Prokaryotes

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 antibacterial activity of psychotherapeutic drugs is independent of the antihistaminic, antihypersecretory, neuroleptic and antidepressant effect of these drugs . The examples chosen of investigations of the antimicrobial effect of psychotherapeutic drugs in vitro and in vivo lead to the conclusion and to the perspectives in the present study . Namely, the need for a general theory of the interplay between host organism, microorganisms and drugs . This proposition is based on a concern to argue against the view that the prokaryotic effect of eukaryote-directed drugs is without major significance, either for scientific research or for clinical treatment.

Immunol Today, 1990 Apr, 11(4), 129 - 36
Heat shock proteins and the immune response; Kaufmann SH; Heat shock proteins (HSPs) or stress proteins are produced by prokaryotic and eukaryotic cells in response to a variety of insults . After this original definition, it has become increasingly clear that HSPs can modify the function and destiny of other proteins and thus play an important role in numerous physiological processes . The heat shock response is one of the most universal reactions known and HSPs are among the most conserved molecules in phylogeny . Here Stefan H.E . Kaufmann discusses the role of HSPs in immunity with respect to both their function and their antigenicity.

Bioessays, 1990 Apr, 12(4), 167 - 72
DNA topoisomerase dysfunction: a new goal for antitumor chemotherapy; Smith PJ; Topoisomerase enzymes--found in prokaryotes to human cells--control conformational changes in DNA and aid the orderly progression of DNA replication, gene transcription and the separation of daughter chromosomes at cell division . Several classes of anti-cancer drugs are now recognised as topoisomerase poisons because of their ability to trap topoisomerase molecules on DNA as 'cleavable complexes' . Understanding how drugs generate such complexes and why they are toxic to actively growing cancer cells is a major challenge for the development of modern approaches to chemotherapy.

DNA Cell Biol, 1990 Apr, 9(3), 149 - 57
Cloning and prokaryotic expression of a biologically active human placental aldose reductase; Grundmann U et al.; cDNA clones coding for human aldose reductase (AR) were isolated by antibody screening of a placental lambda gt11 cDNA library . The cDNA comprises the entire coding region and has a total length of 1,394 bp . The sequence deduced from the open reading frame encodes a protein of 316 amino acids and its amino acid composition is identical to the placental protein 9 (PP9), whose isolation and characterization were described by Bohn et al . (1982) . The amino acid sequence of the placental human AR shows high homology to the rat AR; both proteins belong to the same protein superfamily as human liver AR, frog lens rho-crystallin, and bovine lung prostaglandin F synthase . Northern blot hybridization analysis revealed a size for the AR mRNA of approximately 1,500 bases . In addition to the full-length cDNA, one lambda gt11 clone was isolated which carries a putative intron of 597 bp at nucleotide position 754, corresponding to amino acid position 247 . Expression of the AR cDNA in Escherichia coli resulted in the synthesis of a protein with a molecular weight of approximately 35 kD which can be immunoprecipitated specifically with antiserum raised against PP9 . Despite the absence of a typical signal sequence, the human aldose reductase is partially translocated into the periplasm of the E . coli cells, where it is present in an enzymatically active form.

Biotechnology (N Y), 1990 Apr, 8(4), 343 - 6
Site-directed pegylation of recombinant interleukin-2 at its glycosylation site; Goodson RJ et al.; We have modified recombinant interleukin-2 (rIL-2) to facilitate site-directed covalent attachment of monomethoxy polyethylene glycol (PEG) . The site chosen for modification and subsequent covalent attachment with PEG (PEGylation) was the single glycosylation position found in the native interleukin-2 (IL-2) . The mutant protein was expressed in E . coli, purified, and PEGylated with a PEG-maleimide reagent to obtain PEG-cys3-rIL-2 . The PEG-cys3-rIL-2 had full bioactivity relative to the unmodified molecule and had an increase in hydrodynamic size sufficient to increase its systemic exposure by approximately 4 fold . This method has general applicability for modifying any therapeutic protein at a specific site and thereby alter its potency . In particular, it can be used to attach PEG to prokaryotically expressed recombinant proteins at their glycosylation sites.

Biochem Biophys Res Commun, 1990 Mar 30, 167(3), 1377 - 82
The primary structure of rat ribosomal protein L35; Suzuki K et al.; The amino acid sequence of the rat 60S ribosomal subunit protein L35 was deduced from the sequence of nucleotides in a recombinant cDNA and confirmed from the NH2-terminal amino acid sequence of the protein . Ribosomal protein L35 has 122 amino acids (the NH2-terminal methionine is removed after translation of the mRNA) and has a molecular weight of 14,412 . Hybridization of the cDNA to digests of nuclear DNA suggests that there are 15-17 copies of the L35 gene . The mRNA for the protein is about 570 nucleotides in length . Rat L35 is related to the archaebacterial ribosomal proteins Halobacterium marismortui L33 and Halobacterium halobium L29E; it is also related to Escherichia coli L29 and to other members of the prokaryotic ribosomal protein L29 family . The protein contains a possible internal duplication of 11 residues.

Nucleic Acids Res, 1990 Mar 25, 18(6), 1609 - 12
Conserved DNA structures in origins of replication; Eckdahl TT et al.; According to the model of Bramhill and Kornberg, initiation of DNA replication in prokaryotes involves binding of an initiator protein to origin DNA and subsequent duplex opening of adjacent direct repeat sequences . In this report, we have used computer analysis to examine the higher-order DNA structure of a variety of origins of replication from plasmids, phages, and bacteria in order to determine whether these sequences are localized in domains of altered structure . The results demonstrate that the primary sites of initiator protein binding lie in discrete domains of DNA bending, while the direct repeats lie within well-defined boundaries of an unusual anti-bent domain . The anti-bent structures arise from a periodicity of A3 and T3 tracts which avoids the 10-11 bp bending periodicity . Since DNA fragments which serve as replicators in yeast also contain these two conserved structural elements, the results provide new insight into the universal role of conserved DNA structures in DNA replication.

J Biol Chem, 1990 Mar 25, 265(9), 5113 - 20
Expression of cloned human reticulocyte 15-lipoxygenase and immunological evidence that 15-lipoxygenases of different cell types are related; Sigal E et al.; Cloned 15-lipoxygenase has been expressed for the first time in eukaryotic and prokaryotic cells . Transfection of osteosarcoma cells with a mammalian expression plasmid containing the cDNA for human reticulocyte 15-lipoxygenase resulted in cell lines that were capable of oxidizing body arachidonic acid and linoleic acid . The lipoxygenase metabolites were identified by reverse-phase and straight-phase high pressure liquid chromatography, ultraviolet spectroscopy, and direct mass spectrometry, verifying that the cDNA for 15-lipoxygenase encodes an enzyme with authentic 15-lipoxygenase activity . Incubation of the transformed cells with arachidonic acid generated 15-hydroxyeicosatetraenoic acid (HETE) and 12-HETE in a ratio of 8.6 to 1, demonstrating that 15-lipoxygenase can also perform 12-lipoxygenation . Lesser amounts of 15-keto-ETE, four isomers of 8,15-diHETE, and one isomer of 14,15-diHETE were observed . Incubation with linoleic acid generated predominantly 13-hydroxy linoleic acid . The reaction was inhibited by eicosatetraynoic acid but not by indomethacin . Antibodies to a peptide corresponding to a unique region of the predicted amino acid sequence were generated and shown to react with one major band of 70 kDa on immunoblots of human leukocyte 15-lipoxygenase . To obtain antibodies to the full length enzyme, the cDNA was subcloned into a bacterial expression vector and was expressed as a fusion with the CheY protein . The overexpressed protein was readily purified from bacteria and was shown to be immunoreactive to the peptide-derived antibody . Antibodies raised to this recombinant enzyme did not cross-react with human leukocyte 5-lipoxygenase but did identify 15-lipoxygenase in rabbit reticulocytes, human leukocytes, and tracheal epithelial cells, suggesting that the 15-lipoxygenases from these different cell types are structurally related.

AIDS Res Hum Retroviruses, 1990 Mar, 6(3), 329 - 40
Denaturation/refolding of purified recombinant HIV reverse transcriptase yields monomeric enzyme with high enzymatic activity; Deibel MR Jr et al.; We engineered a prokaryotic expression vector encoding the HIV reverse transcriptase (RT) . We grew Escherichia coli JM109 carrying the vector in a 250-liter stirred tank fermentor and purified RT (p66) under native conditions to apparent homogeneity . Purified p66 (greater than or equal to 5 mg/ml) was not stable, and was rapidly processed to its 51 kD derivative (p51), until p66:p51 levels were approximately 1:1 . These latter RT preparations were chromatographed as heterodimers and had approximately fivefold higher specific RT enzymatic activities compared with those containing predominantly p66 . P66 purified under dilute concentrations (less than or equal to 0.5 mg/ml) was monomeric in solution, resistant to p51 processing for weeks at 4 degrees C, but also had low specific RT enzymatic activities . To attempt the preparation of homogeneous p66 with specific RT enzymatic activities equivalent to p66:p51 heterodimers, purified heterodimers were denatured and p66 was purified and refolded during extensive dialysis (refolded p66) . Refolded p66 (less than or equal to 0.5 mg/ml) was monomeric in solution and had identical specific RT enzymatic activities, Km for dTTP, and inhibition by 3'-azido-3'-deoxythymidine triphosphate compared with heterodimeric p66:p51 RT . The data indicates that HIV RT obtained from recombinant E . coli under native conditions is extensively processed at concentrations promoting dimerization . Moreover, RT denaturation and refolding yields apparently homogeneous monomeric p66, with specific RT enzymatic activities equivalent to heterodimeric RT.

Nucleic Acids Res, 1990 Mar 11, 18(5), 1145 - 52
Cloning, characterization, and expression in Escherichia coli of the gene coding for the CpG DNA methylase from Spiroplasma sp . strain MQ1(M.SssI); Renbaum P et al.; We describe here the cloning, characterization and expression in E . coli of the gene coding for a DNA methylase from Spiroplasma sp . strain MQ1 (M.SssI) . This enzyme methylates completely and exclusively CpG sequences . The Spiroplasma gene was transcribed in E . coli using its own promoter . Translation of the entire message required the use of an opal suppressor, suggesting that UGA triplets code for tryptophan in Spiroplasma . Sequence analysis of the gene revealed several UGA triplets, in a 1158 bp long open reading frame . The deduced amino acid sequence revealed in M.SssI all common domains characteristic of bacterial cytosine DNA methylases . The putative sequence recognition domain of M.SssI showed no obvious similarities with that of the mouse DNA methylase, in spite of their common sequence specificity . The cloned enzyme methylated exclusively CpG sequences both in vivo and in vitro . In contrast to the mammalian enzyme which is primarily a maintenance methylase, M.SssI displayed de novo methylase activity, characteristic of prokaryotic cytosine DNA methylases.

Gene, 1990 Mar 1, 87(1), 7 - 13
Gene duplication in the evolution of the two complementing domains of gram-negative bacterial tetracycline efflux proteins; Rubin RA et al.; The resistance of Gram- bacteria to the broad-spectrum antibiotic tetracycline (Tc) results from energy-dependent drug efflux mediated by the tet gene product, the cytoplasmic membrane Tet protein . Amino acid (aa) sequences deduced from total tet nucleotide sequences of three different resistance determinants (classes A, B and C) indicate that the protein products {Tet(A), Tet(B), and Tet(C)} share a common ancestor . Hydropathic analysis of Tet sequences predicts twelve transmembrane segments in each protein, with six occurring in each half of the molecule . More importantly, the linear distributions of these segments in the N- and C-terminal halves are nearly identical, suggesting that the two halves of each Tet protein are related by a process of tandem gene duplication and divergence . Indeed, a variable but significant conservation of sequence was detected among the N- and C-terminal halves for all possible comparisons of the three proteins . Such conservation was not observed within other prokaryotic integral membrane proteins or when other prokaryotic proteins were compared to Tet halves . Similarity, both in sequence and in predicted transmembrane structural organization, strongly suggests that a common ancestor of Tet(A), Tet(B), and Tet(C) arose by duplication of a gene reading frame specifying a transmembrane protein of approximately 200 aa residues . The two halves of Tet proteins correspond to the two domains, alpha and beta, which have distinct, complementary roles in Tc efflux . Nevertheless, selective constraints to function in the cytoplasmic membrane have apparently led to maintenance of similar patterns of secondary structural organization in these complementary domains.

J Exp Med, 1990 Mar 1, 171(3), 897 - 912
Specialized functions of MHC class I molecules . I . An N-formyl peptide receptor is required for construction of the class I antigen Mta; Shawar SM et al.; Maternally transmitted factor (Mtf) is a mitochondrial gene that controls the antigenic polymorphism of the MHC class I maternally transmitted antigen (Mta) . Synthetic peptides from the NH2 terminus of the mitochondrially encoded NADH dehydrogenase subunit 1 (ND1) mimic Mtf peptide activity in an allele-specific manner . We show that the minimal ND1-alpha peptide length recognized by Mtaa-specific polyclonal CTLs was between 8 and 12 amino acids, while some Mtaa-specific CTL clones recognized a six amino acid peptide . The N-formyl group at the NH2 terminus of ND1 was essential for Mta activity . Competition experiments using N-substituted ND1-alpha peptides showed that an N-formyl peptide receptor on the target cell, which differs from the chemotactic peptide receptor, was required for Mta expression . The specificity of this receptor can account for the distinct immune restriction of Mta in which Mtf peptides are uniquely restricted by Hmt . It is possible that the Hmt gene product is the N-formyl peptide receptor itself and that it represents a class I antigen presentation molecule specialized for binding, transport, and immune presentation of N-formyl-peptide antigens of mitochondrial and prokaryotic origin.

Curr Genet, 1990 Mar, 17(3), 185 - 90
Yeast ribosomal proteins: XI . Molecular analysis of two genes encoding YL41, an extremely small and basic ribosomal protein, from Saccharomyces cerevisiae; Suzuki K et al.; Two genes encoding ribosomal protein YL41 were cloned from Saccharomyces cerevisiae chromosomal DNA . Both genes contain an uninterrupted region of only 75 nucleotides coding for a protein of 3.3 kD . Within the coding regions the nucleotide sequences are virtually identical, whereas in both the 5'- and 3'-flanking regions the two genes differ significantly from each other . The deduced protein shows an arginine and lysine content of 68 percent, i.e., 17 out of 25 residues, and the basic residues are evenly distributed over the molecule . When compared to the ribosomal protein sequences currently available no counterpart to YL41 could be found in prokaryotes and it seems likely that YL41 is a eukaryote-specific ribosomal protein.

Q Rev Biol, 1990 Mar, 65(1), 3 - 22
Evolution in bacterial plasmids and levels of selection; Eberhard WG; Gene flow between different reproductive units such as bacterial plasmids and chromosomes presents unusual problems for evolutionary analysis . Far more than in eukaryotes, reproductive advantages at several levels of selection--genes, transposons, plasmids, cells, and clones--must be considered simultaneously to understand plasmid evolution . No level consistently prevails in conflict situations, and some reproductive units carry genes that restrain their own reproduction or survival, apparently to enhance the reproduction or survival of the higher-level reproductive units that carry them . Despite gene flow between plasmids and chromosomes, genes for certain functions show strong tendencies to occur on plasmids while others consistently occur on chromosomes . Functions generally associated with plasmids are diverse, but all are useful only in locally restricted contexts; it is argued that the selective consequences of the greater horizontal (within generation) transmission of plasmids are responsible for this pattern . The tendency for prokaryote transposons, which are also horizontally mobile, to carry genes similar to those commonly on plasmids supports this argument . The apparent trends in eukaryote plasmids and transposons to lack these same characters also accords with predictions of the local adaptation hypothesis, because genes on these genetic units are generally no more horizontally mobile than chromosomal genes . There are theoretical reasons to expect that plasmid genes tend to evolve more rapidly than chromosomal genes . "The selfish interests of genes have manifestly produced 'vehicles' in the forms of organelles, cells, individuals and yet higher units . If evolution is to predict as well as describe, then selfish interests must be understood in the framework of the constraints and opportunities generated by these 'vehicles'" (Buss, 1987, p . 182).

Trends Genet, 1990 Mar, 6(3), 78 - 85
Translational control of prokaryotic gene expression; McCarthy JE et al.; Awareness of the importance of post-transcriptional control of gene expression in prokaryotes has grown enormously over the past ten years . In particular, translation features as a step where both control over constitutive rates of gene expression, as well as cis and trans regulation are exercised . Recent research has provided us with new insights into the molecular basis of these phenomena.

Res Microbiol, 1990 Mar-Apr, 141(3), 316 - 28
The homologous glucose transport proteins of prokaryotes and eukaryotes; Henderson PJ; Genes or cDNA encoding sugar transport proteins have recently been isolated from a variety of prokaryote and eukaryote organisms . By sequencing the cloned DNA, the amino acid sequences of the transport proteins have been predicted and found to comprise a homologous family extending from cyanobacteria through yeasts, algae and protozoa to mammals, including rat, mouse and man . By aligning all the sequences and comparing them with those of membrane transport proteins for other sugars, we can deduce which features of the primary sequences are critical for maintenance of structure and function . Furthermore, the conservation of extended hydrophobic and hydrophilic regions enables the construction of a consensus model of their arrangement in the membrane . Specific sequence motifs are identified and their occurrence in otherwise apparently unrelated bacterial transport proteins for citrate and tetracycline are discussed.

Photochem Photobiol, 1990 Mar, 51(3), 293 - 8
Singlet molecular oxygen induced mutagenicity in a mammalian SV40-based shuttle vector; Di Mascio P et al.; We have determined the deleterious effects of singlet oxygen (1O2), generated by thermal decomposition of the water-soluble endoperoxide 3,3'-(1,4-naphthylidene)dipropionate (NDPO2), on plasmid DNA . By following the electrophoretic mobility of DNA on agarose gels, we detected single and double strand breaks induced by treatment with NDPO2 . The vector employed was a mammalian shuttle vector and the mutagenic consequences of these damages were investigated, using as mutation target the supF suppressor tRNA gene . A high increase of the mutation frequency, over the background, was observed in plasmids transfected in bacteria or after passage through mammalian cells . Trapping agents and quencher effects and other controls confirm the involvement of 1O2 in DNA damage and mutagenicity . These findings indicate that 1O2 can induce DNA lesions which are repaired by an error-prone process in prokaryotic and eukaryotic cells.

Trans R Soc Trop Med Hyg, 1990 Mar-Apr, 84(2), 181 - 6
Enterocytozoon bieneusi (Microspora): prevalence and pathogenicity in AIDS patients; Canning EU et al.; Microsporidia are unicellular organisms, which lack mitochondria . They have prokaryotic-like ribosomes and characteristic spores containing an extrusible polar tube which serves as a passage for inoculation of the infectious agent (sporoplasm) into host cells . Clinically apparent infections in man appear to be limited to immunoprivileged sites or immunocompromised patients . One species, Encephalitozoon cuniculi, has been reported several times in patients with neurological disorders and once causing a fatal hepatitis in an AIDS patient . The most recently discovered species, Enterocytozoon bieneusi, is known only from the small intestinal enterocytes of patients with the acquired immunodeficiency syndrome, and is easily differentiated from other microsporidia by the precocious development of spore organelles in the sporont and by the poor development of the endospore layer of the spore wall . Although only about 40 cases have been reported, circumstantial evidence suggests that E . bieneusi may be the cause of a severe watery diarrhoea, which responds only temporarily to treatment with metronidazole.

Proc Natl Acad Sci U S A, 1990 Mar, 87(5), 1720 - 4
Reversible ADP-ribosylation is demonstrated to be a regulatory mechanism in prokaryotes by heterologous expression; Fu H et al.; The primary product of biological nitrogen fixation, ammonia, reversibly regulates nitrogenase activity in a variety of diazotrophs by a process called "NH4(+)-switch-off/on." Strong correlative evidence from work in Azospirillum lipoferum and Rhodospirillum rubrum indicates that this regulation involves both the inactivation of dinitrogenase reductase by dinitrogenase reductase ADP-ribosyltransferase and the reactivation by dinitrogenase reductase activating glycohydrolase . The genes encoding these two enzymes, draT and draG, have been cloned from these two organisms, so that direct genetic evidence can be marshaled to test this model in vivo . The draT/G system has been transferred to and monitored in the enteric nitrogen-fixing bacterium Klebsiella pneumoniae, an organism normally devoid of such a regulatory mechanism . The expressed draT and draG genes allowed K . pneumoniae to respond to NH4Cl with a reversible regulation of nitrogenase activity that was correlated with the reversible ADP-ribosylation of dinitrogenase reductase in vivo . Thus, the expression of draT and draG genes in K . pneumoniae is necessary and sufficient to support NH4(+)-switch-off/on, and ADP-ribosylation serves as a reversible regulatory mechanism for controlling nitrogenase activity in prokaryotes.

J Biol Chem, 1990 Feb 25, 265(6), 3417 - 23
Maturation of Escherichia coli maltose-binding protein by signal peptidase I in vivo . Sequence requirements for efficient processing and demonstration of an alternate cleavage site; Fikes JD et al.; Comparative analyses of a number of secretory proteins processed by eukaryotic and prokaryotic signal peptidases have identified a strongly conserved feature regarding the residues positioned -3 and -1 relative to the cleavage site . These 2 residues of the signal peptide are thought to constitute a recognition site for the processing enzyme and are usually amino acids with small, neutral side chains . It was shown previously that the substitution of aspartic acid for alanine at -3 of the Escherichia coli maltose-binding protein (MBP) signal peptide blocked maturation by signal peptidase I but had no noticeable effect or MBP translocation across the cytoplasmic membrane of its biological activity . This identified an excellent system in which to undertake a detailed investigation of the structural requirements and limitations for the cleavage site . In vitro mutagenesis was used to generate 14 different amino acid substitutions at -3 and 13 different amino acid substitutions at -1 of the MBP signal peptide . The maturation of the mutant precursor species expressed in vivo was examined . Overall, the results obtained agreed fairly well with statistically derived models of signal peptidase I specificity, except that cysteine was found to permit efficient processing when present at either -3 and -1, and threonine at -1 resulted in inefficient processing . Interestingly, it was found that substitutions at -1 which blocked processing at the normal cleavage site redirected processing, with varying efficiencies, to an alternate site in the signal peptide represented by the Ala-X-Ala sequence at positions -5 to -3 . The substitution of aspartic acid for alanine at -5 blocked processing at this alternate site but not the normal site . The amino acids occupying the -5 and -3 positions in many other prokaryotic signal peptides also have the potential for constituting alternate processing sites . This appears to represent another example of redundant information contained within the signal peptide.

Nucleic Acids Res, 1990 Feb 25, 18(4), 837 - 44
The methylation of one specific guanosine in a pre-tRNA prevents cleavage by RNase P and by the catalytic M1 RNA; Kahle D et al.; Several modified nucleosides were introduced during in vitro RNA synthesis into a pre-tRNA(Ser) . The pre-tRNAs were used as substrates for RNase P enzymes . No effects were observed with biotin-8-ATP or {alpha-S}-GPT, whereas with m7GTP, the cleavage reaction was completely inhibited . Analysis of pre-tRNAs which contained m7G at various positions has revealed a single base at the 5'-end of the acceptor stem where this modification absolutely prevents cleavage by catalytic M1 RNA, eukaryotic and prokaryotic RNase P holoenzymes . These results suggest that a critical contact must be made between pre-tRNA substrate and enzyme/ribozyme or that the approach of the potential cleaving agent (a positive magnesium ion) is made impossible by the positive charge at N-7 of the guanosine . In addition, we have shown that a pre-tRNA containing only m7G's can still form a complex with M1 RNA in a gel retardation assay.

Science, 1990 Feb 23, 247(4945), 930 - 8
Protein sorting to mitochondria: evolutionary conservations of folding and assembly; Hartl FU et al.; According to the endosymbiont hypothesis, mitochondria have lost the autonomy of their prokaryotic ancestors . They have to import most of their proteins from the cytosol because the mitochondrial genome codes for only a small percentage of the polypeptides that reside in the organelle . Recent findings show that the sorting of proteins into the mitochondrial subcompartments and their folding and assembly follow principles already developed in prokaryotes . The components involved may have structural and functional equivalents in bacteria.

J Immunol Methods, 1990 Feb 20, 127(1), 123 - 30
Rabbit antisera to the variable region domains of an anti-alpha(1----6) dextran using E . coli-produced VL and VH fusion proteins as immunogens; Black A et al.; Bacteria were engineered for the expression of mouse immunoglobulin light chain variable region (VL) and heavy chain variable region (VH) fusion proteins . cDNAs encoding the VL and VH of anti-alpha(1----6)dextran hybridoma protein 19.22.1 were inserted into the pATH 10 prokaryotic expression vector downstream of trp operon sequences . V domains joined to approximately 330 amino acids of the trp E gene product encoded by the expression plasmids accumulated at high levels in E . coli . In addition, the VL domain was expressed with a 15 amino acid extension at low levels in lon mutant bacteria . The trp E-VL and trp E-VH proteins were used to raise antisera in rabbits and the V specificity of the sera demonstrated.

J Biol Chem, 1990 Feb 15, 265(5), 2763 - 7
Plant cytosolic ribosomal protein S11 and chloroplast ribosomal protein CS17 . Their primary structures and evolutionary relationships; Gantt JS et al.; We have isolated cDNA clones specific for Arabidopsis thaliana cytosolic ribosomal protein S11 and plastid ribosomal protein CS17, both of which are encoded in the nuclear genome, through the use of the corresponding soybean and pea cDNAs as probes, respectively . The nucleotide sequences of all four cDNAs were determined . The amino acid sequences derived from these cDNA sequences show that the soybean and A . thaliana S11 cDNAs encode proteins that are homologous to rat ribosomal protein S11 and that the pea and A . thaliana CS17 cDNAs encode proteins that are homologous to Escherichia coli ribosomal protein S17 . The plant S11 cytosolic ribosomal proteins also show significant sequence similarity to both E . coli ribosomal protein S17 and plastid CS17 indicating that these are all related proteins . Comparison of A . thaliana CS17 with A . thaliana S11 and with E . coli S17 suggests that CS17 is more related to S17 than it is to S11 . These results support the idea that the gene encoding CS17 was derived from a prokaryotic endosymbiont and not from a duplication of the eukaryotic S11 gene.

Arch Biochem Biophys, 1990 Feb 15, 277(1), 155 - 9
Inactivation of organophosphorus nerve agents by the phosphotriesterase from Pseudomonas diminuta; Dumas DP et al.; The phosphotriesterase from Pseudomonas diminuta was tested as a catalyst for the hydrolysis of phosphofluoridates . The purified enzyme has been shown to hydrolyze the phosphorus-fluorine bond of diisopropyl fluorophosphate, isopropyl methylphosphonofluoridate, and 1,2,2-trimethylpropylmethylphosphonofluoridate at pH 7.0, 25 degrees C, with turnover numbers of 41, 56, and 5 s-1, respectively . The enzymatic rate enhancement for the hydrolysis of sarin at pH 7.0 is 2.2 X 10(7) . The turnover number for paraoxon hydrolysis is 2100 s-1 . The enzyme does not hydrolyze methanesulfonyl fluoride, phenylmethylsulfonyl fluoride, or O-p-nitrophenyl phenylsulfonate nor do these compounds inactivate or inhibit the ability of the enzyme to hydrolyze diethyl p-nitrophenyl phosphate . The breadth of substrate utility and the efficiency of the hydrolytic reaction exceed the more limited abilities of other prokaryotic and eukaryotic enzymes that catalyze similar reactions . The substantial rate enhancement exhibited by this enzyme for the hydrolysis of a wide variety of organophosphorus nerve agents make this enzyme the prime candidate for the biological detoxification of insecticide and mammalian acetylcholinesterase inhibitors.

Experientia, 1990 Feb 15, 46(2), 180 - 92
Colicins: prokaryotic killer-pores; Pattus F et al.; Colicins are plasmid-encoded protein antibiotics which kill bacteria closely related to the producing strain (generally Escherichia coli) . The study of the function of colicins has revealed many features which reflect common targeting and translocation mechanisms with bacteriophages and toxins . Like many toxins, colicins are composed of structural domains specialized in one of the different steps of the activity, targeting, translocation and killing . The major group comprises those colicins which permeabilize the cytoplasmic membrane, thereby destroying the cell's membrane potential . These colicins form well-defined voltage-gated ion channels in artificial membranes . The scope of this review is to describe some of the more recent findings concerning the structure and mode of action of pore-forming colicins with a special attention to models of membrane insertion and pore structure based on the recently determined three-dimensional structure of the pore-forming domain of colicin A.

Experientia, 1990 Feb 15, 46(2), 174 - 80
Biogenesis of prokaryotic pores; Nikaido H et al.; The prokaryotic pore-forming proteins are synthesized in the cytoplasm, and are assembled in their functional form in the outer membrane . They begin to traverse the cytoplasmic membrane via the SecY/SecA export pathway, which is shared also by periplasmic proteins . The sorting signals that direct these proteins to the outer membrane could be present in the three-dimensional conformations of the proteins, but some results suggest that they may be present in short, contiguous sequences . Outer membrane proteins share a rather hydrophilic amino acid composition, and appear to be rich in beta-sheets (with the exception of lipoproteins) . This observation as well as the demonstration of periplasmic export intermediates favor the secretion pathway through the periplasm, as opposed to export through fusion sites between the inner and the outer membrane, but such intermediates have not yet been observed with the wild type proteins under physiological conditions.

J Gen Virol, 1990 Feb, 71 ( Pt 2), 271 - 9
Localization of antigenic sites of the E2 glycoprotein of transmissible gastroenteritis coronavirus; Correa I et al.; Four antigenic sites of the E2 glycoprotein of transmissible gastroenteritis virus were defined by competitive radioimmunoassays of monoclonal antibodies (MAbs) . Here, we describe the localization of these sites by testing the antigenicity of protein fragments and prokaryotic expression products of E2 gene fragments, and by sequencing of MAb-resistant (mar) mutants . Partial proteolysis of purified E2 protein allowed the isolation of a 28K fragment recognized by both site A- and site C-specific MAbs . An antiserum against this fragment bound to a synthetic peptide containing residues 1 to 18 and to an expression product containing residues 1 to 325 . The same expression product was recognized by site C-specific MAbs . These data indicate that residues within the sequence 1 to 325 contribute to site C and possibly also to site A . Sequencing of mar mutants that escaped neutralization by site A-specific MAbs indicated that residues 538 and 543 also belong to site A . The binding of site-specific MAbs to expression products led directly to the localization of sites B and D, between residues 1 to 325 and 379 to 529, respectively . The first 37% of the polypeptide chain of E2 appears to be more immunogenic than the rest of the sequence.

Semin Cell Biol, 1990 Feb, 1(1), 55 - 63
Role of cytosolic factors in the transport of proteins across membranes; Wiech H et al.; In a review article published in 1986 we emphasized that an unfolded conformation is a prerequisite for the transport of precursor proteins across membranes, and that cytosolic factors exist whose function is to maintain what we termed the transport-competent conformation of precursor proteins . Subsequent observations in a number of different in vitro systems related both the competent conformation and the cytosolic factors to the recent observations on the ATP-requirements for protein transport . Here we review the currently available data on such factors, and their ATP-requirements, for prokaryotic as well as eukaryotic organisms . Furthermore, we discuss possible models for their action and relate them to the so-called molecular chaperones . These were originally defined as being involved in the proper folding and assembly of oligomeric protein complexes, but have since been shown in addition to facilitate the transport of proteins across membranes.

Semin Cell Biol, 1990 Feb, 1(1), 27 - 35
Chaperonins and the immune response; Young DB; Chaperonins are a major target of the immune response to bacteria . Infection, or immunization, with bacteria induces a strong antibody response to chaperonins, and the same proteins also provide a focus for activation of T lymphocyte subsets--including CD4, CD8 and gamma-delta T cells . The high degree of sequence conservation between prokaryotic and eukaryotic chaperonins makes them candidate antigens for models of autoimmunity based on molecular mimicry, and it is possible that the immune response to chaperonins has both protective and pathogenic potential . The interactions between chaperonins and the immune response are reviewed in this article, primarily from the perspective of intracellular bacterial infection.

J Gen Microbiol, 1990 Feb, 136 ( Pt 2), 343 - 52
Cloning, sequencing and expression of the glutamine synthetase structural gene (glnA) from the obligate methanotroph Methylococcus capsulatus (Bath); Cardy DL et al.; The structural gene (glnA) encoding the ammonia-assimulation enzyme glutamine synthetase (GS) has been cloned from the obligate methanotroph Methylococcus capsulatus (Bath) . Complementation of Escherichia coli glnA mutants was demonstrated . In vitro expression analysis revealed that the cloned glnA gene coded for a polypeptide of apparent Mr 60,000, as determined by PAGE . Expression of the M . capsulatus (Bath) glnA gene in E . coli was regulated by nitrogen levels in an Ntr+ but not an Ntr- background . The nucleotide sequence of the M . capsulatus (Bath) glnA gene and flanking sequences was determined . This gene, of 1407 bp, encoded a polypeptide of Mr 51717 containing 468 amino acids . The 5' leader region contained three putative promoters . Promoters P1 and P3 resembled the canonical -10 -35 E . coli-type promoter . Promoter P2, which was located between P1 and P3, resembled the NtrA-dependent promoters of enteric organisms . A potential NtrC-binding site was also determined, flanking the Pribnow box at P1 . Comparisons of nucleotide-derived amino acid sequences of GS enzymes from prokaryotes and eukaryotes with GS from M . capsulatus are made.

Philos Trans R Soc Lond B Biol Sci, 1990 Jan 30, 326(1236), 391 - 410
Homologous sugar transport proteins in Escherichia coli and their relatives in both prokaryotes and eukaryotes; Henderson PJ et al.; Separate proteins for proton-linked transport of D-xylose, L-arabinose, D-galactose, L-rhamnose and L-fucose into Escherichia coli are being studied . By cloning and sequencing the appropriate genes, the amino acid sequences of proteins for D-xylose/H+ symport (XylE), L-arabinose/H+ symport (AraE), and part of the protein for D-galactose/H+ symport (GalP) have been determined . These are homologous, with at least 28% identical amino acid residues conserved in the aligned sequences, although their primary sequences are not similar to those of other E . coli transport proteins for lactose, melibiose, or D-glucose . However, they are equally homologous to the passive D-glucose transport proteins from yeast, rat brain, rat adipocytes, human erythrocytes, human liver, and a human hepatoma cell line . The substrate specificity of GalP from E . coli is similar to that of the mammalian glucose transporters . Furthermore, the activities of GalP, AraE and the mammalian glucose transporters are all inhibited by cytochalasin B and N-ethylmaleimide . Conserved residues in the aligned sequences of the bacterial and mammalian transporters are identified, and the possible roles of some in sugar binding, cation binding, cytochalasin binding, and reaction with N-ethylmaleimide are discussed . Each protein is independently predicted to form 12 hydrophobic, membrane-spanning alpha-helices with a central hydrophilic segment, also comprised of alpha-helix . This unifying structural model of the sugar transporters shares features with other ion-linked transport proteins for citrate or tetracycline.

Philos Trans R Soc Lond B Biol Sci, 1990 Jan 30, 326(1235), 179 - 87
DNA methylation: evolution of a bacterial immune function into a regulator of gene expression and genome structure in higher eukaryotes; Bestor TH; The amino acid sequence of mammalian DNA methyltransferase has been deduced from the nucleotide sequence of a cloned cDNA . It appears that the mammalian enzyme arose during evolution via fusion of a prokaryotic restriction methyltransferase gene and a second gene of unknown function . Mammalian DNA methyltransferase currently comprises an N-terminal domain of about 1000 amino acids that may have a regulatory role and a C-terminal 570 amino acid domain that retains similarities to bacterial restriction methyltransferases . The sequence similarities among mammalian and bacterial DNA cytosine methyltransferases suggest a common evolutionary origin . DNA methylation is uncommon among those eukaryotes having genomes of less than 10(8) base pairs, but nearly universal among large-genome eukaryotes . This and other considerations make it likely that sequence inactivation by DNA methylation has evolved to compensate for the expansion of the genome that has accompanied the development of higher plants and animals . As methylated sequences are usually propagated in the repressed, nuclease-insensitive state, it is likely that DNA methylation compartmentalizes the genome to facilitate gene regulation by reducing the total amount of DNA sequence that must be scanned by DNA-binding regulatory proteins . DNA methylation is involved in immune recognition in bacteria but appears to regulate the structure and expression of the genome in complex higher eukaryotes . I suggest that the DNA-methylating system of mammals was derived from that of bacteria by way of a hypothetical intermediate that carried out selective de novo methylation of exogenous DNA and propagated the methylated DNA in the repressed state within its own genome.(ABSTRACT TRUNCATED AT 250 WORDS)

Eur J Biochem, 1990 Jan 26, 187(2), 425 - 30
Antigenic sites on cytochrome c2 from Rhodospirillum rubrum; Saad B et al.; The antigenic determinants for three monoclonal antibodies against cytochrome c2 from Rhodospirillum rubrum were partially characterized by differential chemical modification of free and antibody-bound cytochrome c2 and by cross-reactivity analysis with different antigens . Circular dichroism spectroscopy was used to probe the effect of antibody binding on the conformation of cytochrome c2 . The binding of two antibodies was strongly dependent on the native folding of the antigen . The first antibody bound to a determinant around the exposed heme edge on the 'front side' of the molecule which is not antigenic in mitochondrial cytochrome c2 . Binding of this antibody to cytochrome c increased the induced CD of the ferric heme in a manner similar to that observed previously when mitochondrial cytochrome-c oxidase bound to the front side of cytochrome c . This observation points to a subtle conformational adaptation of the antigen induced by the antibody . The determinant for the second antibody, which also affected the heme CD spectrum of the antigen, was on a polypeptide loop where cytochrome c2 differs from mitochondrial cytochrome c by an eight-residue insertion . The third antibody, which did not induce a change in CD, bound to a sequential determinant near the amino end of cytochrome c2 . Only this antibody cross-reacted with isolated cytochrome-c-derived peptides and with apo-cytochrome c2 . A preliminary analysis of the polyclonal immune response of five rats against cytochrome c2 indicates that, unlike in eukaryotic cytochrome c, antigenic determinants are distributed over the whole polypeptide chain of the prokaryotic immunogen.

Biochemistry, 1990 Jan 23, 29(3), 632 - 40
Identification and assignment of base pairs in four helical segments of Bacillus megaterium ribosomal 5S RNA and its ribonuclease T1 cleavage fragments by means of 500-MHz proton homonuclear Overhauser enhancements; Kim JH et al.; Three different fragments of Bacillus megaterium ribosomal 5S RNA have been produced by enzymatic cleavage with ribonuclease T1 . Fragment A consists of helices II and III, fragment B contains helix IV, and fragment C contains helix I of the universal 5S rRNA secondary structure . All (eight) imino proton resonances in the downfield region (9-15 ppm) of the 500-MHz proton FT NMR spectrum of fragment B have been identified and assigned as G80.C92-G81.C91-G82.C90-A83.++ +U89-C84.G88 and three unpaired U's (U85, U86, and U87) in helix IV by proton homonuclear Overhauser enhancement connectivities . The secondary structure in helix IV of the prokaryotic loop is completely demonstrated spectroscopically for the first time in any native or enzyme-cleaved 5S rRNA . In addition, G21.C58-A20.U59-G19.C60-A18.U61 in helix II, U32.A46-G31.C47-C30.G48-C29.G49 in helix III, and G4.C112-G5.C111-U6.G110 in the terminal stem (helix I) have been assigned by means of NOE experiments on intact 5S rRNA and its fragments A and C . Base pairs in helices I-IV of the universal secondary structure of B . megaterium 5S RNA are described.

Science, 1990 Jan 19, 247(4940), 315 - 8
Phenotypic switching in mycoplasmas: phase variation of diverse surface lipoproteins; Rosengarten R et al.; The ability of some microorganisms to rapidly alter the expression and structure of surface components reflects an important strategy for adaptation to changing environments, including those encountered by infectious agents within respective host organisms . Mycoplasma hyorhinis, a wall-less prokaryotic pathogen of the class Mollicutes, is shown to undergo high-frequency phase transitions in colony morphology and opacity and in the expression of diverse lipid-modified, cell-surface protein antigens . These proteins spontaneously vary in size, contain highly repetitive structures, and are oriented with their carboxyl-terminal region external to the membrane . Thus, mycoplasma membrane lipoproteins generate microbial surface diversity and may be part of a complex system that controls interactions of these organisms with their hosts.

Biochem Biophys Res Commun, 1990 Jan 15, 166(1), 101 - 8
Human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of iron sulfur (Ip) subunit of liver mitochondria; Kita K et al.; Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex of both the tricarboxylic acid cycle and of the aerobic respiratory chains of mitochondria in eukaryotic cell and prokaryotic organisms . In this study, the amino acid sequence of iron sulfur-subunit in human liver mitochondria was deduced from cDNA which was isolated by immunoscreening a human liver lambda gtll cDNA library . An isolated clone contains an open reading frame of 786 nucleotides and encodes a mature protein of 252 amino acids with a molecular weight of 28,804 . The amino acid sequence was highly homologous with that of bovine heart (94.1%) which has been determined from the purified peptide and that of Escherichia coli sdh B product (50.8%) . Striking sequence conservation was found around the three cysteine-rich clusters which have been thought to comprise the iron-sulfur centers of the enzyme . This is the first report on the cDNA sequence of mitochondrial complex II.

FEMS Microbiol Lett, 1990 Jan 15, 55(1-2), 99 - 105
Immunological characterization of the Prochlorothrix hollandica and Prochloron sp . chlorophyll a/b antenna proteins; Bullerjahn GS et al.; Polyclonal antibodies were prepared against the major antenna chlorophyll (Chl) a/b-binding protein from the prokaryote Prochlorothrix hollandica (Burger-Wiersma et al . (1986) Nature (Lond.) 320, 262-264) . Immunoblotting experiments on Triton X-114 phase-partitioned P . hollandica thylakoids revealed that the antibody recognizes intrinsic membrane polypeptides of 33 and 30 kDa, and immunocytochemistry of P . hollandica thin sections showed that the antibody preferentially decorates the thylakoid . The antibody was immunopurified against a LacZ fusion protein produced in Escherichia coli by an immunopositive phage clone retrieved from a lambda ZAP expression library . This purified antibody crossreacted to both the 33 and 30 kDa polypeptides, indicating that these proteins are either structurally related products of different genes, or modified forms of the same gene product . Whereas immunological crossreactivity of Prochlorothrix antibody to the major LHC-II Chl a/b antenna of maize could not be detected, the immunopurified antibody reacted strongly to the major 34 kDa Chl a/b antenna protein from the prokaryote Prochloron sp . (Lewin (1975) Phycologia 14, 153-160) . These data confirm the structural similarity of the prochlorophyte photosynthetic antenna systems.

Biochem J, 1990 Jan 15, 265(2), 599 - 604
Molecular cloning, sequencing and expression of cytochrome c2 from Rhodospirillum rubrum; Self SJ et al.; Cytochrome c2 (Mr 12,840) of the purple photosynthetic bacterium Rhodospirillum rubrum functions as a mobile electron carrier in the cyclic photosynthetic electron-transport system of this organism . It acts as the electron donor to photochemically oxidized reaction centres and is reduced in turn by electrons from the cytochrome bc1 complex . By using synthetic oligonucleotides based on the known amino acid sequence of the protein, the structural gene (cycA) has been identified and isolated . DNA sequence analysis indicates the presence of a typical prokaryotic 23-residue signal sequence, suggesting that the protein is synthesized as a precursor which is processed during its secretion into the periplasm . Evidence is presented for the production of assembled cytochrome c2 in Escherichia coli, but recombinants grow poorly and are unstable, suggesting toxicity of the gene product in this organism.

Nucleic Acids Res, 1990 Jan 11, 18(1), 143 - 9
Splicing of a C . elegans myosin pre-mRNA in a human nuclear extract; Ogg SC et al.; Splicing of mammalian introns requires that the intron possess at least 80 nucleotides . This length requirement presumably reflects the constraints of accommodating multiple snRNPs simultaneously in the same intron . In the free-living nematode, C . elegans, introns typically are 45 to 55 nucleotides in length . In this report, we determine whether C . elegans introns can obviate the mammalian length requirement by virtue of their structure or sequence . We demonstrate that a 53 nucleotide intron from the unc-54 gene of C . elegans does not undergo splicing in a mammalian (HeLa) nuclear extract . However, insertion of 31 nucleotides of foreign, prokaryotic sequence into the same intron results in efficient splicing . The observed splicing proceeds by the same two-step mechanism observed with mammalian introns, and exploits the same 3' and 5' splice sites as are used in C . elegans . The branch point used lies in the inserted sequence . We conclude that C . elegans splicing components are either fewer in number or smaller than their mammalian counterparts.

Nucleic Acids Res, 1990 Jan 11, 18(1), 163 - 71
Analysis of NADH dehydrogenase proteins, ATPase subunit 9, cytochrome b, and ribosomal protein L14 encoded in the mitochondrial DNA of Paramecium; Pritchard AE et al.; The mitochondrial (mt) encoded ndh1, ndh3, ndh4, ndh5, rpl14, cyt b and atp9 gene products were identified by sequence comparisons with known proteins . Amino acid sequence comparisons between predicted Paramecium mt gene products and proteins in current databases were quantitated approximately by the means of similarity scores for pairs of aligned sequences . The comparisons show that the Paramecium gene products are very divergent from all others with the exception of those from a closely related ciliate, Tetrahymena . The similarity scores of comparisons between a Paramecium mt DNA encoded protein, cytochrome b for example, and the homologous protein from a group of organisms as diverse as other protozoans, vertebrates, fungi, plants, and prokaryotes were all about the same . The Paramecium gene products appear to be equally divergent from proteins representing a number of different kingdoms and organelles.

Mutat Res, 1990 Jan, 240(1), 13 - 8
Mutagenic and genotoxic effects of aqueous extracts of Achyrocline satureoides in prokaryotic organisms; Vargas VM et al.; Aqueous extracts of Achyrocline satureoides (Marcela and/or Macela) were tested for the presence of genotoxic activity in microorganisms . This species belongs to the family Compositae and is used on a large scale by the population of South Brazil . The extracts showed genotoxic activity in the presence of S9 mix in the Ames test TA100, TA98 and TA102 strains, 'SOS' spot chromotest and Microscreen phage-induction assay . The positive results were related to the presence of quercetin and caffeic acid in the aqueous extracts.

Prog Clin Biol Res, 1990, 344, 665 - 82
Genetics and evolution of chloroplast isozymes of triosephosphate isomerase; Feierabend J et al.; Triosephosphate isomerase is an ubiquitous and highly conservative dimeric enzyme, consisting of subunits of Mr 26,000-27,000 . Plants usually contain one cytosolic and one plastid isozyme . While 2x wheats also contain one plastid isozyme, 4x wheats contain 3, and 6x cultural wheats contain five plastid isozymes . The multiplicity of the isozyme pattern in 6x wheats is explainable by the presence of three different genomes (AABBDD), each contributing a distinct triosephosphate isomerase gene (alpha',beta,delta), and by the formation of homodimeric and heterodimeric isozyme forms . While the beta beta-form was, as expected, also found in Aegilops speltoides which is regarded as donor of the B genome, the descent of the other genes for plastid triosephosphate isomerase did not occur in accordance with common contentions on the evolution of 6x cultural wheat and its presumptive ancestors . In the reciprocal intergeneric hybrids between wheat and rye, Secalotricum and Triticale, the patterns of both the cytosolic and the plastid-specific triosephosphate isomerases were biparentally inherited, indicating also that the plastid isozyme was nuclear-encoded . Data which are available about amino acid sequences and gene organization and immunological observations show that the cytosolic triosephosphate isomerase of plants is strongly related to other eukaryotic animal triosephosphate isomerase genes . Multiple evidence has been presented that the plastid- specific isozyme represents a distinct polypeptide and is specified by a distinct gene, relative to the cytosolic isozyme . Immunological comparisons indicate that the plastid isozyme shares homologies with the cytosolic isozyme but is not related to the enzyme from prokaryotic cyanobacteria or bacteria . To enable a more precise comparison, plastid triosephosphate isomerase has been cloned from a cDNA library from rye, and cDNA clones are being sequenced . The plastid enzyme of triosephosphate isomerase appears to have evolved from a duplication on an ancestral nuclear gene of the primordial plant cell . For other plastid-specific isozymes evidence exists that their genes were incorporated into the nucleus by gene transfer from a prokaryotic endosymbiont.

Mol Biol (Mosk), 1990 Jan-Feb, 24(1), 23 - 43
{Possible origin and evolution of enzymatic methylation of eukaryotic DNA . Methylation of cytosine residues in 3 palindromic families: RYRY, YYRR, and YYRYRR}; Mazin AL et al.; Data on nearest neighbors of 5-methylcytosine residues in eukaryotic DNA were analyzed . It was found that the methylation sites C*G and C*NG may be located in three palindromic families: RYRY, YYRR and YYRYRR . It was shown that all the methylated sequences in these DNAs can appear as a result of 5-MeC----T substitutions, proceeding by deamination of 5-MeC residues in the "prototype" sites of each of these families: G*CGC*, C*C*GG and C*C*GCGG . The multiplicity of DNA-methyltransferases in eukaryotic cells and their evolutionary origin from prokaryotic type II methylases, recognizing analogous sequences in DNA, are discussed.

Biosystems, 1990, 23(4), 371 - 84
Open systems living in a closed biosphere: a new paradox for the Gaia debate; Barlow C et al.; While energetically open, the biosphere is appreciably closed from the standpoint of matter exchange . Matter cycling and recycling is hence a necessary and emergent property of the global-scale system known as Gaia . But how can an aggregate of open-system life forms have evolved and persisted for billions of years within a planetary system that is largely closed to matter influx and outflow? The puzzling nature of a closed yet persistent biosphere draws our attention to the course of evolution of fundamental metabolic strategies and matter-capture techniques . It suggests a facet of the Gaia hypothesis, framed in terms of persistence . The oceans, atmosphere, soils and biota constitute a complex system which maintains and adjusts matter cycling and recycling within the constraints of planetary closure such that open-system forms of life can persist . This weaker version of the Gaia hypothesis may be useful because it readily lends itself to at least one form of test . What is the solution to the closed biosphere puzzle, and does it indicate that Gaia merits status as a discrete entity? We suggest several disciplines within the field of biology that might provide tools and perspectives toward reaching a solution . These disciplines include artificial closed ecosystems, prokaryote evolution, the nexus of thermodynamics and evolutionary biology, and hierarchy theory in ecosystem modeling and evolution theory.

Microbiologica, 1990 Jan, 13(1), 79 - 83
Survival of Borrelia burgdorferi in different electroporation buffers; Sambri V et al.; Electroporation consists in the application of an electric field through a cell membrane until the membrane itself develops transient pores . This technique has been used to insert external macromolecules into both eukaryotic and prokaryotic cells . In this study we investigated the survival ratio of Borrelia burgdorferi, the Lyme disease spirochete, under different electroporation conditions.

Proc Natl Acad Sci U S A, 1990 Jan, 87(2), 663 - 7
Higher order structural elements in ribosomal RNAs: pseudo-knots and the use of noncanonical pairs; Gutell RR et al.; The data base of prokaryotic small subunit ribosomal RNAs alone now numbers more than 400 sequences, while that for the large subunit rRNAs numbers more than 70 when eukaryotic, mitochondrial, and plastid sequences are also included . Comparisons among these rRNA sequences reveal a number of positions that covary in composition, suggestive of higher order structural elements; 5 such structures are reported for the small subunit rRNA and 15 for the large subunit rRNA . While some of these are properly (small) secondary structural elements, the majority would have to be classified as more complex "tertiary" interactions, which in some cases bring together diverse areas in the secondary structural diagram . A number of the covariances are not of the canonical type, indicating non-Watson-Crick interactions.

Basic Life Sci, 1990, 53, 417 - 35
Repair of O4-alkylthymine damage in human cells; Wani AA et al.; The capacity of a cell to repair damage is the first step in preventing the deleterious consequences of DNA structural alterations induced by the exposure to mutagenic carcinogens . Mammalian cells, having complex genetic organization, have evolved sophisticated mechanisms for the maintenance of integrity of their genome and normal cell function (Bohr and Hanawalt, 1988; Sancar and Sancar, 1988; Pienta et al., 1989) . However, many DNA repair processes in mammalian cells are similar to those in prokaryotic cells . For example, the unique damage reversal mechanism by transferase, specific for the repair of O6-alkylGua, results in the restoration of intact guanine base in both bacteria and mammalian cells (Olsson and Lindahl, 1980; D'Ambrosio and Wani, 1989) . The proteins involved, however, are different and vary in their specificities . Mammalian transferase specific for O6-alkylGua, more closely resembles the bacterial ogt gene product (Potter et al., 1987; Rebeck et al., 1988) . The main O6-alkylGua specific transferase activity in E . coli resides in the product of the ada gene (Demple et al., 1985; Nakabeppu et al., 1985) . This protein possesses multiple activities including a specificity for the transfer of alkyl group from O4-alkylthy in DNA . Such a transferase activity specific for O4-alkylThy has not been detected in mammalian cells either as an individual activity or part of a multi-activity protein (Brent et al., 1988) . Nevertheless, there is tangible evidence for the active removal of O4-alkylthy in mammalian cells, particularly human cells . The nature, level, and mode of the O4-alkylThy repair activity has not been fully established . Whether the repair occurs by the well known or some novel mechanism(s) has yet to be determined . Recently Boyle et al., (1987) has provided genetic evidence for an alternate mode of repair of O6-alkylGua in mammalian cells . The human cells, that lack O6-MT activity were able to repair O6-nButylGua in cellular DNA . Additional experiments, with mammalian V79 cell lines, indicated a differential specificity for various alkyl groups . It has been suggested that in these cells the repair occurs by an excision process, which is known to recognize the distortions of the DNA duplex rather than the adduct itself (Sancar and Sancar, 1988) . Support for the excision mechanism is also provided by in vitro experiments, showing repair of O6-MeGua by purified E . coli ABC excinuclease enzyme (Voigt et al., 1989) . It is quite likely that the repair of O4-alkylThy in mammalian cells occurs by a similar process.(ABSTRACT TRUNCATED AT 400 WORDS)

Cell Mol Biol, 1990, 36(4), 383 - 93
Partial characterization of a highly conserved aspartyl kinase (AK) in normal human liver; Arenas-Diaz G et al.; Subcellular fractions from human liver were assayed for aspartyl kinase (AK) activity measured by standard spectrophotometric methods . Along the purification procedure three different fractions displayed the expected enzyme activity . Interestingly, two of these fractions were specifically recognized by antibodies raised against E . coli aspartate kinases, suggesting a high degree of evolutionary conservation for these ubiquitous enzymes for prokaryotes . Since their known function in bacteria is not strictly required in eukaryotes, these observation imply that the presence and activity of aspartyl kinase(s) in mammals might represent putative regulatory roles for these enzymes in eukaryotic cell metabolism.

Bull Math Biol, 1990, 52(6), 741 - 72
A model of DNA sequence evolution; Arques DG et al.; Statistical studies of gene populations on the purine/pyrimidine alphabet have shown that the mean occurrence probability of the i-motif YRY(N)iYRY (R = purine, Y = pyrimidine, N = R or Y) is not uniform by varying i in the range, but presents a maximum at i = 6 in the following populations: protein coding genes of eukaryotes, prokaryotes, chloroplasts and mitochondria, and also viral introns, ribosomal RNA genes and transfer RNA genes (Arques and Michel, 1987b, J . theor . Biol . 128, 457-461) . From the "universality" of this observation, we suggested that the oligonucleotide YRY(N)6 is a primitive one and that it has a central function in DNA sequence evolution (Arques and Michel, 1987b, J . theor . Biol . 128, 457-461) . Following this idea, we introduce a concept of a model of DNA sequence evolution which will be validated according to a schema presented in three parts . In the first part, using the last version of the gene database, the YRY(N)6YRY preferential occurrence (maximum at i = 6) is confirmed for the populations mentioned above and is extended to some newly analysed populations: chloroplast introns, chloroplast 5' regions, mitochondrial 5' regions and small nuclear RNA genes . On the other hand, the YRY(N)6YRY preferential occurrence and periodicities are used in order to classify 18 gene populations . In the second part, we will demonstrate that several statistical features characterizing different gene populations (in particular the YRY(N)6YRY preferential occurrence and the periodicities) can be retrieved from a simple Markov model based on the mixing of the two oligonucleotides YRY(N)6 and YRY(N)3 and based on the percentages of RYR and YRY in the unspecified trinucleotides (N)3 of YRY(N)6 and YRY(N)3 . Several properties are identified and prove in particular that the oligonucleotide mixing is an independent process and that several different features are functions of a unique parameter . In the third part, the return of the model to the reality shows a strong correlation between reality and simulation concerning the presence of a large alternating purine/pyrimidine stretches and of periodicities . It also contributes to a greater understanding of biological reality, e.g . the presence or the absence of large alternating purine/pyrimidine stretches can be explained as being a simple consequence of the mixing of two particular oligonucleotides . Finally, we believe that such an approach is the first step toward a unified model of DNA sequence evolution allowing the molecular understanding of both the origin of life and the actual biological reality.

Life Sci, 1990, 47(21), 1875 - 86
Phylogenetic distribution of superoxide dismutase supports an endosymbiotic origin for chloroplasts and mitochondria; Grace SC; Three isozymes of superoxide dismutase (SOD) have been identified and characterized . The iron and manganese isozymes (Fe-SOD and Mn-SOD, respectively) show extensive primary sequence and structural homology, suggesting a common evolutionary ancestor . In contrast, the copper/zinc isozyme (CuZn-SOD) shows no homology with Fe-SOD or Mn-SOD, suggesting an independent origin for this enzyme . The three isozymes are unequally distributed throughout the biological kingdoms and are located in different subcellular compartments . Obligate anaerobes and aerobic diazotrophs contain Fe-SOD exclusively . Facultative aerobes contain either Fe-SOD or Mn-SOD or both . Fe-SOD is found in the cytosol of cyanobacteria while the thylakoid membranes of these organisms contain a tightly bound Mn-SOD . Similarly, most eukaryotic algae contain Fe-SOD in the chloroplast stroma and Mn-SOD bound to the thylakoids . Most higher plants contain a cytosol-specific and a chloroplast-specific CuZn-SOD, and possibly a thylakoid-bound Mn-SOD as well . Plants also contain Mn-SOD in their mitochondria . Likewise, animals and fungi contain a cytosolic CuZn-SOD and a mitochondrial Mn-SOD . The Mn-SOD found in the mitochondria of eukaryotes shows strong homology to the prokaryotic form of the enzyme . Taken together, the phylogenetic distribution and subcellular localization of the SOD isozymes provide strong support for the hypothesis that the chloroplasts and mitochondria of eukaryotic cells arose from prokaryotic endosymbionts.

Subcell Biochem, 1990, 16, 301 - 31
Intracellular trafficking of sterols; Billheimer JT et al.; Cavalier-Smith (1981) has identified 22 characters that are universally present in eukaryotes but absent in prokaryotes . Of these, he argues that one, exocytosis, might have been the driving force behind the evolution of modern eukaryotic cells . Bloom and Mouritsen (1988) further argue that sterols may have removed an evolutionary bottleneck to cytosis . Therefore, the advent of sterols in membranes might have been the single feature that led to eukaryote evolution . The evolutionary advantage conferred by cholesterol is associated primarily with plasma membrane function, since the majority of cellular free cholesterol resides in that membrane . However, sterol synthesis occurs in the ER; therefore, the cell must have a mechanism for transporting sterol to the plasma membrane and its regulation . As has been pointed out in this review, much remains to be elucidated in the study of intracellular sterol trafficking . To date, neither diffusion nor vesicle-mediated transport can be fully confirmed or ruled out . Microtubule and microfilament involvement appears important in some routes (e.g., mitochondria) but not in others . In addition, trafficking roles of cytoplasmic lipoproteinlike particles have not been addressed . Finally, although some "sterol carrier proteins" demonstrate the ability to mediate intervesicular transfer of cholesterol in vitro, the true physiological role of these proteins remains obscure . Future research in this field awaits the refinement of available techniques . Particularly valuable would be cytochemical methods for detection of sterol at the ultrastructural level . Possibly, direct microscopic visualization of radiolabeled components in cells represents the necessary approach . Purification of elements carrying newly synthesized sterols would allow the proteins mediating transport to be identified . Continued analysis of mutants defective in transport, such as in type C Niemann-Pick disease, will shed light on this complex problem . The importance of extracellular trafficking of cholesterol owing to its involvement in the progression of atherosclerosis, has been emphasized in recent years . Little emphasis has been placed on intracellular trafficking of sterol; however, it can be argued that such transport also plays a major role in atherosclerosis, possibly by fueling retrotransport of cholesterol to the liver and secretion in the bile . Therefore, we hope this review will serve to stimulate research interest in this area.

IARC Sci Publ, 1990, (104), 89 - 100
The use of short-term tests in detecting carcinogenicity of complex mixtures; Anderson D; Since it is not feasible to test all substances in long-term animal studies, short-term tests for DNA damage, gene mutation, aneuploidy, chromosomal damage and cell transformation performed in prokaryotes and eukaryotes in vitro and in vivo are used to test pure chemicals and mixtures . Understanding the contribution of mutagens in air, water and food is important to our way of life; methods of sampling and testing such mutagens are discussed in this review.

Adv Inorg Biochem, 1990, 8, 1 - 31
Metalloregulatory proteins and molecular mechanisms of heavy metal signal transduction; Ralston DM et al.; This review has considered what is known about the precise chemical mechanisms involved in the signal transduction of heavy metal ions . By reviewing what is known about general modes of signal transduction, we may draw parallels with the detection of and response to metal ions . In all forms of signal transduction, sensors and transducers are required . Yet, it is apparent that each system has unique features which undoubtedly are critical for the specific signal at hand . Within the context of metal-responsive systems, regardless of whether or not the metal ion is being sequestered, directly utilized, removed or otherwise, several examples of specific metalloregulatory proteins have been elucidated and are summarized in Table II . A close inspection of Table II reveals that in most signal transduction pathways for heavy metals, the presence of the metal ion causes a marked change in the nucleic acid binding capacity of the metalloregulatory protein . For example, the presence of iron results in the dissociation of a protein from iron responsive elements, thereby derepressing ferritin translation . In other instances, metal binding allows a metalloregulatory protein to associate with DNA to activate or repress transcription, as with ACE1 and Fur, respectively . In fact, to the authors' knowledge, it appears that all characterized ligand-responsive transcription factors change nucleic acid binding activity upon ligand binding . This change in affinity is a major feature of the mechanism for activation or repression by these receptors . In contrast, the mercuric ion metalloregulatory protein, MerR, operates by an entirely different transduction mechanism . MerR remains bound to its operator sequence in the presence and absence of mercuric ion, with only a slight increase in the dissociation rate constant in the presence of Hg(II) . Furthermore, the site of MerR binding to the DNA is in a novel position for a prokaryotic activator, directly between the two sets of recognition sequences for RNA polymerase . Analysis of the protein-DNA interactions and transcriptional activity has demonstrated that MerR forms a complex with RNA polymerase in the absence of Hg(II) that is unstable and transcriptionally repressed . When Hg(II) is present in greater than nanomolar concentrations, a highly active transcription complex is formed at PT and a distortion at the center of the palindromic MerR binding site is detectable . Kinetic analysis has determined that, although no change in the binding of RNAP to PT is apparent, the presence of Hg(II) stimulates the rate of isomerization from the closed to the open transcription complex.(ABSTRACT TRUNCATED AT 400 WORDS)

J Gen Microbiol, 1990 Jan, 136 ( Pt 1), 51 - 8
Stable albicidin resistance in Escherichia coli involves an altered outer-membrane nucleoside uptake system; Birch RG et al.; Albicidin blocked DNA synthesis in intact cells of a PolA- EndA- Escherichia coli strain, and in permeabilized cells supplied with all necessary precursor nucleotides, indicating a direct effect on prokaryote DNA replication . Replication of phages T4 and T7 was also blocked by albicidin in albicidin-sensitive (Albs) but not in albicidin-resistant (Albr) E . coli host-cells . All stable spontaneous Albr mutants of E . coli simultaneously became resistant to phage T6 . The locus determining albicidin sensitivity mapped at tsx, the structural gene for an outer-membrane protein used as a receptor by phage T6 and involved in transport through the outer membrane of nucleosides present at submicromolar extracellular concentrations . Albicidin does not closely resemble a nucleoside in structure . However, Albs E . coli strains rapidly accumulated both nucleosides and albicidin from the surrounding medium whereas the Albr mutants were defective in uptake of nucleosides and albicidin at low extracellular concentrations . An insertion mutation blocking Tsx protein production also blocked albicidin uptake and conveyed albicidin resistance . Albicidin supplied at approximately 0.1 microM blocked DNA replication within seconds in intact Albs E . coli cells, but a 100-fold higher albicidin concentration was necessary for a rapid inhibition of DNA replication in permeabilized cells . We conclude that albicidin is effective at very low concentrations against E . coli because it is rapidly concentrated within cells by illicit transport through the tsx-encoded outer-membrane channel normally involved in nucleoside uptake . Albicidin resistance results from loss of the mechanism of albicidin transport through the outer membrane.

Protein Eng, 1990 Jan, 3(3), 161 - 72
Thermitase and proteinase K: a comparison of the refined three-dimensional structures of the native enzymes; Betzel C et al.; We compare the three-dimensional structures of thermitase and of proteinase K determined by X-ray crystallography to a resolution of 1.4 and 1.48 A respectively . Both enzymes are relatively stable towards heat and denaturating agents and are representative of a subgroup of subtilisins characterized by a free SH group close to the active site histidine . Even though they have low sequence homology, the overall tertiary structures are highly conserved . The high resolution structures are compared in terms of the overall fold of the molecules, the active sites, the calcium binding sites, disulphide bridge positions, the positions of the charged residues and the solvent structure . Most subtilisins such as thermitase are of prokaryotic origin and proteinase K is up to now the only known eukaryotic structure.

DNA Cell Biol, 1990 Jan-Feb, 9(1), 11 - 25
Co-expression of a precursor and the mature protein of wheat ribulose-1,5-bisphosphate carboxylase small subunit from a single gene in Escherichia coli; Kaderbhai MA et al.; The cDNA encoding a precursor of wheat ribulose-1,5-bisphosphate carboxylase/oxygenase was inserted in-phase with prokaryotic expression elements in four different vectors . Five expression vectors encoding the small subunit precursors were cloned in Escherichia coli . None of these constructs expressed detectable amounts of the precursor protein, but all directed synthesis of the mature small subunit . The expression of the small subunit was a consequence of an independent, intragenic Shine-Dalgarno sequence optimally located upstream from an ATG specifying the first codon of the mature small subunit portion in the precursor transcript . Similar internal translation signals have been identified in the nuclear-encoded cDNAs of the small-subunit precursors of numerous higher plant genes . The 5' end of the wheat small-subunit precursor was linked with a consensus E . coli DNA sequence such that the modified gene encoded a partial hybrid precursor carrying four additional residues at its amino terminus . The resultant construct, pEI-W3, directed abundant synthesis of both the partially hybrid small-subunit precursor and the mature small subunit, constituting as much as 10% of the total bacterial protein . The bacterially synthesized small subunit precursor was purified to homogeneity . The authenticity of the recombinant protein was verified by its size, immunological properties, amino-terminal sequence, and amino acid composition.

Proteins, 1990, 8(2), 156 - 63
Acid helix-turn activator motif; Zhu QL et al.; A common sequence/structural motif pattern has been identified within the steroid/thyroid hormone receptors and other transcriptional activators using a new massively parallel symbolic learning assistant computer system . The pattern appears nearly diagnostic of transcription activation, including relative activation strength, among nuclear and DNA-binding prokaryotic proteins . In cases where mutation/deletion/chimeric studies have identified the activation domain, the pattern matches within that domain . These facts and the nature of the pattern itself strongly support the idea that the patterned domain is directly involved in a protein-protein transcription activation interaction.

Z Mikrosk Anat Forsch, 1990, 104(1), 61 - 78
Injured mitochondria in cells of Euglena gracilis after DNA gyrase inhibitors treatment; Polonyi J et al.; Five quinolone (ofloxacin, cinoxacin, enoxacin, ciprofloxacin, oxolinic acid) and one non-quinolone (coumermycin A1) inhibitors of prokaryotic DNA gyrase used in clinical practice for treatment of bacterial infections were experimentally examinated . As model organism the flagellate Euglena gracilis was used . Ultrastructural changes in chloroplasts and mitochondria caused by inhibitors were quantitavely evaluated . Simultaneously in all cases injury and hereditary loss of chloroplasts (bleaching) were observed in the cells . In some samples about 45% of cup-shaped mitochondria cumulated in the cytoplasm . In damaged mitochondria some degenerative signs were seen, but after the last subcultivation on drug-free media the number of injured mitochondria in the bleached cells yielded to the normal value.

Genes Dev, 1990 Jan, 4(1), 123 - 36
The E2 trans-activator can act as a repressor by interfering with a cellular transcription factor; Stenlund A et al.; The E2 open reading frame (ORF) of the bovine papillomavirus (BPV-1) encodes a family of site-specific DNA-binding proteins . The full-length protein is a transcriptional activator, whereas the polypeptides that contain only the carboxy-terminal domain are repressors . Here we show that the trans-activator can work as a repressor of transcription for one of the BPV-1 promoters by binding to a DNA sequence required for basal activity of the promoter . This operator site is defined as a 12-bp sequence that lies immediately downstream of the cap site . The operator DNA contains sequences that are defined genetically and biochemically as being important for basal level promoter activity . Furthermore, this site has been shown to be protected in a DNase footprint assay using fractionated HeLa cell extracts . The repression does not simply result from E2 blocking RNA polymerase initiation or elongation, because a strong E2-binding site placed at the operator has no repressive effect on transcription when the basal target sequence is placed independently upstream of the promoter . Thus, this is an interesting parallel to a theme well known in prokaryotes, where some site-specific DNA-binding proteins can work as either activators or repressors . In this system, as well as in the prokaryotic systems, the precise position of the binding site relative to other cis signals at the promoter determines the nature of the effects.

Magnes Trace Elem, 1990, 9(5), 233 - 54
Transport of magnesium across biological membranes; Beyenbach KW; The movement of Mg across biological membranes is reviewed from the perspectives of (1) passive transport, (2) primary active transport and (3) secondary active transport . Since all cells maintain intracellular Mg2+ at a lower electrochemical potential than extracellular Mg2+, active transport pumps bringing Mg2+ into the cell have neither been postulated nor been confirmed . Most evidence points to influx leaks, presumably via membrane channels and carriers which do not perfectly exclude Mg2+ . However, Mg2+ currents have been measured in prokaryotic and eukaryotic cells suggesting the presence of Mg2+ channels . Mg2+ influx through channels is largely driven by the membrane voltage because transmembrane Mg2+ concentration differences are not very large . Efflux mechanisms have attracted most of the investigative focus . Secondary active transport by way of Na/Mg exchange appears to be widely distributed in eukaryotic cells . The early investigations of Na/Mg exchange had Mg2+ efflux driven solely by the Na+ influx (secondary active transport) . However, recent studies have revealed an ATP dependence of Na/Mg exchange which may reflect the operation of an Mg2+ pump (primary active transport) . Similar mechanisms of Mg2+ influx and efflux appear to operate in epithelial tissues which may net absorb or secrete Mg2+ . In recent years, the intact red blood cell has emerged as the model of choice for studies of Mg2+ membrane transport . However, further probing of the details of individual transport mechanisms may be complicated by the presence of multiple parallel Mg2+ influx and efflux systems in intact cells . Accordingly, it would appear that the next round of advances in membrane transport of Mg2+ will come from studies at subcellular levels, which aim at the isolation of transporters and their reconstitution . These studies should now be possible at least for Na/Mg exchange given our fair understanding of this system in intact red blood cells.

Biosystems, 1990, 24(3), 245 - 51
Cyanobacteria or rhodophyta? Interpretation of a precambrian microfossil; Enzien MV; Discovery and interpretation of a filamentous microfossil from the late Proterozoic Narssarssuk Formation in northwest Greenland approximately 770 Ma is reported here . This microfossil is preserved as a single occurrence in a silicified carbonate sequence containing stromatolitic laminae . Based on the absence of other occurrences and its microstratigraphic association with planktonic microfossils, the microfossil is interpreted as allochthonous . The microfossil is similar to two extant taxa representing different kingdoms: one prokaryote, Johannesbaptistia pellucida (cyanobacteria) and one eukaryote, Bangia sp . (rhodophytes) . Definitive identification, due to the lack of distinctive morphology, could not be made.

Orig Life Evol Biosph, 1990-91, 20(6), 499 - 514
Cellular differentiation in the process of generation of the eukaryotic cell; Nakamura H et al.; Primitive atmosphere of the earth did not contain oxygen gas (O2) when the proto-cells were generated successfully as the result of chemical evolution and then evolved . Therefore, they first had acquired anaerobic energy metabolism, fermentation . The cellular metabolisms have often been formed by reorganizing to combine or recombinate between pre-existing metabolisms and newly born bioreactions . Photosynthetic metabolism in eukaryotic chloroplast consists of an electron-transfer photosystem and a fermentative reductive pentose phosphate cycle . On the other hand, O2-respiration of eukaryotic mitochondrion is made of Embden-Meyerhof (EM) pathway and tricarboxylic acid cycle, which originate from a connection of fermentative metabolisms, and an electron-transfer respiratory chain, which has been derived from the photosystem . These metabolisms already are completed in some evolved prokaryotes, for example the cyanobacterium Chlorogloea fritschii and aerobic photosynthetic bacteria Rhodospirillum rubrum and Erythrobacter sp . Therefore, it can be reasonably presumed that the eukaryotic chloroplast and mitochondrion have once been formed as the result of metabolic (and genetic) differentiations in most evolved cyanobacterium . Symbiotic theory has explained the origin of eukaryotic cell as that in which the mitochondrion and chloroplast have been derived from endosymbionts of aerobic bacterium and cyanobacterium, respectively, and has mentioned as one of the most potent supportive evidences that amino acid sequences of the photosynthetic and O2 -respiratory enzymes show similarities to corresponding prokaryotic enzymes . However, as will be shown in this discussion, many examples have shown currently that prokaryotic sequences of informative molecules are conserved well not only in those of the mitochondrial and chloroplast molecules but also in the nuclear molecules . In fact, the similarities in sequence of informative molecules are preserved well among the organisms not only in phylogenetically close relationships but also under highly selective pressure, that is under a physiological constraint for the species in their habitats . Therefore, the similarities in amino acid sequences of proteins between the prokaryotes and the organelles are not necessarily direct evidence for their phylogenetical closeness: it gives still less evidence for a symbiotic relationship between the prokaryotes and the organelles . The metabolic compartmentalization of the membranes is an important tendency in cellular evolution to guarantee high specificity and rate of the metabolisms . It is suggested from the data that the intracellular membranes are not static but undergo dynamic turnover . Furthermore, these facts strongly support the Membrane Evolution Theory which was proposed by one of the authors in 1975.

Allerg Immunol (Leipz), 1990, 36(4), 209 - 23
{Relationship between the immune system and heat shock proteins . A literature review}; Docke WD et al.; Heat shock proteins (HSP) or stress proteins are produced by prokaryotic and eukaryotic cells in response to a variety of environmental stressors . The heat shock response is one of the most universal reactions known and heat shock proteins are among the most conserved molecules in phylogeny . Recent findings concerning the immune response to heat shock proteins are discussed especially with respect to the role of HSPs postulated in septic disease and inflammation, in antipathogenic immunity and in the induction of autoimmune diseases . Results and speculations considering a relationship between HSPs and gamma/delta T cells or polyreactive antibodies, possibly as part of a phylogenetic old immune system, are critically reviewed.

Digestion, 1990, 47 Suppl 1, 35 - 8
Omeprazole and the gastric mucosa; Sachs G et al.; The potential for either omeprazole or its sulphenamide to interact with DNA was investigated by incubating tritiated omeprazole at either neutral or acidic pH with either purified prokaryotic (Escherichia coli) or purified eukaryotic (salmon sperm) DNA . Each incubation was carried out for 30 min . The DNA was separated on agarose gels, stained with ethidium bromide, and the radioactivity in the DNA determined . No radioactivity was associated with the DNA on the gel with either prokaryotic or eukaryotic DNA or with either protocol . No interaction with the DNA was found, thus excluding the possibilities of base modification, DNA strand breakage or intercalation . It was concluded that neither omeprazole nor its gastric metabolites are genotoxic.

Immunol Ser, 1990, 49, 155 - 76
Molecular biology of macrophage colony-stimulating factor; Kawasaki ES et al.; In this chapter we have described one of the more complex hemopoietic factors, M-CSF . The single-copy M-CSF gene is almost 21 kb in length and is arranged into 10 exons and 9 introns . Expression of the gene at the RNA level is heterogeneous, and several species of M-CSF mRNA have been found in human and murine cells and tissues . In human cells the different mRNAs arise from alternative splicing of the nuclear RNA precursor in both coding and noncoding regions . This results in mRNAs encoding two distinct M-CSF proteins, 256 and 554 amino acids in length . In murine cells only a 552-amino-acid form has been found thus far . All forms of M-CSF have a 32-amino-acid signal peptide and a 23-amino-acid hydrophobic region near the carboxy-terminus, which resembles a transmembrane domain . A large portion of the carboxy-terminal end, including the hydrophobic region, is not found in the mature protein . Thus, the primary translation product of M-CSF is a prepropolypeptide, with processing occurring at both amino- and carboxy-terminal ends . The exact size of the mature protein is still somewhat in doubt, but deletion mutagenesis from the carboxy-terminal end indicates that the protein may be as small as 150 amino acids and still be functional . Site-directed mutagenesis has also shown that the first seven cysteines in the mature molecule are probably necessary for biological activity, whereas the next two cysteine residues are not . In spite of the heavy glycosylation found in the native protein, removal of the N-linked glycosylation signals does not seem to affect activity to any great degree . The M-CSF gene and its receptor, C-FMS, are tightly linked on the long arm of chromosome 5, a unique finding in the ligand/receptor field . This region also contains the genes for GM-CSF, IL-3, ECGF, and the receptor for PDGF . A similar situation may exist on chromosome 11 of the mouse . The close linkage of these factors and receptors is the probable cause for the disorders of hemopoiesis that arise when deletions occur in this area . The preceding discussion has shown how quickly the area of M-CSF molecular biology has advanced in the past 2-3 years . A great deal of effort is now being directed toward expressing M-CSF at high levels in a variety of prokaryotic and eukaryotic systems.(ABSTRACT TRUNCATED AT 400 WORDS)

Acta Biochim Pol, 1990, 37(1), 149 - 51
Effect of the extracts from fungus Inonotus obliquus on catalase level in HeLa and nocardia cells; Jarosz A et al.; Growth medium of Inonotus obliquus exerts antimitotic effect on HeLa cells mostly in M, G1 and G2 phases increasing at the same time catalase activity . This effect was not observed in prokaryotic Nocardia . Significant antimitotic effect of mycelium was not associated with stimulation of catalase activity in HeLa cells.

Teratog Carcinog Mutagen, 1990, 10(6), 477 - 501
Interaction of drugs with extranuclear genetic elements and its consequences; Ebringer L; Bacterial ancestry of mitochondria and plastids is now generally accepted . Both organelles contain their own DNA and transcription-translation apparatus of a prokaryotic type . Due to this fact these systems carry bacteria-like properties . Thus organellar DNA and ribosomes are essentially different from nuclear DNA and cytoplasmic ribosomes in physical as well as in functional respects . Due to the bacterial character of both types of organelles they are susceptible to various antibacterial chemicals . Inhibitors of bacterial protein synthesis inhibit mitochondrial (plastidial) biogenesis . Therefore the cellular content of mitochondria (plastids)-made proteins decreases during cytoplasmic turnover or cell division in the presence of these drugs . Such drug activity consequently leads to a reduced capacity for oxidative phosphorylation or photosynthesis . Organellar genomes are less stable and more sensitive to mutagenesis as compared to nuclear genome . It means also that genotoxic agents induce various disorders of mitochondrial (plastidial) functions . Impairments in the respiratory chain are associated with structural as well as functional abnormalities of mitochondria . These are clinically expressed mostly in tissues with a high demand for ATP: brain, heart, skeletal muscle, and retina . On the other hand, some antibacterial inhibitors of mitochondrial biogenesis (e.g., tetracyclines) inhibit selectively tumor cell proliferation . Therefore they may be considered for use in anticancer therapy . The article summarizes the response of mitochondria and plastids in various organisms to drugs and environmental xenobiotics . Various model organisms suitable for detection of xenobiotic effect on mitochondria (plastids) are presented as well as the possible consequences of such interaction.

DNA Cell Biol, 1990 Jan-Feb, 9(1), 57 - 61
A method for the preparation of high-molecular-weight DNA from marine and freshwater triclads (Platyhelminthes, Turbellaria); Hempstead PG et al.; Although the last decade has seen much activity devoted to the phylogeny of the Platyhelminthes, such activity has relied in the main on traditional anatomical, ultrastructural, and developmental data . Extension of these studies to the molecular level has been impeded by the lack of a reliable method for the isolation of DNA from these organisms, especially marine triclads whose DNA is particularly difficult to isolate using methods currently used with other eukaryotic or prokaryotic organisms . We report here the details of a method that results in the rapid isolation of relatively pure DNA in good yields from small amounts of material . The method involves the treatment of the animals with high concentrations of NaDodSO4 followed by phenol extraction, resulting in a product that is an excellent substrate for restriction endonuclease digestion and Southern blotting.

Biomed Biochim Acta, 1990, 49(8-9), 839 - 53
Control of DNA structure and gene expression; Westerhoff HV et al.; In the usual metabolic control theory, the concentrations of enzymes are considered to be parameters rather than variables, i.e., they remain constant as the system relaxes to a new steady state . They can only be reset by interventions . This type of control analysis is useful for understanding principles of metabolic control, and for understanding metabolic changes that are too quick or in too limited a metabolic system to involve changes in gene expression . In actual living systems, metabolic changes are often accompanied by changes in gene expression . In this contribution we shall illustrate how metabolic control analysis is enriched when gene expression is variable . To discuss the new principles emerging in control analysis with variable gene expression, we shall first discuss theoretical model systems . In the first, the number of genes is fixed, but the concentrations of mRNA and enzymes are determined by the activities of RNA polymerase, RNAases, ribosomes and proteases . In a second, there is feedback repression by a metabolite at the level of translation . New coefficients quantifying the strength of regulatory loops will be defined . Also coefficients that indicate to what extent these regulatory strengths themselves are controlled by system parameters, are defined and provided with a summation theorem . The experimental model system we employ, addresses the phenomenon that in prokaryotes, transcription rates are influenced by the extent of supercoiling of the DNA . This includes the transcription of the genes encoding the two enzymes (DNA gyrase and topoisomerase I) involved in the regulation of DNA supercoiling . In vitro the activity of DNA gyrase is influenced by the hydrolytic free energy of ATP . We shall present experimental evidence that the cellular free-energy state influences DNA supercoiling . We shall also discuss experiments inspecting the effect of active transcription on active DNA supercoiling . Also this system will be analyzed in terms of the control analysis with variable gene expression; here the four hierarchical levels (DNA, RNA, enzymes, metabolites) interact, adding complexity to the control analysis.

Arch Virol, 1990, 110(1-2), 1 - 24
Mapping of viral epitopes with prokaryotic expression products; Lenstra JA et al.; Several systems are available for the expression of foreign gene sequences in Escherichia coli . We describe the use of prokaryotic expression products of viral gene fragments in order to identify the regions that specify the binding sites of antibodies . This approach is particularly successful if the antigenicity does not depend on the native protein, but only on the amino acid sequence, i.e., if the epitope is sequential . Combining prokaryotic expression with the use of synthetic peptides often permits a fast and accurate mapping of an epitope . The occurrence of immunodominant sequential epitopes on the surfaces of viruses seems to be a widespread phenomenon.

Gene, 1989 Dec 28, 85(2), 413 - 20
A general method for the induction and screening of antisera for cDNA-encoded polypeptides: antibodies specific for a coronavirus putative polymerase-encoding gene; Zoltick PW et al.; A prokaryotic vector, pGE374, containing the recA and lacZ genes, out-of-frame, was used for the expression of cDNA derived from the putative polymerase-encoding gene of the coronavirus mouse hepatitis virus strain A59 (MHV-A59) . The pGE374/viral recombinant vector generates a tripartite bacterial/viral protein composed of a segment of the RecA protein at the N terminus, the coronaviral sequences in the middle, and an enzymatically active beta-galactosidase at the C terminus . Rabbits immunized with such recombinant proteins generated antibodies to the MHV-A59 portion of the tripartite protein . Because the MHV-A59 polymerase proteins have been difficult to identify during infection, we used a novel method to demonstrate the viral specificity of the antiserum . The viral cDNA was excised from the expression vector, and transferred to a pGem vector, downstream from and in-frame with a portion of the cat gene . This construct contained a bacteriophage RNA polymerase promoter that enabled the cell-free synthesis of a fusion protein that was used to verify that antibodies were generated to the expressed viral DNA . This strategy was shown to successfully result in the specific generation of antibodies to the encoded information of the viral cDNA . Furthermore, this method has general applicability in the generation and characterization of antibodies directed against proteins encoded in cDNAs.

Eur J Biochem, 1989 Dec 22, 186(3), 515 - 21
Frameshifting in the synthesis of Escherichia coli polypeptide chain release factor two on eukaryotic ribosomes; Williams JM 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 . The nucleotide sequence preceding the in-phase stop codon within RF-2 mRNA is complementary to the 3' anti-(Shine-Dalgarno sequence) region found in prokaryotic 16S rRNA and Weiss et al . (1988) have concluded that this pairing triggers the frameshift event . In vitro production of RNA coding for RF-2, suitable for translation on eukaryotic ribosomes, has enabled testing of whether eukaryotic ribosomes can frameshift at this sequence . The 18S rRNA of eukaryotic ribosomes does not contain the 3' anti-(Shine-Dalgarno sequence) region . The prokaryotic RF-2 gene and the gene for the other release factor, RF-1, which does not contain an in-frame stop codon, were subcloned into transcription vectors such that the RNA transcripts produced in vitro would resemble a typical eukaryotic mRNA . These RF-1 and RF-2 RNAs both synthesized a major product of Mr approximately 45,000 when translated in vitro within reticulocyte lysate; the size expected for full length RF-1 and RF-2 molecules . The RF-2 product was immunoprecipitated by RF-2-specific antibodies, including those to regions of the protein encoded in the mRNA downstream from the frameshift site . The putative premature termination product, an oligopeptide of 25 amino acids, was not detected, but a chemically synthesized derivative was shown to be very unstable within the translation system . Although it was not possible therefore to calculate an absolute efficiency of frameshifting, the relative efficiency of the translation of RF-2 RNA was estimated to be 10-20% of that of RF-1 RNA in the reticulocyte system . This was similar to the relative synthesis of the two proteins in a plasmid-DNA-directed prokaryotic transcription/translation system . These results show that in vitro on eukaryotic ribosomes where the Shine-Dalgarno-type interaction is not possible, high efficiency frameshifting around the in-phase stop codon in the RF-2 mRNA can still occur.

Gene, 1989 Dec 21, 85(1), 99 - 108
Correlation between temperature-dependent cytoplasmic solubility and periplasmic export of a heterologous protein in Escherichia coli; Leemans R et al.; The coding sequence of mature human tumor necrosis factor (hTNF) was fused to the signal-encoding sequence of beta-lactamase (Bla) . Mature hTNF was exported into the periplasm of Escherichia coli . A mutant hTNF {Van Ostade et al., FEBS Lett . 238 (1988) 347-352}, which displays a temperature-dependent intracellular solubility, was fused to the same Bla signal-encoding sequence . We found that the export competence of the mutated hTNF was correlated with the intracellular solubility of this protein . We postulate that the secretion proficiency of eukaryotic proteins, when fused to a prokaryotic export signal, depends on the ability of the mature protein to readily fold into a soluble conformation.

EMBO J, 1989 Dec 20, 8(13), 3973 - 84
Saccharomyces cerevisiae STE6 gene product: a novel pathway for protein export in eukaryotic cells; Kuchler K et al.; Saccharomyces cerevisiae MATa cells release a lipopeptide mating pheromone, a-factor . Radiolabeling and immunoprecipitation show that MATa ste6 mutants produce pro-a-factor and mature a-factor intracellularly, but little or no extracellular pheromone . Normal MATa cells carrying a multicopy plasmid containing both MFa1 (pro-a-factor structural gene) and the STE6 gene secrete a-factor at least five times faster than the same cells carrying only MFa1 in the same vector . The nucleotide sequence of the STE6 gene predicts a 1290 residue polypeptide with multiple membrane spanning segments and two hydrophilic domains, each strikingly homologous to a set of well-characterized prokaryotic permeases (including hlyB, oppD, hisP, malK and pstB) and sharing even greater identity with mammalian mdr (multiple drug resistance) transporters . These results suggest that the STE6 protein in yeast, and possibly mdr in animals, is a transmembrane translocator that exports polypeptides by a route independent of the classical secretory pathway.

EMBO J, 1989 Dec 20, 8(13), 3963 - 71
Primary structure of sensory rhodopsin I, a prokaryotic photoreceptor; Blanck A et al.; The gene coding for sensory rhodopsin I (SR-I) has been identified in a restriction fragment of genomic DNA from the Halobacterium halobium strain L33 . Of the 1014 nucleotides whose sequence was determined, 720 belong to the structural gene of SR-I . In the 5' non-coding region two putative promoter elements and a ribosomal binding site have been identified . The 3' flanking region bears a potential terminator structure . The SR-I protein moiety carries no signal peptide and is not processed at its N terminus . The C terminus, however, lacks the last aspartic acid residue encoded by the gene . Analysis of the primary structure of SR-I reveals no consistent homology with the eukaryotic photoreceptor rhodopsin, but 14% homology with the halobacterial ion pumps, bacteriorhodopsin (BR) and halorhodopsin (HR) . Residues conserved in all three proteins are discussed with respect to their contribution to secondary structure, retinal binding and ion translocation . The aspartic acid residue which mediates in BR the reprotonation of the Schiff base (D96) is replaced in SR-I by a tyrosine (Y87) . This amino acid replacement is proposed to be of crucial importance in the evolution of the slow-cycling photosensing pigment SR-I.

J Biol Chem, 1989 Dec 15, 264(35), 21138 - 43
Valyl-tRNA synthetase from rabbit liver . II . The enzyme derived from the high-Mr complex displays hydrophobic as well as polyanion-binding properties; Bec G et al.; The preceding paper (Bec, G., Kerjan, P., Zha, X.D., and Waller, J.P . (1989) J . Biol . Chem . 264, 21131-21137) described the purification to apparent homogeneity from rabbit liver, of a heterotypic complex comprising valyl-tRNA synthetase and Elongation Factor 1H . In the present study, valyl-tRNA synthetase was dissociated and separated from the other components of this complex by hydroxylapatite chromatography in the presence of 0.5 M NaSCN . The properties of the homogeneous mammalian enzyme were compared to those of the corresponding enzyme from yeast . Both behaved as monomeric entities, with apparent molecular masses of 140 and 125 kDa, respectively . Furthermore, both displayed strong affinity toward the polyanionic support heparin-Ultrogel, a property not manifested by the corresponding prokaryotic enzyme . However, unlike the yeast enzyme, that of mammalian origin additionally exhibited hydrophobic properties, as reflected by its affinity toward phenyl-Sepharose . A structural model is proposed according to which mammalian valyl-tRNA synthetase has conserved the polycationic N-terminal domain that distinguishes the corresponding lower eukaryotic enzyme from its prokaryotic counterpart, while acquiring a hydrophobic domain most likely responsible for its association to Elongation Factor 1H.

Gene, 1989 Dec 14, 84(2), 295 - 302
Cloning and characterization of the yeast chaperonin HSP60 gene; Johnson RB et al.; The heat-shock protein, HSP60, is abundant in prokaryotes and eukaryotes and is required in the assembly of specific proteins . We have cloned the Saccharomyces cerevisiae HSP60 gene from a lambda gt11 genomic library using monoclonal antibodies, have obtained its sequence, determined its transcription start point, and shown that it exists as a single copy . The predicted HSP60 contains a mitochondrial target sequence and exhibits striking amino acid sequence similarity to its counterparts in bacteria, plants, and humans . These data indicate a high level of evolutionary conservation and are consistent with the suggestion of evolutionarily conserved function {Hemmingsen et al., Nature 333 (1988), 330-334}.

Nucleic Acids Res, 1989 Dec 11, 17(23), 9583 - 91
An additional promoter within the protein-coding region of the psbD-psbC gene cluster in tobacco chloroplast DNA; Yao WB et al.; Transcription of the psbD-psbC gene cluster in tobacco chloroplasts has been studied . This cluster contains in linear sequence the overlapping genes encoding the D2 and 43 kDa proteins of Photosystem II (psbD and psbC, respectively), and ORF62 . Eight major transcripts ranging from 1.5 to 4.4 kb were detected by northern blot analysis . S1 mapping experiments revealed that these multiple transcripts comprise five distinct 5' ends whose precise positions were further determined by primer extension analysis . Two of the five 5' ends were determined to be the transcriptional initiation sites by in vitro capping assays: the main site is located 905 bp upstream from the ATG codon of psbD and the additional site is 194 bp upstream from the first ATG codon of psbC . The latter site and the preceding prokaryotic promoter motif are within the protein-coding region of psbD . The 3' ends of transcripts were determined by S1 mapping.

Gene, 1989 Dec 7, 84(1), 65 - 72
The block to transcription elongation at the SV40 attenuation site is decreased in vitro by oligomers complementary to segments of the attenuator RNA; Kessler M et al.; We have previously reported that a mechanism resembling attenuation in prokaryotes regulates simian virus 40 (SV40) late gene expression . We have suggested that modulation of the attenuator RNA secondary structure is an integral element regulating the elongation block at the attenuation site {Hay et al., Cell 29 (1982) 183-193} . In the present study, oligodeoxyribonucleotides (oligos), 13-19 nucleotides long, were used to probe the involvement of the attenuator RNA secondary structure in the control of elongation block at the SV40 attenuation site . These oligos are complementary to segments of the attenuator RNA suggested to play a role in the regulation of attenuation . The oligos were added to an in vitro transcription reaction containing SV40 transcription complexes, and their effect on transcription through the attenuation site was measured . As predicted, the three oligos caused specific decreases in the elongation block at the SV40 attenuation site . These results provide direct evidence for the involvement of RNA secondary structure in the attenuation mechanism in SV40.

J Biol Chem, 1989 Dec 5, 264(34), 20552 - 60
Cloning and sequencing of the nuclear gene MIP1 encoding the catalytic subunit of the yeast mitochondrial DNA polymerase; Foury F; The nuclear gene MIP1 is strictly required for mitochondrial DNA replication and mitochondrial DNA polymerase activity (Genga, A., Bianchi, L., and Foury, F . (1986) J . Biol . Chem . 261, 9328-9332) . The MIP1 gene was cloned by genetic complementation of the mip1-1 allele after cell transformation with a yeast genomic library and was mapped to the right arm of chromosome XV about 20 centimorgans distal to the cpa1 gene by Southern blot hybridization and tetrad analysis . The mapping of the 5' ends of the MIP1 transcript and the nucleotide sequence analysis of a 4.7-kilobase DNA fragment complementing the mip1-1 allele allowed the determination of an open reading frame of 3762 nucleotides encoding a basic protein of 143.5 kDa . The following data show that the MIP1 gene encodes the catalytic subunit of the replicative mitochondrial DNA polymerase . 1) The mutant ts71 exhibits both a thermosensitive mitochondrial DNA replication in vivo and a thermosensitive mitochondrial DNA polymerase activity is observed, when compared to that of the wild type strain . 3) Chromosomal disruption of the MIP1 gene by an 80% deletion of the gene and its replacement by URA3 gene is not lethal to the cell but elicits total loss of mitochondrial DNA and mitochondrial DNA polymerase activity . 4) The MIP1 protein exhibits sequence similarities with both eukaryotic nuclear DNA polymerases and reverse transcriptases . There is no significant resemblance with prokaryotic DNA polymerases.

Curr Genet, 1989 Dec, 16(5-6), 461 - 4
The UV excision-repair system of Saccharomyces cerevisiae is involved in the removal of methylcytosines formed in vivo by a cloned prokaryotic DNA methyltransferase; Feher Z et al.; DNA methyltransferase activity is not normally found in yeast . To investigate the response of Saccharomyces cerevisiae to the presence of methylated bases, we introduced the Bacillus subtilis SPR phage DNA-{cytosine-5} methyltransferase gene on the shuttle vector, YEp51 . The methyltransferase gene was functionally expressed in yeast under the control of the inducible yeast GAL 10 promoter . Following induction we observed a time-dependent methylation of yeast DNA in RAD+ and rad2 mutant strains; the rad2 mutant is defective in excision-repair of UV-induced DNA damage . Analysis of restriction endonuclease digestion patterns revealed that the relative amount of methylated DNA was greater in the excision defective rad2 mutant than in the RAD+ strain . These data indicate that the yeast excision-repair system is capable of recognizing and removing m5C residues.

Immunol Rev, 1989 Dec, 112, 133 - 60
Interactions between mycoplasmas and the immune system; Ruuth E et al.; Mycoplasmas are a heterogenous group of prokaryotic organisms causing a wide variety of diseases, including autoimmune disorders . Thus, it is not surprising that various mycoplasmas strains, including Mycoplasma arginini, M . arthritidis, M . neurolyticum and M . pulmonis, are able to regulate the immune response . Though some of the studies of the immunomodulatory action of mycoplasmas have been done in vivo, the majority of the investigations have been conducted in vitro . This has led to the recognition that mycoplasmas are polyclonal activators of both B and T cells from several species, acting through MHC-restricted or -unrestricted pathways . Mycoplasma activation not only induces T-cell proliferation but also leads but to the formation of cytotoxic T cells . We, as well as others, have shown that mycoplasma-mediated B-cell activation induces proliferation as well as Ig secretion, and also that mycoplasma stimulation of lymphocytes may result in the production of cytokines . We communicate here our investigations into the effects of an M . arginini strain on the growth and maturation of preactivated B cells . After an initial biological characterization of the M . arginini effects in vitro, we established the protein nature of the growth-supporting activity and proceeded further on to isolate and identify the responsible proteins . The use of lipid- and lipoglycan-free extracts has allowed us to further extend our studies on the biological activities of the proteins from M . arginini and to compare these results with the effects obtained using live organisms . Furthermore, the study was extended to include a characterization of the in vivo-induced effects of live M . arginini . Altogether, the results from these experiments allow us to conclude that M . arginini is a T-cell independent polyclonal B-cell mitogen, mediated by five identified proteins, inducing growth and Ig secretion of both resting and preactivated B cells.

FASEB J, 1989 Dec, 3(14), 2607 - 14
Molecular and genetic immunoprobes for biotechnology; Conway de Macario E; Recent advances concerning immunoprobes are described and their biotechnologic potential is emphasized . Selected examples of immunoprobes discussed include interleukin 7, genetically engineered vaccines using live bacterial carriers, and applications of monoclonal antibodies (MAb's) for understanding and controlling blood cell malignancies as well as for analysis of prokaryotic cells . The fundamental and practical interconnections of these research areas are highlighted to show, for instance, that definition of lymphoid-cell lineages and identification of their various subsets are aided by MAb's and interleukins . On the other hand, understanding interleukins requires isolation of cell subsets . Likewise, designing vaccines presupposes a knowledge of lymphocyte subsets and of the antigenic mosaics of the pathogens against which the vaccines are directed . Dissection of antigens in cells and in infectious agents depends to a great extent on MAb's, which are also instrumental for cloning the genes encoding antigens for vaccine preparation and those encoding interleukins for large scale production of these molecules that are promising as immunotherapeutic and vaccine adjuvants . Last, brief mention is made of the uses of MAb's in prokaryotic cell biology to illustrate the long-range impact on these immunoprobes and, consequently, how they are opening new avenues for biotechnology.

EMBO J, 1989 Dec 1, 8(12), 3917 - 21
The reaction specificities of the thylakoidal processing peptidase and Escherichia coli leader peptidase are identical; Halpin C et al.; Proteins which are transported across the bacterial plasma membrane, endoplasmic reticulum and thylakoid membrane are usually synthesized as larger precursors containing amino-terminal targeting signals . Removal of the signals is carried out by specific, membrane-bound processing peptidases . In this report we show that the reaction specificities of these three peptidases are essentially identical . Precursors of two higher plant thylakoid lumen proteins are efficiently processed by purified Escherichia coli leader peptidase . Processing of one precursor, that of the 23 kd photosystem II protein, by both the thylakoidal and E . coli enzymes generates the correct mature amino terminus . Similarly, leader (signal) peptides of both eukaryotic and prokaryotic origin are cleaved by partially purified thylakoidal processing peptidase . No evidence of incorrect processing was obtained . Both leader peptidase and thylakoidal peptidase are inhibited by a synthetic leader peptide.

J Biomol Struct Dyn, 1989 Dec, 7(3), 707 - 22
Strong patterns in homooligomer tracts occurrences in non-coding and in potential regulatory sites in eukaryotic genomes; Nussinov R et al.; Previous studies of the dinucleotides flanking both the 5' and 3' ends of homooligomer tracts have shown that some flanks are consistently preferred over others (1,2) . In the first preferred group, the homooligomer tracts are flanked by the same nucleotide and/or the complementary nucleotides, e.g.,ATAn,TTAn,CCGn, where n = 2-5 . Runs flanked by nucleotides with which they cannot base pair are distinctly disfavored . (In this group An/Tn are flanked by C and/or G; Gn/Cn are flanked by A/T, e.g.,CGAn,TnGG,GnAT) . The frequencies of runs flanked by A or T, and G or C ("mixed"group) are as expected . Here we seek the origin of this effect and its relevance to protein-DNA interactions . Surprisingly, within the first group, runs flanked by their complements with a pyrimidine-purine junction (e.g.,TTAn,CnGG) are greatly preferred . The frequencies of their purine-pyrimidine junction mirror-images is just as expected . This effect, as well as additional ones enumerated below, is seen universally in eukaryotes and in prokaryotes, although it is stronger in the former . Detailed analysis of regulatory regions shows these strong trends, particularly in GC sequences . The potential relationship to DNA conformation and DNA-protein interaction is discussed.

J Gen Microbiol, 1989 Dec, 135 ( Pt 12), 3271 - 9
Molecular analysis of a novel glutamine synthetase of the anaerobe Bacteroides fragilis; Hill RT et al.; The nucleotide sequence of a 2777 bp DNA segment containing the Bacteroides fragilis glnA gene was determined . The B . fragilis glnA open reading frame of 2187 bp encoded a glutamine synthetase (GS) subunit of 729 amino acid residues with a calculated Mr of 82,827 . The apparent Mr of the GS subunit determined by SDS-PAGE was approximately 75,000 . A single mRNA transcription start point was identified upstream of the B . fragilis glnA open reading frame . The B . fragilis GS subunit is approximately 270 and 400 amino acids longer than the GSI and GSII subunits, respectively, of other prokaryotes and eukaryotes . The GSI and GSII holoenzymes are dodecamers and octamers respectively, whereas the GS of B . fragilis is a hexamer . Although GSI and GSII subunits show amino acid similarity in five conserved regions, this similarity is not strongly conserved in the B . fragilis GS . The GS of B . fragilis is not regulated by adenylylation and lacks the adenylylation site . It also lacks the Trp residue associated with the active site in GSI and GSII enzymes from other prokaryotes and eukaryotes.

FASEB J, 1989 Dec, 3(14), 2583 - 92
P-glycoprotein: multidrug-resistance and a superfamily of membrane-associated transport proteins; Juranka PF et al.; The study of multidrug resistance (MDR) in tumor cell lines has led to the discovery of the plasma membrane P-glycoprotein (Pgp) molecule . This protein functions as an energy-dependent pump for the efflux of diverse anticancer drugs from MDR cells . It now appears that Pgp-mediated MDR tumor cells do occur in human cancers, and that they are likely to play a role in the ultimate response of patients to chemotherapy . Chemosensitizers, compounds able to reverse the MDR phenotype, have been identified and offer the exciting possibility of improving efficacy for some nonresponsive malignancies . Surprisingly, Pgp-like molecules can be found in evolutionarily distant species among both eukaryotes and prokaryotes . As a group, these proteins form a superfamily of ATP-dependent transport proteins . This finding has broad implications and provides new insights into how living organisms use this fundamental transport system to regulate the trafficking of diverse molecules across biological membranes.

Proc Natl Acad Sci U S A, 1989 Dec, 86(24), 9757 - 61
Multiple functions of human single-stranded-DNA binding protein in simian virus 40 DNA replication: single-strand stabilization and stimulation of DNA polymerases alpha and delta; Kenny MK et al.; The human single-stranded-DNA binding protein (human SSB) is required for simian virus 40 (SV40) DNA replication in vitro . SV40 large tumor antigen and human SSB can support extensive unwinding of SV40 origin-containing DNA in the presence of ATP and a topoisomerase that relieves positive superhelicity . Although SSBs from viral and prokaryotic sources substituted for human SSB in the DNA-unwinding reaction, they did not substitute in the replication of SV40 DNA . The specificity for human SSB in SV40 DNA replication can be explained, at least in part, by the finding that DNA polymerase alpha was stimulated 10-fold by human SSB but not by other SSBs . Human SSB also stimulated proliferating-cell nuclear antigen-dependent DNA polymerase delta; however, other SSBs stimulated this polymerase as well.

New Biol, 1989 Dec, 1(3), 247 - 51
Gene regulation from sites near and far; Magasanik B; Although regulation of gene expression from distant sites would seem to have many advantages, this strategy is utilized far more frequently in eukaryotic cells than in prokaryotic cells . The inherent flexibility of DNA allows factors bound to distant regulatory sites to interact with transcription initiation factors or RNA polymerase components at the promoter of the regulated gene . Regulatory domains that are closely linked to the promoter lose this spatial flexibility, and must lie within a defined region in the vicinity of the promoter . The major drawback to regulation of gene expression from distant sites is that any gene with a promoter located within the effective radius of the regulatory factor will respond to its control . Thus, promoters of independently regulated genes must be separated by ample amounts of nonspecific DNA . Prokaryotic cells may have evolved to favor regulation of transcription from closely linked sites because this mechanism allows them to eliminate excess DNA, which may in turn enable them to achieve rapid growth in an energy-limited environment.

Plant Mol Biol, 1989 Dec, 13(6), 615 - 25
Chloroplast mRNA 3' end maturation is biochemically distinct from prokaryotic mRNA processing; Stern DB et al.; We have characterized a chloroplast processing activity that catalyzes the conversion of the plastid cytochrome b6/f subunit IV (pet D) mRNA 3' end precursor to the mature RNA possessing a 3' inverted repeat (IR) . In a chloroplast soluble protein extract, the activity requires Mg2+ or Mn2+, but not K+ . In the absence of Mg2+, the pet D 3' IR-RNA product does not accumulate, and UV-cross-linking indicates that the 3' IR-RNA precursor binds several new proteins in addition to those previously characterized as part of the 3' IR-RNA: protein complex in vitro . In contrast, high concentrations of Zn2+ or Cu2+ suppress protein binding and inhibit the processing reaction . The purified exoribonuclease polynucleotide phosphorylase (E.C.2.7.7.8) is not efficient in processing the pet D 3' IR-RNA precursor, whereas Escherichia coli ribonuclease II rapidly processes the pet D IR-RNA precursor to a product of a size similar to that of the mature 3' IR-RNA, but also rapidly degrades the mature RNA in the absence of chloroplast extract . We therefore conclude that the maturation of the pet D mRNA in vitro requires specific chloroplast enzymes which process the mRNA 3' end precursor in the absence of efficient transcription termination . The chloroplast enzyme activities are biochemically distinct from their bacterial counterparts . We also note that specific chloroplast components may be required to stabilize the mature pet D mRNA 3' end against further exonucleolytic degradation.

Am J Med, 1989 Nov 30, 87(5A), 2S - 8S
Ciprofloxacin and the fluoroquinolones . New concepts on the mechanism of action and resistance; Fisher LM et al.; Ciprofloxacin, a new fluoroquinolone, is a potent, broad-spectrum antibacterial agent . It rapidly blocks bacterial deoxyribonucleic acid (DNA) replication by inhibiting DNA gyrase, an essential prokaryotic enzyme that catalyzes chromosomal DNA supercoiling . Molecular genetic approaches have been used to study the interaction of 4-quinolones with DNA gyrase from quinolone-sensitive strains and from uropathogenic quinolone-resistant clinical isolates of Escherichia coli . An important mutational locus in the gyrase A gene that confers resistance to ciprofloxacin and other quinolones has been identified, and a new, rapid method to examine clinical isolates for the presence of mutations at this position has been devised . A quinolone resistant gyrA gene has been previously cloned and sequenced from an E . coli clinical isolate . Genetic analysis indicated that resistance resulted from a Ser-83----Trp change in the 875 residue gyrase A protein: two other changes observed in the protein, Asp-678----Glu and Ala-828----Ser, were neutral . GyrA genes carrying these mutations have now been expressed, corresponding mutant gyrase A proteins purified, and their quinolone resistance properties tested by complementing with gyrase B protein and studying the resulting gyrase activity in an adenosine triphosphate-dependent DNA supercoiling assay . The in vitro DNA supercoiling activity of the A (Ser-83----Trp) mutant subunit complemented with wild-type gyrase B subunit was highly resistant to ciprofloxacin and other 4-quinolones . In contrast, A subunit carrying codon 678 and 828 changes reconstituted a quinolone-sensitive gyrase activity . Thus, quinolone-resistant gyrase A proteins may be readily obtained for study by using high-copy gyrA plasmids . In addition, other quinolone-resistant strains of E . coli have been examined for the presence of mutations at gyrase A codons 82 and 83 using a new analytical method based on a restriction fragment length polymorphism (RFLP) . This analysis revealed that seven of eight resistant clinical isolates of E . coli examined carried gyrA mutations at codon 82 or 83, whereas five sensitive strains appeared to possess wild-type sequence . Thus, mutations at codon 83 (and possibly 82) in the gyrA gene frequently confer resistance to 4-quinolones, including ciprofloxacin . The RFLP method described should prove useful in examining strains for such mutations . These results are discussed with regard to the mode of interaction of the 4-quinolones with gyrase.

Gene, 1989 Nov 30, 83(2), 281 - 9
Nucleotide sequence of pho-4+, encoding a phosphate-repressible phosphate permease of Neurospora crassa; Mann BJ et al.; The nucleotide (nt) sequence of the Neurospora crassa pho-4+ gene, which encodes a phosphate-repressible phosphate permease, has been determined . The gene specifies a protein of 590 amino acids (aa) and contains two introns . Two RNA transcripts of 3.3 and 2.4 kb have been identified, and transcription start points (tsp) and termination sites and/or processing sites have been located . The 3.3-kb message is initiated about 890 nt upstream from the tsp for the 2.4-kb transcript . A hydropathy profile of the aa sequence suggests ten to twelve membrane-spanning helices with a large hydrophilic domain between the eighth and ninth helices . This model for the predicted secondary structure of the protein is very similar to models proposed for other sequenced integral membrane proteins from both prokaryotes and eukaryotes . Since very few permease-encoding genes of eukaryotes have been examined in molecular detail, it will be of interest to compare the sequence of pho-4+ with those encoding other anion transport proteins, as they become available.

J Biol Chem, 1989 Nov 25, 264(33), 19587 - 92
Primary structure of the maize NADP-dependent malic enzyme; Rothermel BA et al.; Chloroplast-localized NADP-dependent malic enzyme (EC 1.1.1.40) (NADP-ME) provides a key activity for the carbon 4 fixation pathway . In maize, nuclear encoded NADP-ME is synthesized in the cytoplasm as a precursor with a transit peptide that is removed upon transport into the chloroplast stroma . We present here the complete nucleotide sequence for a 2184-base pair full-length maize NADP-ME cDNA . The predicted precursor protein is 636 amino acids long with a Mr of 69,800 . There is a strong codon bias found in the amino-terminal portion of NADP-ME that is present in genes for the other enzymes of the C-4 photosynthetic pathway . The NADP-ME transit peptide has general features common to other known chloroplast stroma transit peptides . Comparison of mature maize NADP-ME to the amino acid sequences of known malic enzymes shows two conserved dinucleotide-binding sites . There is a third highly conserved region of unknown function . On the basis of amino acid sequence similarity, the maize chloroplastic enzyme is more closely related to eukaryotic cytosolic isoforms of malic enzyme than to prokaryotic isoforms . We discuss the functional and evolutionary relationship between the chloroplastic and cytosolic forms of NADP-ME.

Nucleic Acids Res, 1989 Nov 25, 17(22), 9051 - 62
Transcriptional mapping and nucleotide sequence of a vaccinia virus gene encoding a polypeptide with extensive homology to DNA ligases; Smith GL et al.; Nucleotide sequencing of the vaccinia virus SalI F DNA fragment identified an open reading frame of 552 amino acids encoding a protein of 63.3 kDa . The deduced amino acid sequence shares 30% identity with S . pombe and S . cerevisiae DNA ligases, with homology strongest near the carboxy terminus and around the lysine residue required for ligase-adenylate formation . Prokaryotic DNA ligases are poorly related to the vaccinia sequence . The initiation codon of the ORF forms part of a late transcriptional initiation sequence TAAATG and is preceded by two overlapping early transcriptional termination signals, TTTTTTTAT . Nonetheless, RNA mapping showed that the ligase gene is transcribed early during infection and the 5' end of the mRNA maps to the TAAATG motif . The possible roles of a DNA ligase in vaccinia virus DNA replication and recombination are discussed.

Nature, 1989 Nov 23, 342(6248), 453 - 6
Identification of a novel translation factor necessary for the incorporation of selenocysteine into protein; Forchhammer K et al.; During the biosynthesis of selenoproteins in both prokaryotes and eukaryotes, selenocysteine is cotranslationally incorporated into the nascent polypeptide chain through a process directed by a UGA codon that normally functions as a stop codon . Recently, four genes have been identified whose products are required for selenocysteine incorporation in Escherichia coli . One of these genes, selC, codes for a novel transfer RNA species (tRNAUCA) that accepts serine and cotranslationally inserts selenocysteine by recognizing the specific UGA codon . The serine residue attached to this tRNA is converted to selenocysteine in a reaction dependent on functional selA and selD gene products . By contrast, the selB gene product (SELB) is not required until after selenocysteyl-tRNA biosynthesis . Here we present evidence indicating that SELB is a novel translation factor . The deduced amino-acid sequence of SELB exhibits extensive homology with the sequences of the translation initiation factor-2 (IF-2) and elongation factor Tu (EF-Tu) . Furthermore, purified SELB protein binds guanine nucleotides in a 1:1 molar ratio and specifically complexes selenocysteyl-tRNAUCA, but does not interact with seryl-tRNAUCA . Thus, SELB could be an amino acid-specific elongation factor, replacing EF-Tu in a special translational step.

Gene, 1989 Nov 15, 83(1), 153 - 9
Nucleotide sequence of the Saccharomyces cerevisiae PUT4 proline-permease-encoding gene: similarities between CAN1, HIP1 and PUT4 permeases; Vandenbol M et al.; The complete nucleotide (nt) sequence of the PUT4 gene, whose product is required for high-affinity proline active transport in the yeast Saccharomyces cerevisiae, is presented . The sequence contains a single long open reading frame of 1881 nt, encoding a polypeptide with a calculated Mr of 68,795 . The predicted protein is strongly hydrophobic and exhibits six potential glycosylation sites . Its hydropathy profile suggests the presence of twelve membrane-spanning regions flanked by hydrophilic N- and C-terminal domains . The N terminus does not resemble signal sequences found in secreted proteins . These features are characteristic of integral membrane proteins catalyzing translocation of ligands across cellular membranes . Protein sequence comparisons indicate strong resemblance to the arginine and histidine permeases of S . cerevisiae, but no marked sequence similarity to the proline permease of Escherichia coli or to other known prokaryotic or eukaryotic transport proteins . The strong similarity between the three yeast amino acid permeases suggests a common ancestor for the three proteins.

J Chromatogr, 1989 Nov 10, 496(1), 101 - 10
Assay of ornithine decarboxylase activity by reversed-phase high-performance liquid chromatography; Beeman CS et al.; Ornithine decarboxylase (L-ornithine carboxylase; EC 4.1.1.17; ODCase) is a key enzyme in the biosynthesis of polyamines . It catalyzes the decarboxylation of L-ornithine to putrescine . The high-performance liquid chromatographic (HPLC) method described here for determining ODCase activity combines the sensitivity of radiochemical detection with the separative capacity of HPLC without the necessity of generating a pre-column derivative . In this study, {1,2-3H}putrescine was separated from L-{2,3-3H}ornithine using reversed-phase HPLC eluted isocratically . This method was used to study ODCase from both prokaryotic and mammalian sources . With the ODCase from Escherichia coli we found the reaction rates to be linear for 5 min with an apparent Michaelis constant (KM) of 20 mM . After 1 h this activity had produced approximately four-fold more product at pH 5.0 than at pH 7.3 . In contrast, the initial rate of ODCase from submandibular glands was linear for 60 min . Also, the rate of putrescine synthesis was ten-fold higher in the embryonic gland than in the adult which was 8-80 times lower than that of E . coli.

Cell, 1989 Nov 3, 59(3), 573 - 80
The structure of the Antennapedia homeodomain determined by NMR spectroscopy in solution: comparison with prokaryotic repressors; Qian YQ et al.; The structure of the Antennapedia homeodomain from Drosophila melanogaster was determined by nuclear magnetic resonance spectroscopy in solution . It includes three well-defined helices (residues 10-21, 28-38, and 42-52) and a more flexible fourth helix (residues 53-59) . Residues 30-50 form a helix-turn-helix motif virtually identical to those observed in various prokaryotic repressors . Further comparisons of the homeodomain with prokaryotic repressors showed that there are also significant differences in the molecular architectures . Overall, these studies support the view that the third helix of the homeodomain may function as the DNA recognition site . The elongation of the third helix by the fourth helix is a structured element that so far appears to be unique to the Antennapedia homeodomain.

Cell, 1989 Nov 3, 59(3), 553 - 62
A single amino acid can determine the DNA binding specificity of homeodomain proteins; Treisman J et al.; Many Drosophila developmental genes contain a DNA binding domain encoded by the homeobox . This homeodomain contains a region distantly homologous to the helix-turn-helix motif present in several prokaryotic DNA binding proteins . We investigated the nature of homeodomain-DNA interactions by making a series of mutations in the helix-turn-helix motif of the Drosophila homeodomain protein Paired (Prd) . This protein does not recognize sequences bound by the homeodomain proteins Fushi tarazu (Ftz) or Bicoid (Bcd) . We show that changing a single amino acid at the C-terminus of the recognition helix is both necessary and sufficient to confer the DNA binding specificity of either Ftz or Bcd on Prd . This simple rule indicates that the amino acids that determine the specificity of homeodomains are different from those mediating protein-DNA contacts in prokaryotic proteins . We further show that Prd contains two DNA binding activities . The Prd homeodomain is responsible for one of them while the other is not dependent on the recognition helix.

Nature, 1989 Nov 2, 342(6245), 95 - 8
Induction of the Escherichia coli lactose operon selectively increases repair of its transcribed DNA strand; Mellon I et al.; Nucleotide excision repair helps to ameliorate the lethal and mutagenic consequences of DNA damage by removing helix-distorting lesions from cellular genomes . We have previously analysed the removal of ultraviolet-induced cyclobutane pyrimidine dimers from specific DNA sequences in mammalian cells and demonstrated that transcriptionally active genes are preferentially repaired . Additionally, we found that in rodent and human cells only the transcribed strand of the dihydrofolate reductase gene is selectively repaired . Transcription is blocked by pyrimidine dimers in template DNA and the selective removal of these lesions seems to be important for cell survival after irradiation with ultraviolet light . To determine whether this feature of repair is common to prokaryotes and eukaryotes and better to understand its mechanism, we have investigated repair in the two separate DNA strands of the lactose operon of ultraviolet-irradiated Escherichia coli . We find a dramatic difference in the repair of the two strands only when transcription is induced . Most dimers are removed from the transcribed strand of the induced operon within five minutes of irradiation . In the nontranscribed strand, repair is significantly slower and resembles that found in both strands of the uninduced operon . Thus there seems to be a mechanism that couples nucleotide excision repair and transcription.

Biol Rev Camb Philos Soc, 1989 Nov, 64(4), 409 - 34
Mycetocyte symbiosis in insects; Douglas AE; 1 . Non-pathogenic microorganisms, known as mycetocyte symbionts, are located in specialized 'mycetocyte' cells of many insects that feed on nutritionally unbalanced or poor diets . The insects include cockroaches, Cimicidae and Lygaeidae (Heteroptera), the Homoptera, Anoplura, the Diptera Pupiparia, some formicine ants and many beetles . 2 . Most mycetocyte symbionts are prokaryotes and a great diversity of forms has been described . None has been cultured in vitro and their taxonomic position is obscure . Yeasts have been reported in Cerambycidae and Anobiidae (Coleoptera) and a few planthoppers . They are culturable and those in anobiids have been assigned to the genus Torulopsis . 3 . The mycetocyte cells may be associated with the gut, lie free in the abdominal haemocoel or be embedded in the fat body of the insect . The mycetocytes are large polyploid cells which rarely divide and the symbionts are restricted to their cytoplasm . 4 . The mycetocyte symbionts are transmitted maternally from one insect generation to the next . In many beetles (Anobiidae, Cerambycidae, Chrysomelidae and cleonine Curculionidae), the microoganisms are smeared onto the eggs and consumed by the hatching larvae . In other insects, they are transferred from mycetocytes to oocytes in the ovary, a process known as transovarial transmission . The details of transmission in the different insect groups vary with the age of the mother (adult, larva or embryo) at which symbiont transfer to the ovary is initiated; whether isolated symbionts or intact mycetocytes are transferred; and the site of entry of symbionts to the egg (anterior, posterior or apolar) . 5 . Within an individual insect, the biomass of symbionts varies in a regular fashion with age, weight and sex of the insect . Suppression of symbiont growth rate and lysis of 'excess' microorganisms may contribute to the regulation of symbionts (including freshly-isolated preparations of unculturable forms) are used to investigate interactions between the partners . However, some methods to obtain aposymbiotic insects (e.g . antibiotics and lysozyme) deleteriously affect certain insects and aposymbionts may differ from the symbiont-containing stocks from which they were derived . 7 . The mycetocyte symbionts have been proposed to synthesize various nutrients required by the insect . The symbionts of beetles and haematophagous insects may provide B vitamins and those in cockroaches and the Homoptera essential amino acids . The role of symbionts in the sterol nutrition of insects is equivocal . 8 . Mycetocyte symbionts may have evolved from gut symbionts or guest microorganisms . The association is monophyletic in cockroaches but polyphyletic in many groups, including the sucking lice, beetles and scale insects.(ABSTRACT TRUNCATED AT 400 WORDS)

Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8257 - 61
Energy coupling to periplasmic binding protein-dependent transport systems: stoichiometry of ATP hydrolysis during transport in vivo; Mimmack ML et al.; Periplasmic binding protein-dependent transport systems mediate the accumulation of many diverse substrates in prokaryotic cells . Similar transport systems, including the P-glycoprotein responsible for multidrug resistance in human tumors, are also found in eukaryotes . The mechanism by which energy is coupled to the accumulation of substrate by these transport systems has been controversial . In this paper we demonstrate that ATP hydrolysis occurs in vivo concomitantly with transport . These data strongly suggest that ATP hydrolysis directly energizes substrate accumulation by these transport systems . The apparent stoichiometry is one to two molecules of ATP hydrolyzed per molecule of substrate transported.

Sci China B, 1989 Nov, 32(11), 1318 - 28
Studies on the structure of HBV DNA; Qi ZH et al.; The structure of HBV adr NC-1 DNA is analyzed and compared with another five strains of HBV DNAs . Some of the prokaryotic promoter-like sequences, palindrom sequences and ATAA are found . An enhancer core sequence and some other characteristics are also shown . In considering the frame and its regulatory sequence as a transcriptional unit some of the possible new frames are discussed.

Mol Microbiol, 1989 Nov, 3(11), 1545 - 55
Structure and function of hot spots providing signals for site-directed specific recombination and gene expression in Tn21 transposons; Schmidt FR et al.; Tn21- and Tn3-related transposons are widespread and carry various resistance determinants . The insertion points of different resistance genes were precisely defined in Tn2424, Tn1696, Tn2410, Tn4000 and its derivatives and compared to the corresponding sites in Tn7, pSA, R388, R46, Tn2603, Tn1331 and in Tn3-related elements . Insertional 'hot spots' located at the 3' end of different genes comprised 55 nucleotides and yielded more than 90% homology to the corresponding consensus sequence, termed hs1 . Elements of this class were found to direct recA-independent generation of deletions . Flanking the 5' ends, hs2 (CTAAAACAAAGTTA) comprised the terminal nucleotides of hs1 . Functional properties of hot spots as recognition sites for site-specific recombination and regulation of gene expression indicate that they might be involved in transfer, stable inheritance and expression of prokaryotic genes.

Yakugaku Zasshi, 1989 Nov, 109(11), 802 - 22
{Genetic and biochemical studies on the mechanism of mammalian chromosome replication}; Hanaoka F; It has been clarified that basic mechanism of deoxyribonucleic acid (DNA) replication is conserved from bacteria to higher cells . What distinguishes prokaryotic and eukaryotic modes of DNA replication most clearly is that bacterial chromosomes form a single replicon copied from a single initiation point and eukaryotic chromosomes consist of multiple replicons that initiate at multiple points . Thus, eukaryotes have to coordinate orderly replication of the genome . In order to understand this complex problem as a whole, three approaches were chosen . First approach is a genetic one . Certain number of temperature-sensitive (ts) mutants were isolated from mouse FM3A cells . One of the ts mutants, designated as tsFT20, was shown to contain heat-labile DNA polymerase alpha (pol . alpha) . By the use of this mutant strain, it was proved that pol . alpha is essential for mammalian DNA replication . In addition, the human gene for pol . alpha on the X chromosome was assigned . Second approach is an enzymological one . FM3A cells were used for the identification and characterization of enzymes and proteins supposed to be involved in DNA replication . Four DNA-dependent ATPases, three pol . alpha stimulation factors, DNA topoisomerases I and II have been identified, as well as a stimulation factor for the assembly of nucleosome . DNA helicase activity was detected in two of the DNA-dependent ATPase (B and C1) . Third approach is the reconstitution of DNA replication in cell-free system . By use of polyoma virus DNA as a template, cell-free extract from FM3A cells supported DNA replication in the presence of polyoma virus large T-antigen . This cell-free system will be useful for the analysis of the function of replication enzymes and proteins as well as the characterization of ts mutants.

Somat Cell Mol Genet, 1989 Nov, 15(6), 591 - 603
Long-range activation of transcription by SV40 enhancer is affected by "inhibitory" or "permissive" DNA sequences between enhancer and promoter; Schreiber E et al.; The transcriptional enhancer effect is used in many, if not all, organisms for remote control of gene transcription . An enhancer DNA can dramatically stimulate transcription of a linked gene from positions either 5' or 3' to the gene . Both the proximal promoter and the distal enhancer sequences are binding sites for transcription factors . Interaction between promoter and enhancer is mediated by these factors, presumably via looping out of the intervening DNA . Here we report that the extent of remote activation by an enhancer depends on characteristics of that intervening DNA . Using Beta-globin and SV40 T-antigen test genes, we show that the effect of an SV40 enhancer is transmitted to the responsive promoter, with little or no loss of efficiency, through certain segments of mammalian DNA derived from rabbit beta-globin or mouse alpha-globin gene regions . By contrast, a strong reduction of enhancer activity is observed with certain spacer segments of prokaryotic DNA (from plasmid pBR322 or phage lambda) or sequences of high (G + C) content from eukaryotic genes . We have analyzed more closely sequences that are more or less permissive for transmission of the transcriptional enhancer effect . It appears that these permissive sequences generally have a high (A + T) content and notably a very low abundance of CpG dinucleotides . By contrast, (G + C)-rich DNA segments with high local densities of CpG were the most deleterious for long-range enhancer action . We note that the latter sequence composition is typical for "CpG islands" of many mammalian genes, including housekeeping genes and the human alpha-globin gene . This may be related to the finding that promoters of most cell type-specific genes, whose activity depends on a strong enhancer, do not contain CpG islands . Most likely, the spacer DNAs of typical cell type-specific genes have evolved to allow maximal transmission of the enhancer effect.

Proc Natl Acad Sci U S A, 1989 Nov, 86(21), 8197 - 201
The mRNA encoding a high-affinity cAMP phosphodiesterase is regulated by hormones and cAMP; Swinnen JV et al.; To elucidate the mechanisms by which hormones regulate cAMP phosphodiesterases (PDEs), a group of cDNA clones that had been isolated from a rat Sertoli cell library were characterized . These cDNAs are derived from a single gene (ratPDE3) . The deduced amino acid sequence of the ratPDE3 cDNA corresponds to a 66,200-Da protein homologous to other testicular PDEs, to the Drosophila melanogaster dunce-encoded cAMP PDE, and to bovine and yeast PDEs . Expression of ratPDE3 in eukaryotic and prokaryotic cells leads to the appearance of a cAMP PDE with properties identical to the cAMP PDE purified from Sertoli cells . Although of different size, transcripts corresponding to ratPDE3 were present in all organs studied . In the immature Sertoli cell in culture, the level of mRNA transcripts of ratPDE3 was increased more than 100-fold by follicle-stimulating hormone or N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate treatment . Stimulation of ratPDE3 mRNA by N6,O2'-dibutyryladenosine 3',5'-cyclic monophosphate was also observed in a C6 glioma cell line . These data demonstrate that cAMP regulates the expression of one of its own degrading enzymes by an intracellular feedback mechanism that involves changes in mRNA levels.

Mol Microbiol, 1989 Nov, 3(11), 1579 - 86
Cloning, expression and sequence analysis of the gene encoding the 120 kD surface-exposed protein of Rickettsia rickettsii; Gilmore RD Jr et al.; Rickettsia rickettsii (R strain) genomic DNA was partially digested and cloned into the lambda expression vector gt11 generating a genomic clone bank . Transformant plaques were screened with antisera generated against the 120 kiloDalton protein of R . rickettsii to detect those phage expressing the recombinant protein . The gene encoding the 120 kD protein was localized to a 4.3 kilobase SphI-BamHI fragment from the recombinant phage and subcloned into pUC18 and pUC19 . Full-length expression of the recombinant protein was achieved with both orientations . The gene and flanking regions were sequenced . The p120 gene consists of 3900 base pairs coding for 1300 amino acids . A distinguishable promoter region was not identified, although there are several 5' sequences that resemble classical prokaryotic promoters . Downstream of the termination codon for this gene lies a 726 base pair open reading frame on the opposite strand with the potential to encode a protein of approximately 27 kD . The identity of this putative gene product is unknown . The two open reading frames are separated by a 106 base pair intergenic region that consists of a stretch of dyad symmetry resembling rho-independent transcriptional terminators.

Mol Biol Evol, 1989 Nov, 6(6), 669 - 84
A tree reconstruction method that is economical in the number of pairwise comparisons used; Hein J; A fast method for reconstructing phylogenies from distance data is presented . The method is economical in the number of pairwise comparisons needed . It can be combined with a new phylogenetic alignment procedure to yield an algorithm that gives a complete history of a set of homologous sequences . The method is applicable to very large distance matrices . An auxiliary program was developed that simplifies large phylogenies without ignoring biologically essential features . A set of 213 globins from vertebrates, plants, and Vitreoscilla (a prokaryote) were analyzed using this method.

Mol Biol (Mosk), 1989 Nov-Dec, 23(6), 1732 - 42
{Conformation limited nucleoside-5'-phosphates as termination substrates for DNA-polymerases}; Chidzhavadze ZG et al.; We have investigated the ability of some nucleoside 5'-triphosphate analogues to terminate the DNA synthesis catalyzed by calf thymus DNA polymerase alpha and terminal deoxynucleotidyl transferase, rat liver DNA polymerase beta, E . coli DNA polymerase I (Klenow's fragment) and AMV reverse transcriptase . It has been shown that lyxoanhydronucleoside 5'-triphosphates terminate DNA synthesis catalyzed by reverse transcriptase and terminal deoxynucleotydil transferase . 2',3'-O-Isopropylidenecytidine 5'-triphosphate inhibits the DNA synthesis catalyzed by reverse transcriptase and DNA polymerase beta and its moiety was incorporated in the place of dTMP residue . Riboanhydroadenosine 5'-triphosphate reveals the properties of an effective termination substrate for all the DNA polymerases studied . This is the first attempt to investigate nucleotide analogues with the restricted conformation of the carbohydrate moiety as termination substrates for several prokaryotic and eukaryotic DNA polymerases.

Proc Natl Acad Sci U S A, 1989 Nov, 86(22), 8717 - 21
An immunological determinant of RNase P protein is conserved between Escherichia coli and humans; Mamula MJ et al.; RNase P, an enzyme with RNA and protein subunits, cleaves tRNA precursor molecules to form the 5' termini of mature tRNAs in both prokaryotes and eukaryotes . Rabbit antibodies made against the protein subunit, C5 protein, of Escherichia coli RNase P bound RNase P protein from E . coli and Bacillus subtilis in immunoblots and solid-phase immunoassays . These rabbit anti-C5 antibodies also bound a protein (Mr approximately 40,000) in preparations of RNase P from human (HeLa) cells and depleted the enzymatic activity from preparations of RNase P from both human and E . coli cells . Finally, rabbit anti-C5 antibodies immunoprecipitated from crude extracts of human cells a ribonucleoprotein complex containing H1 RNA, the putative RNA component of human RNase P . These results show that an antigenic determinant is shared by C5 protein from E . coli RNase P and a protein component of RNase P from human cells.

Gene, 1989 Oct 30, 82(2), 357 - 62
Homologous potyvirus and flavivirus proteins belonging to a superfamily of helicase-like proteins; Lain S et al.; Comparison of the nucleoside triphosphate-binding motif(NTBM)-containing proteins of two groups of apparently distantly related positive-strand RNA viruses (potyvirus and flavivirus), revealed significant sequence similarity . In addition, these two groups of viral proteins show amino acid motifs in common with those conserved in a group of five NTBM-containing proteins from prokaryotic and eukaryotic cells, some of which have been experimentally related to helicase activity . Here we propose that the proteins mentioned above constitute a superfamily of helicase-like proteins, distinct from the one previously described {Gorbalenya et al., FEBS Lett . 235 (1988) 16-24; Hodgman, Nature 333 (1988) 22-23; 578}, which includes the NTBM-containing proteins from another group of positive-strand RNA viruses, the 'Sindbis-like' viruses.

J Biol Chem, 1989 Oct 25, 264(30), 17656 - 9
Eukaryotic promoters drive gene expression in Escherichia coli; Antonucci TK et al.; Eight eukaryotic promoters have been tested for their activity in vivo in Escherichia coli . The rat beta-actin, rat amylase, rat chymotrypsin B, mouse metallothionein I, rat insulin I, human insulin, Rous sarcoma virus long terminal repeat (RSV LTR) and hepatitis B viral precore promoter activities were measured by using the bacterial chloramphenicol acetyltransferase coding sequences as the reporter function and by primer extension RNA analysis . All eight promoter-chloramphenicol acetyltransferase constructs produce chloramphenicol acetyltransferase activity with the following relative strengths: RSV LTR greater than rat beta-actin greater than rat insulin I greater than rat amylase greater than hepatitis B virus precore greater than human insulin greater than rat chymotrypsin B greater than mouse metallothionein I . A primer extension analysis indicates that transcription from the RSV LTR, rat insulin I, and rat beta-actin promoters initiates at the sites expected for eukaryotic rather than prokaryotic promoters . Thus the site of initiation is determined by the DNA sequence rather than by the RNA polymerase.

Biochemistry, 1989 Oct 17, 28(21), 8274 - 7
Template-primer activity of 5-(hydroxymethyl)uracil-containing DNA for prokaryotic and eukaryotic DNA and RNA polymerases; Herrala AM et al.; We have utilized Bacillus subtilis phage SPO-1 DNA as a model of irradiated DNA . In this phage, all thymine (Thy) residues are replaced by 5-(hydroxymethyl)uracil (5HmUra), which is a known irradiation-induced derivative of DNA Thy . SPO-1 phage is naturally devoid of other such irradiation-induced DNA lesions . DNase I activated SPO-1 phage DNA served as well as, or even better than, the control DNAs (Bacillus subtilis DNA and calf thymus DNA) as a template-primer for Escherichia coli, Micrococcus luteus, and human HL-60 cell DNA polymerases . Furthermore, the template activity of SPO-1 phage DNA was also superior when transcription with E . coli RNA polymerase was investigated . The results reported here indicated that the replacement of Thy by 5HmUra is not deleterious to template and primer functions during DNA or RNA synthesis.

J Biol Chem, 1989 Oct 15, 264(29), 17032 - 40
Phenomenological theory of gel electrophoresis of protein-nucleic acid complexes; Cann JR; A phenomenological theory of gel electrophoresis is elaborated for protein-DNA complexes involving one, two, or three binding sites on the DNA molecule . The computed electrophoretic patterns simulate experimental patterns shown by both prokaryotic and eukaryotic systems . The mechanism whereby the electrophoretic protein-DNA ladder is generated upon titration of the operator with repressor is embodied in theory of mass transport coupled to reversible interactions under chemical kinetic control . In contrast to strong interactions (association constant greater than 10(12) M-1), patterns observed with weak complexes (K less than 10(10) M-1) could be simulated only by applying the cage effect, a model of which is formulated . Theoretical underpinning is provided for the electrophoretic estimation of equilibrium association constants, and requisite chemical kinetic conditions are elucidated for direct estimation of the rate constant for dissociation of the protein-DNA complex from gel patterns . The theory thus affords an experimenter with a means for determining the conditions required to render the gel retardation method a valid procedure for evaluating equilibrium constants and/or kinetic parameters for the particular protein-nucleic acid system under investigation . These several considerations apply not only to interactions of proteins with nucleic acids (DNA or RNA) but also to a wide range of macromolecular interactions involving peptides, drugs, and other ligands as well as large assemblies such as multienzyme complexes.

J Biol Chem, 1989 Oct 15, 264(29), 17041 - 8
Nuclear magnetic resonance studies of 6-fluorotryptophan-substituted rat cellular retinol-binding protein II produced in Escherichia coli . Analysis of the apoprotein and the holoprotein containing bound all-trans-retinol and all-trans-retinal; Li E et al.; Rat cellular retinol-binding protein II (CRBP II) is a 15.6-kDa intestinal protein which binds all-trans-retinol and all-trans-retinal but not all-trans-retinoic acid . We have previously analyzed the interaction of Escherichia coli-derived rat apoCRBP II with several retinoids using fluorescence spectroscopic techniques . Interpretation of these experiments is complicated, because the protein has 4 tryptophan residues . To further investigate ligand-protein interactions, we have utilized 19F nuclear magnetic resonance (NMR) spectroscopy of CRBP II labeled at its 4 tryptophan residues with 6-fluorotryptophan . Efficient incorporation of 6-fluorotryptophan (93%) was achieved by growing a tryptophan auxotroph of E . coli harboring a prokaryotic expression vector with a full-length rat CRBP II cDNA on defined medium supplemented with the analog . Comparison of the 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II with and without bound all-trans-retinol revealed that resonances corresponding to 2 tryptophan residues (designated WA and WB) undergo large downfield changes in chemical shifts (2.0 and 0.5 ppm, respectively) associated with ligand binding . In contrast, 19F resonances corresponding to two other tryptophan residues (WC and WD) undergo only minor perturbations in chemical shifts . The 19F NMR spectra of 6-fluorotryptophan-substituted CRBP II complexed with all-trans-retinal and all-trans-retinol were very similar, suggesting that the interactions of these two ligands with the protein are similar . Molecular model building, based on the crystalline structures of two homologous proteins was used to predict the positions of the 4 tryptophan residues of CRBP II and to make tentative resonance assignments . The fact that ligand binding produced residue-specific changes in the chemical shifts of resonances in CRBP II suggests that NMR analysis of isotopically labeled retinoid-binding proteins expressed in E . coli will provide an alternate, albeit it complementary, approach to fluorescence spectroscopy for examining the structural consequences of their association with ligand.

Gene, 1989 Oct 15, 82(1), 119 - 26
A site-specific endonuclease and co-conversion of flanking exons associated with the mobile td intron of phage T4; Bell-Pedersen D et al.; The product of the td intron open reading frame (ORF) of phage T4 is required for high-frequency transfer of the intervening sequence from intron-plus (In+) to intron-minus (In-) alleles . In vivo studies have demonstrated that the td ORF product targets cleavage of td In- DNA, and that cleavage is correlated with intron inheritance {Quirk et al., Cell 56 (1989) 455-465} . In the present study we show by in vitro synthesis of the td intron ORF product, that the protein possesses endonuclease activity and efficiently cleaves double-stranded DNA at or near the site of intron integration . In addition, we demonstrate that intron insertion is accompanied by co-conversion of the flanking exon sequences . Co-conversion of markers within 50 nt surrounding the site of intron insertion occurred at a high frequency (80-100%), and decreased at greater distance from the intervening sequence . Co-conversion may provide a mechanism for maintaining exon-intron RNA contacts required for accurate splicing of the relocated intron . Cleavage of target DNA by an intron endonuclease and co-conversion of flanking exon sequences are both features associated with mobile introns of eukaryotes, indicating a common mechanism for intron transfer in the eukaryotic and prokaryotic kingdoms.

Gene, 1989 Oct 15, 82(1), 115 - 8
Mobile introns: definition of terms and recommended nomenclature; Dujon B et al.; A number of introns in mitochondrial, chloroplast, nuclear or prokaryotic genes have recently been shown to encode double-strand sequence-specific endonucleases . Such introns are mobile genetic elements that insert themselves at or near the cleaved sites . A uniform nomenclature to designate the molecular elements involved in the phenomenon of intron mobility is proposed.

J Immunol, 1989 Oct 15, 143(8), 2692 - 8
Analysis of an immunodominant region of infectious bronchitis virus; Kusters JG et al.; We analyzed the antigenic fine-structure of an immunodominant region in the peplomer protein of infectious bronchitis virus . This region near the N-terminus of the S2 subunit is recognized by polyclonal antisera and by the majority of mAb that cross-react with denatured protein . Despite their involvement in neutralization, epitopes in this region were conserved in different serotypes . Epitopes of four mAb and two chicken antisera were localized by using prokaryotic expression of cDNA fragments, and overlapping peptides with lengths increasing from 3 to 12 residues (PEPSCAN) . We found overlapping epitopes with lengths of 6, 9, 11, and more than 17 residues . The results indicate that the expression products are antigenically equivalent to denatured protein fragments . This suggests a general strategy for the localization of sequential epitopes in large proteins . We propose that the immunodominance of the N-terminal region of S2 is explained by features of the protein structure . Presumably, this region is a protruding protein segment of about 20 residues with a high local mobility, as indicated by the antigenicity of the peptides . The conservation of the sequence points to an involvement in a molecular recognition process during infection.

J Biol Chem, 1989 Oct 15, 264(29), 17298 - 308
Archaebacterial histone-like proteins . Purification and characterization of helix stabilizing DNA binding proteins from the acidothermophile Sulfolobus acidocaldarius; Reddy TR et al.; Four DNA binding histone-like proteins have been purified from the nucleoid of the acidothermophilic archaebacterium Sulfolobus acidocaldarius to homogeneity employing DNA-cellulose chromatography and carboxymethylcellulose chromatography . The molecular weights of these proteins are in the range 8,000-12,500 . Immunoblotting results suggest that a few antigenic determinants are common among these proteins which could not be detected by immunodiffusion . Spectroscopic properties of the proteins have been studied . The amino acid compositions of these proteins show both similarities and differences with histones and prokaryotic histone-like proteins . All of the four proteins bind native and denatured DNAs and single stranded RNA with differing affinities . Three of the proteins, denoted by HSNP (helix stabilizing nucleoid protein)-A, HSNP-C, and HSNP-C', show physiologically significant, strong, and synergistic effects in stabilizing duplex DNA against thermal denaturation with Tm increases in the range of 15-30 +/- degrees C.

Nucleic Acids Res, 1989 Oct 11, 17(19), 7609 - 22
The characterization of the EBV alkaline deoxyribonuclease cloned and expressed in E . coli; Baylis SA et al.; Studies of nucleic acid homology suggest the BGLF5 open reading frame of Epstein-Barr virus (EBV) encodes an alkaline deoxyribonuclease (DNase) sharing some homology with that of herpes simplex virus . We report here the expression of the BGLF5 open reading frame in E . coli and the expression of high levels of a novel alkaline DNase activity in induced cells . This alkaline DNase has been purified to apparent homogeneity as a single protein species . This is the first report of the expression of a herpesvirus coded DNase in a prokaryotic system and of the purification of the EBV DNase to demonstrable purity . It has the biochemical characteristics of a typical herpesvirus alkaline exonuclease showing a high pH optimum, an absolute requirement for Mg2+ for activity and sensitivity to high salt concentrations and polyamines . The enzyme activity was neutralized by sera from patients with nasopharyngeal carcinoma and was reactive with these sera in Western blot analysis . Thus the prokaryotic expression system described here provides an economical and efficient source of the EBV DNase for biochemical and seroepidemiological analysis.

Cell, 1989 Oct 6, 59(1), 219 - 28
A conserved 3'----5' exonuclease active site in prokaryotic and eukaryotic DNA polymerases; Bernad A et al.; The 3'----5' exonuclease active site of E . coli DNA polymerase I is predicted to be conserved for both prokaryotic and eukaryotic DNA polymerases based on amino acid sequence homology . Three amino acid regions containing the critical residues in the E . coli DNA polymerase I involved in metal binding, single-stranded DNA binding, and catalysis of the exonuclease reaction are located in the amino-terminal half and in the same linear arrangement in several prokaryotic and eukaryotic DNA polymerases . Site-directed mutagenesis at the predicted exonuclease active site of the phi 29 DNA polymerase, a model enzyme for prokaryotic and eukaryotic alpha-like DNA polymerases, specifically inactivated the 3'----5' exonuclease activity of the enzyme . These results reflect a high evolutionary conservation of this catalytic domain . Based on structural and functional data, a modular organization of enzymatic activities in prokaryotic and eukaryotic DNA polymerases is also proposed.

Cell, 1989 Oct 6, 59(1), 133 - 43
The developmental fate of S . coelicolor hyphae depends upon a gene product homologous with the motility sigma factor of B . subtilis; Chater KF et al.; In the mycelial prokaryote S . coelicolor, whiG is a gene dispensable for growth but needed for the earliest stages of spore formation in aerial hyphae . Nucleotide sequencing indicates that whiG encodes an RNA polymerase sigma factor highly similar to the motility sigma factor (sigma 28) of B . subtilis . High copy number of an intact whiG gene caused sporulation in vegetative hyphae that are usually fated to lyse without sporulating . However, the introduction of many copies of a sigma 28-dependent promoter from B . subtilis into S . coelicolor reduced sporulation, suggesting partial sequestration of the whiG gene product by the foreign promoter sequences . We propose that the level of whiG sigma factor is crucial in determining the developmental fate of hyphae.

Nature, 1989 Oct 5, 341(6241), 456 - 8
Control of topology and mode of assembly of a polytopic membrane protein by positively charged residues; von Heijne G; Positively charged amino acids have been shown to be important elements in targeting-peptides that direct proteins into mitochondria, nuclei, and the secretory pathways of both prokaryotic and eukaryotic cells . The 'positive-inside' rule, which observes that regions of polytopic (multi-spanning) membrane proteins facing the cytoplasm are generally enriched in arginyl and lysyl residues whereas translocated regions are largely devoid of these residues, implies that the distribution of positively charged amino acids may also be a major determinant of the transmembrane topology of integral membrane proteins . If this is indeed the case, it should be possible to predictably alter the topology of a polytopic protein by site-directed insertions and/or deletions of positively charged residues in critical locations . I now describe a derivative of Escherichia coli leader peptidase, a polytopic inner-membrane protein, that switches from sec-gene-dependent membrane insertion with a Nout-Cout transmembrane topology to sec-gene-independent insertion with a Nin-Cin topology in response to the addition of four positively charged lysines to its N terminus.

J Biol Chem, 1989 Oct 5, 264(28), 16727 - 32
Cytovillin, a microvillar Mr 75,000 protein . cDNA sequence, prokaryotic expression, and chromosomal localization; Turunen O et al.; Cytovillin is a microvillar cytoplasmic peripheral membrane protein, with prominent expression in vivo in placental syncytiotrophoblasts and certain human tumors . Cytovillin cDNA was cloned from a human placental lambda gt11 library using affinity purified antibodies . The identity of cytovillin cDNA clones was confirmed by expression of cytovillin in Escherichia coli and using antibodies raised against the expressed fusion protein in comparison with antibodies against cytovillin purified from cultured human choriocarcinoma cells . In these cells Northern blotting analysis identified a major 3.5-kilobase cytovillin mRNA . The cDNA encodes a protein of 575 amino acids corresponding to a molecular weight of 68,084 . According to secondary structure prediction, cytovillin is a hydrophilic protein with an extensive internal alpha-helical region ending in a sequence of 7 consecutive prolines . The predicted alpha-helical region showed limited homology to alpha-helical regions of cytoskeletal proteins and certain other proteins, but no extensive homologies were found in the cytovillin cDNA or the deduced amino acid sequence to other registered DNA or protein sequences . Southern blot analysis of a DNA panel of human mouse somatic cell hybrids localized the cytovillin gene to the end of the long arm of chromosome 6 (6q22-q27) . Our results show that cytovillin is representative of a novel class of microvillar proteins.

J Biol Chem, 1989 Oct 5, 264(28), 16608 - 12
cDNA sequence, predicted primary structure, and evolving amphiphilic helix of human aspartyl-tRNA synthetase; Jacobo-Molina A et al.; Eight of the mammalian aminoacyl-tRNA synthetases associate as a multienzyme complex, whereas prokaryotic and low eukaryotic synthetases occur only as free soluble enzymes . Association of the synthetases may result in effective compartmentalization of synthetases and suggests the association of the entire protein biosynthetic machinery . To elucidate the structural elements and the nature of the molecular interactions involved in the association of the synthetases, we have cloned and sequenced the complementary DNA coding human aspartyl-tRNA synthetase . The full length cDNA encodes an open reading frame of 500 amino acids with 56% identity with yeast aspartyl-tRNA synthetase . The similarity with yeast aspartyl-tRNA synthetase is unevenly distributed with a high percent of identity at the C-terminus and relatively low identity at the N-terminus . The N-terminal sequence strongly prefers an alpha-helical secondary structure and shows amphiphilic characteristics . Further comparison with the yeast synthetases showed that the basic positively charged helixes in yeast synthetases are evolved to a neutral amphiphilic helix in this mammalian synthetase . The mammalian neutral amphiphilic helix is so far unique among all known sequences of bacterial, yeast, and mammalian synthetases and may account for the association of synthetases in the synthetase complex.

Biochemistry, 1989 Oct 3, 28(20), 8129 - 35
Cloning, analysis, and bacterial expression of human farnesyl pyrophosphate synthetase and its regulation in Hep G2 cells; Sheares BT et al.; A partial length cDNA encoding farnesyl pyrophosphate synthetase (hpt807) has been isolated from a human fetal liver cDNA library in lambda gt11 . DNA sequence analysis reveals hpt807 is 1115 bp in length and contains an open reading frame coding for 346 amino acids before reaching a stop codon, a polyadenylation addition sequence, and the first 14 residues of a poly(A+) tail . Considerable nucleotide and deduced amino acid sequence homology is observed between hpt807 and previously isolated rat liver cDNAs for farnesyl pyrophosphate synthetase . Comparison with rat cDNAs suggests that hpt807 is about 20 bp short of encoding the initiator methionine of farnesyl pyrophosphate synthetase . The human cDNA was cloned into a prokaryotic expression vector and Escherichia coli strain DH5 alpha F'IQ was transformed . Clones were isolated that express an active fusion protein which can be readily observed on protein gels and specifically stained on immunoblots with an antibody raised against purified chicken farnesyl pyrophosphate phosphate synthetase . These data confirm the identity of hpt807 as encoding farnesyl pyrophosphate synthetase . Slot blot analyses of RNA isolated from Hep G2 cells show that the expression of farnesyl pyrophosphate synthetase mRNA is regulated . Lovastatin increases mRNA levels for farnesyl pyrophosphate synthetase 2.5-fold while mevalonic acid, low-density lipoprotein, and 25-hydroxycholesterol decrease mRNA levels to 40-50% of control values.

Am J Med Sci, 1989 Oct, 298(4), 278 - 81
In vivo transfection of murine lungs with a functioning prokaryotic gene using a liposome vehicle; Brigham KL et al.; The authors report successful in vivo transfection of lungs of mice with a functioning prokaryotic gene encoding the intracellular enzyme, chloramphenicol acetyltransferase (CAT) . Transfection was accomplished by injecting a plasmid containing the coding region for CAT driven by the SV40 early promoter (pSV2CAT) complexed to specially synthesized cationic liposomes . Intravenous or intratracheal injection of DNA-liposomes resulted in expression of the CAT gene in the lungs, persisting for at least a week, with little enzyme activity detectable in systemic organs . This method should permit either transient or stable in vivo transfection of the lungs with a gene encoding any protein of interest, providing a powerful experimental tool and potentially a novel and broadly applicable clinical therapeutic technique.

Mol Biol Med, 1989 Oct, 6(5), 409 - 24
Genetics and molecular pathogenesis of Legionella pneumophila, an intracellular parasite of macrophages; Cianciotto N et al.; In addition to providing a powerful approach for identifying bacterial factors required for full infectivity and disease production, genetic analysis of Legionella pathogenesis should also lend critical insight into the biology of the macrophage and into the pathogenesis of other intracellular parasites . The interaction between L . pneumophila and the macrophage exhibits many features found in a wide variety of prokaryotic and eukaryotic intracellular human pathogens . For example, binding to complement receptors has been shown to occur for Mycobacterium tuberculosis, M . leprae, Leishmania donovani, Leishmania major and Histoplasma capsulatum . Coiling phagocytosis has been observed during entry of L . donovani . Phagosomes that contain Toxoplasma gondii or M . tuberculosis fail to fuse with lysosomes and, in the case of T . gondii, have been shown to remain close to neutral pH . Although the molecular bases for these phenomena are unknown, their functional similarities to the L . pneumophila-macrophage interaction provide optimism that generally applicable principles are involved . The genetic techniques reviewed here will provide the molecular tools with which such questions of a general biologic nature can be framed and eventually answered . Together with more traditional methods in biochemistry, microbiology and cell biology, molecular genetics offers a robust means toward identifying and understanding the bacterial factors involved in the pathogenesis of Legionnaires' disease . Molecular studies of L . pneumophila can also help address questions concerning the epidemiology, diagnosis and prevention of disease . For example, the distribution of virulence factors might help explain and predict the attack rates of different L . pneumophila strains or Legionella species . Moreover, bacterial genes/factors that are shown to be conserved in Legionella strains could be used to develop such diagnostic tools as DNA probes . Novel types of vaccines consisting of genetically constructed, avirulent L . pneumophila strains or subunit vaccines based on the molecular characterization of virulence factors might be developed and tested as protective immunogens . In this way, the capacity to analyze and to manipulate L . pneumophila genetically may facilitate the use of Legionnaires' disease as a model infection for studying protective cell-mediated immunity . Apart from its clinical significance as the etiologic agent of Legionnaires' disease, L . pneumophila may be a key to broader understandings in microbial pathogenesis and human cell biology and immunology . Although the extremely complex processes of bacterial infection and virulence are best understood when a variety of experimental approaches are employed, we believe that the evolving molecular genetic techniques reviewed here will be critical elements in many important breakthroughs in the future.

Xenobiotica, 1989 Oct, 19(10), 1149 - 60
Evolution of the cytochrome P450 genes; Nebert DW et al.; 1 . The P450 gene superfamily is presently known to contain more than 78 members, divided into 14 families . 2 . The superfamily has undergone divergent evolution, and the ancestral gene is probably more than 2 billion years old . 3 . The recent 'burst' in new P450 genes, particularly in the II family during the past 800 million years, appears to be the result of 'animal-plant warfare' . 4 . Due to the presence or absence of a particular P450 gene in one species but not the other, it may not be correct to extrapolate toxicity or cancer data from rodent to human . 5 . Increases in the P450 gene product (enzyme induction) almost always reflect an elevated rate in gene transcription, although there are several exceptions . 6 . The mechanisms of P450 gene regulation (induction) by classes of inducers might become better understood through the comparison of different phyla that differ in response to a particular class of inducers . 7 . Amongst several carefully selected phyla, delineation between which electron donor (presence of Fe2S2 protein or NADPH-P450 oxidoreductase, or both) interacts with P450 may provide valuable information about the evolution of eukaryotes from prokaryotes.

Zh Mikrobiol Epidemiol Immunobiol, 1989 Oct, (10), 10 - 7
{The multidimensional character of the niches in Gause's law applicable to prokaryotes and the methods for the spatial separation of competing associates}; Kalina GP et al.; The multidimensional niche in Gause's law includes, with respect to prokaryotes, the following factors: the alimentary factor, the symbiosis-antagonism complex, the respiratory function, the concentration of associants, mobility . Cultivation in U-shaped tubes is the optimum method for the detection of spatial separation on account of such factors as respiration and mobility.

J Exp Med, 1989 Oct 1, 170(4), 1271 - 83
Chlamydial disease pathogenesis . The 57-kD chlamydial hypersensitivity antigen is a stress response protein; Morrison RP et al.; Chlamydia trachomatis infection of humans is commonly a localized inflammation that can result in infertility, blindness, and perhaps arthritis . The pathogenic process(es) that cause these sequelae are thought to be immunological . A 57-kD protein that is common among Chlamydia elicits ocular inflammation when introduced onto the conjunctivae of guinea pigs or nonhuman primates previously sensitized by chlamydial infection . This protein is thought to mediate the immunopathology that follows chlamydial infection . To more thoroughly characterize this chlamydial component, we cloned its gene from a C . psittaci strain and identified a particular recombinant that produced the 57-kD polypeptide . The recombinant gene product was immunoreactive with a monospecific anti-57-kD serum, and elicited an ocular inflammation similar to that produced by the 57-kD antigen isolated from chlamydiae . Sequencing identified two ORFs that encode polypeptides of 11.2 and 58.1 kD and are co-transcribed . These two polypeptides show homology with Escherichia coli groE and Coxiella burnetii htp heat-shock proteins . Striking homology (greater than 50%) was found between the 57-kD protein and the HtpB, GroEL, 65-k Mycobacterium tuberculosis and Hsp60 proteins . Thus, the 57-kD chlamydial protein, previously implicated as mediating a deleterious immunologic response to chlamydial infections, is a stress-induced protein similar to those that occur universally in both prokaryotic and eukaryotic organisms.

EMBO J, 1989 Oct, 8(10), 3141 - 8
DNA-directed oligomerization of the monomeric Ner repressor from the Mu-like bacteriophage D108; Kukolj G et al.; We have purified the 8.6 kd ner gene product (a lambda Cro-like protein which negatively regulates transcription from two divergent and overlapping promoters) from the Mu-like transposable bacteriophage D108 . Chemical and enzymatic protection experiments show the D108 ner-operator to contain two perfect 11 bp (5'-CCG-TGAGCTAC-3') inverted repeats separated by an 8 bp AT-rich region . Ner makes base-specific contacts in the major groove spanning the 11 bp repeats and also interacts with regions flanking these sites such that its operator comprises five turns of the DNA helix . Furthermore, gel filtration chromatography and dimethyl suberimidate crosslinking experiments indicate that D108 Ner (at concentrations exceeding 5 microM) is a monomer in solution, yet crosslinks as a dimer when bound to its operator site . As a small (73 amino acids) monomeric protein, Ner does not display strong homology with any known DNA-binding proteins . By virtue of the interactions with its operator it appears to bind DNA in a markedly different manner from other known prokaryotic repressors thus adding to the growing catalog of protein motifs used for specific binding to DNA.

Eur J Biochem, 1989 Oct 1, 184(3), 575 - 81
Molecular and cellular studies of tryptophanyl-tRNA synthetase using monoclonal antibodies . Evaluation of a common antigenic determinant in eukaryotic, prokaryotic and archaebacterial enzymes which maps outside the catalytic domain; Beresten SF et al.; Monoclonal antibodies referred to as Am1, Am2 and Am3 against highly purified bovine tryptophanyl-tRNA synthetase were prepared . Am2 antibodies inhibit the Trp-tRNA synthetase activity and interact with the active truncated enzyme forms (dimers of either 40-kDa or 51-kDa fragments) produced by limited proteolysis . Am1 and Am3 antibodies exert no effect on the Trp-tRNA synthetase activity; epitopes recognized by them are mapped close to one another and reside at the dispensable part of the Trp-tRNA synthetase molecule . Am1 cross-reacts with Trp-tRNA synthetases of eukaryotic, prokaryotic and archaebacterial species, as revealed by immunoblot analysis . A rapid two-step technique was developed for isolating electrophoretically homogeneous Trp-tRNA synthetase from Escherichia coli . The purified enzyme interacted with Am1, but not with Am2 and Am3 antibodies taken at the same concentrations . As in the case of eukaryotic Trp-tRNA synthetase, Am1 did not influence the activity of Trp-tRNA synthetase from E . coli . From the aforementioned results it follows that: (a) the conservation of part of the Trp-tRNA synthetase structure which is not directly involved in the formation of the catalytic centre of prokaryotic and eukaryotic Trp-tRNA synthetases suggests that the dispensable part of the molecule might be involved in some additional biological function(s) of Trp-tRNA synthetase besides tRNA(Trp) charging; (b) the common antigenic determinant in Trp-tRNA synthetase of eukaryotes, prokaryotes and archaebacteria indicates that this enzyme was presumably present in the common ancestor of the above organisms.

Nucleic Acids Res, 1989 Sep 25, 17(18), 7303 - 14
Isolation and characterization of the Drosophila translational elongation factor 2 gene; Grinblat Y et al.; We have isolated a cDNA clone that encodes the Drosophila melanogaster elongation factor 2 (EF2), a protein involved in the elongation step of protein synthesis . This identification was based on the high degree of its amino acid sequence identity (greater than 80%) to that of hamster EF2 . The gene encoding Drosophila EF2 is found at position 39E-F of the 2L chromosomal arm and maybe identical to the M(2)H locus, which produces a Minute phenotype when mutated . The genomic organization of the locus includes four exons . Conserved sequence segments shared with a variety of GTP binding proteins are found in the amino terminal third of the protein, and segments unique to EF2 and its prokaryotic functional homolog, EF-G, are in the carboxy terminal half; these two regions are segregated in two respective exons.

J Mol Biol, 1989 Sep 20, 209(2), 195 - 204
DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells; Romac S et al.; The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells . The gene was the Escherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome . The cells were irradiated with ultraviolet light and gpt- colonies were selected by resistance to 6-thioguanine . The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR), and the amplified DNA sequenced directly by the dideoxy method . Of the 58 sequenced mutants of independent origin 53 were base change mutations . Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair . Only one mutant had a multiple base change mutation with two or more well separated base changes . In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells . Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs . The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes.

FEBS Lett, 1989 Sep 11, 255(1), 37 - 41
Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila; Zhou JH et al.; The nucleotide sequence of the Pseudomonas saccharophila gene encoding maltotetraohydrolase (G4-forming amylase) has been determined . The coding region for the G4-forming amylase precursor contained 1653 nucleotides . The deduced precursor protein included an N-terminal 21-residue putative signal peptide; the deduced mature form of G4-forming amylase contains 530 amino acid residues with a calculated molecular mass of 57 740 Da . Sequence similarities between the G4-forming amylase and other amylolytic enzymes of species ranging from prokaryotes to eukaryotes are quite limited . However, three regions, which are involved in both the catalytic and substrate-binding sites of various amylolytic enzymes, are highly conserved in the G4-forming amylase of P . saccharophila.

J Biol Chem, 1989 Sep 5, 264(25), 14621 - 3
DNA ring closure mediated by protein HU; Hodges-Garcia Y et al.; The histone-like protein HU serves as an accessory factor that can facilitate the interaction of certain proteins with their specific DNA binding sites . Examples occur in different systems for prokaryotic DNA replication, transcription, and gene regulation . The protein-DNA interactions that are stimulated by HU generally involve coiling or looping of the DNA, and the possibility has been considered that HU exerts its effect by contributing flexibility to different DNA binding sites, but there has been no direct demonstration of this . To explore the possibility that HU can mediate tight DNA curvatures, we studied its effect on the formation of DNA circles when DNA ligase cyclizes short linear DNA fragments . It is demonstrated that HU greatly increases the cyclization rates of all fragments that were examined having lengths greater than 98 base pairs . Fragments of 99, 108, 120, or 126 base pairs could not cyclize in the absence of HU, but cyclization went rapidly with HU, showing that HU can mediate very tight DNA curvatures.

Mutat Res, 1989 Sep, 224(1), 105 - 13
In vivo cytogenetic effects of cooked food mutagens; Tucker JD et al.; Using a variety of in vivo cytogenetic endpoints, we have investigated the effects of several compounds formed during the cooking of meat . C57Bl/6 mice were used to test for an increase in the frequency of sister-chromatid exchanges (SCEs), chromosomal aberrations, and micronucleated erythrocytes by 2-amino-3,8-dimethylimidazo{4,5-f}quinoxaline (MeIQx) . 2-amino-3,4,8-trimethylimidazo{4,5-f}quinoxaline (DiMeIQx), and 2-amino-1-methyl-6-phenylimidazo{4,5-b}pyridine (PhIP) . MeIQx and DiMeIQx did not induce SCEs in mouse bone marrow cells . PhIP induced sister-chromatid exchanges, but not chromosomal aberrations in bone marrow . In peripheral blood lymphocytes, PhIP did induce aberrations at 100 mg/kg, the highest dose tested . PhIP induced a low but significantly increased frequency of micronuclei in normochromatic but not polychromatic erythrocytes in bone marrow and peripheral blood . However, dose responses were not observed . With the exception of the SCEs induced by PhIP, these results contrast with observations made in vitro, where these compounds were found to have significant genotoxicity in mammalian cells and a very high mutation frequency in prokaryotic systems.

Microbiologia, 1989 Sep, 5(2), 69 - 77
Transcription in vaccinia virus; Kent R; Transcription of poxvirus genes is temporally regulated and takes place in the cytoplasm of the infected cell . Virus-encoded enzymes produce capped, methylated and polyadenylated mRNA . The virus transcriptional apparatus recognises promoter sequences within the virus genome which bear little resemblance to either eukaryotic or prokaryotic promoter regions . mRNA's produced late in infection possess 5'poly (A) sequences which are not encoded in the genome . The process of transcription in poxviruses is reviewed using vaccinia virus as the prototype.

Yale J Biol Med, 1989 Sep-Oct, 62(5), 481 - 91
Polyamines and their derivatives as modulators in growth and differentiation; Canellakis ZN et al.; The polyamines and their derivatives are essential for life in eukaryotic and most prokaryotic cells, but their exact role in preserving cell function is not clear . These polyamines provide endogenous cations and thus participate in regulation of the intracellular pH; in addition, polyamine derivatives modulate cell growth and differentiation . The naturally occurring monoacetyl derivatives can induce increased activity of ornithine decarboxylase, the first enzyme in polyamine synthesis, and thus produce positive feedback to their production . The diacetyl derivatives of putrescine and of the synthetic analogue, 1,6-diaminohexane, induce differentiation and inhibit growth in many types of cells in vitro . In addition, they inhibit the proliferative and secretory response of normal B lymphocytes to B-cell mitogens and reduce production of antibodies in vitro . They also inhibit the proliferation of chronic lymphocytic leukemia cells (a B-lymphocyte leukemia) . The parent polyamines are post-translational modifiers of proteins, and hypusine, a derivative of spermidine, is a covalently bound constituent of the eukaryotic protein synthetic initiation factor, eIF-4D . Although these various actions do not at present fall into a coherent pattern, they clearly indicate that polyamines and their derivatives play an important part in modulating cell proliferation and differentiation.

Rev Infect Dis, 1989 Sep-Oct, 11 Suppl 6, S1470 - 4
The molecular biology of Borrelia; Barbour AG; Borrelia burgdorferi, the cause of Lyme disease, has two major outer-membrane proteins, OspA and OspB, which act as surface antigens . A 49-kilobase linear plasmid contains the genes that encode for these surface proteins . Direct examination of denatured plasmid molecules has revealed single-stranded circles with a circumference of approximately 100 kilobases (about twice the length of the linear duplex molecule), a finding that indicates the plasmid strands have covalently closed ends . This form of DNA, while present in eukaryotic organisms and their viruses, has not been observed in a prokaryotic organism . Plasmid heterogeneity has been observed in strains with surface proteins of similar molecular weights and similar monoclonal antibody reactivity . Thus, plasmid analysis may prove a sensitive tool for differentiating strains of B . burgdorferi . Furthermore, since loss of plasmids in vitro has been correlated with loss of the ability of many-passaged strains to cause infection, borrelial plasmids may encode for virulence factors as well.

Genes Dev, 1989 Sep, 3(9), 1472 - 9
Effect of the cap structure on pre-mRNA splicing in Xenopus oocyte nuclei; Inoue K et al.; The effect of the 5' cap structure on the splicing of precursor mRNAs was investigated after the RNAs were injected into Xenopus oocyte nuclei . The precursor mRNAs synthesized in vitro in a prokaryotic transcription system with a dinucleotide, ApppG, as a primer, were extremely stable when injected into the nuclei yet behaved like uncapped pre-mRNAs in the in vitro splicing reaction . The ApppG-primed precursor mRNAs served as a control (uncapped) in the injection experiments, and their splicing reactions were compared with those of their capped (m7GpppG-primed) counterparts . The capped precursors were spliced more efficiently than the uncapped precursors . Examination of splicing of the precursor mRNA that contained three exons and two introns with a single molecule has revealed that the cap structure exerts its effect primarily on the 5'-proximal intron . Thus, the cap structure not only stabilizes precursor mRNAs but also plays a positive role in the splicing of precursor mRNAs in cells.

Genetics, 1989 Sep, 123(1), 123 - 9
Sequence analysis of N-ethyl-N-nitrosourea-induced vermilion mutations in Drosophila melanogaster; Pastink A et al.; The mutational specificity of N-ethyl-N-nitrosourea (ENU) was determined in Drosophila melanogaster using the vermilion locus as a target gene . 25 mutants (16 F1 and 9 F2 mutants) were cloned and sequenced . Only base-pair changes were observed; three of the mutants represented double base substitutions . Transition mutations were the most prominent sequence change: 61% were GC----AT and 18% AT----GC substitutions . Both sequence changes can be explained by the miscoding properties of the modified guanine and thymine bases . A strong bias of neighboring bases on the occurrence of the GC----AT transitions or a strand preference of both types of transition mutations was not observed . The spectrum of ENU mutations in D . melanogaster includes a significant fraction (21%) of transversion mutations . Our data indicate that like in other prokaryotic and eukaryotic systems also in D . melanogaster the O6-ethylguanine adduct is the most prominent premutational lesion after ENU treatment . The strong contribution of the O6-ethylguanine adduct to the mutagenicity of ENU possibly explains the absence of distinct difference between the type of mutations observed in the F1 and F2 mutants . Although the latter arise later during development, the spectrum of mosaic mutations is also dominated by GC----AT transition mutations.

Int J Fertil, 1989 Sep-Oct, 34(5), 363 - 7
Ultrastructural analysis of the attachment sites of Escherichia coli to the human spermatozoon after in vitro migration through estrogenic cervical mucus; Sanchez R et al.; The in vitro attachment between Escherichia coli and the human spermatozoon was studied using transmission and scanning electron microscopy, and ultracytochemistry . Samples from estrogenized cervical mucus columns containing migrating spermatozoa revealed two types of contact areas: Type I corresponded to the interaction of the bacterial fimbriae with the spermatozoal surface, and Type II, to the intermingling of the eukaryotic and prokaryotic cell glycocalyx . In both types of associations, the attachment area was lanthanum positive and displayed glycoconjugates . These types of intercellular contacts could represent the morphological basis of a mechanism by which bacteria are attached to spermatozoa and are consequently transported to the upper female genital tract.

Plasmid, 1989 Sep, 22(2), 143 - 50
Chromatin structure of transposon Tn903 cloned into a yeast plasmid; Estruch F et al.; Transposon Tn903 contains the APH gene for kanamycin resistance, which is active in yeast {A . Jimenez and J . Davies (1980) Nature (London) 287, 869-871} and is flanked by two inverted repeats (IR) 1057 bp long . When plasmid pAJ50, carrying Tn903 and the 2-microns circle origin of replication, is cloned into Saccharomyces cerevisiae, nucleosomes are assembled in vivo on the prokaryotic DNA of the transposon . Indirect end labeling revealed that three nucleosomes are preferentially positioned on symmetrical sequences from both IRs . DNase I digestion also confirmed that the chromatin structure is symmetrical in both IRs . This suggests that sequence determinants are decisive for chromatin structure in these regions . We have calculated the rotational and translational fits {H . R . Drew and C . R . Calladine (1987) J . Mol . Biol . 195, 143-173} for the Tn903 sequence and the results indicate that the nucleosome positioning on the IRs is sequence-directed . Nucleosome deposition on the APH gene also occurs, but no clear positioning exists . Some sequence preference for positioning nucleosomes on the promoter can be predicted, especially from the translational fit . Experimental data indicate, however, that nucleosomes are absent from the promoter . Therefore, chromatin can be organized on prokaryotic DNA in a manner that resembles the typical eukaryotic chromatin structure.

Proc Natl Acad Sci U S A, 1989 Sep, 86(17), 6671 - 5
Sequences required for expression of Bordetella pertussis virulence factors share homology with prokaryotic signal transduction proteins; Arico B et al.; The bvg locus of Bordetella pertussis is required for coordinate regulation of several factors associated with virulence . The control system is modulated by various environmental signals, including low temperature, MgSO4, and nicotinic acid . The nucleotide sequence of the bvg region has been determined and three open reading frames, bvgA, bvgB, and bvgC, are present . Twelve-base-pair linker insertion mutations in any of these open reading frames result in a Bvg- phenotype . The predicted protein products of bvgA and bvgC share homology with a family of prokaryotic regulatory proteins that respond to environmental stimuli and are members of two-component sensory transduction systems . We propose a model in which BvgB and the N-terminal portion of BvgC are localized in the periplasm . Environmental signals are recognized, transduced to the cytoplasmic portion of BvgC, and then transmitted to BvgA, a positive regulator of transcription.

Biochimie, 1989 Sep-Oct, 71(9-10), 1021 - 8
Protein phosphorylation and control of excitation energy transfer in photosynthetic purple bacteria and cyanobacteria; Allen JF et al.; The function of phosphorylation of light-harvesting polypeptides is well characterised in chloroplasts of green plants, but the prokaryotic cyanobacteria and purple photosynthetic bacteria have quite different light-harvesting polypeptides whose structure and function cannot be controlled in precisely the same way . Nevertheless, cyanobacteria show light-dependent phosphorylation of membrane polypeptides associated with photosystem II and with the light-harvesting phycobilisome, and purple bacteria show light-dependent phosphorylation of low molecular-weight chromatophore membrane polypeptides . In both cases membrane protein phosphorylation is associated with functional changes observed by chlorophyll fluorescence spectroscopy or chlorophyll fluorescence induction kinetics . Here we report on our recent protein sequence and other data concerning the identities of these phosphoproteins . We also discuss the significance of these findings for regulation by protein phosphorylation of photosynthesis in prokaryotes.

Mol Microbiol, 1989 Sep, 3(9), 1295 - 9
The role of the cell surface in social and adventurous behaviour of myxobacteria; Shimkets LJ; The myxobacteria are an unusually social group of prokaryotic organisms that form fruiting bodies containing dormant myxospores in response to nutritional stress . Social behaviour is controlled by a multigene system known as 'S' and by a series of intercellular signals that are released during development . The genes controlling these communication systems have been identified by mutational analysis and current research is directed toward examining the functions of these genes . S- mutants are generally nondevelopmental and noncohesive . They lack pili, a Congo red receptor, and 50-nm-wide fibrils which extend outward from the cell surface . Changes in the architecture of the cell surface have been studied by means of surface labelling and with monoclonal antibodies directed against cell-surface antigens . The cell surface undergoes dramatic changes during the course of development . Most vegetative antigens decrease in concentration or disappear completely while new development-specific antigens appear.

Clin Exp Rheumatol, 1989 Sep-Oct, 7 Suppl 3, S63 - 8
Epitope mapping of autoantigens using recombinant proteins and synthetic peptides; Elkon KB; In order to evaluate mechanisms of autoantibody production, three hypotheses (random polyclonal B cell activation, molecular mimicry and immune response against restricted self antigens) were considered in relation to autoantibody recognition . Polyclonal B cell activation was considered unlikely because lupus sera recognized a relatively small number of eukaryotic protein antigens on immunoblots and failed to show enhanced reactivity against prokaryotic epitopes . Although two interesting examples of molecular mimicry between autoantigens and two different viruses have been suggested, it is yet to be shown that lupus antibodies bind to the shared epitopes on the viruses and that the viruses induce such antibodies . Finally, epitope mapping of several different autoantigens suggest that the antigens are themselves the focus of the immune response and initiate and/or maintain autoantibody production.

Philos Trans R Soc Lond B Biol Sci, 1989 Aug 31, 324(1224), 477 - 85
Host-vector systems; Kingsman SM et al.; In 1980 it was only possible to express foreign genes in bacteria and a few easily cultured animal cells . During the subsequent eight years specialized vectors have been developed to allow the genetic manipulation of a wide range of both prokaryotes and eukaryotes . One of the major goals of biotechnology in 1980 was to use host cells as 'factories' for the production of proteins that were only available in minute quantities from natural sources . This has already lead to a new generation of pharmaceutical products . Advances in our understanding of host-vector systems have defined new goals . The basic concepts of expression vector design will be illustrated . Some of the new goals are discussed with particular reference to the exploitation of novel host-vector systems to develop vaccines and anti-viral agents against AIDS.

Philos Trans R Soc Lond B Biol Sci, 1989 Aug 31, 324(1224), 447 - 60
Protein engineering and design; Blundell TL et al.; Rapid advances in site-directed mutagenesis and total gene synthesis combined with new expression systems in prokaryotic and eukaryotic cells have provided the molecular biologist with tools for modification of existing proteins to improve catalytic activity, stability and selectivity, for construction of chimeric molecules and for synthesis of completely novel molecules that may be endowed with some useful activity . Such protein engineering can be seen as a cycle in which the structures of engineered molecules are studied by X-ray analysis and two-dimensional nuclear magnetic resonance . The results are used in the improvement of the design by using knowledge-based procedures that exploit facts, rules and observations about proteins of known three-dimensional structure.

Nucleic Acids Res, 1989 Aug 25, 17(16), 6569 - 80
Wheat phosphoglycerate kinase: evidence for recombination between the genes for the chloroplastic and cytosolic enzymes; Longstaff M et al.; We have isolated and sequenced cDNA clones containing the entire coding region of both the chloroplast and cytosolic versions of phosphoglycerate kinase from wheat . Comparison of these sequences reveals a higher than expected level of similarity between the nucleic acids and encoded proteins . Analysis of this data in relation to that for phosphoglycerate kinase sequences of mammals, prokaryotes and yeasts suggests that the wheat genes have recombined . This has resulted in the chloroplast and cytosolic kinases being more similar to each other than would be expected if the chloroplast enzyme had evolved directly from that of a prokaryotic progenitor.

Cell, 1989 Aug 25, 58(4), 695 - 705
SecB functions as a cytosolic signal recognition factor for protein export in E . coli; Watanabe M et al.; A purified 64 kd protein, consisting of four identical subunits of the 16 kd SecB, binds to the signal sequence of preproteins prior to their translocation across inverted vesicles (INV) derived from the E . coli plasma membrane . The purified SecB tetramer competes with canine signal recognition particle (SRP) in signal sequence binding and thus behaves as a prokaryotic equivalent of SRP . As shown by cell fractionation and immunoblot analysis with anti-SecB antibodies, SecB is a cytosolic protein . An E . coli supernatant depleted of SecB after passage through an anti-SecB Sepharose column retains full translation activity but is unable to support translocation into added INV . Translocation into INV is fully restored by readdition of purified SecB.

Nature, 1989 Aug 24, 340(6235), 656 - 9
Similarities between prokaryotic and eukaryotic cyclic AMP-responsive promoter elements; Lin YS et al.; Organisms as diverse as bacteria and man contain genes that show transcriptional induction when the intracellular concentration of cAMP is increased . This regulated transcriptional response is mediated through specific promoter elements located, in general, upstream from the transcription start site . In Escherichia coli the element responsible for cAMP-mediated transcriptional induction is the binding site for the cAMP-receptor protein (CAP) . In mammalian cells the cAMP regulatory element is composed of one or more binding sites for various transcription factors . In many instances the cAMP regulatory element contains binding sites for a family of proteins referred to as ATF . Here we provide evidence that some prokaryotic and mammalian cAMP-response elements are functionally related . First, we show that mammalian ATF binds specifically to some E . coli CAP sites, and conversely E . coli CAP binds specifically to some mammalian ATF sites . Second, we demonstrate that an E . coli CAP binding site can confer cAMP-inducibility onto a mammalian gene when assayed in transfected mammalian cells.

Science, 1989 Aug 18, 245(4919), 725 - 30
Inhibition of DNA binding proteins by oligonucleotide-directed triple helix formation; Maher LJ 3rd et al.; Oligonucleotides that bind to duplex DNA in a sequence-specific manner by triple helix formation offer an approach to the experimental manipulation of sequence-specific protein binding . Micromolar concentrations of pyrimidine oligodeoxyribonucleotides are shown to block recognition of double helical DNA by prokaryotic modifying enzymes and a eukaryotic transcription factor at a homopurine target site . Inhibition is sequence-specific . Oligonucleotides containing 5-methylcytosine provide substantially more efficient inhibition than oligonucleotides containing cytosine . The results have implications for gene-specific repression by oligonucleotides or their analogs.

Biochim Biophys Acta, 1989 Aug 14, 1008(3), 329 - 38
Distinct patterns in homooligomer tract sequence context in prokaryotic and eukaryotic DNA; Nussinov R et al.; The distributions of the junction sequences of homooligomer tracts of various lengths have been examined in prokaryotic DNA sequences and compared with those of eukaryotes . The general trends in the nearest and next to nearest neighbors to the tracts are similar for both groups . In both prokaryotes and eukaryotes A/T runs are preferentially flanked on either the 5' or the 3' ends by A and/or T . G/C runs are preferentially flanked by G and/or C . There is discrimination against A/T runs flanked by G or C and G/C runs flanked by A or T . However, whereas the distribution of prokaryotic homooligomer tract junction sequences was quite homogeneous, large variations were observed in the 5-fold larger eukaryotic database, increasing in magnitude from tracts of length 2 to 3 to 4 base pairs long . Possible DNA conformational implications and in particular DNA curvature and packaging aspects of prokaryotes and eukaryotes are discussed.

J Biol Chem, 1989 Aug 5, 264(22), 12895 - 901
Structure of the canine pancreatic lipase gene; Mickel FS et al.; Identification of three overlapping clones in a canine genomic lambda phage library allowed us to determine a detailed restriction enzyme map of the primary transcriptional unit of the pancreatic lipase gene (15.5 kilobase pairs) as well as 15 and 6 kilobase pairs of 5'- and 3'-flanking regions, respectively . DNA sequence analysis provided the primary structure of (a) 1,345 nucleotides (nt) of 5'-flanking sequence including CAAT and TATA boxes at positions -112 and -35, respectively, and a class 2 glucocorticoid receptor binding sequence at position -97, (b) 13,127 out of approximately 15,500 nt of the transcriptional unit which is organized into 13 exon sequences, and (c) 1,270 nt of 3'-flanking sequence . Exon 1 encodes the entire 5'-nontranslated mRNA sequence; exon 2, the ATG initiation codon and the hydrophobic portion of the signal peptide; and exon 6, Ser154 which shows homology to the active Ser152 in the porcine enzyme . Comparison of the amino acid sequences of human lipoprotein lipase, rat hepatic lipase, and Drosophila yolk proteins 1, 2, and 3 with canine pancreatic lipase shows that the central region of highest homology (encoded by exons 6-8 in the dog gene) contains four highly conserved subregions which may play a critical role in enzyme-substrate and protein-ligand binding for lipases and yolk proteins, respectively . Comparison of the sequences of 10 lipases from prokaryotes and eukaryotes identifies a 9-residue consensus sequence surrounding the active serine which includes the previously identified sequence Gly-X-Ser-X-Gly . The hydrophobic nature of this sequence in the 10 lipases contrasts with the hydrophilic nature of the corresponding sequences in serine proteases and thus defines an active site serine consensus sequence specific for lipases . An analysis of 5'- and 3'-flanking and intron 1-4 sequences in transient expression studies with AR4-2J and 266-6 cells was unable to reveal tissue-specific promoter or enhancer sequences.

J Auton Nerv Syst, 1989 Aug, 27(3), 207 - 19
Effects of isolation and high helium pressure on the nucleolus of sympathetic neurons in the rat superior cervical ganglion; Robaglia A et al.; In prokaryotes, unicellular eukaryotes and cell-free systems, pressure is known to exert an inhibitory effect on protein synthesis and RNA metabolism, the mechanism(s) of which remain to be investigated in detail . The purpose of the present in vitro study was to compare ultrastructural and quantitative changes of the nucleolus, which is the site of ribosome biogenesis, in sympathetic neurons of rat superior cervical ganglia (SCG) maintained for 2, 3 and 5 h in NCTC 109 medium and subjected to pressure or not . In control SCG (left) the nucleolus greatly increased in volume (+ 33%) 2 h after excision, in comparison with SCG fixed immediately . This overall enlargement was found to reflect a marked increase in all nucleolar components (from 16 to 87%) . After 5 h, volumes of nucleolus, fibrillar centers and vacuolar component returned to control values, whereas dense fibrillar and granular components remained affected . Such early and transient changes are regarded as reflecting basic metabolic changes associated with increased nucleolar RNA that should be of primary concern to experiments using SCG transplanted in culture media . Compression under helium up to 180 atmospheric pressure for 1 h of right SCG maintained for 2 h in culture medium, was shown to induce, on the contrary, a marked decrease in nucleolar volume (-39%) and in volumes of all nucleolar components (from -36 to -51%) . When they were kept at constant high pressure for 1 and 3 h a progressive recovery of volumes of nucleoli and nucleolar components was observed . Consequently, compression was shown to exert opposite effects to those of isolation of SCG . Present data are interpreted as an inhibitory effect of pressure on ribosome biogenesis . Such observations on a vertebrate neuron might open a new field in the search for cellular mechanisms underlying the effects of pressure on living organisms and especially on the nervous system.

J Med Chem, 1989 Aug, 32(8), 2002 - 15
Lipophilic analogues of sparsomycin as strong inhibitors of protein synthesis and tumor growth: a structure-activity relationship study; van den Broek LA et al.; Fourteen derivatives of sparsomycin (1) were synthesized . Six of them were prepared following a novel synthetic route starting from the L-amino acid alanine . Some physicochemical properties, viz . lipophilicity and water solubility, of selected derivatives were measured . The biological activity was tested in vitro in cell-free protein synthesis inhibition assays, in bacterial and tumor cell growth inhibition assays, and in the L1210 leukemia in vivo model in mice . Also for selected drugs the acute toxicity in mice was determined . Ribosomes from both an eukaryotic and a prokaryotic organism were used in the protein synthesis inhibition systems . A linear correlation between the lipophilicity parameters measured was observed . Water solubility and drug toxicity in mice were found to be linearly correlated with lipophilicity . All the derivatives studied are more lipophilic than 1 . The deshydroxysparsomycin analogues (30-33) showed an interesting phenomenon: increase in hydrophobicity was accompanied by a considerable increase in water solubility . We found that an increase in hydrophobicity of the drug as a result of replacing the SMe group of 1 with larger alkylthio groups causes an increase in the biological activity of the drug . However, not only the hydrophobicity but also shape and size of the substituent are important; in the homologous series 1-9-10-11-12, 21-22-23-24, and 30-31-32-33, highest protein synthesis inhibitory and in vitro cytostatic activity is found with compounds 11, 23, and 32, respectively, and in comparison with the highly active n-butyl compound 10, the isomeric tert-butyl compound 13 is rather inactive . Polar substituents replacing the SMe group, i.e . Cl in 17 and 35, also render the molecule inactive . Substituting the bivalent sulfur atom for a methylene group decreases the drug's activity . This effect can be compensated for by increasing the length of the alkylsulfinyl side chain . The agreement between the results derived from cell-free and "in vivo" tests is good . The assays using ribosomes of bacterial and eukaryotic organisms give similar results although the latter seem to be more sensitive to changes in hydrophobicity of the drug . Our results confirm the presence of a hydrophobic region at the peptidyl transferase center of the ribosome; the interaction of sparsomycin with this region is more pronounced in the eukaryotic particles . The sparsomycin analogues 11, 23, and 30 show the highest antitumor activity against L1210 leukemia in mice, their median T/C values are 386, 330, and 216%, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Differentiation, 1989 Aug, 41(2), 116 - 26
Regulated expression of muscle-specific genes introduced into mouse embryonal stem cells: inverse correlation with DNA methylation; Shinar D et al.; Pluripotent embryonal stem cell lines (ES) were isolated from cultured normal mouse blastocysts . These cells retained their capacity to differentiate into a great variety of cell types in cell cultures or in tumors formed after subcutaneous injection of the cells into nude mice . A chimeric actin/globin gene containing about two-thirds of the rat skeletal muscle actin gene and 730 bp of its 5' flanking region fused to the 3' end of the human embryonic epsilon-globin gene, was inserted into a plasmid containing a neomycin resistance gene (neor) whose transcription is regulated by the SV40 early control elements . The prokaryotic vector DNA sequences of this plasmid (pAG-Neo) were deleted and the two linked genes were introduced into the ES cells by electroporation . G418-resistant clones were isolated, amplified and injected subcutaneously into nude mice . From the teratocarcinoma-like tumors which developed we isolated myogenic as well as nonmyogenic cell lines . In cell lines derived from three independent transfected ES clones, expression of the actin/globin gene was developmentally regulated in myogenic cells . In contrast, in a number of experiments in which the actin/globin gene or other muscle-specific genes were introduced into the ES cells without the removal of the pBR sequences, no expression could be detected at any stage . Moreover, in the differentiated lines derived from these clones, G418 resistance was lost, and no neor transcripts could be detected . Southern-blot analysis of MSPI- or HpaII-digested DNA revealed extensive methylation in the clones that did not express the foreign DNA, whereas no significant methylation of the inserted DNA was observed in clones which expressed the transfected genes . Examination of the DNA extracted from transgenic mice carrying the same actin/globin gene revealed an inverse correlation between methylation of the exogenous gene and its potential to be expressed in the transgenic strain . However, no tissue-specific differences in methylation, related to the tissue specificity of expression of the exogenous gene, could be detected in these experiments . These results suggest that the process of methylation reported here is causally related to constitutive inactivation of the exogenous genes.

Am J Respir Cell Mol Biol, 1989 Aug, 1(2), 95 - 100
Expression of a prokaryotic gene in cultured lung endothelial cells after lipofection with a plasmid vector; Brigham KL et al.; Transfection of cultured cells with functioning foreign genes can be a powerful tool for delineating mechanisms controlling gene expression and for manipulating cellular synthetic machinery . We transfected cultured bovine pulmonary artery endothelial cells with a plasmid (pRSVCAT) containing the prokaryotic gene, chloramphenicol acetyltransferase (CAT), driven by a Rous sarcoma virus (RSV) promoter . Transfection was accomplished by binding the plasmid DNA to specially synthesized cationic liposomes and incubating cell monolayers with the liposome-DNA complex (a process called lipofection) . We found marked CAT expression in endothelial cells by 24 h after lipofection (complete chloramphenicol acetylation in a 4-h assay incubation with less than 0.1 mg cell protein) . Marked CAT expression persisted after splitting the cells 1:2 at 6 days after lipofection . Detectable CAT activity was present 14 days after lipofection following two 1:2 splits of the cells . CAT expression was related to the quantity of plasmid DNA used for lipofection; 1.0 micrograms DNA per 60-mm dish of cells resulted in less CAT activity at 48 h than did 5 or 10 micrograms DNA per dish . Ten micrograms of DNA-liposome complex per dish caused substantial numbers of endothelial cells to detach from the dish, but little cytotoxic effect was seen with 5 or 1 micrograms DNA-liposome complex per dish . This simple, highly efficient method for introducing functioning foreign genes into cultured endothelial cells will permit a broad range of studies of molecular mechanisms of endothelial cell function and studies of the consequences of highly specific modifications in protein synthesis on endothelial responses.

Gene, 1989 Aug 1, 80(1), 145 - 9
Cloning and expression of mouse-brain calmodulin as an activator of Bordetella pertussis adenylate cyclase in Escherichia coli; Danchin A et al.; Cloning of higher eukaryotic genes has seldom been performed by complementation of a defective prokaryotic function . This is especially true in the case of functions that are normally absent from the prokaryotic host . We demonstrate here that it is possible to identify by complementation the cDNA from mouse brain, which encodes calmodulin (CaM) synthesis, in spite of the fact that the recipient strain, Escherichia coli, does not normally harbour a CaM function . A three-component cloning procedure in which a gene product requiring CaM for activity, adenylate cyclase from the pathogen Bordetella pertussis, was used to screen a cDNA library for cAMP synthesis in E . coli . The nucleotide sequence of the corresponding cDNA is also reported.

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