J Biol Chem, 1993 Apr 5, 268(10), 7514 - 9 SecY, an integral subunit of the bacterial preprotein translocase, is encoded by a plastid genome; Flachmann R et al.; Although the paradigm for the acquisition of photosynthetic organelles is the endocytosis of cyanobacteria-like progenitors by heterotrophic protists, details of this evolutionary process are unclear . The small organellar chromosomes are remnants of the larger bacterial genomes with most genes from the endosymbiont's DNA having been either relocated to the protist's nucleus or entirely lost . As a result of those gene transfers, differences exist between plastids from different algal phyla and higher plants . We report here on the retention of a secY gene in cyanelle (= plastid) DNA of the eukaryotic protist Cyanophora paradoxa . This cyanelle secY encodes a functional protein homologous to SecY of Escherichia coli, identified as a subunit of the preprotein translocase complex . Similarity of the cyanelle and E . coli SecY topology, predicted from sequence information, has been confirmed experimentally through SecY-PhoA fusion protein analysis in E . coli . Cyanelle SecY, expressed in an E . coli secY mutant, substituted for the defective prokaryotic SecY . A plastid-encoded gene for a membrane protein functioning in protein transport across plastid membranes is unprecedented in higher plants . From these results we infer that a functional homolog of the prokaryotic preprotein translocation machinery is retained in some plastids. J Immunol Methods, 1993 Apr 2, 160(2), 261 - 6 An ELISA for blood group specific exoglycosidases; Hobbs L et al.; An enzyme-linked immunosorbent assay (ELISA) for studying erythrocyte A, B and H epitope specific exoglycosidases is described . Human blood type B erythrocyte membranes and Coffea canephora alpha-D-galactosidase were used as a model . Membrane coated microtiter wells were incubated with exoglycosidase, probed with IgM monoclonal antibody, and then with anti-murine mu chain specific alkaline phosphatase conjugate . The assay is useful for studying exoglycosidase modification of the A, B and H epitopes on human erythrocyte membranes as well as in screening prokaryotic and eukaryotic extracts for blood group active enzymes . Furthermore, this technique has the advantage of simplicity, sensitivity, and objectivity of data interpretation. J Biochem (Tokyo), 1993 Apr, 113(4), 456 - 61 Rabbit plasma alpha-1-antiproteinase S-1: cloning, sequencing, expression, and proteinase inhibitory properties of recombinant protein; Saito A et al.; A cDNA clone coding for the isoform S-1 of alpha-1-antiproteinase (also called alpha-1-proteinase inhibitor or alpha-1-antitrypsin) was isolated from rabbit liver cDNA library and sequenced . The cDNA consists of 1,426 nucleotides including 5' and 3' noncoding regions and codes for 413 amino acid residues including a signal peptide of 24 residues . The nucleotide and deduced amino acid sequences show 95.5 and 95.2% homologies, respectively, with the F isoform which occurs more abundantly in the rabbit serum than the S isoform . Of the 20 amino acid differences between the two isoforms, nine are located in a stretch of 15 amino acids encompassing the reactive site region, suggesting that these genes have diverged from each other by a nonrandom mechanism . A hypothesis is proposed that the domestication of animal is responsible for the extremely high evolutionary rate in the serpin reactive site region . Prokaryotic expression plasmids were constructed from the cDNA, transfected into Escherichia coli, and expressed . Partially purified recombinant protein inhibited elastase, but did not inhibit trypsin when a small substrate was used . The recombinant S-1 form, however, protected trypsin from inactivation by soybean trypsin inhibitor, a property characteristic of alpha-macroglobulins or rodent murinoglobulins . It is known that there are two types of interaction between serpin and proteinase: (i) most serpins form a stable equimolar complex with the enzyme, resulting in the enzyme inhibition and (ii) some serpins act as a substrate rather than as an inhibitor, resulting in the loss of inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS) AIDS Res Hum Retroviruses, 1993 Apr, 9(4), 343 - 50 Human endogenous retroviral element K10 (HERV-K10) encodes a full-length gag homologous 73-kDa protein and a functional protease; Mueller-Lantzsch N et al.; The gag-homologous region of the human endogenous retrovirus K10 (HERV-K10) was amplified by PCR from human genomic DNA and was analyzed by DNA cloning, sequencing, and expression of open reading frames in the prokaryotic pATH expression system . The analysis of genomic DNA of three donors provided evidence that HERV-K10 genes contain an open reading frame of 1966 bp spanning the entire gag-homologous region . In the prokaryotic system the entire reading frame of the HERV-K10 gag gene could be expressed as a fusion protein exhibiting a molecular weight of about 110,000 . In addition, when the gag-homologous region and the adjacent HERV-K10 protease gene were prokaryotically expressed, we observed a Gag-protease fusion protein that exhibited specific autoproteolytic activities and processing of HERV-K10 Gag protein . By introducing deletions on the right end of the putative protease gene an autocatalytic site could be localized within 300 bp of the putative HERV-K10 protease gene . For the first time, these results provide evidence that the HERV-K10 encodes a full-length Gag protein and a functional protease. Curr Opin Cell Biol, 1993 Apr, 5(2), 232 - 7 Chromosome segregation and cytokinesis in bacteria; de Boer PA; Substantial progress has recently been made in the understanding of chromosome partitioning and cytokinesis in bacteria . The biochemical properties of some key protein components involved in these processes are beginning to emerge . New evidence supports the recently developed notion that, in prokaryotic cells, basic cell biological processes rely on the activity of previously unidentified cytoskeletal-like elements. Exp Eye Res, 1993 Apr, 56(4), 497 - 507 Structure of the bovine transducin gamma subunit gene and analysis of promoter function in transgenic mice; Tao L et al.; Transducin, the major photoreceptor guanine nucleotide-binding protein (G protein), is composed of three polypeptides: alpha, beta and gamma subunits . The transducin gamma subunit (T gamma) is expressed preferentially in photoreceptors . To study the control mechanisms for photoreceptor-specific expression of the T gamma gene, clones of the bovine T gamma gene were isolated from a bacteriophage genomic library, and the structure of the gene, including a portion of its 5'-flanking region, was characterized . The gene consists of three exons and two introns . The first intron is 91 base pairs (bp) long and is located in the region corresponding to the 5'-untranslated sequence of the T gamma mRNA . The second intron is 5.3 kilobases (kb) long and splits the protein-coding region centrally . A bovine Alu-type repetitive sequence and putative Ret-1 and AP-1 binding site sequences are located in the 5'-flanking region . To investigate promoter function, 1.4 kb of DNA from the 5'-flanking region was joined to the prokaryotic chloramphenicol acetyltransferase (CAT) gene, and the chimeric bovine T gamma gene was used to generate a line of transgenic mice . CAT activity was readily detected in the retinas of the transgenic mice, but was absent in brain, heart, kidney, liver, lung, spleen and other tissues . These results suggest that the 1.4 kb 5'-flanking region of the bovine T gamma gene contains conserved sequence elements that direct tissue-specific expression . Human T gamma cDNA clones were characterized, and a short homologous region of the human T gamma gene promotor was obtained by polymerase chain reaction (PCR) amplification for comparison with the bovine promoter. Biochem J, 1993 Apr 1, 291 ( Pt 1), 179 - 86 Expression of rat liver ketohexokinase in yeast results in fructose intolerance; Donaldson IA et al.; Rat liver ketohexokinase (ATP:D-fructose 1-phosphotransferase; EC 2.7.1.3) was purified to homogeneity and the molecular mass of the protein was found by mass spectrometry to be 32,800 Da . The enzyme was cleaved and the amino acid sequences of seven peptides, comprising 24% of the total sequence, were determined . This sequence information was used to design oligonucleotide primers for a PCR using rat liver single-stranded cDNA as a template . The 224 bp PCR product was used as a probe to screen a rat liver cDNA library . A cDNA sequence of 1342 bp was obtained from three positive clones . This contained the entire coding region for ketohexokinase, and all seven peptides were identified in the predicted amino acid sequence . When ketohexokinase was expressed in Saccharomyces cerevisiae using the yeast expression vector pMA91, the cells became intolerant of the presence of fructose in their growth media . The growth of an exponential-phase culture was completely arrested within 90 min by the addition of fructose to a final concentration as low as 0.1% (w/v) . This response is associated with an accumulation of fructose 1-phosphate . The cDNA for ketohexokinase encodes a protein composed of 299 amino acids with a combined molecular mass of 32,728 Da . This is in close agreement with the value for the isolated protein determined by mass spectrometry . The primary structure does not show any significant homology with those of other eukaryotic hexokinases, but it contains a highly conserved region that is present in three prokaryotic phosphotransferases that have furanose substrates. J Bacteriol, 1993 Apr, 175(8), 2370 - 8 Structures of genes nasA and nasB, encoding assimilatory nitrate and nitrite reductases in Klebsiella pneumoniae M5al; Lin JT et al.; Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources during aerobic growth . Assimilatory nitrate and nitrite reductases convert nitrate through nitrite to ammonium . We report here the molecular cloning of the nasA and nasB genes, which encode assimilatory nitrate and nitrite reductase, respectively . These genes are tightly linked and probably form a nasBA operon . In vivo protein expression and DNA sequence analysis revealed that the nasA and nasB genes encode 92- and 104-kDa proteins, respectively . The NASA polypeptide is homologous to other prokaryotic molybdoenzymes, and the NASB polypeptide is homologous to eukaryotic and prokaryotic NADH-nitrite reductases . The narL gene product positively regulates expression of the structural genes for respiratory nitrate reductase, narGHJI . Surprisingly, we found that the nasBA operon is tightly linked to the narL-narGHJI region in K . pneumoniae, even though the nitrate assimilatory and respiratory enzymes serve different physiological functions. DNA Cell Biol, 1993 Apr, 12(3), 253 - 63 Structure of the rat catechol-O-methyltransferase gene: separate promoters are used to produce mRNAs for soluble and membrane-bound forms of the enzyme; Tenhunen J et al.; The enzyme catechol-O-methyltransferase (COMT) catalyzes the inactivation of catechol-containing molecules by methylation . The cDNAs for the rat and human COMT have recently been cloned and recombinant proteins expressed in prokaryotic and eukaryotic cells . We describe here the structure of the rat COMT gene and its 5'-flanking sequences . The gene spans at least 13 kb and is composed of 5 exons, the first one noncoding . The two ATG codons for the initiation of translation of the membrane-bound (MB-COMT) and soluble (S-COMT) forms of the enzyme reside in the second exon . The gene expresses two mRNA species of 1.6 kb and 1.9 kb that have different tissue distributions . The expression of the transcripts is regulated by at least two promoters, P1 and P2 . The P1 promoter expresses the shorter transcript in a tissue-specific manner and is located between the ATG codons in the coding region of the longer transcript . The P2 promoter is constitutive and responsible for the expression of the longer transcript . The shorter 1.6-kb mRNA (S-mRNA) produces only the S-COMT polypeptide, whereas the longer 1.9-kb mRNA (MB-mRNA) is able to direct synthesis of both forms of the COMT enzyme. Infect Immun, 1993 Apr, 61(4), 1202 - 10 Lipid modification of the 17-kilodalton membrane immunogen of Treponema pallidum determines macrophage activation as well as amphiphilicity; Akins DR et al.; A murine monoclonal antibody specific for a 17-kDa major membrane immunogen of Treponema pallidum was used to select recombinant Escherichia coli clones expressing the molecule from a T . pallidum genomic library . Sequence analysis of the structural gene for the immunogen (designated tpp17) revealed a 468-bp open reading frame encoding a polypeptide of 156 amino acids with a calculated molecular mass of 16,441 Da . The deduced amino acid sequence included a putative leader peptide terminated by a consensus tetrapeptide for the modification and processing of prokaryotic lipoproteins . Immunoprecipitation of the cloned immunogen radiolabeled with {3H}palmitate confirmed that it was a lipoprotein . The amino acid sequence also predicted that the mature protein contains four cysteine residues in addition to the lipid-modified cysteine of the N terminus . The existence of disulfide-bonded multimeric forms of the native immunogen was demonstrated by immunoblotting T . pallidum solubilized in the presence and absence of 2-mercaptoethanol . Triton X-114 phase partitioning of a nonlipidated form of the 17-kDa immunogen cleaved from a glutathione S-transferase fusion protein demonstrated that lipid modification is responsible for the immunogen's hydrophobic character . The same nonlipidated form of the immunogen also was used to demonstrate that lipid modification is essential for the molecule's ability to stimulate production of tumor necrosis factor alpha by murine macrophages . We conclude that covalently attached fatty acids not only anchor T . pallidum lipoproteins to spirochetal membranes but also confer upon these molecules the ability to activate immune effector cells. J Virol, 1993 Apr, 67(4), 2221 - 7 RNA-binding activity of hepatitis delta antigen involves two arginine-rich motifs and is required for hepatitis delta virus RNA replication; Lee CZ et al.; Hepatitis delta antigen (HDAg) is an RNA-binding protein with binding specificity for hepatitis delta virus (HDV) RNA (J . H . Lin, M . F . Chang, S . C . Baker, S . Govindarajan, and M . M . C . Lai, J . Virol . 64:4051-4058, 1990) . By amino acid sequence homology search, we have identified within its RNA-binding domain two stretches of an arginine-rich motif (ARM), which is present in many prokaryotic and eukaryotic RNA-binding proteins . The first one is KERQDHRRRKA and the second is EDEKRERRIAG, and they are separated by 29 amino acids . Deletion of either one of these ARM sequences resulted in the total loss of the in vitro RNA-binding activity of HDAg . Thus, HDAg is different from other RNA-binding proteins in that it requires two ARM-like sequences for its RNA-binding activity . Replacement of the spacer sequence between the two ARMs with a shorter stretch of sequence also reduced RNA binding in vitro . Furthermore, site-specific mutations of the basic amino acid residues in both ARMs resulted in the total loss or reduction of RNA-binding activity . The biological significance of the RNA-binding activity was studied by examining the trans-activating activity of the RNA-binding mutants . The plasmids expressing HDAgs with various mutations in the RNA-binding motifs were cotransfected with a replication-defective HDV dimer cDNA construct into COS cells . It was found that all the HDAg mutants which had lost the in vitro RNA-binding activity also lost the ability to complement the defect of HDV RNA replication . We conclude that the trans-activating function of HDAg requires its binding to HDV RNA. J Protein Chem, 1993 Apr, 12(2), 207 - 13 The role of an active-site lysyl residue of spinach phosphoribulokinase as explored by site-directed mutagenesis; Mural RJ et al.; Based on selective labeling by ATP analogues, Lys68 of the Calvin Cycle enzyme phosphoribulokinase (PRK) from spinach has been assigned to the active-site region {Miziorko et al . (1990), J . Biol . Chem . 265, 3642-3647} . The equivalent position is occupied by lysyl or arginyl residues in the PRK from both prokaryotic and eukaryotic sources, suggesting a requirement for a basic residue at this location . To examine this possibility, we have replaced Lys68 of the spinach enzyme with arginyl, glutaminyl, alanyl, or glutamyl residues by site-directed mutagenesis . All of the mutant enzymes retain substantial kinase activity; and even in the case of the radical substitution by glutamate, the Km values for ATP and ribulose 5-phosphate are not perturbed significantly . Glutamate at position-68 may destabilize tertiary structure, because the yield of this mutant protein from transformed E . coli is quite low compared to that of the other proteins in this series . Despite the active-site proximity of Lys68, our results show that this residue does not play a key role in catalysis or substrate binding. Am J Physiol, 1993 Apr, 264(4 Pt 2), R804 - 10 Electrogenic 2 Na/1 H antiport in crustacean epithelium is inhibited by a monoclonal antibody; Gert de Couet H et al.; We have previously published evidence that suggests that Na/H exchange in crustacean and echinoderm epithelia occurs by an electrogenic antiporter protein with two external cation binding sites that accommodate Na, amiloride, or Ca and display a 2:1 monovalent cation antiport stoichiometry . The present study is an initial investigation into the molecular biology of this invertebrate electrogenic exchanger to ascertain its structural similarity to the analogous vertebrate electroneutral antiport system . A panel of monoclonal antibodies was prepared against components of lobster hepatopancreatic epithelial brush-border membranes and assayed immunohistochemically and by Western blotting . The antibodies were tested further in functional assays for their ability to interfere with electrogenic 2 Na/1 H antiport in isolated hepatopancreatic brush-border membrane vesicles . One cell line was identified producing an antibody that significantly inhibited the electrogenic exchange of cations by these membrane preparations and recognized a single protein band on Western blots of hepatopancreas, antennal gland, and gill epithelia corresponding to a molecular mass of 185 kDa . The existence of such an antibody probe may facilitate the purification of the electrogenic antiporter under denaturing conditions, in in vitro expression systems, or in prokaryotic expression libraries. Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 3009 - 13 Evolution of the glutamine synthetase gene, one of the oldest existing and functioning genes; Kumada Y et al.; We performed molecular phylogenetic analyses of glutamine synthetase (GS) genes in order to investigate their evolutionary history . The analyses were done on 30 DNA sequences of the GS gene which included both prokaryotes and eukaryotes . Two types of GS genes are known at present: the GSI gene found so far only in prokaryotes and the GSII gene found in both prokaryotes and eukaryotes . Our study has shown that the two types of GS gene were produced by a gene duplication which preceded, perhaps by > 1000 million years, the divergence of eukaryotes and prokaryotes . The results are consistent with the facts that (i) GS is a key enzyme of nitrogen metabolism found in all extant life forms and (ii) the oldest biological fossils date back 3800 million years . Thus, we suggest that GS genes are one of the oldest existing and functioning genes in the history of gene evolution and that GSI genes should also exist in eukaryotes . Furthermore, our study may stimulate investigation on the evolution of "preprokaryotes," by which we mean the organisms that existed during the era between the origin of life and the divergence of prokaryotes and eukaryotes. Mol Cell Biol, 1993 Apr, 13(4), 2478 - 85 Two cofactors and cytoplasmic chaperonin are required for the folding of alpha- and beta-tubulin; Gao Y et al.; Though the chaperonins that mediate folding in prokaryotes, mitochondria, and chloroplasts have been relatively well characterized, the folding of proteins in the eukaryotic cytosol is much less well understood . We recently identified a cytoplasmic chaperonin as an 800-kDa multisubunit toroid which forms a binary complex with unfolded actin; the correctly folded polypeptide is released upon incubation with Mg-ATP (Y . Gao, J . O . Thomas, R . L . Chow, G.-H . Lee, and N . J . Cowan, Cell 69:1043-1050, 1992) . Here we show that the same chaperonin also forms a binary complex with unfolded alpha- or beta-tubulin; however, there is no detectable release of the correctly folded product, irrespective of the concentration of added Mg-ATP and Mg-GTP or the presence of added carrier tubulin heterodimers with which newly folded alpha- or beta-tubulin polypeptides might exchange . Rather, two additional protein cofactors are required for the generation of properly folded alpha- or beta-tubulin, which is then competent for exchange into preexisting alpha/beta-tubulin heterodimers . We show that actin and tubulins compete efficiently with one another for association with cytoplasmic chaperonin complexes . These data imply that actin and alpha- and beta-tubulin interact with the same site(s) on chaperonin complexes. Protein Expr Purif, 1993 Apr, 4(2), 95 - 100 One-step affinity isolation of recombinant protein using the baculovirus/insect cell expression system; Peng S et al.; We have developed two baculovirus transfer vectors which allow single-step affinity isolation of recombinant proteins after expression in insect cells . Using these vectors, recombinant proteins are synthesized as fusions with glutathione-S-transferase and are amenable to enrichment from a crude insect cell lysate using glutathione affinity agarose . After affinity isolation, glutathione-S-transferase can be cleaved from the recombinant polypeptides of interest at an engineered thrombin cleavage site . We used this approach to successfully isolate glutathione-S-transferase, the human low density lipoprotein receptor, two large polypeptides containing cytoplasmic domains of the cystic fibrosis transmembrane conductance regulator (CFTR), and the full-length CFTR . The approach has potential advantages over prokaryotic overexpression of foreign polypeptides, including: (i) eukaryotic post-translational modification of expressed protein, (ii) increased solubility of recombinant fusion proteins synthesized in insect cells leading to increased affinity yield under mild conditions, and (iii) production of large and/or complex polypeptides which might be difficult to purify from prokaryotic cells . The method also allows enrichment of recombinant protein representing a small fraction (less than 5%) of total insect cell protein produced and provides a general method for eukaryotic protein synthesis and isolation which is independent of the particular protein being expressed. Proc Natl Acad Sci U S A, 1993 Apr 1, 90(7), 3083 - 7 Synergistic activation of transcription by Escherichia coli cAMP receptor protein; Joung JK et al.; Activation of gene expression in eukaryotes generally involves the action of multiple transcription factors that function synergistically when bound near a particular target gene . Such effects have been suggested to occur because multiple activators can interact simultaneously with one or more components of the basal transcription machinery . In prokaryotes, examples of synergistic effects on transcription are much more limited and can often be explained by cooperative DNA binding . Here we show that the Escherichia coli cAMP receptor protein (CRP) functions synergistically to activate transcription from a derivative of the lac promoter that bears a second CRP-binding site upstream of the natural binding site . We present evidence indicating that cooperative DNA binding of two CRP dimers does not account for the magnitude of the observed cooperative activation . We suggest, instead, that the two dimers stimulate transcription directly by contacting two distinct surfaces of RNA polymerase simultaneously . Thus, synergistic activation by CRP may provide a relatively simple model for examining the molecular basis of such effects in higher organisms. Virology, 1993 Apr, 193(2), 642 - 52 Analysis of proteins encoded by IE regions 1 and 2 of human cytomegalovirus using monoclonal antibodies generated against recombinant antigens; Plachter B et al.; The genomic region of human cytomegalovirus (HCMV) encoding the major immediate-early (IE) proteins was cloned as overlapping fragments into a prokaryotic expression vector . Three recombinant polypeptides were used as antigens to generate monoclonal antibodies specifically reactive with the proteins encoded by IE region 1 and IE region 2 . At least 10 different antigenic regions were identified on the IE proteins of HCMV . One monoclonal generated against an IE-2 polypeptide of 156 amino acids (termed SMX) was found to react with a viral pentapeptide, which was also a constituent of the beta-chain of human HLA-DR . This peptide was contained in the 40-kDa (p40) protein encoded by IE region 2 . This protein appeared to be an abundant product of the major IE region in infected cells at late times after infection . The putative translation initiation site for p40 was mapped to position 3348 of the IE2 sequence using monoclonal antibody SMX . It is therefore proposed that p40 consists of amino acids 242-580 encoded by the IE2 gene of HCMV (strain AD169) . Neither this late protein nor any other of the IE1/IE2-encoded proteins was detectable in extracellular virions. Mutat Res, 1993 Apr, 286(2), 243 - 52 Genotoxicity of the isoquinoline alkaloid berberine in prokaryotic and eukaryotic organisms; Pasqual MS et al.; Berberine, a medically important isoquinoline alkaloid, was tested for the presence of genotoxic, mutagenic and recombinogenic activities in microorganisms . This alkaloid did not show genotoxic activity with or without metabolic activation in the SOS chromotest . It was also unable to induce significant cytotoxic, mutagenic or recombinogenic effects during treatments performed under nongrowth conditions . However, in dividing cells, this alkaloid induced important cytotoxic and cytostatic effects in proficient and repair-deficient Saccharomyces cerevisiae strains . Among the different repair-deficient mutants examined, a mutant blocked in the DNA strand-break repair pathway (rad52-1) was found to be the most sensitive to the cytotoxic effect of berberine . A triple mutant blocked in the excision (rad2-6), in the mutagenic (rad6-1) and in the recombinogenic (rad52-1) repair pathways demonstrated the same sensitivity as the single rad52-1 mutant . In dividing cells, the induction of frameshift and mitochondrial mutations, as well as crossing over, showed that this alkaloid is not a potent mutagenic agent . The possible implication of DNA topoisomerases in berberine toxicity mechanisms is discussed. Science, 1993 Mar 26, 259(5103), 1896 - 9 Similarity of the yeast RAD51 filament to the bacterial RecA filament; Ogawa T et al.; The RAD51 protein functions in the processes of DNA repair and in mitotic and meiotic genetic recombination in the yeast Saccharomyces cerevisiae . The protein has adenosine triphosphate-dependent DNA binding activities similar to those of the Escherichia coli RecA protein, and the two proteins have 30 percent sequence homology . RAD51 polymerized on double-stranded DNA to form a helical filament nearly identical in low-resolution, three-dimensional structure to that formed by RecA . Like RecA, RAD51 also appears to force DNA into a conformation of approximately a 5.1-angstrom rise per base pair and 18.6 base pairs per turn . As in other protein families, its structural conservation appears to be stronger than its sequence conservation . Both the structure of the protein polymer formed by RecA and the DNA conformation induced by RecA appear to be general properties of a class of recombination proteins found in prokaryotes as well as eukaryotes. Nucleic Acids Res, 1993 Mar 25, 21(6), 1351 - 9 Proposed roles for DNA methylation in Alu transcriptional repression and mutational inactivation; Liu WM et al.; Methylation at CpG dinucleotides to produce 5 methyl cytosine (5me-C) has been proposed to regulate the transcriptional expression of human Alu repeats . Similarly, methylation has been proposed to indirectly favor the transpositional activity of young Alu repeats by transcriptionally inactivating older Alu's through the very rapid transition of 5me-C to T . Both hypotheses are examined here by RNA polymerase III (Pol III) in vitro transcription of Alu templates using HeLa cell extracts . A limiting factor represses the template activity of methylated Alu repeats . Competition by methylated prokaryotic vector DNA's relieves repression, showing that the factor is not sequence specific . This competitor has no effect on the activity of unmethylated templates showing that the repressor is highly specific toward methylated DNA . While methylation of a single pair of CpG dinucleotides in the A box of the Poll III promoter is sufficient to cause repression, methylation elsewhere within the template also causes repression . The repressor causing these effects on the Pol III directed transcription of Alu repeats is thought to be a previously reported, repressor for Pol II directed templates . Young Alu repeats are transcriptionally more active templates than a representative older Alu subfamily member . Also, younger Alu's form stable transcriptional complexes faster, potentially giving them an additional advantage . The mutation of three CpG's to CpA's within and near the A box drastically decreases both the template activity and rate of stable complex formation by a young Alu member . The sensitivity of Alu template activity to CpG transitions within the A box partially explains the selective transpositional advantage enjoyed by young Alu members. J Mol Biol, 1993 Mar 20, 230(2), 684 - 8 Prokaryotic members of a new family of putative helicases with similarity to transcription activator SNF2; Kolsto AB et al.; Cloning and sequence analysis of a new open reading frame from Bacillus cereus reveals the relationship to a recently identified family of putative eukaryotic transcription activators similar to the yeast SNF2 gene product . As a result of comparative analysis of sequence features conserved in all members of this family, a gene from a chilo iridescent virus, as well as a putative helicase from Escherichia coli (hepA), can also be grouped into this family . The unexpected presence of prokaryotic and viral sequences in the previously purely eukaryotic SNF2 family suggests a defined subgroup of DNA helicases present in all species, with specific function in transcription activation. Proc Natl Acad Sci U S A, 1993 Mar 15, 90(6), 2547 - 50 An intron within the 16S ribosomal RNA gene of the archaeon Pyrobaculum aerophilum; Burggraf S et al.; The 16S rRNA genes of Pyrobaculum aerophilum and Pyrobaculum islandicum were amplified by the polymerase chain reaction, and the resulting products were sequenced directly . The two organisms are closely related by this measure (over 98% similar) . However, they differ in that the (lone) 16S rRNA gene of Pyrobaculum aerophilum contains a 713-bp intron not seen in the corresponding gene of Pyrobaculum islandicum . To our knowledge, this is the only intron so far reported in the small subunit rRNA gene of a prokaryote . Upon excision the intron is circularized . A secondary structure model of the intron-containing rRNA suggests a splicing mechanism of the same type as that invoked for the tRNA introns of the Archaea and Eucarya and 23S rRNAs of the Archaea . The intron contains an open reading frame whose protein translation shows no certain homology with any known protein sequence. J Biol Chem, 1993 Mar 15, 268(8), 5519 - 23 Purification of glutamine tRNA synthetase from Saccharomyces cerevisiae . A monomeric aminoacyl-tRNA synthetase with a large and dispensable NH2-terminal domain; Ludmerer SW et al.; Glutamine tRNA synthetase from Saccharomyces cerevisiae has been purified to homogeneity and shown to be a monomer of 91 kDa . The size of the polypeptide agrees with that predicted from the previously reported translated DNA sequence . Mild tryptic digestion removes an amino-terminal domain and releases a fragment of 65 kDa which begins at Ser205 . This tryptic fragment is similar in size and sequence to Escherichia coli glutamine tRNA synthetase and shows a modest increase from the full-length yeast enzyme in the Km values for glutamine and ATP and no difference in the kcat for aminoacylation or the Km for tRNA . Thus, the removal of the NH2-terminal domain appears to indirectly affect the ATP- and glutamine-binding sites in the nucleotide-binding fold domain to which the NH2-terminal domain is fused . A monoclonal antibody directed against the NH2-terminal domain of the full-length enzyme has little effect upon the aminoacylation activity . Therefore, over 200 amino acids of the NH2 terminus of the full-length enzyme form a domain that operationally has only a modest influence on the catalytic core of the protein . These studies reinforce the concept that eukaryotic synthetases have quasi-independent domains not found in their prokaryotic counterparts which may confer a function distinct from aminoacylation. Eur J Biochem, 1993 Mar 15, 212(3), 727 - 35 Mitochondrial cardiolipin in diverse eukaryotes . Comparison of biosynthetic reactions and molecular acyl species; Schlame M et al.; Cardiolipin, a unique dimeric phospholipid of bacteria and mitochondria, can be synthesized by two alternative pathways discovered in rat and Escherichia coli, respectively . In mitochondrial preparations from fungi (Saccharomyces cerevisiae, Neurospora crassa), higher plants (Phaseolus aureus), molluscs (Mytilus edulis) and mammals (rat liver, bovine adrenal gland), cardiolipin was synthesized from CDP-diacylglycerol and phosphatidylglycerol, suggesting a common eukaryotic mechanism of cardiolipin formation which is in contrast to the prokaryotic biosynthesis from two molecules of phosphatidylglycerol . All mitochondrial cardiolipin synthases were inhibited by lysophosphatidylglycerol, were insensitive to N-ethylmaleimide and required divalent cations, although they had different cation specificities . The molecular species of cardiolipin from rat liver, bovine heart, S . cerevisiae and N . crassa were analysed by high-performance liquid chromatography of the derivative 1,3-bis{3'-sn-phosphatidyl}-2-benzoyl-sn-glycerol dimethyl ester . Cardiolipins from these organisms contained mainly monounsaturated or diunsaturated chains with 16 or 18 carbon atoms, resulting in a relatively homogeneous distribution of double bonds and carbon numbers among the four acyl positions . About half of the molecular species were symmetrical, i.e . they combined two identical diacylglycerol moieties . In N . crassa, the same species pattern was found at growth temperatures of 25 degrees C and 37 degrees C . Tentative molecular models were created for the most abundant molecular species and subjected to energy minimization . Geometric data, derived from these models, suggested similarities in the gross structure of the major cardiolipin species from different sources. Gene, 1993 Mar 15, 125(1), 97 - 102 Comparison of the rompA gene repeat regions of Rickettsiae reveals species-specific arrangements of individual repeating units; Gilmore RD Jr; Genes containing repeating units have been described for several eukaryotic and prokaryotic proteins, although none exhibit the extraordinary length and conservation seen with the Rickettsia rickettsii 190-kDa protein (OmpA) repeat region . The genetic organization of the repeat regions of the ompA gene for R . conorii and R . akari was examined and compared to that of R . rickettsii . Individual repeating units, which constitute the makeup of the repeat regions, share extensive DNA homology among these species . However, differences were observed in the overall numbers and arrangements of repeating units within each species' repeat region . It appears that the distinctive arrangement of repeating units within the gene may represent the basis for encoding a protective species-specific conformational epitope of the protein. Plant Mol Biol, 1993 Mar, 21(6), 1023 - 33 Sequence analysis of pre-ferredoxin-NADP(+)-reductase cDNA from Cyanophora paradoxa specifying a precursor for a nucleus-encoded cyanelle polypeptide; Jakowitsch J et al.; A cDNA clone for pre-ferredoxin-NADP+ reductase (FNR) was obtained by screening a Cyanophora paradoxa expression library with antibodies specific for cyanelle FNR . The 1.4 kb transcript was derived from a single-copy gene . The precursor (41 kDa) and mature forms (34 kDa) of FNR were identified by western blotting of in vitro translation products and cyanelle extracts, respectively . The derived amino acid sequence of the mature form was corroborated by data from N-terminal protein sequencing and yielded identity scores from 58% to 62% upon comparison with cyanobacterial FNRs . Sequence conservation seemed to be even more pronounced in comparison with enzymes from higher plants, but using the neighbor joining method the C . paradoxa sequence was clearly positioned between the prokaryotic and eukaryotic sequences . The transit peptide of 65 or 66 amino acids appeared to be totally unrelated to those from spinach, pea and ice plant but showed overall characteristics of stroma-targeting peptides. J Gen Microbiol, 1993 Mar, 139 ( Pt 3), 425 - 32 Putrescine oxidase of Micrococcus rubens: primary structure and Escherichia coli; Ishizuka H et al.; The flavin adenine dinucleotide (FAD)-containing putrescine oxidase of Micrococcus rubens catalyses the oxidative deamination of putrescine . The amino acid sequences of the NH2-termini of the mature enzyme and lysyl-endopeptidase-generated fragments were determined for preparation of synthetic oligonucleotides as hybridization probes for cloning . A 4.4 kb BamHI fragment which contained DNA sequences hybridizing to the probes was cloned in pUC19 in Escherichia coli . The nucleotide sequence together with the determined amino acid sequences revealed that this enzyme consists of 480 amino acids (M(r) 52,000) and contains an FAD-binding consensus sequence at its NH2-terminal portion . In front of the transcriptional start point, which is 28 bases upstream of the initiation codon as determined by primer extension, -35 and -10 sequences similar to typical prokaryotic promoter consensus sequences are present . E . coli JM109 containing the putrescine oxidase gene just downstream of the lac promoter in pUC18 produced a large amount of this protein when grown at 37 degrees C but in the enzymically inactive form of inclusion bodies . However, cultivation of the recombinant E . coli cells at temperatures below 30 degrees C led to production of active enzyme (20 times as much as produced by the original M . rubens strain). J Bacteriol, 1993 Mar, 175(6), 1697 - 704 The patB gene product, required for growth of the cyanobacterium Anabaena sp . strain PCC 7120 under nitrogen-limiting conditions, contains ferredoxin and helix-turn-helix domains; Liang J et al.; A mutant of Anabaena sp . strain PCC 7120, called PAT-2, that grows poorly under nitrogen-fixing conditions, has been isolated . The heterocysts of the mutant strain develop much more slowly than those of the wild type and are spaced more closely in an older culture of the mutant than in the wild type . The wild-type gene that complements the mutation in PAT-2, called patB, was isolated and characterized . The predicted 529-amino-acid PatB protein contains a region very similar to the Fe4S4 bacterial-type ferredoxins near its N terminus and a helix-turn-helix motif near its C terminus . This pattern of domains resembles those of transcriptional regulators in both prokaryotes and eukaryotes . The mutation in strain PAT-2 is the deletion of G at nucleotide 1342 in the patB gene, resulting in the loss of a 62-amino-acid fragment from the C terminus of the PatB protein, including the helix-turn-helix motif. Proc Natl Acad Sci U S A, 1993 Mar 1, 90(5), 1642 - 6 Shared thematic elements in photochemical reaction centers; Golbeck JH; The structural, functional, and evolutionary relationships between photosystem II and the purple nonsulfur bacterial reaction center have been recognized for several years . These can be classified as "quinone type" (type II) photosystems because the terminal electron acceptor is a mobile quinone molecule . The analogous relationship between photosystem I and the green sulfur bacterial (and helicobacterial) reaction centers has only recently become clear . These can be classified as "iron-sulfur type" (type I) photosystems because the terminal electron acceptor consists of one or more bound iron-sulfur clusters . At a fundamental level, the quinone type and iron-sulfur type reaction centers share a common photochemical motif in the early process of charge separation, leading to the speculation that all photochemical reaction centers have a common evolutionary origin . This review summarizes the current state of knowledge in comparative reaction center biochemistry between prokaryotic bacteria, cyanobacteria, and green plants. J Bacteriol, 1993 Mar, 175(5), 1344 - 51 The Escherichia coli FtsH protein is a prokaryotic member of a protein family of putative ATPases involved in membrane functions, cell cycle control, and gene expression; Tomoyasu T et al.; The ftsH gene is essential for cell viability in Escherichia coli . We cloned and sequenced the wild-type ftsH gene and the temperature-sensitive ftsH1(Ts) gene . It was suggested that FtsH protein was an integral membrane protein of 70.7 kDa (644 amino acid residues) with a putative ATP-binding domain . The ftsH1(Ts) gene was found to have two base substitutions within the coding sequence corresponding to the amino acid substitutions Glu-463 by Lys and Pro-587 by Ala . Homology search revealed that an approximately 200-amino-acid domain, including the putative ATP-binding sequence, is highly homologous (35 to 48% identical) to the domain found in members of a novel, eukaryotic family of putative ATPases, e.g., Sec18p, Pas1p, CDC48p, and TBP-1, which function in protein transport pathways, peroxisome assembly, cell division cycle, and gene expression, respectively . Possible implications of these observations are discussed. J Cell Biol, 1993 Mar, 120(5), 1101 - 12 The constitutive and stress inducible forms of hsp 70 exhibit functional similarities and interact with one another in an ATP-dependent fashion; Brown CR et al.; Mammalian cells constitutively express a cytosolic and nuclear form of heat shock protein (hsp) 70, referred to here as hsp 73 . In response to heat shock or other metabolic insults, increased expression of another cytosolic and nuclear form of hsp 70, hsp 72, is observed . The constitutively expressed hsp 73, and stress-inducible hsp 72, are highly related proteins . Still unclear, however, is exactly why most eukaryotic cells, in contrast to prokaryotic cells, express a novel form of hsp 70 (i.e., hsp 72) after experiencing stress . To address this question, we prepared antibodies specific to either hsp 72 or hsp 73 and have compared a number of biological properties of the two proteins, both in vivo and in vitro . Using metabolic pulse-chase labeling and immunoprecipitation analysis, both the hsp 72 and hsp 73 specific antibodies were found to coprecipitate a significant number of newly synthesized proteins . Such interactions appeared transient and sensitive to ATP . Consequently, we suspect that both hsp 72 and hsp 73 function as molecular chaperones, interacting transiently with nascent polypeptides . During the course of these studies, we routinely observed that antibodies specific to hsp 73 resulted in the coprecipitation of hsp 72 . Similarly, antibodies specific to hsp 72 were capable of coprecipitating hsp 73 . Using a number of different approaches, we show that the constitutively expressed, pre-existing hsp 73 rapidly forms a stable complex with the newly synthesized stress inducible hsp 72 . As is demonstrated by double-label indirect immunofluorescence, both proteins exhibit a coincident locale within the cell . Moreover, injection of antibodies specific to hsp 73 into living cells effectively blocks the ability of both hsp 73 and hsp 72 to redistribute from the cytoplasm into the nucleus and nucleolus after heat shock . These results are discussed as they relate to the possible structure and function of the constitutive (hsp 73) and highly stress inducible (hsp 72) forms of hsp 70, both within the normal cell as well as in the cell experiencing stress. Curr Genet, 1993 Mar, 23(3), 271 - 80 A novel Euglena gracilis chloroplast operon encoding four ATP synthase subunits and two ribosomal proteins contains 17 introns; Drager RG et al.; The structure of a Euglena gracilis chloroplast operon encoding four subunits of the chloroplast ATP synthase complex and two ribosomal proteins has been determined . These six genes contain 17 introns . This operon is transcribed as a hexacistronic primary transcript which is subsequently processed to monocistronic mRNAs . The linear order of these genes, 5'-rps2-atpI-atpH-atpF-atpA-rps18-3' , encoding ribosomal protein S2, chloroplast ATP synthase subunits CF0IV, CF0III, CF0I, CF1 alpha and ribosomal protein S18, respectively, is similar to the equivalent operons of prokaryotes, cyanelles and land-plant chloroplasts . This operon differs from those of these other organisms in the co-transcription of rps18 and in intron content. Mol Biol (Mosk), 1993 Mar-Apr, 27(2), 358 - 62 {Long terminal repeats of drosophila mobile elements direct transcript ion in Escherichia coli cells}; Abramian LG et al.; Retrotransposons of D . melanogaster are similar to vertebrate retroviruses in their structure and function . Long terminal repeats (LTR) of retrotransposons contain specific eukaryotic promoters and transcriptional control sequences . Recently it has been shown that several retroviral LTRs contain in addition some promoter-like sequences that are functional in E . coli cells . Plasmid constructions containing the mdg1 and mdg4 LTR fragments and the standard reporter chloramphenicol acetyltransferase (cat) gene are also able to direct transcription and expression of the reporter cat gene in E . coli cells . Analysis of the primary structure of corresponding LTR fragments revealed sequences similar to conserved nucleotides of the putative prokaryotic promoter. J Med Virol, 1993 Mar, 39(3), 187 - 95 An ELISA using recombinant proteins for the detection of neutralizing antibodies against human cytomegalovirus; Kropff B et al.; Two prokaryotically expressed fusion proteins encompassing amino acids 484-650 (AD-1) and 27-100 (AD-2) of glycoprotein gp58/116 of human cytomegalovirus (HCMV) were purified from E . coli lysates and used in ELISA to determine antibody levels in human sera . The specificity of the test was established by comparison of 116 randomly selected sera with commercially available HCMV-ELISA tests . The recombinant polypeptides were then used for the analysis of antibody titers in 112 human sera and were compared to the capacity to neutralize HCMV . A strong correlation between the neutralization titer and antibody levels against AD-1 and a weaker correlation for AD-2 was observed . Of 29 sera with a high neutralization titer (> 1:128), 96% and 62% were positive for AD-1 and AD-2, respectively, while 44% and 19% were positive in sera with low neutralization titer (< 1:8) . Serum pools prepared from human sera selected on the basis of recognition of the recombinant antigens had a 10-fold higher neutralization capacity than pools prepared from sera with a high titer in commercially available HCMV tests . A synchronous increase in neutralization capacity and titer against recombinant antigens was observed in transplant patients. J Gen Virol, 1993 Mar, 74 ( Pt 3), 495 - 500 The glycoprotein B homologue of human herpesvirus 6; Ellinger K et al.; The gene for the homologue of herpesvirus glycoprotein B (gB) has been identified in the genome of human herpesvirus 6 (HHV-6), strain U1102, and the nucleotide sequence was determined . The open reading frame encodes a protein of 830 amino acids (93.2K) with the characteristics of a transmembrane glycoprotein and close similarity to the gp58/116 complex of human cytomegalovirus (HCMV) . Monoclonal antibodies 2D10 and 2B9 have been shown previously to react with an HHV-6 glycoprotein of apparent M(r) 112K, and its proteolytic cleavage products of M(r) 64K and 58K . We show that both monoclonal antibodies detect prokaryotically expressed carboxy-terminal fragments of the HHV-6 gB homologue . This indicates that the HHV-6 gB homologue is probably processed by proteolytic cleavage similar to its equivalents in HCMV and various other herpesviruses. J Bacteriol, 1993 Mar, 175(5), 1264 - 71 Implications of Tn5-associated adjacent deletions; Jilk RA et al.; The prokaryotic transposable element Tn5 has been found to promote the formation of adjacent deletions . The frequency of adjacent deletion formation is much lower than that of normal transposition events . Like normal transposition, however, adjacent deletion formation requires the activity of the transposase protein . The deletions can be divided into two classes, as distinguished by their endpoints . The occurrence of one of the two deletion classes is increased when the frequency of normal transposition is reduced by the introduction of a deletion or a certain base substitution at one of the two outside ends (OEs) . The nature of the base substitution at the mutant OE influences the class of deletion found adjacent to the wild-type OE, even though these two ends are about 12 kbp apart . By studying the formation of these deletions, we have gained some insight into the way in which the transposase interacts with the OEs . Our observations suggest that there is a protein-mediated interaction between the two ends, that different end base pairs are involved in different transposition-related processes, and that the adjacent deletions are the result of nonproductive attempts at transposition. J Lipid Mediat, 1993 Mar-Apr, 6(1-3), 353 - 60 Functional aspects of eicosanoid hydroxylation by lung and kidney cytochromes P450 . Expression of cDNAs in mammalian cells and E . coli; Masters BS et al.; A gene subfamily of cytochromes P450 with catalytic activity toward various eicosanoid substrates has been studied with a variety of techniques in this laboratory, including purification and characterization, localization at the tissue and subcellular levels, physiological function, and cloning and expression in prokaryotic and eukaryotic systems . This paper reports experiments directed toward determining the function of the cytochrome P4504A metabolite, 20-hydroxyarachidonic acid (20-hydroxyeicosatetraenoic acid; 20-HETE), in cellular ion flux, immunohistochemical localization in lung, the effects of a mechanism-based inhibitor, 12-hydroxy-16-heptadecynoic acid (12-HHDYA) on PGE1 omega-hydroxylation, and the structure-function determinants which govern the activities of the enzymes encoded by this gene subfamily. Bioessays, 1993 Mar, 15(3), 157 - 64 The evolutionary history of the first three enzymes in pyrimidine biosynthesis; Davidson JN et al.; Some metabolic pathways are nearly ubiquitous among organisms: the genes encoding the enzymes for such pathways must therefore be ancient and essential . De novo pyrimidine biosynthesis is an example of one such metabolic pathway . In animals a single protein called CAD carries the first three steps of this pathway . The same three enzymes in prokaryotes are associated with separate proteins . The CAD gene appears to have evolved through a process of gene duplication and DNA rearrangement, leading to an in-frame gene fusion encoding a chimeric protein . A driving force for the creation of eukaryotic genes encoding multienzymatic proteins such as CAD may be the advantage of coordinate expression of enzymes catalyzing steps in a biosynthetic pathway . The analogous structure in bacteria is the operon . Differences in the translational mechanisms of eukaryotes and prokaryotes may have dictated the different strategies used by organisms to evolve coordinately regulated genes. Lett Appl Microbiol, 1993 Mar, 16(3), 136 - 41 Use of charge-coupled device (CCD) image-enhancement for rapid screening and monitoring of prokaryotic promoter expression; Waterhouse RN et al.; Charge-coupled device (CCD) image-enhancement was used for rapid screening of genomic libraries and allowed selection of promoters with differing characteristics . In addition, both the CCD and luminometry were used to monitor and characterize the expression from two Pseudomonas syringae pv . phaseolicola promoters . The same pattern of gene expression was indicated by the two methods but the CCD enabled the rapid non-destructive in situ monitoring of a microbial population, over a prolonged period. Bull Acad Natl Med, 1993 Mar, 177(3), 371 - 80; discussion 380-1 {The cystic fibrosis gene, its product CFTR protein and its mutations}; Goossens M et al.; Cystic fibrosis (CF) is a fatal genetic disease primarily affecting Caucasians . Its etiology is complex, but it is chiefly a disease of electrolyte transport characterized by defects in fluid secretion by several epithelia . In this review are analyzed the data obtained since the cloning of the CF gene and the characterization of its product, the CF transmembrane conductance regulator (CFTR) protein, which has been shown to act like a cAMP-regulated chloride channel . This protein is a member of a family of ATP-binding proteins that are membrane-spanning, are found in a number of prokaryotic and eucaryotic cells, and have two ATP-binding domains . Unique to this family of proteins, the CFTR possesses an additional highly charged domain (the R domain) . The majority of CF chromosomes (70%) have a single Phenylalanine codon deletion at position 508 of the protein (delta F508) . A large number of other rare mutations (more than 230) have also been identified . This rapid accumulation of data is essential to genetic diagnosis and will aid in understanding the structure and function of the protein. Mol Microbiol, 1993 Mar, 7(5), 777 - 83 Chlamydia trachomatis Mip-like protein has peptidyl-prolyl cis/trans isomerase activity that is inhibited by FK506 and rapamycin and is implicated in initiation of chlamydial infection; Lundemose AG et al.; The Mip-like protein of Chlamydia trachomatis has sequence similarity with both the Mip protein of Legionella pneumophila, a virulence factor necessary for optimal intracellular infection, and FK506-binding proteins (FKBPs) of both prokaryotic and eukaryotic origin . FKBPs contain a site for peptidyl-prolyl cis/trans isomerase activity, which is blocked upon binding of the drugs, FK506 or rapamycin . In this paper we report that the recombinant chlamydial Mip-like protein exhibits a peptidyl-prolyl cis/trans isomerase activity which is inhibited by either rapamycin or FK506 . To assess the role of the Mip-like protein in chlamydial infection, rapamycin or FK506 (25 microM), were used in either treatment of chlamydial organisms prior to inoculation, or were present at different intervals through the infection . Pretreatment of organisms alone reduced infectivity for McCoy cells by 30%, with inhibition rising to 80% on more prolonged exposure from 0 to 8h and 8 to 16h post-inoculation and declining thereafter . When drug was present during the developmental cycle at intervals from 0 to 24h post-inoculation abnormal chlamydiae were induced in residual inclusions . The results suggest that inhibition of the isomerase of the Mip-like protein interferes with one or more early events in the infective process that determine productive intracellular infection. Exp Parasitol, 1993 Mar, 76(2), 101 - 14 Schistosoma mansoni: a Cu/Zn superoxide dismutase is glycosylated when expressed in mammalian cells and localizes to a subtegumental region in adult schistosomes; Hong Z et al.; We previously isolated a gene from Schistosoma mansoni with the capacity to encode a 20-kDa polypeptide that had homology to Cu/Zn superoxide dismutase from 19 other species . The predicted protein sequence contained a hydrophobic N-terminus as well as a site for N-linked glycosylation, suggesting that the protein was a secreted or membrane-associated form of the enzyme . To study this protein further, we first expressed it in a prokaryotic system and used the gene product to make both monoclonal and polyclonal antibodies . We then expressed the protein in CMT3 cells, a monkey kidney cell line, to investigate possible post-translational modifications . Our results demonstrated that the schistosomal protein expressed in CMT3 cells migrated on an SDS-polyacrylamide gel as multiple glycosylated species with molecular masses of 20, 22, and 23 kDa . Glycosylation was inhibited by tunicamycin, resulting in the expression of an unglycosylated product which migrated with a molecular mass of 18 kDa . The CMT3-cell expressed protein eluted from a gel filtration column with a molecular mass larger than 200 kDa, suggesting that it was membrane-associated or bound to a high-molecular-weight component . The product could not be detected in the medium of the CMT3 cell culture and apparently was not secreted . Comparison between the protein expressed in CMT3 cells and that found in adult schistosomes showed that the parasite-derived gene product was also heterogeneous but had different molecular masses (20, 23, and 25 kDa) . The protein was localized in frozen sections of adult worms to the subtegumental area as detected by indirect immunofluorescence using monoclonal antibodies . Since the protein was glycosylated but not secreted we suggest it be called signal peptide-containing superoxide dismutase. J Virol, 1993 Mar, 67(3), 1185 - 94 Identification of an immunodominant linear neutralization domain on the S2 portion of the murine coronavirus spike glycoprotein and evidence that it forms part of complex tridimensional structure; Daniel C et al.; Numerous studies have demonstrated that the spike glycoprotein of coronaviruses bears major determinants of pathogenesis . To elucidate the antigenic structure of the protein, a panel of monoclonal antibodies was studied by competitive ELISA, and their reactivities were assayed against fragments of the murine coronavirus murine hepatitis virus strain A59 S gene expressed in prokaryotic vectors . An immunodominant linear domain was localized within the predicted stalk, S2, of the peplomer . It is recognized by several neutralizing antibodies . Other domains were also identified near the proteolytic cleavage site, in the predicted globular head, S1, and in another part of the stalk . Furthermore, competition results suggest that the immunodominant functional domain forms part of a complex three-dimensional structure . Surprisingly, some antibodies which have no antiviral biological activities were shown to bind the immunodominant neutralization domain. Nucleic Acids Res, 1993 Feb 25, 21(4), 807 - 10 The XylS/AraC family of regulators; Gallegos MT et al.; At least twenty-seven proteins belong to the XylS/AraC family of prokaryote transcriptional regulators . All members of this family except CelD and TetD are positive transcriptional factors . Three subgroups were distinguished within the family in accordance with the Needleman and Wunsch algorithm . Multiple alignment of these proteins revealed that they shared a high degree of sequence homology at their C-terminal end, where a characteristic conserved motif, whose consensus sequence is I-DIA--GF-S--YF--F---G-TPS--R (where - means any aminoacid), was found . Within the homologous C-terminal region, but outside the above consensus motif, a putative DNA-binding domain organized as a helix-turn-helix motif was located in all regulators . For regulators recognizing chemical signals, the non-homologous N-terminal region of these regulators is presumed to contain binding sites for activator molecules that confer specificity. J Biol Chem, 1993 Feb 25, 268(6), 4494 - 8 Construction of Zn2+/Cd2+ hypersensitive cyanobacterial mutants lacking a functional metallothionein locus; Turner JS et al.; Eukaryotic metallothioneins (MTs) have been extensively studied, but the precise functions of most of these molecules are not yet fully understood . Prokaryotes are often more tractable for the analysis of gene function and we report here the generation of mutants of Synechococcus PCC 7942 (strain R2-PIM8) deficient in the MT locus, smt . Viability of these mutants, designated R2-PIM8 (smt), reveals that prokaryotic MT performs no "vital" role (such as donation of metals to metallo-proteins) in Synechococcus . R2-PIM8 (smt) has reduced (approximately 5-fold) tolerance to elevated Zn2+, with detectable hypersensitivity to Cd2+ . Restoration of Zn2+ tolerance was used as a selectable marker to isolate recombinants derived from R2-PIM8(smt) after reintroduction of a linear DNA fragment containing an uninterrupted smt locus . These smt-complemented cells also exhibited restored Cd2+ tolerance . Hypersensitivity to Cu2+ was not detected in R2-PIM8(smt) indicating independence of Cu2+ resistance from smt mediated metal (Zn2+/Cd2+) tolerance. J Biol Chem, 1993 Feb 25, 268(6), 4499 - 503 Characterization of a distinct binding site for the prokaryotic chaperone, GroEL, on a human granulocyte ribonuclease; Rosenberg HF et al.; Although ribonucleases fold into correct tertiary conformation in vitro guided solely by information contained in the primary amino acid sequence (Sela, M., White, F . H., and Anfinsen, C . B . (1957) Science 124, 691-693), it is not clear whether folding of these proteins proceeds unassisted in a complex intracellular environment . We describe here the specific and high affinity binding of groEL, the prokaryotic homolog of the heat shock protein 60 family of molecular chaperones, to recombinant eosinophil cationic protein and eosinophil-derived neurotoxin, two members of the human ribonuclease gene family . We have determined that groEL binds to a unique peptide sequence near the amino terminus of nascent eosinophil cationic protein that includes the first of eight cysteine residues . This binding site functions independently and can confer groEL binding activity on an unrelated carrier protein . GroEL dissociates from the binding site upon addition of ATP and Mg2+; no other cations or cofactors are necessary . These findings suggest the possibility that interaction with a groEL-like molecular chaperone may be a requirement for correct folding and/or translocation of eukaryotic ribonucleases in vivo. J Biol Chem, 1993 Feb 15, 268(5), 3389 - 95 An interaction between replication protein A and SV40 T antigen appears essential for primosome assembly during SV40 DNA replication; Melendy T et al.; Replication protein A from human cells (hRPA) is a multisubunit single-stranded DNA-binding protein (ssb) and is essential for SV40 DNA replication in vitro . The related RPA from Saccharomyces cerevisiae (scRPA) is unable to substitute for hRPA in SV40 DNA replication . To understand this species specificity, we evaluated human and yeast RPA in enzymatic assays with SV40 T antigen (TAg) and human DNA polymerase alpha/primase, the factors essential for initiation of SV40 DNA replication . Both human and yeast RPA stimulated the polymerase and (at subsaturating levels of RPA) the primase activities of human DNA polymerase alpha/primase on homopolymer DNA templates . In contrast, both human and yeast RPA inhibited synthesis by DNA polymerase alpha/primase on natural single-stranded DNA (ssDNA) templates . T antigen reversed the inhibition of DNA polymerase alpha/primase activity on hRPA-coated natural ssDNA, as previously described, but was unable to reverse the inhibition on scRPA or Escherichia coli ssb-coated templates . Therefore, the ability of an ssb to reconstitute SV40 DNA replication correlated with its ability to allow the TAg stimulation of polymerase alpha/primase in this assay . Enzyme-linked immunoassays demonstrated that hRPA interacts with TAg, as previously described; however, scRPA does not bind to TAg in this assay . These and other recent results suggest that T antigen contains a function analogous to some prokaryotic DNA replication proteins that facilitate primosome assembly on ssb-coated template DNAs. Biochem Biophys Res Commun, 1993 Feb 15, 190(3), 724 - 31 Expression of extracellular ligand-binding domain of murine guanylate cyclase/atrial natriuretic factor receptor cDNA in Escherichia coli; Pandey KN et al.; The membrane-bound form of guanylate cyclase/atrial natriuretic factor receptor (GC/ANF-R) is a 135 kDa transmembrane glycoprotein which binds ANF with high affinity . We have expressed the extracellular ligand-binding domain of murine guanylate cyclase ANF-R (GC/ANFR-LBD) cDNA in Escherichia coli . The cDNA encoding the extracellular ANF-binding domain (nucleotide positions covering from 432-1755 base pair) of GC/ANF-R was amplified by polymerase chain reaction, cloned into BamHI site of pGEX-3X prokaryotic expression vector and was transfected into E . coli, strain JM101 . After isopropyl-beta-D-thiogalactopyranoside (IPTG) induction of bacterial cells, the GC/ANFR-LBD was expressed as the glutathione-S-transferase (GST) fusion protein, yielding a molecular mass of 70 kDa . The expressed fusion protein was characterized for binding affinity to both full length and truncated ANF molecules . After expression in E . coli, the binding of 125I-ANF to the extracellular region of GC/ANF-R was similar and corresponded to the pharmacological class of native receptor protein . The 70 kDa fusion product was purified as a predominant single protein band by glutathione-affinity chromatography . These findings establish that E . coli may be utilized as an effective heterologous model system to delineate the structure-function analysis of guanylate cyclase-coupled ANF receptor molecules. Gene, 1993 Feb 14, 124(1), 1 - 11 Topology, structure and evolution of two families of proteins involved in antibiotic and antiseptic resistance in eukaryotes and prokaryotes--an analysis; Paulsen IT et al.; Analysis of deduced amino acid sequences has demonstrated that the sequences of eukaryotic and prokaryotic proteins mediating resistance to antibiotics and antiseptics are highly related . Hydropathy analysis and alignment of conserved motifs revealed that these proteins can be divided into two separate families with either 12 or 14 transmembrane segments (TMS) . Conserved motifs have been identified which are either characteristic for each family or conserved in both families . The conservation of these motifs suggested that they may be essential for the function of these proteins . Phylogenetic and structural analysis revealed that the two families may have evolved from a common ancestor with six TMS. Nucleic Acids Res, 1993 Feb 11, 21(3), 555 - 60 Ease of DNA unwinding is a conserved property of yeast replication origins; Natale DA et al.; Autonomously replicating sequence (ARS) elements function as plasmid replication origins . Our studies of the H4 ARS and ARS307 have established the requirement for a DNA unwinding element (DUE), a broad easily-unwound sequence 3' to the essential consensus that likely facilitates opening of the origin . In this report, we examine the intrinsic ease of unwinding a variety of ARS elements using (1) a single-strand-specific nuclease to probe for DNA unwinding in a negatively-supercoiled plasmid, and (2) a computer program that calculates DNA helical stability from the nucleotide sequence . ARS elements that are associated with replication origins on chromosome III are nuclease hypersensitive, and the helical stability minima correctly predict the location and hierarchy of the hypersensitive sites . All well-studied ARS elements in which the essential consensus sequence has been identified by mutational analysis contain a 100-bp region of low helical stability immediately 3' to the consensus, as do ARS elements created by mutation within the prokaryotic M13 vector . The level of helical stability is, in all cases, below that of ARS307 derivatives inactivated by mutations in the DUE . Our findings indicate that the ease of DNA unwinding at the broad region directly 3' to the ARS consensus is a conserved property of yeast replication origins. Nucleic Acids Res, 1993 Feb 11, 21(3), 401 - 6 Differential response to frameshift signals in eukaryotic and prokaryotic translational systems; Garcia A et al.; The genomic RNA of beet western yellows virus (BWYV) contains a potential translational frameshift signal in the overlap region of open reading frames ORF2 and ORF3 . The signal, composed of a heptanucleotide slippery sequence and a downstream pseudoknot, is similar in appearance to those identified in retroviral RNAs . We have examined whether the proposed BWYV signal functions in frameshifting in three translational systems, i.c . in vitro in a reticulocyte lysate or a wheat germ extract and in vivo in E . coli . The efficiency of the signal in the eukaryotic system is low but significant, as it responds strongly to changes in either the slip sequence or the pseudoknot . In contrast, in E . coli there is hardly any response to the same changes . Replacing the slip sequence to the typical prokaryotic signal AAAAAAG yields more than 5% frameshift in E . coli . In this organism the frameshifting is highly sensitive to changes in the slip sequence but only slightly to disruption of the pseudoknot . The eukaryotic assay systems are barely sensitive to changes in either AAAAAAG or in the pseudoknot structure in this construct . We conclude that eukaryotic frameshift signals are not recognized by prokaryotes . On the other hand the typical prokaryotic slip sequence AAAAAAG does not lead to significant frameshifting in the eukaryote . In contrast to recent reports on the closely related potato leafroll virus (PLRV) we show that the frameshifting in BWYV is pseudoknot-dependent. J Biol Chem, 1993 Feb 5, 268(4), 2984 - 8 Expression of biologically active recombinant keratinocyte growth factor . Structure/function analysis of amino-terminal truncation mutants; Ron D et al.; Keratinocyte growth factor (KGF) is a newly identified member of the fibroblast growth factor (FGF) family (FGF-7) . KGF is expressed by stromal fibroblasts and acts on epithelial cells in a paracrine mode . To facilitate structure/function studies, we utilized the T7 prokaryotic expression system to synthesize this growth factor . Recombinant KGF (rKGF) was mitogenic with a specific activity around 10-fold higher than native KGF . By in vitro mutagenesis, we generated a series of KGF mutants with sequential deletions of the amino-terminal domain, the most divergent region among different FGF members . Mutant proteins, produced in bacteria, were tested for their ability to bind heparin, bind and activate the KGF receptor, and induce DNA synthesis . Heparin binding properties were preserved with deletion of up to 28 amino-terminal residues of the mature KGF but lost by the deletion of an additional 10 residues . Biological activity of mutants with deletions of up to 10 residues was comparable to that of rKGF . However, deletion of 29 residues resulted in significantly reduced ability to stimulate KGF receptor tyrosine-kinase activity and DNA synthesis, although this mutant bound the receptor at high affinity . These characteristics of a partial agonist may be useful in the development of competitive antagonists of KGF action. J Biol Chem, 1993 Feb 5, 268(4), 2348 - 52 N-myristylation of the catalytic subunit of cAMP-dependent protein kinase conveys structural stability; Yonemoto W et al.; Coexpression of the yeast N-myristyltransferase with the murine catalytic subunit of cAMP-dependent protein kinase in prokaryotic cells results in the N-myristylation of the recombinant catalytic subunit . The acylated recombinant catalytic subunit was purified following in vitro holoenzyme formation with a mutant form of the regulatory subunit and compared to the non-myristylated recombinant enzyme and to the mammalian porcine enzyme . All three enzymes are very similar in terms of their kinetic properties and their capacity to reassociate in vitro with the regulatory subunit to form holoenzyme . In contrast, the myristylated recombinant catalytic subunit is significantly more stable to thermal denaturation than the non-myristylated enzyme . Its thermal stability is now comparable to the mammalian enzyme . All three catalytic subunits are significantly more stable to thermal denaturation when they are part of the holoenzyme complex . Each shows an increase in T1/2 of 10 degrees C . This study demonstrates that one function for the myristic acid at the NH2 terminus of the catalytic subunit is to provide structural stability. J Biol Chem, 1993 Feb 5, 268(4), 2269 - 72 Helicase-catalyzed DNA unwinding; Lohman TM; DNA helicases are ubiquitous and multiple helicases have been identified in a number of prokaryotes and eukaryotes . Although it is clear that not all helicases function identically, many of these enzymes possess similar properties that appear to be of general importance for their mechanism of action . For example, the assembly states of most (possibly all) helicases are oligomeric . The prime consequence of an oligomeric helicase is that it possesses multiple DNA binding sites, a feature that is required for any "active" mechanism of DNA unwinding, since it enables a helicase to bind both ss- and duplex DNA or two strands of ss-DNA simultaneously at an unwinding fork . Modulation of the relative affinities of ss- versus duplex DNA for these multiple binding sites through ATP binding and hydrolysis, as has been observed for the E . coli Rep dimer, can provide a mechanism for translocation and processive unwinding of DNA . Along with studies of DNA unwinding, further understanding of helicase mechanisms requires quantitative studies of the equilibria and kinetics of the multiple, linked reactions among protein, DNA, and nucleotide cofactors, including the protein-protein interactions involved in assembly of the oligomeric helicase. Biokhimiia, 1993 Feb, 58(2), 202 - 10 {Phosphorus-containing polysaccharides from the cell wall of Actinoplanes sp . INA 3697 cell wall}; Shashkov AS et al.; A phosphorus-containing polysaccharide has been isolated from defatted cells of Actinoplanes sp . INA 3697 . The structure of the polymer has been established by a non-destructive method including one- and two-dimensional NMR 1H-spectroscopy as well as by NMR 13C-spectroscopy and confirmed by chemical analysis of the monosaccharide composition and the repeating link . Association of the repeating units-2-acetamido-2-deoxy-beta-D-mannopyranosyl-(1-->3)-2-acetamido -2-deoxy-D-glucopyranoses, is provided by phosphodiester bonds between C1 of N-acetylglucosamine and C6 of N-acetylmannosamine . The polymer molecule is made up of about 12 disaccharides and is localized in the cell wall of the actinomycete . No structurally identical phosphorylated polysaccharides have as yet been found in prokaryotes. Mol Microbiol, 1993 Feb, 7(3), 343 - 50 Protein HU binds specifically to kinked DNA; Pontiggia A et al.; We have purified the main four-way junction DNA-binding protein of Escherichia coli, and have found it to be the well-known HU protein . HU protein recognizes with high-affinity one of the angles present in the junction, a molecule with the shape of an X . Other DNA structures characterized by sharp bends or kinks, like bulged duplex DNAs containing unpaired bases, are also bound . HU protein appears to inhibit cruciform extrusion from supercoiled inverted repeat (palindromic) DNA, either by constraining supercoiling or by trapping a metastable interconversion intermediate . All these properties are analogous to the properties of the mammalian chromatin protein HMG1 . We suggest that HU is a prokaryotic HMG1-like protein rather than a histone-like protein. Cell Mol Neurobiol, 1993 Feb, 13(1), 25 - 38 Expression and reconstitution of biologically active human acetylcholinesterase from Escherichia coli; Fischer M et al.; 1 . Authentic human acetylcholinesterase (AChE) was expressed in Escherichia coli under regulation of the constitutive deo promoter or the thermo-inducible lambda PL promoter . 2 . To facilitate expression in the prokaryotic system, recombinant human AChE (rhAChE) cDNA was modified at the N terminus by oligonucleotide substitutions in order to replace some of the GC-rich regions by AT . These modifications did not alter the amino acid sequence but resulted in ample production of the protein . 3 . rhAChE accumulated in the cells and reached a level of 10% of total bacterial proteins . A partially purified inactive recombinant protein was recovered from inclusion bodies . 4 . Active rhAChE was obtained after solubilization, folding, and oxidation, although the recovery of the active enzyme was low . A 20- to 40-fold increase in enzymatically active rhAChE was achieved by replacing Cys580 by serine . 5 . The recombinant enzyme analogue was indistinguishable from native AChE isolated from erythrocytes in terms of substrate specificity and inhibitor selectivity. Trends Genet, 1993 Feb, 9(2), 56 - 60 Control of photosystem genes in Rhodobacter capsulatus; Bauer C et al.; Two environmental factors, oxygen and high light intensity, are known to repress synthesis of the Rhodobacter capsulatus photosystem . One level of regulation is the control of light harvesting and reaction centre gene expression at the point of transcription initiation . This has recently been shown to involve transcriptional activators which exhibit sequence similarity to members of the 'two-component' class of prokaryotic regulators . An additional level of regulation involves the formation of 'superoperons' that transcriptionally link pigment biosynthesis operons with operons that code for the light harvesting and reaction centre structural genes . A final level of regulation involves the selective degradation of reaction centre mRNA transcripts which influence the stoichiometric synthesis of the light harvesting and reaction centre complexes. Plant Mol Biol, 1993 Feb, 21(3), 487 - 502 Sorghum phosphoenolpyruvate carboxylase gene family: structure, function and molecular evolution; Lepiniec L et al.; Although housekeeping functions have been shown for the phosphoenolpyruvate carboxylase (EC 4.1.1.31, PEPC) in plants and in prokaryotes, PEPC is mainly known for its specific role in the primary photosynthetic CO2 fixation in C4 and CAM plants . We have shown that in Sorghum, a monocotyledonous C4 plant, the enzyme is encoded in the nucleus by a small multigene family . Here we report the entire nucleotide sequence (7.5 kb) of the third member (CP21) that completes the structure of the Sorghum PEPC gene family . Nucleotide composition, CpG islands and GC content of the three Sorghum PEPC genes are analysed with respect to their possible implications in the regulation of expression . A study of structure/function and phylogenetic relationships based on the compilation of all PEPC sequences known so far is presented . Data demonstrated that: (1) the different forms of plant PEPC have very similar primary structures, functional and regulatory properties, (2) neither apparent amino acid sequences nor phylogenetic relationships are specific for the C4 and CAM PEPCs and (3) expression of the different genes coding for the Sorghum PEPC isoenzymes is differently regulated (i.e . by light, nitrogen source) in a spatial and temporal manner . These results suggest that the main distinguishing feature between plant PEPCs is to be found at the level of genes expression rather than in their primary structure. Regul Toxicol Pharmacol, 1993 Feb, 17(1), 19 - 34 Evaluation of the carcinogenic potential of pesticides . 4 . Chloroalkylthiodicarboximide compounds with fungicidal activity; Quest JA et al.; The Health Effects Division of the Office of Pesticide Programs (OPP) assessed the carcinogenic potential of three structurally related chloroalkylthiodicarboximide fungicides using a consensus peer review process and EPA's 1986 guidelines for cancer risk assessment . All of the fungicides were categorized as Group B2 (probable human) carcinogens based upon findings of an increased incidence of malignant tumors, or combined malignant and benign tumors, in multiple experiments involving different strains of mice and rats . The primary sites of tumor formation with the chloroalkylthiodicarboximide fungicides in male and/or female mice (CD-1 and B6C3F1) were the gastrointestinal tract (captan, folpet, and captafol), the lymph system (folpet and captafol), and the vascular system (captafol) . The main sites of tumor formation in rats of one or both sexes (CR CD, Wistar, or F344 strains) were the kidney (Captan and captafol), uterus (captan), mammary gland and liver (captafol) . In addition, positive trends for thyroid, testicular, mammary gland, and lymph node tumors were observed with folpet in the same strains of rats . All three of the compounds exhibited positive mutagenic activity in a variety of in vitro short-term tests for gene mutation, DNA repair, and chromosomal aberrations in prokaryotic and eukaryotic cells, but were not genotoxic in available studies performed under in vivo conditions . The assessment of human cancer risk for captan, folpet, and captafol was made using low-dose extrapolation models. EMBO J, 1993 Feb, 12(2), 563 - 71 Chloroplast rps15 and the rpoB/C1/C2 gene cluster are strongly transcribed in ribosome-deficient plastids: evidence for a functioning non-chloroplast-encoded RNA polymerase; Hess WR et al.; Transcription of plastid genes and transcript accumulation were investigated in white leaves of the albostrians mutant of barley (Hordeum vulgare) and in heat-bleached leaves of rye (Secale cereale) as well as in normal green leaves of both species . Cells of white leaves of the mutant and cells of heat-bleached leaves bear undifferentiated plastids lacking ribosomes and, consequently, plastid translation products, among them the subunits of a putative chloroplast RNA polymerase encoded by the plastid genes rpoA, B, C1 and C2 . The following results were obtained . (i) Plastid genes are transcribed despite the lack of chloroplast gene-encoded RNA polymerase subunits . The plastid origin of these transcripts was proven . This finding provides evidence for the existence of a plastid RNA polymerase encoded entirely by nuclear genes . (ii) Transcripts of the rpo genes and of rps15, but not of genes involved in photosynthesis and related processes (psbA, rbcL, atpI-H), were abundantly accumulated in ribosome-deficient plastids . In contrast, chloroplasts accumulated transcripts of photosynthetic, but not of the rpo genes . (iii) Differences in transcript accumulation between chloroplasts and ribosome-deficient plastids are due to different relative transcription rates and different transcript stability . (iv) The observed differences in transcription are not caused by an altered pattern of methylation of plastid DNA . Thus, the prokaryotic plastid genome of higher plants is transcribed by two RNA polymerases . The observed differences in transcription between chloroplasts and undifferentiated plastids might reflect different functions of the two enzymes. EMBO J, 1993 Feb, 12(2), 413 - 21 Construction and characterization of a mercury-independent MerR activator (MerRAC): transcriptional activation in the absence of Hg(II) is accompanied by DNA distortion; Parkhill J et al.; The MeR regulatory protein of transposon Tn501 controls the expression of the mercury resistance (mer) genes in response to the concentration of mercuric ions . MerR is unique among prokaryotic regulatory proteins so far described in that it acts as a repressor {-Hg(II)} and an activator {+Hg(II)} of transcription of the mer genes, but binds to a single site on the DNA in both cases . This transcriptional activation process has been postulated to involve a protein-induced conformational change in the DNA that allows RNA polymerase more readily to form an open complex at the promoter . It has been shown {Frantz and O'Halloran (1990) Biochemistry, 29, 4747-4751} that activation of transcription by MerR in the presence of mercury is accompanied by hypersensitivity of the operator to chemical nucleases that are sensitive to local distortion in DNA structure . Here we describe specific mutations in MerR that allow the protein to stimulate transcription in the absence of the allosteric activator Hg(II) . We demonstrate that the degree of activation caused by these mutants directly correlates with the degree of DNA distortion as measured by the hypersensitivity of MerR-DNA complexes to the nuclease Cu-5-phenyl-o-phenanthroline . These results support the model described above. Carcinogenesis, 1993 Feb, 14(2), 303 - 5 A method for selection of forward mutations in supF gene carried by shuttle-vector plasmids; Ariza RR et al.; The supF gene of Escherichia coli has been widely used as a mutagenic target in several shuttle-vector plasmids . Mutations in this gene are usually screened by a colony colour assay based on the suppression of a lacZ amber mutation in an appropriate bacterial indicator strain . This screening method cannot measure the low mutation frequencies usually detected in prokaryotes, and therefore precludes the use of supF gene for studying mutational spectra in bacteria . In this paper we report the development of a simple method for the selection of supF forward mutations in shuttle-vector plasmids . The method has implied the construction of an araD- araC(Am) mutant strain (MBL50) of E.coli . The L-arabinose sensitivity caused by the accumulation of a toxic intermediate in araD- mutants is abolished in MBL50 because the araC(Am) mutation blocks the L-arabinose catabolic pathway . Strain MBL50 becomes sensitive to L-arabinose when transformed with a supF+ plasmid but remains resistant upon transformation with a supF- mutant . This new L-arabinose resistance selection method was able to detect supF- mutant fractions up to three orders of magnitude below those determined with the colony colour screening assay . The method was further validated by carrying out in vivo mutagenesis experiments with N-methyl-N-nitrosourea (MNU) and a shuttle-vector-bearing strain (UC2109) completely defective in O6-methylguanine (O6meG) alkyltransferase repair capacity . The DNA sequence alterations of 22 independent supF- mutants induced by MNU were determined . All mutations were G:C-->A:T transitions in agreement with the predicted significance of the mispairing potential of the O6meG lesion . A preference for the sequence 5'-GG-3' was detected, revealing a 5'-flanking base influence . The accumulation of all 22 MNU-induced mutations in three sites of the supF genes might be related to the lack of O6meG alkyltransferase repair capacity of strain UC2109 . The L-arabinose resistance method described in this paper allows rapid scoring and sequencing of forward mutations in the supF gene on shuttle-vectors, thus permitting its use as a genetic target for repair and mutagenesis studies in bacteria . Since shuttle-vectors replicate both in bacteria and mammalian cells, this method makes it possible to compare prokaryotic and eukaryotic mutational spectra. J Mol Evol, 1993 Feb, 36(2), 121 - 6 Accumulation of adenine and thymine in a groE-homologous operon of an intracellular symbiont; Ohtaka C et al.; As a result of the nucleotide sequence analysis of an aphid endosymbiont's operon homologous to the Escherichia coli groE, we noted that directional base substitutions tending toward an increase of A + T content represent an obvious evolutionary trend in this prokaryotic operon, housed for a long period by an eukaryotic cell . This result, when taken together with previous reports, raised the possibility that genomic DNA of prokaryotes residing in an eukaryotic cell is subject to A/T-biased directional mutation pressure and/or both negative and positive selection operating under conditions specific to the intracellular environments. J Bacteriol, 1993 Feb, 175(4), 1061 - 8 Analysis of feedback-resistant anthranilate synthases from Saccharomyces cerevisiae; Graf R et al.; The initial step of tryptophan biosynthesis is catalyzed by the enzyme anthranilate synthase, which in most microorganisms is subject to feedback inhibition by the end product of the pathway . We have characterized the TRP2 gene from a mutant Saccharomyces cerevisiae strain coding for an anthranilate synthase that is unresponsive to tryptophan . Sequence analysis of this TRP2(Fbr) (feedback-resistant) allele revealed numerous differences from a previously published TRP2 sequence . However, TRP2(Fbr) was found to differ in only one single-point mutation from its own parent wild type, a C-to-T transition resulting in a serine 76-to-leucine 76 amino acid substitution . Therefore, serine 76 is a crucial amino acid for proper regulation of the yeast enzyme . We constructed additional feedback-resistant enzyme forms of the yeast anthranilate synthase by site-directed mutagenesis of the conserved LLES sequence in the TRP2 gene . From analysis of these variants, we propose an extended sequence, LLESX10S, as the regulatory element in tryptophan-responsive anthranilate synthases from prokaryotic and eukaryotic organisms. Hum Mol Genet, 1993 Feb, 2(2), 133 - 8 A large inverted duplicated DNA region associated with an amplified oncogene is stably maintained in a YAC; Hayashi Y et al.; In the polyoma virus (Py) transformed 3B rat cell line the Py oncogene and adjacent cellular DNA are amplified in arrays of very large inverted duplications . A region of the 3B amplified DNA was cloned as a 550 kb insert in a Yeast Artificial Chromosome (YAC) vector, designated y3B01 . Analysis of the y3B01 cloned insert revealed it contained a large inverted duplicated DNA region which was approximately 400 kb in size (two palindromic arms of about 200 kb) . At least 420 kb of the 550 kb YAC insert has been identified as being derived from the 3B amplified DNA and the amplicon in 3B cells is at least 220 kb in size . No DNA instability of the y3B01 YAC clone was detected . The y3B01 DNA replicated as efficiently as yeast chromosomes and was structurally stable in yeast cells during more than 30 cell divisions . Comparison of the restriction endonuclease maps of the inverted duplicated region of the y3B01 DNA insert and the amplified 3B genomic DNA did not reveal any gross differences suggesting that no rearrangements had occurred during or after the cloning into the YAC vector . These results suggest that large inverted duplications, which can show instability in prokaryotic cloning systems, can be stably cloned and maintained in YAC vectors in yeast. Mol Microbiol, 1993 Feb, 7(4), 497 - 503 Translational frameshifting in the control of transposition in bacteria; Chandler M et al.; The expression of an increasing number of genes of both prokaryotic and eukaryotic origin has been shown to be regulated at the translational level by programmed (sequence-specific) ribosomal frameshifting . Among these are the bacterial insertion sequences IS1 and two members of the widely distributed IS3-family, IS150 and IS911 . Frameshifting provides a means of specifying several proteins with different functions using a minimum of genetic information . In this review, we survey present understanding of the way in which frameshifting is integrated into the overall control of transposition activity in these elements. Immunol Lett, 1993 Feb, 35(2), 163 - 8 Detection of a circulating antibody against a peptide epitope on a mucin core protein, MUC1, in ulcerative colitis; Hinoda Y et al.; This study aims at clarifying whether the humoral immune response to the tandem repeat domain of MUC1 can be induced or not in vivo . The expression of MUC1 mRNA in the colon was revealed by Northern blot analysis, and cDNA cloning of an extracellular tandemly repeated domain of MUC1 was then performed to prepare the recombinant MUC1 protein . A cDNA clone coding for ten repeat domains was ligated into an expression vector in prokaryotes, resulting in a recombinant protein which could react with the MAb MUSE11 against an adenocarcinoma-associated antigen whose epitope has been shown to be localized in the tandem repeat domain on MUC1 . The reactivity of sera from patients with ulcerative colitis with the recombinant protein was evaluated by SDS-PAGE and Western blot analysis to detect the antibodies against this tandem repeat domain . Five out of 19 serum samples tested positive, and these reactions were totally inhibited by MAb MUSE11, suggesting that the epitope recognized by these antibodies in sera is almost identical to that recognized by MAb MUSE11 . The data represent the first demonstration of antibody production against a peptide epitope of the tandem repeat domain of MUC1. Bioessays, 1993 Feb, 15(2), 113 - 20 RNA processing in prokaryotic cells; Apirion D et al.; RNA processing in Escherichia coli and some of its phages is reviewed here, with primary emphasis on rRNA and tRNA processing . Three enzymes, RNase III, RNase E and RNase P are responsible for most of the primary endonucleolytic RNA processing events . The first two are proteins, while RNase P is a ribozyme . These three enzymes have unique functions and in their absence, the cleavage events they catalyze are not performed . On the other hand a relatively large number of exonucleases participate in the trimming of the 3' ends of tRNA precursor molecules and they can substitute for each other . Primary processing is the first event that happens to the nascent RNA molecule, while in secondary RNA processing, the substrate is a product of a primary processing event . Although most RNA processing occurs in RNP particles, it seems that only in secondary RNA processing is the RNP particle required for the reaction . Bacteria and especially bacteriophages contain self-splicing introns which in cases were probably acquired from other species. J Gen Microbiol, 1993 Feb, 139 ( Pt 2), 317 - 23 DNA sequence determination and biochemical analysis of the immunogenic protein P36, the lactate dehydrogenase (LDH) of Mycoplasma hyopneumoniae; Haldimann A et al.; The DNA sequence of the gene encoding the early and specific immunogenic protein P36 of Mycoplasma hyopneumoniae has been determined . Comparison of the DNA sequence and the deduced amino acid sequence of P36 with known genes and proteins in data banks indicated that P36 is a L-lactate dehydrogenase (LDH) (EC 1.1.1.27) . Biochemical analysis of protein P36 expressed from the cloned gene in Escherichia coli confirmed that P36 has L-lactate dehydrogenase activity . Protein P36 of M . hyopneumoniae therefore is termed LDH and its gene ldh . M . hyopneumoniae LDH was shown to contain the typical domains of LDH of other bacterial species . Immunologically however, we have shown that polyclonal antibodies against M . hyopneumoniae LDH do not cross-react with related LDH and show high specificity for M . hyopneumoniae . The ldh gene is preceded by several typical -10 sequences found in promoters of prokaryotes, but lacks the -35 sequence . Sequences rich in A+T, however, precede the -10 boxes, suggesting that factors involved in transcription initiation and their regulation may be different in M . hyopneumoniae compared to other bacterial species, but the putative ribosome binding site seems to be conserved. Mutat Res, 1993 Feb, 301(2), 87 - 92 Ciprofloxacin: mammalian DNA topoisomerase type II poison in vivo; Mukherjee A et al.; Ciprofloxacin (CF), a fluoroquinolone widely used as a potent antimicrobial drug, was evaluated in vivo in mouse bone marrow cells for its ability to induce clastogenicity and DNA damage in terms of increased sister-chromatid exchange (SCE) frequencies . Doses of 0.6, 6 and 20 mg/kg body weight of CF given intraperitoneally induced a positive dose-dependent significant clastogenicity (trend test alpha < or = 0.05), though the effects were not specific for specific phases of the cell cycle . The DNA-damaging effect observed as increased SCE frequencies using doses of 0.15, 0.30, 0.60, 1.2 and 6 mg/kg body weight showed a significant dose-dependent increase (trend test alpha < or = 0.05; lowest effective concentration 1.2 mg/kg of body weight) . Compared to a potent eukaryotic DNA topoisomerase type II poison, etoposide (VP-16, 0.5, 1 and 5 mg/kg body weight, given intraperitoneally), ciprofloxacin produced comparable dose-dependent SCE frequency increases . Ciprofloxacin was postulated to be specific for the target DNA gyrase, the prokaryotic homologue of DNA topoisomerase type II enzyme . The present paper along with the existing earlier data strongly suggest that topoisomerase type II and DNA gyrase are physiological targets for the drug action . In view of the present significant in vivo mammalian DNA topoisomerase type II-mediated genotoxicity and clastogenicity data, ciprofloxacin should be administered with caution. Biochem Biophys Res Commun, 1993 Jan 15, 190(1), 1 - 7 Cloning of a functional cDNA for human cytidine deaminase (CDD) and its use as a marker of monocyte/macrophage differentiation; Kuhn K et al.; We have identified a cDNA clone for human cytidine deaminase (EC 3.5.4.5) during an investigation which aimed at cloning novel gene expression products related to monocyte/macrophage differentiation . The derived amino acid sequence of the clone comprises 145 residues yielding a molecular mass for the polypeptide of 16.1 kDa and exhibits a nearly 50% homology to cytidine deaminase from Bacillus subtilis . Cytidine deaminase activity of the cloned sequence could be demonstrated in a prokaryotic expression system . The mRNA is highly expressed in granulocytes while expression is very low in fibroblasts, chondrocytes, monocytes, and T- as well as B-cell lines . The mRNA can be induced in monocytes, the monocytoid cell line U937 and the myeloblastic line HL 60 by the differentiation inducer calcitriol. J Biol Chem, 1993 Jan 15, 268(2), 1479 - 87 Hsp90 chaperonins possess ATPase activity and bind heat shock transcription factors and peptidyl prolyl isomerases; Nadeau K et al.; Heat shock proteins of the 82-90 kDa class (hsp82 and hsp90) are abundant, conserved, and ubiquitous from prokaryotes to eukaryotes . Although proposed to be chaperones, they had not been reported to possess enzymatic activity until our recent observation that pure trypanosomatid hsp83 had potent ATPase activity (Nadeau, K., Sullivan, M., Engman, D., and Walsh, C . T . (1992) Protein Sci . 1, 970-979) . We have now purified the hsp90 homolog from Escherichia coli (HtpG) and from Saccharomyces cerevisiae (hsp82) to homogeneity and observe ATPase activity with kcat values of 3 min-1 and 140 min-1 . In addition, examinations of purified rat hsp90 and human hsp90 detect ATPase activity with a kcat of 0.6 min-1 and 10 min-1 . Each of these hsp90s undergoes autophosphorylation on serine or threonine residues . In prokaryotes and eukaryotes, the induction of hsps during heat shock is controlled, respectively, by the binding of an alternate sigma 32 or a transcriptional activator (heat shock factor or HSF) at heat shock promoter elements . Here we show that E . coli HtpG immobilized to Affi-Gel beads selectively retains sigma 32 while the yeast hsp90 and rat hsp90 retain HSF . The peptidyl prolyl isomerase hsp59 of the FK506 binding class is known to bind to hsp90 . We also detect binding of the other family of PPIases, the cyclophilins, to immobilized hsp90, consistent with a functional convergence of protein foldases. Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 745 - 9 Acidic C terminus of vaccinia virus DNA-binding protein interacts with ribonucleotide reductase; Davis RE et al.; Evidence from prokaryotic systems suggests that enzymes of dNTP synthesis are organized near the DNA replication apparatus, allowing direct utilization of dNTPs at their sites of synthesis . To investigate whether similar interactions exist within a eukaryotic environment, we have prepared anti-idiotypic antibodies to the small subunit of vaccinia virus ribonucleotide reductase, and we used these antibodies to search for proteins that interact with this enzyme . This approach identified a 34-kDa viral phosphoprotein, which, like ribonucleotide reductase itself, is localized within infected cells at DNA replication sites . After expression of its structural gene in Escherichia coli, the recombinant protein was purified and found (i) to bind tightly to single-stranded DNA and (ii) to stimulate enzymatic activity of vaccinia ribonucleotide reductase . These observations suggest a physical association between dNTP synthesis and DNA replication in this viral system. Proc Natl Acad Sci U S A, 1993 Jan 15, 90(2), 620 - 4 The ATP-binding component of a prokaryotic traffic ATPase is exposed to the periplasmic (external) surface; Baichwal V et al.; The membrane-bound complex of bacterial periplasmic permeases consists of two hydrophobic integral membrane proteins and two copies of a hydrophilic ATP-binding protein . The ATP-binding proteins from all periplasmic permeases display a high level of sequence similarity and are referred to as "conserved components." The conserved component from the histidine permease, HisP, has been postulated on the basis of genetic evidence to be accessible at the exterior membrane surface, in contrast to the commonly postulated association with the interior membrane surface as peripheral membrane proteins . We have used proteolysis and biotinylation of membrane vesicles to show that HisP is accessible to these reagents at the external surface and that this orientation depends on the presence of the two hydrophobic components, HisQ and HisM . Several binding-protein-independent hisP mutants are shown to produce HisP proteins that are more susceptible to proteases from the external membrane surface . Since the hydrophilic component is well conserved also in a group of eukaryotic transporters, which together with many prokaryotic systems form the superfamily of traffic ATPases, this insight about its membrane topology has general implications for understanding the molecular mechanism of action of this large superfamily, which includes the cystic fibrosis transmembrane conductance regulator and multidrug-resistance proteins. J Mol Biol, 1993 Jan 5, 229(1), 268 - 76 Isolation and sequencing of Escherichia coli gene proP reveals unusual structural features of the osmoregulatory proline/betaine transporter, ProP; Culham DE et al.; Transporters encoded in genetic loci putP, proP and proU mediate proline and/or betaine accumulation by Escherichia coli K-12 . The ProP and ProU systems are osmoregulatory . Activation of ProP in response to hyperosmotic stress has been demonstrated both in vivo and in vitro . It therefore serves as a model experimental system for the analysis of osmosensory and osmoregulatory mechanisms . We developed methodologies which will facilitate the identification of proline transporter genes by functional complementation of putP proP proU bacteria . E . coli gene proP was isolated and located within a chromosomal DNA fragment . Deletion, complementation and sequence analysis revealed putative promoter and transcription termination signals flanking a 1500 base-pair open reading frame . The predicted 55 kDa ProP protein was hydrophobic . In vitro expression of proP yielded a protein whose apparent molecular mass was determined to be 42 kDa by polyacrylamide gel electrophoresis under denaturing conditions . Database searches and cluster analysis defined relationships among the ProP sequence and those of integral membrane proteins that comprise a transporter superfamily . Members of the superfamily catalyze facilitated diffusion or ion linked transport of organic solutes in prokaryotes and eukaryotes . Multiple alignment revealed particularly close correspondence among the ProP protein, citrate transporters from E . coli and Klebsiella pneumoniae and an alpha-ketoglutarate transporter from E . coli . The predicted ProP sequence differed from those closely similar sequences in possessing an extended central hydrophilic loop and a carboxyl terminal extension . Unlike other protein sequences within the transporter superfamily, the carboxyl terminal extension of ProP was strongly predicted to participate in formation of an alpha-helical coiled coil . These data suggest that the ProP protein catalyzes solute-ion cotransport . Its unusual structural features may be related to osmoregulation of its activity. J Biol Chem, 1993 Jan 5, 268(1), 653 - 7 m-Calpain requires DNA for activity on nuclear proteins at low calcium concentrations; Mellgren RL et al.; m-Calpain (calpain II, m-CANP), which normally requires millimolar Ca2+ for activity in vitro, was capable of proteolyzing a number of matrix proteins in isolated rat liver nuclei at Ca2+ concentrations as low as 3 microM (Mellgren, R . L . (1991) J . Biol . Chem . 266, 13920-13924) . Treatment of nuclei with deoxyribonuclease I eliminated the activity of m-calpain at low Ca2+ concentrations, while ribonuclease A and phospholipase C had no effect . Addition of DNA to DNase-treated nuclei restored m-calpain activity at low Ca2+ . RNA had little if any effect . Eukaryotic and prokaryotic DNA were equally effective, and synthetic polydeoxyribonucleotides were also activators . m-Calpain did not bind to a DNA-cellulose column in the presence of 200 microM Ca2+, and m-calpain preincubated in the presence of DNA and 200 microM Ca2+ was not activated at low Ca2+ concentrations following removal of the DNA . DNA did not alter the Ca2+ requirement for m-calpain-catalyzed cleavage of casein . These results demonstrate that the Ca2+ requirement for proteolysis of nuclear matrix proteins by m-calpain can be dramatically decreased in the presence of DNA . Activation did not seem to be a result of DNA binding directly to calpain but appeared to require interaction of DNA, calpain, and calpain substrates in the nuclear matrix. Crit Rev Microbiol, 1993, 19(1), 43 - 59 The prochlorophytes: are they more than just chlorophyll a/b-containing cyanobacteria? Bullerjahn GS, Post AF. The prochlorophytes are a diverse group of photosynthetic prokaryotes that fall within the cyanobacterial lineage, yet lack phycobilisomes as light harvesting structures . Instead, the prochlorophytes have a light-harvesting apparatus composed of the higher plant pigments chlorophylls a and b . This review discusses the evolutionary relationships among these bacteria, with focus on the structure and function of the photosynthetic apparatus . This analysis yields a consensus from studies both on Prochloron sp . and Prochlorothrix hollandica as to how the thylakoid membrane is organized . Overall, we propose that the structure of the light-harvesting complexes (LHC) from prochlorophytes is very different from those of chloroplast systems, and is evolutionarily very ancient . The functional association of the light-harvesting apparatus with photosystem I (PSI) in both Prochlorothrix and Prochloron, as well as a demonstrated capacity for PSI-dependent anoxygenic photosynthesis in Prochlorothrix, may indicate that there is an increased dependence on cyclic photophosphorylation in these organisms . Finally, the structure of the prochlorophyte thylakoid membrane is discussed with respect to the forces that drive thylakoid membrane stacking in prochlorophytes and chloroplasts . We suggest that the light-harvesting structures in prochlorophytes play little, if any, role in this process. Crit Rev Microbiol, 1993, 19(1), 17 - 42 Biomass growth rate during the prokaryote cell cycle; Koch AL; The rate of biomass growth throughout the cell cycle of prokaryotes is important in the study of global regulation . Two limiting cases have generally been considered: the exponential model and the linear model . The exponential model is a logical expectation because protein, the main component of biomass of a bacterial cell, increases continuously during the cell cycle and therefore the means for synthesis of other cell components and metabolites also increases . In addition, during the cell cycle, ribosomes, the means of production of proteins, increase monotonically . As a consequence, the increase of all should be autocatalytic and the content of cell substance should be an exponential function of time . Two cellular components would not be expected to increase exponentially: the DNA and the cell envelope . The former because of the intermittent synthesis of the chromosome, and the latter because of changes in the surface-to-volume ratio with growth and division . In contrast to the exponential model, the linear model of Kubitschek postulates that the cell only increases its membrane transport capability over a brief period during the cell cycle, and, thus limited by transport, all cell components can increase only at a constant linear rate during most of the cell cycle . Other proposed models are intermediate and assume that the growth rate of the cell depends on some cell cycle event, such as the initiation of chromosome replication . The models have relevance to prokaryotes undergoing balanced growth; they may not be relevant to eukaryotic microbes or to eukaryotic cells in tissue culture that have endogenous rhythms or are controlled by protein growth factors . Logically, the models could possibly apply to a free-living cell that does not respond to environmental cues . Even under rigidly constant conditions, however, cells may try to respond to a stimulus that was periodic or regulatory under natural conditions, but is present at a constant level under the experimental culture condition . There are four classes of experiments that have been used to measure the accumulation of dry biomass or its components during the cell cycle of a bacterium, as typified by Escherichia coli . For the first class of experiments, the dimensions of living cells are measured under the microscope . So far, the experiments have been limited by the resolving power of the phase microscope, but adequate resolution should be possible with the confocal scanning light microscope or various video computer systems . Such experiments are called integral because augmentation of cell constituents is followed . The second class involves pulse-chase labeling of cells and then their separation into different phases of the cycle or age groups and measurement of the radioactivity per cell in the fractions . Such experiments are called differential in that the rate is measured directly instead of being deduced by comparing the total size at different times.(ABSTRACT TRUNCATED AT 400 WORDS) Bioessays, 1993 Jan, 15(1), 25 - 32 Bending of DNA by transcription factors; van der Vliet PC et al.; An increasing number of transcription factors both from prokaryotic and eukaryotic sources are found to bend the DNA upon binding to their recognition site . Bending can easily be detected by the anomalous electrophoretic behaviour of the DNA-protein complex or by increased cyclization of DNA fragments containing the protein-induced bend . Induction of DNA bending by transcription factors could regulate transcription in various ways . Bending may bring distantly bound transcription factors closer together by facilitating DNA-looping or it could mediate the interaction between transcription factors and the general transcription machinery by formation of large nucleoprotein structures in which the DNA is wrapped around the protein complex . Alternatively, the energy stored in a protein-induced bend could be used to favour formation of an open transcription complex or to dissociate the RNA polymerase in the transition from initiation to elongation . Modification of the bend angles and bending centers, caused by homodimerization or heterodimerization of transcription factors, may well turn out to be an important way to enlarge the range of interactions required for regulation of gene expression. Mol Microbiol, 1993 Jan, 7(2), 189 - 95 Deletion within the metallothionein locus of cadmium-tolerant Synechococcus PCC 6301 involving a highly iterated palindrome (HIP1); Gupta A et al.; Genomic rearrangements involving amplification of metallothionein (MT) genes have been reported in metal-tolerant eukaryotes . Similarly, we have recently observed amplification and rearrangement of a prokaryotic MT locus, smt, in cells of Synechococcus PCC 6301 selected for Cd tolerance . Following the characterization of this locus, the altered smt region has now been isolated from a Cd-tolerant cell line, C3.2, and its nucleotide sequence determined . This has identified a deletion within smtB, which encodes a trans-acting repressor of smt transcription . Two identical palindromic octanucleotides (5'-GCGATC-GC-3') traverse both borders of the excised element . This palindromic sequence is highly represented in the smt locus (7 occurrences in 1326 nucleotides) and analysis of the GenBank/EMBL/DDBJ DNA Nucleotide Sequence Data Libraries reveals that this is a highly iterated palindrome (HIP1) in other known sequences from Synechococcus strains (estimated to occur at an average frequency of once every c . 664 bp) . HIP1 is also abundant in the genomes of other cyanobacteria . The functional significance of smtB deletion and the possible role of HIP1 in genome plasticity and adaptation in cyanobacteria are discussed. Mol Microbiol, 1993 Jan, 7(2), 177 - 87 Isolation of a prokaryotic metallothionein locus and analysis of transcriptional control by trace metal ions; Huckle JW et al.; In eukaryotes, metallothioneins (MTs) are involved in cellular responses to elevated concentrations of certain metal ions . We report the isolation and analysis of a prokaryotic MT locus from Synechococcus PCC 7942 . The MT locus (smt) includes smtA, which encodes a class II MT, and a divergently transcribed gene, smtB . The sites of transcription initiation of both genes have been mapped and features within the