Carcinogenesis, 1990 Sep, 11(9), 1509 - 15 Lonidamine: a non-mutagenic antitumor agent; Forster R et al.; Lonidiamine is a novel indazole-carboxylic acid with antitumour properties; it has been studied for potential mutagenicity in a comprehensive battery of tests . In assays for the induction of gene mutations in prokaryotes (Ames test) and eukaryotes (induction of HPRT mutations in CHO cells), negative results were obtained . There was no evidence of the induction of chromosomal damage in cultured mammalian cells in vitro . No mutagenic activity was observed in tests for chromosomal damage in vivo, in somatic cells (micronucleus test) or in germinal cells (dominant lethal test) . These negative results are consistent with observations indicating that lonidamine affects cellular energy processes, rather than the mechanisms of cell division . The lack of mutagenic properties suggests that lonidamine may present significant advantages in treatment of some tumours, offering a reduced risk of resistant clones, secondary cancer and heritable genetic damage. J Biochem Biophys Methods, 1990 Sep-Oct, 21(3), 185 - 96 The use of competitive affinity chromatography for the isolation of proteins which promote transcription; Sanzo MA; A method is described for isolating proteins which bind preferentially to specific sequences of DNA and is used to enrich preparations in proteins promoting eukaryotic gene transcription . An ion-exchange fraction which promotes the transcription of the ovalbumin gene in in vitro runoff assays was isolated from the oviducts of diethylstilbesterol-stimulated chicks . This fraction was recirculated between two coupled columns, one containing random sequences of prokaryotic DNA and the other a specific cloned DNA fragment from the 5' region of the ovalbumin gene . Recirculation was performed in the presence of a decreasing salt gradient, after which columns were disconnected and separately eluted . The eluate obtained from the column containing cloned DNA showed a preference for binding to DNA fragments derived from the ovalbumin gene when examined in nitrocellulose filter binding assays . This fraction retained the ability of its precursor to promote gene transcription in a concentration-dependent manner . Active preparations were also examined in assays measuring total RNA synthesis from native DNA templates . Although the fraction isolated from the column of specific DNA had no detectable RNA polymerase activity itself, it enhanced the activity of calf thymus RNA polymerase II . The method presented should find general application in the purification of factors which regulate biological processes by binding to specific sequences of DNA. Mol Biol Evol, 1990 Sep, 7(5), 399 - 406 Evolution of isopenicillin N synthase genes may have involved horizontal gene transfer; Landan G et al.; The isopenicillin N synthase genes from three fungal species, three Gram-positive species, and one Gram-negative bacterial species share an unusually high sequence similarity . A phylogenetic analysis was carried out to determine which type of evolutionary scenario best accounts for this similarity . The most plausible scenario is one in which a horizontal gene-transfer event, from the prokaryotes to the eukaryotes, occurred at a time close to the divergence between the Gram-positive and the Gram-negative bacteria. Genes Dev, 1990 Sep, 4(9), 1623 - 36 Amino acid changes in conserved regions of the beta-subunit of Escherichia coli RNA polymerase alter transcription pausing and termination; Landick R et al.; Control of transcription at pause and termination sites is common in bacteria . Many transcriptional pause and termination events are thought to occur in response to formation of an RNA hairpin in the nascent transcript . Some mutations in the beta-subunit of Escherichia coli RNA polymerase that confer resistance to the transcription inhibitor rifampicin also alter the response to transcriptional pause and termination signals . Here, we report isolation of termination-altering mutations that do not confer rifampicin resistance and show that such mutations occur predominantly in limited regions of the beta-subunit polypeptide . One region is between amino acid residues 500 and 575, which encompasses the locations of almost all known rifampicin-resistance mutations . Many termination-altering mutations also occur in two other regions: between amino acid residues 740 and 840 and near the carboxyl terminus of the beta-subunit (amino acid residues 1225-1342) . Amino acid sequences in these three regions of the beta-subunit are conserved between prokaryotic and eukaryotic beta-subunit homologs . Several mutations that alter transcription termination in vitro affect amino acid residues that are identical in prokaryotic and eukaryotic RNA polymerase beta-subunit homologs, suggesting that they alter an important function common to multisubunit RNA polymerases . We propose that these three regions of the beta-subunit may contact the nascent RNA transcript, the RNA-DNA heteroduplex, or the DNA template in the transcription complex and that mutations in these regions alter transcription pausing and termination by affecting these contacts. J Antimicrob Chemother, 1990 Sep, 26(3), 307 - 17 A comparative study on the inhibitory actions of chloramphenicol, thiamphenicol and some fluorinated derivatives; Cannon M et al.; Chloramphenicol, thiamphenicol and three fluorinated derivatives, Sch 24893, Sch 25298 and Sch 25393, were studied with respect to inhibition of the growth of selected bacterial strains and cell-free translation systems . Thiamphenicol was the least potent inhibitor in the former experiments, but behaved similarly to chloramphenicol and Sch 25298 in the latter, thereby displaying selective inhibition of prokaryotic protein synthesis . Thiamphenicol and Sch 25298 were shown to be like chloramphenicol in inhibiting peptidyl transferase activity specifically on 70 S ribosomes, but the antibiotics bound to their common ribosomal-receptor site with different efficiencies in the order chloramphenicol greater than thiamphenicol greater than Sch 25298 . Selected bacterial strains highly resistant to chloramphenicol and thiamphenicol because of chloramphenicol acetyltransferase production were, in contrast, highly sensitive to inhibition by the fluorinated antibiotics . Thus Sch 24893, Sch 25298 and Sch 25393 may have important uses in veterinary and clinical medicine. Mutat Res, 1990 Sep-Nov, 236(2-3), 147 - 60 DNA photolyases: physical properties, action mechanism, and roles in dark repair; Sancar GB; DNA photolyases catalyze the light-dependent repair of cis,syn-cyclobutane dipyrimidines (pyrimidine dimers) . Although the phenomenon of enzymatic photoreactivation was first described 40 years ago and photolyases were the first enzymes shown unequivocally to effect DNA repair, it has only been in the last 8 years that sufficient quantities of the enzymes have been purified to permit detailed studies of their physical properties, identification of their intrinsic chromophores, and elucidation of the mechanisms of dimer recognition and photolysis . In addition several of the genes encoding these enzymes have now been cloned and sequenced . These studies have revealed remarkable functional and structural conservation among these evolutionarily ancient enzymes and have identified a new role for photolyases in dark-repair processes which has implications for the mechanism of nucleotide excision repair in both prokaryotes and eukaryotes. New Biol, 1990 Sep, 2(9), 771 - 7 Ribonuclease H: from discovery to 3D structure; Crouch RJ; Ribonucleases H (RNases H) from Escherichia coli and retroviruses share common features at the primary amino acid sequence and activity levels . RNase H is involved in selection of the origins of replication in E . coli and in DNA synthesis of the positive strand of retroviruses . Crystallographic studies of E . coli RNase H indicate that several amino acids, conserved in both cellular and retroviral RNases H, form an active site for hydrolysis of the RNA of RNA-DNA hybrids . Multiple forms of RNase H are present in both prokaryotes and eukaryotes . It is suggested that these RNases H may be part of larger polypeptides and, as has been shown for reverse transcriptase RNase H derived from retroviruses, that the location and/or activity of the RNase H may be influenced by other regions of the polypeptides. Mol Gen Genet, 1990 Sep, 223(3), 517 - 20 Cauliflower mosaic virus P35S promoter activity in Escherichia coli; Assaad FF et al.; We present evidence that the cauliflower mosaic virus promoter P35S can direct expression of the bacterial neomycin phosphotransferase II (NPTII) gene in Escherichia coli . Transcription is initiated at several sites, the major one being located approximately 315 bases upstream of the plant start site . The nucleotide sequence directly preceding this start site is strongly homologous to the prokaryotic promoter consensus sequence . Thus constructs designed for introduction into plants can be expressed in E . coli. J Gen Virol, 1990 Sep, 71 ( Pt 9), 2023 - 31 Prokaryotic expression of the major capsid protein of human cytomegalovirus and antigenic cross-reactions with herpes simplex virus type 1; Rudolph SA et al.; The major capsid protein (MCP) of human cytomegalovirus (HCMV) was expressed in three portions as beta-galactosidase fusion proteins, covering about 75% of the open reading frame (ORF) . Fusion protein SH 1 contained nucleotides 101 to 1243 of the ORF, fusion protein FS 1 contained nucleotides 1944 to 3089 and fusion protein SS 1 covered nucleotides 2624 to 3793 . The recombinant proteins were tested for their immunoreactivity with human sera . Fusion protein FS 1 was found to represent the immunodominant region . The recombinant proteins were used to generate polyvalent rabbit antisera to investigate cross-reactivities with the major capsid protein (VP5) of herpes simplex virus type 1 (HSV-1) . A monospecific antiserum raised against the fusion protein close to the N terminus of the MCP, as well as a monoclonal antibody and a monospecific rabbit antiserum directed against the viral MCP, cross-reacted with the VP5 as shown by immunoblotting and immunofluorescence . In order to detect common epitopes of the major capsid proteins of HCMV and HSV-1, the recombinant proteins were conjugated to CNBr-activated Sepharose and taken for purification of MCP antibodies from HCMV and HSV-1 seropositive individuals . Using this affinity chromatography method, cross-reactivity could be observed with HCMV- and HSV-positive human antisera in immunoblot experiments. Biochem Cell Biol, 1990 Sep, 68(9), 1075 - 82 Kinetic characterization of dihydrofolate reductase from Drosophila melanogaster; Rancourt SL et al.; The kinetic characteristics of a purified insect dihydrofolate reductase (DHFR) have been described . The Km values for the substrate dihydrofolate and the cofactor NADPH have been estimated by primary and secondary Hanes plots to be 0.3 and 5.2 microM, respectively . Drosophila melanogaster DHFR can use folate and NADH at acidic pH values, but at a much lower rate than the preferred substrate and cofactor . Folic acid is a partial competitive inhibitor of Drosophila DHFR (Ki = 0.4 microM) and trimethoprim is a complete competitive inhibitor (Ki = 5.4 microM) . Methotrexate binds less tightly to the Drosophila enzyme than to many other DHFRs (Kd = 0.9 nM) . Drosophila DHFR is inhibited by KCl and organic mercurials and is slightly activated by urea . These data indicate that Drosophila DHFR has some characteristics which are typical of vertebrate DHFRs and others which are typical of prokaryotic DHFRs . The study of this enzyme, therefore, should aid in the definition of the structural features that are responsible for the kinetic characteristics in different DHFRs. Genomics, 1990 Sep, 8(1), 71 - 82 Construction of a facsimile data set for large genome sequence analysis; Seely O Jr et al.; A test was devised for exploring the question of whether it will be possible to identify genes in large-scale genome studies solely by sequence comparison with current sequence collections . To this end, a facsimile data set was constructed by dividing GenBank Release 56 randomly into two halves, one to serve as a reference set and the other intended to simulate raw data anticipated from large genome sequence projects . All supplementary information and identifying marks were removed from the test set after assignment of random identification numbers to each entry and their encryption . Because noncoding intervening sequences (introns) are underrepresented in GenBank, a program that introduced (simulated) introns into mRNA and prokaryotic sequences was devised . In a further attempt to make the problem of identification more realistic, random base substitutions and single-base deletions were also incorporated . The randomly ordered entries were concatenated, along with random intergenic flanking sequences, into a single long "chromosome" 33 Mb in length and then cut into "cosmids" 50-100 kb long . The chopping process was conducted in such a way that terminal overlaps would allow the order of the entries in the chromosome to be reconstituted . Finally, the sequences of a substantial fraction of the cosmids were converted to their complements . Preliminary searching of 10 test cosmids revealed that more than two-thirds of the entries in the test set should be readily identifiable by type of gene product solely on the basis of comparison with the reference set . These preliminary results suggest that existing computer regimens and sequence collections would be able to identify the majority of eukaryotic genes in any new raw data set, the existence of introns not withstanding . Moreover, the analysis can be conducted in pace with the data collection so that the search results and summary identifications will be instantly available to the research community at large. Mol Microbiol, 1990 Sep, 4(9), 1595 - 601 Tandem DNA-bound cAMP-CRP complexes are required for transcriptional repression of the deoP2 promoter by the CytR repressor in Escherichia coli; Sogaard-Andersen L et al.; We have studied the deoP2 promoter in Escherichia coli to define features important for its interaction with the CytR repressor . As is characteristic for CytR-regulated promoters, deoP2 encodes tandem binding sites for the activating complex cAMP-CRP . One of these sites, CRP-1, overlaps the -35 region, and is sufficient for activation; the second site, CRP-2, centred around -93, is indispensable for repression . Here we demonstrate, by means of in vivo titration, that CytR interaction with deoP2 depends not only on CRP-2, but also on CRP-1 and the length and possibly the sequence separating these two sites . Also, point mutations in either CRP site reduce or abolish CytR titration; however, no co-operativity is observed in the interaction of CytR with the two CRP binding sites . Furthermore, the reduction in CytR titration parallels the reduction in binding of cAMP-CRP to the mutated CRP sites in vitro . These observations are not easily explained by current models for the action of prokaryotic repressors; instead we favour a model in which the interaction of CytR with deoP2 depends on the presence of tandem DNA-bound cAMP-CRP complexes. FEMS Microbiol Rev, 1990 Sep, 7(1-2), 91 - 102 Microbes and membrane biology; Maloney PC; General principles of membrane function have been elucidated by the study of lactic acid bacteria . In this review, the operation and function of ion pumps, secondary transport systems and solute ATPases will be discussed . Despite their differences in kinetics and mechanisms between the transport systems, structural similarities can be recognized among these proteins irrespective of whether they originate from prokaryotes, lower or higher eukaryotes. FEMS Microbiol Immunol, 1990 Sep, 2(2), 103 - 10 Characterization of monoclonal antibodies to human immunodeficiency virus type 1 gp41 by HIV-1 polypeptides expressed in Escherichia coli; Larcher C et al.; Two monoclonal antibodies (MAbs) were produced in Balb/c mice by immunization with recombinant gp41 derived from expression of lambda-BH10 cDNA of the human immunodeficiency virus-1 (HIV-1) in the prokaryotic expression vector pEX-41 . Characterization of the epitopes recognized by these MAbs was done with HIV-1 envelope (env) fusion proteins expressed in Escherichia coli encoding ten distinct segments of the env proteins . In comparison, another mouse MAb, M25, a human MAb directed against gp41, which was produced by the xeno hybridoma line 3D6 and a pool of human patient sera containing antibodies to HIV-1 were tested . We were able to demonstrate that the epitopes recognized by our MAbs are located between arg732 and ser759 of the HIV-1 env glycoprotein gp160 of HTLV-III strain B . M25 reacted with epitopes between ser647 and pro731, which includes the hydrophobic transmembrane region of gp41 . The human MAb against gp41, 3D6 reacts with epitopes between ile474 and trp646, a polypeptide stretch consisting of gp120 and gp41 specific amino acids . The human serum pool, positive for HIV-1 antibodies, reacted predominantly with antigenic determinants located between ile474 and leu863 . The recombinant env fusion proteins were initially produced to test the immunoreactivity with patient sera and to characterize epitopes which are relevant for immunodiagnostic purposes . In this study, we showed that the set of recombinant env proteins is also a simple and accurate tool for the characterization of MAbs directed to the HIV envelope proteins. Trends Biochem Sci, 1990 Sep, 15(9), 347 - 51 RNA polymerase II: subunit structure and function; Woychik NA et al.; RNA polymerase II is the core of the complex apparatus that is responsible for the regulated synthesis of mRNA . A comprehensive knowledge of RNA polymerase II is essential to our understanding of the molecular mechanisms through which a variety of transcription factors regulate eukaryotic gene expression . The recent cloning of genes for all ten subunits of yeast RNA polymerase II has revealed intriguing similarities and differences between the eukaryotic RNA polymerase and its simpler prokaryotic counterpart . Epitope tagging and other experiments made possible by the cloning of these genes have provided a clearer picture of RNA polymerase II subunit composition, stoichiometry and function, and set the stage for further investigating the dialogue between RNA polymerase II and transcription factors. Biochim Biophys Acta, 1990 Aug 27, 1050(1-3), 283 - 7 Frameshifting at the internal stop codon within the mRNA for bacterial release factor-2 on eukaryotic ribosomes; Donly C et al.; A translational frameshift is necessary in the synthesis of Escherichia coli release factor 2 (RF-2) to bypass an in-frame termination codon within the coding sequence . High-efficiency frameshifting around this codon can occur on eukaryotic ribosomes as well as prokaryotic ribosomes . This was determined from the relative efficiency of translation of RF-2 RNA compared with that for the other release factor RF-1, which lacks the in-frame premature stop codon . Since the termination product is unstable an absolute measure of the efficiency of frameshifting has not been possible . A gene fusion between trpE and RF-2 was carried out to give a stable termination product as well as the frameshift product, thereby allowing a direct determination of frameshifting efficiency . The extension of RF-2 RNA near its start codon with a fragment of the trpE gene, while still allowing high efficiency frameshifting on prokaryotic ribosomes, surprisingly gives a different estimate of frameshifting on the eukaryotic ribosomes than that obtained with RF-2 RNA alone . This paradox may be explained by long distance context effects on translation rates in the frameshift region created by the trpE sequences in the gene fusion, and may reflect that pausing and translation rate are fundamental factors in determining the efficiency of frameshifting. Nucleic Acids Res, 1990 Aug 11, 18(15), 4471 - 8 Organization and expression of the 16S, 23S and 5S ribosomal RNA genes from the archaebacterium Thermoplasma acidophilum; Ree HK et al.; To elucidate the organization of the transcription units encoding the 16S, 23S and 5S rRNAs in the archaebacterium Thermoplasma acidophilum, the nucleotide sequences flanking the three rRNA genes were determined, and the 5' and 3' termini of the rRNA transcripts were mapped by primer extension and nuclease S1 protection . The results show that each of the rRNAs is transcribed separately, consistent with the lack of physical proximity among them in the T . acidophilum genome . The transcription initiation sites are preceded at an interval of approximately 25 base pairs by conserved A + T-rich sequences of the form CTTATATA, which strongly resemble the archaebacterial promoter consensus, TTTAT/AATA . In all three cases, transcription termination occurs within T-rich tracts just downstream from inverted repeats which can be folded into relatively stable stem-loop structures . While no partially processed intermediates of the 16S or 5S rRNA transcripts were detected, the 23S rRNA transcript appears to be processed by a RNase III-like activity prior to final maturation . This is the only organism known in the prokaryotic world in which the 16S, 23S and 5S rRNAs are all expressed from separate transcription units. Nature, 1990 Aug 9, 346(6284), 534 - 9 Sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the SV40 DNA replication origin; Tsurimoto T et al.; Enzymatic synthesis of DNA from the simian virus 40 origin of DNA replication has been reconstituted in vitro with eight purified components . DNA polymerase alpha-primase complex first initiates DNA synthesis at the replication origin and continues as the lagging strand polymerase . Subsequently, the DNA polymerase delta complex initiates replication on the leading strand template . Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase delta complex . A model for polymerase switching during initiation of DNA replication is presented. J Biol Chem, 1990 Aug 5, 265(22), 13066 - 73 Compartmentalization of mammalian proteins produced in Escherichia coli; Rosenwasser TA et al.; We have examined the patterns of compartmentalization of several mammalian proteins in Escherichia coli which do not have signal peptides or functional signal peptide equivalents . These proteins include (i) human proapolipoprotein A-I (proapoA-I), a 249-residue protein which contains a hexapeptide NH2-terminal prosegment plus a mature domain of 243 residues comprised of tandemly arrayed, docosapeptide repeats with predicted amphipathic alpha-helical structure; (ii) the mature apoA-I molecule without its prosegment; (iii) mouse interleukin-1 beta (IL-1 beta), a 17-kDa protein which is composed of 12 beta strands that form a tetrahedral structure; and (iv) the 31-kDa precursor of IL-1 beta, proIL-1 beta . Efficient expression of these proteins in E . coli was achieved using a plasmid that contains the nalidixic acid-inducible recA promoter and ribosome binding site from the gene 10 leader of bacteriophage T7 . In induced cultures the mammalian proteins represented up to 20% of the total bacterial protein mass . Surprisingly, cell fractionation using cold (osmotic) shock indicated that proapoA-I, apoA-I, and IL-1 beta, but not its 31-kDa precursor, were segregated into the periplasmic space with high efficiency: the ratio of periplasmic space/spheroplast distribution ranged from 0.6 to 1.1 in cells harvested 60-180 min after nalidixic acid induction . Not only was this compartmentalization efficient but it was also selective: analysis of the osmotic shock fractions revealed that the periplasmic space preparations were not contaminated with cytoplasmic proteins (e.g . phosphoglycerate dehydrogenase) . Sequential Edman degradation showed that these proteins had not undergone any NH2-terminal proteolytic processing . The mammalian proteins did not affect the export of a prototypic bacterial preprotein, beta-lactamase . Together the data suggest that osmotic shock fractionation of E . coli may facilitate the purification of functional foreign proteins produced in this prokaryote . They also raise the possibility that structural elements in these proteins other than conventional signal peptides may effect periplasmic targeting in E . coli. Bioorg Khim, 1990 Aug, 16(8), 1052 - 9 {Construction of a complete "library" of mutants at region -35 of a model prokaryotic promotor}; Drutsa VL et al.; A novel approach to study the variability of consensus sites of regulatory regions of DNA is proposed . The overall strategy includes chemical synthesis of representative series of all possible DNA structures under investigation, cloning and screening according to their function . The chemical-enzymatic synthesis of a complete library of 40-bp DNA duplexes, corresponding to the model prokaryotic promoter and differing in 6-membered segments at -35 region, is described. J Protein Chem, 1990 Aug, 9(4), 427 - 32 The primary structure of rabbit muscle enolase; Chin CC; The primary amino acid sequence of rabbit muscle enolase has been determined by standard spinning-cup sequencing techniques applied to peptides produced by chemical (cyanogen bromide and mild acid hydrolysis) and enzymatic fragmentation of the enzyme . The 433 amino acid sequence has been compared to other available enolase sequences from eukaryotic and prokaryotic sources, confirming a high degree of conserved sequence identity; the three mammalian muscle sequences (mouse and rat deduced from c-DNA sequences and rabbit) show 94% identity. FEBS Lett, 1990 Aug 1, 268(2), 408 - 14 Insertion and translocation of proteins into and through membranes; Lazdunski CJ et al.; In prokaryotic and eukaryotic organisms, proteins are efficiently sorted to reach their final destinations in a whole range of subcellular compartments . Targeting is mediated by hydrophobic signal sequences or hydrophilic targeting sequences depending upon the compartment, these sequences being often processed . Proteins cannot be translocated through a membrane in a tightly folded stage, they must have a loose conformation, the so-called 'translocation competent state', which is usually kept through interactions with chaperones . In addition to these cytosolic receptor-like components, receptors are also present on the target membranes . Depending upon the organelles and organisms, two different energy sources have been identified, energy rich phosphate bonds (ATP and GTP) and a potential across the target membrane . Besides the signal peptides, various classes of signals have been identified to account for topologies of membrane proteins . Protein secretion in bacterial organisms has been extensively studied . Various classes of proteins use different strategies, some of these may also be used in eukaryotic cells. Proc Natl Acad Sci U S A, 1990 Aug, 87(15), 5589 - 93 Ribosomes as sensors of heat and cold shock in Escherichia coli; VanBogelen RA et al.; Nearly all cells respond to an increase in temperature by inducing a set of proteins, called heat shock proteins (HSPs) . Because a large number of other stress conditions induce the HSPs (or at least the most abundant ones), this response is often termed the universal stress response . However, a careful study of conditions that truly mimic a temperature shift suggested that these proteins are induced in response to a change in the translational capacity of the cell . To test this directly, Escherichia coli cells were treated with antibiotics that target the prokaryotic ribosome . Two-dimensional gels were used to evaluate the ability of these drugs to alter the rate of synthesis of the HSPs . One group of antibiotics induced the HSPs, whereas a second group repressed the HSPs and induced another set of proteins normally induced in response to a cold shock . Depending on the concentration used, the induction of the heat or cold shock proteins mimicked a mild or severe temperature shift . In addition, antibiotics of the cold shock-inducing group were found to block high temperature induction of the HSPs . The results implicate the ribosome as a prokaryotic sensor for the heat and cold shock response networks, a role it may serve in eukaryotes as well. J Cell Sci, 1990 Aug, 96 ( Pt 4), 675 - 82 The F1 ATP synthetase beta-subunit: a major yeast novobiocin binding protein; Jenkins JR et al.; Novobiocin affects DNA metabolism in both prokaryotes and eukaryotes, resulting in cell death . In prokaryotes, the drug is a specific inhibitor of DNA gyrase, a type II topoisomerase that can be purified on a novobiocin-Sepharose column . The yeast type II topoisomerase is neither the biochemical, nor the genetic target of the antibiotic . We have purified the major yeast novobiocin binding proteins and identified one of them as the beta-subunit of the yeast mitochondrial F1 ATP synthetase, a protein highly conserved throughout evolution . The inactivation of this protein might explain the toxic effects of novobiocin on higher eukaryotic cells. FEMS Microbiol Rev, 1990 Aug, 6(4), 429 - 46 Bacterial periplasmic permeases belong to a family of transport proteins operating from Escherichia coli to human: Traffic ATPases; Ames GF et al.; Bacterial periplasmic transport systems are complex permeases composed of a soluble substrate-binding receptor and a membrane-bound complex containing 2-4 proteins . Recent developments have clearly demonstrated that these permeases are energized by the hydrolysis of ATP . Several in vitro systems have allowed a detailed study of the essential parameters functioning in these permeases . Several of the component proteins have been shown to interact with each other and the actual substrate for the transport process has been shown to be the liganded soluble receptor . The affinity of this substrate for the membrane complex is approximately 10 microM . The involvement of ATP in energy coupling is mediated by one of the proteins in the membrane complex . For each specific permease, this protein is a member of a family of conserved proteins which bind ATP . The similarity between the members of this family is high and extends itself beyond the consensus motifs for ATP binding . Interestingly, over the last few years, several eukaryotic membrane-bound proteins have been discovered which bear a high level of homology to the family of the conserved components of bacterial periplasmic permeases . Most of these proteins are known to, or can be inferred to participate in a transport process, such as in the case of the multidrug resistance protein (MDR), the STE6 gene product of yeast, and possibly the cystic fibrosis protein . This homology suggests a similarity in the mechanism of action and possibly a common evolutionary origin . This exciting development will stimulate progress in both the prokaryotic and eukaryotic areas of research by the use of overlapping procedures and model building . We propose that this universal class of permeases be called 'Traffic ATPases' to distinguish them from other types of transport systems, and to highlight their involvement in the transport of a vast variety of substrates in either direction relative to the cell interior and their use of ATP as energy source. Biochimie, 1990 Aug, 72(8), 589 - 98 Recognition of tRNA(Tyr) by tyrosyl-tRNA synthetase; Bedouelle H; In this review, I have brought together and compared the available data on the interaction between tRNA(Tyr) and tyrosyl-tRNA synthetases (TyrTS) of prokaryotic origins . The amino acid sequences of the heterologous TyrTS that can charge Escherichia coli tRNA(Tyr), show that the residues involved in the binding and recognition of tyrosine are strictly conserved whereas those involved in the interaction with tRNA(Tyr) are only weakly similar . The results of in vivo genetic complementation experiments indicate that the identity elements of tRNAs and the recognition mechanisms of such elements by the synthetases have been conserved during evolution . Heterologous or mutant tRNA(Tyr) are quantitatively charged by E coli TyrTS; the set of their common residues contains less than 10 elements if one excludes the invariant and semi-invariant residues of tRNAs . The residues of this set are candidates for a specific recognition by TyrTS . So far, adenosine-73 is the only residue for which a specific recognition of the base has been demonstrated . The residues that might serve as identity elements for E coli tRNA(Tyr) {McClain WH, Nicholas Jr HB (1987) J Mol Biol 194, 635-642} do not belong to the above set of conserved residues and therefore probably play negative roles, enabling tRNA(Tyr) to avoid non-cognate synthetases . Comparison of the charging and stability properties of mutant tRNA(Tyr) su +3 shows that bases 1 and 72 must pair (either by Watson-Crick or non-canonical hydrogen bonds) and adopt a geometry which is compatible with the helical structure of the acceptor stem in order for the mutant tRNA(Tyr) to be charged with tyrosine . If bases 1 and 72 or bases 2 and 71 cannot form such pairings, the suppressor phenotype of the mutant tRNA(Tyr)su +3 becomes thermosensitive . The weakening of base pair 1/72 by mutation or the change of adenosine-73 into guanosine results in the charging of tRNA(Tyr)su +3 with glutamine . Comparison of the structural model of the TyrTS/tRNA(Tyr) complex with the crystallographic structure of the GlnTS/tRNA(Gln) complex indicates that the mechanisms for the recognition of the acceptor arm are different in the 2 cases . Chemical attack and molecular modeling experiments have indicated that the acceptor end of tRNA(Tyr) .. . CCCA3'-OH, remains mobile after the initial binding of tRNA(Tyr) to TyrTS. Mol Microbiol, 1990 Aug, 4(8), 1303 - 10 Nucleotide sequence and codon usage of the elongation factor Tu(EF-Tu) gene from Mycoplasma pneumoniae; Yogev D et al.; The Mycoplasma pneumoniae tuf gene, encoding the elongation factor protein Tu, was cloned and sequenced . The nucleotide sequence of the mycoplasmal gene showed about 60% homology to the sequences of tuf genes of other prokaryotes, yeast mitochondria and Euglena gracilis chloroplasts, and about 75% similarity was found when comparing the deduced amino acid sequences of the various Tu proteins . The relatively low G + C content (40%) of the M . pneumoniae DNA was reflected in a low G + C content (44.6%) of the tuf gene, and in a preferential use of adenine and uracil at the third position of codons, yet codon usage analysis revealed the presence of almost all of the codons of the genetic code in the mycoplasmal gene . Southern blot hybridization of digested DNAs of 11 Mollicutes species with the entire M . pneumoniae tuf gene and with its 5' part suggested the presence of one copy only of this gene in the representative species of the Mollicutes . In this respect, the Mollicutes resemble Gram-positive bacteria and differ from the Gram-negative bacteria, which carry two copies of the tuf gene. Genes Dev, 1990 Aug, 4(8), 1396 - 403 Development-specific sigma-factor essential for late-stage differentiation of Myxococcus xanthus; Apelian D et al.; The gene for a developmentally expressed sigma-factor, sigB, has been isolated from Myxococcus xanthus by use of the sigA gene (formerly rpoD) of the vegetative sigma-factor as a probe . The sequence of sigB has been determined, and an open reading frame of 193 amino acid residues (Mr = 21,551) was identified . The amino-terminal region of SigB contains 69 residues, of which 35 are identical (50% identity) to the region of SigA required for core RNA polymerase binding and initiation of RNA polymerization . SigB also possesses many features commonly found in other prokaryotic sigma-factors . Analysis of an M . xanthus strain carrying a sigB-lacZ fusion gene revealed that sigB is expressed from a middle to late stage of differentiation corresponding to the period from the onset of sporulation to late development . A sigB deletion mutant displayed normal mound formation and sporulation; however, production of the ops gene product in myxospores of the delta sigB strain was shown to be blocked . Myxospores from the sigB deletion strain also exhibited severe defects in stability and viability during late development . Our data indicate that sigB encodes a sigma-factor essential for the maturation of myxospores at a late stage of M . xanthus differentiation . Our results also suggest that differentiation of M . xanthus is regulated by development-specific sigma-factors. EMBO J, 1990 Aug, 9(8), 2639 - 47 Eukaryotic coupled translation of tandem cistrons: identification of the influenza B virus BM2 polypeptide; Horvath CM et al.; Previous nucleotide sequence analysis of RNA segment 7 of influenza B virus indicated that, in addition to the reading frame encoding the 248 amino acid M1 protein, there is a second overlapping reading frame (BM2ORF) of 585 nucleotides that has the coding capacity for 195 amino acids . To search for a polypeptide product derived from BM2ORF, a genetically engineered beta-galactosidase-BM2ORF fusion protein was expressed in Escherichia coli and a polyclonal rabbit antiserum was raised to the purified fusion protein . This antiserum was used to identify a polypeptide, designated BM2 protein (Mr approximately equal to 12,000), that is synthesized in influenza B virus-infected cells . To understand the mechanism by which the BM2 protein is generated from influenza B virus RNA segment 7, a mutational analysis of the cloned DNA was performed and the altered DNAs were expressed in eukaryotic cells . The expression patterns of the M1 and BM2 proteins from the altered DNAs indicate that the BM2 protein initiation codon overlaps with the termination codon of the M1 protein in an overlapping translational stop-start pentanucleotide, TAATG, and that the expression of the BM2 protein requires 5'-adjacent termination of M1 synthesis . Our data suggest that a termination-reinitiation scheme is used in translation of a bicistronic mRNA derived from influenza B virus RNA segment 7, and this strategy has some analogy to prokaryotic coupled stop-start translation of tandem cistrons. Plant Mol Biol, 1990 Aug, 15(2), 207 - 23 Analysis of the genes encoding the largest subunit of RNA polymerase II in Arabidopsis and soybean; Dietrich MA et al.; We have cloned and sequenced the gene encoding the largest subunit of RNA polymerase II (RPB1) from Arabidopsis thaliana and partially sequenced genes from soybean (Glycine max) . We have also determined the nucleotide sequence for a number of cDNA clones which encode the carboxyl terminal domains (CTDs) of RNA polymerase II from both soybean and Arabidopsis . The Arabidopsis RPB1 gene encodes a polypeptide of approximately 205 kDa, consists of 12 exons, and encompasses more than 8 kb . Predicted amino acid sequence shows eight regions of similarity with the largest subunit of other prokaryotic and eukaryotic RNA polymerases, as well as a highly conserved CTD unique to RNA polymerase II . The CTDs in plants, like those in most other eukaryotes, consist of tandem heptapeptide repeats with the consensus amino acid sequence PTSPSYS . The portion of RPB1 which encodes the CTD in plants differs from that of RPB1 of animals and lower eukaryotes . All the plant genes examined contain 2-3 introns within the CTD encoding regions, and at least two plant genes contain an alternatively spliced intron in the 3' untranslated region . Several clustered amino acid substitutions in the CTD are conserved in the two plant species examined, but are not found in other eukaryotes . RPB1 is encoded by a multigene family in soybean, but a single gene encodes this subunit in Arabidopsis and most other eukaryotes. J Bioenerg Biomembr, 1990 Aug, 22(4), 593 - 618 Genetic basis of multidrug resistance of tumor cells; Kane SE et al.; Multidrug resistance in animal cells is defined as the simultaneous resistance to a variety of compounds which appear to be structurally and mechanistically unrelated . One type of multidrug resistance is characterized by the decreased accumulation of hydrophobic natural product drugs, a phenotype which is mediated by an ATP-dependent integral membrane multidrug transporter termed P-glycoprotein or P170 . The gene coding for P170 is called MDR . The nucleotide-binding domain of P-glycoprotein shares sequence homology with a family of bacterial permease ATP-binding components . In addition, P170 as a whole is structurally very similar to a number of prokaryotic and eukaryotic proteins believed to be involved in transport activities . This review summarizes our current knowledge of the molecular biology and clinical significance of MDR expression and P-glycoprotein transport activity, as well as some theories about the function of this protein in normal cells. J Ind Microbiol, 1990 Aug, 5(6), 355 - 63 Introduction and maintenance of prokaryotic DNA in Ustilago violacea; Perlin MH et al.; A strain of the basidiomycete, Ustilago violacea, was transformed with a prokaryotic plasmid, pMP4-1, which confers resistance to neomycin . U . violacea transformants were selected at a frequency of 5 per microgram pMP4-1 DNA . Such transformants were at least 8-fold more resistant to neomycin than was the untransformed recipient U . violacea . Enzyme activity associated with the neomycin resistance gene was also found in the transformants . Southern DNA-DNA hybridization detected pMP4-1-derived sequences in both nuclear and mitochondrially-associated DNAs from transformants . The patterns of hybridization suggested integration of pMP4-1 sequences into the respective genomes . DNA from the nuclear fraction of U . violacea transformants failed to produce E . coli transformants resistant to neomycin or to carbenicillin . In contrast, DNA from the mitochondrially-associated fraction in U . violacea transformants produced E . coli transformants resistant to neomycin . The E . coli transformants contained a pMP4-1-derivative, pWP8, which was subsequently shown by Southern blot analysis to harbor U . violacea mitochondrial DNA . Thus, a prokaryotic plasmid can be used to transform the eukaryote U . violacea and acquire endogenous sequences from this organism. Nature, 1990 Jul 26, 346(6282), 376 - 9 Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae; Thorsness PE et al.; The migration of genetic information from ancestral prokaryotic endosymbionts into eukaryotic nuclei is thought to have had an important role in the evolution of mitochondria and chloroplasts . Here we describe an assay for the detection of movement of DNA between mitochondria and the nucleus in yeast . Because recombinant plasmid DNA replicates after transformation into mitochondria of yeast strains lacking endogenous mitochondrial DNA we were able to propagate the nuclear genetic marker URA3 in mitochondria . As expected, the wild-type URA3 gene in mitochondria failed to complement the uracil auxotrophy (Ura-) caused by a nuclear ura3 mutation . But selection of Ura+ prototrophs from a Ura- strain carrying URA3 on a plasmid in its mitochondria enabled us to detect plasmid movement to the nucleus . Conversely, as the plasmid used also contained the mitochondrial gene COX2 required for respiratory growth, we were able to set up corresponding selections to detect migration of DNA from the nucleus to the mitochondria . Our results show that, in yeast, DNA escapes from mitochondria and appears in the nucleus at a surprisingly high frequency (approximately 2 x 10(-5) per cell per generation) . But the rate at which DNA makes the journey in the opposite direction--nucleus to mitochondria--is apparently at least 100,000 times less. Nature, 1990 Jul 26, 346(6282), 362 - 5 Structural model of ATP-binding proteins associated with cystic fibrosis, multidrug resistance and bacterial transport; Hyde SC et al.; The ATP-binding cassette (ABC) superfamily of transport systems now includes over thirty proteins that share extensive sequence similarity and domain organization . This superfamily includes the well characterized periplasmic binding protein-dependent uptake systems of prokaryotes, bacterial exporters, and eukaryotic proteins including the P-glycoprotein associated with multidrug resistance in tumours (MDR), the STE6 gene product that mediates export of yeast a-factor mating pheromone, pfMDR that is implicated in chloroquine resistance of the malarial parasite, and the product of the cystic fibrosis gene (CFTR) . Here we present a tertiary structure model of the ATP-binding cassettes characteristic of this class of transport system, based on similarities between the predicted secondary structures of members of this family and the previously determined structure of adenylate kinase . This model has implications for both the molecular basis of transport and cystic fibrosis and provides a framework for further experimentation. Eur J Biochem, 1990 Jul 20, 191(1), 177 - 85 Biochemical and genetic analysis of a maltopentaose-producing amylase from an alkaliphilic gram-positive bacterium; Candussio A et al.; Two amylases have been purified from the culture fluid of an alkaliphilic bacterium . Amylase A-60 consists of a single type of polypeptide chain of 60 kDa and exhibits an alpha-amylase-type of starch cleavage . Amylase A-180 is approximately 180 kDa in size, represents the largest exoenzyme so far identified in prokaryotes and in the initial enzyme reaction cleaves starch exclusively to maltopentaose . A-60 and A-180 are immunologically unrelated enzymes . The structural gene for amylase A-180 has been cloned and its nucleotide sequence was determined . An open reading frame was identified for a putative protein of 182 kDa whose amino-terminal sequence, deduced from the nucleotide sequence, was identical in 23 out of 25 positions to that determined for the protein . The amino-terminus of the mature protein, at the gene level, is preceded by a sequence segment showing all the characteristics of a signal peptide from Gram-positive bacteria . Analysis of the deduced amino acid sequence revealed that the 70-kDa N-terminal part is similar to classical alpha-amylases . The C-terminal part contains three repeated sequence blocks of 99 amino acid residues each which are also present in two bacterial beta-amylases . It appears, therefore, that A-180 has arisen by gene fusion events. J Biol Chem, 1990 Jul 15, 265(20), 12104 - 10 Expression and assembly of mature apotransacylase (E2b) of bovine branched-chain alpha-keto acid dehydrogenase complex in Escherichia coli . Demonstration of transacylase activity and modification by lipoylation; Griffin TA et al.; A cDNA clone encoding the entire transacylase (E2b) precursor of the bovine branched-chain alpha-keto acid dehydrogenase complex (Griffin, T . A., Lau, K . S., and Chuang, D . T . (1988) J . Biol . Chem . 263, 14008-14014) was used to construct a prokaryotic expression vector for recombinant mature E2b . The overexpression in Escherichia coli correlates with the presence near the 5'-terminus of the mature E2b coding region (nucleotides 20 to 28) of the sequence 5'-TCAAACT-CT-3' . It has been proposed that this sequence is involved in secondary mRNA recognition through interaction with the 5'-terminus of the bacterial 16 S rRNA . The mature E2b protein has transacylase activity when assayed with exogenous dihydrolipoamide and {1-14C} isovaleryl-CoA as substrates . However, the recombinant protein has no attached lipoic acid . This was established by the absence of radiolabel incorporation when transformed E . coli cells were grown in a medium containing DL-{2-3H}lipoic acid . The recombinant mature E2b protein was purified to greater than 95% purity in one step using Sepharose 4B column chromatography . The purified recombinant protein was shown to have a cubic 24-mer structure by electron microscopy and to possess a specific activity similar to that of the purified natural bovine E2b . The purified recombinant mature E2b was lipoylated in vitro in the presence of 2 mM ATP using a mitochondrial extract prepared from bovine liver . The above results provide the first evidence that the proper folding and assembly of mature bovine E2b is independent of the attachment of lipoyl moieties and that mammalian lipoylation activity is present in mitochondria. Biochim Biophys Acta, 1990 Jul 6, 1039(3), 261 - 8 The purification of dihydrofolate reductase from Drosophila melanogaster; Rancourt SL et al.; Dihydrofolate reductase (DHFR) has been purified over 30,000-fold from Drosophila adults with a yield of 35%, using a combination of low pH extraction, (NH4)2SO4 precipitation, Sephadex gel filtration, Affi-Gel blue affinity chromatography, ion exchange and gel filtration FPLC . The Drosophila enzyme is a soluble, 17-22 kDa monomeric protein displaying the two pH optima characteristic of eukaryotic DHFRs . The sequence of the first 23 amino acids from the amino-terminal end of the protein shows that Drosophila DHFR is more homologous to the mosquito and vertebrate DHFRs than to the prokaryotic enzymes . However, the percent similarity between the two insect enzymes is not as close as expected when compared to the virtually identical initial sequence conservation of mammalian DHFRs. J Biol Chem, 1990 Jul 5, 265(19), 10988 - 92 Expression and secretion of Mirabilis antiviral protein in Escherichia coli and its inhibition of in vitro eukaryotic and prokaryotic protein synthesis; Habuka N et al.; Mirabilis antiviral protein (MAP), a ribosome-inactivating protein, exhibits inhibitory effects on both plant virus infection and protein synthesis . To study these functions by site-specific mutagenesis, the total synthetic gene of MAP was constructed and expressed in Escherichia coli . However, the growth of the host was inhibited by the products, and the yield of MAP was very low . To improve the system for expressing MAP, an expression vector, pSH7, was constructed . This vector is based on the high copy number plasmid pUC19 and includes PL promoter and temperature-sensitive cI857 repressor . The plasmid also contains the ompA signal sequence and the total synthetic MAP gene . The MAP gene was expressed and its product was secreted into the culture medium after E . coli transformants were cultivated at 30 degrees C and the temperature was raised to 42 degrees C . The secreted MAP was then purified and characterized . This protein was identical to native MAP as determined by its mobility in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the amino acid sequence at the NH2 terminus, and its inhibitory effect on in vitro protein synthesis . MAP was found to inhibit the in vitro protein synthesis of rabbit reticulocyte and wheat germ . It further showed an IC50 concentration of approximately 200 nM in an E . coli in vitro translation system in contrast to ricin A-chain, a well known ribosome-inactivating protein. Eur J Biochem, 1990 Jul 5, 190(3), 463 - 7 Potent inhibitory effects of the 5'-triphosphates of (E)-5-(2-bromovinyl)-2'-deoxyuridine and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil on DNA polymerase gamma; Ono K et al.; (E)-5-(2-Bromovinyl)-2'-deoxyuridine 5'-triphosphate (BrVdUTP) and (E)-5-(2-bromovinyl)-1-beta-D-arabinofuranosyluracil 5'-triphosphate (BrVarafUTP), which are known as specific inhibitors of herpes simplex viral (type 1 and 2) DNA polymerase, were found to be strong inhibitors of DNA polymerase gamma from human KB and murine myeloma cells . In fact BrVdUTP and BrVarafUTP were found to be stronger inhibitors of DNA polymerase gamma than of other DNA polymerases having viral (herpes simplex virus or retrovirus) origin or cellular (eukaryotic alpha and beta, or prokaryotic) origin . The mode of inhibition of DNA polymerase gamma by BrVdUTP and BrVarafUTP was competitive with respect to dTTP, the normal substrate . Whereas BrVdUTP was an efficient substrate for DNA polymerase gamma and other DNA polymerases that were examined, BrVarafUTP failed to serve as a substrate for DNA synthesis . Ki values for BrVdUTP (40 nM) and BrVarafUTP (7 nM) with DNA polymerase gamma, as determined with (rA)n.(dT) as the template.primer, were much smaller than the Km values for dTTP (0.16 microM and 0.71 microM for murine and human DNA polymerase gamma, respectively) . Thus, the affinity of BrVdUTP or BrVarafUTP for DNA polymerase gamma was much stronger than that of dTTP. J Mol Biol, 1990 Jul 5, 214(1), 183 - 97 Determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution by 1H nuclear magnetic resonance spectroscopy; Billeter M et al.; The determination of the three-dimensional structure of the Antennapedia homeodomain from Drosophila in solution is described . The techniques used are 1H nuclear magnetic resonance spectroscopy for the data collection, and calculation of the protein structure with the program DISMAN followed by restrained energy minimization with a modified version of the program AMBER . A group of 19 conformers characterizes a well-defined structure for residues 7 to 59, with an average root-mean-square distance from the backbone atoms of 0.6 A relative to the mean of the 19 structures . The structure contains a helix from residues 10 to 21, a helix-turn-helix motif from residues 28 to 52, which is similar to those reported for several prokaryotic repressor proteins, and a somewhat flexible fourth helix from residues 53 to 59, which essentially forms an extension of the presumed recognition helix, residues 42 to 52 . The helices enclose a structurally well-defined molecular core of hydrophobic amino acid side-chains. Health Phys, 1990 Jul, 59(1), 29 - 34 T cell potentiation by low dose ionizing radiation: possible mechanisms; Makinodan T et al.; The phenomenon of a stimulatory response induced by an exposure to a low dose of an otherwise toxic agent has been observed in a wide variety of organisms, ranging from the simplest prokaryotes to higher eukaryotes and with a spectrum of stimuli . This would suggest that the phenomenon is evolutionarily conserved and biologically important . However, we do not understand the mechanism responsible for the phenomenon, although it has been known for over 100 y . A reasonable assumption would be that adequate models and challenging paradigms are lacking to resolve this fundamental problem . Evidence is presented to show that the potentiation of T cell response by exposing them to single or multiple low doses of ionizing radiation is a feasible cellular model to understand the phenomenon . In addition, several possible mechanisms are discussed, including the participation of stress proteins and prostaglandins in stabilizing the signal transducing, transcriptional and translational machineries, and the possible role of a more efficient mechanism of DNA repair. Biokhimiia, 1990 Jul, 55(7), 1266 - 75 {DNA-bound lipids from eukaryotic (loach spermatozoa, pigeon erythrocytes) and prokaryotic (E . coli B, phage T2) cells}; Struchkov VA et al.; Using thin-layer chromatography, some specific DNA-bound neutral lipids and phospholipids of loach spermatozoa, pigeon erythrocytes, E . coli B and phage T2 cells were studied . These lipids are represented by loosely and firmly bound components . The content of neutral lipids in the above DNAs (per mg of DNA) is 10.6, 4.8, 7.81 and 1.43 micrograms, respectively; that of phospholipids is 4.31, 1.28, 1.14 and 0.54 micrograms, respectively . The eucaryotic DNA-bound lipids are highly deficient of free cholesterol, phosphatidylcholine, phosphatidylinositol and phosphatidylserine but are rich in cardiolipin, phosphatidylethanolamine, cholesterol esters, diglycerides and free fatty acids . The quantitative and qualitative composition of DNA-bound lipids of loach spermatozoa changes during the transition from the superhelical to the relaxed conformation of DNA . Procaryotic DNA-bound neutral lipids are also represented by the free cholesterol, diglyceride and free fatty acid fractions, whereas the DNA-bound phospholipids of procaryotes consist of only two fractions, i.e., cardiolipin and phosphatidylethanolamine . The role of DNA-bound lipids in the structural and functional organization of eucaryotic and procaryotic genomes is discussed. Mutat Res, 1990 Jul, 239(1), 1 - 16 Studies on mammalian mutants defective in rejoining double-strand breaks in DNA; Jeggo PA; Mutants with defects in the rejoining of DNA double-strand breaks (dsbs) have been identified and characterised from E . coli and the yeast, Saccharomyces cerevisiae . More recently, 3 mammalian cell mutants with defective dsb rejoining have also been described . These mutants are xrs, XR-1 and L5178Y/S, and they are derived from at least two distinct complementation groups . The aim of this article is to review the current status of the studies with these mammalian cell mutants which are defective in dsb rejoining and, in particular, to compare their properties with those mutants identified from lower organisms . Possible mechanistic differences in the process of dsb rejoining between prokaryotes and lower and higher eukaryotes are discussed . All the mammalian mutants defective in dsb rejoining, are sensitive primarily to ionising radiation with little cross-sensitivity to UV-radiation . This is similar to the rad52 mutants of S . cerevisiae but contrasts to the majority of the E . coli mutants with defective dsb rejoining . Where studied, the mammalian cell mutants show enhanced resistance to ionizing radiation in late S/G2 phase, which, in one case, correlates with an enhanced ability to rejoin dsbs . This, together with other evidence, suggests that two mechanisms of dsb rejoining may exist in higher eukaryotes, one which operates uniquely in S/G2 phase and a second mechanism operating throughout the cell cycle and dependent upon the xrs and XR-1 gene products (although whether the xrs and XR-1 dependent pathways are distinct cannot at present be ascertained) . Since duplicate homologues will be present in late S/G2 phase cells, this pathway may involve a recombinational mechanism . The xrs-dependent pathway might involve illegitimate recombination, but the xrs mutants do not appear to have a major defect in homologous recombination (involving plasmid DNA) and in this respect are distinct from rad52 mutants. EMBO J, 1990 Jul, 9(7), 2321 - 9 An essential Saccharomyces cerevisiae single-stranded DNA binding protein is homologous to the large subunit of human RP-A; Heyer WD et al.; Single-stranded DNA binding proteins (SSBs) are known to play a role in DNA replication and recombination in prokaryotes . An SSB was previously purified from the yeast Saccharomyces cerevisiae . This SSB stimulated the activity of a cognate strand exchange protein (SEP1) in vitro suggesting a role in recombination . We have cloned and functionally analyzed the gene encoding this protein . DNA sequencing of the cloned DNA revealed a 621 amino acid open reading frame with a coding potential for a Mr 70,269 polypeptide . Highly significant amino acid homology was detected between this S.cerevisiae gene and the Mr 70,000 subunit polypeptide of human RP-A, a cellular protein essential for SV40 DNA replication in vitro . Therefore, we named the S.cerevisiae gene RPA1 . RPA1 encodes an essential function in this organism as shown by tetrad analysis of heterozygous insertion mutants and is continuously required for mitotic growth . Cells lacking RPA1 accumulate as multiply budded cells with a single nucleus suggesting a defect in DNA replication. Plant Cell, 1990 Jul, 2(7), 659 - 71 Complex RNA maturation pathway for a chloroplast ribosomal protein operon with an internal tRNA cistron; Christopher DA et al.; We have studied the expression of a large chloroplast ribosomal protein operon from Euglena gracilis that resembles the Escherichia coli S10 and spc ribosomal protein operons . We present evidence that 11 ribosomal protein genes, a tRNA gene, and a new locus, orf214/orf302, are expressed as a single transcription unit . The primary transcript also contains at least 15 group II and group III introns . Gene-specific probes for each ribosomal protein gene, orf214/orf302, and for trnl hybridized to a common pre-mRNA of an estimated size of 8.3 kilobases . This is the RNA size predicted for a full-length transcript of the entire operon after splicing of all 15 introns . Polycistronic ribosomal protein mRNAs accumulated primarily as spliced hepta-, hexa-, penta-, tetra-, tri-, and dicistronic mRNAs, which were presumed to arise by stepwise processing of the 8.3-kilobase pre-mRNA . A novel finding was the cotranscription of the trnl gene as an internal cistron within the ribosomal protein operon . Several combined mRNA/tRNA molecules, such as the pentacistronic rpl5-rps8-rpl36-trnl-rps14, were characterized . The occurrence of the orf214/orf302 is a unique feature of the Euglena operon, distinguishing it from all chloroplast and prokaryotic ribosomal protein operons characterized to date . The orf214/orf302 are not similar to any known genes but are cotranscribed with the ribosomal protein loci and encode stable RNA species of 2.4, 1.8, and 1.4 kilobases. J Struct Biol, 1990 Jul-Sep, 104(1-3), 84 - 90 TF1, a bacteriophage-specific DNA-binding and DNA-bending protein; Geiduschek EP et al.; We review and discuss the biological and DNA-binding properties of the bacteriophage SPO1 transcription factor 1 (TF1), a DNA-binding protein belonging to the ubiquitous prokaryotic family of Type II DNA-binding proteins . We review recent information on the effects of certain mutations in TF1 on DNA-binding and on its ability to sharply bend DNA . We also compare the DNA-binding properties of the three best-studied type II DNA-binding proteins, Escherichia coli HU, E . coli integration host factor, and TF1, and discuss them in the context of the structure and properties of chromatin in prokaryotes. Mutat Res, 1990 Jul, 231(1), 31 - 45 Comparison of dose-response relationships for ethyl methanesulfonate and 1-ethyl-1-nitrosourea in Drosophila melanogaster spermatozoa; Lee WR et al.; The relative importance of different sites of alkylation on DNA was determined by comparing two ethylating agents . 1-Ethyl-1-nitrosourea (ENU) ethylates DNA with a higher proportion of total adducts on ring oxygens than ethyl methanesulfonate, which ethylates with a higher proportion of total adducts on the N-7 of guanine . Research with somatic cells in culture and prokaryotes strongly suggests that O6-guanine (O6-G) is the principal genotoxic site . To determine the importance in germ-line mutagenesis of the O6-G site relative to the N-7 of guanine, dose-response curves were constructed for both ENU and EMS, where dose was measured as total adducts per deoxynucleotide (APdN) and response as sex-linked recessive lethals (SLRL) induced in Drosophila melanogaster spermatozoa . For both mutagens the dose response curve was linear and extrapolated to the origin . The dose-response curve for ENU was fit to an equation m = 6.2D, and the dose response curve for EMS, from this and previous experiments, was m = 3.2D where m = %SLRL and D = APdN X 10(-3) . Therefore, ENU is 1.9 times more efficient per adduct in inducing SLRL mutations than EMS . In vitro studies showed that ENU induced 9.5% of its total adducts on O6-G while EMS induced 2.0% of its adducts on O6-G . If O6-G was the sole genotoxic site, then ENU should be 4.8 times more efficient per adduct than EMS . In contrast, if N-7 G was the sole genotoxic site, ENU would be only 0.19 as effective as EMS . It was concluded that while O6-G was the principal genotoxic site, N-7 G made a significant contribution to germ-line mutagenesis. J Struct Biol, 1990 Jul-Sep, 104(1-3), 107 - 11 Mechanisms in microbial evolution; Arber W; Molecular genetic studies with prokaryotic microorganisms reveal that many different molecular processes contribute to the formation of spontaneous mutations . Besides infidelities in DNA replication and the consequences of environmental mutagens, enzyme-mediated DNA rearrangements bring about important, evolutionarily relevant alterations in the genetic information . Particular attention is given in this article to site-specific recombination at secondary crossover sites and to the transposition of mobile genetic elements with relaxed target specificity . Besides these diverse processes of genomic mutation the acquisition of genetic information from other organisms plays an uncontested role in microbial evolution . Enzymes and organelles mediating any of these mutational processes can be looked at as biological functions acting at the level of populations for the needs of biological evolution, rather than to fulfill the needs of individual living organisms. J Neurochem, 1990 Jul, 55(1), 97 - 105 Molecular cloning, structure, and expression of dopamine-beta-hydroxylase from bovine adrenal medulla; Wu HJ et al.; Dopamine-beta-hydroxylase (DBH), the enzyme that catalyzes the conversion of dopamine to norepinephrine, remains the topic of many unanswered questions . We isolated DBH cDNA clones from a bovine adrenal medulla cDNA library in the vector lambda gt10 . The longest cDNA had an open reading frame encoding an entire mature DBH 578 amino acid (64,808 dalton) polypeptide chain, though lacking a portion of the signal peptide . Additional 5' clones, obtained by the polymerase chain reaction, established the sequence of a 19 amino acid signal peptide . The mature protein sequence was 84% homologous to that of human pheochromocytoma DBH, including preservation of four potential copper ligand sites (HH or HXH) and substrate binding domains . There were no hydrophobic (putative membrane spanning) domains, other than the signal peptide . All available DBH peptide and protein sequence data can be accounted for by the cDNA-deduced 578 amino acid mature protein primary structure . Prokaryotic DBH expression yielded a 65-kilodalton DBH-immunoreactive peptide that differed from eukaryotic adrenal DBH only in N-linked, endoglycosidase F-sensitive glycosylation in the latter . Southern analysis suggested one DBH gene, whereas Northern analysis suggested a single 2.6 kbp tissue-specific DBH transcript . Comparison of the DBH primary structure with other reported sequences {Protein Identification Resource (PIR), New Atlas (NEWAT)} did not indicate that DBH is a member of any known gene family . The results suggest that a single DBH gene encodes a message specifying a single DBH polypeptide chain. J Mol Biol, 1990 Jun 20, 213(4), 671 - 6 Mutational analysis of the inverted repeats of Tn3; Nissley DV et al.; The transposase protein and the terminal inverted repeat sequences of the prokaryotic transposon Tn3 are essential for transposition . In order to determine the sequences within the inverted repeat necessary for transposition and interaction with transposase, we have constructed a series of mini-Tn3s in which specific mutations have been introduced into the inverted repeats . The effects of these mutations on transposition have been assayed in vivo using a mating-out transposition assay . Several single base-pair mutations within the transposase binding site reduce transposition frequency . Mutations that affect transposition show a greater effect when present in both inverted repeats than when present in only one inverted repeat. FEBS Lett, 1990 Jun 18, 266(1-2), 51 - 4 Prokaryotic 20 beta-hydroxysteroid dehydrogenase is an enzyme of the 'short-chain, non-metalloenzyme' alcohol dehydrogenase type; Marekov L et al.; The primary structure of 20 beta-hydroxysteroid dehydrogenase from Streptomyces hydrogenans was determined after FPLC purification of a commercial preparation . Peptides obtained from different proteolytic cleavages were purified by reverse phase HPLC . The 255-residue structure deduced was found to be distantly homologous to those of Drosophila alcohol dehydrogenase and several other dehydrogenases, establishing that prokaryotic 20 beta-hydroxysteroid dehydrogenase as a member of the 'short-chain alcohol dehydrogenase family' . With the enzymes characterized, the identity is greatest (31-34%) towards 4 other prokaryotic dehydrogenases, but the family also includes mammalian steroid and prostaglandin dehydrogenases . These enzymes are low in Cys and have a strictly conserved Tyr residue that appears to be important. Experientia, 1990 Jun 15, 46(6), 586 - 92 Lipid transport in microorganisms; Daum G et al.; Microorganisms are useful model systems for the study of intracellular transport of lipids . Eukaryotic microorganisms, such as the yeast Saccharomyces cerevisiae, are similar to higher eukaryotes with respect to organelle structure and membrane assembly . Experiments in vivo showed that transport of phosphatidylcholine between yeast microsomes and mitochondria is energy independent; transfer of phosphatidylinositol to the plasma membrane and the flux of secretory vesicles take place by different mechanisms . Linkage of transfer and biosynthesis of phospholipids was demonstrated in the case of intramitochondrial phospholipid transfer . A yeast phosphatidylinositol/phosphatidylcholine transfer protein, which is essential for cell viability, was isolated and characterized . Another phospholipid transfer protein present in yeast cytosol, which has a different specificity, is currently under investigation . Transfer of phospholipids between cellular membranes was also demonstrated with prokaryotes . The cytoplasm and the periplasma of the gram-negative facultative photosynthetic bacterium Rhodopseudomonas sphaeroides contain phospholipid transfer proteins; these seem to be involved in the biosynthesis of prokaryotic membranes. Int J Cancer, 1990 Jun 15, 45(6), 1048 - 53 Antibodies against the large subunit of the EBV-encoded ribonucleotide reductase in patients with nasopharyngeal carcinoma; Ginsburg M; The open reading frame corresponding to BORF2 and encoding the large subunit of the Epstein-Barr virus (EBV) ribonucleotide reductase has been inserted into the prokaryotic expression vector pUC19 . A 90-kDa protein was produced when the intact plasmid was used as a template for in vitro DNA-directed protein synthesis . Using templates generated by restriction digests within the BORF2 open reading frame, in the same system, truncated polypeptides resulted confirming the identity of the 90-kDa protein . The protein was then produced in a heterologous expression system and used in protein immunoblotting to screen for antibodies in sera from nasopharyngeal carcinoma (NPC), Burkitt's lymphoma (BL) or control subjects . Twenty out of 33 NPC sera were positive for antibodies against the large subunit, 13 of these were positive for both IgG and IgA, whilst 7 were positive for IgG only . Out of 15 BL sera and 10 control sera, none were positive . These results are similar to those observed for other EBV-encoded enzymes, including the DNase which had been used as an early marker for the development of NPC . The results presented here indicate that antibodies against the large subunit of ribonucleotide reductase could serve as an additional marker for NPC. J Biol Chem, 1990 Jun 15, 265(17), 9659 - 63 Stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments; Koch KW et al.; The stereochemical course of the reaction catalyzed by guanylate cyclase from bovine retinal rod outer segments was investigated using phosphorothioate analogs of GTP as chiral probes . (Sp)-Guanosine 5'-O-(1-thiotriphosphate) (Sp-GTP alpha S) is a substrate, whereas (Rp)-GTP alpha S is a competitive inhibitor (K1 = 0.1 mM), but not a substrate . (Sp)-GTP alpha S is converted into (Rp)-guanosine 3':5'-monophosphorothioate, showing that the reaction proceeds with inversion of configuration at the alpha-phosphorus atom . Km and Vmax for (Sp)-GTP alpha S (at low {Ca2+}, 20 nM) are 3.7 mM and 1.1 nmol/min/mg of rhodopsin, respectively, compared with 1.1 mM and 23.1 nmol/min/mg of rhodopsin for GTP . Vmax for the cyclization of (Sp)-GTP alpha S, as for GTP, increases 10-20-fold when the calcium level is lowered . This activity change is centered at approximately 90 nM and has a Hill coefficient of 4.8 . The configuration of the metal-substrate complex was determined by measuring the effectiveness of the Sp and Rp isomers of GTP alpha S and guanosine 5'-O-(2-thiotriphosphate) (GTP beta S) in the presence of Mg2+ or Mn2+ . (Sp)-GTP alpha S is a substrate with either Mg2+ or Mn2+, whereas (Rp)-GTP beta S is a substrate with only Mn2+ . These findings suggest that the substrate is a metal-beta, gamma-bidentate complex with delta screwsense . We also found that the cyclization reaction catalyzed by the membrane-bound guanylate cyclase from sea urchin sperm proceeds with inversion of configuration at the alpha-phosphorus atom . The stereochemical course of the reactions catalyzed by all prokaryotic and eukaryotic adenylate cyclases and guanylate cyclases studied thus far is the same. J Biol Chem, 1990 Jun 5, 265(16), 9272 - 9 Evidence for the presence of a phosphatidylinositol anchor on the lipoarabinomannan and lipomannan of Mycobacterium tuberculosis; Hunter SW et al.; The recent availability (Hunter, S.W., Gaylord, H., and Brennan, P.J . (1986) J . Biol . Chem . 261, 12345-12351) of the well known arabinomannan of Mycobacterium leprae and Mycobacterium tuberculosis as the pure native lipoarabinomannan has resulted in its implication in key aspects of the immunopathogenesis of leprosy and tuberculosis . We had indicated that the lipid moiety of lipoarabinomannan is probably based on a diacylglycerol unit in that glycerol and the two fatty acids, hexadecanoate and 10-methyloctadecanoate, were identified . In addition, lipoarabinomannan was also shown to contain myo-inositol 1-phosphate . Evidence is now presented, based on selective radiolabeling and analysis of various cleavage fragments, that the inositol phosphate exists as both an alkalilable phosphodiester and as part of a phosphatidylinositol "membrane anchor." The mannan of M . tuberculosis was also isolated as the native lipomannan . It also apparently contains a phosphatidylinositol unit but is devoid of the alkali-labile inositol phosphate residues . These lipopolysaccharides are apparently multiglycosylated versions of the well known myocobacterial mannosyl phosphatidylinositols and are prokaryotic versions of the growing list of phosphatidylinositol-anchored macromolecules . Immunogold labeling demonstrates that lipoarabinomannan is a true antigenic capsular or extracellular product of M . tuberculosis . The presence of a phosphatidylinositol residue on lipoarabinomannan may explain its interaction with macrophage membranes and role in mycobacterial pathogenesis. FEMS Microbiol Lett, 1990 Jun 1, 57(3), 239 - 43 Effects of sinefungin and S-adenosylhomocysteine on DNA and protein methyltransferases from Streptomyces and other bacteria; Barbes C et al.; Sinefungin is a naturally occurring nucleoside isolated from cultures of Streptomyces griseolus and S . incarnatus . It is structurally related to S-adenosyl-methionine (SAM) and S-adenosyl-L-homocysteine (SAH) . Its effect and level of action on prokaryotes has not been studied with the same detail as with eukaryotic cells . In this report we describe the effect of sinefungin and SAH on several Streptomyces methyltransferases (DNA and protein MTases) and on other bacterial DNA-MTases . Protein MTases are resistant to sinefungin, whereas DNA-MTases are inhibited . Adenine MTases however, seem more sensitive to this analogue than cytosine MTases. J Bioenerg Biomembr, 1990 Jun, 22(3), 451 - 71 Lipoproteins in bacteria; Hayashi S et al.; Covalent modification of membrane proteins with lipids appears to be ubiquitous in all living cells . The major outer membrane (Braun's) lipoprotein of E . coli, the prototype of bacterial lipoproteins, is first synthesized as a precursor protein . Analysis of signal sequences of 26 distinct lipoprotein precursors has revealed a consensus sequence of lipoprotein modification/processing site of Leu-(Ala, Ser)-(Gly, Ala)-Cys at -3 to +1 positions which would represent the cleavage region of about three-fourth of all lipoprotein signal sequences in bacteria . Unmodified prolipoprotein with the putative consensus sequence undergoes sequential modification and processing reactions catalyzed by glyceryl transferase, O-acyl transferase(s), prolipoprotein signal peptidase (signal peptidase II), and N-acyl transferase to form mature lipoprotein . Like all exported proteins, the export of lipoprotein requires functional SecA, SecY, and SecD proteins . Thus all precursor proteins are exported through a common pathway accessible to both signal peptidase I and signal peptidase II . The rapidly increasing list of lipid-modified proteins in both prokaryotic as well as eukaryotic cells indicates that lipoproteins comprise a diverse group of structurally and functionally distinct proteins . They share a common structural feature which is derived from a common biosynthetic pathway. Oncogene, 1990 Jun, 5(6), 839 - 43 Tumour-related expression of a translation-elongation factor-like protein; Koch I et al.; We have identified a tumour-related 43 kd cytoplasmic protein (LC/p43) using a monoclonal antibody against the total proteins of human hepatoma cell line PLC/PRF/5 . LC/p43 is preferentially expressed in a variety of tumours of human and animal origin, whereas no expression was detected in several normal adult tissues tested . LC/p43 expression was induced in rodent fibroblasts upon transfection with several viral oncogenes . Expression in non-transformed peripheral blood lymphocytes could be induced by treatment with phytohaemagglutinin (PHA) and subsequent culture with interleukin II, whereas retinoic acid treatment of transformed cells caused a drastic reduction of the antigen in the cells . Sequence analysis of three tryptic peptides of LC/p43 revealed 50-70% homology to different domains of the eukaryotic and prokaryotic translation-elongation factors EF1-alpha and EF-Tu, respectively. Oncogene, 1990 Jun, 5(6), 777 - 85 Transcription elongation and eukaryotic gene regulation; Spencer CA et al.; Each step in the synthesis of functional transcript by RNA polymerase II provides a level at which gene expression can be regulated . Control over the elongation phase of transcription is a recognized regulatory mechanism in prokaryotes; however, only recently have examples of conditional transcription elongation blockage been reported in eukaryotic cellular genes . In several cases, control over transcription elongation clearly contributes to the regulated expression of these genes . Indeed, reports that transcription by RNA polymerase II is initiated and paused on many Drosophila promoters, prior to induction of gene expression, suggests that release of an arrested polymerase, as opposed to polymerase recruitment to a disengaged promoter, may be the key regulatory step for many genes thought to be controlled by transcription initiation (Rougvie & Lis, 1988) . RNA polymerase II undergoes modifications, such as association with ancillary elongation factors and phosphorylation of its large subunit carboxy terminal domain (CTD), at stages subsequent to recruitment to a promoter and establishment of a pre-initiation complex (Reinberg & Roeder, 1987; Rappaport et al., 1987; Payne et al., 1989; Laybourn & Dahmus, 1989) . It is possible that modifications such as these, or others occurring prior to, during or following transcription initiation, may alter the holoenzyme's transcription elongation properties, to allow recognition or read-through of elongation block signals within a transcription unit . In this review, we will present features of transcription elongation blockage in several eukaryotic cellular genes in the context of our understanding of attenuation and premature transcription termination in prokaryotic and viral genes . We will also present evidence supporting the model that modifications to the RNA polymerase II transcription complex are pivotal to the control of transcriptional at the level of elongation. Proc Natl Acad Sci U S A, 1990 Jun, 87(12), 4610 - 4 The highly conserved amino acid sequence motif Tyr-Gly-Asp-Thr-Asp-Ser in alpha-like DNA polymerases is required by phage phi 29 DNA polymerase for protein-primed initiation and polymerization; Bernad A et al.; The alpha-like DNA polymerases from bacteriophage phi 29 and other viruses, prokaryotes and eukaryotes contain an amino acid consensus sequence that has been proposed to form part of the dNTP binding site . We have used site-directed mutants to study five of the six highly conserved consecutive amino acids corresponding to the most conserved C-terminal segment (Tyr-Gly-Asp-Thr-Asp-Ser) . Our results indicate that in phi 29 DNA polymerase this consensus sequence, although irrelevant for the 3'----5' exonuclease activity, is essential for initiation and elongation . Based on these results and on its homology with known or putative metal-binding amino acid sequences, we propose that in phi 29 DNA polymerase the Tyr-Gly-Asp-Thr-Asp-Ser consensus motif is part of the dNTP binding site, involved in the synthetic activities of the polymerase (i.e., initiation and polymerization), and that it is involved particularly in the metal binding associated with the dNTP site. Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4028 - 32 Peptidyl-prolyl cis-trans-isomerase from Escherichia coli: a periplasmic homolog of cyclophilin that is not inhibited by cyclosporin A; Liu J et al.; The prokaryotic peptidyl-prolyl cis-trans-isomerase called "rotamase", a homolog of the human cyclophilin, has been identified in Escherichia coli . The E . coli rotamase, a product of the gene we suggest be called "rot," has been purified to homogeneity after cloning of the gene by the polymerase chain reaction and its overexpression in E . coli . Based on the chymotrypsin-coupled assay using the tetrapeptide substrate succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, the purified protein has rotamase activity identical to human cyclophilin with a catalytic efficiency close to the upper diffusional limit (kcat/Km approximately 1.0 x 10(7) M-1 x S-1 at 10 degrees C) . Unlike the human cyclophilins, however, the E . coli rotamase is not significantly inhibited by the immunosuppressant drug cyclosporin A . By spheroplast fractionation of cells harboring the expression vector for the complete rot gene, the rotamase is located in the periplasm, where it could function in refolding of secreted proteins. Mol Gen Genet, 1990 Jun, 222(1), 71 - 6 The Mu1 transposable element of maize contains two promoter signals recognized by the Escherichia coli RNA polymerase; Del Giudice L et al.; The galactokinase (GalK) expression plasmid vector system pKO-1 has been used to screen for promoter elements in the maize transposable element Mu1 that function in Escherichia coli . Two transcriptional start points, named S1 and S2, were identified, which are located in the two direct repeats of the transposable element . This paper demonstrates that sequence elements exist in a plant transposable element which function as prokaryotic promotors. Mol Microbiol, 1990 Jun, 4(6), 881 - 93 DNA from diverse sources manifests cryptic low-level transcription in Escherichia coli; Miller WG et al.; We present evidence that DNA from diverse prokaryotic and eukaryotic sources gives rise to low-level fusion expression in Escherichia coli promoter-probe vectors . This expression may be as high as approximately 10% of the E . coli lacUV5 promoter . Although expression does not correlate with the presence of obvious E . coli promoter-like sequences, it is blocked by transcriptional terminators . Furthermore, transcription across the fusion junction is detected at levels that correlate with fusion expression . We suggest that this 'low-level transcription' (LLT) results from infrequent initiation by RNA polymerase at random sites and/or weak promoters . We propose that LLT has biological significance . In some instances, it may provide an advantageous basal level of gene expression, and we suggest that this may be true for the E . coli lacY gene . In other instances, LLT may be detrimental, in which case it may be blocked by mechanisms such as RNA secondary structure or transcriptional polarity . We present evidence to show that activation of the IS10 transposase gene by LLT is blocked at the translational level. Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4093 - 7 DNA binding properties of the purified Antennapedia homeodomain; Affolter M et al.; The in vitro DNA binding properties of a purified 68-amino acid Antennapedia homeodomain (Antp HD) peptide have been analyzed . Equilibrium and kinetic binding studies showed that stable DNA-protein complexes are formed with a Kd of 1.6 x 10(-9) M and 1.8 x 10(-10) M, respectively . Heterodimer analysis led to the conclusion that Antp HD interacts in vitro as a monomer with the DNA target sites used in our study . The results of methylation and ethylation interference studies indicated that the Antp HD closely approaches the target DNA primarily from one side in a region extending across three phosphate backbones . The DNA binding properties of the Antp HD and prokaryotic DNA binding domains that share a helix-turn-helix motif are compared. Proc Natl Acad Sci U S A, 1990 Jun, 87(11), 4193 - 7 Yeast structural gene (APN1) for the major apurinic endonuclease: homology to Escherichia coli endonuclease IV; Popoff SC et al.; DNA damage generated by oxygen radicals includes base-free apurinic/apyrimidinic (AP) sites and strand breaks that bear deoxyribose fragments . The yeast Saccharomyces cerevisiae repairs such DNA lesions by using a single major enzyme . We have cloned the yeast structural gene (APN1) encoding this AP endonuclease/3'-repair diesterase by immunological screening of a yeast genomic DNA expression library in lambda gt11 . Gene disruption experiments confirm that the Apn1 protein accounts for greater than or equal to 97% of both AP endonuclease and DNA 3'-repair diesterase activities in yeast cell-free extracts . The DNA and predicted amino acid sequences for the APN1 gene are homologous to those for the nfo gene encoding DNA endonuclease IV of Escherichia coli . This conservation of structure between a eukaryotic enzyme and its prokaryotic counterpart underscores the fundamental nature of their roles in DNA repair. Gene, 1990 May 31, 90(1), 93 - 8 Isolation and sequence analysis of CDC43, a gene involved in the control of cell polarity in Saccharomyces cerevisiae; Johnson DI et al.; The Saccharomyces cerevisiae CDC43 gene product is involved in establishing cell polarity during the cell-division cycle . When grown at restrictive temperatures, temperature-sensitive cdc43 mutants are unable to form buds and display delocalized cell-surface deposition {Adams et al., J . Cell Biol . (1990) in press} . We have isolated a cdc43-complementing plasmid from a yeast genomic-DNA library and localized the CDC43 gene, by subcloning and transposon-mutagenesis experiments, to a 1.2-kb region of DNA that contained only one significant ATG-initiated open reading frame of 213 codons . The putative CDC43 gene product contains a possible nuclear-localization signal sequence, a cysteine-rich domain and a histidine-rich domain, and a region that is similar in structure to alpha-helix-turn-alpha-helix structural domains present in some prokaryotic and eukaryotic DNA-binding proteins. J Biol Chem, 1990 May 25, 265(15), 8966 - 70 Inhibition of EcoRI DNA methylase with cofactor analogs; Reich NO et al.; Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase . The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3' . Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively . In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet . The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM) . Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively . In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM) . Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific . The ternary complex is the first sequence-specific complex detected for any DNA methylase . Potential applications to structural studies of methylase-DNA interactions are discussed. Nature, 1990 May 24, 345(6273), 361 - 4 A mechanism for synergistic activation of a mammalian gene by GAL4 derivatives; Carey M et al.; In prokaryotes and eukaryotes many gene activators work synergistically . For example, two dimers of lambda repressor interact to promote binding of these proteins to DNA, a reaction that is crucial at the repressor concentrations found in lysogens . In this case one of the bound dimers activates transcription, evidently by touching RNA polymerase . In another example, the yeast transcriptional activator GAL4, which can stimulate transcription in many eukaryotes, binds to multiple sites on DNA to activate transcription synergistically; the presence of two such sites can elicit a level of transcription more than twice that found with a single site . In this paper we show that synergistic activation by each of several GAL4 derivatives involves a mechanism different from that illustrated by the lambda repressor: multiple activator molecules can work synergistically under conditions in which their binding sites on DNA are saturated . The accompanying paper shows that under similar conditions of activator excess, GAL4 derivatives work synergistically with a heterologous mammalian gene activator . These results support the idea that eukaryotic activators can cooperate not by directly interacting but by simultaneously touching some component(s) of the transcriptional machinery. Blood, 1990 May 15, 75(10), 2030 - 4 Tumor necrosis factor-alpha exhibits greater proinflammatory activity than lymphotoxin in vitro; Desch CE et al.; Tumor necrosis factor-alpha/cachectin (TNF-alpha) and lymphotoxin (LT, TNF-beta) are primarily products of monocytes and lymphocytes, respectively . The proteins are 51% homologous in their primary structure, cause necrosis of Meth A sarcoma in vivo, are toxic to selected tumor cells in vitro, and bind to the same receptor on cells in vitro . However, some recent studies have indicated both qualitative and quantitative differences between recombinant human (rh) LT and rhTNF with respect to inducing human umbilical vein endothelial cell (HEC) adhesiveness for neutrophils and release of hematopoietic growth factor and interleukin-1 (IL-1) from HEC . The rhLT used in these studies was expressed in bacteria and was consequently not glycosylated, whereas natural LT is glycosylated . Therefore, we have compared various preparations of glycosylated and nonglycosylated rhLT with two preparations of rhTNF with respect to their proinflammatory characteristics . We now report that the same LT cDNA, when expressed in mammalian cell line and appropriately glycosylated, is also markedly less potent than rhTNF on a molar basis in inducing endothelial adhesiveness for neutrophils and in directly activating neutrophil adherence to albumin-coated plastic . All recombinant proteins, however, were equipotent on a molar basis in producing cytotoxicity in L929 cells . We conclude that differences in the primary structure of rhTNF and rhLT, rather than alterations induced by prokaryote protein processing, account for the disparate proinflammatory activity in vitro. J Biol Chem, 1990 May 5, 265(13), 7472 - 7 Purification and properties of the bacteriophage P2 ogr gene product . A prokaryotic zinc-binding transcriptional activator; Lee TC et al.; The bacteriophage P2 ogr gene product, a 72-residue basic protein rich in cysteine and histidine, is a positive regulatory factor for phage late gene transcription in both P2 and satellite phage P4 . We have developed a simple purification procedure for Ogr protein synthesized from an overproducing plasmid . Inclusion bodies formed upon overproduction were denatured using 8 M urea, and the overproduced protein was purified by gel filtration . The purified Ogr was allowed to refold under optimized conditions and was subsequently shown to be able to transactivate the phage P4 late promoter Psid in an in vitro coupled transcription-translation system . Using a 65Zn blotting method and absorption spectroscopy, we show that Ogr is a zinc-binding protein and that the conserved cysteine residues are involved in forming a complex with Zn(II) . The purification procedure described allows Ogr to be obtained in both high purity and yield. J Biol Chem, 1990 May 5, 265(13), 7632 - 7 ATP synthase complex from bovine heart mitochondria . Passive H+ conduction through F0 does not require oligomycin sensitivity-conferring protein; Pringle MJ et al.; Oligomycin sensitivity-conferring protein (OSCP) is a water-soluble subunit of bovine heart mitochondrial H(+)-ATPase (F1-F0) . In order to investigate the requirement of OSCP for passive proton conductance through mitochondrial F0, OSCP-depleted membrane preparations were obtained by extracting purified F1-F0 complexes with 4.0 M urea . The residual complexes, referred to as UF0, were found to be deficient with respect to OSCP, as well as alpha, beta, and gamma subunits of F1-ATPase, but had a full complement of coupling factor 6 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting techniques . These UF0 complexes had no intrinsic ATPase activity and were able to bind nearly the same amount of F1-ATPase in the presence of either OSCP or NH4+ ions alone, or a combination of the two . However, the preparations exhibited an absolute dependency on OSCP for conferral of oligomycin sensitivity to membrane-bound ATPase . The passive proton conductance in UF0 proteoliposomes was measured by time-resolved quenching of 9-amino-6-chloro-2-methoxyacridine or 9-aminoacridine fluorescence following a valinomycin-induced K(+)-diffusion potential . The data clearly establish that OSCP is not a necessary component of the F0 proton channel nor is its presence required for conductance blockage by the inhibitors oligomycin or dicyclohexylcarbodiimide . Furthermore, OSCP does not prevent or block passive H+ leakage . Comparisons of OSCP with the F1-F0 subunits from Escherichia coli and chloroplast lead us to suggest that mitochondrial OSCP is, both structurally and functionally, a hybrid between the beta and delta subunits of the prokaryotic systems. Science, 1990 May 4, 248(4955), 573 - 8 An RNA polymerase-binding protein that is required for communication between an enhancer and a promoter; Herendeen DR et al.; Although bacteriophage T4 late promoters are selectively recognized by Escherichia coli RNA polymerase bearing a single protein encoded by T4 gene 55 (gp55), efficient transcription at these promoters requires enhancement by the three T4 DNA polymerase accessory proteins, bound to distal "mobile enhancer" sites . Two principles are shown to govern this transcriptional enhancement: (i) Promoter recognition and communication between the enhancer and the promoter require separate phage-coded proteins . Only RNA polymerase that has the T4 gene 33 protein (gp33) bound to it is subject to enhancement by the three DNA replication proteins . (ii) Transcriptional enhancement in this prokaryotic system is promoter-specific . Promoter specificity is generated by a direct competition of phage T4 gp33 and gp55 with the E . coli promoter recognition protein, sigma 70, for binding to the E . coli RNA polymerase core . Thus, polymerase that contains sigma 70 is competent to transcribe T4 early and middle genes, but lacks the ability to be enhanced by the DNA replication proteins, while polymerase that contains gp55 and gp33 is capable of enhancement via gp33, but its activity is restricted to T4 late promoters by gp55. Biokhimiia, 1990 May, 55(5), 911 - 6 {The role of protein binding with single-stranded DNA (SSB-protein) in DNA replication in Ehrlich ascites carcinoma cells}; Koterov AN et al.; Possible involvement of the single-strand DNA-binding protein (SSB-protein) in DNA replication in Ehrlich ascite tumour (EAT) cells was studied . There was a direct correlation between the content of SSB-protein in chromatin and the intensity of replicative synthesis of DNA in various preparations of EAT in vitro and in vivo (the computed value of the correlation coefficient was equal to 0.9) . It was shown that the addition of exogenous SSB-protein to permeable EAT cells increased the replicative synthesis . It was concluded that although eukaryotic SSB-proteins are not complete analogs of prokaryotic ones, they may participate in DNA replication in eukaryotic cells and, possibly, are intracellular regulators of proliferation. Mol Gen Genet, 1990 May, 221(3), 339 - 46 Tandem promoters, tsrp1 and tsrp2, direct transcription of the thiostrepton resistance gene (tsr) of Streptomyces azureus: transcriptional initiation from tsrp2 occurs after deletion of the -35 region; Janssen GR et al.; Nuclease S1 protection experiments indicated that the thiostrepton resistance gene (tsr) of Streptomyces azureus is transcribed from tandem promoters, tsrp1 and tsrp2, that initiate transcription 45 and 173 nucleotides, respectively, upstream of the presumptive translational start codon . The -10 regions of both promoters show similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 regions do not, although they show some similarity to each other . Replacement of sequences upstream of position -22 relative to the tsrp2 start site with two different DNA segments affected the levels of the tsrp2 transcript but did not alter the tsrp2 initiation site . In vitro transcription assays using RNA polymerase from Streptomyces coelicolor A3(2) also confirmed the location of tsrp2 and identified additional start sites near tsrp2 that were barely detectable with in vivo synthesised RNA . Transcripts corresponding to initiation in vitro at trsp1 could not be detected, suggesting that additional factors are required for utilisation of this promoter. Neuron, 1990 May, 4(5), 807 - 12 Estrogen induction of a small, putative K+ channel mRNA in rat uterus; Pragnell M et al.; Estrogen causes dramatic long-term changes in the activity of the uterus . Here we report the molecular cloning of a small (700 base) uterine mRNA species capable of inducing a slow K+ current in Xenopus oocytes . The 130 amino acid protein encoded by this mRNA species has a predicted structure that does not resemble that of previously described voltage-dependent channels from mammalian sources . It is, however, similar to structural motifs found in certain prokaryotic ion channels . The induction of this mRNA by estrogen is rapid; this uterine mRNA species is not detectable in uteri from estrogen-deprived rats, but is substantially induced after 3 hr of estrogen treatment . These results support a critical role for regulation of ion channel expression by estrogen in the uterus. Mutat Res, 1990 May, 238(3), 223 - 33 Possible mutagens derived from lipids and lipid precursors; Esterbauer H et al.; Free radicals can initiate the oxidative decomposition of cellular membranes by lipid peroxidation . In this process a great variety of reactive aldehydes are produced intracellularly . Some of them, such as 4-hydroxynonenal or malonaldehyde, are biologically very active and might be involved in free radical-mediated DNA damage . A short review of the effects of aldehydic lipid peroxidation products on isolated DNA, their genotoxic effect in prokaryotes and eukaryotes and their in vivo carcinogenicity is given . Additionally own experiments on cytotoxic and genotoxic effects of 4-hydroxynonenal, 2-nonenal and nonanal in primary cultures of rat hepatocytes are reported . 4-Hydroxynonenal was highly cytotoxic at 100 microM, at subcytotoxic concentrations of 0.1-10 microM 4-hydroxynonenal increased the frequency of micronuclei, chromosomal aberrations and sister-chromatid exchange . 2-Nonenal and nonanal were not cytotoxic at 100 microM, the maximum dose tested . At 100 microM 2-nonenal led to a slight increase in micronuclei; chromosomal aberrations were not significantly altered . Nonanal had no detectable genotoxic effects . The level of endogenous 4-hydroxynonenal in tissues is in the range of 0.1-3.0 microM and can increase to 10 microM in conditions of oxidative stress; such levels appear to be sufficiently high to produce DNA damages, whether such damages are transient or irreversible is not known. New Biol, 1990 May, 2(5), 441 - 9 Targeted insertion of exogenous DNA into the eukaryotic genome by the Cre recombinase; Sauer B et al.; Cre is a 38-kD protein from bacteriophage P1 that catalyzes site-specific recombination between 34-bp loxP sequences . Our previous work has shown that Cre can perform site-specific excisive recombination not only in prokaryotes, but also in eukaryotes such as yeast and cultured mammalian cells . In this work we show that intermolecular Cre-mediated recombination can specifically direct the integration of a loxP-containing circular DNA into a chromosomal loxP site, both in yeast and in mammalian cells . The resulting integrants are predominantly simple single-copy insertions . Cre-mediated recombination thus provides a simple way to direct single-copy site-specific integration of exogenous DNA into the eukaryotic genome. Bioessays, 1990 May, 12(5), 231 - 6 DNA polymerase delta: a second eukaryotic DNA replicase; Downey KM et al.; During the past few years significant progress has been made in our understanding of the structure and function of the proteins involved in eukaryotic DNA replication . Data from several laboratories suggest that, in contrast to prokaryotic DNA replication, two distinct DNA polymerases are required for eukaryotic DNA replication, i.e . DNA polymerase delta for the synthesis of the leading strand and DNA polymerase alpha for the lagging strand . Several accessory proteins analogous to prokaryotic replication factors have been identified and some of these are specific for pol delta whereas others affect both DNA replicases . The replicases and their accessory proteins appear to be highly conserved in eukaryotes, as homologous proteins have been found in species ranging from humans to yeast. Proc Natl Acad Sci U S A, 1990 May, 87(10), 3846 - 50 Ultrasensitivity and heavy-metal selectivity of the allosterically modulated MerR transcription complex; Ralston DM et al.; The MerR metalloregulatory protein is a heavy-metal receptor that functions as the repressor and Hg(II)-responsive transcription activator of the prokaryotic mercury-resistance (mer) genes . We demonstrate that this allosterically modulated regulatory protein is sensitive to HgCl2 concentrations of 1.0 +/- 0.3 x 10(-8) M in the presence of 1.0 x 10(-3) M dithiothreitol for half-maximal induction of transcription of the mer promoter by Escherichia coli RNA polymerase in vitro . Transcription mediated by MerR increases from 10% to 90% of maximum in response to a 7-fold change in concentration of HgCl2, consistent with a threshold phenomenon known as ultrasensitivity . In addition, MerR exhibits a high degree of selectivity . Cd(II), Zn(II), Ag(I), Au(I), and Au(III) have been found to partially stimulate transcription in the presence of MerR, but concentrations at least two to three orders of magnitude greater than for Hg(II) are required . The molecular basis of the ultrasensitivity and selectivity phenomena are postulated to arise from the unusual topology of the transcription complex and a rare trigonal mercuric ion coordination environment, respectively . This mercuric ion-induced switch is to our knowledge the only known example of ultrasensitivity in a signal-responsive transcription mechanism. Proc Natl Acad Sci U S A, 1990 May, 87(9), 3513 - 7 Conservation of the regulatory subunit for the Clp ATP-dependent protease in prokaryotes and eukaryotes; Gottesman S et al.; Bacteria, tomatoes, and trypanosomes all contain genes for a large protein with extensive homology to the regulatory subunit, ClpA, of the ATP-dependent protease of Escherichia coli, Clp . All members of the family have between 756 and 926 amino acids and contain two large regions, of 233 and 192 amino acids, each containing consensus sequences for nucleotide binding . Within these regions there is at least 85% similarity between the most distant members of the family . The high degree of similarity among the ClpA-like proteins suggests that Clp-like proteases are likely to be important participants in energy-dependent proteolysis in prokaryotic and eukaryotic cells. Proc Natl Acad Sci U S A, 1990 May, 87(9), 3574 - 8 Characterization of the restriction site of a prokaryotic intron-encoded endonuclease; Chu FK et al.; The 1016-base-pair (bp) intron in the T4 bacteriophage thymidylate synthase gene (td) contains a 735-bp open reading frame that encodes a protein product with endonucleolytic activity . The endonuclease shows specificity for the intronless form of the td gene . Highly purified endonuclease cleaves the DNA of the intronless form of the td gene in vitro at 24 bp upstream of the exon 1-exon 2 junction, generating a 2-base staggered cut with 3'-hydroxyl overhangs . Although the endonuclease cleaves in exon 1, it requires some exon 2 sequence for recognition . The maximum recognition sequence lies in an 87-bp stretch, from 52 bp upstream to 35 bp downstream of the cleavage site, ending at 11 bp into exon 2 . The td intron endonuclease appears involved in the conversion of the intronless form of td to intron-containing td gene in the T-even phages . A role for intron mobility is discussed. Biochimie, 1990 May, 72(5), 337 - 43 Human bisphosphoglycerate mutase expressed in E coli: purification, characterization and structure studies; Calvin MC et al.; Bisphosphoglycerate mutase (EC 5.4.2.4.) is an erythrocyte-specific enzyme whose main function is to synthesize 2,3-diphosphoglycerate (glycerate-2,3-P2) an effector of the delivery of O2 in the tissues . In addition to its main synthase activity the enzyme displays phosphatase and mutase activities both involving 2,3-diphosphoglycerate in their reaction . Using a prokaryotic expression system, we have developed a recombinant system producing human bisphosphoglycerate mutase in E coli . The expressed enzyme has been extracted and purified to homogeneity by 2 chromatographic steps . Purity of this enzyme was checked with sodium dodecyl sulfate polyacrylamide gel and Cellogel electrophoresis and structural studies . The bisphosphoglycerate mutase expressed in E coli was found to be very similar to that of human erythrocytes and showed identical trifunctionality, thermostability, immunological and kinetics' properties . However, the absence of a blocking agent on the N-terminus results in a slight difference of the electrophoretic mobility of the enzyme expressed in E coli compared to that of the erythrocyte. Plant Mol Biol, 1990 May, 14(5), 697 - 706 Sequence conservation among the glucose transporter from the cyanobacterium Synechocystis sp . PCC 6803 and mammalian glucose transporters; Schmetterer GR; Synechocystis sp . PCC 6803 is capable of facultative photoheterotrophy with glucose as the sole carbon source . Eight mutants that were unable to take up glucose were transformed with plasmids from pooled gene banks of wild-type Synechocystis DNA prepared in an Escherichia coli vector that does not replicate in Synechocystis . One mutant (EG216) could be complemented with all gene banks to restore ability for photoheterotrophic growth . One of the gene banks was fractionated into single clones and plasmid DNA from each clone used to complement EG216 . This yielded a 1.5 kb DNA fragment that was sequenced . It contained one complete open reading frame (gtr) whose putative gene product displayed high sequence conservation with the xylose transporter of E . coli and the mammalian glucose transporters . Further, the isolated gtr gene interrupted in vitro by a kanamycin resistance cassette could be used to construct mutants from wild-type Synechocystis sp . PCC 6803 that lacked a functional glucose transporter, thus confirming the identity of the gtr gene with the glucose transporter gene . This is the first prokaryotic glucose transporter known to share a sequence relationship with mammalian glucose transporters and the first sugar transporter from a cyanobacterium characterized at the sequence level. Gene, 1990 Apr 16, 88(2), 279 - 83 Construction of a neo fusion gene for expression in both prokaryotic and eukaryotic cells; Harwood AJ et al.; A high-copy-number plasmid, pLink, was constructed to allow the direct selection in Escherichia coli of a neo fusion gene capable of conferring Geneticin (G418) resistance on mouse L cells . pLink was derived from pdMmtneo by insertion of a KpnI linker within the 5'-coding region of the neo gene . This created a minus-one frameshift mutation resulting in a translational termination within the N-terminal region of the protein . The Neo activity was restored by insertion into the modified neo gene of a piece of coding sequence derived from human HPRT cDNA . The resulting plasmid, pAH, was microinjected into mouse A9 cells and shown to confer resistance to G418. J Theor Biol, 1990 Apr 5, 143(3), 307 - 18 Periodicities in coding and noncoding regions of the genes; Arques DG et al.; Gene population statistical studies of protein coding genes and introns have identified two types of periodicities on the purine/pyrimidine alphabet: (i) the modulo 3 periodicity or coding periodicity (periodicity P3) in protein coding genes of eukaryotes, prokaryotes, viruses, chloroplasts, mitochondria, plasmids and in introns of viruses and mitochondria, and (ii) the modulo 2 periodicity (periodicity P2) in the eukaryotic introns . The periodicity study is herein extended to the 5' and 3' regions of eukaryotes, prokaryotes and viruses and shows: (i) the periodicity P3 in the 5' and 3' regions of eukaryotes . Therefore, these observations suggest a unitary and dynamic concept for the genes as for a given genome, the 5' and 3' regions have the genetic information for protein coding genes and for introns: (1) In the eukaryotic genome, the 5' (P2 and P3) and 3' (P2 and P3) regions have the information for protein coding genes (P3) and for introns (P2) . The intensity of P3 is high in 5' regions and weak in 3' regions, while the intensity of P2 is weak in 5' regions and high in 3' regions . (2) In the prokaryotic genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3) . (3) In the viral genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3) and for introns (P3) . The absence of P2 in viral introns (in opposition to eukaryotic introns) may be related to the absence of P2 in 5' and 3' regions of viruses. Int J Parasitol, 1990 Apr, 20(2), 141 - 7 Phylogenetic relationships of Cryptosporidium determined by ribosomal RNA sequence comparison; Johnson AM et al.; Reverse transcription of total cellular RNA was used to obtain a partial sequence of the small subunit ribosomal RNA of Cryptosporidium, a protist currently placed in the phylum Apicomplexa . The semi-conserved regions were aligned with homologous sequences in a range of other eukaryotes, and the evolutionary relationships of Cryptosporidium were determined by two different methods of phylogenetic analysis . The prokaryotes Escherichia coli and Halobacterium cuti were included as outgroups . The results do not show an especially close relationship of Cryptosporidium to other members of the phylum Apicomplexa. Mol Microbiol, 1990 Apr, 4(4), 669 - 76 Ureaplasma urealyticum urease genes; use of a UGA tryptophan codon; Blanchard A; Nucleotide sequence analysis of a Ureaplasma urealyticum DNA fragment, homologous to cloned urease genes of other prokaryotes, revealed three consecutive open reading frames . The molecular weights of the three deduced polypeptides are 11.2 kD, 13.6 kD and 66.6 kD . These values are consistent with the size of the three subunits previously reported for purified native urease . A significant sequence homology was found between the three polypeptides of the ureaplasmal urease and the single polypeptide of jack bean (Canavalia ensiformis) urease . Codon usage indicates that UGA is a tryptophan codon in this mollicute . Use of polymerase chain reactions has disclosed the existence of genetic polymorphism among the urease genes of different serotypes of U . urealyticum. Dan Med Bull, 1990 Apr, 37(2), 165 - 82 The antimicrobial activity of psychotherapeutic drugs and stereo-isomeric analogues; Kristiansen JE; The aim of the investigation was to throw light on the question whether drugs other than antibiotics and chemotherapeutic agents exert an antimicrobial effect . In order to elucidate this, the antimicrobial effect of selected psychotherapeutic drugs and their stereo-isomeric analogues was studied . The development of psychotherapeutic drugs from aniline dyes has been reviewed against the background of its history considered as a scientific idea . It is demonstrated that psychotherapeutic drugs have an antimicrobial effect . Psychotherapeutic drugs show antimicrobial activity at high concentrations . Stereo-isomeric analogues of known psychotherapeutic drugs also have an antimicrobial effect . The selectivity of the various stereo-isomeric compounds depends on which microorganism and which chemical compound is investigated . Synergism is found between psychotherapeutic drugs (CPZ) and penicillin in vitro, and between a non-neuroleptic stereo-isomeric compound trans-CPT and penicillin in vivo, using infected mice as material . The a