Prokaryotes

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 smt operator-promoter region identified . Elevated concentrations of the ionic species of Cd, Co, Cr, Cu, Hg, Ni, Pb and Zn elicited an increase in the abundance of smtA transcripts . There was no detectable effect of elevated metal (Cd) on smtA transcript stability . Sequences upstream of smtA, fused to a promoterless lacZ gene, conferred metal-dependent beta-galactosidase activity in Synechococcus PCC 7942 (strain R2-PIM8) . At maximum permissive concentrations, Zn was the most potent elicitor in vivo, followed by Cu and Cd with slight induction by Co and Ni . The deduced SmtB polypeptide has similarity to the ArsR and CadC proteins involved in resistance to arsenate/arsenite/antimonite and to Cd, contains a predicted helix-turn-helix DNA-binding motif and is shown to be a repressor of transcription from the smtA operator-promoter.

Mol Gen Genet, 1993 Jan, 236(2-3), 309 - 14
Cloning and expression analysis of beta-isopropylmalate dehydrogenase from potato; Jackson SD et al.; A full-length cDNA clone for beta-isopropylmalate dehydrogenase from potato has been isolated and sequenced . The open reading frame is 1071 bp in length encoding a protein of 357 amino acids which includes a 29 amino acid, putative chloroplastic transit peptide . The amino acid sequence shows 33.3% and 28.6% identity to beta-isopropylmalate dehydrogenases from rape and Bacillus subtilis, respectively . Southern analysis shows that the gene is present in low copy number in potato, and in single copy in tomato and Arabidopsis . The gene is expressed in all tissues of the potato plant and its expression is increased by leucine, and leucine plus threonine, in contrast to the situation in yeast and prokaryotes . The gene is also induced by sucrose in a manner similar to that seen with genes involved in carbohydrate metabolism, which indicates that there may be some interaction at the transcriptional level between genes involved in carbon and nitrogen metabolism.

J Cell Biochem, 1993 Jan, 51(1), 7 - 13
ATP-dependent protein kinases in bacteria; Cozzone AJ; Protein phosphorylation has been shown to occur in over fifty different bacterial species and, therefore, seems to be a universal device among prokaryotes . Most of the protein kinases responsible for this modification of proteins share the common property of using adenosine triphosphate as phosphoryl donor . However, they differ from one another in a number of structural and functional aspects . Namely, they exhibit a varying acceptor amino acid specificity and can be classified, on this basis, in three main groups: protein-histidine kinases, protein-serine/threonine kinases and protein-tyrosine kinases.

J Cell Biochem, 1993 Jan, 51(1), 29 - 33
Eukaryotic-like protein serine/threonine kinases in Myxococcus xanthus, a developmental bacterium exhibiting social behavior; Munoz-Dorado J et al.; Myxococcus xanthus, a gram-negative bacterium exhibits a spectacular life cycle and social behavior . Its developmental cycle and multicellular morphogenesis resemble those of eukaryotic slime molds such as Dictyostelium discoideum . On the basis of this resemblance, we explored the existence of eukaryotic-like protein serine/threonine kinases which are known to play important roles in signal transduction during development of D . discoideum . It was indeed found that M . xanthus contains a large family of protein serine/threonine kinases related to the eukaryotic enzymes . This is the first unambiguous demonstration of eukaryotic-like serine/threonine kinases in the prokaryotes.

Plant Mol Biol, 1993 Jan, 21(2), 363 - 73
DNA amplification fingerprinting of the Azolla-Anabaena symbiosis; Eskew DL et al.; The Azolla-Anabaena symbiosis has been used for centuries as a nitrogen biofertilizer in rice paddies . Genetic improvement of the symbiosis has been limited by the difficulty in identifying Azolla-Anabaena accessions and Anabaena azollae strains . The recently developed technique of DNA amplification fingerprinting (DAF) was applied to this problem . DAF uses single, short, oligonucleotide primers of arbitrary sequence to direct amplification of a characteristic set of DNA products by a thermostable DNA polymerase in a thermocycling reaction . The products are separated in polyacrylamide gels and detected by silver staining . DAF could easily distinguish and positively identify accessions of Azolla-Anabaena with DNA extracted from the intact symbiosis . The contribution of prokaryotic Anabaena sequences to the fingerprint of the intact symbiosis, however, ranged from 0 to 77%, depending on the primer sequence . Therefore, DNA extracted from the intact symbiosis would not be suitable for Azolla taxonomy studies . The fingerprints of Anabaena strains isolated by sucrose gradient centrifugation from different species of Azolla could be easily distinguished, and DAF patterns were used to confirm the maternal pattern of transmission of Anabaena in a sexual hybrid . Template DNA extracted from roots was used to produce fingerprints for Azolla without interference from the microsymbiont . Comparison of the patterns from the parents and a hybrid gave strong evidence confirming sexual hybridization.

Plant Mol Biol, 1993 Jan, 21(1), 47 - 58
Organization, expression and nucleotide sequence of the operon encoding R-phycoerythrin alpha and beta subunits from the red alga Polysiphonia boldii; Roell MK et al.; The characterization of the operon encoding the alpha and beta subunits of rhodophytan (R)-phycoerythrin (PE) from the macrophytic red alga Polysiphonia boldii is reported . This plastid-encoded operon was cloned, its nucleotide sequence determined, and its expression characterized by northern and primer extension analyses . The arrangement and expression of the PE alpha and beta genes, named rpeA and rpeB, are similar to those of the cyanobacterial (C)-PE genes: rpeB is located 5' of rpeA, with an intergenic region of 64 nucleotides . The two genes are transcribed on a 1.25 kb dicistronic transcript, and each coding region is preceded by a prokaryotic ribosome binding site consensus sequence . Transcription is initiated 95 nucleotides upstream of the initiating methionine codon of rpeB . The promoter region resembles that of prokaryotic genes, with an AT-rich -10 sequence . A direct pentanucleotide repeat (5'-TGTTA-3') was found in the -35 region . This pentanucleotide is present upstream of all PE operons that have been characterized thus far . An extensive inverted repeat is present 3' of rpeA; inverted repeats are found downstream of all PE operons sequenced to date, although the sequence is not conserved . The deduced amino acid sequences from these genes provide complete sequences for an R-PE . Of the amino acid residues 85% are identical to those of bangeophycean (B)-PE from the unicellular red alga Porphyridium cruentum . Conserved residues include cysteines at the bilin attachment sites of C- and B-PEs, aspartates at positions postulated to interact with bilin chromophores, and an apparent consensus sequence for N-methylation of an asparagine residue in C-PEs.

Arch Biochem Biophys, 1993 Jan, 300(1), 213 - 22
O-acetylserine(thiol)lyase from spinach (Spinacia oleracea L.) leaf: cDNA cloning, characterization, and overexpression in Escherichia coli of the chloroplast isoform; Rolland N et al.; The last enzymatic step for L-cysteine biosynthesis is catalyzed by O-acetylserine(thiol)lyase (OASTL, EC 4.2.99.8) which synthesizes L-cysteine from O-acetylserine and "sulfide." We have isolated and characterized a full-length cDNA (1432 bp) from a lambda gt11 library of spinach leaf encoding the complete precursor of the chloroplast isoform . The 1149-nucleotide open reading frame coding for O-acetylserine(thiol)lyase was in the direction opposite that of the lambda gt11 beta-galactosidase gene . The derived amino acid sequence indicates that the protein precursor consists of 383 amino acid residues including a N-terminal presequence peptide of 52 residues . The amino acid sequence of mature spinach chloroplast O-acetylserine(thiol)lyase shows 40 and 57% homology with its bacterial counterparts . Sequence comparison with several pyridoxal 5'-phosphate-containing proteins reveals the presence of a lysine residue assumed to be involved in cofactor binding . A synthetic cDNA was constructed, coding for the entire 331-amino-acid mature O-acetylserine(thiol)lyase and for an initiating methionine . A high level of expression of the active mature chloroplast isoform was achieved in an Escherichia coli strain carrying the T7 RNA polymerase system (F . W . Studier, A . H . Rosenberg, J . J . Dunn, and J . W . Dubendorff, 1990, in Methods in Enzymology, D . V . Goeddel, Ed., Vol . 185, pp . 60-89, Academic Press, San Diego, CA) . Addition of pyridoxine to the bacterial growth medium enhanced the enzyme activity due to the recombinant protein . The extent of production is 25-fold higher than in chloroplast from spinach leaves and the recombinant protein presents the relative molecular mass and immunological properties of the natural enzyme from spinach leaf chloroplast . This work, together with our previous biochemical studies, are in accordance with a prokaryotic type enzyme for L-cysteine biosynthesis in higher plant chloroplasts . Southern blot analysis indicated that O-acetylserine(thiol)lyase is encoded by multiple genes in the spinach leaf genomic DNA.

FASEB J, 1993 Jan, 7(1), 223 - 31
Unique phylogenetic position of Diplomonadida based on the complete small subunit ribosomal RNA sequence of Giardia ardeae, G . muris, G . duodenalis and Hexamita sp; van Keulen H et al.; Complete small-subunit rRNA (SSU-rRNA) coding region sequences were determined for two species of the intestinal parasite Giardia: G . ardeae and G . muris, both belonging to the order Diplomonadida, and a free-living member of this order, Hexamita sp . These sequences were compared to published SSU-rDNA sequences from a third member of the genus Giardia, G . duodenalis (often called G . intestinalis or G . lamblia) and various representative organisms from other taxa . Of the three Giardia sequences analyzed, the SSU-rRNA from G . muris is the smallest (1432 bases as compared to 1435 and 1453 for G . ardeae and G . duodenalis, respectively) and has the lowest G+C content (58.9%) . The Hexamita SSU-rRNA is the largest in this group, containing 1550 bases . Because the sizes of the SSU-rRNA are prokaryotic rather than typically eukaryotic, the secondary structures of the SSU-rRNAs were constructed . These structures show a number of typically eukaryotic signature sequences . Sequence alignments based on constraints imposed by secondary structure were used for construction of a phylogenetic tree for these four taxa . The results show that of the four diplomonads represented, the Giardia species form a distinct group . The other diplomonad Hexamita and the microsporidium Vairimorpha necatrix appear to be distinct from Giardia.

Infect Immun, 1993 Jan, 61(1), 109 - 16
Complete nucleotide sequence of the Actinomyces viscosus T14V sialidase gene: presence of a conserved repeating sequence among strains of Actinomyces spp; Yeung MK; The nucleotide sequence of the Actinomyces viscosus T14V sialidase gene (nanH) and flanking regions was determined . An open reading frame of 2,703 nucleotides that encodes a predominately hydrophobic protein of 901 amino acids (M(r), 92,871) was identified . The amino acid sequence at the amino terminus of the predicted protein exhibited properties characteristic of a typical leader peptide . Five 12-amino-acid units that shared between 33 and 67% sequence identity were noted within the central domain of the protein . Each unit contained the sequence Ser-X-Asp-X-Gly-X-Thr-Trp, which is conserved among other bacterial and trypanosoma sp . sialidases . Thus, the A . viscosus T14V nanH gene and the other prokaryotic and eukaryotic sialidase genes evolved from a common ancestor . Southern hybridization analyses under conditions of high stringency revealed the existence of DNA sequences homologous to A . viscosus T14V nanH in the genomes of 18 strains of five Actinomyces species that expressed various levels of sialidase activity . The data demonstrate that the sialidase genes from divergent groups of Actinomyces spp . are highly conserved.

Genetics, 1993 Jan, 133(1), 127 - 32
Do deleterious mutations act synergistically? Metabolic control theory provides a partial answer; Szathmary E; Metabolic control theory is used to derive conditions under which two deleterious mutations affecting the dynamics of a metabolic pathway act synergistically . It is found that two mutations tend to act mostly synergistically when they reduce the activity of the same enzyme . If the two mutations affect different enzymes, the conclusion depends on the way that fitness is determined by aspects of the pathway . The cases analyzed are: selection for (1) maximal flux, (2) maximal equilibrium concentration (pool size) of an intermediate, (3) optimal flux, (4) optimal pool size . The respective types of epistasis found are: (1) antagonistic, (2) partly synergistic, (3-4) synergism is likely to predominate over antagonism . This results in somewhat different predictions concerning the effect of metabolic mutations on fitness in prokaryotes and eukaryotes . The fact that bacteria are largely clonal but have often a mosaic gene structure is consistent with expectations from the model.

Mol Cell Biol, 1993 Jan, 13(1), 506 - 20
GCD11, a negative regulator of GCN4 expression, encodes the gamma subunit of eIF-2 in Saccharomyces cerevisiae; Hannig EM et al.; The eukaryotic translation initiation factor eIF-2 plays a critical role in regulating the expression of the yeast transcriptional activator GCN4 . Mutations in genes encoding the alpha and beta subunits of eIF-2 alter translational efficiency at the GCN4 AUG codon and constitutively elevate GCN4 translation . Mutations in the yeast GCD11 gene have been shown to confer a similar phenotype . The nucleotide sequence of the cloned GCD11 gene predicts a 527-amino-acid polypeptide that is similar to the prokaryotic translation elongation factor EF-Tu . Relative to EF-Tu, the deduced GCD11 amino acid sequence contains a 90-amino-acid N-terminal extension and an internal cysteine-rich sequence that contains a potential metal-binding finger motif . We have identified the GCD11 gene product as the gamma subunit of eIF-2 by the following criteria: (i) sequence identities with mammalian eIF-2 gamma peptides; (ii) increased eIF-2 activity in extracts prepared from cells cooverexpressing GCD11, eIF-2 alpha, and eIF-2 beta; and (iii) cross-reactivity of antibodies directed against the GCD11 protein with the 58-kDa polypeptide present in purified yeast eIF-2 . The predicted GCD11 polypeptide contains all of the consensus elements known to be required for guanine nucleotide binding, suggesting that, in Saccharomyces cerevisiae, the gamma subunit of eIF-2 is responsible for GDP-GTP binding.

Plant J, 1993 Jan, 3(1), 143 - 50
Molecular characterization of a 70 kDa heat-shock protein of bean mitochondria; Vidal V et al.; A bean cDNA clone that specifies a 70 kDa heat-shock protein (hsp70) has been isolated and sequenced . The nucleotide sequence analysis shows that the cDNA could encode a 72 kDa protein that is highly related to prokaryotic and mitochondrial members of the hsp70 family . The predicted protein was found to contain an amino-terminal extension typical of transit sequences . The in vitro transcription/translation product of the cDNA behaved as a 72 kDa polypeptide as predicted from the longest open reading frame . This polypeptide could be imported into isolated mitochondria and recovered as a 68 kDa product . The imported protein is identical in size to a mitochondrial protein that cross-reacts with hsp70-specific antibodies . The import data and Western blot analysis suggest that the cDNA clone encodes a mitochondrial member of the hsp70 family . Electrophoretic and immunoblot analysis reveal that the protein is loosely associated to the mitochondrial envelope and also exists as discrete soluble protein aggregates of about 270 and 420 kDa . Hsp70 of bean mitochondria can be in vitro phosphorylated on threonine residues in a calcium-dependent manner, and the modified protein was detected as an oligomer of about 160 kDa only . The data are discussed with respect to the chaperone function of hsp70 in mitochondria.

Folia Microbiol (Praha), 1993, 38(2), 147 - 9
Activity of the AIB factor observed in prokaryotic and eukaryotic microorganisms; Pospisil S et al.; Streptomyces cinnamonensis produces a new substance named AIB (for anti-isobutyrate) factor which, on a solid medium, efficiently counteracts toxic concentrations not only of isobutyrate but also of other salts of short-chain monocarboxylic acids . In the present study we demonstrate that the AIB factor activity is widely spread because this effect was positively detected in 25 of 31 randomly chosen microorganisms (streptomycetes, ascomycetes, zygomycetes and basidiomycetes) . The AIB factor produced by the tested microorganisms on an agar media allows for germination, growth, and sporulation of the testing Streptomyces coelicolor on an agar medium containing 20 mmol/L acetate, propionate, butyrate, isobutyrate, valerate, isovalerate, and 2-methylbutyrate . The activity of the AIB factor from different sources towards these substances differs.

Annu Rev Nutr, 1993, 13, 65 - 81
Regulation of selenoproteins; Burk RF et al.; Selenium exerts its biological activity largely through selenoproteins, which contain the element in the form of selenocysteine . Five selenoproteins have been characterized in animal tissues and there is evidence that a number of others exist . Selenoprotein synthesis is a complex process that has been well characterized in prokaryotic systems but incompletely characterized in eukaryotic systems . Selenium deficiency causes a decrease in selenoproteins, but the decrease is not uniform and some selenoproteins are maintained better than others . The selenoprotein most sensitive to selenium deficiency is liver cGSH-Px . It contains a significant fraction of the selenium in the body, and decreased synthesis of it under deficiency conditions might serve to increase the selenium available for synthesis of selenoproteins that are more important to the survival of the animal than is cGSH-Px . The regulation of individual selenoproteins in selenium deficiency appears to be at the mRNA level . Factors that affect mRNA levels have not been completely characterized, but the fall in cGSH-Px mRNA in rat liver is not accompanied by decreased transcription, which suggests that it is regulated through changes in degradation.

Folia Microbiol (Praha), 1993, 38(3), 171 - 5
Characterization of two "Metabacterium" sp . from the gut of rodents . 2 . Heteroxenic cultivation and proof of dipicolinic acid in "M . polyspora"; Stunkel S et al.; The vegetative cell of "Metabacterium polyspora" is "cucumber-shaped", about 21 x 5.7 microns, Gram-negative . Cylindrical endospores are best stained by Rakette and Ziehl-Neelsen staining . The bacterium reproduces by sporulation (2 to 8 endospores per cell) and by binary fission . Lateral, bow-like "hatching" of the endospores was seen . About 52% of guinea pigs harbor 5 x 10(6), 36% below 2 x 10(6) and 1% more than 1 x 10(8) "M . polyspora" in 1 g of caecal content . Dipicolinic acid was demonstrated using HPLC in the caecum homogenate from a guinea pig . The amount of it was proportional to the number of spores . Cultivation under strict anaerobic conditions did not succeed . It was possible to cultivate this giant endosymbiont in vitro in a heteroxenic culture incubated in a 5% CO2 atmosphere using liquid medium supplemented with cell-free filtrate of the caecum . The caecum filtrate containing undefined growth factor(s) is necessary for long-term culture . The replication rate was low . These findings suggest that the giant endosymbiont "M . polyspora" is a spore-forming prokaryote without the attributes of a strict anaerobe.

Adv Enzyme Regul, 1993, 33, 255 - 65
Molecular cloning of the branched-chain alpha-keto acid dehydrogenase kinase and the CoA-dependent methylmalonate semialdehyde dehydrogenase; Harris RA et al.; The complete amino acid sequence of rat liver CoA-dependent methylmalonate semialdehyde dehydrogenase, the enzyme responsible for the oxidative decarboxylation of malonate- and methylmalonate semialdehydes to acetyl- and propionyl-CoA in the distal portions of the valine and pyrimidine catabolic pathways, has been deduced from overlapping cDNAs obtained by screening a lambda gt11 library with nondegenerate oligonucleotide probes synthesized according to PCR-amplified portions coding for the N-terminal amino acid sequence of the enzyme . Although unique because of its requirement for coenzyme A, the methylmalonate semialdehyde dehydrogenase clearly belongs to the aldehyde dehydrogenase superfamily of enzymes . Quantitation of mRNA and protein levels indicates tissue-specific expression of methylmalonate semialdehyde dehydrogenase . A large increase in expression of methylmalonate semialdehyde dehydrogenase occurs during 3T3-L1 preadipocyte differentiation into adipocytes . The complete amino acid sequence of rat liver branched-chain alpha-ketoacid dehydrogenase kinase, the enzyme responsible for phosphorylation and inactivation of the branched-chain alpha-ketoacid dehydrogenase complex, was deduced from a cDNA cloned by a procedure similar to that described above for the methylmalonate semialdehyde dehydrogenase . Expression of the cDNA in E . coli yielded a protein that phosphorylated and inactivated the branched-chain alpha-ketoacid dehydrogenase complex . Very little sequence similarity between branched-chain alpha-ketoacid dehydrogenase kinase and other eukaryotic protein kinases could be identified . However, a high degree of similarity within subdomains characteristic of prokaryotic histidine protein kinases was apparent . Thus, this first mitochondrial protein kinase to be cloned appears closer, evolutionarily, to the prokaryotic histidine protein kinases than eukaryotic ser/thr protein kinases.

Cell Mol Biol Res, 1993, 39(4), 311 - 7
The role of the sigma subunit in promoter recognition by RNA polymerase; Dombroski AJ et al.; Prokaryotic transcription initiation factor sigma is required for sequence-specific promoter recognition by RNA polymerase holoenzyme . Genetic and physiological studies have indicated that sigma interacts with promoter DNA sequences but biochemical analysis did not demonstrate DNA binding by the sigma subunit itself . We have investigated both the DNA binding properties and the regulation of DNA binding for several sigma factors using partial polypeptides . In this report we demonstrate that partial sigmas can bind to promoter DNA in the absence of the core subunits of RNA polymerase and the binding is regulated by an N-terminal inhibitory domain.

Crit Rev Biochem Mol Biol, 1993, 28(6), 515 - 42
The physical biochemistry and molecular genetics of sulfate activation; Leyh TS; This article is an overview of current research in the area of sulfate activation . Emphasis is placed on presenting unresolved issues in an appropriate context for critical evaluation by the reader . The energetics of sulfate activation is reevaluated in light of recent findings that demonstrate that the synthesis of activated sulfate is thermodynamically driven by GTP hydrolysis . The structural and functional bases of this GTPase activation are discussed in detail . The bonding and hydrolysis of the high-energy, phosphoric-sulfuric acid anhydride bond of activated sulfate are presented along with an analysis of the importance of the divalent cation and pyrophosphate protonation in the equilibria governing activated sulfate formation . The molecular genetics of sulfate assimilation in prokaryotes is reviewed with an emphasis on the regulation of the pathway . Recent discoveries connecting sulfate activation to plant/microbe symbiogenesis are presented, as are several examples of the importance of activated sulfate in human metabolism and disease.

Adv Exp Med Biol, 1993, 336, 51 - 4
A new approach to the molecular characterization of the c-ANCA antigen in Wegener's granulomatosis; Bleil L et al.; Classic anti-neutrophil cytoplasmic antibodies (c-ANCA) specific for constituents of neutrophil primary granules and monocyte lysosomes have been implicated in the pathogenesis of Wegener's Granulomatosis (WG) . The revised amino-terminal sequence of Proteinase 3 (PR-3) as ANCA antigen, suggested that PR-3 is identical to myeloblastin (MBN) . As it has been proposed that autoantibodies recognize a conformational epitope on c-ANCA, prokaryotic expressed protein might not be recognized by the patients sera . Therefore we set up an in vitro translation in an eukaryotic cell-free system using an internal ATG (amino acid 15 of the mature protein).

Crit Rev Eukaryot Gene Expr, 1993, 3(4), 255 - 77
Replitase: a complex integrating dNTP synthesis and DNA replication; Reddy GP et al.; Replitase is a multienzyme complex of mammalian cells that produces deoxynucleoside triphosphates and delivers them to the DNA polymerase activity, which also resides in the complex . Structural interactions within this complex form the basis of internal controls to keep these key biosynthetic processes efficient and in balance . The active complex is formed in the nuclear region in only the S phase of the cell cycle, when the cell's DNA is being replicated . Replitase is a member of the growing family of structured, multienzyme, biosynthetic complexes for which very similar complexes are found in prokaryotes and eukaryotes . Logically, the most basic of all biosynthetic pathways should show the efficiency and precise controls that even lesser pathways possess and, in fact, this seems to be so . In this article, we have outlined a broad range of evidence supporting the existence of the replitase complex in mammalian cells, a complex for dNTP synthesis and polymerase that exists only in the S phase and only in the nuclear region . This is consistent with localization studies in intact cells and after various forms of cell fractionation and, particularly, with experiments of incorporation of precursors into DNA in isolated complexes and S phase permeabilized cells . A most forceful argument for replitase is the existence of three striking phenomena--channeling, compartmentation, and cross-inhibition . These are very difficult, perhaps impossible, to explain without replitase; with replitase, their explanation is beautifully simple.

Insect Mol Biol, 1993, 1(3), 133 - 8
Genomic subtractive hybridization to isolate species-specific DNA sequences in insects; Clapp JP et al.; Selective enrichment has been used in a number of instances for the isolation of species-specific sequences in prokaryotes . This paper reports the successful application of the technique to insects . Genomic probes were derived to the target species D . funebris and D . simulans . The method involves the biotinylation of non-target 'driver' DNA prepared from the closely related species D . melanogaster and its hybridization to homologous sequences in the target DNA . Hybrid molecules were removed from the reaction by incubation with streptavidin followed by phenol extraction, leaving a preparation enriched for target fragments . All DNA fragments isolated in the D . funebris experiments proved to be specific to that species . Five out of twenty-four fragments screened in the D . simulans experiments were specific when screened with homologous DNA and genomic DNA from its sibling species, D . melanogaster.

Annu Rev Microbiol, 1993, 47, 597 - 626
Molecular biology of the LysR family of transcriptional regulators; Schell MA; The LysR family is composed of > 50 similar-sized, autoregulatory transcriptional regulators (LTTRs) that apparently evolved from a distant ancestor into subfamilies found in diverse prokaryotic genera . In response to different coinducers, LTTRs activate divergent transcription of linked target genes or unlinked regulons encoding extremely diverse functions . Mutational studies and amino acid sequence similarities of LTTRs identify: (a) a DNA-binding domain employing a helix-turn-helix motif (residues 1-65), (b) domains involved in coinducer recognition and/or response (residues 100-173 and 196-206), (c) a domain required for both DNA binding and coinducer response (residues 227-253) . DNA footprinting studies suggest that in the absence of coinducer many LTTRs bind to regulated promoters via a 15-bp dyadic sequence with a common structure and position (near -65) . Coinducer causes additional interactions of LTTRs with sequences near the -35 RNA polymerase binding site and/or DNA bending that results in transcription activation.

Annu Rev Microbiol, 1993, 47, 535 - 64
Antibiotics synthesized by posttranslational modification; Hansen JN; Peptides that have antimicrobial activity are synthesized by many prokaryotic and eukaryotic organisms . Antimicrobial peptides commonly contain unusual amino acids that contribute to their properties and functions . Although bacteria synthesize most of these peptides by nonribosomal mechanisms, this review focuses on those that are synthesized by pathways that involve posttranslational modification of ribosomally synthesized precursor peptides . A particularly interesting class of these antimicrobial peptides is the lantibiotics, of which nisin and subtilin are the longest-known examples, although nearly a dozen new lantibiotics have been discovered in recent years . The fact that the lantibiotic structures are derived from gene-encoded peptides means that structural analogs of natural lantibiotics can be constructed by mutagenesis of their structural genes . Recent advances in our understanding of the molecular genetics of lantibiotics has made the construction of novel lantibiotics with enhanced chemical and antimicrobial properties possible . This review describes these advances and proposes future trends of research, as well as potential application of engineered lantibiotics, in the context of the general field of antimicrobial peptides.

Folia Microbiol (Praha), 1993, 38(6), 486 - 90
Inhibitory action of palmitic acid on the growth of Saccharomyces cerevisiae; Dell'Angelica EC et al.; High concentrations of long-chain fatty acids have been found to be harmful to mammalian cells and prokaryotic organisms . This effect was investigated in Saccharomyces cerevisiae . Addition of 3 mmol/L palmitate to a yeast extract-peptone medium caused a significant inhibition of cell growth during the first 2 d of incubation, followed by renewed growth and palmitate utilization . Inhibition was also observed with palmitate concentrations down to 0.1 mmol/L . As inferred from catalase activity determinations, this effect was found to correlate with the absence of peroxisome proliferation . Finally, no inhibition was observed in exponential-phase cultures or in the presence of 0.1 g/L glucose, this suggesting that the physiological state of the cell may determine whether its growth will be inhibited by fatty acids.

Growth Factors, 1993, 9(4), 269 - 78
Subcellular localization and biological activity of M(r) 18,000 basic fibroblast growth factor: site-directed mutagenesis of a putative nuclear translocation sequence; Presta M et al.; Residues 27-31 (Lys-Asp-Pro-Lys-Arg) of the 155-amino acid form of basic fibroblast growth factor (bFGF) are in good agreement with a consensus sequence for nuclear translocation . To evaluate the role of this sequence in mediating the intracellular localization and biological activity of bFGF, basic residues Lys-27, Lys-30, and Arg-31 were changed to neutral glutamine residues by site-directed mutagenesis of the human bFGF cDNA . The bFGF mutant (M1Q-bFGF) was expressed in eukaryotic cells and in prokaryotic cells, from which it was purified to homogeneity . Transient expression of bFGF cDNA and of M1Q-bFGF cDNA in simian COS-1 cells followed by immunolocalization and by subcellular fractionation indicated that both molecules localize in the nucleus, as well as in the cytoplasm of transfected cells, and interact with nuclear chromatin and with eukaryote DNA in a similar manner . Prokaryotic expression of M1Q-bFGF cDNA yields a polypeptide endowed with a receptor-binding capacity and mitogenic activity similar to that exerted by wild-type bFGF . However, recombinant M1Q-bFGF showed a drastically reduced capacity to induce the production of urokinase-type plasminogen activator (uPA) in endothelial cells . The uPA-inducing activity of M1Q-bFGF was fully restored by the presence of soluble heparin in the culture medium . In conclusion, the sequence bFGF(27-31) does not appear to represent a nuclear translocation and/or retention sequence for bFGF . However, neutralization of its basic residues seems to modify the tertiary structure of the growth factor, thus affecting some of its biological properties.

Receptor, 1993 Winter, 3(4), 247 - 55
DNA bending by nuclear receptors; Nardulli AM et al.; Although steroid hormone receptors constitute an intensively studied family of ligand-regulated transcription factors, the mechanism by which these receptors activate transcription has not been defined . Evidence has accumulated from prokaryotic and eukaryotic systems that many transcription factors are capable of binding to their cognate recognition sequences and causing DNA to bend . Therefore, it has been hypothesized that DNA bending and transcription activation may be functionally coupled . We have utilized circular permutation analysis to examine the ability of the estrogen receptor DNA binding domain and the intact estrogen receptor to bend DNA fragments containing estrogen response elements (EREs) . The DNA binding domain, which is a less potent activator of transcription, bent ERE containing DNA fragments less (34 degrees) than the intact estrogen receptor (56 degrees), which is a more potent activator of transcription . In addition, when two EREs were present in a DNA fragment, the degree of DNA bending observed was greater than when one ERE was present . These data suggest that DNA bending may play a role in transcription activation of estrogen responsive genes.

Annu Rev Genet, 1993, 27, 235 - 56
Lateral transfer in natural populations of eukaryotes; Kidwell MG; Although there are several likely instances of trans-kingdom lateral transfer of genomic sequences involving eukaryotes and prokaryotes, almost all well-documented cases of eukaryote to eukaryote transfer seem to involve mobile elements or other parasitic sequences . Consistent with general observations of phylogenetic regularity, the limited molecular evidence suggests that lateral transfer of eukaryotic genomic sequences is at best very rare . However, due to limited data, the possibility of rare transfers that could have considerable evolutionary significance cannot be ruled out . A possible propensity for lateral transfer by mobile elements may reflect their innate capacity for genomic wandering . In addition, occasional cross-species mobility may play a critical role in the long-term evolutionary survival of these elements and have been subject to natural selection . Much work is needed to fully understand the dynamics of TEs and other multigene families . Problems of paralogy, recombination, and variation in evolutionary rates currently present important difficulties in distinguishing conclusively between occasional lateral transfer and strictly vertical transfer . The importance of lateral transfers for host organisms must await answers to more general questions about the long-term evolutionary significance of mobile elements and the extent to which they can act as vectors for host genomic sequences.

J Mol Neurosci, 1993 Summer, 4(2), 125 - 39
Deletion mutagenesis of rat PC12 tyrosine hydroxylase regulatory and catalytic domains; Ribeiro P et al.; The functional organization of rat tyrosine hydroxylase was investigated by deletion mutagenesis of the regulatory and catalytic domains . A series of tyrosine hydroxylase cDNA deletion mutants were amplified by PCR, cloned into the pET3C prokaryotic expression vector, and the mutant proteins were partially purified from E . coli . The results show that the deletion of up to 157 N-terminal amino acids activated the enzyme, but further deletion to position 184 completely destroyed catalytic activity . On the carboxyl end, the removal of 43 amino acids decreased but did not eliminate activity, suggesting that this region may play a different role in the regulation of the enzyme . These findings place the amino end of the catalytic domain between residues 158 and 184 and the carboxyl end at or prior to position 455 . Deletions within the first 157 amino acids in the N-terminus caused an increase in hydroxylating activity, a decrease in the apparent Km for tyrosine and phenylalanine substrates, and a substantial increase in the Ki for dopamine inhibition . The results define this region of the N-terminus as the regulatory domain of tyrosine hydroxylase, whose primary functions are to restrict the binding of amino acid substrates and to facilitate catecholamine inhibition . The results also suggest that the well-established role of the regulatory domain in restricting cofactor binding may be secondary to an increase in catecholamine binding, which in turn lowers the affinity for the cofactor . These findings provide new insight into the functional organization and mechanisms of regulation of tyrosine hydroxylase.

J Med Entomol, 1993 Jan, 30(1), 214 - 6
Patterns of erythrocyte digestion by bloodsucking insects: constraints on vector competence; Vaughan JA et al.; Two general patterns of erythrocyte digestion were observed in representative species from four insect orders . Ingested erythrocytes were hemolyzed rapidly, and blood meals remained liquefied within body lice, Pediculus humanus L . and the fleas Ctenocephalides felis (Bouche) and Xenopyslla cheopis (Rothschild) . Peritrophic membrane was absent . In contrast, there was a lag time of 6-18 h before substantial degradation of erythrocytes within the blood meals of bed bugs, Cimex lectularius L.; the sand fly Phlebotomus papatasi Scopoli; and the mosquitoes Anopheles stephensi Liston and Culex pipiens L . Blood meals of sand flies and mosquitoes were clotted and surrounded by peritrophic membrane at 18-24 h after feeding . Clotting and peritrophic membrane were less pronounced in bed bugs . It is proposed that acquisition and maintenance of pathogen types (i.e., prokaryotic versus eukaryotic) within insects are constrained by the general pattern of bloodmeal processing.

Gene Expr, 1993, 3(3), 317 - 23
Remarks on the mechanism of ribosome binding to eukaryotic mRNAs; Sonenberg N; It is evident from the data discussed here that the mechanism and the rules for mRNA binding in eukaryotes are complex and not well defined . The major points of this review are (1) ribosome binding could be preceded by the unwinding of mRNA secondary structure; (2) there is no obligatory ribosome entry through the 5' end of the mRNA; (3) there is no obligatory linear "scanning" of the 5'UTR; and (4) there are some interesting similarities between prokaryotes and eukaryotes in the mode of ribosome binding to mRNA, particularly in the ability of the small ribosomal subunit to diffuse or "scan" on the mRNA, and in the requirement for a minimally structured RNA for efficient ribosome binding.

Biosystems, 1993, 31(2-3), 185 - 91
Gut microbe of aphid closely related to its intracellular symbiont; Harada H et al.; Pea aphid, Acyrthosiphon pisum harbors prokaryotic intracellular symbionts and, at least, two species of gut microbes . It has been shown that the aphid symbiont is more closely related to Escherichia coli than to any other free-living bacterium . Analysis of RFLP of their 16S rDNA and groE homologues suggested that one of the gut microbes is a close relative of E . coli and that the aphid symbiont is still closer to this microbe than to E . coli . In addition, the optimal temperature for the growth of this gut microbe was significantly lower than that of E . coli suggesting that this microbe has been adapted to the ambient temperature of the host insect and shares a very close ancestor with the aphid symbiont in common.

Mol Microbiol, 1993 Jan, 7(1), 21 - 8
Isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic Mycoplasma-like organisms; Neimark H et al.; We describe the isolation and characterization of full-length chromosomes from non-culturable plant-pathogenic, mycoplasma-like organisms (MLOs) . MLO chromosomes are circular and their sizes (640 to 1185 kbp) are heterogeneous . Divergence in the range of chromosome sizes is apparent between MLOs in the two major MLO disease groups, and chromosome size polymorphism occurs among some related agents . MLO chromosome sizes overlap those of culturable mycoplasmas; consequently, small genome size alone cannot explain MLO non-culturability . Hybridization with cloned MLO-specific chromosomal and 16S rRNA probes detected two separate chromosomes in some MLO 'type' strains . Large DNA molecules that appear to be MLO megaplasmids were also demonstrated . The ability to characterize full-length chromosomes from virtually any non-culturable prokaryote should greatly facilitate the molecular and genetic analysis of these difficult bacteria.

FASEB J, 1993 Jan, 7(1), 208 - 13
Secondary structure of RNase MRP RNA as predicted by phylogenetic comparison; Schmitt ME et al.; RNase MRP is a ribonucleoprotein endoribonuclease that has been shown to cleave mitochondrial primer RNA sequences from a variety of sources . The bulk of RNase MRP activity is found in the nucleus where its function remains unknown . Two different approaches have resulted in predictions of distinct secondary structures for RNase MRP RNA . In order to analyze more definitively the higher-order structure of RNase MRP RNA, we have conducted a phylogenetic comparison of the available RNase MRP RNA sequences from human, mouse, rat, cow, toad, and yeast . The resulting secondary structure shares features in common with previously described structures for prokaryotic and eukaryotic RNase P RNAs (1) and RNase MRP RNAs (2, 3) . In addition, the phylogenetic structure is consistent with available chemical modification data on RNase MRP RNA and with the detailed analysis of the To antigen binding domain located near the 5' end of the RNase MRP RNA . The structure is not limited to RNase MRP RNAs, but can be expanded to cover both eukaryotic RNase P RNAs and RNase P/MRP RNAs from plants.

DNA Cell Biol, 1993 Jan-Feb, 12(1), 1 - 51
The P450 superfamily: update on new sequences, gene mapping, accession numbers, early trivial names of enzymes, and nomenclature; Nelson DR et al.; We provide here a list of 221 P450 genes and 12 putative pseudogenes that have been characterized as of December 14, 1992 . These genes have been described in 31 eukaryotes (including 11 mammalian and 3 plant species) and 11 prokaryotes . Of 36 gene families so far described, 12 families exist in all mammals examined to date . These 12 families comprise 22 mammalian subfamilies, of which 17 and 15 have been mapped in the human and mouse genome, respectively . To date, each subfamily appears to represent a cluster of tightly linked genes . This revision supersedes the previous updates {Nebert et al., DNA 6, 1-11, 1987; Nebert et al., DNA 8, 1-13, 1989; Nebert et al., DNA Cell Biol . 10, 1-14 (1991)} in which a nomenclature system, based on divergent evolution of the superfamily, has been described . For the gene and cDNA, we recommend that the italicized root symbol "CYP" for human ("Cyp" for mouse), representing "cytochrome P450," be followed by an Arabic number denoting the family, a letter designating the subfamily (when two or more exist), and an Arabic numeral representing the individual gene within the subfamily . A hyphen should precede the final number in mouse genes . "P" ("p" in mouse) after the gene number denotes a pseudogene . If a gene is the sole member of a family, the subfamily letter and gene number need not be included . We suggest that the human nomenclature system be used for all species other than mouse . The mRNA and enzyme in all species (including mouse) should include all capital letters, without italics or hyphens . This nomenclature system is identical to that proposed in our 1991 update . Also included in this update is a listing of available data base accession numbers for P450 DNA and protein sequences . We also discuss the likelihood that this ancient gene superfamily has existed for more than 3.5 billion years, and that the rate of P450 gene evolution appears to be quite nonlinear . Finally, we describe P450 genes that have been detected by expressed sequence tags (ESTs), as well as the relationship between the P450 and the nitric oxide synthase gene superfamilies, as a likely example of convergent evolution.

Proc Int Conf Intell Syst Mol Biol, 1993, 1, 319 - 27
Object-oriented knowledge bases for the analysis of prokaryotic and eukaryotic genomes; Perriere G et al.; The amount of biological sequences introduced in the general collections, and the growing complexity of the biological knowledge require the construction of models to formalize this knowledge and particularly the relationships between several data types . Two examples of such situations are presented here, they result from the biological research lead in our team in the field of molecular evolution . ColiGene is a modelling of E . coli genetics devoted to the analysis of relationships between genomic sequences and gene expressivity . MultiMap implements a new formalization of genome maps allowing manipulation of "maps of maps" in two species . Application of ColiGene and MultiMap are not restricted to molecular evolution and, for instance, MultiMap offers new capabilities for infering data on a genome from knowledge on another species . This could be essential for many mapping projects (human, mouse but also other mammals like pig) . Development and implementation of those models have been done using an object-oriented knowledge base management system (SHIRKA) interfaced with a dedicated genomic data base management system (ACNUC) . Graphical interfaces have been designed to give an environment similar to the biological representations used by biologists.

Biochimie, 1993, 75(12), 1027 - 39
Mammalian tryptophanyl-tRNA synthetases; Kisselev LL; Aminoacyl-tRNA synthetases of higher organisms are far less studied compared to their prokaryotic and unicellular eukaryotic counterparts . However, many aminoacyl-tRNA synthetases from multi-cellular organisms exhibit certain features not yet described for the same enzymes of bacteria or yeast . Tryptophanyl-tRNA synthetases (TrpRS) are among the most thoroughly studied mammalian enzymes of this group . TrpRS are Zn(2+)-dependent, dimeric, class I aminoacyl-tRNA synthetases with known amino acid sequence for four different mammalian orders . TrpRS is not associated in a stable multi-synthetase complex, although it exhibits a long N-terminal extension absent from bacterial TrpRS . The human gene encoding TrpRS belongs to the interferon-responsive gene family and TrpRS activity drastically increases after interferon gamma induction . For unknown reasons TrpRS is overproduced in pancreas of Ruminantia . Other data on TrpRS available so far are summarized and briefly discussed here.

J Cardiovasc Pharmacol, 1993, 22 Suppl 8, S4 - 6
Extracellular cysteine residues 174 and 255 are essential for active expression of human endothelin receptor ETB in Escherichia coli; Haendler B et al.; The coding sequences for the non-isopeptide-selective human endothelin receptor ETB were introduced into the prokaryotic expression vector pKK233-2, and the resulting construct was used for transformation of competent E . coli JM105 cells . Specific binding was observed for bacterial membrane fractions, using labeled ET-1 as a ligand . Site-directed mutagenesis was employed to individually modify the triplets for the cysteines of the first and second extracellular loops of ETB to alanine codons . E . coli JM105 transformed with the mutated plasmids no longer displayed specific ET-1 binding to membranes, which suggests a crucial role for these extracellular cysteines in agonist binding or receptor stability.

Annu Rev Microbiol, 1993, 47, 291 - 319
ATP-dependent transport systems in bacteria and humans: relevance to cystic fibrosis and multidrug resistance; Doige CA et al.; The prokaryotic permeases are members of a superfamily of membrane transporters called traffic ATPases, which includes the medically important eukaryotic multidrug resistance (MDR) protein and cystic fibrosis transmembrane regulator (CFTR) . Members of this superfamily have extensive sequence and structural similarity, in particular in an ATP-binding motif, and are believed to use ATP to energize translocation of substrates across biological membranes . The prokaryotic histidine permease is well-characterized and serves as a convenient model system . In this review, we highlight some of the biochemical and molecular biological approaches used to study the functional and architectural organization of this permease and relate the results of these approaches to what is known about other traffic ATPases . We have identified specific regions that we believe critical for the function of the histidine permease and propose that the corresponding regions in the eukaryotic traffic ATPases are also important for their function . In light of the fact that CFTR (and possibly the MDR protein) is an ion channel, we compare the properties of channels and transporters; in addition, we discuss the possibility that other members of the traffic ATPases may also have channel-like activity.

Biochem Biophys Res Commun, 1992 Dec 30, 189(3), 1444 - 9
Cysteine protects Na,K-ATPase and isolated human lymphocytes from silver toxicity; Hussain S et al.; Metal-binding proteins are important components of retroviruses such as human immunodeficiency virus (HIV) . Therefore, metals could be used as antiviral agents . However, most metals are toxic for humans with the exception of silver which is toxic only to prokaryotic cells and viruses . In addition, HIV infection causes a decrease in body cysteine . We formed a complex of silver and cysteine, named silver-cysteine . Healthy human lymphocytes were incubated with silver-nitrate or silver-cysteine . Negligible cell survival was seen at 50 microM silver-nitrate . However, in presence of 1 mM cysteine, the viability remained unaffected up to 1 mM of silver . Further, silver inhibition of isolated Na,K-ATPase was easily reversed by cysteine . Thus, non-toxic silver-cysteine could be used as an anti-viral and cysteine-replenishing agent.

FEMS Microbiol Lett, 1992 Dec 15, 79(1-3), 423 - 31
Peculiar properties of mycoplasmas: the smallest self-replicating prokaryotes; Razin S; Mycoplasmas are the smallest and simplest prokaryotes capable of self-replication, with information provided by a genome which may be as small as 600 kb, estimated to carry less than 500 genes . Keeping the number of structural elements, metabolic pathways and components of the protein synthesizing machinery to an essential minimum places mycoplasmas closest to the concept of 'minimum cells' . Mycoplasmas are, therefore, most adequate candidates for the complete deciphering of the machinery of a self-replicating organism, and studies towards this goal are already underway . Living as 'minimum cells' was made possible by adopting a parasitic mode of life, securing from the host the many nutrients which cannot be synthesized by the mycoplasmas themselves . When pathogenic, infections by mycoplasmas usually follow a chronic course, with host immune reactions playing an important role in symptom production . Recent studies on the possible association of mycoplasmas with rheumatoid arthritis and AIDS are reviewed.

Proc Natl Acad Sci U S A, 1992 Dec 15, 89(24), 12170 - 4
Expression cloning of a human dual-specificity phosphatase; Ishibashi T et al.; Using an expression cloning strategy, we isolated a cDNA encoding a human protein-tyrosine-phosphatase . Bacteria expressing the kinase domain of the keratinocyte growth factor receptor (bek/fibroblast growth factor receptor 2) were infected with a fibroblast cDNA library in a phagemid prokaryotic expression vector and screened with a monoclonal anti-phosphotyrosine antibody . Among several clones showing decreased anti-phosphotyrosine recognition, one displayed phosphatase activity toward the kinase in vitro . The 4.1-kilobase cDNA encoded a deduced protein of 185 amino acids with limited sequence similarity to the vaccinia virus phosphatase VH1 . The purified recombinant protein dephosphorylated several activated growth factor receptors, as well as serine-phosphorylated casein, in vitro . Both serine and tyrosine phosphatase activities were completely abolished by mutagenesis of a single cysteine residue conserved in VH1 and the VH1-related (VHR) human protein . These properties suggest that VHR is capable of regulating intracellular events mediated by both tyrosine and serine phosphorylation.

FASEB J, 1992 Dec, 6(15), 3410 - 20
Structural and evolutionary relationships among the immunophilins: two ubiquitous families of peptidyl-prolyl cis-trans isomerases; Trandinh CC et al.; The immunophilins, protein receptors for the immunosuppressing drugs cyclosporin A and FK506 and related proteins from plants, fungi, and bacteria, have been analyzed structurally and evolutionarily . The cyclosporin A binding proteins (cyclophilins) represent one ubiquitous family of homologous proteins, and the FK506- and rapamycin-binding proteins (FKBPs) constitute a second, unrelated family . Multiple sequence alignments of members of each of these two protein families define the highly conserved residues that are likely to play important structural and functional roles, and mutations in representative members of these two families that abolish or alter function have been evaluated . FKBPs have undergone greater evolutionary divergence than the cyclophilins . Evolutionary trees were constructed using two distinct programs, and these trees establish the structural relationships that allow division of each of these families into subgroups . The results lead to the suggestion that several genes encoding isozymic forms of the FKBPs and possibly also of the cyclophilins existed in prokaryotes before the emergence of eukaryotes on earth and that representatives of these genes were transmitted to both kingdoms to give rise to current subfamilies of these proteins . By contrast, compartmentalization of both classes of immunophilins appears to have arisen independently in prokaryotes and eukaryotes, late in evolutionary history.

Nucleic Acids Res, 1992 Dec 11, 20(23), 6339 - 46
The yeast nuclear gene MRF1 encodes a mitochondrial peptide chain release factor and cures several mitochondrial RNA splicing defects; Pel HJ et al.; We report the molecular cloning, sequencing and genetic characterization of the first gene encoding an organellar polypeptide chain release factor, the MRF1 gene of the yeast Saccharomyces cerevisiae . The MRF1 gene was cloned by genetic complementation of a respiratory deficient mutant disturbed in the expression of the mitochondrial genes encoding cytochrome c oxidase subunit 1 and 2, COX1 and COX2 . For COX1 this defect has been attributed to an impaired processing of several introns . Sequence analysis of the MRF1 gene revealed that it encodes a protein highly similar to prokaryotic peptide chain release factors, especially RF-1 . Disruption of the gene results in a high instability of the mitochondrial genome, a hallmark for a strict lesion in mitochondrial protein synthesis . The respiratory negative phenotype of mrf1 mutants lacking all known mitochondrial introns and the reduced synthesis of mitochondrial translation products encoded by unsplit genes confirm a primary defect in mitochondrial protein synthesis . Over-expression of the MRF1 gene in a mitochondrial nonsense suppressor strain reduces suppression in a dosage-dependent manner, shedding new light on the role of the '530 region' of 16S-like ribosomal RNA in translational fidelity.

Nature, 1992 Dec 3, 360(6403), 488 - 91
Control of RNase E-mediated RNA degradation by 5'-terminal base pairing in E . coli; Bouvet P et al.; Despite the variety of messenger RNA half-lives in bacteria (0.5-30 min in Escherichia coli) and their importance in controlling gene expression, their molecular basis remains obscure . The lifetime of an entire mRNA molecule can be determined by features near its 5' end, but no 5' exoribonuclease has been identified in any prokaryotic organism . A mutation that inactivates E . coli RNase E also increases the average lifetime of bulk E . coli mRNA and of many individual messages, suggesting that cleavage by this endonuclease may be the rate-determining step in the degradation of most mRNAs in E . coli . We have investigated the substrate preference of RNase E in E . coli by using variants of RNA I, a small untranslated RNA whose swift degradation in vivo is initiated by RNase E cleavage at an internal site . We report here that RNase E has an unprecedented substrate specificity for an endoribonuclease, as it preferentially cleaves RNAs that have several unpaired nucleotides at the 5' end . The sensitivity of RNase E to 5'-terminal base pairing may explain how determinants near the 5' end can control rates of mRNA decay in bacteria.

Microbiol Rev, 1992 Dec, 56(4), 592 - 621
Regulation by proteolysis: energy-dependent proteases and their targets; Gottesman S et al.; A number of critical regulatory proteins in both prokaryotic and eukaryotic cells are subject to rapid, energy-dependent proteolysis . Rapid degradation combined with control over biosynthesis provides a mechanism by which the availability of a protein can be limited both temporally and spatially . Highly unstable regulatory proteins are involved in numerous biological functions, particularly at the commitment steps in developmental pathways and in emergency responses . The proteases involved in energy-dependent proteolysis are large proteins with the ability to use ATP to scan for appropriate targets and degrade complete proteins in a processive manner . These cytoplasmic proteases are also able to degrade many abnormal proteins in the cell.

Mol Microbiol, 1992 Dec, 6(23), 3455 - 60
Peroxisome biogenesis in yeast; Aitchison JD et al.; Eukaryotic cells have evolved a complex set of intracellular organelles, each of which possesses a specific complement of enzymes and performs unique metabolic functions . This compartmentalization of cellular functions provides a level of metabolic control not available to prokaryotes . However, it presents the eukaryotic cell with the problem of targeting proteins to their specific location(s) . Proteins must be efficiently transported from their site of synthesis in the cytosol to their specific organelle(s) . Such a process may require translocation across one or more hydrophobic membrane barriers and/or asymmetric integration into specific membranes . Proteins carry cis-acting amino acid sequences that serve to act as recognition motifs for protein sorting and for the cellular translocation machinery . Sequences that target proteins to the endoplasmic reticulum/secretory pathway, mitochondria, and chloroplasts are often present as cleavable amino-terminal extensions . In contrast, most peroxisomal proteins are synthesized at their mature size and are translocated to the organelle without any post-translational modification . This review will summarize what is known about how yeast solve the problem of specifically importing proteins into peroxisomes and will suggest future directions for investigations into peroxisome biogenesis in yeast.

Trends Biochem Sci, 1992 Dec, 17(12), 489 - 93
Evolution by acquisition: the case for horizontal gene transfers; Smith MW et al.; One of the most debated questions in the field of molecular evolution is the possible role of horizontal transfer in evolution . Of all the claims that have been made over the years, those reporting transfers between eukaryotes and prokaryotes are the most controversial . Here we present the cases for and against several such possible gene acquisitions.

EMBO J, 1992 Dec, 11(13), 5121 - 7
Virulence in bacteriophage Mu: a case of trans-dominant proteolysis by the Escherichia coli Clp serine protease; Geuskens V et al.; The importance of proteases in gene regulation is well documented in both prokaryotic and eukaryotic systems . Here we describe the first example of genetic regulation controlled by the Escherichia coli Clp ATP-dependent serine protease . Virulent mutants of bacteriophage Mu, which carry a particular mutation in their repressor gene (vir mutation), successfully infect Mu lysogens and induce the resident Mu prophage . We show that the mutated repressors have an abnormally short half-life due to an increased susceptibility to Clp-dependent degradation . This susceptibility is communicated to the wild type repressor present in the same cell, which provides the Muvir phages with their trans-dominant phenotype . To our knowledge this is the first case where the instability of a mutant protein is shown to trigger the degradation of its wild type parent.

EMBO J, 1992 Dec, 11(13), 4843 - 50
Molecular cloning of human growth inhibitory factor cDNA and its down-regulation in Alzheimer's disease; Tsuji S et al.; In previous studies, we discovered a growth inhibitory factor (GIF) that was abundant in normal human brain, but greatly reduced in Alzheimer's disease (AD) brain . Molecular cloning of a full-length cDNA for human GIF revealed that the GIF had striking homology to metallothioneins . Furthermore, it was determined that the GIF gene was on chromosome 16, as are the metallothionein genes . GIF, in contrast to metallothioneins, was found to be expressed exclusively in the nervous system . The GIF protein produced by Escherichia coli harboring the GIF cDNA in a prokaryotic expression vector inhibited the growth of neonatal rat cortical neurons . These results indicate that GIF is a new member of the metallothionein family with distinct tissue-specific expression and functions . Northern blot analysis revealed that expression of the GIF mRNA is drastically decreased in AD brains . The result raises the possibility that down-regulation of the GIF gene in AD brain plays an important role in the pathogenesis of AD.

Plant Mol Biol, 1992 Dec, 20(6), 1111 - 9
Adenine depurination and inactivation of plant ribosomes by an antiviral protein of Mirabilis jalapa (MAP); Kataoka J et al.; Mirabilis antiviral protein (MAP) is a single-chain ribosome-inactivating protein (RIP) isolated from Mirabilis jalapa L . It depurinates the 28S-like rRNAs of prokaryotes and eukaryotes . A specific modification in the 25S rRNA of M . jalapa was found to occur during isolation of ribosomes by polyacrylamide/agarose composite gel electrophoresis . Primer extension analysis revealed the modification site to be at the adenine residue corresponding to A4324 in rat 28S rRNA . The amount of endogenous MAP seemed to be sufficient to inactivate most of the homologous ribosomes . The adenine of wheat ribosomes was also found to be removed to some extent by an endogenous RIP (tritin) . However, the amount of endogenous tritin seemed to be insufficient for quantitative depurination of the homologous ribosomes . Endogenous MAP could shut down the protein synthesis of its own cells when it spreads into the cytoplasm through breaks of the cells . Therefore, we speculate that MAP is a defensive agent to induce viral resistance through the suicide of its own cells.

Plant Mol Biol, 1992 Dec, 20(6), 1097 - 110
Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp . PCC6803; Ellersiek U et al.; The plastid DNA of higher plants contains eleven reading frames that are homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I) . The genes are expressed, but a plastid NAD(P)H dehydrogenase has not yet been isolated and the function of the enzyme in plastid metabolism is unknown . Cyanobacteria also contain a NADH dehydrogenase that is homologous to the mitochondrial complex I . The enzyme is sensitive to rotenone and is located on the cytoplasmic and the thylakoid membrane . We report here the sequence of five subunits (ndhA, -I, G, -E and -D) of the NADH dehydrogenase from the unicellular cyanobacterium Synechocystis sp . PCC6803 . As in plastid DNA, the genes ndh(A-I-G-E) are clustered and probably constitute an operon . The ndhD gene is associated with a gene encoding an iron-sulphur protein of photosystem I (psaC) as in plastid DNA . In contrast to the situation in plastids, psaC and ndhD are not cotranscribed but transcribed from opposite strands . The deduced amino acid sequence of the cyanobacterial polypeptides is more similar to the corresponding plastid (40-68% identity) than to the corresponding mitochondrial subunits (17-39% identity) . Thus, the cyanobacterial NADH-dehydrogenase provides a prokaryotic model system which is more suitable to genetic analysis than the enzyme of plastids.

J Bioenerg Biomembr, 1992 Dec, 24(6), 529 - 46
Energetics of methanogenesis studied in vesicular systems; Blaut M et al.; Methanogenesis is restricted to a group of prokaryotic microorganisms which thrive in strictly anaerobic habitats where they play an indispensable role in the anaerobic food chain . Methanogenic bacteria possess a number of unique cofactors and coenzymes that play an important role in their specialized metabolism . Methanogenesis from a number of simple substrates such as H2 + CO2, formate, methanol, methylamines, and acetate is associated with the generation of transmembrane electrochemical gradients of protons and sodium ions which serve as driving force for a number of processes such as the synthesis of ATP via an ATP synthase, reverse electron transfer, and solute uptake . Several unique reactions of the methanogenic pathways have been identified that are involved in energy transduction . Their role and importance for the methanogenic metabolism are described.

Arch Biochem Biophys, 1992 Dec, 299(2), 340 - 3
Studies on the holoenzyme biogenesis of the spinach ferredoxin-NADP+ reductase; Aliverti A et al.; An expression plasmid, pPreFNR, in which the DNA sequence coding for the spinach ferredoxin-NADP+ reductase precursor was under the control of prokaryotic transcription and translation initiation signals has been constructed . The plasmid directed the synthesis in Escherichia coli of a 43-kDa immunoreactive polypeptide which could be identified with the reductase preprotein . Analyses of bacterial extracts showed that the precursor was unstable and devoid of catalytic activities, suggesting that the presence of the transit peptide would not allow the assembly in E . coli of an active preholoenzyme . Furthermore, the reductase precursor was found to undergo a processing in E . coli . The proteolysed form, which retained 13 of the 55 residues of the transit peptide, was active, suggesting that removal of the first 42 residues of the presequence enabled the protein to properly fold and to bind the FAD prosthetic group in the bacterial host, as it was previously shown in the case of the mature form of the spinach reductase.

J Bacteriol, 1992 Dec, 174(23), 7585 - 94
A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp; Ishizuka H et al.; A DNA fragment stimulating actinorhodin, undecylprodigiosin, and A-factor production in Streptomyces lividans 66 was cloned from Streptomyces coelicolor A3(2) . Nucleotide sequencing revealed the presence of an open reading frame of 225 codons, named afsQ1, that showed great similarity in amino acid sequence to the response regulators of typical prokaryotic two-component regulatory systems responsible for adaptive responses . The termination codon, TGA, of afsQ1 overlapped the initiation codon, GTG, of a second open reading frame, afsQ2, of 535 codons . The afsQ2 gene product showed homology with the sensory histidine protein kinases of two-component systems . In agreement with the assumption that the AfsQ1 and AfsQ2 proteins comprise an aspartate-histidine phosphotransfer system, an amino acid replacement from Asp to Glu at residue 52 of AfsQ1, generated by site-directed mutagenesis, resulted in loss of the protein's ability to stimulate antibiotic production in S . lividans . Primer extension experiments indicated that transcription of the afsQ1 and afsQ2 genes initiates at the translational start codon (GTG) of the afsQ1 gene . The afsQ1 and afsQ2 genes were physically mapped at a chromosomal position near the actinorhodin biosynthetic gene cluster (act) by hybridization to Southern blots of restriction fragments separated by pulsed-field gel electrophoresis . Disruption of either afsQ1 or afsQ2 on the S . coelicolor chromosome by use of phage phi C31KC515 led to no detectable change in secondary metabolite formation or morphogenesis . The afsQ1 gene on pIJ922 suppressed the S . coelicolor absA mutation and caused actinorhodin production but did not suppress the absB mutation . Southern blot hybridization showed that sequences homologous to afsQ1 and afsQ2 are present in almost all of the actinomycetes examined.

Proc Natl Acad Sci U S A, 1992 Dec 1, 89(23), 11461 - 5
Positive supercoiling of DNA greatly diminishes mRNA synthesis in yeast; Gartenberg MR et al.; In Saccharomyces cerevisiae cells harboring a GAL1 promoter-linked beta-galactosidase gene, the simultaneous expression of Escherichia coli DNA topoisomerase I and inactivation of yeast DNA topoisomerases I and II reduces the cellular level of beta-galactosidase to an undetectable level . Analysis of intracellular mRNA level and the density of RNA polymerase along DNA indicates that this reduction is due to the suppression of transcription and that both plasmid-borne and chromosomally located genes are affected . These results are interpreted in terms of inhibition of transcription in vivo due to positive supercoiling of the DNA template: preferential removal of transcription-generated negative supercoils by E . coli DNA topoisomerase I in the absence of both yeast DNA topoisomerases I and II results in the accumulation of positive supercoils in intracellular DNA . In normal prokaryotic or eukaryotic cells, accumulation of positive supercoils is presumably avoided through the balanced actions of DNA topoisomerases.

Biochemistry, 1992 Dec 1, 31(47), 11874 - 80
Protein-tyrosyl radical interactions in photosystem II studied by electron spin resonance and electron nuclear double resonance spectroscopy: comparison with ribonucleotide reductase and in vitro tyrosine; Hoganson CW et al.; The stable tyrosine radical in photosystem II, YD*, has been studied by ESR and ENDOR spectroscopies to obtain proton hyperfine coupling constants from which the electron spin density distribution can be deduced . Simulations of six previously published ESR spectra of PSII (one at Q band; five at X band, of which two were after specific deuteration and two others were of oriented membranes) can be achieved by using a single set of magnetic parameters that includes anisotropic proton hyperfine tensors, an anisotropic g tensor, and noncoincident axis systems for the g and A tensors . From the spectral simulation of the oriented samples, the orientation of the phenol head group of YD* with respect to the membrane plane has been determined . A similar orientation for YZ*, the redox-active tyrosine in PSII that mediates electron transfer between P680 and the oxygen-evolving complex, is expected . ENDOR spectra of YD* in PSII preparations from spinach and Synechocystis support the set of hyperfine coupling constants but indicate that small differences between the two species exist . Comparison with the results of spectral simulations for tyrosyl radicals in ribonucleotide reductase from prokaryotes or eukaryotes and with in vitro radicals indicates that the spin density distribution remains that of an odd-alternant radical but that interactions with the protein can shift spin density within this basic pattern . The largest changes in spin density occur at the tyrosine phenol oxygen and at the ring carbon para to the oxygen, which indicates that mechanisms exist in the protein environment for fine-tuning the chemical and redox properties of the radical species.

Biochem Int, 1992 Dec, 28(5), 887 - 93
Nucleotide sequence of cDNA for porcine heme oxygenase and its expression in Escherichia coli; Suzuki T et al.; The nucleotide sequence of a cDNA for porcine heme oxygenase was determined . The open reading frame encoded a polypeptide of 288 amino acid residues with a molecular mass of 33,074 Da . A prokaryotic expression plasmid carrying porcine heme oxygenase cDNA was constructed and transfected into Escherichia coli cells . The full-length heme oxygenase expressed was localized in the bacterial membranes . Two small-sized heme oxygenases with no membrane-bound properties were also detected, suggesting that in E . coli cells a considerable amount of the enzyme expressed was degraded.

Protein Eng, 1992 Dec, 5(8), 729 - 38
Quantile distributions of amino acid usage in protein classes; Karlin S et al.; A comparative study of the compositional properties of various protein sets from both cellular and viral organisms is presented . Invariants and contrasts of amino acid usages have been discerned for different protein function classes and for different species using robust statistical methods based on quantile distributions and stochastic ordering relationships . In addition, a quantitative criterion to assess amino acid compositional extremes relative to a reference protein set is proposed and applied . Invariants of amino acid usage relate mainly to the central range of quantile distributions, whereas contrasts occur mainly in the tails of the distributions, especially contrasts between eukaryote and prokaryote species . Influences from genomic constraint are evident, for example, in the arginine:lysine ratios and the usage frequencies of residues encoded by G + C-rich versus A + T-rich codon types . The structurally similar amino acids, glutamate versus aspartate and phenylalanine versus tyrosine, show stochastic dominance relationships for most species protein sets favoring glutamate and phenylalanine respectively . The quantile distribution of hydrophobic amino acid usages in prokaryote data dominates the corresponding quantile distribution in human data . In contrast, glutamate, cysteine, proline and serine usages in human proteins dominate the corresponding quantile distributions in Escherichia coli . E . coli dominates human in the use of basic residues, but no dominance ordering applies to acidic residues . The discussion centers on commonalities and anomalies of the amino acid compositional spectrum in relation to species, function, cellular localization, biochemical and steric attributes, complexity of the amino acid biosynthetic pathway, amino acid relative abundances and founder effects.

Biochem Int, 1992 Dec, 28(5), 805 - 11
Specific incorporation of kinetin into eukaryotic and prokaryotic transfer ribonucleic acid molecules; Barciszewski J et al.; We show that kinetin, a non-natural product with strong cytokinin activity, is incorporated into prokaryotic and eukaryotic tRNAs in the exchange reaction catalysed by a putative tRNA-kinetin transglycosylase . We also show that kinetin is specifically incorporated into E . coli tRNA(Tyr) and most probably at position 37 . To our knowledge, this is the first report of a nucleic acid base exchange reaction occurring at this position.

J Gen Virol, 1992 Dec, 73 ( Pt 12), 3289 - 94
Bovine coronavirus spike glycoprotein: localization of an immunodominant region at the amino-terminal end of S2; Vautherot JF et al.; We have identified the binding site of monoclonal antibodies (MAbs) against the S2 subunit of the bovine coronavirus spike (S) glycoprotein . The location of this site was first investigated by using prokaryotic expression of DNA restriction fragments covering the entire S gene . The amino acid sequence containing the antibody binding site was shortened from 70 to 20 amino acids by digestion of plasmid DNA with exonuclease III, followed by sequencing of the smallest digestion product encoding an immunoreactive fusion protein . Finally we synthesized a set of nonapeptides covering the 20 amino acid sequence extending from the N-terminal residue of the S2 subunit (Ala 769 to Tyr 798) . MAbs reacted mainly with six consecutive overlapping peptides with the sequence TTGYRFTNFEPFTV . Polyclonal antibodies from hyperimmunized or convalescent animals reacted only with the recombinant proteins identified by MAbs, and the hyperimmune serum bound to the same set of peptides . This suggests that this highly conserved linear antigenic determinant corresponds to an immunodominant region . This region resembles both in location and immunodominance the linear determinant defined on the infectious bronchitis virus S2 subunit . The presence of similar regions in the N-terminal region of the S2 subunit of other coronaviruses is discussed.

FEBS Lett, 1992 Nov 30, 313(3), 243 - 7
Mode of action of the Spiroplasma CpG methylase M.SssI; Renbaum P et al.; The cytosine DNA methylase from the wall-less prokaryote, Spiroplasma strain MQ1 (M.SssI) methylates completely and exclusively CpG-containing sequences, thus showing sequence specificity which is similar to that of mammalian DNA methylases . M.SssI is shown here to methylate duplex DNA processively as judged by kinetic analysis of methylated intermediates . The cytosine DNA methylases, M.HpaII and M.HhaI, from other prokaryotic organisms, appear to methylate in a non-processive manner or with a very low degree of processivity . The Spiroplasma enzyme interacts with duplex DNA irrespective to the presence of CpG sequences in the substrate DNA . The enzyme proceeds along a CpG-containing DNA substrate molecule methylating one strand of DNA at a time.

J Biol Chem, 1992 Nov 25, 267(33), 23963 - 71
Identification of nuclear encoded precursor tRNAs within the mitochondrion of Trypanosoma brucei; Hancock K et al.; RNAs that function in mitochondria are typically encoded by the mitochondrial DNA . However, the mitochondrial tRNAs of Trypanosoma brucei are encoded by the nuclear DNA and therefore must be imported into the mitochondrion . It is becoming evident that RNA import into mitochondria is phylogenetically widespread and is essential for cellular processes, but virtually nothing is known about the mechanism of RNA import . We have identified and characterized mitochondrial precursor tRNAs in T . brucei . The identification of mitochondrially located precursor tRNAs clearly indicates that mitochondrial tRNAs are imported as precursors . The mitochondrial precursor tRNAs hybridize to cloned nuclear tRNA genes, label with {alpha-32P}CTP using yeast tRNA nucleotidyltransferase and in isolated mitochondria via an endogenous nucleotidyltransferase-like activity, and are processed to mature tRNAs by Escherichia coli and yeast mitochondrial RNase P . We show that T . brucei mitochondrial extract contains an RNase P activity capable of processing a prokaryotic tRNA precursor as well as the T . brucei tRNA precursors . Precursors for tRNA(Asn) and tRNA(Leu) were detected on Northern blots of mitochondrial RNA, and the 5' ends of these RNAs were characterized by primer extension analysis . The structure of the precursor tRNAs and the significance of nuclear encoded precursor tRNAs within the mitochondrion are discussed.

J Mol Biol, 1992 Nov 20, 228(2), 450 - 9
Two tRNA-binding sites in addition to A and P sites on eukaryotic ribosomes; Rodnina MV et al.; The interaction of tRNA with 80 S ribosomes from rabbit liver was studied using biochemical as well as fluorescence techniques . Besides the canonical A and P sites, two additional sites were found which specifically bind deacylated tRNA . One of the sites is analogous to the E site of prokaryotic ribosomes, in that binding of tRNA is labile, does not depend on codon-anticodon interaction, does not protect the anticodon loop from solvent access, and requires the presence of the 3'-terminal adenosine of the tRNA . In contrast, the stability of the tRNA complex with the second site (S site) is high . tRNA binding to the S site is also codon-independent; nevertheless, the anticodon loop is shielded from solvent access . Removal of the 3'-terminal adenosine decreases the affinity of tRNA(Phe) for the S site approximately 50-fold . tRNA(Phe) is retained at the S site during translocation and through poly(Phe) synthesis . Thus, the S site does not seem to be an intermediate site for the tRNA during the elongation cycle . Rather, the tRNA bound to the S site may allosterically modulate the function of the ribosome.

Biochemistry, 1992 Nov 17, 31(45), 11224 - 30
Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase: characterization of the isolated recombinant protein and investigation of the enzyme's cation requirements; Narasimhan C et al.; Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-CoA lyase has been expressed in an active form in Escherichia coli and purified to homogeneity . Enzyme activity in crude extracts is 30-fold higher than reported for a homologous expression system . After Q-Sepharose fast-flow anion-exchange chromatography, the enzyme, which represents the first homogeneous preparation of a prokaryotic form of the protein, exhibits a specific activity of 70 units/mg . The purified enzyme is stable when stored in 20% glycerol at -80 degrees C . The recombinant bacterial enzyme cross reacts with antiserum produced against avian liver lyase, indicating some sequence homology between the two proteins . The enzyme exhibits a Km = 20 microM for (S)-HMG-CoA . Divalent cations (Mg2+ and Mn2+) markedly stimulate the enzyme activity under assay conditions; activity is only modestly increased by exogenous mercaptans . The activator constant, K(a), for Mg2+ (6.9 mM) is 3 orders of magnitude greater than that for Mn2+ (2.0 microM) . While EDTA does not affect activity, o-phenanthroline treatment markedly inhibits the enzyme . In contrast, m-phenanthroline is ineffective, suggesting that the ortho isomer's effect is attributable to chelation of a tightly bound metal ion . Atomic absorption and EPR analyses of isolated enzyme indicate the presence of tightly bound copper . In enzyme expressed using standard LB broth, copper is detected at stoichiometries of only 0.07-0.10 . When the growth medium is supplemented with 1 mM CuSO4, stoichiometry of copper binding increases to over 0.7 per enzyme subunit . Copper-enriched lyase displays enhanced thermal stability in comparison with enzyme that is low in metal content.(ABSTRACT TRUNCATED AT 250 WORDS)

Gene, 1992 Nov 16, 121(2), 321 - 4
Affinity purification of histidine-tagged proteins transiently produced in HeLa cells; Janknecht R et al.; In order to produce eukaryotic proteins in a functional state, it is often necessary to use eukaryotic instead of prokaryotic expression systems . We have designed vectors which can be employed to express either N- or C-terminally histidine-tagged proteins in transiently transfected eukaryotic cells . The histidine tag allows the rapid enrichment of these proteins by metal chelate affinity chromatography in a native and functional state . Yields of up to 5 micrograms protein/5 x 10(7) cells were achieved.

FEMS Microbiol Lett, 1992 Nov 15, 78(1), 89 - 94
Detection of bacterial and mycoplasma contamination in cell cultures by polymerase chain reaction; Spaepen M et al.; A fast and simple method to detect bacterial and especially mycoplasma contamination in tissue culture by means of polymerase chain reaction (PCR) amplification is described . In a first step the universal primer pairs P1/P2 (190-bp fragment) and P3/P4 (120-bp fragment) directed to different conserved parts of the prokaryotic 16S rRNA gene are used . A positive signal after amplification on cell culture DNA with these primers provides an indication of bacterial infection . Using the internal primers IP1, IP3 and IP'3 complementary to a part of the V4 and V8 variable regions of the 16S rRNA gene, in combination with a universal primer, cultures contaminated with mycoplasma could be identified . Six mycoplasma species, typical contaminants in tissue cultures, were investigated: Mycoplasma orale, M . fermentans, M . arginini, M . hyorhinis, M . hominis and Aeromonas laidlawii . This mycoplasma test is an easy, specific and sensitive assay which should be extremely useful in any tissue culture setting.

Biochim Biophys Acta, 1992 Nov 15, 1171(1), 1 - 10
Characterization of recombinant human and rat pancreatic phospholipases A2 secreted from Saccharomyces cerevisiae: difference in proteolytic processing; Kanda A et al.; An expression plasmid for human pancreatic phospholipase A2 in Saccharomyces cerevisiae was constructed by insertion of cDNA encoding its preprophospholipase A2 into a yeast expression vector pAM82 . The resulting product secreted in the yeast culture medium was mainly prophospholipase A2, which was the same as the natural proenzyme in all aspects examined, including the higher order structure . However, when the rat preprophospholipase A2 cDNA was manipulated in the same manner, the active phospholipase A2 of the intact mature form was secreted with the proenzyme being hardly detected in the medium . This unexpected favorable result would occur due to cleavage of rat phospholipase A2 pro-peptide by a trypsin-like proteinase in S . cerevisiae . Based on this finding, we constructed a plasmid carrying the sequence coding for the prepro-peptide of rat pancreatic phospholipase A2 behind the PHO5 promoter in the pAM82 vector, which leads to the secretion of heterologous proteins as their mature form . The use of this plasmid led to secretion of biologically active human pancreatic secretory trypsin inhibitor and a glutamic acid-specific endopeptidase from Staphylococcus aureus ATCC 12600, which are eukaryote and prokaryote proteins, respectively, in the culture medium of S . cerevisiae.

Proc Natl Acad Sci U S A, 1992 Nov 15, 89(22), 10955 - 9
Structure, expression, and functional analysis of a Na(+)-dependent glutamate/aspartate transporter from rat brain; Storck T et al.; Transport systems specific for L-glutamate and L-aspartate play an important role in the termination of neurotransmitter signals at excitatory synapses . We describe here the structure and function of a 66-kDa glycoprotein that was purified from rat brain and identified as an L-glutamate/L-aspartate transporter (GLAST) . A GLAST-specific cDNA clone was isolated from a rat brain cDNA library . The cDNA insert encodes a polypeptide with 543 amino acid residues (59,697 Da) . The amino acid sequence of GLAST suggests a distinctive structure and membrane topology, with some conserved motifs also present in prokaryotic glutamate transporters . The transporter function has been verified by amino acid uptake studies in the Xenopus laevis oocyte system . GLAST is specific for L-glutamate and L-aspartate, shows strict dependence on Na+ ions, and is inhibited by DL-threo-3-hydroxy-aspartate . In situ hybridization reveals a strikingly high density of GLAST mRNA in the Purkinje cell layer of cerebellum, presumably in the Bergmann glia cells, and a less dense distribution throughout the cerebrum . These data suggest that GLAST may be involved in the regulation of neurotransmitter concentration in central nervous system.

Science, 1992 Nov 6, 258(5084), 942 - 7
The nuclear membrane; Dingwall C et al.; The nuclear membrane forms a major barrier within the cell, permitting levels of regulation not found in prokaryotes . The dynamics and diverse functions of the nuclear membrane and its associated structures are considered in this review . The role of the nuclear pore complex in selective transport across the nuclear membrane has been studied to a considerable degree; however, many crucial questions remain . Components of a signal transduction mechanism are associated with the nucleus, suggesting that nuclear functions may be influenced directly by this system . The involvement of the heat shock cognate protein Hsc70 in nuclear protein import is discussed, and a specific signal-presentation role for this protein is proposed.

J Mol Biol, 1992 Nov 5, 228(1), 41 - 57
Characterization of the nucleic acid-binding activities of the isolated amino-terminal head domain of the intermediate filament protein vimentin reveals its close relationship to the DNA-binding regions of some prokaryotic single-stranded DNA-binding proteins; Traub P et al.; In order to demonstrate that the nucleic acid-binding activities of vimentin are dictated by its Arg-rich N-terminal head domain, this was cut off at position Lys96 with lysine-specific endoproteinase and analysed for its capacity to associate with a variety of synthetic and naturally occurring nucleic acids . The isolated polypeptide (vim NT) showed a preference for single-stranded (ss) polynucleotides, particularly for ssDNAs of high G-content . A comparison of the sequence and predicted secondary structure of vim NT with that of two prokaryotic ssDNA-binding proteins, G5P and G32P of bacteriophages fd and T4, respectively, revealed that the nucleic acid-binding region of all three polypeptides is almost entirely in the beta-conformation and characterized by a very similar distribution of aromatic amino acid residues . A partial sequence of vim NT can be folded into the same beta-loop structure as the DNA-binding wing of G5P of bacteriophage fd and related viruses . As in the case of G5P, nitration of the Tyr residues with tetranitromethane was blocked by single-stranded nucleic acids . This and spectroscopic data indicate intercalation of the Tyr aromatic ring systems between the bases of the nucleic acids and thus the contribution of a stacking component to the binding reaction . The binding was accompanied by significant changes in the ultraviolet absorption spectra of both vim NT and single-stranded nucleic acids . Upon mixing of vim NT with nucleic acids, massive precipitation of the reactants occurred, followed by the quick rearrangement of the aggregates with the formation of specific and soluble association products . Even at very high ionic strengths, at which no electrostatic reaction should be expected, a distinct fraction of vim NT incorporated naturally occurring ssRNAs and ssDNAs into fast sedimenting complexes, suggesting co-operative interaction of the polypeptide with the nucleic acids . In electron microscopy, the complexes obtained from 28 S rRNA appeared as networks of extended nucleic acid strands densely covered with vim NT, in contrast to the compact random coils of uncomplexed RNA . The networks produced from fd DNA were heterogeneous in appearance and their nucleoprotein strands in rare cases were very similar to the rod-like structures of G5P-fd DNA complexes.

J Biol Chem, 1992 Nov 5, 267(31), 22407 - 13
Molecular cloning of AMP deaminase isoform L . Sequence and bacterial expression of human AMPD2 cDNA; Bausch-Jurken MT et al.; Human AMPD2 cDNA clones have been isolated from T-lymphoblast and placental lambda gt11 libraries utilizing a previously cloned rat partial AMPD2 cDNA as the probe . Alignment analysis of all cDNA clones indicates the presence of intervening sequences in several placental isolates . This has been confirmed by sequencing human AMPD2 genomic clones . Intervening sequences can be removed from the cDNA clones by restriction with endonucleases at unique sites within the proposed open reading frame . This results in a 3292-base pair cDNA proposed to contain the entire AMPD2 open reading frame, which would encode a 760-amino acid polypeptide with a predicted subunit molecular mass of 88.1 kDa . Nucleotide and predicted amino acid comparisons with the 264 base pairs of proposed coding sequences in the rat AMPD2 cDNA demonstrate 91% similarity and identity, respectively . A comparison of the predicted human AMPD1 and AMPD2 polypeptides demonstrates homology in their C-terminal domains . Included in this region is the conserved motif, SLSTDDP, proposed to be part of the catalytic site of all AMP deaminases . In contrast, the predicted N-terminal domains of the human AMPD1 and AMPD2 polypeptides are unique . When placed in a prokaryotic expression vector, the human AMPD2 cDNA expresses AMP deaminase activity which can be precipitated with polyclonal antisera specific for isoform L.

Mol Biol (Mosk), 1992 Nov-Dec, 26(6), 1263 - 73
{SLS--a new type of polynucleotide chain folding}; Gorgoshidze MZ et al.; Short tandem repeats (5-8 base pairs) are not uncommon in the prokaryotic and eukaryotic DNA . Regions with such sequence motifs, when under superhelical stress, manifest unusual sensitivity to single-strand specific nuclease . To explain this, it has been suggested that one DNA thread should be shifted relatively to another, so that they could form two single-stranded loops protruding from the opposite chains and separated on the DNA helix by the length of a direct repeat . The structure was proposed to play a role in the regulation of transcription, organization of chromatin and in the recombination . Such type of folding could have been extra-stabilized by base pairing between the loops . This attractive possibility of the interloop minihelix formation requires a delicate stereochemical analysis and direct experimental support . Formation of the interloop minihelix in the Slipped Loop Structure (SLS) was tested by a chemical modification method at one nucleotide level resolution . The results show that bases located within the proposed interloop helix are well protected from the probes used . This fact encourages us to publish a 3-D model for the SLS-form DNA (and RNA) . The SLS is characterized by a remarkable symmetry having three mutually perpendicular dyad axes . Scanning the bank of nucleotide sequences has revealed more than 500 sites, the transcripts of which are capable of folding into the SLS form, which allow us to regard the SLS form as a novel universal structural form . Remarkably, the abundance of SLS in intrones three times exceeds that of the coding sequences . This may reflect a functional role (or roles) of the SLS conformation.

Plasmid, 1992 Nov, 28(3), 262 - 6
Construction of recA mutants of Acholeplasma laidlawii by insertional inactivation with a homologous DNA fragment; Dybvig K et al.; Mycoplasmas (class Mollicutes) are wall-less prokaryotes phylogenetically related to gram-positive bacteria . This study describes the construction of recA mutants of the mycoplasma Acholeplasma laidlawii . An internal fragment of the recA gene from A . laidlawii was cloned into a plasmid that does not replicate in this organism . When this plasmid construct was used to transform A . laidlawii, it inserted into the chromosome, disrupting the recA gene . The phenotype of the resulting recA mutant was compared to that of wild-type cells and to that of a strain that has a naturally occurring ochre mutation in its recA gene . As found in other bacterial systems, loss of RecA activity resulted in cells deficient in DNA repair.

Trends Biochem Sci, 1992 Nov, 17(11), 474 - 8
Signal peptidases in prokaryotes and eukaryotes--a new protease family; Dalbey RE et al.; Signal peptidases remove targeting peptides from pre-proteins and play central roles in the secretory pathway, as well as in the delivery of proteins to the mitochondrial intermembrane space and to the lumen of thylakoids . The catalytic mechanism of pre-protein cleavage has long been an enigma, but recent data from site-directed mutagenesis and sequence alignment studies suggest that signal peptidases may constitute a new type of serine protease, mechanistically related to the beta-lactamases.

Curr Genet, 1992 Nov, 22(5), 421 - 7
Identification of a chloroplast-encoded secA gene homologue in a chromophytic alga: possible role in chloroplast protein translocation; Scaramuzzi CD et al.; SecA is one of seven Sec proteins that comprise the prokaryotic protein translocation apparatus . A chloroplast-encoded secA gene has been identified from the unicellular chromophytic alga Pavlova lutherii . The gene predicts a protein that is related to the SecA proteins of Escherichia coli and Bacillus subtilis . The presence of secA, as well as the previously described secY and hsp70 genes, on the chloroplast genome of P . lutherii suggests that this eukaryotic organism utilises protein translocation mechanisms similar to those of bacterial cells.

EMBO J, 1992 Nov, 11(11), 4227 - 37
Site-directed mutagenesis at the Exo III motif of phi 29 DNA polymerase; overlapping structural domains for the 3'-5' exonuclease and strand-displacement activities; Soengas MS et al.; In this report we present the alignment of one of the most conserved segments (Exo III) of the 3'-5' exonuclease domain in 39 DNA polymerase sequences, including prokaryotic and eukaryotic enzymes . Site-directed substitutions of the two most conserved residues, which form the Exo III motif Tyr-(X)3-Asp of phi 29 DNA polymerase, did not affect single-stranded DNA binding, DNA polymerization, processivity or protein-primed initiation . In contrast, substitution of the highly conserved Tyr residue by Phe or Cys decreased the 3'-5' exonuclease activity to 7.5 and 4.1%, respectively, of the wild-type activity . Change of the highly conserved Asp residue into Ala resulted in almost complete inactivation (0.1%) of the 3'-5' exonuclease . In accordance with the contribution of the 3'-5' exonuclease to the fidelity of DNA replication, the three mutations in the Exo III motif (Y165F, Y165C and D169A) produced enzymes with an increased frequency of misinsertion and extension of DNA polymerization errors . Surprisingly, the three mutations in the Exo III motif strongly decreased (80- to 220-fold) the ability to replicate phi 29 DNA, this behaviour being due to a defect in the strand displacement activity, an intrinsic property of phi 29 DNA polymerase required for this process . Taking these results into account, we propose that the strand displacement activity of phi 29 DNA polymerase resides in the N-terminal domain, probably overlapping with the 3'-5' exonuclease active site.

Curr Microbiol, 1992 Nov, 25(5), 283 - 90
Sequence analysis of an aphid endosymbiont DNA fragment containing rpoB (beta-subunit of RNA polymerase) and portions of rplL and rpoC; Clark MA et al.; The aphid Schizaphis graminum is dependent on an association with a prokaryotic endosymbiont (Buchnera aphidicola) . The nucleotide (nt) sequence of a 5040 base pair (bp) DNA fragment of B . aphidicola, homologous to the rplL-rpoB-rpoC portion of the Escherichia coli beta operon, was determined . The DNA coded for the terminal 35 amino acids of RplL (large ribosomal subunit protein L7/L12), the complete RpoB (beta-subunit of RNA polymerase), and the first 209 amino acids of RpoC (beta'-subunit of RNA polymerase) . The deduced sequences of B . aphidicola RplL, RpoB, and RpoC were 71, 84, and 91% identical, respectively, to the homologous proteins of E . coli . The sequences of two portions of the intergenic region between rplL and rpoB were nearly identical in both B . aphidicola and E . coli . One sequence constituted an inverted repeat that could be an RNase III-messenger RNA processing site; the other sequence preceded RpoB . A compilation of the codon usage for RpoB, RpoC, and other B . aphidicola proteins indicated a major preference for A or T in the first and third positions, a result consistent with the low guanine plus cytosine (G + C) content of the DNA of this organism.

Anal Biochem, 1992 Nov 1, 206(2), 389 - 93
High efficiency cell-free synthesis of proteins: refinement of the coupled transcription/translation system; Kudlicki W et al.; Two modifications are introduced to convert the Escherichia coli cell-free extract ("S30") into a high efficiency system for coupled transcription/translation of exogenously added genes . (a) The ribosome fraction collected from the S30 by ultracentrifugation is used . It contains all the proteins necessary for gene expression but has lost the vast majority of soluble proteins that might interfere with purification and enzymatic activity of product formed . (b) Plasmids containing coding sequences to be expressed are not linearized thus enhancing their stability by avoiding their degradation . These two modifications not only improve protein synthesis in a static system but allow gene expression over 20-40 h in the continuous-flow cell-free system . Both prokaryotic and eukaryotic proteins have been synthesized in this system.

J Gen Virol, 1992 Nov, 73 ( Pt 11), 2933 - 40
Glycoprotein 300 is encoded by gene 28 of equine herpesvirus type 1: a new family of herpesvirus membrane proteins?
Whittaker GR, Bonass WA, Elton DM, Halliburton IW, Killington RA, Meredith DM.
A portion of equine herpesvirus type 1 (EHV-1) gene 28, which is homologous to herpes simplex virus type 1 gene UL32, was expressed using a prokaryotic system to yield a fusion protein which reacted on Western blots with P19, a monoclonal antibody (MAb) that reacts with EHV-1 glycoprotein 300 (gp300), confirming that this gene encodes gp300 . Hydrophobicity analysis showed that gp300 is a glycoprotein with multiple hydrophobic domains that might interact with, or span, the membrane several times . As such, it may represent the first member of a new family of herpesvirus glycoproteins to be identified as a virus structural component . Gp300 was also shown to be modified by palmitic acid residues, and a second MAb (1G12) directed against gp300 inhibited fusion between EHV-1-infected cells.

Boll Soc Ital Biol Sper, 1992 Nov, 68(11), 699 - 705
Multi-system approach to study mutagenesis induced by chemical carcinogens; Campomenosi P et al.; To understand molecular mechanisms of the mutation fixation process induced by a mutagen and carcinogen, a multi-system approach is suggested to reduce the probability that the results are biased by the assay used . In this light we described our different approaches to answer basic questions on the mutagenesis induced by the chemical carcinogen 4-Nitroquinoline-1-oxide . We determined mutations at the molecular level in three experimental systems: a) in prokaryotes (ss M13mp19 lacZ'/E . coli F'lacZ delta M15); b) in eukaryotes (i) ss and ds pZ189 supF/CV1-P/E.coli lacZam and (ii) HPRT in CHO cells with different repair capacity . We think this type of approach can be used to study the genetic effects of new cancer drugs for which the molecular mechanisms of action at the molecular level are still not well understood . We think to apply the know-how to study mutational spectra in tumor derived tumor suppressor genes.

Biochem Biophys Res Commun, 1992 Oct 30, 188(2), 597 - 603
Comparison of the aflatoxin B1-8,9-epoxide conjugating activities of two bacterially expressed alpha class glutathione S-transferase isozymes from mouse and rat; Buetler TM et al.; The complementary DNAs of rat glutathione S-transferase (GST, EC 2.5.1.18) Yc1 and of mouse Yc were expressed from a prokaryotic expression vector in E . coli . The purified proteins were analyzed for their activity toward aflatoxin B1-8,9-epoxide (AFBO), the reactive intermediate of the fungal mycotoxin aflatoxin B1 (AFB) . The mouse Yc isozyme had about 50-fold higher conjugating activity toward AFBO than the rat Yc1 isozyme (144 nmol/mg/min versus 3.3 nmol/mg/min) . The rat Yc1 isozyme had specific activities toward 1-chloro-2,4-dinitrobenzene, cumene hydroperoxide and ethacrynic acid of 10.7, 0.98 and 0.92 mumol/mg/min, respectively, whereas the mouse Yc isozyme had specific activities of 5.7, 2.1 and 0.1 mumol/mg/min for these substrates, respectively . These data provide further support for the hypothesis that the constitutive presence of the alpha class GST Yc isozyme in mouse liver protects mice from the hepatocarcinogenic effects of aflatoxin B1.

J Med Chem, 1992 Oct 30, 35(22), 3991 - 4000
Synthesis and biological evaluation of 5'-sulfamoylated purinyl carbocyclic nucleosides; Peterson EM et al.; The first series of 5'-sulfamoylated carbocyclic purinyl nucleosides was synthesized and tested for antitumor and antibacterial activities . The target compounds were formed by reacting the 2',3'-acetonide-protected carbocyclic nucleosides with sulfamoyl chloride, followed by deprotection . The agents were tested for cytotoxic activity against P388 mouse leukemia cells . Two compounds, 5'-sulfamoyl carbocyclic adenosine (2) and 5-sulfamoyl-8-aza carbocyclic adenosine (6) exhibited IC50 values as low as 62 and 15 nM, respectively . These analogs inhibited protein biosynthesis and slowed down DNA and RNA biosyntheses in the P388 cells . None of the target molecules were as potent against Escherichia coli as they were against the tumor cells . Also, in cell-free systems, agents 2 and 6 were more effective inhibitors of protein synthesis in rabbit reticulocyte lysate than in E . coli . These new carbocyclic derivatives appear to be somewhat selective for eukaryotic over prokaryotic cells in affecting translation.

Ann N Y Acad Sci, 1992 Oct 28, 660, 251 - 62
Antisense RNA . History and perspective; Pestka S; Antisense RNA was first an in vitro curiosity that was found to shut off protein synthesis in cell-free extracts . It was later shown to function in prokaryotic cells as a natural modulator of the synthesis of some proteins . Artificial antisense constructs can inhibit protein synthesis in prokaryotic and eukaryotic cells . To inhibit synthesis of proteins effectively, high ratios of antisense to sense RNAs are required . Thus, the challenge is to develop strategies to locate suitable targets and provide for amplification of the antisense RNA . This report provides a summary of our original work on antisense RNA.

Nature, 1992 Oct 22, 359(6397), 744 - 6
The E . coli ffh gene is necessary for viability and efficient protein export; Phillips GJ et al.; Homologues of the gene encoding the 54K (M(r) 54,000) subunit of the mammalian signal recognition particle have been identified in different organisms . The Escherichia coli homologue, termed ffh (for fifty-four homologue), specifies a protein (Ffh) that shares many properties with its eukaryotic counterpart, including association with mammalian 7S RNA and the ability to bind signal sequences specifically . Ffh also associates with E . coli 4.5S RNA, showing that it can form a ribonucleoprotein complex in prokaryotes . These results are intriguing because extensive genetic and biochemical characterization of E . coli failed to identify a signal recognition particle-like mechanism for protein export . Here we address this issue directly by construction of a strain in which ffh expression is arabinose-dependent . Results of depletion experiments indicate that Ffh is important in protein translocation.

Nature, 1992 Oct 22, 359(6397), 741 - 3
Signal-sequence recognition by an Escherichia coli ribonucleoprotein complex; Luirink J et al.; Hydrophobic signal-sequences direct the transfer of secretory proteins across the inner membrane of prokaryotes and the endoplasmic reticulum membranes of eukaryotes . In mammalian cells, signal-sequences are recognized by the 54K protein (M(r) 54,000) of the signal recognition particle (SRP) which is believed to hold the nascent chain in a translocation-competent conformation until it contacts the endoplasmic reticulum membrane . The SRP consists of a 7S RNA and six different polypeptides . The 7S RNA and the 54K signal-sequence-binding protein (SRP54) of mammalian SRP exhibit strong sequence similarity to the 4.5S RNA and P48 protein (Ffh) of Escherichia coli which form a ribonucleoprotein particle . Depletion of 4.5S RNA or overproduction of P48 causes the accumulation of the beta-lactamase precursor, although not of other secretory proteins . Whether 4.5S RNA and P48 are part of an SRP-like complex with a role in protein export is controversial . Here we show that the P48/4.5S RNA ribonucleoprotein complex interacts specifically with the signal sequence of a nascent secretory protein and therefore is a signal recognition particle.

J Biol Chem, 1992 Oct 15, 267(29), 20589 - 93
CDC43 and RAM2 encode the polypeptide subunits of a yeast type I protein geranylgeranyltransferase; Mayer ML et al.; The question regarding the identity of the alpha and beta subunits of the yeast type I protein geranylgeranyltransferase was explored using prokaryotic expression of candidate genes . The Saccharomyces cerevisiae CDC43 and RAM2 genes were expressed in Escherichia coli and cell extracts examined for the ability to transfer {3H}geranylgeranyl diphosphate to an appropriate CaaX protein substrate . Individual expression of each gene yielded no activity; however, co-expression of the two genes resulted in high levels of {3H} geranylgeranyl incorporation into the substrate protein Ras-Cys-Val-Val-Leu . The activity was partially purified yielding approximately 12,600 units/liter . The partially purified enzyme geranylgeranylated the Ras-Cys-Val-Val-Leu, Ras-Cys-Ala-Ile-Leu, Ras-Cys-Ile-Ile-Leu, and Ras-Cys-Thr-Ile-Leu substrates but not the Ras-Cys-Val-Leu-Ser or Ras-Ser-Val-Leu-Ser substrates . The protein geranylgeranyltransferase was highly specific for geranylgeranyl diphosphate and poorly transferred farnesyl . The recombinant enzyme was indistinguishable from the native type I geranylgeranyltransferase in yeast extracts . As has been reported for the protein farnesyltransferase, the yeast type I protein geranylgeranyltransferase is also a magnesium-requiring, zinc metalloenzyme . Interestingly, the recombinant enzyme functioned with calcium as the only divalent cation, although addition of zinc increased calcium-dependent activity 2-fold.

Biochem Biophys Res Commun, 1992 Oct 15, 188(1), 20 - 7
Analysis of antibody response to ovine lentivirus by using viral gene products expressed in a prokaryotic system; Kwang J et al.; The polymerase chain reaction (PCR) was used to amplify, clone, and express eight DNA fragments encoding p25, p16, reverse transcriptase (RT) core, C'-terminal RT, N'- and C'-terminals of external (gp70), and transmembrane (gp40) envelope proteins from visna virus infectious recombinant DNA . Efforts were focused on characterizing the nature of the humoral immune response of ovine progressive pneumonia (OPP) virus infected animals and identifying the conserved and prime-reactive antigenic determinants that have potential diagnostic value . This communication reports that the N'-terminal region of gp40 appeared to be the most immunoreactive of the bacterially expressed proteins and could serve as a sensitive immunodiagnostic antigen for the detection of OPP infection.

J Bacteriol, 1992 Oct, 174(20), 6488 - 97
Alterations in the hydrophilic segment of the maltose-binding protein (MBP) signal peptide that affect either export or translation of MBP; Puziss JW et al.; Mutations that reduce the net positive charge within the hydrophilic segments of the signal peptides of several prokaryotic exported proteins can result in a reduction in the rate of protein export, as well as a reduction in protein synthesis (M . N . Hall, J . Gabay, and M . Shwartz, EMBO J . 2:15-19, 1983; S . Inouye, X . Soberon, T . Franceschini, K . Nakamura, K . Itakura, and M . Inouye, Proc . Natl . Acad . Sci . USA 79:3438-3441, 1982; J . W . Puziss, J . D . Fikes, and P . J . Bassford, Jr., J . Bacteriol . 171:2302-2311, 1989) . This result has been interpreted as evidence that the hydrophilic segment is part of a mechanism that obligatorily couples translation to protein export . We have investigated the role of the hydrophilic segment of the Escherichia coli maltose-binding protein (MBP) signal peptide in the export and synthesis of MBP . Deletion of the entire hydrophilic segment from the MBP signal peptide resulted in a defect in MBP export, as well as a dramatic reduction in total MBP synthesis . Suppressor mutations that lie upstream of the malE coding region were isolated . These mutations do not affect MBP export but instead were shown to partially restore MBP synthesis by increasing the efficiency of MBP translational initiation . In addition, analysis of a series of substitution mutations in the second codon of certain malE alleles demonstrated that MBP export and synthesis can be independently affected by mutations in the hydrophilic segment . Finally, analysis of alterations in the hydrophilic segment of the ribose-binding protein signal peptide fused to the mature moiety of the MBP has revealed that the role of the hydrophilic segment in the export process can be functionally separated from any role in translation . Taken together, these results strongly suggest that the hydrophilic segment of the MBP signal peptide is not involved in a mechanism that couples MBP translation to export and argue against the presence of a mechanism that obligatorily couples translation to protein export in Escherichia coli.

J Theor Biol, 1992 Oct 7, 158(3), 271 - 91
Evolution of metabolic pathways by chance assembly of enzyme proteins generated from sense and antisense strands of pre-existing genes; Fukuchi S et al.; In order to get an insight into the evolutionary aspect of metabolic pathways, especially of the ubiquitous glycolytic pathway, we have carried out an extensive search of sense-sense and sense-antisense similarities for enzyme proteins in the glycolytic pathway, the pentose phosphate cycle, alcohol and lactate fermentation pathways and the TCA cycle . This investigation of amino acid sequences reveals a curious pattern of similarity relations; no similarity can be found between the enzyme proteins in a section of the glycolytic pathway where the glyceraldehyde-3-phosphate or even glycerol-3-phosphate is converted into the pyruvate while many examples of sense-sense and sense-antisense similarities are found even between enzyme proteins in distant blocks, e.g . between the proteins in the TCA cycle and those in the pentose phosphate cycle, as well as between the functionally associated proteins in each of these blocks . Complementary to this characteristic pattern of amino acid sequence similarity, the search for similarities of nucleotide sequences also finds that the similarities of glycolytic enzyme genes, some sense-sense and others sense-antisense similarities, are concentrated on the nucleotide sequences of prokaryotic 16S or eukaryotic 18S ribosomal RNA gene with its flanks, although some of the copy sequences are also found in transfer RNA genes as well as in 23S or 26S ribosomal RNA gene . These results strongly suggest that the metabolic pathways have been developed by the chance assembly of enzyme proteins generated from the sense and antisense strands of pre-existing genes, e.g . the fermentation pathways and pentose phosphate cycle by the proteins from the genes of enzymes in the glycolytic pathway and the TCA cycle from all these successively increased genes, ascribing the origin of metabolic enzyme genes to the close relation between the glycolytic enzyme protein genes and the RNA gene cluster.

Mol Microbiol, 1992 Oct, 6(20), 2975 - 88
Determinants of an unusually stable mRNA in the bacterium Myxococcus xanthus; Romeo JM et al.; Myxococcus xanthus is a Gram-negative bacterium which has a complex life cycle that includes development (fruiting body formation) . The gene for myxobacterial haemagglutinin, mbhA, is developmentally regulated and highly expressed . In this report we show that the mbhA mRNA is exceptionally stable for a prokaryotic organism, exhibiting a chemical half life (t1/2) of 150 min at 18 h of development . The mbhA mRNA was not stable in vegetatively growing cells nor was it stable when expressed in Escherichia coli . We have used site-directed mutagenesis of the mbhA gene to analyse some of the determinants which mediate the stability of the mbhA transcript . Sequences within the 3'-untranslated region (3'-UTR) were found to be crucial for mRNA stability . This region of mRNA can potentially form an extremely stable stem-loop structure immediately adjacent to the translational stop codon . A deletion within this region caused a 10-fold increase in the decay rate of the transcript . Furthermore, conditions which were associated with reduced mbhA translation or mutations that caused premature termination of translation drastically reduced mRNA stability even in the presence of the wild type 3'-UTR . These results suggest that a significant aspect of mbhA mRNA stability involves a synergistic interaction of the translational machinery with sequence elements within the 3'-UTR.

Curr Opin Genet Dev, 1992 Oct, 2(5), 799 - 804
Sporulation in prokaryotes and lower eukaryotes; Strauch MA et al.; During the past year, highlights in sporulation research include the demonstration that phosphorylation of SpoOA is a critical factor in Bacillus subtilis development; the identification of C alpha proteins, adenylyl cyclase and protein kinase A genes in Dictyostelium; proof that an endogenous antisense RNA regulates gene expression in Dictyostelium; and characterization of a second type of differentiated cell in Myxococcus.

Curr Opin Genet Dev, 1992 Oct, 2(5), 756 - 67
Flagella in prokaryotes and lower eukaryotes; Blair DF et al.; During the past year, significant advances have been made in the understanding of both prokaryotic and eukaryotic flagella . About 50 genes are dedicated to the assembly and operation of bacterial flagella . Recent discoveries have advanced our understanding of how these genes are regulated and how their products assemble into a functional, rotating organelle . The dynein arms of eukaryotic flagella are now also better understood . Several genes that are found in the mechanochemical macroassemblies have been cloned, and other loci have been identified, suggesting that there is even greater complexity than first expected.

Curr Opin Genet Dev, 1992 Oct, 2(5), 683 - 90
Genetic analysis of recombination in prokaryotes; Lloyd RG et al.; Bacteria provide a simple system for the genetic analysis of homologous recombination . More than twenty genes have been identified in Escherichia coli . The enzymatic activities associated with the products of many of these genes have been revealed by studies with model DNA substrates . It is now possible to pair homologous molecules in vitro and process these through defined intermediates into mature recombinants of the types predicted by genetic crosses.

Genetics, 1992 Oct, 132(2), 375 - 86
The yeast omnipotent suppressor SUP46 encodes a ribosomal protein which is a functional and structural homolog of the Escherichia coli S4 ram protein; Vincent A et al.; The accurate synthesis of proteins is crucial to the existence of a cell . In yeast, several genes that affect the fidelity of translation have been identified (e.g., omnipotent suppressors, antisuppressors and allosuppressors) . We have found that the dominant omnipotent suppressor SUP46 encodes the yeast ribosomal protein S13 . S13 is encoded by two similar genes, but only the sup46 copy of the gene is able to fully complement the recessive phenotypes of SUP46 mutations . Both copies of the S13 genes contain introns . Unlike the introns of other duplicated ribosomal protein genes which are highly diverged, the duplicated S13 genes have two nearly identical DNA sequences of 25 and 31 bp in length within their introns . The SUP46 protein has significant homology to the S4 ribosomal protein in prokaryotic-type ribosomes . S4 is encoded by one of the ram (ribosomal ambiguity) genes in Escherichia coli which are the functional equivalent of omnipotent suppressors in yeast . Thus, SUP46 and S4 demonstrate functional as well as sequence conservation between prokaryotic and eukaryotic ribosomal proteins . SUP46 and S4 are most similar in their central amino acid sequences . Interestingly, the alterations resulting from the SUP46 mutations and the segment of the S4 protein involved in binding to the 16S rRNA are within this most conserved region.

Int J Biol Macromol, 1992 Oct, 14(5), 249 - 56
Construction of nucleosome cores from defined DNA sequences of prokaryotic origin; Seger R et al.; A procedure for the de novo construction of nucleosome core particles from defined DNA sequences of prokaryotic origin is described . Efficient de novo reconstitution without added carrier DNA is demonstrated . DNase I and exonuclease III analysis of a nucleosome core prepared from a 154 base pair fragment extending from base 853 to base 1006 of pBR322 indicates a non-random positioning of the histone core along the DNA . As bacteria have no histones, their DNA cannot be expected to have a histone core positioning signal encoded in it, the efficient formation of a uniquely positioned core particle is not self evident . The possibility that a phosphate end group positions DNA fragments on the histone is considered . The de novo reconstitution of carrier-less defined nucleosome core particles should facilitate the physicochemical study of nucleosomes on the fine structural level.

Mol Cell Biol, 1992 Oct, 12(10), 4590 - 600
Sequences in the human c-myc P2 promoter affect the elongation and premature termination of transcripts initiated from the upstream P1 promoter; Meulia T et al.; A conditional block to transcription elongation provides one mechanism for controlling the steady-state levels of c-myc RNA in mammalian cells . Although prematurely terminated c-myc RNAs are not detectable in mammalian cells, truncated c-myc RNAs with 3' ends that map near the end of the first exon are transcribed from human c-myc templates injected into Xenopus oocytes germinal vesicles . A series of linker scanner and deletion mutants within the c-myc P2 promoter was tested in the Xenopus oocyte injection assay to determine the potential contribution of promoter elements to the elongation or premature termination of c-myc transcription . Although this analysis failed to identify sequences in the P2 promoter that significantly affect the elongation or termination of P2-initiated transcripts, our results suggest that sequences within the P2 promoter contribute to the premature termination of transcripts initiated at the upstream P1 promoter . A subset of these sequences is essential for the efficient elongation of P1-initiated transcripts through intrinsic sites of termination at the end of exon 1 . These sequences affect P1 elongation when they are downstream of the site of initiation, and we hypothesize that they may be analogous to a class of prokaryotic elements required for antitermination.

J Bacteriol, 1992 Oct, 174(19), 6215 - 20
Two promoters for the whiB sporulation gene of Streptomyces coelicolor A3(2) and their activities in relation to development; Soliveri J et al.; Two transcripts corresponding to the whiB sporulation gene of Streptomyces coelicolor A3(2) that differed in length at their 5' ends by 164 nucleotides were identified by S1 mapping . Their presumptive promoters differed from each other; the more downstream, P2, resembled typical prokaryotic promoters (i.e., those recognized by the major form of RNA polymerase) at five of six positions in its -10 and -35 regions; and the more upstream, P1, was comparably similar, instead, to the previously described hrdDp1 promoter (M . J . Buttner, K . F . Chater, and M . J . Bibb, J . Bacteriol . 172:3367-3378, 1990) around -40, -10, and +1 . In surface cultures of the wild-type strain, the abundance of transcripts from the weak P1 promoter showed no obvious correlation with the developmental stage, whereas transcripts from P2 were barely detectable until aerial mycelium was present and then became relatively abundant, consistent with the developmental role of whiB . Both types of transcript were detected during and, to a lesser extent, after rapid growth in liquid culture . In addition, both promoters were utilized in vitro by RNA polymerase purified from a liquid culture of S . coelicolor . Transcription from P1 and P2 was observed during surface culture in strains carrying mutations blocking aerial mycelium formation (bldA and bldB) or the formation of spores in aerial mycelium (whiA, whiB, whiG, and whiH) . Thus, whiB transcription is not severely dependent on any of these developmental genes, among which whiG is the determinant of a putative sigma factor specific for, and crucial to, sporulation.

Eur J Biochem, 1992 Oct 1, 209(1), 233 - 9
Short-chain dehydrogenases . Proteolysis and chemical modification of prokaryotic 3 alpha/20 beta-hydroxysteroid, insect alcohol and human 15-hydroxyprostaglandin dehydrogenases; Krook M et al.; Prokaryotic 3 alpha/20 beta-hydroxysteroid dehydrogenase exhibits one segment sensitive to proteolysis with Glu-C protease and trypsin (cleaving after Glu192 and Arg196, respectively) . Cleavage is associated with dehydrogenase inactivation; the presence of NADH offers almost complete protection and substrate (cortisone) gives some protection . Distantly related insect alcohol dehydrogenase is more resistant to proteolysis, but cleavage in a corresponding segment is detectable with Asp-N protease (cleaving before Asp198), while a second site (at Glu243) is sensitive to cleavage with both Glu-C and Asp-N proteases . Combined, the results suggest the presence of limited regions especially sensitive to proteolysis and the possibility of some association between the enzyme active site and the sensitive site(s) . Modification of the hydroxysteroid dehydrogenase with tetranitromethane is paralleled by enzyme inactivation . With a 10-fold excess of reagent, labeling corresponds to 1.2 nmol Tyr/nmol protein chain and is recovered largely in Tyr152, with lesser amounts in Tyr251 . Tetranitromethane also rapidly inhibits the other two dehydrogenases, but they contain Cys residues, preventing direct correlation with Tyr modification . Together, the proteolysis and chemical modifications highlight three segments of short-chain dehydrogenase subunits, one mid-chain, containing Tyr152 of the steroid dehydrogenase (similar numbers in the other enzymes), strictly conserved and apparently close to the enzyme active site, the other around position 195, sensitive to proteolysis and affected by coenzyme binding, while the third is close to the C-terminus.

Eur J Biochem, 1992 Oct 1, 209(1), 151 - 6
Efficiency of the 5'-terminal sequence (omega) of tobacco mosaic virus RNA for the initiation of eukaryotic gene translation in Escherichia coli; Ivanov IG et al.; Recent studies have demonstrated that the 5' leader (omega sequence) of tobacco mosaic virus RNA has a certain enhancing capacity for translation of mRNA in both prokaryotes and eukaryotes . In order to estimate the efficiency of omega to initiate translation of mRNA in Escherichia coli, in comparison to the Shine-Dalgarno (S/D) sequence, we have inserted eight different eukaryotic genes into two types of E . coli expression vectors containing one constitutive promoter (P1) but different translation-initiation sites (S/D or omega delta 3 sequence, respectively) . The efficiency of transcription and translation in vivo was evaluated for these vectors by measuring the yield of protein and both the level and stability of mRNA . We report that substitution of omega delta 3 for S/D decreases the yield of expressed protein 4-1900-fold and the content of gene-specific mRNA is decreased by about sevenfold . However, in comparison with the S/D sequence, the level of protein expressed under the translational control of omega delta 3 is less sensitive to changes in the 5' coding region . We also report that the omega sequence contains a region of 10-12 nucleotides complementary to the small ribosomal subunit RNA (rRNA) of E . coli, Eikenella corrodens and Xenopus laevis, and to the rRNA of the (small ribosomal) subunit of Oryza sativa.

Radiat Res, 1992 Oct, 132(1), 30 - 9
Transfection of the Escherichia coli nth gene into radiosensitive Chinese hamster cells: effects on sensitivity to radiation, hydrogen peroxide, and bleomycin sulfate; Harrison L et al.; The Escherichia coli nth gene encodes endonuclease III, which catalyses the glycolytic removal of various oxidized thymine residues from DNA . A truncated version of nth, with the prokaryotic regulatory sequences removed, was ligated into the retrovirus-based vector pZipneoSV(X)1 and transfected into the radiosensitive Chinese hamster ovary cell line, xrs7 . Following selection with G418, two clones (x7nth1 and x7nth6) were shown by Southern analysis to contain the nth gene . No substantial difference in gamma-ray sensitivity was detected between xrs7, clones x7nth1 and x7nth6, and the parent vector transfected clone (x7neo1) . However, clones containing the nth gene were more resistant to hydrogen peroxide cytotoxicity {D0's for x7nth1 and x7nth6 were 0.072 microgram/ml (4 microM) and 0.046 microgram/ml, respectively, compared with D0's of 0.034 and 0.027 microgram/ml for xrs7 and x7neo1, respectively} but markedly more sensitive to bleomycin sulfate cytotoxicity than xrs7 and x7neo1 (e.g., 1D0's for x7nth6 and xrs7 were 0.05 and 0.12 microgram/ml, while 2D0's for x7nth1 and xrs7 were 0.35 and 0.48 microgram/ml, respectively) . Alterations in sensitivity to hydrogen peroxide and bleomycin sulfate could not be explained by differences in the distribution of the cell-cycle phases and growth rate of nth-containing clones and control cell lines . These results are consistent with the hypothesis that modified thymine lesions are potentially cytotoxic . Hence, when cells incur a high level of endonuclease III-repairable damage relative to strand breakage, such as after treatment with hydrogen peroxide, increased repair capacity increases survival . Gamma radiation produces a lower level of endonuclease III-repairable damage relative to all the other types of lesions produced; hence increased repair capacity has no measurable effect on cell survival . The increased sensitivity of x7nth1 and x7nth6 to bleomycin sulfate toxicity may indicate that, when thymine damage and single-strand breaks are in close proximity on opposite strands of the DNA, endonuclease III, which incises DNA at the site of damaged residues, can increase the number of double-strand breaks and hence decrease the level of cell survival.

Biotechnology (N Y), 1992 Oct, 10(10), 1143 - 7
Effect of glycosylation on properties of soluble interferon gamma receptors produced in prokaryotic and eukaryotic expression systems; Fountoulakis M et al.; We investigated the influence of glycosylation on solubility, chromatographic behavior and resistance to heat- and chaotrope-dependent denaturation and proteolytic digestion of three recombinant human interferon gamma receptors produced in Escherichia coli, Spodoptera frugiperda and Chinese hamster ovary cells . The proteins produced in the eukaryotic expression systems were glycosylated, carrying different, heterogeneous carbohydrate moieties . They were assayed fully glycosylated and after removal of the oligosaccharides . Although glycosylation influenced the chromatographic behavior of the tested proteins, it did not protect against proteolysis and heat- or chaotrope-induced denaturation . The glycosylated receptors were slightly more sensitive to certain proteolytic cleavages and slightly less resistant to chaotropes, than the soluble receptor produced in Escherichia coli.

Lik Sprava, 1992 Oct, (10), 29 - 32
{The determination of the antiviral activity of the sorbent Evirkhip (in vitro studies)}; Boiko II et al.; It was found that "Evirkhip" possesses a marked antiviral activity confirmed on a model of enteroviruses (coxsackie) . The authors also studied the resistance to the drug in the chain: eukaryotic cell-viral particle-prokaryotic cell . The resistance decreased with the shift to right.

Bioorg Khim, 1992 Oct-Nov, 18(10-11), 1394 - 402
{Gene expression in cell-free systems on a preparative scale}; Spirin AS; In vivo expression of foreign or synthetic genes can be subject to certain restrictions such as protein aggregation, degradation and toxicity . Conventional in vitro systems can overcome these problems, but in turn suffer from other limitations, in particular short life-time and low protein yield . In this review, two types of gene expression system are described . Both are based on the novel concept of the enhanced expression from cell-free extracts where incubation is performed in the continuous flow of a feeding solution rather than in a fixed volume of a test-tube . The first type makes use of cell-free translation of mRNA templates in either prokaryotic or eukaryotic cell lysates . The second utilizes the coupled transcription--translation of DNA templates, with genes transcribed by either endogenous or bacteriophage RNA polymerases . In both systems, translation can be carried out over tens or hundreds of hours resulting in high protein yields.

Boll Soc Ital Biol Sper, 1992 Oct, 68(10), 619 - 24
Single stranded DNA-vectors for analyzing processing of DNA damage induced by 4-nitroquinoline-1-oxide in prokaryotes and eukaryotes; Iannone R et al.; Previous studies indicate that single stranded DNA vectors could be used in different organisms to study mutagenesis induced by DNA damaging agents . We applied this approach to study mutagenesis induced by 4NQO lesions . The use of ssDNA, on which the ultimate metabolite of 4NQO (Ac-4HAQO) induces mainly C8-guanine adducts, allowed us to find a correlation between G-transversions and the dGuo-C8-AQO adduct . This correlation was established in two independent assay-systems, based on prokaryotic and eukaryotic cells.

Curr Opin Genet Dev, 1992 Oct, 2(5), 739 - 47
Turnover of mRNA in prokaryotes and lower eukaryotes; Higgins CF et al.; The turnover of mRNA plays an important role in the regulation of gene expression . The two best understood model systems are those of the prokaryote Escherichia coli and the lower eukaryote Saccharomyces cerevisiae . Considerable progress in recent years has helped define the general pathways by which mRNA is degraded in E coli . Much less is known about the pathways of decay, or the enzymes involved, in eukaryotic cells . However, both cis-acting sequences and trans-acting factors have recently been characterized in S . cerevisiae and an indispensable role for translation has been identified . A comparison of these model species highlights both similarities and differences in mRNA turnover between prokaryotic and eukaryotic systems.

Curr Opin Genet Dev, 1992 Oct, 2(5), 720 - 6
Diversity of mechanisms in the regulation of translation in prokaryotes and lower eukaryotes; Lindahl L et al.; Regulation of translation is used to control the expression of many essential and highly expressed genes . The known repertoire of molecular mechanisms for translational regulation is expanding . Recently elucidated mechanisms involve alterations in mRNA structure and modulation of the activity of translation initiation factors.

Biochem Pharmacol, 1992 Sep 25, 44(6), 1123 - 9
In vitro DNA synthesis by DNA polymerase I and DNA polymerase alpha on single-stranded DNA containing either purine or pyrimidine monoadducts; Hoffmann JS et al.; The capacity of the large fragment of DNA polymerase I from Escherichia coli and of DNA polymerase alpha from Drosophila embryo to replicate single-stranded M13mp10 DNA containing either purine or pyrimidine monoadducts was compared . The monoadducts were respectively induced by cisplatinum and by furocoumarin photoaddition . For both types of lesions, it is observed that the eukaryotic enzyme is more inhibited than the prokaryotic one . By mapping the arrest sites produced by furocoumarin monoadducts on the synthesis catalysed by DNA polymerase alpha, we show that, in contrast with the photoreaction observed with double-stranded DNA, these compounds do not show a strong sequence specificity in reacting with single-stranded DNA.

Nucleic Acids Res, 1992 Sep 25, 20(18), 4741 - 6
Involvement of the size and sequence of the anticodon loop in tRNA recognition by mammalian and E . coli methionyl-tRNA synthetases; Meinnel T et al.; The rates of the cross-aminoacylation reactions of tRNAs(Met) catalyzed by methionyl-tRNA synthetases from various organisms suggest the occurrence of two types of tRNA(Met)/methionyl-tRNA synthetase systems . In this study, the tRNA determinants recognized by mammalian or E . coli methionyl-tRNA synthetases, which are representative members of the two types, have been examined . Like its prokaryotic counterpart, the mammalian enzyme utilizes the anticodon of tRNA as main recognition element . However, the mammalian cytoplasmic elongator tRNA(Met) species is not recognized by the bacterial synthetase, and both the initiator and elongator E . coli tRNA(Met) behave as poor substrates of the mammalian cytoplasmic synthetase . Synthetic genes encoding variants of tRNAs(Met), including the elongator one from mammals, were expressed in E . coli . tRNAs(Met) recognized by a synthetase of a given type can be converted into a substrate of an enzyme of the other type by introducing one-base substitutions in the anticodon loop or stem . In particular, a reduction of the size of the anticodon loop of cytoplasmic mammalian elongator tRNA(Met) from 9 to 7 bases, through the creation of an additional Watson-Crick pair at the bottom of the anticodon stem, makes it a substrate of the prokaryotic enzyme and decreases its ability to be methionylated by the mammalian enzyme . Moreover, enlarging the size of the anticodon loop of E . coli tRNA(Metm) from 7 to 9 bases, by disrupting the base pair at the bottom of the anticodon stem, renders the resulting tRNA a good substrate of the mammalian enzyme, while strongly altering its reaction with the prokaryotic synthetase . Finally, E . coli tRNA(Metf) can be rendered a better substrate of the mammalian enzyme by changing its U33 into a C . This modification makes the sequence of the anticodon loop of tRNA(Metf) identical to that of cytoplasmic initiator tRNA(Met).

Nucleic Acids Res, 1992 Sep 25, 20(18), 4721 - 5
Determination of lysine residues affinity labeled in the active site of yeast RNA polymerase II(B) by mutagenesis; Treich I et al.; In a previous study, yeast RNA polymerase II(B) was affinity labeled with two nucleotide derivatives (III and VIII) (1) . In both cases, the labeled site was localized to the C-terminal part of the B150 subunit . The potential target lysyl residues of derivative III were mapped to the conserved domain H, between Asn946 and Met999 . In the present work, we have mutagenized to arginine the five lysines present in domain H . Three lysines can be replaced, individually or simultaneously, without affecting cell growth, and each mutated enzyme can still be affinity labeled . Hence one or both of the other two lysyl residues, Lys979 and Lys987, is the target of the affinity reagent . These two lysines were each found to be essential for cell viability . Derivative VIII labeled another domain in addition to domain H . Supported by analogous results obtained for E . coli RNA polymerase using derivative VIII (2), we hypothesized that the second domain labeled by this derivative in the B150 subunit was domain I . Mutagenesis of the unique lysine present in domain I demonstrated that Lys 1102 was the target of derivative VIII . These results indicate that in both prokaryotic and eukaryotic RNA polymerases, domains H and I are in close proximity and participate to the active site.

Gene, 1992 Sep 21, 119(1), 107 - 11
Translational initiation factors IF-1 and eIF-2 alpha share an RNA-binding motif with prokaryotic ribosomal protein S1 and polynucleotide phosphorylase; Gribskov M; Initiation of translation is a complicated process involving numerous accessory factors whose functions remain incompletely understood . Bacterial ribosomal protein S1 is known to contain a repeated sequence motif (S1-RM), also found in polynucleotide phosphorylase, that is thought to be involved in binding to RNA . Using the technique of profile analysis, the S1-RM can also be found in bacterial and chloroplast translation initiation factor IF-1 sequences, and in the sequences of eukaryotic translation initiation factor eIF-2 alpha chains . The significance of the similarity of the sequences is very high suggesting that the occurrence of the S1-RM in these diverse proteins represents homology . The similarity of S1 to IF-1 further suggests that S1 has evolved from an IF-1 like ancestor, and therefore that the two proteins have a similar or competitive function . The most obvious common function of the proteins containing the S1-RM seems to be RNA binding, suggesting that IF-1 and eIF-2 alpha may bind to RNA.

J Mol Biol, 1992 Sep 20, 227(2), 510 - 31
Solution structure of a DNA octamer containing the Pribnow box via restrained molecular dynamics simulation with distance and torsion angle constraints derived from two-dimensional nuclear magnetic resonance spectral fitting; Schmitz U et al.; The DNA octamer {d(GTATAATG}.{(CATATTAC)}, containing the prokaryotic upstream consensus recognition sequence, has been examined via proton homonuclear two-dimensional nuclear Overhauser effect (2D NOE) and double-quantum-filtered correlation (2QF-COSY) spectra . All proton resonances, except those of H5' and H5" protons, were assigned . A temperature dependence study of one-dimensional nuclear magnetic resonance (NMR) spectra, rotating frame 2D NOE spectroscopy (ROESY), and T1 rho measurements revealed an exchange process that apparently is global in scope . Work at lower temperatures enabled a determination of structural constraints that could be employed in determination of a time-averaged structure . Simulations of the 2QF-COSY cross-peaks were compared with experimental data, establishing scalar coupling constant ranges of the individual sugar ring protons and hence pucker parameters for individual deoxyribose rings . The rings exhibit a dynamic equilibrium of N and S-type conformers with 80 to 100% populations of the latter . A program for iterative complete relaxation matrix analysis of 2D NOE spectral intensities, MARDIGRAS, was employed to give interproton distances for each mixing time . According to the accuracy of the distance determination, upper and lower distance bounds were chosen . The distance bounds define the size of a flat-well potential function term, incorporated into the AMBER force-field, which was employed for restrained molecular dynamics calculations . Torsion angle constraints in the form of a flat-well potential were also constructed from the analysis of the sugar pucker data . Several restrained molecular dynamics runs of 25 picoseconds were performed, utilizing 184 experimental distance constraints and 80 torsion angle constraints; three different starting structures were used: energy minimized A-DNA, B-DNA, and wrinkled D-DNA, another member of the B-DNA family . Convergence to similar structures obtained with root-mean-square deviations between resulting structures of 0.37 to 0.92 A for the central hexamer of the octamer . The average structure from the nine different molecular dynamics runs was subjected to final restrained energy minimization . The resulting final structure was in good agreement with the structures derived from different molecular dynamics runs and exhibited a substantial improvement in the 2D NOE sixth-root residual index in comparison with the starting structures . An approximation of the structure in the terminal base-pairs, which displayed experimental evidence of fraying, was made by maintaining the structure of the inner four base-pairs and performing molecular dynamics simulations with the experimental structural constraints observed for the termini.(ABSTRACT TRUNCATED AT 400 WORDS)

J Photochem Photobiol B, 1992 Sep 15, 15(4), 277 - 98
Photochemistry of nucleic acids in cells; Cadet J et al.; A survey of the recent aspects of the main photoreactions induced by far-UV radiation in cellular DNA is reported . This mostly includes the formation of cyclobutadipyrimidines, pyrimidine(6-4)pyrimidone photoadducts and related Dewar valence isomers in various eukaryotic and prokaryotic cells, as monitored by using either specific or more general assays . Information is also provided on mechanistic aspects regarding the formation of 5,6-dihydro-5-(alpha-thyminyl) thymine, the so-called "spore photoproduct" within far-UV-irradiated bacterial spores . The second major topic of the review deals with the effects of near-UV radiation and visible light on cellular DNA which are mostly mediated by photosensitizers . The main photoreactions of furocoumarins with DNA, one major class of photosensitizers used in the phototherapy of skin diseases, involve a {2 + 2} cycloaddition to the thymine bases according to an oxygen-independent mechanism . In contrast a second type of photosensitized reaction which appears to play a major role in the genotoxic effects of both near-UV and visible light requires the presence of oxygen . The photodynamic effects which are mediated by either still unidentified endogenous photosensitizers or defined exogenous photosensitizers lead to the formation of a wide spectrum of DNA modifications including base damage, oligonucleotide strand breaks and DNA-protein cross-links.

Eur J Biochem, 1992 Sep 15, 208(3), 781 - 7
Stoichiometry of interaction between interferon gamma and its receptor; Fountoulakis M et al.; The biological response of interferon gamma is mediated by binding to a specific cell-surface receptor . We investigated the stoichiometry of this binding using soluble receptors produced in prokaryotic and eukaryotic expression systems comprising the extracellular ligand-binding domain of the native protein . The ligand-receptor complexes were analyzed by cross-linking, chromatography, analytical ultracentrifugation and laser-light scattering . Cross-linking and chromatography showed that the stoichiometry of the interaction between ligand and receptor depends on the molar ratios of the two components mixed . All approaches confirmed that mixtures of ligand-receptor complexes are formed with one interferon-gamma dimer bound by one or two receptors . The soluble receptor produced in Escherichia coli mainly showed a ligand/receptor stoichiometry of 1:1, while the receptors produced in eukaryotic cells showed a stoichiometry of binding of 1:2 . This apparent discrepancy is most likely due to the conformational heterogeneity of the Escherichia-coli-derived protein.

J Biol Chem, 1992 Sep 15, 267(26), 18961 - 5
Cloning and characterization of the 5'-flanking region of the human topoisomerase II alpha gene; Hochhauser D et al.; Topoisomerases are essential enzymes for DNA metabolism in prokaryotes and eukaryotes . In human cells, DNA topoisomerase II enzyme activity can be modulated by both viral transformation and changes in proliferation status . To identify elements important for regulation of topoisomerase II alpha gene expression, genomic DNA clones covering the 5'-end of the gene were isolated . The intron/exon structure of a 2.5-kilobase region encompassing the translation start site was determined . Transcription was found to initiate at multiple sites clustered around 90 base pairs 5' to the ATG initiation codon . Transient expression of chimeric topoisomerase II-reporter gene constructs in HeLa cells revealed that the 5'-flanking region exhibited promoter activity . The region -90 to -1 upstream of the major transcription start site was shown by deletion analysis to include a promoter . This minimal promoter lacks a TATA box, is moderately GC-rich, and contains a high frequency of CpG dinucleotides; characteristic of a "housekeeping" gene promoter . Maximal promoter activity was observed using a fragment extending to position -562 . Putative regulatory elements are contained within and immediately upstream of the minimal promoter region . The regulatory region of the topoisomerase II alpha gene identified here is similar in basic structure to those of the human thymidine kinase and DNA polymerase alpha genes, which are also controlled by proliferation-specific factors.

J Biol Chem, 1992 Sep 15, 267(26), 18929 - 39
Characterization of the structure of a low Km, rolipram-sensitive cAMP phosphodiesterase . Mapping of the catalytic domain; Jin SL et al.; Considerable structural similarities are present in a region of approximately 270 amino acids in most known cyclic nucleotide phosphodiesterase (PDE) sequences, opening the possibility that this region encodes the catalytic domain of the enzyme . To test this hypothesis, the structure of a high affinity cAMP PDE (cAMP-PDE) was analyzed by deletion mutations and site-directed mutagenesis . A ratPDE3 cDNA was mutated using a strategy based on fragment amplification by polymerase chain reaction . The effect of the introduced mutations was determined by expressing wild type and mutated proteins in prokaryotic and eukaryotic cells . The level of expression of the PDE protein was monitored by immunoblot analysis using two specific cAMP-PDE polyclonal antibodies and by measuring the PDE activity . After removal of a 99-amino acid region at the carboxyl terminus flanking the conserved domain, the protein retains its catalytic activity even though its Km and velocity were changed . Internal deletions at the amino terminus of this PDE showed that the enzyme activity was increased when a 97-amino acid fragment (from Tyr49 to Lys145) was removed . Further deletions within the amino terminus produced inactive proteins . Within the domain that appears essential for catalysis, 1 threonine and 2 serine residues are conserved in all PDEs . Substitutions of the invariant threonine (Thr349) present in the most conserved region with alanine, proline, or serine yielded proteins of the correct size and a level of expression comparable to the wild type PDE . However, in both expression systems used, proteins were completely devoid of the ability to hydrolyze cyclic nucleotides, except when the threonine was substituted with a serine . Conversely, mutations of 2 other conserved serine residues (Ser305 and Ser398) present in the catalytic domain either had no effect or produced changes only in Km and Vmax, but did not abolish catalytic activity . In addition, 2 histidine residues (His278 and His311) present in proximity to Thr349 appeared to be essential for the structure of the catalytic domain, since any substitution performed in these residues yielded an inactive enzyme . Mutations of a serine residue (Ser295) in the region homologous to the cAMP binding site of the regulatory subunit of the cAMP-dependent protein kinase demonstrated that this region does not have the same function in the two proteins . These data provide direct evidence that a 37-kDa domain, which in part corresponds to the region of conservation in all PDEs, contains the catalytic domain, and that threonine and histidine residues are probably involved in catalysis and/or are essential for the conformation of an active enzyme.

J Biol Chem, 1992 Sep 15, 267(26), 18612 - 22
Polar arrest of the simian virus 40 tumor antigen-mediated replication fork movement in vitro by the tus protein-terB complex of Escherichia coli; Amin AA et al.; The effect of the tus protein-terB sequence complex of Escherichia coli on the movement of the SV40 large tumor antigen (T antigen)-mediated replication fork during SV40 DNA replication in vitro has been examined . In the monopolymerase and dipolymerase systems, the tus protein-terB complex efficiently blocked the replication fork movement in a polar fashion, as observed in prokaryotic replication systems . With crude cytosolic extracts of HeLa cells, the same polarity of fork arrest was observed, but the block of replication fork movement was inefficient . These results indicate that the structure of the prokaryotic tus protein-terB complex allows it to block replication fork movement in an orientation-dependent manner . We also show that the tus protein-terB complex blocks the 3'----5' helicase action of T antigen in a polar fashion, using substrates comprised of single-stranded M13 DNA with either a 52-base pair (bp) or 29-bp duplex containing the terB sequence . The tus protein-terB complex formed on the 52-bp duplex was less effective than the complex formed on the 29-bp duplex in blocking the helicase action of T antigen . With the 52-bp duplex substrate, T antigen movement was only partially (30%) blocked by the tus protein-terB sequence complex in the active orientation, whereas the E . coli dnaB helicase moving 5'----3' was blocked more than 90% by the complex in the active orientation . However, with the shorter 29-bp duplex substrate, the complex blocked the T antigen helicase activity about 75%, whereas the dnaB helicase activity was completely blocked . Altogether, these results suggest that the T antigen helicase activity, when coupled to DNA replication, is more susceptible to arrest by the tus protein-terB complex than the T antigen functioning as a helicase alone.

Nucleic Acids Res, 1992 Sep 11, 20(17), 4631 - 8
Identification of the motifs within the tobacco mosaic virus 5'-leader responsible for enhancing translation; Gallie DR et al.; The leader (called omega) of tobacco mosaic virus RNA enhances translation in both eukaryotes and prokaryotes . Although little secondary structure is predicted to exist within omega, the primary sequence of the 68 base leader is highly organized . Three copies of an eight base direct repeat and a (CAA)n region represent the two motifs found in the leaders of many TMV strains, and together these comprise 72% of omega . In previous deletion studies, no mutants exhibited loss-of-function, suggesting that functional redundancy exists within omega . We report here that a more comprehensive deletion analysis identified the motifs involved in translational enhancement . In a separate approach, oligonucleotides containing the sequence of each motif were used to construct leaders that varied in the number and configuration of the motifs . beta-Glucuronidase mRNA constructs containing these mutant leaders were synthesized in vitro and their translational efficiency measured in vivo following mRNA delivery to carrot protoplasts via electroporation . A combination of one copy of the 8 base direct repeat and a 25 base (CAA)n region was identified as the core regulatory element, although the (CAA)n motif is more critical . Two copies of the (CAA)n region are sufficient to confer a high level of enhancement and a leader composed of multiple copies of the direct repeat is moderately enhancing . Thus, these two motifs are functionally redundant.

Gene, 1992 Sep 10, 118(2), 217 - 22
Genomic organization and tissue expression of the murine gene encoding the protein beta-aspartate methyltransferase; Romanik EA et al.; Two overlapping clones containing the entire 684-nucleotide (nt) sequence encoding murine protein beta-aspartate methyltransferase (EC 2.1.1.77) were isolated from a genomic library . Partial nt sequence analysis of the two clones revealed that the protein carboxyl methyltransferase (PCMT)-encoding sequence is distributed among seven exons, ranging from 32 to 339 bp in length, within 25 kb of genomic DNA . Three exons correspond to regions of primary structure which are strongly conserved among a number of eukaryotic and prokaryotic enzymes which utilize S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) . The 5'-flanking region of the PCMT-encoding gene (PCMT) contains an 800-bp G+C-rich region with potential binding sites for transcription factor ETF, but lacks a TATA box and binding sites for other known transcription factors . Multiple PCMT mRNAs were detected on Northern blots of RNA extracted from murine brain, testis, liver and kidney . The overall abundance of PCMT mRNAs in each tissue paralleled the measured specific activity of the PCMT . Comparison of the genomic sequence information with the 3'-untranslated regions (UTRs) of two cDNA clones from a murine testis library indicated that PCMT mRNA precursors undergo alternative splicing . The structure and widespread expression of PCMT are characteristics of vertebrate housekeeping genes.

Biochemistry, 1992 Sep 8, 31(35), 8369 - 76
Parameters affecting the elongation block by RNA polymerase II at the SV40 attenuator 1 in vitro; Goldring NB et al.; We have previously suggested that folding of the SV40 attenuator RNA 1 into two hairpin elements leads to a block of transcription elongation . Using site-directed mutagenesis, we created three templates that either strengthened or weakened the proposed hairpin structures . The mutated and wild-type templates were cloned downstream from the adenovirus 2 MLP, and transcription patterns were compared among the templates, in cell-free extracts . In this report, we show that at the standard temperature (30 degrees C) the elongation block occurs at elevated KCl concentrations (0.5-1.2 M KCl) while at high temperature (65 degrees C), although the transcription elongation rate increases, the block to elongation occurs at lower KCl concentrations (less than 0.5 M KCl) as well . Since under the conditions tested the extent of the elongation block is directly dependent on the stability of the hairpin structure of the RNA, as assessed by a comparison of transcription of the wild-type and mutated templates, we suggest that elevated KCl concentrations and high temperatures are favored conditions for optimizing the processes whereby the hairpin structures are recognized by the polymerase as an attenuation signal . Moreover, the present experiments indicate that cellular elongation factors are not directly involved in modulating the extent of the elongation block at the SV40 attenuator 1 in vitro . The SV40 attenuator 1 is the only eukaryotic system known to date which has been shown to have structural characteristics so similar to rho-independent terminator in prokaryotes . We discuss the similarities between the mechanisms which lead to elongation blocks at these eukaryotic and prokaryotic sites.

Biochemistry, 1992 Sep 8, 31(35), 8171 - 9
Sequential NMR resonance assignment and secondary structure of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenborough; Marion D et al.; Two-dimensional nuclear magnetic resonance spectroscopy was used to assign the proton resonances of ferrocytochrome c553 from Desulfovibrio vulgaris Hildenbourough at 37 degrees C and pH = 5.9 . Only a few side-chain protons were not identified because of degeneracy or overlap . The spin systems of the 79 amino acids were identified by DQF-COSY and HOHAHA spectra in H2O and D2O . Sequential assignments were obtained from NOESY connectivities between adjacent amide, C alpha H, and C beta H protons . From sequential NH(i)----NH(i + 1) and long-range C alpha H(i)----NH(i + 3) connectivities, four stretches of helices were identified (2----8, 34----46, 53----59, 67----77) . Long-range NOE between residues in three different helices provide qualitative information on the tertiary structure, in agreement with the general folding pattern of cytochrome c . The heme protons, including the propionate groups, were assigned, and the identification of Met 57 as sixth heme ligand was established . The dynamical behavior of the ring protons of the six tyrosines was analyzed in detail in terms of steric hindrance . The NMR data for ferrocytochrome c553 are consistent with the X-ray structure for the homologous cytochrome from D . vulgaris Miyazaki . On the basis of the secondary structure element and of observed chemical shift due to the heme ring current, a structural alignment of eukaryotic and prokaryotic cytochromes c is proposed.

J Protozool, 1992 Sep-Oct, 39(5), 642 - 4
Codon usage in Giardia lamblia; Char S et al.; A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons . Codon usage revealed a biased use of synonymous codons with a preference for NNC codons (42.1%) . The codon usage of G . lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e . Trypanosoma brucei (r = 0.434) and Plasmodium falciparum (r = -0.31) . These observations are consistent with the view that G . lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.

Clin Infect Dis, 1992 Sep, 15(3), 434 - 8
Fatal septicemia due to Mycoplasma arginini: a new human zoonosis; Yechouron A et al.; A 64-year-old slaughterhouse worker with advanced non-Hodgkin's lymphoma developed septicemia and pneumonia . Mycoplasma arginini, a wall-free prokaryote found in a variety of domestic animal hosts, was repeatedly isolated from blood and bronchial washings from the patient . Immunosuppression, in part caused by hypogammaglobulinemia, probably played a key role in predisposing the patient to a fatal infection . This case suggests that animal mycoplasmas should be considered in the list of infectious agents acquired by immunosuppressed hosts.

Proc Natl Acad Sci U S A, 1992 Sep 1, 89(17), 8135 - 9
Photosynthetic reaction center genes in green sulfur bacteria and in photosystem 1 are related; Buttner M et al.; Oxygenic photosynthesis of chloroplasts and cyanobacteria involves two photosystems, which originate from different prokaryotic ancestors . The reaction center of photo-system 2 (PS2) is related to the well-characterized reaction center of purple bacteria, while the reaction center of photosystem 1 (PS1) is related to the green sulfur bacteria, as is convincingly documented here . An operon encoding the P840 reaction center of Chlorobium limicola f.sp . thiosulfatophilum has been cloned and sequenced . It contains two structural genes, coding for proteins of 730 and 232 amino acids . The first protein resembles the large subunits of the PS1 reaction center . Putative binding elements for the primary donor, P840 in Chlorobium and P700 in PS1, and for the acceptors A0, A1, and FeS center X are conserved . The second protein is related to the PS1 subunit carrying the FeS centers A and B . An adjacent third gene, not belonging to the reaction center, encodes a protein related to dolichyl-phosphate-D-mannose synthase from yeast . The different origins of PS1 and PS2 are discussed.

J Mol Evol, 1992 Sep, 35(3), 217 - 22
Evidence that mammalian glutamine-dependent carbamyl phosphate synthetase arose through gene fusion; Kern CB et al.; On the basis of homology, the mammalian CAD (glutamine-dependent carbamyl phosphate synthetase-aspartate transcarbamylase-dihydroorotase) gene appears to have arisen from the fusion of four separate ancestral genes . Evidence for two of these precursor genes is found in the carbamyl phosphate synthetase (CPSase) domain of CAD . In prokaryotes, such as Escherichia coli CPSase is encoded by two distinct cistrons of the carAB operon . Whereas carA and carB are separated by a short noncoding intercistronic region, the homologous sequences of the CAD gene encode an amino acid bridge . This bridge connects the subdomains of the CAD CPSase . We constructed a bacterial carAB fusion gene in which the intercistronic region codes for a hamster bridgelike sequence . The fused carAB gene directs the synthesis of a stable bifunctional polypeptide whose glutamine-dependent CPSase activity is comparable to the E . coli CPSase holoenzyme . The fusion in E . coli of the single gene counterparts of CAD demonstrates a potential model system to study the genetic events that lead to gene fusion and the creation of multienzymatic proteins.

Gene, 1992 Sep 1, 118(1), 87 - 91
Development of a prokaryotic expression vector that exploits dicistronic gene organization; Ito W et al.; For unknown reasons, levels of expression of foreign genes inserted into expression vectors in Escherichia coli have frequently been undetectable . The most critical step in the successful production of foreign proteins seems to be the initiation of translation . Since most prokaryotic genes are transcribed in a polycistronic form, we have devised a new prokaryotic expression system utilizing dicistronic gene organization . Downstream from a strong promoter and the gene encoding glutathione S-transferase from Schistosoma japonicum, various foreign genes were connected via a ribosome-binding site, a stop codon and a start codon . The VH domain of an immunoglobulin fused to the alpha subunit of tryptophan synthase, FK506-binding protein, cyclophilin, and a domain of a major histocompatibility complex antigen were successfully produced in E . coli as discrete polypeptides by this method.

Mol Cell Biol, 1992 Sep, 12(9), 4038 - 45
Establishment of a system for conditional gene expression using an inducible tRNA suppressor gene; Dingermann T et al.; We investigated the use of the prokaryotic tetracycline operator-repressor system as a regulatory device to control the expression of Dictyostelium discoideum tRNA genes . The tetO1 operator fragment was inserted at three different positions in front of a tRNA(Glu) (Am) suppressor gene from D . discoideum, and the tetracycline repressor gene was expressed under the control of a constitutive actin 6 promoter . The effectiveness of this approach was determined by monitoring the expression of a beta-galactosidase gene engineered to contain a stop codon that could be suppressed by the tRNA . When these constructs were introduced into Dictyostelium cells, the repressor bound to the operator in front of the tRNA gene and prevented expression of the suppressor tRNA . Addition of tetracycline (30 micrograms/ml) to the growth medium prevented repressor binding, allowed expression of the suppressor tRNA, and resulted in beta-galactosidase synthesis . The operator-repressor complex interfered with tRNA gene transcription when the operator was inserted immediately upstream (position +1 or -7) of the mature tRNA coding region . Expression of a tRNA gene carrying the operator at position -46 did not respond to repressor binding . This system could be used to control the synthesis of any protein, provided the gene contained a translational stop signal.

Biologicals, 1992 Sep, 20(3), 197 - 202
The use of bovine fibrin-streptokinase films for the determination of recombinant human plasminogen; Dodd I et al.; Plasminogen is a key component of the haemostatic system in man and the plasma-derived protein molecule has been actively investigated . Within the last few years cDNA and the gene encoding plasminogen have been cloned and the protein has been expressed in a number of eukaryotic or prokaryotic systems . Yields of expressed plasminogen are frequently low . Currently available assays for plasminogen generally rely on the determination of antigen or utilize tripeptide substrates for measuring functional activity, and they have certain limitations . Assays employing relevant protein substrates offer an alternative way to measure function and overcome the drawbacks associated with the other tests . The use of fibrin films for the assay of low levels of recombinant plasminogen has not been described fully before . The two fibrin film-based assays described in this paper are significant additions to the array of assays available for plasminogen molecules.

Clin Investig, 1992 Sep, 70(9), 773 - 9
Progress towards a molecular understanding of cyclosporin A-mediated immunosuppression; Schumacher A et al.; Immunosuppressive drugs like cyclosporin A (CsA), FK506 and rapamycin exert their immunosuppressive potential primarily by interfering with the activation and proliferation of T cells . These substances bind to intracellular receptor proteins, called immunophilins . Immunophilins are ubiquitous and abundant proteins, found in all prokaryotic and eukaryotic cells investigated, as well as in many subcellular compartments . Immunophilins display an inherent enzymatic activity, the prolyl-peptidyl cis-trans isomerase (PPIase) . The eukaryotic PPIases are inhibited upon the binding of immunosuppressants . In addition, the complex of immunophilins and CsA or FK506 acquires a new activity, namely the binding and inhibition of the cytoplasmic Ca(2+)-binding phosphatase calcineurin . This inhibition is postulated to prevent the proper assembly and nuclear positioning of the transcription factor NF-AT (nuclear factor of activated T cells) . The correct DNA binding of NF-AT to regulatory elements of the interleukin 2 (IL-2) promoter normally contributes to the transcriptional activation of this gene . Thus, immunosuppressive drugs prevent T-cell activation by interfering with Ca(2+)-dependent signal transduction pathways which regulate gene activities.

Appl Environ Microbiol, 1992 Sep, 58(9), 3007 - 11
Identification of individual prokaryotic cells by using enzyme-labeled, rRNA-targeted oligonucleotide probes; Amann RI et al.; A method to microscopically detect and identify individual cells of members of the domains Bacteria and Archaea is presented . rRNA-targeted oligonucleotides were 5' end labeled with the enzyme horseradish peroxidase and used for whole-cell hybridization . Specifically bound probe was visualized by the enzymatic formation of an intracellular precipitate from the substrate diaminobenzidine . Permeation of the enzyme-labeled probe into whole fixed cells of gram-negative bacteria required their pretreatment with lysozyme-EDTA, whereas permeability of some archaebacterial cells was improved by addition of detergent to the hybridization buffer . Hitherto we had not achieved penetration of enzyme-labeled probe into gram-positive bacteria and yeast cells . This method should be a valuable tool for identification of suitable prokaryotic cells in environments with elevated background fluorescence or in situations in which an epifluorescence microscope is not available.

Vet Immunol Immunopathol, 1992 Sep, 33(4), 285 - 300
The potential of molecular biology for the production of monoclonal antibodies derived from outbred veterinary animals; Suter M; The protein structure of immunoglobulins and the genetics on the regulation of immunoglobulin expression are reviewed . This basic knowledge has led to the development of systems to produce monoclonal antibodies in eukaryotic or prokaryotic cells . The potential and limitations of molecular biology for the understanding of immunoglobulin regulation and for the production of monoclonal antibodies derived from animals of veterinary importance are discussed.

FEMS Microbiol Immunol, 1992 Sep, 5(1-3), 45 - 53
The HlyB/HlyD-dependent secretion of toxins by gram-negative bacteria; Koronakis V et al.; Hemolysin (HlyA) and related toxins are secreted across both the cytoplasmic and outer membranes of Escherichia coli and other pathogenic Gram-negative bacteria in a remarkable process which proceeds without a periplasmic intermediate . It is directed by an uncleaved C-terminal targetting signal and the HlyD and HlyB translocator proteins, the latter of which are members of a transporter superfamily central to import and export of a wide range of substrates by prokaryotic and eukaryotic cells . Our mutational analyses of the HlyA targetting signal and definition for the first time of stages and intermediates in the HlyB/HlyD-dependent translocation allow a discussion of the hemolysin export process in the wider context of protein translocation.

Mol Microbiol, 1992 Sep, 6(17), 2539 - 48
Chlamydia trachomatis Mip-like protein; Lundemose AG et al.; A 27 kDa Chlamydia trachomatis Mip-like protein with homology of a 175-amino-acid C-terminal fragment to the surface-exposed Legionella pneumophila mip-gene product has previously been described . In this paper the entire chlamydia Mip-like sequence of C . trachomatis serovar L2 (lymphogranuloma venereum (LGV) biovar) is presented . The sequence shows high similarity to the legionella Mip protein and its C-terminal region, like that of the legionella Mip, has high amino acid similarity to eukaryotic and prokaryotic FK506-binding proteins . The chlamydial mip-like gene was detected by polymerase chain reaction (PCR) in other C . trachomatis serovars and by sequencing of the mip-like genes of serovars B and E (trachoma biovar) was shown to be highly conserved within the two major biovars of C . trachomatis . Monoclonal and polyclonal antibodies raised against the recombinant Mip-like protein failed to demonstrate surface-exposed epitopes on infectious elementary bodies or reproductive reticulate body forms either by immunofluorescence or immuno-gold electron microscopy . However, a complement-dependent inhibition of up to 91% of infectivity for cell cultures was observed with antibodies to the N-terminal fragment of the Mip-like protein suggesting that antibody-accessible epitopes are present on infectious EBs.

J Gen Virol, 1992 Sep, 73 ( Pt 9), 2375 - 83
Glycoprotein gp116 of human cytomegalovirus contains epitopes for strain-common and strain-specific antibodies; Meyer H et al.; Glycoprotein gp116 of human cytomegalovirus (HCMV) is a target for neutralizing antibodies . Gp116 is a component of the gCI complex which consists of gp58 and gp116 . Like its homologue, glycoprotein B of herpes simplex virus type 1, gp116 contains a highly antigenic region in the N-terminal part of the molecule, between amino acids 28 and 84 . Prokaryotic expression plasmids and synthetic peptides were used to define binding sites for mouse and human monoclonal antibodies (MAbs) as well as HCMV convalescent sera . Site I, located between amino acids 68 and 77, contains an epitope recognized by the human MAb C23, which is capable of neutralizing HCMV independently of complement and the site is conserved between HCMV strains . Of HCMV-positive human sera, 53% recognized site I . Site II was mapped using mouse MAbs as well as human sera . It is located between residues 50 and 54, an area which is not conserved between strains AD169 and Towne, the two laboratory strains of known sequence . Strain-specific antibodies were detected in 25% of human sera . Site II-specific antibodies, purified from human sera by affinity chromatography, were found to be incapable of neutralizing HCMV in tissue culture.

Trends Genet, 1992 Sep, 8(9), 317 - 22
Secretion across the bacterial outer membrane; Wandersman C; Many bacteria secrete extracellular proteins such as hydrolytic enzymes or toxins . In Gram-negative bacteria, secreted proteins must cross the two membranes that constitute the cell envelope . Recent studies have identified several specific secretion systems that can be classified in three distinct pathways, and related systems have been discovered in a wide range of prokaryotic and eukaryotic cells.

Mol Biochem Parasitol, 1992 Sep, 54(2), 201 - 11
Primary sequences of two P-glycoprotein genes of Entamoeba histolytica; Descoteaux S et al.; Two P-glycoprotein genes (EhPgp1 and EhPgp2) from the protozoan parasite Entamoeba histolytica were sequenced from a genomic library made with the DNA of an emetine-resistant ameba mutant, which overexpresses mRNAs homologous to segments of the human mdr1 (P-glycoprotein) gene . The open reading frames for EhPgp1 and EhPgp2 were 1302 and 1310 amino acids long, respectively, and showed a 67% positional identity with each other and 41% and 40% positional identities, respectively, with human mdr1 gene . Within each ameba P-glycoprotein were the ATP-binding sites found twice in eukaryotic P-glycoproteins and once in prokaryotic transport proteins . Hydropathy plots of the ameba P-glycoproteins were nearly superimposable on that of the human mdr 1, showing 2 homologous halves, each containing an ATP-binding site and 6 hydrophobic transmembrane domains that form the putative channel . A phylogenetic tree showed that the Entamoeba P-glycoproteins are more related to the human and mouse P-glycoproteins than to the Plasmodium and Leishmania P-glycoproteins . Also identified in the E . histolytica genomic library were 2 P-glycoprotein pseudogenes, each with a frame shift and stop codons in identical places within the amino ATP-binding site . In conclusion, the 2 E . histolytica P-glycoproteins encoded by the EhPgp1 and EhPgp2 genes are similar in structure to the mammalian P-glycoproteins and so may be involved in energy-dependent drug efflux by this human parasite.

Chem Phys Lipids, 1992 Sep, 62(2), 93 - 103
Rational design and synthesis of phospholipids for the two-dimensional crystallization of DNA gyrase, a key element in chromosome organization; Lebeau L et al.; Properties required of lipids for two-dimensional crystallization of proteins on lipid layers at the air/water interface are discussed in terms of molecular structure . These properties are related to essential features of the overall system such as (i) the fluidity and stability of the lipid film, (ii) the affinity of the protein to be crystallized for the lipids and (iii) the accessibility of the protein to the ligand in the lipid layer as well as (iv) technical constraints of the crystallization technique . The resulting ideas were tested through the rational design and synthesis of original phospholipid structures linked to novobiocin subsequently used in the production of two-dimensional crystals of DNA gyrase (B subunit), a prokaryotic type II DNA topoisomerase.

Am J Respir Cell Mol Biol, 1992 Sep, 7(3), 349 - 56
Gene expression from adeno-associated virus vectors in airway epithelial cells; Flotte TR et al.; Lung diseases such as cystic fibrosis (CF) might be treated by gene therapy using viral vectors delivered to the airway . One potential vector is the defective human parvovirus, adeno-associated virus (AAV) . We examined the AAV p5 transcription promoter for gene expression in immortalized cell lines derived from the airway (IB3-1) or pancreas (CFPAC-1) of CF patients . AAV vectors expressing the prokaryotic genes cat (pAAVp5cat) or neo (pAAVp5neo) from the p5 promoter were evaluated after introduction into IB3-1 or CFPAC-1 cells by lipofection . In transient assays in both cell lines, the cat gene was expressed 5- to 10-fold more efficiently from the p5 promoter than from a simian virus 40 early gene promoter (pSVcat) . IB3-1 cells were transformed stably to geneticin resistance by pAAVp5neo at a 5-fold higher efficiency than by an SVneo vector . The AAV inverted terminal repeat (ITR) region immediately upstream of the p5 promoter appears to have an enhancer effect and the promoter also contains a CREB site which confers a response to forskolin . In IB3-1 cells, expression of the cat gene from a p5 promoter was decreased about 5-fold by deletion of both the upstream ITR and the CREB site . The AAVp5neo vector was also packaged into AAV particles and used to infect IB3-1 cells as a transducing virus . Under these conditions, 60 to 70% of the cells could be stably transformed to geneticin resistance . Thus, AAV transducing vectors appear to be a highly efficient delivery system for stable integration and expression of genes in cultured airway epithelial cells.

Cell, 1992 Aug 7, 70(3), 501 - 12
Polypeptides containing highly conserved regions of transcription initiation factor sigma 70 exhibit specificity of binding to promoter DNA; Dombroski AJ et al.; The sigma 70 subunit of E . coli RNA polymerase is required for sequence-specific recognition of promoter DNA . Genetic studies and sequence analysis have indicated that sigma 70 contains two specific DNA-binding domains that recognize the two conserved portions of the prokaryotic promoter . However, intact sigma 70 does not bind to DNA . Using C-terminal and internal polypeptides of sigma 70, carrying one or both putative DNA-binding domains, we demonstrate that sigma 70 does contain two DNA-binding domains, but that N-terminal sequences inhibit the ability of intact sigma 70 to bind to DNA . Thus, we propose that sigma 70 is a sequence-specific DNA-binding protein that normally functions through an allosteric interaction with the core subunits of RNA polymerase.

Biochem J, 1992 Aug 15, 286 ( Pt 1), 117 - 23
Nucleotide sequence and expression of a cDNA encoding rabbit liver cytosolic serine hydroxymethyltransferase; Byrne PC et al.; A rabbit liver cDNA library in phage lambda gt10 was screened using a portion of the coding sequences for rabbit cytosolic serine hydroxymethyltransferase (amino acids 244-420) that had been amplified by PCR, with total rabbit liver RNA as a template . A clone of 2.3 kb (pUS1203) was isolated and the nucleotide sequence showed that it contained an open reading frame of 1452 bp, which coded for serine hydroxymethyltransferase and was flanked by 155 bp at the 5' end and 653 bp at the 3' end . The full-length cDNA was cloned into an expression vector and transfected into COS-1 cells . Serine hydroxymethyltransferase activity was increased by 33% in the transfected cells and a new protein band of the appropriate size was seen by SDS/PAGE analysis of proteins extracted from transfected cells . The protein sequence for rabbit cytosolic serine hydroxymethyltransferase derived from the cDNA nucleotide sequence was compared with three other derived or known prokaryotic and eukaryotic sequences . An overall sequence similarity of 34% was noted between all four sequences, whereas the similarity between the rabbit cytosolic and mitochondrial isoforms was 62%.

Proc Natl Acad Sci U S A, 1992 Aug 15, 89(16), 7290 - 4
An ATPase domain common to prokaryotic cell cycle proteins, sugar kinases, actin, and hsp70 heat shock proteins; Bork P et al.; The functionally diverse actin, hexokinase, and hsp70 protein families have in common an ATPase domain of known three-dimensional structure . Optimal superposition of the three structures and alignment of many sequences in each of the three families has revealed a set of common conserved residues, distributed in five sequence motifs, which are involved in ATP binding and in a putative interdomain hinge . From the multiple sequence alignment in these motifs a pattern of amino acid properties required at each position is defined . The discriminatory power of the pattern is in part due to the use of several known three-dimensional structures and many sequences and in part to the "property" method of generalizing from observed amino acid frequencies to amino acid fitness at each sequence position . A sequence data base search with the pattern significantly matches sugar kinases, such as fuco-, glucono-, xylulo-, ribulo-, and glycerokinase, as well as the prokaryotic cell cycle proteins MreB, FtsA, and StbA . These are predicted to have subdomains with the same tertiary structure as the ATPase subdomains Ia and IIa of hexokinase, actin, and Hsc70, a very similar ATP binding pocket, and the capacity for interdomain hinge motion accompanying functional state changes . A common evolutionary origin for all of the proteins in this class is proposed.

J Photochem Photobiol B, 1992 Aug 14, 15(1-2), 113 - 40
Structure, function and organization of antenna polypeptides and antenna complexes from the three families of Rhodospirillaneae; Brunisholz RA et al.; Comparative primary structural analysis of polypeptides from antenna complexes from species of the three families of Rhodospirillaneae indicates the structural principles responsible for the formation of spectrally distinct light-harvesting complexes . In many of the characterized antenna systems the basic structural minimal unit is an alpha/beta polypeptide pair . Specific clusters of amino acid residues, in particular aromatic residues in the C-terminal domain, identify the antenna polypeptides to specific types of antenna systems, such as B880 (strong circular dichroism (CD)), B870 (weak CD), B800-850 (high), B800-850 (low) or B800-820 . The core complex B880 (B1020) of species from Ectothiorhodospiraceae and Chromatiaceae apparently consists of four (alpha 1 alpha 2 beta 1 beta 2) or three (2 alpha beta 1 beta 2) chemically dissimilar antenna polypeptides respectively . There is good evidence that the so-called variable antenna complexes, such as the B800-850 (high), B800-850 (low) or B800-820 of Rp . acidophila, Rp . palustris and Cr . vinosum, are comprised of multiple forms of peripheral light-harvesting polypeptides . Structural similarities between prokaryotic and eukaryotic antenna polypeptides are discussed in terms of similar pigment organization . The structural basis for the strict organization of pigment molecules (bacteriochlorophyll (BChl) cluster) in the antenna system of purple bacteria is the hierarchical organization of the alpha- and beta-antenna polypeptides within and between the antenna complexes . On the basis of the three-domain structure of the antenna polypeptides with the central hydrophobic domain, forming a transmembrane alpha helix, possible arrangements of the antenna polypeptides in the three-dimensional structure of core and peripheral antenna complexes are discussed . Important structural and functional features of these polypeptides and therefore of the BChl cluster are the alpha/beta heterodimers, the alpha 2 beta 2 basic units and cyclic arrangements of these basic units . Equally important for the formation of the antenna complexes or the entire antenna are polypeptide-polypeptide, pigment-pigment and pigment-polypeptide interactions.

J Biol Chem, 1992 Aug 5, 267(22), 15687 - 91
Histamine N-methyltransferase from rat kidney . Cloning, nucleotide sequence, and expression in Escherichia coli cells; Takemura M et al.; Complementary DNA clones encoding rat kidney histamine N-methyltransferase have been isolated using synthetic oligonucleotide probes based on partial amino acid sequences of tryptic peptides of the purified enzyme . The 1.3-kilobase cDNA consisted of a 5'-noncoding region of 8 nucleotides, a coding region of 885 nucleotides, and a 3'-noncoding region of 369 nucleotides . The encoded protein of 295 amino acid residues had a calculated molecular weight of 33,940.2 . After introduction of a prokaryotic expression vector containing the isolated cDNA, Escherichia coli cells expressed histamine N-methyltransferase activity . The enzyme expressed in these cells was isolated and purified as a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, whose mobility was identical to the natural enzyme purified from rat kidney . The recombinant enzyme had Vmax and Km values for both histamine and S-adenosylmethionine identical to those of the natural enzyme . All of the inhibitors of the natural enzyme tested showed similar Ki values on both recombinant and natural enzyme.

J Mol Biol, 1992 Aug 5, 226(3), 867 - 82
Crystal structure of Escherichia coli malate dehydrogenase . A complex of the apoenzyme and citrate at 1.87 A resolution; Hall MD et al.; The crystal structure of malate dehydrogenase from Escherichia coli has been determined with a resulting R-factor of 0.187 for X-ray data from 8.0 to 1.87 A . Molecular replacement, using the partially refined structure of porcine mitochondrial malate dehydrogenase as a probe, provided initial phases . The structure of this prokaryotic enzyme is closely homologous with the mitochondrial enzyme but somewhat less similar to cytosolic malate dehydrogenase from eukaryotes . However, all three enzymes are dimeric and form the subunit-subunit interface through similar surface regions . A citrate ion, found in the active site, helps define the residues involved in substrate binding and catalysis . Two arginine residues, R81 and R153, interacting with the citrate are believed to confer substrate specificity . The hydroxyl of the citrate is hydrogen-bonded to a histidine, H177, and similar interactions could be assigned to a bound malate or oxaloacetate . Histidine 177 is also hydrogen-bonded to an aspartate, D150, to form a classic His.Asp pair . Studies of the active site cavity indicate that the bound citrate would occupy part of the site needed for the coenzyme . In a model building study, the cofactor, NAD, was placed into the coenzyme site which exists when the citrate was converted to malate and crystallographic water molecules removed . This hypothetical model of a ternary complex was energy minimized for comparison with the structure of the binary complex of porcine cytosolic malate dehydrogenase . Many residues involved in cofactor binding in the minimized E . coli malate dehydrogenase structure are homologous to coenzyme binding residues in cytosolic malate dehydrogenase . In the energy minimized structure of the ternary complex, the C-4 atom of NAD is in van der Waals' contact with the C-3 atom of the malate . A catalytic cycle involves hydride transfer between these two atoms.

Biochemistry, 1992 Aug 4, 31(30), 6917 - 24
Modified, large-scale purification of the cytochrome o complex (bo-type oxidase) of Escherichia coli yields a two heme/one copper terminal oxidase with high specific activity; Minghetti KC et al.; The cytochrome o complex is a bo-type ubiquinol oxidase in the aerobic respiratory chain of Escherichia coli . This complex has a close structural and functional relationship with the eukaryotic and prokaryotic aa3-type cytochrome c oxidases . The specific activity, subunit composition, and metal content of the purified cytochrome o complex are not consistent for different preparative protocols reported in the literature . This paper presents a relatively simple preparation of the enzyme starting with a strain of Escherichia coli which overproduces the oxidase . The pure enzyme contains four subunits by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) . Partial amino acid sequence data confirm the identities of subunit I, II, and III from the SDS-PAGE analysis as the cyoB, cyoA, and cyoC gene products, respectively . A slight modification of the purification protocol yields an oxidase preparation that contains a possible fifth subunit which may be the cyoE gene product . The pure four-subunit enzyme contains 2 equivs of iron but only 1 equiv of copper . There is no electron paramagnetic resonance detectable copper in the purified enzyme . Hence, the equivalent of CuA of the aa3-type cytochrome c oxidases is absent in this quinol oxidase . There is also no zinc in the purified quinol oxidase . Finally, monoclonal antibodies are reported that interact with subunit II . One of these monoclonals inhibits the quinol oxidase activity of the detergent-solubilized, purified oxidase . Hence, although subunit II does not contain CuA and does not interact with cytochrome c, it still must have an important function in the bo-type ubiquinol oxidase.

Infect Immun, 1992 Aug, 60(8), 3079 - 86
Presence of parasite antigen on the surface of P388D1 cells infected with Ehrlichia risticii; Messick JB et al.; Indirect immunofluorescence staining of macrophages infected with Ehrlichia risticii by anti-E . risticii serum revealed a punctate staining pattern on the surface of the host cell . This pattern was distinguishable by fluorescence microscopy from E . risticii bound to the surface of the macrophage and from intracellular E . risticii . The surface localization of ehrlichial antigen on infected macrophages was confirmed by electron microscopy with immunoferritin labeling . As the intracellular ehrlichial burden increased, the amount of ehrlichial antigen on the host cell surface increased . Prokaryotic protein synthesis was necessary for the maintenance of ehrlichial antigen on the host cell surface, as demonstrated by disappearance of the surface antigen following treatment with oxytetracycline . However, host cell protein synthesis was not required, as demonstrated by the continued presence of ehrlichial antigen on the surface of host cells after cycloheximide treatment . Pronase treatment abolished the ehrlichial antigen present on the cell surface, indicating that this antigen is a protein . Anti-E . risticii serum or immunoglobulin G-mediated antibody-dependent cellular cytotoxicity of infected cells was demonstrated in a chromium release assay . These results imply that the parasite antigen on the host cell surface has a role in the pathogenesis of ehrlichiosis.

EMBO J, 1992 Aug, 11(8), 3129 - 34
HSD restriction-modification proteins partake in latent anticodon nuclease; Amitsur M et al.; Phage T4-induced anticodon nuclease triggers cleavage-ligation of the host tRNA(Lys) . The enzyme is encoded in latent form by the optional Escherichia coli locus prr and is activated by the product of the phage stp gene . Anticodon nuclease latency is attributed to the masking of the core function prrC by flanking elements homologous with type I restriction-modification genes (prrA-hsdM and prrD-hsdR) . Activation of anticodon nuclease in extracts of uninfected prr+ cells required synthetic Stp, ATP and GTP and appeared to depend on endogenous DNA . Stp could be substituted by a small, heat-stable E . coli factor, hinting that anticodon nuclease may be mobilized in cellular situations other than T4 infection . Hsd antibodies recognized the anticodon nuclease holoenzyme but not the prrC-encoded core . Taken together, these data indicate that Hsd proteins partake in the latent ACNase complex where they mask the core factor PrrC . Presumably, this masking interaction is disrupted by Stp in conjunction with Hsd ligands . The Hsd-PrrC interaction may signify coupling and mutual enhancement of two prokaryotic restriction systems operating at the DNA and tRNA levels.

J Bacteriol, 1992 Aug, 174(15), 5072 - 8
Occurrence of novel groups of the domain Bacteria as revealed by analysis of genetic material isolated from an Australian terrestrial environment; Liesack W et al.; A molecular ecological study was performed on an Australian soil sample to unravel a substantial portion of the bacterial diversity . A large fragment of the 16S rRNA gene was amplified, using DNA isolated by lysing the microorganisms directly within the soil matrix, and a clone library was generated . Comparative sequence analysis of 30 clones and dot blot hybridization of 83 additional clones with defined oligonucleotide probes revealed the presence of three major groups of prokaryotes of the domain Bacteria . The first one comprises 57 clones that indicate relatives of nitrogen-fixing bacteria of the alpha-2 subclass of the class Proteobacteria; the second group of 7 clones originates from members of the order Planctomycetales that, however, reveal no close relationship to any of the described Planctomycetales species; 22 clones of the third group are indicative of members of a novel main line of descent, sharing a common ancestry with members of planctomycetes and chlamydiae.

Genomics, 1992 Aug, 13(4), 983 - 90
Dissecting (CAC)5/(GTG)5 multilocus fingerprints from man into individual locus-specific, hypervariable components; Zischler H et al.; Individual components of multilocus fingerprints from man produced by (CAC)5/(GTG)5 oligonucleotides have been scrutinized to characterize their peculiar properties . Successful cloning and changes occurring during the propagation of recombinant simple repetitive DNA in prokaryotic hosts are described . The isolated locus-specific probes were characterized with respect to their formal (and population genetic) properties and their usefulness for individualization and linkage studies . The localization was determined on chromosomes 8, 9, 11, and 22 . Repeat flanking sequences were characterized and analyzed for their coding potential because of significant open reading frames and apparent evolutionary conservation among vertebrates . The organization of the repeats and their flanking regions in the human genome is discussed with respect to the sequence (fine) architecture that developed during evolution . Classical "minisatellite" sequences were not detected near hypervariable (cac)n/(gtg)n repeats . The single-copy probes described herein are a convenient complement to the oligonucleotides employed for multilocus fingerprinting . Many practical applications are apparent.

Proc Natl Acad Sci U S A, 1992 Aug 1, 89(15), 7060 - 4
Glutaredoxin homolog encoded by vaccinia virus is a virion-associated enzyme with thioltransferase and dehydroascorbate reductase activities; Ahn BY et al.; Glutaredoxins (GRXs), also known as thioltransferases, use glutathione as a cofactor for reduction of disulfides in prokaryotes and eukaryotes . We demonstrate that the vaccinia virus O2L open reading frame encodes a functional GRX, as predicted by Johnson et al . {Johnson, G . P., Goebel, S . J., Perkus, M . E., Davis, S . W., Winslow, J . P . & Paoletti, E . (1991) Virology 181, 378-381} from sequence homology . The 12-kDa protein product of the O2L open reading frame was synthesized after viral DNA replication, coincident with a major increase in cytoplasmic glutathione-dependent thioltransferase activity . The protein was associated with purified vaccinia virions and was not released by treatment with a nonionic detergent unless dithiothreitol was added . The virion-derived protein, as well as a recombinant form expressed in Escherichia coli, exhibited thioltransferase and dehydroascorbate reductase activities indicative of a functional GRX . The postreplicative synthesis of vaccinia virus GRX and its association with virions suggest that the enzyme may have novel roles in the virus growth cycle.

Eur J Cell Biol, 1992 Aug, 58(2), 319 - 30
Assembly of a tail-less mutant of the intermediate filament protein, vimentin, in vitro and in vivo; Eckelt A et al.; Recent reports on the possible contribution of the non-alpha-helical carboxy-terminal domain ("tail") of type III intermediate filament (IF) proteins to IF assembly have been controversial . To examine the importance and role of this domain, we have therefore engineered a Xenopus laevis vimentin cDNA to code for a tail-less polypeptide and have used it in combination with prokaryotic and eukaryotic expression systems . Here we show that tail-less vimentin, isolated from transfected bacteria (Escherichia coli), when used for assembly in vitro, forms normal-looking, loosely packed IFs . By viscometry we demonstrate that this tail-less vimentin assembles at an even higher rate and into longer IFs than wild-type vimentin . In vivo, i.e., by forced expression in transfected type III IF-free cultured epithelial cells, tail-less vimentin was also recovered in short fibrillar structures, in rodlets and in small as well as large spheroidal aggregates ("granules") that did not reveal any IF substructure . Surprisingly, however, spheroidal aggregate structures formed from the tail-deleted vimentin, were seen not only in the cytoplasm but also in the nucleus, indicating a role of the tail in higher order organization and compartmentalization of the vimentin IF system.

Endocr Rev, 1992 Aug, 13(3), 499 - 514
Secretion of peptides and proteins lacking hydrophobic signal sequences: the role of adenosine triphosphate-driven membrane translocators; Kuchler K et al.; In this review, we will emphasize the role of ATP-dependent membrane transporters in protein export and intracellular protein trafficking in prokaryotic and eukaryotic cells . ATP-binding-cassette (ABC)-transport proteins, also termed "traffic ATPases," belong to a superfamily of ubiquitous ATP-driven membrane transporters that share extensive sequence similarity and highly conserved domain organization . They are implicated in a remarkable variety of transmembrane transport processes, including the transport of ions, heavy metals, sugars, anticancer drugs, amino acids, oligopeptides, and proteins . Bacterial ABC-proteins include the well-characterized periplasmic permeases involved in nutrient uptake, but also include protein secretion systems, such as the exporter for the Escherichia coli enterotoxin hemolysin A . Prominent eukaryotic members of this superfamily include the human P-glycoprotein (which is associated with the phenomenon of multiple drug resistance in tumor cells), the product of the cystic fibrosis gene (CFTR), the gene (pfmdr) implicated in chloroquine resistance of the malarial parasite, putative peptide transporters encoded at the locus for the class II major histocompatibility complex (MHC), and the yeast Ste6 transporter which mediates export of a peptide hormone that lacks a classical hydrophobic signal peptide . The well-established function of prokaryotic ABC-transporters in the secretion of proteins without typical signal sequences, and the example set by the Ste6 transporter, have led to the reasonable hypothesis that certain ABC-proteins in animal cells may be operating by a similar mechanism to mediate the export of a new class of secretory proteins, those lacking a classical hydrophobic signal peptide.

Antimicrob Agents Chemother, 1992 Aug, 36(8), 1782 - 4
Increased susceptibility of transfected prokaryotic and eukaryotic cells to antibiotic selection; Asselbergs FA et al.; At 39 to 40 degrees C, selection of antibiotic-resistant transfected mammalian cell lines or Escherichia coli required lower aminoglycoside antibiotic concentrations than at 37 degrees C . The thermosensitivity of antibiotic susceptibility was much more manifest during genetic selection experiments than in conventional growth inhibition assays.

Mol Microbiol, 1992 Aug, 6(16), 2267 - 78
A metalloprotease gene from Streptomyces coelicolor 'Müller' and its transcriptional activator, a member of the LysR family; Dammann T et al.; A metalloprotease gene (mprA) and its regulatory gene (mprR) from Streptomyces coelicolor 'Muller' DSM3030 were isolated and their DNA sequences determined . The protease secreted by the heterologous host Streptomyces lividans was characterized biochemically as a metalloprotease with a M(r) of 20,000, which is in good agreement with data derived from DNA sequence analysis . The mprA gene is transcribed divergently from mprR, the deduced protein of which displays homology to prokaryotic transcriptional regulators of the LysR family . The regulatory protein (MprR) was shown to bind to the intergenic region between mprR and mprA . It was found to activate transcription of mprA in S . lividans and also in Escherichia coli.

Mol Microbiol, 1992 Aug, 6(15), 2065 - 72
Homologous catalytic domains in a rumen fungal xylanase: evidence for gene duplication and prokaryotic origin; Gilbert HJ et al.; A cDNA (xynA), encoding xylanase A (XYLA), was isolated from a cDNA library, derived from mRNA extracted from the rumen anaerobic fungus, Neocallimastix patriciarum . Recombinant XYLA, purified from Escherichia coli harbouring xynA, had a M(r) of 53,000 and hydrolysed oat-spelt xylan to xylobiose and xylose . The enzyme did not hydrolyse any cellulosic substrates . The nucleotide sequence of xynA revealed a single open reading frame of 1821 bp coding for a protein of M(r) 66,192 . The predicted primary structure of XYLA comprised an N-terminal signal peptide followed by a 225-amino-acid repeated sequence, which was separated from a tandem 40-residue C-terminal repeat by a threonine/proline linker sequence . The large N-terminal reiterated regions consisted of distinct catalytic domains which displayed similar substrate specificities to the full-length enzyme . The reiterated structure of XYLA suggests that the enzyme was derived from an ancestral gene which underwent two discrete duplications . Sequence comparison analysis revealed significant homology between XYLA and bacterial xylanases belonging to cellulase/xylanase family G . One of these homologous enzymes is derived from the rumen bacterium Ruminococcus flavefaciens . The homology observed between XYLA and a rumen prokaryote xylanase could be a consequence of the horizontal transfer of genes between rumen prokaryotes and lower eukaryotes, either when the organisms were resident in the rumen, or prior to their colonization of the ruminant . It should also be noted that Neocallimastix XYLA is the first example of a xylanase which consists of reiterated sequences . It remains to be established whether this is a common phenomenon in other rumen fungal plant cell wall hydrolases.

Am J Physiol, 1992 Aug, 263(2 Pt 1), C267 - 86
CFTR!
Fuller CM, Benos DJ.
Cystic fibrosis (CF) is a fatal genetic disease primarily affecting Caucasians, although cases have been reported from other ethnic groups . CF has a complex etiology, but it is chiefly a disease of electrolyte transport and is characterized by defects in fluid secretion by several epithelia, including the sweat duct, exocrine pancreas, and the pulmonary airways . The link between CF and a defect in cAMP-mediated Cl- transport in secretory epithelia was established in the early 1980s . Since then, numerous electrophysiological studies have focused on the characterization and regulation of individual Cl- channels underlying the macroscopic Cl- currents of secretory epithelia in the airways, sweat ducts, and gut . In this review the results of these studies in the light of current knowledge of the function of the CF gene product, the CF transmembrane conductance regulator (CFTR) protein, will be analyzed . The CFTR protein is a member of a family of ATP-binding proteins that act as unidirectional solute pumps . These proteins are membrane spanning, are found in both prokaryotic and eukaryotic cells, and have two ATP-binding domains . The family includes the p-glycoproteins that are involved with the expression of multidrug resistance in certain tumor cells . The majority of CF chromosomes (70%) have a single codon deletion that translates to a missing phenylalanine residue at position 508 (delta F508) of the protein . Unique for this family of proteins, the CFTR protein possesses an additional highly charged domain (the R domain) containing several consensus polypeptide sequences for kinase phosphorylation . Although CFTR bears structural resemblance to this family of ATP-dependent pumps, overexpression of the protein in a variety of different cell types is associated with the appearence of a cAMP-sensitive Cl- channel . We critically examine current information concerning the structure-function relationships of the CFTR protein obtained from both electrophysiological and biochemical approaches . We also summarize recent evidence suggesting that the CFTR protein may act as a pump and a channel, a hypothesis in keeping with the multifaceted nature of the disease.

Mutat Res, 1992 Aug, 280(2), 109 - 15
Genotoxic potential of crown ethers in mammalian cells: induction of sister-chromatid exchanges; Arenaz P et al.; Crown ethers are macrocyclic polyethers which possess ionophoric properties . These compounds are currently being studied for potential use as pharmaceutical agents as well as antibacterials . Though crown ethers have been shown to be highly toxic in prokaryotes and eukaryotes, there have been few investigations into the potential genotoxicity of these compounds . When sister-chromatid exchanges (SCEs) were quantitated after exposure to crown ethers, the results reflected no significant genotoxic effects on Chinese hamster V79 cells at any of the crown ether concentrations utilized . One crown ether, dicyclohexyl 21-crown-7, did appear to possess antigenotoxic activity . The data on the induction of SCEs by crown ethers reported herein suggest that these compounds are not genotoxic in mammalian cells despite their cytotoxicity.

Bioessays, 1992 Aug, 14(8), 519 - 25
E . coli hemolysin interactions with prokaryotic and eukaryotic cell membranes; Hughes C et al.; The hemolysin toxin (HlyA) is secreted across both the cytoplasmic and outer membranes of pathogenic Escherichia coli and forms membrane pores in cells of the host immune system, causing cell dysfunction and death . The processes underlying the interaction of HlyA with the bacterial and mammalian cell membranes are remarkable . Secretion of HlyA occurs without a periplasmic intermediate and is directed by an uncleaved C-terminal targetting signal and the HlyB and HlyD translocator proteins, the former being a member of a transporter superfamily central to import and export of a wide range of substrates by prokaryotic and eukaryotic cells . The separate process by which HlyA is targetted to mammalian cell membranes is dependent upon fatty acylation of a non-toxic precursor, proHlyA . This is achieved by a novel mechanism directed by the activator protein HlyC, which binds to an internal proHlyA recognition sequence and provides specificity for the transfer of fatty acid from cellular acyl carrier protein.

Mol Cell Biol, 1992 Aug, 12(8), 3449 - 59
Murine helix-loop-helix transcriptional activator proteins binding to the E-box motif of the Akv murine leukemia virus enhancer identified by cDNA cloning; Nielsen AL et al.; The enhancer region of Akv murine leukemia virus contains the sequence motif ACAGATGG . This sequence is homologous to the E-box motif originally defined as a regulatory element in the enhancers of immunoglobulin mu and kappa genes . We have used double-stranded oligonucleotide probes, corresponding to the E box of the murine leukemia virus Akv, to screen a randomly primed lambda gt11 cDNA expression library made from mouse NIH 3T3 fibroblast RNA . We have identified seven lambda clones expressing DNA-binding proteins representing two different genes termed ALF1 and ALF2 . The results of sequencing ALF2 cDNA suggests that we have recovered the gene for the basic-helix-loop-helix transcription factor A1, the murine analog of the human transcription factor E47 . The cDNA sequence of ALF1 codes for a new member of the basic-helix-loop-helix protein family . Two splice variants of ALF1 cDNA have been found, differing by a 72-bp insertion, coding for putative proteins of 682 and 706 amino acids . The two ALF1 mRNAs are expressed at various levels in mouse tissues . In vitro DNA binding assays, using prokaryotically expressed ALF1 proteins, demonstrated specific binding of the ALF1 proteins to the Akv murine leukemia virus E-box motif ACAGATGG . Expression in NIH 3T3 fibroblasts of GAL4-ALF1 chimeric protein stimulated expression from a minimal promoter linked to a GAL4 binding site, indicating the existence of a transcriptional activator domain in ALF1.

Science, 1992 Jul 31, 257(5070), 678 - 9
Specialized role for a murine class I-b MHC molecule in prokaryotic host defenses; Kurlander RJ et al.; Although nonclassical (class I-b) gene products represent the majority of murine major histocompatibility complex (MHC) genes, the role of these relatively nonpolymorphic molecules remains uncertain . Recently, one such protein, H-2M3 (formerly designated Hmt), was shown to bind and specifically present N-formylated peptides to cytotoxic T lymphocytes . Because N-formylation is characteristic of prokaryotic proteins, this MHC molecule may be especially adapted for a role in the mammalian defense against bacterial attack . The current studies demonstrate that an MHC molecule, indistinguishable from H-2M3, presents antigens derived from the intracellular pathogen Listeria monocytogenes to Listeria-specific CD8+ cells.

FEBS Lett, 1992 Jul 27, 307(1), 62 - 5
Prokaryotic polyprotein precursors; Thony-Meyer L et al.; Polyproteins have been found only recently in prokaryotes . The four known examples of single bacterial genes encoding precursors that are posttranslationally processed into two mature proteins are addressed here with respect to (i) their genomic arrangement, (ii) the sites of proteolytic processing, (iii) the relevant proteases, (iv) their maturation pathway, and (v) the function of the mature proteins . How these polyproteins may have evolved is also discussed.

FEBS Lett, 1992 Jul 27, 307(1), 81 - 6
How do eukaryotic activator proteins stimulate the rate of transcription by RNA polymerase II?
Ham J, Steger G, Yaniv M.
A large number of activator proteins have now been identified in higher and lower eukaryotes, which bind to the regulatory regions of protein-encoding genes and increase the rate at which they are transcribed by RNA polymerase II . The mechanism by which activators function is being intensively studied and some of the targets of transcriptional activation domains have now been identified . These studies have also revealed novel classes of regulatory factors, which were not anticipated by extrapolating from the principles obtained with prokaryotic promoters.

J Biol Chem, 1992 Jul 25, 267(21), 15205 - 9
Ligand binding properties of the human erythropoietin receptor extracellular domain expressed in Escherichia coli; Harris KW et al.; We developed an assay to directly measure the ligand binding properties of the cloned human erythropoietin receptor (EpoR) . The cDNA encoding the extracellular domain of the human EpoR was amplified by polymerase chain reaction and ligated into the prokaryotic expression vector pGEX3X . Synthesis in Escherichia coli was induced and a soluble glutathione S-transferase fusion protein, EREx, was purified by erythropoietin affinity chromatography . Purified EREx was bound to GSH agarose beads and used in a solid phase ligand binding assay . Specific binding of 125I-erythropoietin to EREx beads was demonstrated . A single affinity class (Kd = 1.5 nM) of the binding site was evident on Scatchard analysis . The Kd of this site is quantitatively equivalent to that of the "low" affinity cellular binding site . Kinetic analysis of ligand binding to EREx revealed both the on and off rates to be rapid, with t1/2 of 60 and 40 s, respectively . EREx ligand binding exhibits no obvious metal ion dependence or cross-competition by other hemopoietins . Antibodies to EREx block the binding of erythropoietin to the cellular EpoR . We conclude that the 66-kDa EpoR protein is capable of specific ligand binding and that no covalent modifications or associated molecules are required for this interaction . We speculate that the "high" affinity cellular binding site (Kd less than 0.2 nM) results from the interaction of the EpoR with another molecule, either additional EpoR or associated subunits, that decreases the ligand off rate.

J Mol Biol, 1992 Jul 20, 226(2), 411 - 24
Ribosomal RNA identity elements for ricin A-chain recognition and catalysis . Analysis with tetraloop mutants; Gluck A et al.; Ricin is a cytotoxic protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond between the base and the ribose of the adenosine at position 4324 in eukaryotic 28 S rRNA . Ricin A-chain will also catalyze depurination in naked prokaryotic 16 S rRNA; the adenosine is at position 1014 in a GAGA tetraloop . The rRNA identity elements for recognition by ricin A-chain and for the catalysis of cleavage were examined using synthetic GAGA tetraloop oligoribonucleotides . The RNA designated wild-type, an oligoribonucleotide (19-mer) that approximates the structure of the ricin-sensitive site in 16 S rRNA, and a number of mutants were transcribed in vitro from synthetic DNA templates with phage T7 RNA polymerase . With the wild-type tetraloop oligoribonucleotide the ricin A-chain-catalyzed reaction has a Km of 5.7 microM and a Kcat of 0.01 min-1 . The toxin alpha-sarcin, which cleaves the phosphodiester bond on the 3' side of G4325 in 28 S rRNA, does not recognize the tetraloop RNA, although alpha-sarcin does affect a larger synthetic oligoribonucleotide that has a 17-nucleotide loop with a GAGA sequence; thus, there is a clear divergence in the identity elements for the two toxins . Mutants were constructed with all of the possible transitions and transversions of each nucleotide in the GAGA tetraloop; none was recognized by ricin A-chain . Thus, there is an absolute requirement for the integrity of the GAGA sequence in the tetraloop . The helical stem of the tetraloop oligoribonucleotide can be reduced to three base-pairs, indeed, to two base-pairs if the temperature is decreased, without affecting recognition; the nature of these base-pairs does not influence recognition or catalysis by ricin A-chain . If the tetraloop is opened so as to form a GAGA-containing hexaloop, recognition by ricin A-chain is lost . This suggests that during the elongation cycle, a GAGA tetraloop either exists or is formed in the putative 17-member single-stranded region of the ricin domain in 28 S rRNA and this bears on the mechanism of protein synthesis.

Experientia, 1992 Jul 15, 48(7), 629 - 34
Mammalian heat shock protein families . Expression and functions; Burel C et al.; When prokaryotic or eukaryotic cells are submitted to a transient rise in temperature or to other proteotoxic treatments, the synthesis of a set of proteins called the heat shock proteins (hsp) is induced . The structure of these proteins has been highly conserved during evolution . The signal leading to the transcriptional activation of the corresponding genes is the accumulation of denatured and/or aggregated proteins inside the cells after stressful treatment . The expression of a subset of hsp is also induced during early embryogenesis and many differentiation processes . Two different functions have been ascribed to hsp: a molecular chaperone function: chaperones mediate the folding, assembly or translocation across the intracellular membranes of other polypeptides, and a role in protein degradation: some of the essential components of the cytoplasmic ubiquitin-dependent degradative pathway are hsp . These functions of hsp are essential in every living cell . They are required for repairing the damage resulting from stress.

J Biol Chem, 1992 Jul 15, 267(20), 14193 - 203
Cytochrome P-450terp . Isolation and purification of the protein and cloning and sequencing of its operon; Peterson JA et al.; Cytochromes P-450 are extremely important in the oxidative metabolism of a variety of endogenous and exogenous compounds in pro- and eukaryotic organisms . Progress in understanding the structure and mechanism of action of this superfamily of enzymes has been hampered by the properties of the eukaryotic enzymes and the availability of only one well-characterized prokaryotic enzyme as a model . We report here the isolation of a Pseudomonas species which will utilize a monoterpene natural product, alpha-terpineol, as its sole source of carbon and energy . Approximately 1% of the soluble protein in the cell-free extract is a novel cytochrome P-450 (P-450terp) . This enzyme and its associated iron sulfur protein electron carrier (terpredoxin) have been purified to homogeneity and their NH2-terminal amino acid sequences determined . The amino acid sequences of six tryptic peptide fragments of cytochrome P-450terp have also been determined . This sequence information was used to clone the gene encoding cytochrome P-450terp . Three clones representing approximately 8 kilobase pairs of unique sequences were selected and sequenced . Five non-overlapping open reading frames (ORFs) were found in the sequences, and the translated sequences were used to search the Protein Identification Resource for comparable proteins . The ORFs were identified as: 1) an alcohol dehydrogenase, 2) an aldehyde dehydrogenase, 3) cytochrome P-450terp, 4) terpredoxin reductase, and 5) terpredoxin . The identification of both the cytochrome P-450terp and terpredoxin DNA sequence was confirmed by the presence of each of the corresponding amino acid sequences found in the purified proteins . The five ORFs were bounded on both the 5' and 3' ends by consensus factor-independent terminator sequences . A consensus promoter sequence was found immediately 5' to the first ORF . These results indicate that we have sequenced the complete terp operon . Comparison of the amino acid sequence of cytochrome P-450terp to that of all other cytochromes P-450 has shown that it is the first member of the gene family CYP108 . Preliminary characterization of the chemical and physical properties and the preparation of crystals of this new cytochrome P-450, suitable for x-ray diffraction analysis, indicate that it will be useful in comparison studies with other members of this class of proteins.

J Biol Chem, 1992 Jul 15, 267(20), 14266 - 9
Requirement of hydrophilic amino-terminal residues for granulocyte-macrophage colony-stimulating factor bioactivity and receptor binding; Meropol NJ et al.; Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a glycoprotein required for the proliferation and differentiation of granulocyte and macrophage precursors . Previous investigations have identified regions in human and murine GM-CSF that are required for bioactivity . In the present study, alanine substitution mutagenesis was undertaken to define more precisely specific amino-terminal residues in murine GM-CSF that are involved in bioactivity and receptor binding . Five double alanine mutants were identified that showed at least 10-fold reductions in bioactivity (K14AK20A, K14AE21A, H15AK20A, H15AE21A, K20AE21A) . Each of these mutants maintained a normal N-linked glycosylation pattern when expressed in COS-1 cells, suggesting that native polypeptide backbone conformation was preserved . The purified prokaryotic expression products of two mutants (K14AE21A and H15AE21A) had a 100-fold decrease in bioactivity and a decrease in receptor binding, indicating that the side chains of K14, H15, and E21 are required for optimal receptor binding and maximal bioactivity.

Toxicol Appl Pharmacol, 1992 Jul, 115(1), 137 - 45
The genetic toxicology of cobalt; Beyersmann D et al.; Genetic and related effects of cobalt compounds are reviewed and discussed with respect to mechanisms . In prokaryotic assays, Co(II) salts generally are nonmutagenic . In Saccharomyces cerevisiae, CoCl2 is mutagenic to mitochondrial genes and weakly mutagenic or nonmutagenic to chromosomal genes . In plants, Co(II) salts induced gene mutations and chromosomal aberrations . In mammalian cells in vitro, Co(II) compounds caused DNA strand breaks, sister-chromatid exchanges and aneuploidy, but not chromosomal aberrations . In two cell lines, CoCl2 was weakly mutagenic . Interestingly, the poorly soluble compound CoS caused DNA strand breaks and morphological transformation of mammalian cell lines . In contrast to its weak clastogenic and mutagenic properties, cobalt(II) exerts pronounced antimutagenicity in bacteria and mostly comutagenic effects in mammalian cells . In Escherichia coli CoCl2 lowered the frequency of mutations induced by MNNG, uv or X rays . In Chinese hamster V79 cells, CoCl2 enhanced the mutagenicity and clastogenicity of uv light but not of gamma rays . Regarding direct genotoxic mechanisms, Co(II) induces the formation of reactive oxygen species when combined with hydrogen peroxide in cell-free systems . At high (i.e., millimolar) concentrations, Co(II) also decreases the fidelity of DNA synthesis . Regarding anti- and co-mutagenic mechanisms, evidence for the interference of Co(II) with DNA repair processes is discussed . These mechanisms are regarded as relevant for the risk assessment of human exposure to cobalt in combination with other agents.

Proc Natl Acad Sci U S A, 1992 Jul 1, 89(13), 5769 - 73
Gene expression in cotton (Gossypium hirsutum L.) fiber: cloning of the mRNAs; John ME et al.; Cotton, an important natural fiber, is a differentiated epidermal cell . The number of genes that are active in fiber cells is similar to those in leaf, ovule, or root tissues . Through differential screening of a fiber cDNA library, we isolated five cDNA clones that are preferentially expressed in fiber . One of the cDNA clones, pCKE6, corresponded to an abundant mRNA in fiber . Transcripts for E6 were detected throughout the development of the fiber . Immunoprecipitation of in vitro translation products and Western blot analysis of fiber proteins showed two polypeptides in the range of 30-32 kDa as the products of E6 mRNA . Sequence analysis and hybrid-selected RNA translation also suggest that E6 mRNAs encode two polypeptides . Concentrations of E6 mRNA and protein are highest during the late primary cell wall and early secondary cell wall synthesis stages . Sequence comparison of E6 with other known eukaryotic and prokaryotic genes reveals no significant homology (GenBank; December 1991) . E6 or a homologous gene(s) is conserved in several members of Malvaceae as well as in one other fiber-producing plant, kapok, but is not found in several other plants examined or in Acetobacter xylinum . A genomic clone corresponding to pCKE6 was isolated, and the promoter element of the E6 gene was shown to direct the expression of a carrot extensin mRNA in a tissue-specific and developmentally regulated fashion in transgenic cotton plants.

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