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 Condensation of DNA does not occur with prokaryotic nucleoids, but does take place when DNA is withdrawn from the vegetative phage pool to become packaged into phage heads .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 dep