospirillum rubrum by the beta subunit of the chloroplast coupling factor 1; Richter ML et al.; A method is described for isolating the beta subunit from spinach chloroplast F1 (CF1) . The isolated beta subunit reconstituted an active F1 hybrid with the F1 of Rhodospirillum rubrum chromatophores from which the beta subunit had been removed . The CF1 beta subunit was similar to the isolated beta subunit of Escherichia coli F1 (Gromet-Elhanan, Z., Khananshivili, D., Weiss, S., Kanazawa, H., and Futai, M . (1985) J . Biol . Chem . 260, 12635-12640) in that it restored a substantial rate of ATP hydrolysis and low, but significant light-dependent ATP synthesis to the beta-less chromatophores . The low rate of photophosphorylation observed with the hybrid enzyme probably resulted from a looser coupling of the CF1 beta subunit to proton translocation in the R . rubrum Fo-F1 complex . The hybrid enzyme exhibited a high specificity for Mg2+-ATP as substrate for ATP hydrolysis and both ATP synthesis and hydrolysis were strongly inhibited by the antibiotic tentoxin . In contrast, chromatophores reconstituted with the native R . rubrum beta subunit actively hydrolyzed both Mg2+-ATP and Ca2+-ATP and were insensitive to tentoxin . These results indicate a close functional homology between the beta subunits of the prokaryotic and eukaryotic H+-ATPases and suggest a role for the beta subunit in conferring the different metal ion specificities and inhibitor sensitivities upon the enzymes . They also demonstrate the feasibility of isolating the beta subunit from CF1 in a reconstitutively active form.

J Biol Chem, 1986 Sep 5, 261(25), 11744 - 50
A new DNA-dependent ATPase which stimulates yeast DNA polymerase I and has DNA-unwinding activity; Sugino A et al.; Two forms of DNA-dependent ATPase activity were previously purified from the yeast Saccharomyces cerevisiae and characterized (Plevani, P., Badaracco, G., and Chang, L . M . S . (1980) J . Biol . Chem . 255, 4957-4963) . Here, an additional DNA-dependent ATPase (ATPase III) has been purified from S . cerevisiae to near homogeneity . This ATPase differs from those described previously in its chromatographic properties, molecular weight, reaction properties and immunological relatedness . Its molecular weight is about 63,000 in the presence of sodium dodecyl sulfate . It hydrolyzes ATP to ADP and orthophosphate in the presence of DNA as an effector . In addition, yeast DNA polymerase I, which is a true DNA replicase of yeast, is stimulated severalfold by this ATPase . Neither yeast DNA polymerase II nor prokaryotic DNA polymerases are stimulated . This stimulation is intrinsic to the ATPase activity, since both activities copurified in the last four steps of purification, showed the same heat stability and showed dependence on and hydrolysis of ATP . The ATPase III preparation also contains a DNA-unwinding (DNA helicase) activity, which unwinds double-stranded DNA in the presence of ATP . In the S . cerevisiae radiation-sensitive mutant rad3, no significant ATPase III activity could be detected, suggesting that the RAD3 gene, which codes for a different polypeptide, regulates the expression of ATPase III activity.

Tsitologiia, 1986 Sep, 28(9), 899 - 910
{The origin of the eukaryotic cell . IV . The general hypothesis of the autogenous origin of eukaryotes}; Seravin LN; The general hypothesis of autogenous (non-symbiotic) origin of the eukaryotic cell summarises some hypotheses explaining possible ways of the origin of main components and organelles of such a cell (the primary unicellular protist) . Six hypothesises are suggested . Arising of the eukaryotic surface membrane of protist (cell) as a result of modification of its lipidoacidic composition, when most of synblocks and ensembles of eukaryotic enzymes sink into the cytoplasm (due to membrane vesiculation) . Establishment of eukaryotic cytoplasm on the basis of successive formation of two locomotory-supporting apparates: the primary one (microtrabecular system), and the second one (cytoskeleton) . Arising of the nucleus from a polyheteronomous nucleoid of proeukaryotes . A combinatorical hypothesis of mitosis formation . Polyheteronucleoid hypothesis of the origin of the mitochondria and chloroplasts . Arising of the flagellum from the contractile tentacle-like organelle, whose axoneme is made of single microtubules . A close interrelation and interaction in the process of evolution is noted between surface membranes, the cytoplasm and the nucleus . In accord a principles of block-construction and heterochrony (see: Seravin, 1986r), the author explains the preservation of prokaryotic signs of organization in some components (and organelles) of eukaryotic cell (and protists).

Mutat Res, 1986 Sep, 166(2), 143 - 7
Evaluation of homology between cloned Escherichia coli and yeast DNA photolyase genes and higher eukaryotic genomes; Meechan PJ et al.; Repair of ultraviolet-induced pyrimidine dimers by photoreactivation is catalyzed by a single enzyme, DNA photolyase . However, the process of photoreactivation is difficult to detect reproducibly in cultured mammalian cells . We have used clones containing yeast and Escherichia coli DNA photolyase genes to determine whether their sequences are conserved and whether there is homology between either cloned sequence and chick or human genomic DNA and mRNA sequences . The cloned sequences failed to hybridize to each other even under nonstringent conditions, indicating little conservation of sequence between the yeast and E . coli genes . Furthermore, only weak hybridization under nonstringent conditions was found between the cloned photoreactivating genes and human or chick genomic DNA or mRNA . This indicates that there is negligible homology between the cloned probes and mammalian DNA, but we are unable to conclude whether this indicates sequence divergence for prokaryotic and eukaryotic photoreactivation genes or the absence of such genes from the mammalian genome.

Proc Natl Acad Sci U S A, 1986 Sep, 83(17), 6292 - 6
Reduction of the toxicity and mutagenicity of alkylating agents in mammalian cells harboring the Escherichia coli alkyltransferase gene; Brennand J et al.; The toxic, mutagenic, and carcinogenic effects of alkylating agents have been attributed to their ability to damage DNA . Reaction at the O6 position of guanine results in miscoding during DNA replication, has been shown to be mutagenic in both bacteriophage and bacteria, and may be responsible for malignant transformation . In common with many other prokaryotes and eukaryotes the Escherichia coli B strain contains a protein that repairs O6-alkylation damage in DNA by transferring the alkyl group to one of its own cysteine residues . We have recently cloned the E . coli O6-alkylguanine alkyltransferase gene and shown it to encode a 37-kDa protein containing an additional activity that removes alkyl groups from alkylphosphotriesters in DNA . To examine the biological effects of this gene in mammalian cells, we have now inserted the coding sequence into a retrovirus-based selectable expression vector and transfected it into Chinese hamster V79 cells that lack endogenous alkyltransferase activity . A clone expressing high levels of the bacterial protein was selected and shown to produce a 37-kDa alkyltransferase protein and to rapidly repair O6-methylguanine produced in the host genome following exposure to N-methyl-N-nitrosourea . In comparison with a control population, this clone is considerably more resistant to the toxic and mutagenic effects of alkylating agents that react extensively with oxygen atoms in DNA . The usefulness of these clones in examining the role of DNA alkylation and other biological effects of alkylating agents is discussed.

Microbiol Sci, 1986 Sep, 3(9), 268 - 74
The myxobacteria: common organisms with uncommon behaviour; Reichenbach H; The gliding myxobacteria, common and easily obtainable soil organisms, exhibit a fascinating social organization which culminates in the formation of fruiting bodies by an ordered cooperation of hundreds of thousands of cells . Inside the fruiting body, the vegetative cells transform themselves into dormant myxospores . The myxobacteria have become potent model systems for studying morphogenetical problems at the prokaryotic organizational level, particularly since the methods of modern genetics now may be readily applied to them.

Eur J Biochem, 1986 Sep 1, 159(2), 219 - 25
Structure and drug inducibility of the human cytochrome P-450c gene; Kawajiri K et al.; A human genomic clone (lambda hP-450mc-1), highly homologous to the rat cytochrome P-450c gene, was isolated and analyzed for the complete nucleotide sequence . The gene structure coincides with that of a recently reported human gene isolated from genomic DNA of a human breast carcinoma cell line, MCF-7 {Jaiswal, A.K., Gonzalez, F.J . & Nebert, D.W . (1985) Nucleic Acids Res . 13, 4503-4520} with notable exceptions in the first intron: a 320-base-pair fragment is inserted and a 650-base-pair fragment is deleted in the gene examined in the present study . The 320-base-pair insert appears to contain a moderately repetitive sequence (approx . 140 copies) in the human genome . The 650-base-pair fragment, present in intron 1 of the reported sequence, is dislocated in the lambda hP-450mc-1 to about 10(4) base pairs upstream from the putative transcription initiation site . The results of Southern blot analysis using human total DNA were compatible with the gene structure of lambda hP-450mc-1 . A fusion gene, which was constructed by ligating the 5' flanking region of the gene to the structural gene for prokaryotic chloramphenicol acetyltransferase (CAT), inducibly expressed the CAT activity in mouse Hepa-1 cells in response to administered methylcholanthrene, indicating that the isolated human gene is indeed of methylcholanthrene inducibility.

Nucleic Acids Res, 1986 Aug 26, 14(16), 6357 - 73
The TRP4 gene of Saccharomyces cerevisiae: isolation and structural analysis; Furter R et al.; The TRP4 gene of Saccharomyces cerevisiae, encoding the anthranilate phosphoribosyl transferase, was isolated and subcloned by functional complementation in yeast . A 2 kb fragment containing information for a polypeptide of 380 amino acids and the 5'- and 3'-flanking regions was sequenced . The TRP4 transcript was identified and mapped with S1 nuclease . Homologies to two prokaryotic genes encoding the same function, and sequences potentially involved in transcription start and termination and in regulation of TRP4 gene expression are discussed.

Science, 1986 Aug 22, 233(4766), 889 - 92
Upstream operators enhance repression of the lac promoter; Mossing MC et al.; To study regulation of transcription by distant elements, a wild-type lac operator was inserted upstream of a promoter-constitutive operator control region . The upstream operator is shown to aid in repression of transcription from the mutant control region . The effectiveness of the upstream operator as a function of its distance from the mutant control region parallels the length dependence observed for DNA cyclization . A quantitative model is proposed for action-at-a-distance of DNA control sites in which protein-protein and protein-DNA interactions are mediated by DNA looping . In this model, the effective concentrations of interacting proteins that are tethered by DNA are determined by the length of the intervening DNA and by its inherent bending and torsional stiffness . This model makes a number of predictions for both eukaryotic and prokaryotic control sequences located far from their sites of action.

J Biol Chem, 1986 Aug 15, 261(23), 10931 - 5
Uncoupling of the DNA breaking and rejoining steps of Escherichia coli type I DNA topoisomerase . Demonstration of an active covalent protein-DNA complex; Tse-Dinh YC; DNA topoisomerases have been shown to cleave DNA phosphodiester bond and simultaneously become linked to the DNA at the cleavage site via a phosphotyrosine linkage (Tse, Y.-C., Kirkegaard, K., and Wang, J . C . (1980) J . Biol . Chem . 255, 5560-5565) . For prokaryotic DNA topoisomerases, this is observed only when denaturant or protease is added to the topoisomerase-DNA incubation mixture . Previous attempts to reform DNA phosphodiester bonds from the covalent protein-DNA complex have been unsuccessful . Using oligonucleotides as substrates, the cleavage reaction of Escherichia coli DNA topoisomerase I occurs spontaneously (Tse-Dinh, Y.-C., McCarron, B . G . H., Arentzen, R., and Chowdhry, V . (1983) Nucleic Acids Res . 11, 8691-8701) . Upon reaction with oligo(dA) labeled with 32P using terminal transferase and {alpha-32P}dATP, the enzyme becomes covalently linked to the 32P-labeled oligonucleotide . This 32P label can then be transferred to the 3'-OH end of a linear or nicked duplex DNA molecule subsequently added to the reaction mixture . This phosphodiester bond rejoining reaction can occur at a recessed, blunt, or protruding 3'-end of double-stranded DNA . It requires magnesium ions . These observations suggest that the covalent protein-DNA complex is a true intermediate during topoisomerization . Implications on the structure of prokaryotic type I DNA topoisomerases as compared to their eukaryotic counterparts are discussed.

Nucleic Acids Res, 1986 Aug 11, 14(15), 6215 - 26
Nucleotide sequence of the hemC locus encoding porphobilinogen deaminase of Escherichia coli K12; Thomas SD et al.; Porphobilinogen deaminase, the product of the hemC locus in Escherichia coli K12, catalyses the tetrapolymerisation of porphobilinogen (PBG) into the hydroxymethylbilane, preuroporphyrinogen . The hemC locus has been subcloned from the Clarke and Carbon plasmid pLC41-4 . The sequence of the hemC structural gene and flanking DNA was determined by the dideoxy chain termination method of Sanger . The structural gene for hemC is located within a 942bp sequence encoding the monomeric PBG deaminase, molecular weight 33,857 . The extent of the coding region was confirmed by sequencing the N-terminus of the purified enzyme and by determination of the molecular weight . The hemC locus is closely linked to the cyaA locus, the genes being transcribed in a divergent manner . Upstream of the hemC coding region, a possible promoter and three repeated GGATG sequences were identified . This is the first report of a complete DNA sequence for a structural gene specifying an enzyme of the heme biosynthetic pathway in prokaryotes.

Can J Genet Cytol, 1986 Aug, 28(4), 483 - 96
Evidence for local DNA influences on patterns of substitutions in the human alpha-interferon gene family; Golding GB et al.; The evolutionary history of genes can be used to examine patterns of spontaneous mutation if the sequences are sufficiently extensive to provide reliable data . Many human alpha-interferon genes have been sequenced and they form a large multigene family including several pseudogenes . A phylogenetic history for 15 human interferon sequences was reconstructed and their ancestral sequences inferred using a maximum parsimony method . This evolutionary history provided a record of more than 738 spontaneous mutations that have occurred in man's recent evolution . Of these mutations, more than 267 base substitution and deletion-insertion events were analyzed to determine the possible effects of nearby DNA sequences . Many substitutions occur at the end of long runs of identical bases and some dinucleotide pairs may mutate more often than others . Because templating by local DNA sequences has been implicated in prokaryotic mutation, the sequences were also examined for nearby repeats that include the substituted nucleotide and hence are potentially capable of templating the substitution . The majority of sequence alterations examined have either a similar direct repeat or palindrome nearby . Often such templates can account for simultaneous multiple mutations . These results suggest that sequence-directed events may occur occasionally in eukaryotes and that neighbouring DNA sequences can influence both the occurrence and types of mutations in several different ways.

Tsitologiia, 1986 Aug, 28(8), 796 - 801
{Electron microscopic research on the extrachromosomal genetic elements of Escherichia coli}; Kiseleva EV et al.; The structural organization of extrachromosomal genetic elements were studied in a subfraction obtained after centrifugation of the lysate of E . coli spheroplasts . With this method of isolation, the tertiary structure of the extrachromosomal genetic elements was preserved . The majority of DNA macromolecules were released in the form of single and connected rosettes . Typical rosettes composed of radial loops of DNA clustered around the central dense core (the diameter is about 60 nm) . The mean length of the rosette loops was 1.06 +/- 0.4 micron . Both relaxed folded and supercoiled folded forms of DNA were observed on the preparation . Sometimes the rosettes were connected with large aggregates of DNA (possibly the material of bacterial chromosomes) and had the appearance of thick fibers with numerous lateral loops . Linear, cyclic and various replicative forms of DNA have also been observed . It is assumed that rosettes of the extrachromosomal elements of E . coli reflect one of the levels of organization of prokaryotic genetic material.

Tsitologiia, 1986 Aug, 28(8), 779 - 89
{The origin of the eukaryotic cell . III . Principles of the morphofunctional organization of the eukaryotic cell}; Seravin LN; The eukaryotic plasmalemma, eukaryotic cytoplasm with its usual cytomembranes, and eukaryotic nucleus are obligatory components of the eukaryotic cell . All other structural elements (organelles) are only derivates of the aforesaid cell components and they may be absent sometimes . There are protozoans having simultaneously no flagelles, mitochondria and chloroplasts (all the representatives of phylum Microspora, amoeba Pelomyxa palustris, and others) . The following five general principles play the main role in the morphofunctional organization of the cell . The principle of hierarchy of block organization of living systems . Complex morphofunctional blocks (organelles) specific for the eukaryotic cell are formed . The compartmentalization principle . The main cell organelles (nuclei, flagellae, mitochondria, chloroplasts, etc.) undergo a relative morphological isolation from each other and other cell organelles by means of the total or partial surrounding by membranes; this may ensure the originality of their evolution and function . The principle of poly- and oligomerization of morphofunctional blocks . It permits the cell to enlarge its sizes and to raise the level of integration . The principle of heterochrony, including three subprinciples: conservatism of useful signs; a strong acceleration of evolutionary development of the separate blocks; simplification of the structure, reduction or total disappearance of some blocks . It explains a preservation of prokaryotic signs in the eukaryotic cell or in its organelles . The principle of independent origin of similar morphofunctional blocks in the process of evolution of living systems . The parallelism of the signs in unrelated groups of cells (or protists) arises due to this principle.

EMBO J, 1986 Aug, 5(8), 2031 - 6
The bacteriophage T4 gene for the small subunit of ribonucleotide reductase contains an intron; Sjoberg BM et al.; The bacteriophage T4 gene nrdB codes for the small subunit of the enzyme ribonucleotide reductase . The T4 nrdB gene was localized between 136.1 kb and 137.8 kb in the T4 genetic map according to the deduced structural homology of the protein to the amino acid sequence of its bacterial counterpart, the B2 subunit of Escherichia coli . This positions the C-terminal end of the T4 nrdB gene approximately 2 kb closer to the T4 gene 63 than earlier anticipated from genetic recombinational analyses . The most surprising feature of the T4 nrdB gene is the presence of an approximately 625 bp intron which divides the structural gene into two parts . This is the second example of a prokaryotic structural gene with an intron . The first prokaryotic intron was reported in the nearby td gene, coding for the bacteriophage T4-specific thymidylate synthase enzyme . The nucleotide sequence at the exon-intron junctions of the T4 nrdB gene is similar to that of the junctions of the T4 td gene: the anticipated exon-intron boundary at the donor site ends with a TAA stop codon and there is an ATG start codon at the putative downstream intron-exon boundary of the acceptor site . In the course of this work the denA gene of T4 (endonuclease II) was also located.

Proc Natl Acad Sci U S A, 1986 Aug, 83(16), 5875 - 9
Processing of phage T4 td-encoded RNA is analogous to the eukaryotic group I splicing pathway; Ehrenman K et al.; Several features of the split td gene of phage T4 suggest an RNA processing mechanism analogous to that of the self-splicing rRNA of Tetrahymena and other group I eukaryotic introns . Previous work has revealed conserved sequence elements and the ability of td-encoded RNA to self-splice in vitro . We show here that a noncoded guanosine residue is covalently joined to the 5' end of the intron during processing . Further, we demonstrate the existence of linear and circular intron forms in RNA extracted from T4-infected cells and from uninfected Escherichia coli expressing the cloned td gene . Sequence analysis of the intron cyclization junction indicates that the noncoded guanosine and one additional nucleotide are lost from the 5' end of the intron upon cyclization . This analysis places a uridine residue upstream of the cyclization site, in analogy to three other group I cyclization junctions . These striking similarities to the splicing intermediates of eukaryotic group I introns point not only to an analogous processing pathway and conserved features of cyclization site recognition but also to a common ancestry between this prokaryotic intervening sequence and the group I eukaryotic introns.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5587 - 91
Transfection and homologous recombination involving single-stranded DNA substrates in mammalian cells and nuclear extracts; Rauth S et al.; We have examined the ability of single-stranded DNA to participate in homologous recombination reactions in mammalian cells and nuclear extracts derived from them . We have inserted a fragment of the neo gene into the single-stranded DNA phage vector M13 mp11 . The neo fragment was derived from a deletion derivative of the prokaryotic-eukaryotic shuttle vector pSV2neo . The resulting single-stranded DNA was mixed with a double-stranded deletion derivative of pSV2neo and tested for recombination in human cells, monkey cells, and nuclear extracts obtained from human cells . We were able to obtain recombinant molecules containing wild-type neo genes in all three systems . Examination of the products of recombination indicated that they resulted from correction of the deletion in the double-stranded DNA substrate . We were unable to detect any extensive conversion of single-stranded DNA into its double-stranded counterpart before it participated in the recombination reaction . We have also tested the ability of single-stranded DNA to yield transfectants . When a single-stranded DNA derivative of the herpes simplex virus thymidine kinase (TK) gene was introduced into mouse L-M(TK-) cells, we were able to obtain TK+ colonies . From these results, we conclude that single-stranded DNA can participate in transfection as well as homologous recombination reactions in mammalian cells.

Proc Natl Acad Sci U S A, 1986 Aug, 83(15), 5387 - 91
Bifunctional thymidylate synthase-dihydrofolate reductase from Leishmania tropica: sequence homology with the corresponding monofunctional proteins; Grumont R et al.; In protozoa, thymidylate synthase (TS; EC 2.1.1.45) and dihydrofolate reductase (DHFR; EC 1.5.1.3) exist as a bifunctional protein . Full-length cDNA and genomic DNA clones encoding the bifunctional TS-DHFR from Leishmania tropica have been isolated, characterized, and sequenced . There are no intervening sequences within the coding region for TS-DHFR, and the G+C content is high, with a strong bias for codons possessing guanine or cytosine in the third base . From the predicted protein sequence for TS-DHFR, we conclude that the enzymes exist as separate structural domains with DHFR located at the amino terminus and TS at the carboxyl terminus; in between, there is a junctional peptide predicted to consist of a random coil flanked by two alpha-helices that corresponds to the site of a putative gene fusion . Comparison of the predicted amino acid sequence of L . tropica TS-DHFR with sequences of the monofunctional enzymes from other sources shows that the TS domain is much more conserved than is the DHFR domain and that both enzymes are more related to their monofunctional counterparts in vertebrates than in prokaryotes . Most of the recognized elements of secondary structure of other DHFRs, as ascertained by x-ray crystallography, are found in the predicted structure of the DHFR domain of the bifunctional protein.

Theor Popul Biol, 1986 Aug, 30(1), 1 - 16
Distribution of transposable elements in prokaryotes; Sawyer S et al.; We consider models for the distribution of the number of elements per host genome for families of transposable elements (TEs) . The hosts are assumed to be prokaryotes . These models assume a constant rate of infection of uninfected hosts by TEs, replicative transposition within each host, and a reduction of the fitness of a host dependent on the number of TEs it contains . No provision was made for the deletion of individual TEs within a host or for recombination, since both are relatively rare events in prokaryotes . These models mostly assume that the TE performs no function for the host, and that the reduction in fitness with increased copy number is due to effects such as the impairment of beneficial genes by transposition or homologous recombination . We also consider a model in which the TEs can convey a selective advantage to the host . The equilibrium distributions of copy number are determined for these models, and are of a variety of classical types . Relevant parameters of the models are estimated using data on the distribution of insertion sequences in natural isolates of Escherichia coli.

Am J Physiol, 1986 Aug, 251(2 Pt 1), E164 - 71
Prokaryotic adenylate cyclase toxin stimulates anterior pituitary cells in culture; Cronin MJ et al.; Bordetella pertussis synthesizes a variety of virulence factors including a calmodulin-dependent adenylate cyclase (AC) toxin . Treatment of anterior pituitary cells with this AC toxin resulted in an increase in cellular cAMP levels that was associated with accelerated exocytosis of growth hormone (GH), prolactin, adrenocorticotropic hormone (ACTH), and luteinizing hormone (LH) . The kinetics of release of these hormones, however, were markedly different; GH and prolactin were rapidly released, while LH and ACTH secretion was more gradually elevated . Neither dopamine agonists nor somatostatin changed the ability of AC toxin to generate cAMP (up to 2 h) . Low concentrations of AC toxin amplified the secretory response to hypophysiotrophic hormones . We conclude that bacterial AC toxin can rapidly elevate cAMP levels in anterior pituitary cells and that it is this response that explains the subsequent acceleration of hormone release.

Virology, 1986 Jul 30, 152(2), 432 - 45
Antibodies to distal carboxyl terminal epitopes in the v-fms-coded glycoprotein do not cross-react with the c-fms gene product; Furman WL et al.; The product of the v-fms oncogene is an integral transmembrane glycoprotein that is closely related to the cell surface receptor for the macrophage colony stimulating factor, CSF-1 . A fragment of the v-fms gene encoding a major portion of the extracellular amino terminal domain, the membrane-spanning segment, and the entire carboxyl terminal tyrosine kinase domain of the glycoprotein was molecularly cloned into an inducible prokaryotic expression plasmid . Polypeptide products consisting only of v-fms-coded amino acids were produced in bacteria and were used to prepare immune reagents that precipitated the v-fms-coded glycoproteins expressed in transformed cells . Whereas rabbit antisera to recombinant polypeptides detected antigenic determinants of the c-fms proto-oncogene product, seven mouse monoclonal antibodies to these same antigens reacted only with v-fms-specific epitopes . Proteolytic mapping experiments and studies with a mutant v-fms-coded glycoprotein lacking the 37 carboxyl terminal amino acids of the wild-type product showed that the monoclonal antibodies were restricted in their reactivity to epitopes at the extreme carboxyl terminus of the glycoprotein . The v-fms and c-fms gene products must differ significantly in this region.

Nucleic Acids Res, 1986 Jul 25, 14(14), 5761 - 76
Site-directed mutagenesis of the binding site for ribosomal protein S8 within 16S ribosomal RNA from Escherichia coli; Gregory RJ et al.; Twelve specific alterations have been introduced into the binding site for ribosomal protein S8 in Escherichia coli 16S rRNA . Appropriate rDNA segments were first cloned into bacteriophage M13 vectors and subjected to bisulfite and oligonucleotide-directed mutagenesis in vitro . Subsequently, the mutagenized sequences were placed within the rrnB operon of plasmid pNO1301 and the mutant plasmids were used to transform E . coli recipients . The growth rates of cells containing the mutant plasmids were determined and compared with that of cells containing the wild-type plasmid . Only those mutations which occurred at highly conserved positions, or were expected to disrupt the secondary structure of the binding site, increased the doubling time appreciably . The most striking changes in growth rate resulted from mutations that altered a small internal loop within the S8 binding site . This structure is phylogenetically conserved in prokaryotic 16S rRNAs and may play a direct role in S8-16S rRNA recognition and interaction.

Nature, 1986 Jul 17-23, 322(6076), 279 - 81
Overlapping transcription units in the dopa decarboxylase region of Drosophila; Spencer CA et al.; The many examples of overlap in the genes of various viruses and bacteria illustrate that the parsimonious utilization of the coding capacity of DNA is relatively common amongst prokaryotes . The recent discovery of a pupal cuticle gene within an intron of the completely unrelated Gart locus in Drosophila shows that overlapping transcription units also exist in higher organisms . However, the prevalence of such phenomena in unknown . We report here a quite different situation of overlap between the 3' termini of a pair of convergent transcription units in another region of the Drosophila genome . This 88-base-pair (bp) genomic region encodes the 3' terminus of the messenger RNA for the enzyme dopa decarboxylase (Ddc) and, in opposite orientation, the 3' terminus of the adjacent gene whose function is unknown . An analysis of the temporal and spatial distribution of the two transcripts within the organism shows that high levels of both transcripts are never concordant . However, within the testes, where the 3' transcript is maximally expressed, low levels of Ddc transcript were detected . This result raises the possibility that a hybrid molecule involving the two transcripts forms in vivo or that transcription interference occurs, with concomitant regulatory implications.

Nature, 1986 Jul 10-16, 322(6075), 187 - 9
Newly replicated DNA is associated with DNA topoisomerase II in cultured rat prostatic adenocarcinoma cells; Nelson WG et al.; DNA topoisomerases have been proposed to function in a variety of genetic processes in both prokaryotes and eukaryotes . Here, we have assessed the role of DNA topoisomerase II in mammalian DNA replication by determining the proximity of newly synthesized DNA to covalent enzyme-DNA complexes generated by treating cultured rat prostatic adenocarcinoma cells with teniposide . Teniposide (VM-26), an epipodophyllotoxin, is known to interact with mammalian DNA topoisomerase II so as to trap the enzyme in a covalent complex with DNA . We have found that the teniposide-induced trapping of such complexes requires MgCl2, is stimulated by ATP and is inhibited by novobiocin . The formation of covalent complexes seems to be reversible on removal of teniposide . Furthermore, analysis of the covalent complexes formed between 3H-thymidine pulse-labelled DNA and topoisomerase II following teniposide treatment reveals a direct association of the enzyme with nascent DNA fragments . Our results suggest that DNA topoisomerase II may interact with newly replicated daughter DNA molecules near DNA replication forks in mammalian cells.

Anticancer Res, 1986 Jul-Aug, 6(4), 525 - 42
Interference with polyamine biosynthesis and/or function by analogs of polyamines or methionine as a potential anticancer chemotherapeutic strategy; Porter CW et al.; The obvious goal in cancer chemotherapy is selectivity . Highly cytotoxic agents abound but their usefulness as anticancer agents extends only so far as their specificity for tumor cells and tissues . In this context, we have reviewed those aspects of polyamine and AdoMet metabolism and function which might contribute to their potential as target sites for chemotherapeutic intervention . Although largely untested to date and far from unequivocal, these various considerations seem to provide sufficient rationale for continued evaluation of the therapeutic potential of these sites . Polyamine analogs and methionine analogs designed to modulate polyamine biosynthesis directly or through AdoMet formation have been discussed as strategies to effect this goal and previous studies with similar analogs have been reviewed . Progress achieved thus far with analogs derived from our own laboratories provides novel insights into polyamine and AdoMet metabolism and/or function as well as new leads towards the design of more effective agents and drug combinations . More detailed reading of the biochemistry of polyamines in eukaryotes and prokaryotes is available in several very excellent current reviews (6-9, 77).

Proc Natl Acad Sci U S A, 1986 Jul, 83(14), 5199 - 203
Sequence homology requirements for intermolecular recombination in mammalian cells; Ayares D et al.; We have examined the homology requirements for intermolecular recombination between plasmids introduced into human, monkey, and bacterial cells . Variable-size-deletion derivatives of the prokaryotic-eukaryotic shuttle vector pSV2neo were constructed . Each of these plasmids was mixed with another pSV2neo plasmid containing a different, nonoverlapping deletion . Recombination was measured in mammalian cells and bacteria by the frequency of reconstruction of an intact neo gene . We observed that 25 base pairs of homologous sequence is sufficient to yield recombinant products, implying that synapsis and homologous pairing can occur with this level of homology . Examination of the products revealed that nonreciprocal recombination played a role in the generation of normal neo genes . In addition coconversion of linked markers was observed . Exonucleolytic action seems to play a role in gene conversion.

Int J Radiat Biol Relat Stud Phys Chem Med, 1986 Jul, 50(1), 1 - 30
The use of recombinant DNA techniques to study radiation-induced damage, repair and genetic change in mammalian cells; Thacker J; A brief Introduction is given to appropriate elements of recombinant DNA techniques and applications to problems in radiobiology are reviewed with illustrative detail . Examples are included of studies with both 254 nm ultraviolet light (u.v.) and ionizing radiation (i.r.) and the review progresses from the molecular analysis of DNA damage in vitro through to the nature of consequent cellular responses . The section on the Molecular distribution of DNA damage (section 2) focuses on the use of defined DNA molecules to assess the nature, sites and frequency of radiation damage . Recombinant DNA techniques have also been used in the study of enzyme-DNA interactions, to comment upon the role of specific types and sites of damage in producing cellular responses . The use of DNA-mediated gene transfer to assess damage and repair (section 3) indicates that recombinant DNA molecules can be used to implicate (or reject) specific types of DNA damage in gene inactivation . Some gene-transfer assays may also be able to confirm the presence of specific repair functions in mammalian cells . Restriction endonucleases are essential for the construction of recombinant DNA molecules, but their ability to cut DNA at specific sequences is also being exploited to implicate the double-strand break as an important type of damage leading to the well-characterized responses of irradiated cells . The DNA double strand break: use of restriction endonucleases to model radiation damage (section 4) documents experiments showing that blunt-ended cuts introduced into cellular DNA are able to produce chromosome aberrations and cell death . Assays based upon the introduction of restriction endonuclease-cut plasmids into radiosensitive and normal cells suggest that sensitivity is in some instances, e.g . the radiosensitive disorder ataxia-telangiectasia, a result of excessive degradation of DNA around broken ends . Identification and cloning of DNA repair genes (section 5) reviews the successful cloning of one human repair gene and the putative identification of others, as well as the lack of success in identifying genes complementing radiosensitive human disorders . Analysis of radiation-induced genetic change (section 6) links the types of DNA damage observed in defined DNA molecules with the types of mutations occurring in irradiated prokaryotes . In mammalian cells recombinant DNA techniques have allowed the nature of mutational changes to be determined for the first time: to date it seems that u.v . produces mainly small (point) mutations while i.r . produces mainly large changes (deletions/rearrangements).(ABSTRACT TRUNCATED AT 400 WORDS)

Mol Biol Evol, 1986 Jul, 3(4), 313 - 21
Sequence of a cDNA for mouse thymidylate synthase reveals striking similarity with the prokaryotic enzyme; Perryman SM et al.; We report the nucleotide sequence of a cloned cDNA, pMTS-3, that contains a 1-kb insert corresponding to mouse thymidylate synthase (E.C . 2.1.1.45) . The open reading frame of 921 nucleotides from the first AUG to the termination codon specifies a protein with a molecular mass of 34,962 daltons . The predicted amino acid sequence is 90% identical with that of the human enzyme . The mouse sequence also has an extremely high degree of similarity (as much as 55% identity) with prokaryotic thymidylate synthase sequences, indicating that thymidylate synthase is among the most highly conserved proteins studied to date . The similarity is especially pronounced (as much as 80% identity) in the 44-amino-acid region encompassing the binding site for deoxyuridylic acid . The cDNA sequence also suggests that mouse thymidylate synthase mRNA lacks a 3' untranslated region, since the termination codon, UAA, is followed immediately by a poly(A) segment.

Tsitologiia, 1986 Jul, 28(7), 659 - 69
{The origin of the eukaryotic cell . II . A critical analysis of the symbiotic (exogenous) concept}; Seravin LN; The exogenous (symbiotic) conception of the eukaryotic cell origin is unable to explain satisfactory the structure of mitochondria and chloroplasts . Either of these organelles possess its genome that can be compared with the viral one rather than with the bacterial one, judging by the dimensions and quantity of coding genes . The mitochondria resemble a little prokaryotes in the number of their proteins, chemical composition of their inner membrane and peculiarities of the protein-synthesizing apparatus . The primitive structure of mt DNA, the lesser quantity and greater unspecifity of the mitochondrial tRNA prove, additionally, the non-bacterial origin of this organelles . The deflexion of the genetic code from the universal one in the mitochondrial nucleoids also testify in favour of this point of view . The results of micropaleontological and paleobiochemical investigations evidence towards initial ability of the primary eukaryotes (primary protists) to photosynthesis . In this case, they did not need to acquire plastids from outside by symbiotic way . The autogenous origin of the flagellum of the primary protists was reported earlier (Seravin, 1985) . The accumulated data permit us to consider that the cell organelles formed endogenously in the process of evolution of the cell.

Proc Natl Acad Sci U S A, 1986 Jul, 83(14), 5282 - 5
Expression of the protein encoded by the 3' open reading frame of human T-cell lymphotropic virus type III in bacteria: demonstration of its immunoreactivity with human sera; Franchini G et al.; The genome of human T-cell lymphotropic virus type III/lymphadenopathy-associated virus (HTLV-III/LAV), the infectious agent etiologically associated with the acquired immunodeficiency syndrome, contains, in addition to the genes for the polymerase, core, and envelope proteins, several open reading frames . To investigate whether the 3' open reading frame (3' orf) located between the envelope gene and the 3' long terminal repeat is a gene expressed in vivo in infected individuals, we inserted a fragment of 3' orf in a prokaryotic expression vector . The protein product synthesized in bacteria was purified and allowed to react with sera from individuals infected with human T-cell lymphotropic virus type III as indicated by seropositivity for other viral proteins . Two-thirds of the sera, regardless of the clinical status of the individuals, reacted with the purified protein indicating that 3' orf is a viral gene the product of which is immunogenic in vivo . A polyclonal rabbit antiserum reacting against the 3' orf gene product was obtained by serial injection of rabbits with the purified bacterial protein . The antiserum recognized a 27-kDa protein in the human T-cell lymphotropic virus type III-infected lymphocytes.

Proc Natl Acad Sci U S A, 1986 Jul, 83(14), 5091 - 5
The fusion-related hydrophobic domain of Sendai F protein can be moved through the cytoplasmic membrane of Escherichia coli; Davis NG et al.; Recent work on a prokaryotic membrane protein, gene III protein (pIII) of coliphage f1, showed that polypeptide segments of sufficient hydrophobicity functioned to stop transfer of the polypeptide across the cell membrane: strings of 16 or more hydrophobic amino acids sufficed . A fusion-related hydrophobic domain (FRHD) of Sendai F protein, a sequence of 26 consecutive uncharged residues, has been implicated in the fusion of the viral membrane envelope and the target-cell membrane through a hydrophobic interaction . As it is located on the exterior of the viral membrane, this sequence must be transferred across the host-cell membrane during synthesis . We have inserted either the FRHD or the F protein membrane anchor (the COOH-terminal region of the F protein) into an internal site of a secreted pIII, which lacks its natural membrane anchor . These two hydrophobic sequences behave in the bacteria just as they do in their natural eukaryotic cell host . The F protein membrane anchor functions to stop transfer, conferring a membrane-spanning topology to the F-pIII hybrid protein; however, the FRHD is moved through the cytoplasmic membrane and derivatives carrying this sequence are secreted to the periplasm . We discuss how the FRHD is compatible with passage through the membrane and yet is still able to mediate membrane fusion through a presumed hydrophobic interaction.

J Gen Virol, 1986 Jul, 67 ( Pt 7), 1461 - 7
Mapping of the major glycoprotein gene of human cytomegalovirus; Mach M et al.; The gene coding for the most abundant glycoprotein (gp58) of human cytomegalovirus (HCMV), strain AD169, was physically mapped on the viral genome . A monospecific rabbit antiserum against gp58 was used to screen a cDNA library that was constructed from poly(A)+ RNA of HCMV-infected cells in the prokaryotic expression vector lambda gt11 . A cDNA clone was identified which synthesized part of the glycoprotein . It allowed localization of the coding region within the right terminal sequence of the HindIII-F fragment between map coordinates 0.344 and 0.380 of HCMV virion DNA.

J Infect, 1986 Jul, 13 Suppl A, 61 - 71
Novel hepatitis B vaccines; Zuckerman AJ; Development of vaccines against hepatitis B has proceeded along four main lines . Human plasma-derived vaccines are safe, effective, and in general use . Subunit polypeptide vaccines formulated in micelles have reached the stage of clinical trials . Recombinant DNA vaccines have been produced in prokaryotic and eukaryotic cells, notably in yeast . The yeast-derived recombinant vaccine has proved safe and effective in extensive clinical trials, eliciting antibodies of equal quantity and quality of specificity to those elicited by plasma-derived vaccine . DNA recombination has also been applied to the development of hybrid vaccinia virus vaccines which are capable of immunological 'priming' . Finally, chemical synthesis has succeeded in producing small peptides which include specific epitopes eliciting antibody responses in experimental animals . This last approach offers a prospect of ultimately producing multivalent synthetic vaccines against several microbial agents.

Biochimie, 1986 Jul-Aug, 68(7-8), 981 - 90
Transcription of the chloroplast DNA: a review; Briat JF et al.; The transcription systems of chloroplasts and bacteria share different properties . The genetic material of chloroplasts is organized in the same way as bacterial nucleoids . The regulatory DNA sequences for transcription have a strong homology with their E . coli counterparts and some regulatory mechanisms could be conserved . The RNA polymerase subunits and some transcription factors also share similarities with prokaryotes . However, the chloroplast core-enzyme seems to be synthesized in the cytoplasm from nuclear encoded messages.

J Theor Biol, 1986 Jun 21, 120(4), 457 - 65
A hypothesis for the mechanism of mycoplasma evolution; Sladek TL; Mycoplasmas are wall-less prokaryotes which have small genomes and are known to have evolved from ancestors of Gram-positive bacteria . A model is proposed to explain how mycoplasmas may have evolved from these ancestors which had cell walls and large genomes . It is proposed that the initial step in this process was loss of the cell wall and conversion of the ancestral bacterium to an L-form . Fusion of L-forms would have resulted in a single cell that contained two or more complete genomes . It is thought that this bringing together of multiple genomes by cell fusion resulted in genetic recombination between genomes and loss of DNA segments from the cell . Data from bacterial systems are cited in support of this model.

Nature, 1986 Jun 12-18, 321(6071), 706 - 8
Idealization of the hydrophobic segment of the alkaline phosphatase signal peptide; Kendall DA et al.; Proteins secreted by prokaryotic cells are synthesized as precursors containing an amino-terminal extension sequence or signal peptide . Although these signal peptides share little primary sequence homology, recent studies suggest that they function via common pathways during the transport process and that a common element may reside in their secondary structural characteristics . We are investigating the role of an idealized hydrophobic sequence with high potential for alpha-helix formation in the Escherichia coli alkaline phosphatase signal peptide . Here, amino-acid substitutions were made using site-directed mutagenesis to produce a mutant signal sequence containing nine consecutive leucine residues in the hydrophobic core segment . Transport studies with this mutant precursor indicate that mature alkaline phosphatase is correctly targeted to the E . coli periplasm and that processing of the precursor to the mature form of the enzyme is extremely rapid . In contrast, processing is slowed when the mutant signal sequence is lengthened by the insertion of five additional leucine residues and one serine.

Nucleic Acids Res, 1986 Jun 11, 14(11), 4683 - 90
A new method for predicting signal sequence cleavage sites; von Heijne G; A new method for identifying secretory signal sequences and for predicting the site of cleavage between a signal sequence and the mature exported protein is described . The predictive accuracy is estimated to be around 75-80% for both prokaryotic and eukaryotic proteins.

Am J Physiol, 1986 Jun, 250(6 Pt 2), R991 - 5
Behavioral fever and therapy in a rickettsia-infected Orthoptera; Louis C et al.; Gryllus bimaculatus were infected with an intracellular prokaryote, Rickettsiella grylli, then reared either at fixed temperatures or in a temperature gradient (22-36 degrees C) where they could select the temperature they preferred . Only 50% of the infected insects reared at 28 degrees C or less survived after 20 days, against 75% of those reared at 30 degrees C or more and 90% of those in the temperature gradient . Examination of smears of insect tissue showed that all (100%) of the infected insects reared between 23 and 29 degrees C had developed a strong rickettsial infection . Only 20% of the insects reared in a gradient of temperature showed signs of strong infection . Body temperature of crickets in the temperature gradient, recorded using thin thermocouples, was 33 degrees C in infected crickets and 26.6 degrees C in controls . It is concluded that thermoregulatory behavior was used by the insects to produce a fever when infected with Rickettsiella grylli . This protected them and increased survival capacity.

Tsitologiia, 1986 Jun, 28(6), 563 - 75
{The origin of the eukaryotic cell . I . Historical sources and current state of the concept of symbiotic and autogenic origins of the cell}; Seravin LN; The exogenous (symbiotic) conception of the eukaryotic origin is now widely spread . It is based on the recognition of the principle of combination (addition or enclosing) of diverse prokaryotic organisms; so the complicated unicellular eukaryotic organism (eukaryotic cell) was resulted . the principle of combination takes its historical scientific sources from the ideas of Buffon . With reference to the cell this principle was claimed for the first time . In our time the exogenous conception is characterized as a "symbiotic boom", because it is widely used in attempts to explain the origin of all the main organelles of the cell (right up to the micro-bodies) . The autogenetic (endogenous) conception is based on the principle of straight phyliation, on the recognition of a successive evolutionary transformation of prokaryotic forms into eukaryotic ones . In this way all the cell organelles may have an endogenous origin . This principle springing from Lamarck has got a contemporary meaning in the doctrine of Darwin . In the next papers the author will present his own analysis and generation of the present day relevant facts to find out which of these two conceptions based on quite different scientific methodological principles may be correct.

Mol Biol Med, 1986 Jun, 3(3), 279 - 92
Cloning of cDNA coding for human tissue-type plasminogen activator and its expression in Escherichia coli; Harris TJ et al.; cDNA clones of the mRNA coding for tissue-type plasminogen activator (t-PA) have been obtained and their nucleotide sequences compared to those reported previously . A gene coding for t-PA has been reconstructed and inserted into vectors for expression in prokaryotic cells . Relatively high levels of t-PA accumulated in inclusion bodies in Escherichia coli containing an optimized expression plasmid, but only a small proportion of the insoluble protein was recovered as active enzyme using a variety of solubilization procedures.

Proc Natl Acad Sci U S A, 1986 Jun, 83(11), 3924 - 8
Genes encoding major light-harvesting polypeptides are clustered on the genome of the cyanobacterium Fremyella diplosiphon; Conley PB et al.; The polypeptide composition of the phycobilisome, the major light-harvesting complex of prokaryotic cyanobacteria and certain eukaryotic algae, can be modulated by different light qualities in cyanobacteria exhibiting chromatic adaptation . We have identified genomic fragments encoding a cluster of phycobilisome polypeptides (phycobiliproteins) from the chromatically adapting cyanobacterium Fremyella diplosiphon using previously characterized DNA fragments of phycobiliprotein genes from the eukaryotic alga Cyanophora paradoxa and from F . diplosiphon . Characterization of two lambda-EMBL3 clones containing overlapping genomic fragments indicates that three sets of phycobiliprotein genes--the alpha- and beta-allophycocyanin genes plus two sets of alpha- and beta-phycocyanin genes--are clustered within 13 kilobases on the cyanobacterial genome and transcribed off the same strand . The gene order (alpha-allophycocyanin followed by beta-allophycocyanin and beta-phycocyanin followed by alpha-phycocyanin) appears to be a conserved arrangement found previously in a eukaryotic alga and another cyanobacterium . We have reported that one set of phycocyanin genes is transcribed as two abundant red light-induced mRNAs (1600 and 3800 bases) . We now present data showing that the allophycocyanin genes and a second set of phycocyanin genes are transcribed into major mRNAs of 1400 and 1600 bases, respectively . These transcripts are present in RNA isolated from cultures grown in red and green light, although lower levels of the 1600-base phycocyanin transcript are present in cells grown in green light . Furthermore, a larger transcript of 1750 bases hybridizes to the allophycocyanin genes and may be a precursor to the 1400-base species.

EMBO J, 1986 Jun, 5(6), 1291 - 8
Characterization of the gene for the microbody (glycosomal) triosephosphate isomerase of Trypanosoma brucei; Swinkels BW et al.; To determine how microbody enzymes enter microbodies, we are studying the genes for glycosomal (microbody) enzymes in Trypanosoma brucei . Here we present our results for triosephosphate isomerase (TIM), which is found exclusively in the glycosome . We found a single TIM gene without introns, having one major polyadenylated transcript of 1500 nucleotides with a long untranslated tail of approximately 600 nucleotides . By a novel method, suitable for low abundance transcripts, we demonstrate that TIM mRNA contains the 35-nucleotide leader sequence (mini-exon) also found on several other trypanosome mRNAs . The TIM gene and a DNA segment of at least 6 kbp upstream of the gene are transcribed at an equal rate in isolated nuclei, suggesting that the gene is part of a much larger transcription unit . The predicted protein is of the same size as TIMs from other organisms and shares approximately 50% amino acid homology with other eukaryote TIMs, somewhat less with prokaryote TIMs . Trypanosome TIM is the most basic of all TIMs sequenced thus far . This is, in part, due to the presence of two clusters of positively charged residues in the molecule which may act as a signal for entry into glycosomes.

Vaccine, 1986 Jun, 4(2), 75 - 6
Vaccines made from recombinant yeast cells; Hilleman MR et al.; Production of polypeptide and protein antigens through recombinant DNA technology in prokaryotic and certain eukaryotic cells in culture is facilitating the development of new vaccines that are safe, efficacious, and economically feasible to manufacture . A current example is that of human hepatitis B vaccine that, to the present, has been produced commercially using hepatitis B viral surface antigen (HBsAg) purified from the plasma of human carriers chronically infected with the virus . Production of plasma-derived vaccine is limited by the available supply of suitable carrier plasma and by the need to apply highly technical procedures to purify the antigen as well as to ensure inactivation of all infectious agents that might be present in human plasma.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4307 - 11
Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation; Cherington V et al.; We have constructed infectious retroviral vectors, derived from Moloney murine leukemia virus, that efficiently transduce the polyoma virus tumor (T) antigens individually . The parental vector we have chosen {pZIP-NeoSV(X)1} expresses a dominant selectable marker for neomycin resistance and is a shuttle vector capable of propagation in both eukaryotic and prokaryotic cells, thus facilitating its use in structure-function studies . To address the relationship between polyoma T-antigen tumorigenesis and the effects of individual T antigens on growth control and differentiation, we used these vectors to introduce and stably express large, middle-sized, or small T antigens into mouse fibroblasts and preadipocytes . All cDNAs introduced into the vector are expressed stably even in the absence of selective pressure . The stable expression of small T antigen is noted particularly because cell lines expressing small T antigen have not been readily available prior to the use of retroviral vectors . Small T antigen-induced increase in saturation density of NIH 3T3 cells can be scored on the basis of the morphology of drug-resistant colonies . Middle-sized T antigen eliminates the growth requirement of NIH 3T3 cells for epidermal growth factor in a defined medium and permits growth in platelet-poor plasma, indicating elimination of the platelet-derived growth factor requirement as well . Large T antigen suppresses mouse preadipocyte (3T3-F442A) differentiation . These vectors and these functional assays of T-antigen activity permit genetic analysis of the relationship between tumorigenesis by T antigens and the alteration of cellular growth and differentiation.

Proc Natl Acad Sci U S A, 1986 Jun, 83(11), 3604 - 8
The A+T-rich genome of Herpesvirus saimiri contains a highly conserved gene for thymidylate synthase; Honess RW et al.; Herpesvirus saimiri (HVS) is the prototype member of a distinctive subset of lymphotropic herpesviruses (the gamma 2 subgroup) with A+T-rich coding sequences . In this paper, we show that cells productively infected with HVS contain high concentrations of a virus-specified thymidylate synthase (5,10-methylenetetrahydrofolate:dUMP C-methyltransferase, EC 2.1.1.45); we identify the active polypeptide and present the sequence of the virus gene . The predicted amino acid sequence of the 294-residue subunit of the virus enzyme is 70% homologous with the sequence of the human enzyme and about 50% homologous with prokaryotic thymidylate synthases, illustrating the remarkable structural constraints imposed by the thymidylate synthase function . However, the presence of the enzyme is not a conserved property of herpesviruses . We find no evidence for a virus-encoded thymidylate synthase activity (or a homology to a thymidylate synthase sequence) in G+C-rich representatives of alpha 1 (e.g., herpes simplex viruses, 66-68% G+C), beta (i.e., human cytomegalovirus, 58-59% G+C), and gamma 1 (i.e., Epstein-Barr virus, 60% G+C) herpesvirus subgroups . The production of excess thymidylate by a virus thymidylate synthase in cells infected with an A+T-rich herpesvirus would provide one plausible source of biased mutations by the virus-encoded replicative enzymes, which we have previously suggested as the likely general cause of differences in the mean nucleotide compositions of herpesvirus genomes.

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