Genetics, 1994 Oct, 138(2), 317 - 27 Sequenced alleles of the Caenorhabditis elegans sex-determining gene her-1 include a novel class of conditional promoter mutations; Perry MD et al.; In the control of Caenorhabditis elegans sex determination, the her-1 gene must normally be activated to allow male development of XO animals and deactivated to allow hermaphrodite development of XX animals . The gene is regulated at the transcriptional level and has two nested male-specific transcripts . The larger of these encodes a small, novel, cysteine-rich protein responsible for masculinizing activity . Of the 32 extant mutant alleles, 30 cause partial or complete loss of masculinizing function (lf), while 2 are gain-of-function (gf) alleles resulting in abnormal masculinization of XX animals . We have identified the DNA sequence changes in each of these 32 alleles . Most affect the protein coding functions of the gene, but six are in the promoter region, including the two gf mutations . These two mutations may define a binding site for negative regulators of her-1 . Three of the four remaining promoter mutations are single base changes that cause, surprisingly, temperature-sensitive loss of her-1 function . Such conditional promoter mutations have previously not been found among either prokaryotic or eukaryotic mutants analyzed at the molecular level. Oral Microbiol Immunol, 1994 Oct, 9(5), 318 - 20 Absence of Helicobacter pylori in subgingival samples determined by polymerase chain reaction; Asikainen S et al.; The polymerase chain reaction was used for the detection of Helicobacter pylori from subgingival plaque in 336 periodontitis patients . A pair of primers derived from the H . pylori urease gene A served to amplify a targeted 411-bp fragment of genomic DNA . This technique permitted the detection of as few as 60 H . pylori cells . Paper point samples from 3 deep periodontal pockets per patient were immersed in 1 ml of phosphate-buffered saline or distilled water, DNA was solubilized by detergent/protease method, 3.7 microliters or 37 microliters of lysate supernatant was used as template, and the amplification product was analyzed in 1% agarose gel containing ethidium bromide . Each experiment included purified DNA and cell lysate of H . pylori as positive controls . The presence of bacteria in the sample was verified by a primer pair common to prokaryote 16S rRNA . The present study did not reveal the specific polymerase chain reaction amplification product characteristic of H . pylori . We conclude that periodontal pockets do not constitute a natural reservoir for H . pylori. Curr Opin Biotechnol, 1994 Oct, 5(5), 540 - 5 The cellular response to unfolded proteins: intercompartmental signaling; McMillan DR et al.; Both prokaryotic and eukaryotic cells respond to the accumulation of unfolded proteins by increasing the transcription of genes encoding molecular chaperones and other stress-responsive proteins . Different sets of genes are activated when particular cellular compartments are burdened with unfolded proteins . Cells thus maintain mechanisms to monitor changes in the concentration of unfolded proteins not only in the cytosol, but also in membrane-bound extracytoplasmic compartments . During the past year, work in yeast has identified a transmembrane receptor that appears to play a pivotal role in the regulation of protein folding . This receptor monitors the concentration of available chaperone molecules in the endoplasmic reticulum and transmits a signal to the cytosol to activate the transcription of nuclear genes encoding chaperones that are localized in the endoplasmic reticulum . Work using Escherichia coli suggests that prokaryotes also contain an intercompartmental 'unfolded protein' signaling pathway, in this case from the periplasmic space or outer membrane to the cytoplasm. Curr Opin Biotechnol, 1994 Oct, 5(5), 516 - 20 Inducible gene expression systems for higher eukaryotic cells; Gossen M et al.; Of the wide variety of regulatory systems that have been developed to control gene expression in higher eukaryotes, those utilizing elements from prokaryotic systems presently achieve the highest specificity . In particular, systems based on the repressor/operator elements of an Escherichia coli tetracycline resistance operon appear broadly applicable . During the past year, the usefulness of these systems has been demonstrated not only at the cellular level, but also at the organismal level (i.e . in transgenic animals and plants). Immunobiology, 1994 Oct, 191(4-5), 451 - 60 The Bcg gene story; Skamene E; The genetic influences on the course of mycobacterial infections during epidemics and in endemic areas have always been suspected, but the precise nature of such genetic control and of the inherited mechanisms of susceptibility have been unknown . We have used methods of population genetics in the mouse to discover a single dominant autosomal gene (Bcg), which controls the susceptibility to various species of mycobacteria as well as to other intracellular parasites . The phenotypic expression of the Bcg gene has been defined as nonspecific macrophage activation for bactericidal function, resulting in the destruction of ingested intracellular parasites early following infection . Using recombinant inbred strains of mice, we have mapped this gene to the centromeric part of chromosome 1 and we have created a high resolution linkage map and, subsequently, a physical map in the close vicinity of this locus . A 400 kb bacteriophage and cosmid contig assembled within the genomic interval overlapping Bcg contained six novel transcription units . RNA expression studies showed that one of these genes (designated Nramp for "natural resistance associated macrophage protein"), was expressed exclusively in macrophages . Nramp encodes an integral membrane protein that has structural homology with known prokaryotic and eukaryotic transport systems, suggesting a macrophage-specific membrane transport function . Susceptibility to infection (Bcgs) in 27 Bcgs and Bcgr strains tested is associated with a Gly-105 to Asp-105 substitution within predicted transmembrane domain 2 of Nramp, making this gene a strong candidate for Bcg . The chromosomal segment in the vicinity of the Bcg gene has been conserved in the human genome (chromosome 2q) . Linkage analysis between the phenotype of disease during a tuberculosis outbreak in an extended multisib Canadian Indian family and allelic variants of chromosome 2 has revealed a significant LOD score . This finding, together with the emerging information on almost total sequence homology between the murine and human Nramp genes suggests that this gene may be responsible for the phenotype of resistance or susceptibility to tuberculosis. J Virol Methods, 1994 Oct, 49(3), 343 - 51 Truncating the putative membrane association region circumvents the difficulty of expressing hepatitis C virus protein E1 in Escherichia coli; Yan BS et al.; The putative E1 of hepatitis C virus (HCV) was expressed in Escherichia coli using a glutathione-S-transferase (GST) fusion protein system . The full length E1 protein is difficult to express . A series of E1 DNA fragments was generated and used for expression vector construction . Fusion proteins containing the E1 C-terminal region could not be expressed . When this region was truncated, the fusion proteins were synthesized to high levels . The possibility of this C-terminal region hampering the production of fusion protein was further explored . A construct with this segment directly fused to the C-terminus of GST indeed generated no detectable recombinant protein . According to the predicted structure of E1, this region may have membrane-associating properties . The expression results suggest a general approach to facilitate the production of viral membrane proteins in prokaryotes . Furthermore, these recombinant E1 proteins generated as antigens were used for Western blotting with sera from HCV-infected individuals . It was found that E1 is antigenic during HCV natural infection. Mutat Res, 1994 Oct, 318(2), 73 - 114 Genetic toxicity of 2-acetylaminofluorene, 2-aminofluorene and some of their metabolites and model metabolites; Heflich RH et al.; 2-Acetylaminofluorene and 2-aminofluorene are among the most intensively studied of all chemical mutagens and carcinogens . Fundamental research findings concerning the metabolism of 2-acetylaminofluorene to electrophilic derivatives, the interaction of these derivatives with DNA, and the carcinogenic and mutagenic responses that are associated with the resulting DNA damage have formed the foundation upon which much of genetic toxicity testing is based . The parent compounds and their proximate and ultimate mutagenic and carcinogenic derivatives have been evaluated in a variety of prokaryotic and eukaryotic assays for mutagenesis and DNA damage . The reactive derivatives are active in virtually all systems, while 2-acetylaminofluorene and 2-aminofluorene are active in most systems that provide adequate metabolic activation . Knowledge of the structures of the DNA adducts formed by 2-acetylaminofluorene and 2-aminofluorene, the effects of the adducts on DNA conformation and synthesis, adduct distribution in tissues, cells and DNA, and adduct repair have been used to develop hypotheses to understand the genotoxic and carcinogenic effects of these compounds . Molecular analysis of mutations produced in cell-free, bacterial, in vitro mammalian, and intact animal systems have recently been used to extend these hypotheses. Nucleic Acids Res, 1994 Sep 25, 22(19), 3936 - 42 Phylogenetically preserved inter-rRNA base pairs: involvement in ribosomal subunit association; Thanaraj TA; Intermolecular complementary base pairs, that can be formed between the bases from single-stranded and weak stem regions on 16S rRNA and those on 23S rRNA, were located and checked for preservation in a variety of species covering the complete phylogenetic spectrum . Putative base pairs that exhibited two 'compensatory base pair changes' (a requisite as dictated by the approach of 'comparative sequence data analysis') were picked up . Potential base pairs were selected by assessing the frequency and taxonomy specificity of the occurrence of compensatory base pair changes against those of other types of base pair changes . The selected base pairs were classified as universal, non-mitochondrial, and prokaryote-specific . The positions of the proposed base pairs occur near the structurally and functionally important regions on rRNAs. Science, 1994 Sep 23, 265(5180), 1863 - 6 Synergistic activation of transcription by bacteriophage lambda cI protein and E . coli cAMP receptor protein; Joung JK et al.; Two heterologous prokaryotic activators, the bacteriophage lambda cI protein (lambda cI) and the Escherichia coli cyclic AMP receptor protein (CRP), were shown to activate transcription synergistically from an artificial promoter bearing binding sites for both proteins . The synergy depends on a functional activation (positive control) surface on each activator . These results imply that both proteins interact directly with RNA polymerase and thus suggest a precise mechanism for transcriptional synergy: the interaction of two activators with two distinct surfaces of RNA polymerase. J Biol Chem, 1994 Sep 23, 269(38), 23484 - 90 Activation of the membrane glucolipid synthesis in Acholeplasma laidlawii by phosphatidylglycerol and other anionic lipids; Karlsson OP et al.; In membrane lipids of the prokaryote Acholeplasma laidlawii similar phase equilibria and a nearly constant spontaneous curvature are maintained by an extensive metabolic regulation of especially the major polar lipids monoglucosyldiacylglycerol (MGlcDAG) and diglucosyldiacylglycerol (DGlcDAG), forming nonlamellar and lamellar phases, respectively . A constant surface charge density is maintained by the anionic phospholipid fraction . These lipids are synthesized from phosphatidic acid in two competing pathways . The in vitro synthesis of MGlcDAG and DGlcDAG were totally lost upon delipidation of the membrane proteins by detergent solubilization or solvent extraction of lyophilized cells . Activities were restored by critical concentrations of anionic lipids, but not by bilayer or nonbilayer zwitterionic phospholipids or glucolipids . Phosphatidylglycerol (PG), and to a lesser extent certain other anionic lipids, could activate the synthesis of MGlcDAG in lipid bilayers, whereas the synthesis of DGlcDAG was similarly dependent upon PG only . Two endogenous phosphoglucolipids with no activating potency could partially replace the PG activator for the MGlcDAG synthesis but less so for DGlcDAG formation . A change of inert matrix from phosphatidylcholine to DGlcDAG lowered the apparent cooperativity, but enhanced the efficiency, of activation by PG for both glucolipid synthesizing enzymes, most strongly the synthesis of DGlcDAG . These results indicate that the enzymatic formation of MGlcDAG is regulated by the lipid surface charge density, whereas the consecutive step to DGlcDAG is more dependent upon the specific properties of PG . The modulating effect of the surrounding matrix on the activator efficiencies and cooperativity may constitute part of the bilayer-nonbilayer lipid regulation mechanism. Nature, 1994 Sep 22, 371(6495), 297 - 300 The ancient regulatory-protein family of WD-repeat proteins; Neer EJ et al.; WD proteins are made up of highly conserved repeating units usually ending with Trp-Asp (WD) . They are found in all eukaryotes but not in prokaryotes . They regulate cellular functions, such as cell division, cell-fate determination, gene transcription, transmembrane signalling, mRNA modification and vesicle fusion . Here we define the common features of the repeating units, and criteria for grouping such proteins into functional subfamilies. Gene, 1994 Sep 15, 147(1), 111 - 4 Expression in yeast of an Escherichia coli gene encoding a phospholipid biosynthetic enzyme; Kelly BL et al.; Cardiolipin (CL) is a structurally unique phospholipid having important functional roles in both prokaryotic and eukaryotic cells . The genes encoding CL biosynthetic enzymes have been identified and extensively studied in Escherichia coli, and manipulation of CL biosynthesis in this organism has elucidated a great deal about CL function in prokaryotes . In contrast, little is known about CL biosynthesis or its regulation in eukaryotic cells . We sought to determine whether we could utilize E . coli genes to manipulate expression of CL biosynthetic enzymes and CL content in yeast . The E . coli pgsA gene encodes phosphatidylglycerophosphate synthase (PGPS), catalyzing the first step in the CL biosynthetic pathway . We constructed plasmids with pgsA under the control of the yeast CUP1 promoter . Extracts of Saccharomyces cerevisiae cells transformed with this plasmid contained high levels of E . coli PGPS activity . However, when compared to cells transformed with a control plasmid, pgsA-transformed cells did not exhibit differences in phospholipid composition . The most likely explanation is that the in vitro activity of the E . coli pgsA product is not indicative of its activity in vivo, due to mislocalization of the enzyme and/or inaccessibility of the enzyme to the substrates . To our knowledge, this is the first demonstration of expression of a bacterial phospholipid biosynthetic enzyme in yeast. Int J Cancer, 1994 Sep 15, 58(6), 836 - 40 Expression of an endogenous retroviral gene product in human placenta; Kitamura M et al.; To investigate the presence and potential pathophysiological role of endogenous retroviruses in humans, we prepared a recombinant protein using clone 4-I, a proviral sequence . DNA fragments containing the env region of clone 4-I were subcloned into a prokaryotic expression vector (pET3), and 2 fusion proteins, SU413 and SU415, were then expressed in Escherichia coli after treatment with isopropyl-beta-thiogalactopyranoside (IPTG) . By sonicating lysates of the transformed E . coli, the recombinant protein SU413 was successfully separated from the native bacterial components, and was used to raise an antiserum in rabbits . In immunoblot analysis, this antiserum specifically recognized the recombinant protein, but did not react with other components of E . coli . This antiserum was then used for an immunofluorescence study of human placenta, in which the env gene transcript has been reported . As a result, the anti-SU413 serum detected substances in syncytiotrophoblasts and vascular endothelia from a human placenta . No such reactivity was detected in human kidney or human liver . Immunoblot analysis revealed that this antiserum reacted to a single molecule of 38-kDa in placenta, and its reactivity was reduced by the antiserum absorbed with SU413 antigen . These findings suggest that human placental syncytiotrophoblasts and vascular endothelia preferentially express a molecule encoded by human endogenous retrovirus clone 4-I. Biochim Biophys Acta, 1994 Sep 13, 1219(1), 115 - 20 Binding of a bZip protein to the estrogen-inducible apoVLDL II promoter; Smidt MP et al.; Activation of the very low density apolipoprotein II (apoVLDL II) gene in chicken liver by estrogen results in the binding of a variety of nuclear proteins including members of the steroid receptor superfamily and the bZip superfamily to the immediate 5' flanking region . In the present study, we have identified a bZip protein from chicken liver as one of the potential binding activities . Its cognate cDNA was cloned from an expression library using a recognition site DNA probe corresponding to part of the apoVLDL II promoter region . By footprinting and gel shift analysis with the recombinant protein from a prokaryotic expression system we have established that the protein binds to at least three different sites in the apoVLDLII promoter region . One of these sites partially overlaps with the major estrogen response element of the gene . Despite the proximity of their binding sites, the estrogen receptor and the bZip protein can bind simultaneously to the very region . Possible implications of this intimate arrangement of binding sites for the activation of the apoVLDL II promoter are discussed. Gene, 1994 Sep 2, 146(2), 297 - 301 Gene expression, purification and characterization of recombinant human neutrophil collagenase; Ho TF et al.; Human neutrophil collagenase (HNC) is a member of a family of matrix metalloproteinases (MMP) . HNC is capable of cleaving all three alpha-chains of types I, II and III collagens . In rheumatoid and osteo-arthritis, MMP members have been implicated in the pathology associated with these diseases due to the accelerated breakdown of the extracellular matrix of articular cartilage . A cDNA coding for the HNC catalytic domain (lacking both the propeptide and C-terminal fragments) was sub-cloned into the pETlla prokaryotic expression vector . The cloned fragment encodes a protein that extends from amino acids (aa) Met100 through Gly262 of the full-length proenzyme, which as a result, would not require proteolytic or chemical activation . The HNC construct was expressed in Escherichia coli and recombinant mature, truncated neutrophil collagenase (re-mNC-t) was produced at high levels (approx . 30% of total bacterial protein) . The re-mNC-t protein was extracted from inclusion bodies by solubilization in 6 M urea, followed by ion-exchange chromatography . The protein was refolded to an active conformation in the presence of Ca2+ and Zn2+ . A final purification step on size-exclusion chromatography yielded 30 mg per liter of active re-mNC-t with minor autodegradative products . Alternatively, hydroxamate affinity chromatography was used to obtain pure, non-degraded re-mNC-t (20-25 mg per liter) . The catalytic activity of re-mNC-t was abolished by known MMP inhibitors and the Ki measurement against actinonin was similar to that of HNC prepared from human blood. Gene, 1994 Sep 2, 146(2), 251 - 6 Extensive sequence divergence in the 3' inverted repeat of the chloroplast rbcL gene in non-flowering land plants and algae; Calie PJ et al.; A stem-loop region is present at the 3' terminus of the chloroplast rbcL mRNA in all taxa surveyed to date . In spinach, this structure has been shown by others to be involved in modulating transcript stability and correct 3' terminus processing, and is a conserved feature of other flowering plant rbcL mRNAs . In Chlamydomonas reinhardtii, an analogous structure has been shown by others to serve as a transcription terminator . Our sequencing data have shown that this region is highly divergent in several non-flowering land plants, as evidenced by representatives from the ferns, conifers, 'fern-allies' and liverworts . To extend our analysis, a computer-assisted survey of the stem-loop region of the 3' flanking region of published chloroplast rbcL genes was undertaken . The flowering plant rbcL inverted repeats (IR) were remarkably conserved in sequence, allowing for precise multiple alignments of both monocot and dicot sequences within a single matrix . Surprisingly, sequences obtained from non-flowering land plants, algae, photosynthetic protists and photosynthetic prokaryotes were extremely variant, in terms of both sequence composition and thermodynamic parameters. Nature, 1994 Sep 1, 371(6492), 75 - 80 Mutations of two PMS homologues in hereditary nonpolyposis colon cancer; Nicolaides NC et al.; Hereditary nonpolyposis colorectal cancer (HNPCC) is one of man's commonest hereditary diseases . Several studies have implicated a defect in DNA mismatch repair in the pathogenesis of this disease . In particular, hMSH2 and hMLH1 homologues of the bacterial DNA mismatch repair genes mutS and mutL, respectively, were shown to be mutated in a subset of HNPCC cases . Here we report the nucleotide sequence, chromosome localization and mutational analysis of hPMS1 and hPMS2, two additional homologues of the prokaryotic mutL gene . Both hPMS1 and hPMS2 were found to be mutated in the germline of HNPCC patients . This doubles the number of genes implicated in HNPCC and may help explain the relatively high incidence of this disease. J Bacteriol, 1994 Sep, 176(17), 5459 - 65 Membrane topology of Escherichia coli diacylglycerol kinase; Smith RL et al.; The topology of Escherichia coli diacylglycerol kinase (DAGK) within the cytoplasmic membrane was elucidated by a combined approach involving both multiple aligned sequence analysis and fusion protein experiments . Hydropathy plots of the five prokaryotic DAGK sequences available were uniform in their prediction of three transmembrane segments . The hydropathy predictions were experimentally tested genetically by fusing C-terminal deletion derivatives of DAGK to beta-lactamase and beta-galactosidase . Following expression, the enzymatic activities of the chimeric proteins were measured and used to determine the cellular location of the fusion junction . These studies confirmed the hydropathy predictions for DAGK with respect to the number and approximate sequence locations of the transmembrane segments . Further analysis of the aligned DAGK sequences detected probable alpha-helical N-terminal capping motifs and two amphipathic alpha-helices within the enzyme . The combined fusion and sequence data indicate that DAGK is a polytopic integral membrane protein with three transmembrane segments with the N terminus of the protein in the cytoplasm, the C terminus in the periplasmic space, and two amphipathic helices near the cytoplasmic surface. Mol Biol (Mosk), 1994 Sep-Oct, 28(5), 1183 - 90 {A model prokaryotic promotor . III . Cloning and study of functional activity in vivo}; Koroleva ON et al.; The efficiency of two schemes of oligonucleotide-directed insertion mutagenesis was studied in comparison with standard cloning of DNA duplexes based on blunt-end ligation . Using new approaches, the 30-bp consensus-like prokaryotic promoter was inserted in proper orientation in a promoter-testing plasmid at an almost 100% frequency . Data on the marker gal operon expression and S1-nuclease mapping of the transcription initiation points indicate formation of an active promoter in the region of the insertion. Parasitology, 1994 Sep, 109 ( Pt 3), 265 - 72 Codon usage and bias among individual genes of the coccidia and piroplasms; Ellis JT et al.; Codon usage has been analysed in individual gene sequences, derived from a variety of parasitic protozoa in the class Sporozoa of the phylum Apicomplexa using metric multidimensional scaling . The two groups of codon usage patterns detected reflect the two main subgroups of organisms studied (the coccidia and the piroplasms), and it is the pattern of usage of synonymous codons that has the largest influence on overall codon usage in the individual genes, rather than being the pattern of amino acid composition of the gene product . The magnitude of the codon usage bias in the sequences was determined using three commonly used indices-NC, GC3S and B . In general, although relatively low levels of codon usage bias were detected in these gene sequences, codon usage bias does explain at least some of the codon usage patterns observed . Codon usage bias was observed to be dependent on the overall base composition of the genes analysed, which in turn was reflected in the types of codons that were either over- or under-represented in the nucleotide sequences . In keeping with observations on prokaryotic organisms, it is speculated that the codon usage patterns detected in these parasitic protozoa are the result of directional mutation pressure on the base composition of the genomic DNA. Microbiol Rev, 1994 Sep, 58(3), 387 - 400 Do prokaryotes contain microtubules? Bermudes D, Hinkle G, Margulis L. In eukaryotic cells, microtubules are 24-nm-diameter tubular structures composed of a class of conserved proteins called tubulin . They are involved in numerous cell functions including ciliary motility, nerve cell elongation, pigment migration, centrosome formation, and chromosome movement . Although cytoplasmic tubules and fibers have been observed in bacteria, some with diameters similar to those of eukaryotes, no homologies to eukaryotic microtubules have been established . Certain groups of bacteria including azotobacters, cyanobacteria, enteric bacteria, and spirochetes have been frequently observed to possess microtubule-like structures, and others, including archaebacteria, have been shown to be sensitive to drugs that inhibit the polymerization of microtubules . Although little biochemical or molecular biological information is available, the differences observed among these prokaryotic structures suggest that their composition generally differs among themselves as well as from that of eukaryotes . We review the distribution of cytoplasmic tubules in prokaryotes, even though, in all cases, their functions remain unknown . At least some tend to occur in cells that are large, elongate, and motile, suggesting that they may be involved in cytoskeletal functions, intracellular motility, or transport activities comparable to those performed by eukaryotic microtubules . In Escherichia coli, the FtsZ protein is associated with the formation of a ring in the division zone between the newly forming offspring cells . Like tubulin, FtsZ is a GTPase and shares with tubulin a 7-amino-acid motif, making it a promising candidate in which to seek the origin of tubulins. Math Biosci, 1994 Sep, 123(1), 103 - 25 Analytical expression of the purine/pyrimidine autocorrelation function after and before random mutations; Arques DG et al.; The mutation process is a classical evolutionary genetic process . The type of mutations studied here is the random substitutions of a purine base R (adenine or guanine) by a pyrimidine base Y (cytosine or thymine) and reciprocally (transversions) . The analytical expressions derived allow us to analyze in genes the occurrence probabilities of motifs and d-motifs (two motifs separated by any d bases) on the R/Y alphabet under transversions . These motif probabilities can be obtained after transversions (in the evolutionary sense; from the past to the present) and, unexpectedly, also before transversions (after back transversions, in the inverse evolutionary sense, from the present to the past) . This theoretical part in Section 2 is a first generalization of a particular formula recently derived . The application in Section 3 is based on the analytical expression giving the autocorrelation function (the d-motif probabilities) before transversions . It allows us to study primitive genes from actual genes . This approach solves a biological problem . The protein coding genes of chloroplasts and mitochondria have a preferential occurrence of the 6-motif YRY(N)6YRY (maximum of the autocorrelation function for d = 6, N = R or Y) with a periodicity modulo 3 . The YRY(N)6YRY preferential occurrence without the periodicity modulo 3 is also observed in the RNA coding genes (ribosomal, transfer, and small nuclear RNA genes) and in the noncoding genes (introns and 5' regions of eukaryotic nuclei) . However, there are two exceptions to this YRY(N)6YRY rule: the protein coding genes of eukaryotic nuclei, and prokaryotes, where YRY(N)6YRY has the second highest value after YRY(N)0YRY (YRYYRY) with a periodicity modulo 3 . When we go backward in time with the analytical expression, the protein coding genes of both eukaryotic nuclei and prokaryotes retrieve the YRY(N)6YRY preferential occurrence with a periodicity modulo 3 after 0.2 back transversions per base . In other words, the actual protein coding genes of chloroplasts and mitochondria are similar to the primitive protein coding genes of eukaryotic nuclei and prokaryotes . On the other hand, this application represents the first result concerning the mutation process in the model of DNA sequence evolution we recently proposed . According to this model, the actual genes on the R/Y alphabet derive from two successive evolutionary genetic processes: an independent mixing of a few nonrandom types of oligonucleotides leading to genes called primitive followed by a mutation process in these primitive genes.(ABSTRACT TRUNCATED AT 400 WORDS) Eur J Biochem, 1994 Sep 1, 224(2), 317 - 28 Isolation and 1H-NMR spectroscopic identification of poly(3-hydroxybutanoate) from prokaryotic and eukaryotic organisms . Determination of the absolute configuration (R) of the monomeric unit 3-hydroxybutanoic acid from Escherichia coli and spinach; Seebach D et al.; Trace amounts of poly{(R)-3-hydroxybutanoate} were isolated from competent Escherichia coli, spinach, bovine serum albumin, beef heart mitochondria, and aortal tissues, all sources in which it is not accumulated as storage material . Its identity was in all cases proved by 1H-NMR spectroscopy . In some runs, the poly{(R)-3-hydroxybutanoate} isolated from competent E . coli also contained some 3-hydroxyvalerate, an observation confirmed by 1H-NMR spectroscopy and gas chromatography . The absolute configuration of the polymers isolated from E . coli and spinach was shown to be (all-R) by gas chromatography on chiral columns. Mol Cell Biochem, 1994 Sep, 138(1-2), 107 - 12 Vertebrate mono-ADP-ribosyltransferases; Zolkiewska A et al.; Mono-ADP-ribosylation appears to be a reversible modification of proteins, which occurs in many eukaryotic and prokaryotic organisms . Multiple forms of arginine-specific ADP-ribosyltransferases have been purified and characterized from avian erythrocytes, chicken polymorphonuclear leukocytes and mammalian skeletal muscle . The avian transferases have similar molecular weights of approximately 28 kDa, but differ in physical, regulatory and kinetic properties and subcellular localization . Recently, a 38-kDa rabbit skeletal muscle ADP-ribosyltransferase was purified and cloned . The deduced amino acid sequence contained hydrophobic amino and carboxy termini, consistent with known signal sequences of glycosylphosphatidylinositol (GPI)-anchored proteins . This arginine-specific transferase was present on the surface of mouse myotubes and of NMU cells transfected with the cDNA and was released with phosphatidylinositol-specific phospholipase C . Arginine-specific ADP-ribosyltransferases thus appear to exhibit considerable diversity in their structure, cellular localization, regulation and physiological role. Int J Neural Syst, 1994 Sep, 5(3), 159 - 63 Classification of genetic sequences with backpropagation; Lende R et al.; A backpropagation algorithm is used to train a neural net with the goal of distinguishing between two groups of biological species: prokaryotic and eukaryotic, based on frequencies of all 16 doublets in DNA sequences . An improvement of about 15% is obtained compared to statistical analysis based on one doublet only . This is done first by presenting sequences of species to the network with known classification (the training phase) and then showing species which the neural net has never seen before, and looking for the response . A brief discussion of the speed of training is given. Mol Phylogenet Evol, 1994 Sep, 3(3), 187 - 91 The SecY protein family: comparative analysis and phylogenetic relationships; Rensing SA et al.; We have cloned and sequenced the plastome encoded secY homologue of the cryptomonad alga Pyrenomonas salina . In this study we have carried out a comparative analysis of all but one fully sequenced proteins of the SecY family . We present an alignment of 16 SecY family proteins, containing family signatures, putative transmembrane domains, consensus sequence, and conservation grade . A phylogenetic tree derived from the conserved blocks of the alignment reveals the relationships among this protein family . The tree shows division into two broad subfamilies, one consisting of prokaryotic and plastidal sequences and the other of eukaryotic as well as archaeal sequences. Mol Microbiol, 1994 Sep, 13(5), 765 - 73 Signal peptides: exquisitely designed transport promoters; Izard JW et al.; Prokaryotic proteins destined for transport out of the cytoplasm typically contain an N-terminal extension sequence, called the signal peptide, which is required for export . It is evident that many secretory proteins utilize a common export system, yet the signal sequences themselves display very little primary sequence homology . In attempting to understand how different signal peptides are able to promote protein secretion through the same pathway, the physical features of natural signal sequences have been extensively examined for similarities that might play a part in function . Experimental data have confirmed statistical analyses which highlighted dominant features of natural signal sequences in Escherichia coli: a net positive charge in the N-terminus increases efficiency of transport; the core region must maintain a threshold level of hydrophobicity within a range of length limitations; the central portion adopts an alpha-helical conformation in hydrophobic environments; and the signal cleavage region is ideally six residues long, with small side-chain amino acids in the -1 and -3 positions . This review focuses on the parallels between signal peptide physical features and their functions, which emerge when the results of a variety of experimental approaches are combined . The requirement for each property may be ascribed to a potential interaction that is critical for efficient protein export . The summation of the key physical features produces signal peptides with the flexibility to function in multiple roles in order to expedite secretion . In this way, nature has indeed evolved exquisitely tuned signal sequences. Chin Med J (Engl), 1994 Sep, 107(9), 658 - 63 Discovery of extracellular multiple form of Chlamydia trachomatis in the tissue culture; Li ZH et al.; A strain of Chlamydia trachomatis was isolated from a patient with nongonococcal urethritis (NGU) . Alternate passages between chick embryo and McCoy cell culture were examined . From the Giemsa stained coverslips taken from the cell culture 96 hours after inoculation, we found, to our surprise, that elementary bodies (EBs) distributed over a large area, and several intact cells embedded in them . These pure EB particles are round, fairly uniform in size and often appeared in pair . According to their morphology, distribution, arrangement and relationship with host cells, they are not the remains after cell lysis or directly released from host cells . We considered that they consisted of EBs which continued to divide by binary fission after their release . The name "Extracellular Multiply Form" was designated and their formation mechanism was proposed . This discovery gives a great challenge to primary theory, i.e . Chlamydias are obligate intracellular prokaryotic parasites . If we can further reveal the law of their formation, it will be of great significance both theoretically and practically. Mol Microbiol, 1994 Sep, 13(5), 855 - 61 Universal barrier to lateral spread of specific genes among microorganisms; Diaz E et al.; A genetic circuit to suppress the lateral spread of cloned genes from recombinant to indigenous microorganisms in the environment has been developed . It is based on the endonucleolytic activity of the bacterial toxin colicin E3, which has a distinct target at the 3' end of the 16S ribosomal RNA; this sequence is conserved in virtually all prokaryotic and many eukaryotic genera . Cleavage at this sequence separates the mRNA binding sites from the remainder of the 16S rRNA, thereby inhibiting protein synthesis . While host bacteria carrying the genes for both colicin production and colicin immunity are perfectly viable, lateral transfer of the E3 gene to non-immune recipients results in killing of such recipients . This genetic circuit decreases operational transfer frequencies of cloned genes linked to the E3 gene among a variety of bacterial genera by four to five orders of magnitude . In combination with transposon cloning vectors, the circuit is predicted to reduce the rate of lateral spread of specific genes to ecologically insignificant levels . This system therefore represents a useful tool both to explore the evolutionary and ecological consequences of experimentally reducing lateral gene spread among microorganisms, and to increase the ecological predictability of novel recombinant microorganisms. Eur J Biochem, 1994 Sep 1, 224(2), 589 - 96 Tyr394 and Tyr505 are autophosphorylated in recombinant Lck protein-tyrosine kinase expressed in Escherichia coli; Jullien P et al.; The activity of the Src family protein-tyrosine kinase p56lck is regulated by phosphorylation and dephosphorylation of two critical tyrosine residues Tyr394 and Tyr505 . Tyr394 is autophosphorylated after p56lck activation, whereas phosphorylation of Tyr505 is believed to be due to p50csk which negatively modulates p56lck activity . To determine whether Tyr505 could be autophosphorylated, we used the prokaryotic glutathione S-transferase expression system to express wild-type Lck, the mutants {Y394F}Lck and {Y505F}Lck, a kinase-deficient p56lck with a mutation of the ATP-binding site {K273E}Lck and a double mutant {Y394F, Y505F}Lck . We studied the kinase activities and the patterns of autophosphorylation for tyrosine residues in these mutants and wild-type Lck both in vivo and in vitro . Wild-type Lck, {Y505F}Lck and {Y394F}Lck were phosphorylated on tyrosine . Both the kinase-deficient mutant{K273E}Lck and the double mutant {Y394F, Y505F}Lck did not react with monoclonal anti-phosphotyrosine antibody {anti-Y(P) mAb}, thus providing evidence that (a) the bacterial strains used lacked intrinsic protein-tyrosine kinase activities, and therefore tyrosine phosphorylations of wild-type Lck, {Y505F}Lck and {Y394F}Lck are due to autophosphorylation occurring in vivo in bacteria, and (b) that p56lck can only be autophosphorylated on two tyrosine residues, namely Tyr394 and Tyr505 . Phosphopeptide mapping analysis confirmed that p56lck can undergo autophosphorylation on these two tyrosine residues . We propose that autophosphorylation at Tyr505 of p56lck may represent an accessory mechanism for the down-regulation of the tyrosine kinase activity of p56lck. Clin Immunol Immunopathol, 1994 Sep, 72(3), 380 - 9 The immunoreactive region in a novel autoantigen contains a nuclear localization sequence; Bloch DB et al.; Antibodies in the serum of patients with autoimmune diseases have been used to identify human autoantigens . Because autoantibodies often recognize active sites within corresponding protein antigens, autoantibodies have facilitated the functional characterization of these polypeptides . In the present study, serum from a patient with Sjogren's syndrome was used to identify a novel autoantigen which was designated Ge-1 . Using the patient's serum, a 4.8-kb cDNA encoding Ge-1 was identified . Fragments of the cDNA were ligated into prokaryotic expression vectors, expressed in Escherichia coli, and used to produce recombinant Ge-1 fusion proteins . Fusion proteins containing different portions of Ge-1 were used to identify a 58 amino acid immunoreactive region within the protein . This immunoreactive region contained the protein's putative nuclear localization sequence (NLS) . To demonstrate that the immunoreactive region was capable of functioning as a NLS, a eukaryotic expression plasmid was constructed to encode the immunoreactive region fused to the cytoplasmic protein, chicken muscle pyruvate kinase . After transfection of this plasmid into COS-1 cells, the fusion protein was detected in the nucleus . The presence of the NLS motif within the immunoreactive region of Ge-1 and other nuclear autoantigens suggests that the NLS may be a target of human autoantibodies. J Biol Chem, 1994 Aug 26, 269(34), 21650 - 6 Human liver arylacetamide deacetylase . Molecular cloning of a novel esterase involved in the metabolic activation of arylamine carcinogens with high sequence similarity to hormone-sensitive lipase; Probst MR et al.; Microsomal arylacetamide deacetylase (DAC) competes against the activity of cytosolic arylamine N-acetyltransferase, which catalyzes one of the initial biotransformation pathways for arylamine and heterocyclic amine carcinogens in many species and tissues . Activity determination and immunoblot analysis of DAC in human target tissues for arylamine carcinogens revealed that in extrahepatic tissues, additional enzymes are responsible for any deacetylation activity, whereas a single enzyme predominantly catalyzes this hydrolytic reaction in liver . We isolated and characterized a full-length cDNA from a human liver lambda gt11 library . This clone encodes an open reading frame of 400 amino acids with a deduced molecular mass of 45.7 kDa and contains two putative glycosylation sites . The 3'-untranslated region contains two putative polyadenylation signals . The cDNA was confirmed to be that for DAC in tryptic peptides from the purified human liver protein . Highest sequence similarity of DAC was found in a series of prokaryotic esterases encompassing the putative active site . Two extended regions of significant sequence homology with hormone-sensitive lipase and with lipase 2 from Moraxella TA144 were identified, whereas similarity to carboxyl esterases was restricted to the region encompassing the putative active site, indicating that DAC should be classified as esterase . This cDNA provides an important tool to study deacetylation and its effects on the metabolic activation of arylamine and heterocyclic amine carcinogens. J Mol Biol, 1994 Aug 19, 241(3), 492 - 7 The DIM1 gene responsible for the conserved m6(2)Am6(2)A dimethylation in the 3'-terminal loop of 18 S rRNA is essential in yeast; Lafontaine D et al.; Biogenesis of cytoplasmic ribosomes universally involves methylation of ribosomal RNA . Little genetic evidence is available about the functional role(s) of this conserved posttranscriptional modification . The only known methylase gene involved in rRNA maturation is ksgA in Escherichia coli, which directs dimethylation of two adjacent adenosines (m6(2)A1518m6(2)A1519) in the loop of a conserved hairpin near the 3'-end of 16 S rRNA . This tandem methylation is the only rRNA modification common to pro and eukaryotes . Disruption of ksgA confers resistance to the aminoglycoside antibiotic kasugamycin without significantly impairing viability . Here we report the cloning of the DIM1 gene encoding the homolog 18 S rRNA dimethylase in Saccharomyces cerevisiae . The yeast enzyme is evolutionary related to the ksgA protein . It carries a distinctive lysine-rich-N-terminal extension with a potential protein kinase C phosphorylation site . Like ksgA, DIM1 belongs to the erm family of prokaryotic 23 S rRNA dimethylases responsible for erythromycin resistance . Surprisingly, disruption of DIM1 turns out to be lethal in yeast. Gene, 1994 Aug 19, 146(1), 133 - 4 Analysis of the sequences within and flanking the cyanoglobin-encoding gene, glbN, of the cyanobacterium Nostoc commune UTEX 584; Angeloni SV et al.; A 3.5-kb segment of DNA containing nifU glbN nifH nifD was cloned from a gene library of Nostoc 584 and sequenced . The nifU-glbN intergenic region contains short tandemly repeated repetitive sequences (5'-AATTACG) . A sequence corresponding to a NifA-like upstream activator sequence (with the consensus recognition sequence for BifA in Anabaena 7120), elements of a nifH promoter and a sequence that may function as a transcription terminator, were identified downstream from glbN . GlbN, unique to certain Nostoc spp., is more homologous to protozoan myoglobins than to any other prokaryotic, vertebrate or plant globins. Biochem J, 1994 Aug 15, 302 ( Pt 1), 11 - 4 Cloning of cDNA encoding the bifunctional dehydroquinase.shikimate dehydrogenase of aromatic-amino-acid biosynthesis in Nicotiana tabacum; Bonner CA et al.; Nicotiana tabacum cDNA encoding a bifunctional protein having catalytic domains for dehydroquinase and shikimate dehydrogenase was cloned and sequenced . Complementation of Escherichia coli aroD and aroE auxotrophs was successful . Amino acid sequencing located the N-terminus of the mature protein . The two catalytic domains exhibited greater amino acid identity with prokaryote homologues than with yeast and fungal homologues. J Biol Chem, 1994 Aug 12, 269(32), 20691 - 9 Cloning and expression of cyclosporin A- and FK506-sensitive nuclear factor of activated T-cells: NF45 and NF90; Kao PN et al.; Nuclear Factor of Activated T-cells (NF-AT) is a crucial transcription factor required for T-cell expression of interleukin 2 . Purified NF-AT contains 45-kDa and 90-kDa subunits (Corthesy, B., and Kao, P . N . (1994) J . Biol . Chem . 269, 20682-20690) . Partial internal amino acid sequences derived from each subunit indicate that these proteins are novel . The amino acid sequences were used to clone the cDNAs encoding each subunit . The cDNAs predict proteins of novel structures: NF45 has limited similarity to prokaryotic transcription factor sigma-54 and to human DNA topoisomerase II; NF90 has limited similarity to Drosophila Staufen in a domain predicted to bind double-stranded RNA . RNA encoding NF45 and NF90 exists in nonstimulated Jurkat T-cells and in all other cell types examined (HeLa, HepG2, K562) . Immunofluorescence microscopy was used to demonstrate that both proteins are located in the nucleus of Jurkat T-cells . Clones NF45 and NF90 with a polyhistidine fusion tag were transiently expressed and processed in the native environment of Jurkat T-cells . Histidine-tagged NF45 and NF90 proteins, affinity-purified on nickel chelate columns, encode a NF-AT DNA-binding activity that is enhanced following T-cell stimulation, and this enhancement is blocked when T-cells are stimulated in the presence of cyclosporin A or FK506. Gene, 1994 Aug 5, 145(2), 289 - 92 Human RNA polymerase II subunit hRPB14 is homologous to yeast RNA polymerase I, II, and III subunits (AC19 and RPB11) and is similar to a portion of the bacterial RNA polymerase alpha subunit; Pati UK; The cDNA cloning of the human polII 14-kDa subunit, hRPB14, and the comparison of its aa sequence with those of other pol subunits are described . The aa sequence of hRPB14 has homology to yeast poIII subunit RPB11 (44%), to a common subunit of yeast polI and polIII AC19 (24%) and to a Caenorhabditis elegans sequence (33%) . hRPB14 contains a 19-aa motif, located in its N terminus, which was also found in human polII 33-kDa subunit hRPB33, yeast pol subunits (AC40, AC19, RPB3 and RPB11), and in the bacterial pol alpha subunit, which was involved in subunit assembly . This motif was also conserved in the conjugation-specific gene products of Tetrahymena (CnjC), Merchantia polymorpha chloroplast DNA (RNLVA) and C . elegans DNA (CEF58A4; deduced from the nucleotide sequence and of unknown function) . The evolutionary emergence of a probable eukaryotic heterodimer, hRPB14/hRPB33, from a prokaryotic homodimer, alpha 2, is hypothesized. Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7708 - 11 Coordination of selenium to molybdenum in formate dehydrogenase H from Escherichia coli; Gladyshev VN et al.; Formate dehydrogenase H from Escherichia coli contains multiple redox centers, which include a molybdopterin cofactor, an iron-sulfur center, and a selenocysteine residue (SeCys-140 in the polypeptide chain) that is essential for catalytic activity . Here we show that addition of formate to the native enzyme induces a signal typical of Mo(V) species . This signal is detected by electron paramagnetic resonance (EPR) spectroscopy . Substitution of 77Se for natural isotope abundance Se leads to transformation of this signal, indicating a direct coordination of Se with Mo . Mutant enzyme with cysteine substituted at position 140 for the selenocysteine residue has decreased catalytic activity and exhibits a different EPR signal . Since determination of the Se content of wild-type enzyme indicates approximately 1 gram atom per mol, we conclude that it is the Se atom of the SeCys-140 residue in the protein that is coordinated directly with Mo . The amino acid sequence flanking the selenocysteine residue in formate dehydrogenase H is similar to a conserved sequence found in several other prokaryotic molybdopterin-dependent enzymes . In most of these other enzymes a cysteine residue, or in a few cases a serine or a selenocysteine residue, occurs in the position corresponding to SeCys-140 of formate dehydrogenase H . By analogy with formate dehydrogenase H in these other enzymes, at least one of the ligands to Mo should be provided by an amino acid residue of the protein . This ligand could be the Se of a selenocysteine residue, sulfur of a cysteine residue, or, in the case of a serine residue, oxygen. Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7648 - 52 Protein synthesis elongation factor EF-1 alpha is essential for ubiquitin-dependent degradation of certain N alpha-acetylated proteins and may be substituted for by the bacterial elongation factor EF-Tu; Gonen H et al.; Targeting of different cellular proteins for conjugation and subsequent degradation via the ubiquitin pathway involves diverse recognition signals and distinct enzymatic factors . A few proteins are recognized via their N-terminal amino acid residue and conjugated by a ubiquitin-protein ligase that recognizes this residue . Most substrates, including the N alpha-acetylated proteins that constitute the vast majority of cellular proteins, are targeted by different signals and are recognized by yet unknown ligases . We have previously shown that degradation of N-terminally blocked proteins requires a specific factor, designated FH, and that the factor acts along with the 26S protease complex to degrade ubiquitin-conjugated proteins . Here, we demonstrate that FH is the protein synthesis elongation factor EF-1 alpha . (a) Partial sequence analysis reveals 100% identity to EF-1 alpha . (b) Like EF-1 alpha, FH binds to immobilized GTP (or GDP) and can be purified in one step using the corresponding nucleotide for elution . (c) Guanine nucleotides that bind to EF-1 alpha protect the ubiquitin system-related activity of FH from heat inactivation, and nucleotides that do not bind do not exert this effect . (d) EF-Tu, the homologous bacterial elongation factor, can substitute for FH/EF-1 alpha in the proteolytic system . This last finding is of particular interest since the ubiquitin system has not been identified in prokaryotes . The activities of both EF-1 alpha and EF-Tu are strongly and specifically inhibited by ubiquitin-aldehyde, a specific inhibitor of ubiquitin isopeptidases . It appears, therefore, that EF-1 alpha may be involved in releasing ubiquitin from multiubiquitin chains, thus rendering the conjugates susceptible to the action of the 26S protease complex. Proc Natl Acad Sci U S A, 1994 Aug 2, 91(16), 7435 - 9 Human cytoplasmic isoleucyl-tRNA synthetase: selective divergence of the anticodon-binding domain and acquisition of a new structural unit; Shiba K et al.; We show here that the class I human cytoplasmic isoleucyl-tRNA synthetase is an exceptionally large polypeptide (1266 aa) which, unlike its homologues in lower eukaryotes and prokaryotes, has a third domain of two repeats of an approximately 90-aa sequence appended to its C-terminal end . While extracts of Escherichia coli do not aminoacrylate mammalian tRNA with isoleucine, expression of the cloned human gene in E . coli results in charging of the mammalian tRNA substrate . The appended third domain is dispensable for detection of this aminoacylation activity and may be needed for assembly of a multisynthetase complex in mammalian cells . Alignment of the sequences of the remaining two domains shared by isoleucyl-tRNA synthetases from E . coli to human reveals a much greater selective pressure on the domain needed for tRNA acceptor helix interactions and catalysis than on the domain needed for interactions with the anticodon . This result may have implications for the historical development of an operational RNA code for amino acids. Appl Environ Microbiol, 1994 Aug, 60(8), 2766 - 71 Isolation, sequence, and characterization of the Cercospora nicotianae phytoene dehydrogenase gene; Ehrenshaft M et al.; We have cloned and sequenced the Cercospora nicotianae gene for the carotenoid biosynthetic enzyme phytoene dehydrogenase . Analysis of the derived amino acid sequence revealed it has greater than 50% identity with its counterpart in Neurospora crassa and approximately 30% identity with prokaryotic phytoene dehydrogenases and is related, but more distantly, to phytoene dehydrogenases from plants and cyanobacteria . Our analysis confirms that phytoene dehydrogenase proteins fall into two groups: those from plants and cyanobacteria and those from eukaryotic and noncyanobacter prokaryotic microbes . Southern analysis indicated that the C . nicotianae phytoene dehydrogenase gene is present in a single copy . Extraction of beta-carotene, the sole carotenoid accumulated by C . nicotianae, showed that both light- and dark-grown cultures synthesize carotenoids, but higher levels accumulate in the light . Northern (RNA) analysis of poly(A)+ RNA, however, showed no differential accumulation of phytoene dehydrogenase mRNA between light- and dark-grown fungal cultures. Plant Mol Biol, 1994 Aug, 25(5), 771 - 90 The use of a hybrid genetic system to study the functional relationship between prokaryotic and plant multi-enzyme fatty acid synthetase complexes; Kater MM et al.; Fatty acid synthesis in bacteria and plants is catalysed by a multi-enzyme fatty acid synthetase complex (FAS II) which consists of separate monofunctional polypeptides . Here we present a comparative molecular genetic and biochemical study of the enoyl-ACP reductase FAS components of plant and bacterial origin . The putative bacterial enoyl-ACP reductase gene (envM) was identified on the basis of amino acid sequence similarities with the recently cloned plant enoyl-ACP reductase . Subsequently, it was unambiguously demonstrated by overexpression studies that the envM gene encodes the bacterial enoyl-ACP reductase . An anti-bacterial agent called diazaborine was shown to be a specific inhibitor of the bacterial enoyl-ACP reductase, whereas the plant enzyme was insensitive to this synthetic antibiotic . The close functional relationship between the plant and bacterial enoyl-ACP reductases was inferred from genetic complementation of an envM mutant of Escherichia coli . Ultimately, envM gene-replacement studies, facilitated by the use of diazaborine, demonstrated for the first time that a single component of the plant FAS system can functionally replace its counterpart within the bacterial multienzyme complex . Finally, lipid analysis of recombinant E . coli strains with the hybrid FAS system unexpectedly revealed that enoyl-ACP reductase catalyses a rate-limiting step in the elongation of unsaturated fatty acids. J Nutr, 1994 Aug, 124(8 Suppl), 1499S - 1502S Regulation of branched-chain amino acid catabolism; Harris RA et al.; Catabolism of the branched-chain amino acids is regulated in part at the step catalyzed by the branched-chain alpha-ketoacid dehydrogenase complex . Previous work suggests both short-term and long-term control mechanisms are involved in regulation of the kinase responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex . Recent work of this laboratory has focused on the isolation, characterization and molecular cloning of the branched-chain alpha-ketoacid dehydrogenase kinase . The cDNA obtained encodes the complete mature protein of 412 amino acids among with a mitochondrial entry sequence of 30 amino acids . Analysis of the deduced amino acid sequence revealed little similarity with eukaryotic Ser/Thr protein kinases . However, the kinase shows considerable sequence similarity with prokaryotic histidine protein kinases . The availability of this cDNA will facilitate gene expression studies of this important regulatory enzyme for the branched-chain alpha-ketoacid dehydrogenase complex. EMBO J, 1994 Aug 1, 13(15), 3472 - 80 Streptomyces chrysomallus FKBP-33 is a novel immunophilin consisting of two FK506 binding domains; its gene is transcriptionally coupled to the FKBP-12 gene; Pahl A et al.; The nucleotide sequence of the region 5' to the fkbA gene, encoding the Streptomyces chrysomallus FK506 binding protein (FKBP-12), revealed an open reading frame (fkbB) encoding a protein of 312 amino acids, with an M(r) of approximately 33,000 . FkbB and fkbA appear to be co-transcribed under the control of a promoter upstream of fkbB . The presumptive protein encoded by fkbB would be an FKBP (designated FKBP-33) consisting of two FK506 binding domains with 43 and 32% sequence identity to FKBP-12 and a signal peptide sequence characteristic of bacterial membrane lipoproteins . The portion of the gene comprising the two FKBP domains, as well as each individual domain, were expressed as fusion proteins in Escherichia coli and purified . Each expressed domain, as well as FKBP-33 itself, possesses peptidyl-prolyl cis-trans isomerase activity, though with much lower specific activities than FKBP-12 . FKBP-33 is located in the cell membrane of S.chrysomallus and of other streptomycetes, as predicted from the presence of the signal peptide sequence . Pulse-chase experiments with radioactive palmitate in whole cells revealed significant labelling of FKBP-33, which probably carries palmitate at its N-terminus and an additional diacylglycerol residue attached to the N-terminal cysteine in thioether linkage . The two domains of FKBP-33 showed considerable homology with numerous eukaryotic and prokaryotic FKB domains . Calculations of phylogenetic relationships indicate with high probability that the two domains of the protein have arisen by a double gene duplication of fkbA lying in tandem to fkbB. Eur J Biochem, 1994 Aug 1, 223(3), 917 - 25 Cloning of a wheat 15-kDa grain softness protein (GSP) . GSP is a mixture of puroindoline-like polypeptides; Rahman S et al.; The wheat starch 15-kDa protein (called grain softness protein or GSP) consists of a major polypeptide and several minor polypeptides . An antiserum raised against GSP was used to screen a wheat cDNA library . A cDNA family encoding approximately 15-kDa proteins that included a heptapeptide sequence previously isolated from protease digests of GSP was identified . A partial cDNA was used in a prokaryotic expression system to produce a fusion protein which reacted strongly against the original anti-GSP serum . A new antiserum raised against the fusion protein produced a weak reaction against a 15-kDa polypeptide extracted from wheat seeds . The results suggest that the proteins encoded by the cDNA family form a minor component of the mixture of 15-kDa polypeptides defined as GSP . RNA complementary to the cDNAs could be extracted from both soft and hard wheat grains from about half-way through grain filling . The encoded proteins are novel members of the 2S superfamily of seed proteins, a diverse family of proteins which maintain a characteristic framework of cysteine residues . The deduced proteins show the highest similarity to the oat 16-kDa avenin and to wheat puroindoline (a lipid-binding 15-kDa protein from wheat) . Review of previously published data shows that puroindoline is also closely related to the major polypeptide of GSP, suggesting that the lipid-binding properties of GSP polypeptides may influence grain softness. J Bacteriol, 1994 Aug, 176(15), 4770 - 3 The yeast Saccharomyces kluyveri as a recipient eukaryote in transkingdom conjugation: behavior of transmitted plasmids in transconjugants; Inomata K et al.; The prokaryote Escherichia coli successfully conjugated with the eukaryote Saccharomyces kluyveri, which is relatively distant from the species S . cerevisiae . To achieve this transkingdom conjugation, we constructed three types of conjugative plasmids, namely integrative, replicative, and centromere vectors, for S . cerevisiae . By transfer of any of the three plasmids from E . coli, an S . kluyveri Ura- mutant was converted to the Ura+ phenotype . This phenotype was easily lost under nonselective conditions . Southern analysis of the transconjugants clearly indicated the presence of the plasmids in many different structures and sizes. Mol Cell Biol, 1994 Aug, 14(8), 5043 - 55 Mapping initiation sites for simian virus 40 DNA synthesis events in vitro; Bullock PA et al.; Primer RNA-DNA, a small (approximately 30-nucleotide) RNA-DNA hybrid molecule, was identified in recent studies of simian virus 40 DNA synthesis in vitro . The available evidence indicates that primer RNA-DNA is the product of the polymerase alpha-primase complex . Primer RNA-DNA is formed exclusively on lagging-strand DNA templates; it is synthesized initially in the vicinity of the simian virus 40 origin and at later times at sites progressively distal to the origin . To further characterize initiation events, template sequences encoding the 5' ends of both primer RNA and primer DNA, formed during a 5-s pulse, have been determined . Analyses of these sequences demonstrate the existence of an initiation signal for lagging-strand synthesis . At any given position, the initiation signal is located within those template sequences encoding primer RNA, situated proximal to the nucleotide encoding the 5' end of the RNA primer . In most instances, the sequence 5'-TTN-3' (where N encodes the nucleotide at the 5' end of the primer) is a feature of the initiation signal . Initiation signals are present, on average, once every 19 nucleotides . These results are discussed in terms of the mechanism of Okazaki fragment formation and possible links between prokaryotic and eukaryotic initiation events. Biochem Mol Biol Int, 1994 Aug, 33(5), 909 - 15 Transfer RNA binding to 80S ribosomes from yeast: evidence for three sites; Triana F et al.; The number of tRNA binding sites in 80S ribosomes from Saccharomyces cerevisiae was assessed by means of tRNA saturation and translocation experiments . In the absence of cognate mRNA yeast ribosomes could bind 0.6 {32P}tRNA(Phe) per 80S while poly(U) programmed ribosomes accepted up to 1.7 tRNA(Phe) molecules per 80S or 0.5 molecules of Ac{14C}Phe-tRNA(Phe) per 80S . Compared with the known features of E . coli ribosomes these binding values indicated both the presence of three tRNA binding sites and the validity of the exclusion principle for peptidyl-tRNA binding to yeast ribosomes . Upon EF-2 dependent translocation of a complex containing deacyl-tRNA in the P-site and AcPHe-tRNA in the A-site, the deacylated tRNA does not leave the ribosome quantitatively . This observation suggests the presence of an E site in 80S ribosomes which is functionally equivalent to the one previously characterized in prokaryotic systems. Curr Opin Cell Biol, 1994 Aug, 6(4), 571 - 82 Bacterial transporters; Maloney PC; Recent experiments in bacterial systems have established an extended database of sequences broadly relevant to all membrane transporters, allowing serious study of evolutionary relationships . The database will be especially useful in integrating conclusions derived from work with proteins in the major facilitator superfamily, because this kinship includes both eukaryotic and prokaryotic model systems . Even among carriers not linked by evolution, clear hints of functional homology have been note . Advances are also evident in the structural analysis of membrane carriers . Site-directed mutagenesis in a bacterial antiporter has shown how the translocation pathway might be identified; this should complement recent progress in preparing two-dimensional crystals of the eukaryotic anion-exchange protein, band 3 . Together, these studies could soon verify or reject the idea that the transport pathway lies at the interface between the amino-terminal and carboxy-terminal helical bundles found in the hydrophobic core of most carrier proteins . If verified, the argument might allow construction of informed three-dimensional models in the absence of crystallographic evidence. Trends Biochem Sci, 1994 Aug, 19(8), 325 - 30 The cytochrome oxidase superfamily of redox-driven proton pumps; Calhoun MW et al.; Most respiratory oxidases of eukaryotic and prokaryotic organisms are members of a superfamily of enzymes that couple the redox energy available from the reduction of molecular oxygen to the mechanism of pumping protons across the membrane . The recent applications of site-directed mutagenesis and of a variety of spectroscopic techniques have allowed major advances in our understanding of the structure and function of these proteins. J Parasitol, 1994 Aug, 80(4), 580 - 90 Presence and cellular distribution of a 60-kDa protein related to mitochondrial hsp60 in Giardia lamblia; Soltys BJ et al.; Giardia lamblia trophozoites contain a 60-kDa protein recognized in immunoblots by antibody to mammalian hsp60, a protein localized in mitochondria in eukaryotic cells . The cellular distribution of this protein is evaluated by immunofluorescence microscopy using monoclonal antibody to human hsp60, polyclonal antibody to rodent hsp60, and 2 monoclonal antibodies to mycobacterial 65-kDa antigen, a prokaryotic hsp60 homolog . All of these antibodies, except 1, which is specific for prokaryotic hsp60, give a punctate labeling pattern throughout the cytoplasm, indicating that the 60-kDa protein is concentrated at discrete sites in the cytoplasm . The polyclonal hsp60 antiserum reveals additional punctate labeling colocalized on axonemes of the anterior flagella . Postembedment immunogold labeling and electron microscopy confirm that the antigen is clustered at foci in the cytoplasm but show no evidence of association with a membranous organelle . The hsp60 reactivity is also observed on anterior axonemes within the cytoplasm and on the adhesive disc . Hoechst 33258 DNA staining as well as electron microscopy give no evidence of endosymbionts, so a false positive due to a prokaryotic hsp60 homolog is unlikely . The presence of an hsp60-related protein in G . lamblia raises interesting questions concerning its origin. Insect Mol Biol, 1994 Aug, 3(3), 131 - 42 A prokaryotic dnaA sequence in Drosophila melanogaster: Wolbachia infection and cytoplasmic incompatibility among laboratory strains; Bourtzis K et al.; Using oligonucleotide primers derived from the aligned polypeptide sequences of several prokaryotic dnaA genes, we amplified from Drosophila melanogaster DNA a 557 bp fragment containing a single open reading frame . The predicted peptide sequence shows a significant similarity to previously characterized protein sequences that are encoded by the dnaA genes of several prokaryotes . The dnaA sequences are also detectable by PCR in DNA from Drosophila simulans and Nasonia vitripennis flies which are infected by a symbiotic bacterium assigned to the type species Wolbachia pipientis . A tetracycline treatment that eradicates bacterial parasites from insects, abolishes the dnaA sequences from Drosophila and Nasonia DNA . In addition, dnaA-positive Drosophila melanogaster contain numerous rod-shaped bacteria in embryos, which are abolished in subsequent generations after treatment with tetracycline . Combined with phylogenetic analysis of DnaA and 16S rRNA sequences, these results show that the dnaA cognate comes from Wolbachia . A survey of Drosophila stocks using PCR amplification of dnaA and 16S rRNA sequences showed that Wolbachia is widely spread among D . melanogaster laboratory strains but absent from several established strains of the Mediterranean fruit fly Ceratitis capitata . Evidence is also presented that presence of the bacterium can cause partial cytoplasmic incompatibility between infected and non-infected D . melanogaster strains. Protein Sci, 1994 Aug, 3(8), 1296 - 304 Mouse liver NAD(P)H:quinone acceptor oxidoreductase: protein sequence analysis by tandem mass spectrometry, cDNA cloning, expression in Escherichia coli, and enzyme activity analysis; Chen S et al.; The amino acid sequence of mouse liver NAD(P)H:quinone acceptor oxidoreductase (EC 1.6.99.2) has been determined by tandem mass spectrometry and deduced from the nucleotide sequence of the cDNA encoding for the enzyme . The electrospray mass spectral analyses revealed, as previously reported (Prochaska HJ, Talalay P, 1986, J Biol Chem 261:1372-1378), that the 2 forms--the hydrophilic and hydrophobic forms--of the mouse liver quinone reductase have the same molecular weight . No amino acid sequence differences were found by tandem mass spectral analyses of tryptic peptides of the 2 forms . Moreover, the amino-termini of the mouse enzymes are acetylated as determined by tandem mass spectrometry . Further, only 1 cDNA species encoding for the quinone reductase was found . These results suggest that the 2 forms of the mouse quinone reductase have the same primary sequences, and that any difference between the 2 forms may be attributed to a labile posttranslational modification . Analysis of the mouse quinone reductase cDNA revealed that the enzyme is 273 amino acids long and has a sequence homologous to those of rat and human quinone reductases . In this study, the mouse quinone reductase cDNA was also ligated into a prokaryotic expression plasmid pKK233.2, and the constructed plasmid was used to transform Escherichia coli strain JM109 . The E . coli-expressed mouse quinone reductase was purified and characterized . Although mouse quinone reductase has an amino acid sequence similar to those of the rat and human enzymes, the mouse enzyme has a higher NAD(P)H-menadione reductase activity and is less sensitive to flavones and dicoumarol, 2 known inhibitors of the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS) Mutat Res, 1994 Aug 1, 309(1), 53 - 61 Expression of genes carried by pR plasmid in damaged E . coli and mouse cells; Marcucci L et al.; In LTA mouse cells pR plasmid constitutively expresses itself resulting in protection against typical SOS inducers (UV, 4NQO) and in sensitization to different DNA-damaging agents (MNNG, cisDDP, BLM and geneticin (G418) . The pR sensitizing effect is specific to mammalian cells, since the plasmid can only protect prokaryotic cells against the damaging agents tested . The pR protecting effect requires the expression of both the uvp1 and uvp2 (mucAB) regions in bacteria as well as in mouse cells . The coordinated function of these regions could result in protection against typical SOS inducers through an SOS/SOS-like pathway . The sensitization conferred by pR plasmid depends mostly on the expression of the mucAB genes, as shown by the survival of mouse cells transfected with different pR::Tn5 mutants . In particular, BLM and G418 survival data demonstrate that, inserted into the pR plasmid, the ble and neo genes of the Tn5 transposon express themselves . This was confirmed by the presence of Tn5 transcripts in untreated mouse cells . The comparison between the pR effects in bacterial and mouse cells shows that during evolution the repair pathways against UV damage are better conserved than those against other kinds of damage. Gene, 1994 Jul 22, 145(1), 153 - 4 Sequence of the flavodoxin-encoding gene from the cyanobacterium Synechocystis PCC6803; Poncelet M et al.; The flavodoxin-encoding gene of the unicellular, facultatively heterotrophic and transformable cyanobacterium Synechocystis PCC6803 was cloned and sequenced . This single-copy gene is highly conserved in prokaryotes, irrespective of their ability to perform photosynthesis. Proc Natl Acad Sci U S A, 1994 Jul 19, 91(15), 6870 - 4 Snapshot blotting: transfer of nucleic acids and nucleoprotein complexes from electrophoresis gels to grids for electron microscopy; Jett SD et al.; We present a technique, "snapshot blotting," for the electrophoretic transfer of nucleic acids and nucleoprotein complexes in gel electrophoresis bands onto highly stable carbon film-coated grids for imaging by electron microscopy . The method permits structural analysis of macromolecular species that have been resolved by a gel mobility-shift assay . To demonstrate the efficiency and integrity of the transfer process for a multiprotein-DNA assembly, we have imaged various species of a prokaryotic transcription complex, using the cleavage-defective EcoRI(Q111) protein as an orientation marker and as a blockade of transcription elongation . Snapshot blotting should be of great utility in the structural characterization of nucleic acids and protein-nucleic acid interactions. Proc Natl Acad Sci U S A, 1994 Jul 19, 91(15), 6735 - 42 Disparate rates, differing fates: tempo and mode of evolution changed from the Precambrian to the Phanerozoic; Schopf JW; Over the past quarter century, detailed genus- and species-level similarities in cellular morphology between described taxa of Precambrian microfossils and extant cyanobacteria have been noted and regarded as biologically and taxonomically significant by numerous workers world-wide . Such similarities are particularly well documented for members of the Oscillatoriaceae and Chroococcaceae, the two most abundant and widespread Precambrian cyanobacterial families . For species of two additional families, the Entophysalidaceae and Pleurocapsaceae, species-level morphologic similarities are supported by in-depth fossil-modern comparisons of environment, taphonomy, development, and behavior . Morphologically and probably physiologically as well, such cyanobacterial "living fossils" have exhibited an extraordinarily slow (hypobradytelic) rate of evolutionary change, evidently a result of the broad ecologic tolerance characteristic of many members of the group and a striking example of G . G . Simpson's {Simpson, G.G . (1944) Tempo and Mode in Evolution (Columbia Univ . Press, New York)} "rule of the survival of the relatively unspecialized." In both tempo and mode of evolution, much of the Precambrian history of life--that dominated by microscopic cyanobacteria and related prokaryotes--appears to have differed markedly from the more recent Phanerozoic evolution megascopic, horotelic, adaptationally specialized eukaryotes. Proc Natl Acad Sci U S A, 1994 Jul 19, 91(15), 6860 - 4 Wheat acetyl-coenzyme A carboxylase: cDNA and protein structure; Gornicki P et al.; cDNA fragments encoding part of wheat (Triticum aestivum) acetyl-CoA carboxylase (ACC; EC 6.4.1.2) were cloned by PCR using primers based on the alignment of several biotin-dependent carboxylases . A set of overlapping clones encoding the entire wheat ACC was then isolated by using these fragments as probes . The cDNA sequence contains a 2257-amino acid reading frame encoding a 251-kDa polypeptide . The amino acid sequence of the most highly conserved domain, corresponding to the biotin carboxylases of prokaryotes, is 52-55% identical to ACC of yeast, rat, and diatom . Identity with the available C-terminal amino acid sequence of maize ACC is 66% . The biotin attachment site has the typical eukaryotic EVMKM sequence . The cDNA does not encode an obvious chloroplast targeting sequence . Various cDNA fragments hybridize in Northern blots to a 7.9-kb mRNA . Southern analysis with cDNA probes revealed multiple hybridizing fragments in hexaploid wheat DNA . Some of the wheat cDNA probes also hybridize with ACC-specific DNA from other plants, indicating significant conservation among plant ACCs. FEBS Lett, 1994 Jul 18, 348(3), 244 - 8 Functional analysis of individual brain myosin II isoforms through hybrid formation; Wang Y et al.; We have used a scallop hybrid myosin test system in an attempt to determine the regulatory properties of an individual myosin II isoform from rat brain . The complete coding region of cDNA corresponding to a regulatory light chain isoform previously shown to be expressed in brain {Feinstein, Durand and Milner (1991) Mol . Brain Res . 10, 97-105} was ligated within the prokaryotic expression vector, pAED4, overexpressed in bacteria, and the purified light chain incorporated within a scallop hybrid myosin . Actin activation was calcium insensitive for all hybrids tested, irrespective of whether light chain phosphorylation had taken place before, or subsequent to, hybrid formation . We discuss the implications of these results, including the possibility that these results constitute evidence for a myosin II isoform within brain that is regulated at the level of the thin filament . In addition, evidence is presented for the presence of an additional, novel isoform of regulatory light chain expressed in rat brain. Eur J Biochem, 1994 Jul 15, 223(2), 447 - 53 Mechanism of resistance to the antibiotic trichothecin in the producing fungi; Iglesias M et al.; Trichothecium roseum, an imperfecti fungus producer of the translation inhibitor trichothecin, is constitutively resistant to its product . Fusarium oxysporum, a fungi not described as a toxin producer, is sensitive to trichothecin but becomes resistant when grown in the presence of the drug . In both cases, the resistance occurs at the level of the ribosomes . In cell-free polypeptide polymerization systems, trichothecin resistance is associated with the presence of 60S subunits from the resistant organisms . Resistant ribosomes can be prepared in vitro by incubating sensitive ribosomes, from either non-induced F . oxysporum or Saccharomyces cerevisiae, with cell extracts from the resistant cells in the presence of S-adenosylmethionine . An in-vitro specific differential methylation is detected in the sensitive ribosomes but not in resistant particles using radioactive S-adenosylmethionine . The results indicate for the first time the existence in eukaryotic organisms of an antibiotic-resistance mechanism involving a ribosomal methylation similar to that described previously in prokaryotic systems. Eur J Biochem, 1994 Jul 15, 223(2), 339 - 44 Expression, purification and functional properties of a soluble form of Bradyrhizobium japonicum TlpA, a thioredoxin-like protein; Loferer H et al.; The TlpA protein of Bradyrhizobium japonicum was previously identified genetically as a membrane-anchored, periplasmic thioredoxin-like protein . Here we describe the heterologous expression in Escherichia coli, subsequent purification and biochemical characterization of TlpA . A soluble form of TlpA, which lacks its N-terminal membrane anchor, was overexpressed in E . coli and purified by a two-step procedure . Pure TlpA was shown to be a monomer in solution and was active in reducing the disulfides of insulin and in reactivating reduced, denatured RNaseA . Evidence is presented that two non-active-site cysteine residues form an intramolecular disulfide bond, a feature that is not normally found in other prokaryotic thioredoxins. EMBO J, 1994 Jul 15, 13(14), 3389 - 94 A functional site of the GTPase-associated center within 28S ribosomal RNA probed with an anti-RNA autoantibody; Uchiumi T et al.; An anti-RNA autoantibody (anti-28S) was employed to identify structural and functional elements characteristic of a domain termed the 'GTPase center' in eukaryotic 28S ribosomal RNA . This antibody, an inhibitor of ribosome-associated GTP hydrolysis, has a unique property: it binds to the RNA domain of eukaryotes but not to that of prokaryotes . The antibody binding occurred in the presence of Mg2+ and protected from chemical modification three conserved bases (U1958, G1960 and A1990) and the base G1959 which is replaced by A in prokaryotic 23S rRNA (A1067 in Escherichia coli) . In vitro substitution of G1959 to A drastically weakened the antibody binding, and the reciprocal substitution, A1067-->G of the E.coli domain conferred the binding ability . This suggests that the G base determines the specificity of antibody binding . The G1959 was also protected by the association of ribosomes with elongation factor EF-2 . The result, together with protection of E.coli base A1067 by EFG {D.Moazed, I.M.Robertson and H.F.Noller (1988) Nature, 334, 362-364}, suggests that the position of G1959 in eukaryotes and A1067 in prokaryotes constitutes at least part of the factor binding site irrespective of the base replacement during evolution. Biochem J, 1994 Jul 15, 301 ( Pt 2), 557 - 61 Expression of rat liver S-adenosylmethionine synthetase in Escherichia coli results in two active oligomeric forms; Alvarez L et al.; A cDNA containing the complete coding sequence for rat liver S-adenosylmethionine synthetase was cloned into the prokaryotic expression vector pT7-7 and expressed in Escherichia coli BL21(DE3) . A major additional band corresponding to a protein of 48 kDa was detected on SDS/PAGE after induction with isopropyl beta-D-thiogalactopyranoside . This protein was distributed in both the soluble and insoluble fractions and accounted for approx . 30% of the total bacterial protein . The soluble enzyme was fully active, as revealed by assays in vitro of S-adenosylmethionine synthetase activity . In addition, transformed bacteria exhibited highly increased levels of intracellular S-adenosylmethionine . Two active forms of the recombinant enzyme, with apparent molecular masses of 210 kDa and 110 kDa, were detected when cytosolic extracts of the transformed cells were fractionated by gel-filtration chromatography . It is concluded that the expressed S-adenosylmethionine synthetase polypeptide assemble as tetramers and dimers. EMBO J, 1994 Jul 15, 13(14), 3378 - 88 An S18 ribosomal protein gene copy at the Arabidopsis PFL locus affects plant development by its specific expression in meristems; Van Lijsebettens M et al.; In Arabidopsis, mutation at PFL causes pointed first leaves, reduced fresh weight and growth retardation . We have cloned the wild-type PFL gene by T-DNA tagging, and demonstrate that it complements the mutant phenotype . PFL codes for ribosomal protein S18, based on the high homology with rat S18 and on purification of S18-equivalent peptides from plant ribosomes . pfl represents the first mutation in eukaryotic S18 proteins or their S13 prokaryotic counterparts, involved in translation initiation . Arabidopsis contains three S18 gene copies dispersed in the genetic map; they are all transcribed and code for completely identical proteins . No transcript is detected from the mutated gene, S18A . The activity of the S18A promoter is restricted to meristems, with a markedly high expression at the embryonic heart stage, and to wounding sites . This means that plants activate an extra copy of this ribosomal protein gene in tissues with cell division activity . We postulate that in meristematic tissues plants use transcriptional control to synthesize extra ribosomes to increase translational efficiency . In analogy with this, an additional, developmentally regulated gene copy might be expected for all ribosomal proteins. J Biol Chem, 1994 Jul 15, 269(28), 18572 - 5 Mapping of cystic fibrosis transmembrane conductance regulator membrane topology by glycosylation site insertion; Chang XB et al.; Technical difficulties in obtaining three-dimensional structures of intrinsic membrane proteins continues to limit understanding of their function . However, considerable insight can be gained from their two-dimensional topological arrangement in the lipid bilayer . Efficient molecular genetic approaches are available to discern the topology of prokaryotic but not of eukaryotic membrane proteins . The absolute asymmetry of the sidedness of their N-glycosylation was employed here to develop such a method using the cystic fibrosis transmembrane conductance regulator (CFTR) . Insertion by in vitro mutagenesis of N-glycosylation consensus sequences (NXS/T) in predicted cytoplasmic and extracytoplasmic loops between hydrophobic sequences capable of traversing the membrane established the membrane topology of CFTR . This provides the first experimental evaluation of the original topological model of CFTR based solely on hydropathy algorithms and a method which may be generally applicable for the in vivo evaluation of the topology of other mammalian membrane proteins. Nucleic Acids Res, 1994 Jul 11, 22(13), 2476 - 8 Prediction of an rRNA methyltransferase domain in human tumor-specific nucleolar protein P120; Koonin EV; Using computer methods for identification of amino acid motifs in sequence databases and multiple alignment, it is shown that human proliferation-associated nucleolar protein P120 contains a putative methyltransferase domain that is conserved in a group of bacterial proteins . It is hypothesized that P120 and the related prokaryotic proteins are rRNA methylases required for division of all types of cells. J Biol Chem, 1994 Jul 8, 269(27), 18118 - 27 The mechanism of translational coupling in Escherichia coli . Higher order structure in the atpHA mRNA acts as a conformational switch regulating the access of de novo initiating ribosomes; Rex G et al.; Bacterial genes are commonly transcribed to form polycistronic mRNAs bearing reading frames whose respective translational efficiencies are not independently determined . As in many bacterial operons, expression of the atp genes of Escherichia coli is strongly influenced by translational coupling . The gene pair atpHA is tightly coupled, whereby atpA is translated at least three times more efficiently than atpH . However, there is no fixed stoichiometry of coupling: mutations in atpH lead to increases in the translation ratio (atpA/atpH) of up to approximately 40-fold . We have demonstrated that secondary structure sequestering the atpA translational initiation region (TIR) is important to the coupling mechanism in that it inhibits de novo translational initiation at the atpA start codon . Genetic and structural analyses indicate that this inhibitory structure can be induced to refold into a less inhibitory conformation either by introducing two single-base substitutions or as a result of ribosomes translating atpH . We propose a model in which the secondary structure of the atpA TIR acts analogously to a "gating device" in that it restricts de novo ribosomal initiation until it is "switched" into a more open conformation . This contrasts with the function of a stem-loop structure located immediately downstream of atpA and upstream of the Shine-Dalgarno region of atpG, which was found to inhibit translation, but not to mediate tight coupling . Results obtained using the "specialized" ribosome system of Hui and de Boer ((1987) Proc . Natl . Acad . Sci . U.S.A . 84, 4762-4766) indicate that primarily ribosomes reinitiating after termination on atpH are responsible for inducing refolding of the atpA TIR . The principle of alternative mRNA conformations with different functional properties embodied in the model presented here can only be fulfilled by certain types of structure . It is likely to operate in several steps of prokaryotic gene expression, underlying a range of regulatory events including transcriptional attenuation and translational activation. Proc Natl Acad Sci U S A, 1994 Jul 5, 91(14), 6481 - 5 A role for a eukaryotic GrpE-related protein, Mge1p, in protein translocation; Laloraya S et al.; The 70-kDa heat shock proteins (hsp70s) function as molecular chaperones in a wide variety of cellular processes through cycles of binding and release from substrate proteins coupled to cycles of ATP hydrolysis . In the prokaryote Escherichia coli, the hsp70 DnaK functions with two other proteins, DnaJ and GrpE, which modulate the activity of DnaK . While numerous hsp70s and DnaJ-related proteins have been identified in eukaryotes, to our knowledge no GrpE-related proteins have been reported . We report the isolation and characterization of a eukaryotic grpE-related gene, MGE1 . MGE1, an essential nuclear gene of the yeast Saccharomyces cerevisiae, encodes a soluble protein of the mitochondrial matrix . Cells with reduced expression of Mge1p accumulate the precursor form of a mitochondrial protein . Since mitochondrial hsp70 is required for translocation of precursors of mitochondrial proteins from the cytosol into the matrix of mitochondria, these data suggest that Mge1p acts in concert with mitochondrial hsp70 in protein translocation. Genetics, 1994 Jul, 137(3), 791 - 801 The Drosophila molybdenum cofactor gene cinnamon is homologous to three Escherichia coli cofactor proteins and to the rat protein gephyrin; Kamdar KP et al.; Essentially all organisms depend upon molybdenum oxidoreductases which require a molybdopterin cofactor for catalytic activity . Mutations resulting in a lack of the cofactor show a pleiotropic loss of molybdoenzyme activities and thereby define genes involved in cofactor biosynthesis or utilization . In prokaryotes, two operons are directly associated with biosynthesis of the pterin moiety and its side chain while additional loci play a role in the acquisition of molybdenum and/or activation of the cofactor . Here we report the cloning of cinnamon, a Drosophila molybdenum cofactor gene encoding a protein with sequence similarity to three of the prokaryotic cofactor proteins . In addition, the Drosophila cinnamon protein is homologous to gephyrin, a protein isolated from the rat central nervous system . Our results suggest that some portions of the prokaryotic cofactor biosynthetic pathway composed of monofunctional proteins have evolved into a multifunctional protein in higher eukaryotes. Plant Mol Biol, 1994 Jul, 25(4), 735 - 8 Synechocystis sp . PCC6803 fusB gene, located outside of the str operon, encodes a polypeptide related to protein synthesis factor EF-G; Welcsh PL et al.; Synechocystis sp . PCC6803, a cyanobacterium, possesses an unusual gene (fusB) which encodes a protein with strong homology to protein synthesis elongation factor G (EF-G), although it is not linked to the classical str operon . The fusB gene is redundant, since a Synechocystis gene similar to str operon-encoded fusA genes of other bacteria is also present (based on PCR and hybridization results) . There is no evidence for the presence of a fusB homologue in other bacteria . The Synechocystis fusB gene encodes unusual amino acids at some positions that are highly conserved in fusA genes of other prokaryotes. J Mol Biol, 1994 Jul 1, 240(1), 20 - 7 The influence of adenine-rich motifs in the 3' portion of the ribosome binding site on human IFN-gamma gene expression in Escherichia coli; Chen H et al.; The ribosome binding site (RBS) of prokaryotic mRNA is divided into 5' and 3' portions by the translation initiation codon . Although it is well known that the presence of an appropriate RBS containing only the 5' portion is sufficient to direct the initiation of protein synthesis, the 3' portion appears to play a significant role in modulating the initiation process as well . Here we examine the influence of adenine-rich motifs frequently found in the 3' portion of highly expressed prokaryotic mRNAs . Two synthetic DNA fragments, GAGAAAAAAATC (corresponding to the first 12 nucleotides following the initiation codon of the chloramphenicol acetyltransferase gene), and AAAAAAATTAA were used to modify the beginning of the coding region of the human immune interferon-gamma (IFN-gamma) gene . The level of the protein synthesis in Escherichia coli directed by plasmids containing these constructs was quantitated . We found that placing either adenine-rich motif in the 3' portion of the RBS strongly enhanced gene expression, probably through an effect on translation initiation . We have also compared the protein expression levels of these gene constructs containing different series of 5'-RBSs with varying precistronic lengths and Shine-Dalgarno sequence lengths . The results suggest a positive functional role for the 3' adenine-rich motif . A possible mechanism for these effects is discussed. J Biol Chem, 1994 Jul 1, 269(26), 17397 - 400 A heteroduplex template circumvents the energetic requirement for ATP during activated transcription by RNA polymerase II; Tantin D et al.; Unlike most eukaryotic and prokaryotic RNA polymerases, promoter-specific transcription by RNA polymerase II requires hydrolysis of the ATP beta-gamma phosphoanhydride bond . Here we show that a template containing a 10-base pair DNA mismatch encompassing the start site circumvents this requirement such that the non-hydrolyzable ATP analogues, ATP gamma S (adenosine 5'-O-(thiotriphosphate)) and AMP-PNP (adenyl-5'-yl imidodiphosphate), support both basal and GAL4-VP16-activated transcription in a reconstituted HeLa cell in vitro transcription system . The results imply that ATP regulates either opening of the template at the start site or a closely associated step. J Bacteriol, 1994 Jul, 176(13), 4025 - 33 Nucleotide sequence and mutational analysis of the gene encoding KpsD, a periplasmic protein involved in transport of polysialic acid in Escherichia coli K1; Wunder DE et al.; The 17-kb kps gene cluster encodes proteins necessary for the synthesis, assembly, and translocation of the polysialic acid capsule of Escherichia coli K1 . We previously reported that one of these genes, kpsD, encodes a 60-kDa periplasmic protein that is involved in the translocation of the polymer to the cell surface . The nucleotide sequence of the 2.4-kb BamHI-PstI fragment accommodating the kpsD gene was determined . Sequence analysis showed an open reading frame for a 558-amino-acid protein with a typical N-terminal prokaryotic signal sequence corresponding to the first 20 amino acids . KpsD was overexpressed, partially purified, and used to prepare polyclonal antiserum . A chromosomal insertion mutation was generated in the kpsD gene and results in loss of surface expression of the polysialic acid capsule . Immunodiffusion analysis and electron microscopy indicated that polysaccharide accumulates in the periplasmic space of mutant cells . A wild-type copy of kpsD supplied in trans complemented the chromosomal mutation, restoring extracellular expression of the K1 capsule . However, a kpsD deletion derivative (kpsD delta C11), which results in production of a truncated KpsD protein lacking its 11 C-terminal amino acids, was nonfunctional . Western blot (immunoblot) data from cell fractions expressing KpsD delta C11 suggest that the truncated protein was inefficiently exported into the periplasm and localized primarily to the cytoplasmic membrane. J Bacteriol, 1994 Jul, 176(13), 3903 - 10 Cloning and nucleotide sequences of the genes encoding triose phosphate isomerase, phosphoglycerate mutase, and enolase from Bacillus subtilis; Leyva-Vazquez MA et al.; The Bacillus subtilis genes tpi, pgm, and eno, encoding triose phosphate isomerase, phosphoglycerate mutase (PGM), and enolase, respectively, have been cloned and sequenced . These genes are the last three in a large putative operon coding for glycolytic enzymes; the operon includes pgk (coding for phosphoglycerate kinase) followed by tpi, pgm, and eno . The triose phosphate isomerase and enolase from B . subtilis are extremely similar to those from all other species, both eukaryotic and prokaryotic . However, B . subtilis PGM bears no resemblance to mammalian, fungal, or gram-negative bacterial PGMs, which are dependent on 2,3-diphosphoglycerate (DPG) for activity . Instead, B . subtilis PGM, which is DPG independent, is very similar to a DPG-independent PGM from a plant species but differs from the latter in the absolute requirement of B . subtilis PGM for Mn2+ . The cloned pgm gene has been used to direct up to 25-fold overexpression of PGM in Escherichia coli; this should facilitate purification of large amounts of this novel Mn(2+)-dependent enzyme . Inactivation of pgm plus eno in B . subtilis resulted in extremely slow growth either on plates or in liquid, but growth of these mutants was enhanced by supplementation of media with malate . However, these mutants were asporogenous with or without malate supplementation. Mol Microbiol, 1994 Jul, 13(1), 17 - 23 When replication forks stop; Bierne H et al.; DNA synthesis is an accurate and very processive phenomenon, yet chromosome replication does not proceed at a constant rate and progression of the replication fork can be impeded . Several structural and functional features of the template can modulate the rate of progress of the replication fork . These include DNA secondary structures, DNA damage and occupied protein-binding sites . In addition, prokaryotes contain sites where replication is specifically arrested . DNA regions at which the replication machinery is blocked or transiently slowed could be particularly susceptible to genome rearrangements . Illegitimate recombination, a ubiquitous phenomenon which may have dramatic consequences, occurs by a variety of mechanisms . The observation that some rearrangements might be facilitated by a pause in replication could provide a clue in elucidating these processes . In support of this, some homologous and illegitimate recombination events have already been correlated with replication pauses or arrest sites. Genomics, 1994 Jul 1, 22(1), 205 - 10 The identification of novel gene sequences of the human adult testis; Affara NA et al.; To facilitate the characterization of genetic expression in human adult testis, expressed sequence tag analysis of cDNAs from this tissue has been undertaken . Over 180 kb of DNA sequence has been determined and used to search the GenBank database . The results from the first 359 cDNA clones analyzed indicate that the sequences could be sorted into several categories with a high proportion being novel . Twenty-five clones (7%) showed 100% identity with human genes, 11 (3%) with prokaryotic sequences, 21 (5%) with between 60 and 95% similarity to human genes, 27 (8%) with between 60 and 95% similarity to genes from other species, and 33 (9%) with matches to human repeat sequences . Two hundred forty-two (67%) showed no significant matches and thus are likely to represent novel transcripts . In comparison to similar studies on human brain tissue and a hepatoma cell line, the findings indicate that the matches in the testis transcript population appear to be identifying a different spectrum of gene sequences. Bioessays, 1994 Jul, 16(7), 497 - 502 Oxygen and the control of gene expression; Pahl HL et al.; The respiration of oxygen, while essential to aerobic organisms for the generation of energy, leads to the formation of reactive oxygen intermediates (ROIs) as harmful byproducts . ROIs damage nucleic acids, lipids and proteins . Therefore, protective mechanisms against elevated intracellular ROI levels, referred to as oxidative stress, have evolved . These include the activation of transcription factors which elevate the expression of protective enzymes . Eukaryotic cells have also evolved the ability to specifically generate ROIs following stimulation with various agents . In these cases, ROIs are used as second messengers to activate gene expression . Here we will discuss both prokaryotic and eukaryotic transcription factors that respond to ROIs . In addition, transcription factors will be described that are activated by either exposure to antioxidants, which reduce the intracellular ROI concentration, or by hypoxia, the absence of oxygen. Pediatr Res, 1994 Jul, 36(1 Pt 1), 1 - 6 How the cell copes with stress and the function of heat shock proteins; Schlesinger MJ; Virtually all cells, including the prokaryotic microorganisms and the highly differentiated eukaryotic cells in human tissues, contain a small set of normally silent genes that are rapidly activated by a heat shock that raises the temperature only 5 to 10% above that of the normal physiologic range for that organism . Concomitantly, many active genes are turned off . Other kinds of stress, such as exposure to alcohol or other organic agents, heavy metals, oxidants, and agents capable of perturbing protein structure, produce a similar response, and many of these activate the same set of genes . The proteins encoded by these stress-activated genes are called heat shock proteins (hsp) . They are strongly conserved in structure among widely divergent biologic species, and many function as "molecular chaperones" by forming transient complexes with partially folded or misfolded polypeptides so as to prevent their irreversible denaturation . Most hsp are members of gene/protein families, and isoforms are frequently found under normal physiologic conditions in many compartments of the cell where they act also as chaperones, binding to a variety of polypeptides to facilitate folding, oligomerization, transport, metabolic activity, and degradation . Few of the polypeptide "targets" that complex with stress-induced forms of hsp have been identified, but a number of cellular components have been shown to be particularly stress sensitive.(ABSTRACT TRUNCATED AT 250 WORDS) J Clin Microbiol, 1994 Jul, 32(7), 1710 - 7 General primer-mediated PCR for detection of Aspergillus species; Melchers WJ et al.; A PCR assay was developed for the diagnosis of invasive aspergillosis in immunocompromised patients . For this purpose, the complete nucleotide sequences of the genes encoding the 18S rRNA of Aspergillus nidulans, Aspergillus terreus, Aspergillus niger, and Aspergillus flavus were elucidated and aligned to the sequences of Aspergillus fumigatus and other clinically relevant prokaryotic and eukaryotic microorganisms . Genus-specific sequences could be identified in the V7 to V9 region of 18S rRNA . By using hot-start PCR, Southern blot hybridization, and restriction enzyme analysis, Aspergillus-specific and -sensitive determination was achieved . Five of six immunosuppressed mice experimentally infected with A . fumigatus developed infection, and rRNA could be detected in each case, even in livers with the absence of positive cultures . Aspergillus species were detected by PCR in four neutropenic patients with proven aspergillosis, although Aspergillus species had been isolated from only one bronchoalveolar lavage (BAL) fluid sample . Aspergillus species were detected by PCR in two more patients suspected of having infection . Positive PCR signals were obtained from the BAL samples of 3 of 8 neutropenic patients who had developed pulmonary infiltrates, but none were obtained from the samples of 14 nonimmunosuppressed patients . These results indicate the potential value of PCR to detect Aspergillus species in BAL samples and, therefore, to identify neutropenic patients at risk for invasive aspergillosis. Braz J Med Biol Res, 1994 Jul, 27(7), 1517 - 25 Mutagenic and genotoxic effects of mate (Ilex paraguariensis) in prokaryotic organisms; Leitao AC et al.; 1 . The mutagenic and genotoxic effects of mate (Ilex paraguariensis) aqueous solutions were analyzed in bacterial cells . 2 . Mate solutions showed mutagenic activity in the Ames test (TA97, TA98, TA100 and TA102 strains) at concentrations of 20 to 50 mg/plate (mutagenic factors of 3.5 to 5.6) and genotoxic activity in the inductest (WP2s (lambda) strain), with a maximal phage induction at concentrations of 10 to 20 mg/plate . Above these concentrations the mate solutions were cytotoxic . 3 . Addition of 5 U/ml catalase, 20 microliters/ml S9 rat liver microsomal fraction, 100 mM thiourea or 10 mM dipyridyl completely inhibited the lysogenic induction produced by mate; however, the addition of 1,000 U/ml superoxide dismutase was almost ineffective . 4 . Oxygen reactive species may be present in mate solutions playing an essential role in its genotoxicity. J Biochem (Tokyo), 1994 Jul, 116(1), 221 - 7 Human complex II (succinate-ubiquinone oxidoreductase): cDNA cloning of the flavoprotein (Fp) subunit of liver mitochondria; Hirawake H et al.; Complex II (succinate-ubiquinone oxidoreductase) is an important enzyme complex in both the tricarboxylic acid cycle, and the aerobic respiratory chains of mitochondria in eukaryotic cells and prokaryotic organisms . In this study, homology probing with mixed primers for the polymerase chain reaction and subsequent sequence analysis were successfully applied to clone cDNA for the flavoprotein (Fp) subunit of human liver complex II . The isolated clone contains an open reading frame of 1,992 nucleotides and encodes a mature protein of 621 amino acids with a molecular weight of 68,011 . The amino acid sequence was highly homologous with that of bovine heart Fp (93.2%) and was quite different from the partial sequence of human placental Fp reported previously {Malcovati et al . (1991) in Flavins and Flavoproteins 1990, pp . 727-730}, which showed striking homology to that of Bacillus subtilis . To solve this discrepancy, the partial cDNA sequences of the stomach and placental Fp subunits of human complex II were determined in addition to the full length cDNA of liver . The sequence data, sensitivity to thiol reagents and antigenic properties indicated that the major from of FP subunit in human complex II is unique at least among the three tissues analyzed, and is more similar to the Fp subunit of bovine heart than to that of B . subtilis. J Clin Invest, 1994 Jul, 94(1), 228 - 36 Cystic fibrosis transmembrane conductance regulator mutations that disrupt nucleotide binding; Logan J et al.; Increasing evidence suggests heterogeneity in the molecular pathogenesis of cystic fibrosis (CF) . Mutations such as deletion of phenylalanine at position 508 (delta F508) within the cystic fibrosis transmembrane conductance regulator (CFTR), for example, appear to cause disease by abrogating normal biosynthetic processing, a mechanism which results in retention and degradation of the mutant protein within the endoplasmic reticulum . Other mutations, such as the relatively common glycine-->aspartic acid replacement at CFTR position 551 (G551D) appear to be normally processed, and therefore must cause disease through some other mechanism . Because delta F508 and G551D both occur within a predicted nucleotide binding domain (NBD) of the CFTR, we tested the influence of these mutations on nucleotide binding by the protein . We found that G551D and the corresponding mutation in the CFTR second nucleotide binding domain, G1349D, led to decreased nucleotide binding by CFTR NBDs, while the delta F508 mutation did not alter nucleotide binding . These results implicate defective ATP binding as contributing to the pathogenic mechanism of a relatively common mutation leading to CF, and suggest that structural integrity of a highly conserved region present in over 30 prokaryotic and eukaryotic nucleotide binding domains may be critical for normal nucleotide binding. Mutat Res, 1994 Jul, 315(1), 75 - 84 Recent advances in DNA repair, mutation and recombination . A report of the meeting of the British Photobiology Society, London, 20 December 1993; Strike P; The 1993 British Photobiology Society DNA Repair meeting was held at City University, London, on December 20th . The well supported meeting heard presentations covering diverse aspects of repair, mutation and recombination in both prokaryotes and eukaryotes . The meeting particularly aims to provide a forum for presentations from postgraduate and postdoctoral workers, and contributions were received from many of the British laboratories engaged in these fields. Biochim Biophys Acta, 1994 Jun 28, 1186(1-2), 27 - 34 Isolation of Tn917 insertional mutants of Bacillus subtilis that are resistant to the protonophore carbonyl cyanide m-chlorophenylhydrazone; Quirk PG et al.; Tn917 transposition libraries prepared from Bacillus subtilis were screened for mutants that had insertions in the chromosome resulting in resistance to the protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) . Five such strains were characterized . Three of these were found to have distinct insertion sites that resulted in changes in fatty acid composition of the membrane lipids . The lipid changes were qualitatively similar to changes observed earlier in CCCP-resistant strains of B . subtilis that had been isolated after chemical mutagenesis . However, the extent of the changes was more modest, correlating with a lower level of protonophore-resistance . One of these mutants was disrupted in a gene homologous to the Escherichia coli rho gene, as reported earlier (Quirk et al . (1993) J . Bacteriol . 175, 647-654), one was disrupted in a new member of the two-component signalling systems, and the third was disrupted in a new gene of unknown function that apparently forms an operon with transporter genes . The other two CCCP-resistant mutants were disrupted in genes that are likely to encode membrane transporters; the disruption of these genes may have reduced the transmembrane ion leaks during growth, thus conferring modest protonophore-resistance . In one of these strains, the disrupted gene is part of an apparent operon that is a homologue of iron uptake operons from other prokaryotes. FEBS Lett, 1994 Jun 27, 347(2-3), 268 - 72 Isolation and characterization of a cDNA clone encoding the pokeweed antiviral protein II from Phytolacca americana and its expression in E . coli; Poyet JL et al.; Three distinct ribosome-inactivating proteins (RIPs) were isolated from pokeweed (Phytolacca americana) . We identified and sequenced for the first time a complete cDNA encoding the pokeweed antiviral protein II (PAP II), which is expressed in the late summer leaves of pokeweed . The cDNA of PAP II consists of 1,187 nucleotides and encodes a mature protein of 285 amino acids . Its predicted amino acid sequence is only 33% similar to PAP and PAP-S . The NH2 terminal extrapeptide (25 amino acid residues) was similar but not identical to that of PAP's extrapeptide . The cDNA of PAP II was expressed in E . coli . The growth of the transformants was strongly inhibited after induction of the gene . Furthermore, PAP II, which was produced in E . coli, inhibited protein synthesis in a rabbit reticulocyte translation system . Thus, recombinant PAP II would appear to be as functional as native PAP in inhibiting protein synthesis in both prokaryotes and eukaryotes. Nucleic Acids Res, 1994 Jun 25, 22(12), 2228 - 33 Eukaryotic selenocysteine inserting tRNA species support selenoprotein synthesis in Escherichia coli; Baron C et al.; Although the tRNA species directing selenocysteine insertion in prokaryotes differ greatly in their primary structure from that of their eukaryotic homologues they share very similar three-dimensional structures . To analyse whether this conservation of the overall shape of the molecules reflects a conservation of their functional interactions it was tested whether the selenocysteine inserting tRNA species from Homo sapiens supports selenoprotein synthesis in E . coli . It was found that the expression of the human tRNA(Sec) gene in E.coli can complement a lesion in the tRNA(Sec) gene of this organism . Transcripts of the Homo sapiens and Xenopus laevis tRNA(Sec) genes synthesised in vitro were amino-acylated by the E.coli seryl-tRNA ligase although at a very low rate and the resulting seryl-tRNA(Sec) was bound to and converted into selenocysteyl-tRNA(Sec) by the selenocysteine synthase of this organism . Selenocysteyl-tRNA(Sec) from both eukaryotes was able to form a complex with translation factor SELB from E.coli . Although the mechanism of selenocysteine incorporation into seleno-proteins appears to be rather different in E.coli and in vertebrates, we observe here a surprising conservation of functions over an enormous evolutionary distance. Gene, 1994 Jun 24, 144(1), 37 - 43 The ASP1 gene of Saccharomyces cerevisiae, encoding the intracellular isozyme of L-asparaginase; Sinclair K et al.; Saccharomyces cerevisiae produces two L-asparaginases (ASPs), intracellular ASP I and cell-wall ASP II . In this report, the ASP-I-encoding gene, ASP1, has been identified by homology cloning based on the structures of ASPs from other organisms . Its deduced protein product has a subunit M(r) of 41,414, and shows substantial sequence homology to the bacterial amidohydrolase family . The product of the S . cerevisiae ASP3 gene, a further member of this family, encoding the nitrogen catabolite-regulated cell-wall ASP II, has 46% overall sequence identity to ASP1 . Duplication of ancestral asparaginase genes, resulting in separate intra- and extracellular isozymes, appears to have occurred independently in the prokaryotic and eukaryotic lineages . Exact physical mapping of the new cloned ASP1 gene locates it 73% of the distance from the left telomere of chromosome IV, at a position precisely matching the known genetic map location of ASP1 . This, along with the structural features of the clone, confirms that ASP1 is the structural gene encoding cytoplasmic ASP I in S . cerevisiae . Sequence analysis of the ethylmethanesul