Prokaryotes

Gene, 1996 Oct 24, 177(1-2), 261 - 3
Cloning and sequencing of the gene for the Thiobacillus ferrooxidans ATCC33020 glutamate synthase (GOGAT) small subunit and complementation of an Escherichia coli gltD mutant; Deane SM et al.; A recombinant plasmid which contains the gltD gene coding for the glutamate synthase (GOGAT) small subunit was isolated from a Thiobacillus ferrooxidans ATCC33020 gene bank by complementation of an Escherichia coli gltD mutant . The sequence of gltD was determined . The deduced amino acid sequence shows strong similarity to the two other prokaryote gltD sequences available, namely those of E . coli and A . brasilense (53% and 45% identity, respectively) . A cosmid containing the gltBD region was isolated from a T . ferrooxidans cosmid gene bank, but was unable to complement an E . coli gltB mutant.

Mol Biochem Parasitol, 1996 Oct 18, 81(1), 41 - 51
Cloning and sequence analysis of a novel member of the ATP-binding cassette (ABC) protein gene family from Plasmodium falciparum; Bozdech Z et al.; We have employed oligonucleotide primers directed against the Walker A and B ATP-binding consensus motifs in a PCR-approach to clone a novel member of the eukaryotic ABC protein family of genes from Plasmodium falciparum . The novel gene is predicted to encode a 95.5-kDa protein with two ATP-binding folds each containing a Walker A and B consensus motif and an ABC protein signature sequence . The predicted protein is highly hydrophilic and contains numerous phosphorylation consensus sites but does not contain any potential membrane spanning domains . The gene is present on chromosome 11 and is expressed as a 3.3-kb transcript . The closest homologue with known function to the plasmodial gene is the yeast GCN20 gene which is part of the translation initiation pathway in amino acid starved yeast cells . We have therefore tentatively named the gene Plasmodium falciparum GCN20 homologue (pfgcn20) . The pfgcn20 encoded Pfgcn20 protein is also highly homologous to a number of ATP-binding subunits of prokaryotic ABC transporters . We speculate that Pfgcn20 may be an example of a eukaryotic ATP-binding cytosolic subunit of a multipeptide ABC transporter.

J Biol Chem, 1996 Oct 18, 271(42), 25738 - 41
A novel bifunctional fusion enzyme catalyzing ethylene synthesis via 1-aminocyclopropane1-carboxylic acid; Li N et al.; A C terminus truncated soybean 1-aminocyclopropane-1-carboxylic acid (ACC) synthase (466 aa) was fused to an N terminus truncated tomato ACC oxidase (312 aa) to create a 778-amino acid fusion polypeptide . This ACC synthase-ACC oxidase fusion enzyme (ACSO) was expressed in a heterologous prokaryotic Escherichia coli system, which is capable of converting endogenous S-adenosyl-L-methionine (AdoMet) to ethylene . The molecular weight of the fusion enzyme, ACSO, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, was 90 +/- 3 kDa . Gel filtration analysis indicates that the native ACSO is oligomeric and is capable of converting exogenously supplied AdoMet to ethylene . The ethylene production rate of ACSO fusion enzyme was determined to be 6.0 nmol h-1 mg-1 under our assaying conditions using the partially purified enzyme extract . In the enzyme reaction mixture, an increase in ethylene production catalyzed by the bifunctional ACSO was accompanied by a decrease in ACC accumulation . Similarly, in E . coli cells, the level of ACC, produced as an intermediate during the sequential reactions from AdoMet to ethylene, was also found to arise earlier than that of ethylene . Because ACSO could produce ethylene from the ubiquitous AdoMet in living cell and the method commonly used to measure gaseous ethylene is simple, fast, and sensitive, we anticipate this bifunctional fusion enzyme to be useful as a reporter and for research in molecular biology, developmental biology, fermentation, and genetic engineering.

Genomics, 1996 Oct 15, 37(2), 177 - 82
Cloning, tissue expression, and mapping of a human photolyase homolog with similarity to plant blue-light receptors; van der Spek PJ et al.; Enzymatic photoreactivation is a DNA repair mechanism that removes UV-induced pyrimidine dimer lesions by action of a single enzyme, photolyase, and visible light . Its presence has been demonstrated in a wide variety of organisms, ranging from simple prokaryotes to higher eukaryotes . We have isolated a human gene encoding a 66-kDa protein that shows clear overall homology to known bacterial photolyase genes . The human gene product is more similar to plant blue-light receptors within class I photolyases than to higher eukaryote class II photolyases . Northern blot analysis showed two transcripts with constitutive expression in all tissues examined and an elevated expression in testis . In situ hybridization with a cDNA-derived probe localized this human gene to chromosome 12q23-q24.1 . Southern analysis of the cloned human gene suggests a wide distribution of the gene family in various species.

Proc Natl Acad Sci U S A, 1996 Oct 15, 93(21), 11597 - 602
p21Cip1/Waf1 disrupts the recruitment of human Fen1 by proliferating-cell nuclear antigen into the DNA replication complex; Chen U et al.; Fen1 or maturation factor 1 is a 5'-3' exonuclease essential for the degradation of the RNA primer-DNA junctions at the 5' ends of immature Okazaki fragments prior to their ligation into a continuous DNA strand . The gene is also necessary for repair of damaged DNA in yeast . We report that human proliferating-cell nuclear antigen (PCNA) associates with human Fen1 with a Kd of 60 nM and an apparent stoichiometry of three Fen1 molecules per PCNA trimer . The Fen1-PCNA association is seen in cell extracts without overexpression of either partner and is mediated by a basic region at the C terminus of Fen1 . Therefore, the polymerase delta-PCNA-Fen1 complex has all the activities associated with prokaryotic DNA polymerases involved in replication: 5'-3' polymerase, 3'-5' exonuclease, and 5'-3' exonuclease . Although p21, a regulatory protein induced by p53 in response to DNA damage, interacts with PCNA with a comparable Kd (10 nM) and a stoichiometry of three molecules of p21 per PCNA trimer, a p21-PCNA-Fen1 complex is not formed . This mutually exclusive interaction suggests that the conformation of a PCNA trimer switches such that it can either bind p21 or Fen1 . Furthermore, overexpression of p21 can disrupt Fen1-PCNA interaction in vivo . Therefore, besides interfering with the processivity of polymerase delta-PCNA, p21 also uncouples Fen1 from the PCNA scaffold.

Biochem Biophys Res Commun, 1996 Oct 14, 227(2), 489 - 93
An unusual bacterial reverse transcriptase having LVDD in the YXDD box from Escherichia coli; Mao JR et al.; A minor population of wild strains of Escherichia coli contains a gene for reverse transcriptase (RT) which is responsible for the synthesis of multicopy single-stranded DNA (msDNA), a branched DNA-RNA complex . A DNA fragment capable of synthesizing msDNA was cloned from strain ECOR-58, one of the 72 wild strains in the ECOR collection . The complete open reading frame of a novel reverse transcriptase, designated ECOR-58 RT, was identified . ECOR-58 RT consisted of 408 amino acid residues, and its 227-residue polymerase domain from residue 43 to 269 showed significant homologies to all the other bacterial RTs so far identified . Most significantly, its YXDD box, the most highly conserved sequence in all RTs from prokaryotes to eukaryotes, was found to be replaced with LVDD . ECOR-58 RT was found to be most distantly related in a phylogenetic tree to all 9 other bacterial RTs so far identified.

Gene, 1996 Oct 10, 175(1-2), 65 - 70
Cloning and characterization of the gene encoding Halobacterium halobium adenylate kinase; Song S et al.; The gene (AK) encoding adenylate kinase (AK) of Halobacterium halobium was cloned . AK consisted of 648 bp and coded for 216 amino acids (aa) . S1 mapping and primer extension experiments indicated that the transcription start point (tsp) was located immediately upstream from the start codon . The TAT-like promoter sequence was found at a position 20-24 bp upstream from tsp . The most striking property of the enzyme was a putative Zn finger-like structure with four cysteines . It might contribute to the structural stability of the molecule in high-salt conditions . Phylogenetic analysis indicated two lineages of the AK family, the short and long types which diverged a long time ago, possibly before the separation of prokaryotes and eukaryotes . Although the H . halobium AK belongs to the long-type AK lineage, it is located in an intermediary position between the two lineages of the phylogenetic tree, indicating early divergence of the gene along the long-type lineage.

Semin Cancer Biol, 1996 Oct, 7(5), 229 - 40
Xpa knockout mice; de Vries A et al.; The xeroderma pigmentosum group A correcting (XPA) gene encodes a DNA binding zinc-finger protein that recognizes DNA damage . As such the XPA protein participates in the initial step of the process of nucleotide excision repair . The multicomponent nucleotide excision repair pathway is one of the most thoroughly studied mechanisms that defends both eukaryotic and prokaryotic cells against the deleterious effects of UV-B and several chemical components . In the absence of nucleotide excision repair common cellular processes like transcription and replication are disturbed by persisting (unrepaired) DNA lesions (adducts), which may lead to the accumulation of gene mutations and ultimately to cancer . Xeroderma pigmentosum patients have a > 2000 fold increased risk to develop skin cancer at sun-exposed areas . Here we describe that XPA-deficient transgenic mice show features that mimic the phenotype found in humans . Furthermore, the possible use of Xpa- and other nucleotide excision repair deficient mice in cancer research will be outlined in more detail.

Oral Microbiol Immunol, 1996 Oct, 11(5), 319 - 25
Lipopolysaccharide isolated from Porphyromonas gingivalis grown in hemin-limited chemostat conditions has a reduced capacity for human neutrophil priming; Champagne CM et al.; One way prokaryotes respond to environmental stresses is by modifying selected outer membrane components . Iron, in the form of hemin, has been shown to be a significant regulator of Porphyromonas gingivalis growth and virulence and of the expression of outer membrane proteins and lipopoly saccharide . Since lipopoly saccharide has profound effects on host immune cells, this study compared the effect of hemin-restricted and hemin-normal P . gingivalis growth conditions on lipopolysaccharide priming of N-formylmethionyl-leucyl-phenylalanine-induced superoxide generation by human neutrophils . P . gingivalis was grown in a chemostat under normal (5 micrograms hemin/ml) and hemin-restricted (0.08 microgram hemin/ml) conditions . Purified lipopolysaccharide from both P . gingivalis normal and hemin-limited environments increased N-formylmethionyl-leucyl-phenylalanine-induced superoxide release by neutrophils in a dose-dependent manner . Lipopolysaccharide isolated from the hemin-normal conditions was a significantly more potent neutrophil priming agent than the lipopolysaccharide isolated from hemin-restricted conditions . Addition of normal human serum enhanced the priming effect of both lipopolysaccharide preparations; this effect, however, was more evident with the hemin-normal lipopolysaccharide . Further, this enhancing effect of serum was partly reduced in the presence of antibodies raised against the serum lipopolysaccharide-binding protein . The differences in the biological activity of the two lipopolysaccharide preparations could be associated with structural differences detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis . These results indicate that hemin availability affects regulation of an aspect of P . gingivalis virulence, lipopolysaccharide-human neutrophils priming . The reduced capacity for neutrophil priming by hemin-restricted lipopolysaccharide appears to be related to lipopolysaccharide-neutrophil interactions and not to serum factors Targeting bacterial cell-surface components involved in hemin transport might be effective therapy for P . gingivalis-associated periodontal diseases.

Plant Mol Biol, 1996 Oct, 32(1-2), 315 - 26
Regulation of gene expression in chloroplasts of higher plants; Sugita M et al.; Chloroplasts contain their own genetic system which has a number of prokaryotic as well as some eukaryotic features . Most chloroplast genes of higher plants are organized in clusters and are cotranscribed as polycistronic pre-RNAs which are generally processes into many shorter overlapping RNA species, each of which accumulates of steady-state RNA levels . This indicates that posttranscriptional RNA processing of primary transcripts is an important step in the control of chloroplast gene expression . Chloroplast RNA processing steps include RNA cleavage/trimming, RNA splicing, ENA editing and RNA stabilization . Several chloroplast genes are interrupted by introns and therefore require processing for gene function . In tobacco chloroplasts, 18 genes contain introns, six for tRNA genes and 12 for protein-encoding genes . A number of specific proteins and RNA factors are believed to be involved in splicing and maturation of pre-RNAs in chloroplasts . Processing enzymes and RNA-binding proteins which could be involved in posttranscriptional steps have been identified in the last several years . Our current knowledge of the regulation of gene expression in chloroplasts of higher plants is overviewed and further studies on this matter are also considered.

Plant Mol Biol, 1996 Oct, 32(1-2), 107 - 44
The plant translational apparatus; Browning KS; Protein synthesis in both eukaryotic and prokaryotic cells is a complex process requiring a large number of macromolecules: initiation factors, elongation factors, termination factors, ribosomes, mRNA, amino-acylsynthetases and tRNAs . This review focuses on our current knowledge of protein synthesis in higher plants.

Genet Anal, 1996 Oct, 13(4), 95 - 8
Application of repetitive sequence-based PCR (inter-LINE PCR) for the analysis of genomic rearrangements and for the genome characterization on different taxonomic levels; Smida J et al.; Oligonucleotide primers derived from consensus LINE-sequences generated highly reproducible, species-specific PCR product patterns suitable for the identification of genomic rearrangements and for the discrimination on different taxonomic levels of higher and lower eukaryotes and even prokaryotes.

Curr Opin Biotechnol, 1996 Oct, 7(5), 494 - 9
Optimization of heterologous protein production in Escherichia coli; Weickert MJ et al.; Escherichia coli has long been the primary prokaryotic host for the synthesis of heterologous proteins . Recent advances have been made in the expression of complex proteins as soluble, functional molecules, complete with prosthetic groups, disulfide bonds, and quaternary structure . The development of alternative promoter and induction strategies has improved the options available for manipulating the expression conditions, which are frequently critical to soluble yield.

Infect Immun, 1996 Oct, 64(10), 4154 - 62
Dual flaA1 flaB1 mutant of Serpulina hyodysenteriae expressing periplasmic flagella is severely attenuated in a murine model of swine dysentery; Rosey EL et al.; The motility imparted by the periplasmic flagella (PF) of Serpulina hyodysenteriae is thought to play a pivotal role in the enteropathogenicity of this spirochete . The complex PF are composed of multiple class A and class B polypeptides . Isogenic strains containing specifically disrupted flaAl or flaB1 alleles remain capable of expressing PF, although such mutants display aberrant motility in vitro . To further examine the role that these proteins play in the maintenance of periplasmic flagellar structural integrity, motility, and fitness for intestinal colonization, we constructed a novel strain of S . hyodysenteriae which is deficient in both FlaA1 and FlaB1 . To facilitate construction of this strain, a chloramphenicol gene cassette, with general application as a selectable marker in prokaryotes, was developed . The cloned flaAl and flaB1 genes were disrupted by replacement of internal fragments with chloramphenicol and kanamycin gene cassettes, respectively . The inactivated flagellar genes were introduced into S . hyodysenteriae, and allelic exchange at the targeted chromosomal flaA1 and flaB1 loci was verified by PCR analysis . Immunoblots or cell lysates with antiserum raised against purified FlaA or FlaB confirmed the absence of the corresponding sheath and core proteins in this dual flagellar mutant . These mutations selectively abolished the expression of the targeted genes without affecting the synthesis of other immunologically related FlaB proteins . The resulting flaA1 flaB1 mutant exhibited altered motility in vitro . Surprisingly, it was capable of assembling periplasmic flagella that were morphologically normal as evidenced by electron microscopy . The virulence of this strain was assessed in a murine model of swine dysentery by determining the incidence of cecal lesions and the persistence of S . hyodysenteriae in the gut . Mice challenged with the wild-type strain or a passage control strain showed a dose-related response to the challenge organism . The dual flagellar mutant was severely attenuated in murine challenge experiments, suggesting that the FlaA1 and FlaB1 proteins are dispensable for flagellar assembly but critical for normal flagellar function and colonization of mucosal surfaces of the gastrointestinal tract . This strain represents the first spirochete engineered to contain specifically defined mutations in more than one genetic locus.

Infect Immun, 1996 Oct, 64(10), 4000 - 7
The hemagglutinin gene A (hagA) of Porphyromonas gingivalis 381 contains four large, contiguous, direct repeats; Han N et al.; Porphyromonas gingivalis is a gram-negative anaerobic bacterial species strongly associated with adult periodontitis . One of its distinguishing characteristics and putative virulence properties is the ability to agglutinate erythrocytes . We have previously reported the cloning of multiple hemagglutinin genes from P . gingivalis 381 . Subsequent sequencing of clone ST 2 revealed that the cloned fragment contained only an internal portion of the gene which lacked both start and stop codons . We here report the cloning and sequencing of the entire gene, designated hagA, as well as its relationship to other genes of this species . By use of inverse PCR technology and the construction of several additional genomic libraries, the complete open reading frame of hagA was found to be 7,887 bp in length, encoding a protein of 2,628 amino acids with a molecular mass of 283.3 kDa, which is among the largest genes ever cloned from a prokaryote to date . Within its open reading frame, four large, contiguous, direct repeats (varying from 1,318 to 1,368 bp) were identified . The repeat unit (HArep), which is assumed to contain the hemagglutinin domain, is also present in other recently reported protease and hemagglutinin genes in P . gingivalis . Thus, we propose that hagA and the other genes which share the HArep sequence form a multigene family with hagA as a central member.

Immunopharmacology, 1996 Oct, 35(1), 1 - 21
Microbial/host interactions in health and disease: who controls the cytokine network?
Henderson B, Poole S, Wilson M.
The interacting cellular and molecular systems which we classify as immunity and inflammation evolved to protect the organism from exogenous parasites including viruses and bacteria . Cytokines play a pivotal, but paradoxical, role both in immunity and inflammation . These local peptide hormone-like molecules form a major arm of the organisms, defenses against infectious microorganisms but they are also implicated as potent mediators of the pathology of infectious diseases . The apparently lethal effects of interleukin-1 and tumor necrosis factor in experimental septic shock testify to the latter . In the current paradigm, cytokine induction, as a protective or pathological mechanism, is a direct response to the presence of infectious microorganisms . Evidence is now accumulating that cytokines play a much more complex role in the interplay between exogenous microorganisms and the host . For example, it has been established that viruses have evolved pro-active methods of subverting the cytokine network by producing: (i) soluble cytokine receptors which bind and inactivate cytokines, (ii) immunomodulatory cytokine homologues, and (iii) ICE inhibitors . The possibility exists that the major role of these 'viral cytokines' is to neutralize certain host responses . Recent cytokine transgenic knockouts demonstrate that the normal benign response to commensal gut microflora becomes a lethal inflammatory state in the absence of the cytokines interleukin 2 or interleukin 10 . The human body contains an enormous number of microorganisms which constitute the normal microflora . It is estimated that the average human contains 10(13) eukaryotic cells but 10(14) bacteria . We propose that the ability of the multicellular organism to live harmoniously with its commensal microflora must depend on mutual signalling involving eukaryotic cytokines and prokaryotic cytokine-like molecules . Such interactive signalling sets up non-inflammatory cytokine networks in tissues which form the background on which responses to infectious microorganisms must be built and related . The capacity of bacteria to induce cytokine synthesis was believed to be due to a small number of components, such as lipopolysaccharide (LPS), which is only active as a complex with host factors (lipopolysaccharide binding protein and CD14) . However, it is now clear that bacteria contain and produce a large number of diverse molecules which can selectively induce the synthesis of both pro-inflammatory and immunomodulatory/anti-inflammatory cytokines . Many toxins are potent inducers of cytokine release or synthesis and some can inhibit LPS-induced cell activation . We have introduced the term bacteriokine to describe these bacterial cytokine inducers . The question that has to be addressed therefore is - who controls the cytokine network (eukaryotic or prokaryotic cells) and how is it controlled? It is proposed that an understanding of this question will bring with it an understanding of how to control the pathological inflammatory response and may allow the development of truly effective anti-inflammatory agents.

Mol Cell Probes, 1996 Oct, 10(5), 359 - 70
Detection of rRNA from four respiratory pathogens using an automated Q beta replicase assay; Stone BB et al.; Ribosomal RNA targets from Mycobacterium avium complex (23S), Mycoplasma pneumoniae (16S), Pneumocystis carinii (18S) and Legionella pneumophila (16S) were detected in four separate assays on a model automated Q-beta amplification instrument . Sandwich hybridization, reversible target capture, detector probe amplification and fluorescent signal detection occurred in closed, disposable packs at 38 degrees C . Packs were injected with 0.5 ml samples in 3.06 M guanidine thiocyanate . Ten samples per run were read after 7 h, requiring only 4 min loading time . Synthetic RNA transcripts and purified, natural RNAs from up to four different strains per assay were diluted to 10(6) or fewer molecules per sample (approximately 100 cells for prokaryotes, 10 cells for Pneumocystis) . All analytes were detected at 10(6) targets . The limits of detection were found at 10(5) to 10(4) . Discrimination against competitor RNA was tested using up to 10(9) molecules (1000 X excess) of appropriate test strains . Samples containing either zero targets or 10(7) competitors produced negative results in 95 to 100% of the samples, depending on the assay . Closely related Legionella and Mycoplasma species cross-reacted at high challenge levels of 10(9) molecules as a result of sequence similarities in the target regions . These results demonstrate the utility and versatility of an automated, high sensitivity, closed system for amplified analysis of direct-from-sample testing of respiratory pathogens.

Chronobiol Int, 1996 Oct, 13(4), 239 - 50
Heat shock proteins and circadian rhythms; Rensing L et al.; Significant circadian rhythms in heat shock gene expression were observed in a prokaryotic species (Synechocystis) . In eukaryotes, in contrast, several heat shock genes (constitutive and inducible) were shown to be constantly expressed . A few cases of circadian expression of heat shock proteins (HSPs), however, have been reported . Significant circadian changes of thermotolerance were observed in yeast and several plant species . Higher thermotolerance can be attributed to a higher abundance of HSPs, but also to other adaptive mechanisms . Zeitgeber effects of temperature changes can be explained on the basis of their direct effects on the state variables of the clock gene (per,frq) expression and its negative feedback loop . Effects of increased HSP concentrations, as observed after heat shock, but also after light and serotonin (5HT), appear possible, in particular with respect to nuclear localization of the clock (PER) protein, but these effects have not been documented yet . Thus, the role of HSPs in the circadian clock system is little understood and, from our point of view, deserves more attention.

Proc Natl Acad Sci U S A, 1996 Oct 1, 93(20), 11126 - 30
Coexistence of phycoerythrin and a chlorophyll a/b antenna in a marine prokaryote; Hess WR et al.; Prochlorococcus marinus CCMP 1375, a ubiquitous and ecologically important marine prochlorophyte, was bound to possess functional genes coding for the alpha and beta subunits of a phycobiliprotein . The latter is similar to phycoerythrins (PE) from marine Synechococcus cyanobacteria and bind a phycourobilin-like pigment as the major chromophore . However, differences in the sequences of the alpha and beta chains compared with known PE subunits and the presence of a single bilin attachment site on the alpha subunit designate it as a novel PE type, which we propose naming PE-III . P . marinus is the sole prokaryotic organisms known so far that contains chlorophylls a and b as well as phycobilins . These data strongly suggest that the common ancestor of prochlorophytes and the Synechococcus cyanobacteria contained phycobilins . Flow cytometric data from the tropical Pacific Ocean provide evidence that deep populations of Prochlorococcus possess low amounts of a PE-like pigment, which could serve either in light harvesting or nitrogen storage or both.

RNA, 1996 Oct, 2(10), 1022 - 32
An antisense/target RNA duplex or a strong intramolecular RNA structure 5' of a translation initiation signal blocks ribosome binding: the case of plasmid R1; Malmgren C et al.; Antisense RNAs in prokaryotic systems often inhibit translation of mRNAs . In some cases, this involves sequestration of Shine-Dalgarno (SD) sequences and start codons . In other cases, antisense/target RNA duplexes do not overlap these signals, but form upstream . We have performed toeprinting analyses on repA mRNA of plasmid R1, both free and in duplex with the antisense RNA, CopA . An intermolecular RNA duplex 2 nt upstream of the tap SD prevents ribosome binding . An intrastrand stem-loop at this location yields the same inhibition . Thus, stable secondary structures immediately upstream of the tap SD sequence inhibit translation, as shown by toeprinting in vitro and repA-lacZ expression in vivo . Previous work showed that repA (initiator protein) expression requires tap (leader peptide) translation . Toeprinting data confirm that the tap ribosome binding site (RBS) is accessible, whereas the repA RBS, which is sequestered by a stable stem-loop, is weakly recognized by the ribosome . Truncated CopA RNA (CopI) is unable to pair completely with target RNA, but proceeds normally to a kissing intermediate . This mutant RNA species inhibits repA expression in vivo . By a kinetic toeprint inhibition protocol, we have shown that the structure of the kissing complex is sufficient to sterically prevent ribosome binding . These results are discussed in comparison with the effect of RNA structures elsewhere in the ribosome-binding region of an mRNA.

J Med Microbiol, 1996 Oct, 45(4), 258 - 62
Opsonin-independent adherence and phagocytosis of Listeria monocytogenes by murine peritoneal macrophages; Pierce MM et al.; Listeria monocytogenes adhered to and multiplied intracellularly in murine peritoneal macrophages in the absence of opsonins . The infective process in these cells was evaluated by viable bacterial cell colony counts of intracellular organisms and documented by transmission and scanning electron microscopy . Adherence of listeriae to macrophages involved surface interactions of the prokaryotic cell surface and eukaryotic cell membranes . Subsequent phagocytosis was seen to occur through a process in which host cell-derived pseudopodia surrounded and engulfed organisms leaving them within phagosomes in the cytoplasm of infected cells . This process of uptake of L . monocytogenes by macrophages occurred at 4 degrees C . Following invasion of the cell, escape of L . monocytogenes from the phagosome into the cytoplasm was initiated as early as 10 min into the infective process . Intracellular multiplication of bacteria continued for 8 h after inoculation at which point loss of adherent macrophages due to cell lysis was evident . The mean generation time of the organism in these cells was 58 min . The cellular and ultrastructural events of L . monocytogenes adherence to and phagocytosis by murine macrophages in the absence of antibody or complement have been defined.

Appl Environ Microbiol, 1996 Oct, 62(10), 3620 - 31
Physiological ecology of Methanobrevibacter cuticularis sp . nov . and Methanobrevibacter curvatus sp . nov., isolated from the hindgut of the termite Reticulitermes flavipes; Leadbetter JR et al.; Two morphologically distinct, H2- and CO2-utilizing methanogens were isolated from gut homogenates of the subterranean termite, Reticulitermes-flavipes (Kollar) (Rhinotermitidae) . Strain RFM-1 was a short straight rod (0.4 by 1.2 micron), whereas strain RFM-2 was a slightly curved rod (0.34 by 1.6 microns) that possessed polar fibers . Their morphology, gram-positive staining reaction, resistance to cell lysis by chemical agents, and narrow range of utilizable substracts were typical of species belonging to the family Methanobacteriaceae . Analysis of the nearly complete sequences of the small-subunit rRNA-encoding genes confirmed this affiliation and supported their recognition as new species of Methanobrevibacter: M . cuticularis (RFM-1) and M . curvatus (RFM-2) . The per cell rates of methanogenesis by strains RFM-1 and RFM-2 in vitro, taken together with their in situ population densities (ca . 10(6) cells.gut-1; equivalent to 10(9) cells . ml of gut fluid-1), could fully account for the rate of methane emission by the live termites . UV epifluorescence and electron microscopy confirmed that RFM-1- and RFM-2-type cells were the dominant methanogens in R.flavipes collected in Michigan (but were not the only methanogens associated with this species) and that they colonized the peripheral, microoxic region of the hindgut, i.e., residing on or near the hindgut epithelium and also attached to filamentous prokaryotes associated with the gut wall . An examination of their oxygen tolerance revealed that both strains possessed catalase-like activity . Moreover, when dispersed in tubes or agar medium under H2-CO2-O2 (75: 18.8:6.2, vol/vol/vol), both strains grew to form a thin plate about 6 mm below the meniscus, just beneath the oxic-anoxic interface . Such growth plates were capable of mediating a net consumption of O2 that otherwise penetrated much deeper into uninoculated control tubes . Similar results were obtained with an authentic strain of Methanobrevibacter arboriphilicus . This is the first detailed description of an important and often cited but poorly understood component of the termite gut microbiota.

Mol Cell Biol, 1996 Oct, 16(10), 5764 - 71
Molecular cloning of Drosophila mus308, a gene involved in DNA cross-link repair with homology to prokaryotic DNA polymerase I genes; Harris PV et al.; Mutations in the Drosophila mus308 gene confer specific hypersensitivity to DNA-cross-linking agents as a consequence of defects in DNA repair . The mus308 gene is shown here to encode a 229-kDa protein in which the amino-terminal domain contains the seven conserved motifs characteristic of DNA and RNA helicases and the carboxy-terminal domain shares over 55% sequence similarity with the polymerase domains of prokaryotic DNA polymerase I-like enzymes . This is the first reported member of this family of DNA polymerases in a eukaryotic organism, as well as the first example of a single polypeptide with homology to both DNA polymerase and helicase motifs . Identification of a closely related gene in the genome of Caenorhabditis elegans suggests that this novel polypeptide may play an evolutionarily conserved role in the repair of DNA damage in eukaryotic organisms.

Mol Cell Biol, 1996 Oct, 16(10), 5754 - 63
Induction of DNA replication by transcription in the region upstream of the human c-myc gene in a model replication system; Ohba R et al.; An important relationship between transcription and initiation of DNA replication in both eukaryotes and prokaryotes has been suggested . In an attempt to understand the molecular mechanism of this interaction, we examined whether transcription can induce DNA replication in vitro by constructing a system in which both replication and transcription were combined . Relaxed circular DNA possessing a replication initiation zone located upstream of the human c-myc gene and a T7 promoter near the P1 promoter of the gene was replicated in the presence of T7 RNA polymerase . In our model system, replication was carried out with the proteins required for simian virus 40 DNA replication . DNA synthesis, which was dependent on both T7 RNA polymerase and the replication proteins, was detected mainly in the promoter and upstream regions of the c-myc gene . Blocking RNA synthesis at the initial stage of the reaction severely reduced DNA synthesis, suggesting that RNA chain elongation is required to induce DNA synthesis . The results indicated that transcription can induce DNA replication in the upstream region of the transcribed gene, most likely by introducing negative supercoiling into the region, which results in unwinding of the DNA duplex.

J Virol, 1996 Oct, 70(10), 7085 - 91
A hydrophobic heptad repeat of the core protein of woodchuck hepatitis virus is required for capsid assembly; Yu M et al.; The capsid particle of hepadnaviruses is assembled from its dimer precursors . However, the mechanism of the protein-protein interaction is still poorly understood . A small region in the capsid protein of woodchuck hepatitis virus (WHV) contains four hydrophobic residues, including leucine 101, leucine 108, valine 115, and phenylalanine 122, that are conserved and spaced every seventh residue in the primary sequence to form a hydrophobic heptad repeat (hhr) . A hydrophobic force often plays an important role in the interaction of proteins . Therefore, to investigate the role of this region in capsid assembly, we individually changed the codons specifying these four hydrophobic amino acids to codons specifying alanine or proline . In addition, we examined the in vivo infectivity of a WHV genome bearing a naturally occurring single amino acid change (histidine 104-->proline) in the hhr region . The phenotype of each altered genome was determined in both eukaryotic and prokaryotic systems by a capsid protein assay and electron microscopic examination . We show that replacement of any one of the four hydrophobic residues with alanine did not prevent capsid assembly . However, assembled capsid particles were not detected if combinations of any two of the four residues were substituted with alanines or if the spacing of these four hydrophobic residues was changed . An individual introduction of a proline (which dramatically changes the secondary structure of proteins) into different positions of this small region also abolished capsid assembly in vitro or viral replication in vivo . These results suggested that the hhr region of the core protein of WHV was critical for capsid assembly.

FEBS Lett, 1996 Sep 30, 394(2), 206 - 12
Identification of a second Mycobacterium tuberculosis gene cluster encoding proteins of an ABC phosphate transporter; Braibant M et al.; Following the identification of a M . tuberculosis phosphate transporter belonging to the superfamily of ABC transporters, we report on the cloning and sequencing of two additional genes, called pstS-3 and pstC-2, encoding proteins homologous to PstS and PstC of Escherichia coli, respectively . Together with the previously isolated M . tuberculosis gene similar to the E . coli pstA, these are included in a cluster encoding a second putative phosphate transport system . We demonstrate that pstS-3 encodes the previously described Ag 88, a 40 kDa M . bovis BCG culture filtrate antigen (immunodominant in H-2b haplotype type mice) . Finally, a signature motif identifying integral transmembrane proteins of prokaryotic phosphate binding-dependent permeases is proposed.

Gene, 1996 Sep 26, 174(1), 175 - 9
Insertion of a short Alu sequence into the hMSH2 gene following a double cross over next to sequences with chi homology; Marshall B et al.; Alu repeat sequences and other multiple copy repetitive elements are present throughout the human genome and are active in promoting recombination . It is believed that reverse transcription of transcribed Alu repeats followed by chromosomal integration has been responsible for the wide dispersion and high copy number of these sequences . During studies on the hMSH2 gene we have used RT-PCR to amplify from peripheral blood lymphocytes a cDNA species in which 553 base pairs of hMSH2 cDNA have been deleted to be replaced by a short 36 base pair Alu sequence as a result of a genomic insertion/deletion event . The 36 base pair Alu insert is homologous to a 26 base pair Alu sequence previously implicated in the promotion of recombination and contains the GCTGG motif which is part of the prokaryotic chi sequence . A second chi-like sequence is also located within the deleted hMSH2 region . Both chi-like sequences are located within 4 bp of the two 4-bp regions of cross over containing the insertion/deletion breakpoints . This suggest that a double recombination event has occurred, providing direct evidence for the recombinogenic activity of this Alu element . Furthermore, it suggests that chi-like sequences may define recombination hotspots as in prokaryotes.

Biochem Biophys Res Commun, 1996 Sep 24, 226(3), 626 - 30
Expression of the petE gene encoding plastocyanin in the photosynthetic prokaryote, Prochlorothrix hollandica; Arudchandran A et al.; The expression of the petE gene encoding plastocyanin (PC) in the prokaryote Prochlorothrix hollandica is dependent on the presence of copper in the medium . PC protein and petE mRNA are detectable only under copper (Cu) replete conditions, suggesting that control of PC accumulation can occur at the level of transcription or transcript stability . Addition of Cu (0.3 microM) to log-phase Cu-deficient cultures yields accumulation of PC to detectable levels within 12 h; transfer of Cu-replete cells to Cu-deficient medium results in a slow decrease in the level of protein likely due to dilution by cell growth . By contrast, addition of high-affinity Cu-specific chelators to rapidly deplete Cu-grown cells of copper yields a rapid loss of PC with 2 h . These data suggest that Cu-free apoPC is turned over rapidly by proteolysis . Overall, these data demonstrate that regulation of PC levels as Cu levels change involve events occurring at the level of both RNA and protein turnover.

Cell, 1996 Sep 20, 86(6), 865 - 75
Yeast HOG1 MAP kinase cascade is regulated by a multistep phosphorelay mechanism in the SLN1-YPD1-SSK1 "two-component" osmosensor; Posas F et al.; An osmosensing mechanism in the budding yeast (Saccharomyces cerevisiae) involves both a two-component signal transducer (Sln1p, Ypd1p and Ssk1p) and a MAP kinase cascade (Ssk2p/Ssk22p, Pbs2p, and Hog1p) . The transmembrane protein Sln1p contains an extracellular sensor domain and cytoplasmic histidine kinase and receiver domains, whereas the cytoplasmic protein Ssk1p contains a receiver domain . Ypd1p binds to both Sln1p and Ssk1p and mediates the multistep phosphotransfer reaction (phosphorelay) . This phosphorelay system is initiated by the autophosphorylation of Sln1p at His576 . This phosphate is then sequentially transferred to Sln1p-Asp-1144, then to Ypd1p-His64, and finally to Ssk1p-Asp554 . We propose that the multistep phosphorelay mechanism is a universal signal transduction apparatus utilized both in prokaryotes and eukaryotes.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10222 - 7
Rad51 expression and localization in B cells carrying out class switch recombination; Li MJ et al.; Rad51 is a highly conserved eukaryotic homolog of the prokaryotic recombination protein RecA, which has been shown to function in both recombinational repair of DNA damage and meiotic recombination in yeast . In primary murine B cells cultured with lipopolysaccharide (LPS) to stimulate heavy chain class switch recombination, Rad51 protein levels are dramatically induced . Immunofluorescent microscopy shows that anti-Rad51 antibodies stain foci that are localized within the nuclei of switching B cells . Immunohistochemical analysis of splenic sections shows that clusters of cells that stain brightly with anti-Rad51 antibodies are evident within several days after primary immunization and that Rad51 staining in vivo is confined to B cells that are switching from expression of IgM to IgG antibodies . Following switch recombination, B cells populate splenic germinal centers, where somatic hypermutation and clonal proliferation occur . Germinal center B cells are not stained by anti-Rad51 antibodies . Rad51 expression is therefore not coincident with somatic hypermutation, nor does Rad51 expression correlate simply with cell proliferation . These data suggest that Rad51, or a highly related member of the conserved RecA family, may function in class switch recombination.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10183 - 8
Circadian gating of cell division in cyanobacteria growing with average doubling times of less than 24 hours; Mori T et al.; To ascertain whether the circadian oscillator in the prokaryotic cyanobacterium Synechococcus PCC 7942 regulates the timing of cell division in rapidly growing cultures, we measured the rate of cell division, DNA content, cell size, and gene expression (monitored by luminescence of the PpsbAI::luxAB reporter) in cultures that were continuously diluted to maintain an approximately equal cell density . We found that populations dividing at rates as rapid as once per 10 h manifest circadian gating of cell division, since phases in which cell division slows or stops recur with a circadian periodicity . The data clearly show that Synechococcus cells growing with doubling times that are considerably faster than once per 24 h nonetheless express robust circadian rhythms of cell division and gene expression . Apparently Synechococcus cells are able to simultaneously sustain two timing circuits that express significantly different periods.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10162 - 6
Determinants of RNA polymerase alpha subunit for interaction with beta, beta', and sigma subunits: hydroxyl-radical protein footprinting; Heyduk T et al.; Escherichia coli RNA polymerase (RNAP) alpha subunit serves as the initiator for RNAP assembly, which proceeds according to the pathway 2 alpha-->alpha 2-->alpha 2 beta-->alpha 2 beta beta'-->alpha 2 beta beta' sigma . In this work, we have used hydroxyl-radical protein footprinting to define determinants of alpha for interaction with beta, beta', and sigma . Our results indicate that amino acids 30-75 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta (i.e., in alpha 2 beta, alpha 2 beta beta', and alpha 2 beta beta' sigma), and amino acids 175-210 of alpha are protected from hydroxyl-radical-mediated proteolysis upon interaction with beta' (i.e., in alpha 2 beta beta' and alpha 2 beta beta' sigma) . The protected regions are conserved in the alpha homologs of prokaryotic, eukaryotic, archaeal, and chloroplast RNAPs and contain sites of substitutions that affect RNAP assembly . We conclude that the protected regions define determinants of alpha for direct functional interaction with beta and beta' . The observed maximal magnitude of protection upon interaction with beta and the observed maximal magnitude of protection upon interaction with beta' both correspond to the expected value for complete protection of one of the two alpha protomers of RNAP (i.e., 50% protection) . We propose that only one of the two alpha protomers of RNAP interacts with beta and that only one of the two alpha protomers of RNAP interacts with beta'.

Proc Natl Acad Sci U S A, 1996 Sep 17, 93(19), 10151 - 5
Dual-function regulators: the cAMP receptor protein and the CytR regulator can act either to repress or to activate transcription depending on the context; Rasmussen PB et al.; Studies of gene regulation have revealed that several transcriptional regulators can switch between activator and repressor depending upon both the promoter and the cellular context . A relatively simple prokaryotic example is illustrated by the Escherichia coli CytR regulon . In this system, the cAMP receptor protein (CRP) assists the binding of RNA polymerase as well as a specific negative regulator, CytR . Thus, CRP functions either as an activator or as a corepressor . Here we show that, depending on promoter architecture, the CRP/CytR nucleoprotein complex has opposite effects on transcription . When acting from a site close to the DNA target for RNA polymerase, CytR interacts with CRP to repress transcription, whereas an interaction with CRP from appropriately positioned upstream binding sites can result in formation of a huge preinitiation complex and transcriptional activation . Based on recent results about CRP-mediated regulation of transcription initiation and the finding that CRP possesses discrete surface-exposed patches for protein-protein interaction with RNA polymerase and CytR, a molecular model for this dual regulation is discussed.

Gene, 1996 Sep 16, 173(2), 129 - 35
Secondary structures and features of the 18S, 5.8S and 26S ribosomal RNAs from the Apicomplexan parasite Toxoplasma gondii; Gagnon S et al.; The two major subunits of the ribosomal RNA (rRNA) of Toxoplasma gondii, 18S and 26S, as well as 5.8S, have been sequenced and folded according to known consensus and established secondary structures . Conserved and variable nucleotide (nt) regions were identified using multiple alignments with rRNA sequences of selected organisms . The 18S rRNA showed a well conserved core structure of 48 stems and a hypervariable V4 region identified four additional stems including a pseudoknot . The 18S rRNA contained an additional helix in the V2 region located between nt 204 to 258 . We noted that T . gondii 18S does not have a true V6 region, but was organized as a motif of a simple stem . T . gondii 26S had a conserved core structure of 83 stems and its expansion segments, so-called divergent domains, demonstrated a high degree of similarity with secondary structures from rRNA of dinoflagellates and ciliates . For the T . gondii 26S sequence, we found two additional stems, D3d and D3e, composed of 140 nt having a higher deltaG value . These segments are absent from the prokaryotic rRNA structures, whereas the hypervariable V4 region of the small subunit is not as variable . The well preserved structures could indicate an additional function for the eukaryotic ribosome.

FEBS Lett, 1996 Sep 16, 393(2-3), 147 - 50
The primary structure of UK114 tumor antigen; Ceciliani F et al.; UK114 is a tumor antigen expressed by various malignant neoplasms . The complete amino acid sequence of UK114 purified from goat liver has been determined by automated Edman degradation of CNBr and endoproteinase Lys-C peptides . The protein contains 137 amino acid residues . which corresponds to a molecular mass of 14,229 Da . MALDI-TOF analysis resulted in a molecular weight of 14,290, suggesting that the N-terminal Met residue is acetylated . Sequence comparison shows that UK114 from goat liver (1) has 77% identity with a previously described 23 kDa protein from rat liver (Levy-Favatier et al . (1993) Eur . J . Biochem . 212, 665-673), (2) shares a very high degree of similarity with a family of prokaryotic and eukaryotic hypothetic proteins whose function have not yet been characterized, and (3) exhibits a significant similarity to a group of tumor-associated antigens which belongs to a superfamily of heat shock proteins, acting as possible targets for the host's antitumor immunity.

Cell Immunol, 1996 Sep 15, 172(2), 262 - 8
Recombinant Brucella abortus proteins that induce proliferation and gamma-interferon secretion by CD4+ T cells from Brucella-vaccinated mice and delayed-type hypersensitivity in sensitized guinea pigs; Oliveira SC et al.; Optimal protective immunity to Brucella abortus infection is dependent on a coordinate interaction between different T-cell subsets which leads to an antigen-specific T-lymphocyte-mediated activation of macrophages, the main cellular reservoir for the bacterium . As an initial step in the identification of bacterial proteins that mediate cellular immunity, we have subcloned the B . Abortus ssb, uvrA, GroES, and GroEL genes into the prokaryotic expression vector pMAL-c2 using PCR . Escherichia coli DH5 alpha was transformed with the pMAL-ssb, pMAL-uvrA, pMAL-GroES, and pMAL-GroEL constructs separately, and gene expression was induced by isopropyl-beta-D-thiogalactopyranoside . The resulting fusion proteins were purified by affinity chromatography and confirmed by Western blot analysis using an anti-maltose-binding protein antibody . Furthermore, we have examined the pattern of T helper (Th) cell response from vaccinated BALB/c mice after in vitro stimulation with the recombinant (r) fusion proteins . In addition to T-cell proliferative responses, CD4+ T cells were tested for interleukin-2 (IL-2), IL-4, and gamma interferon (IFN-gamma) secretion . Primed CD4+ T cells proliferated to the rUvrA, rGroES, and rGroEL, but not to rSsb . The cytokine profile of the proliferating cells was characteristic of a Th1 type, as we detected IL-2 and IFN-gamma but not IL-4 in the T-cell culture supernatants . The recombinant B . abortus proteins were also screened in vivo to their ability to elicit DTH reaction in Brucella-sensitized guinea pigs . Moreover, the results of this study suggest that B . abortus rUvrA, rGroES, and rGroEL might be important sources of potentially protective molecules.

Structure, 1996 Sep 15, 4(9), 1093 - 104
Structural evidence for specific S8-RNA and S8-protein interactions within the 30S ribosomal subunit: ribosomal protein S8 from Bacillus stearothermophilus at 1.9 A resolution; Davies C et al.; BACKGROUND . Prokaryotic ribosomal protein S8 is an important RNA-binding protein that occupies a central position within the small ribosomal subunit . It interacts extensively with 16S rRNA and is crucial for the correct folding of the central domain of the rRNA . S8 also controls the synthesis of several ribosomal proteins by binding to mRNA . It binds specifically to very similar sites in the two RNA molecules . RESULTS . S8 is divided into two tightly associated domains and contains three regions that are proposed to interact with other ribosomal components: two potential RNA-binding sites, and a hydrophobic patch that may interact with a complementary hydrophobic region of S5 . The N-terminal domain fold is found in several proteins including two that bind double-stranded DNA . CONCLUSIONS . These multiple RNA-binding sites are consistent with the role of S8 in organizing the central domain and agree with the latest models of the 16S RNA which show that the S8 location coincides with a region of complicated nucleic-acid structure . The presence in a wide variety of proteins of a region homologous to the N-terminal domain supports the idea that ribosomal proteins must represent some of the earliest protein molecules.

Mutat Res, 1996 Sep 13, 370(2), 75 - 80
Genotoxic effects of metronidazole; Elizondo G et al.; Metronidazole (MTZ) is an effective agent used in the treatment of parasitic infections . Its genotoxic effects have been shown in a variety of prokaryotic systems; however, negative results have been reported in human in vivo studies . Due to its wide spread use, a study was performed to evaluate the chromosomal aberration frequencies in peripheral blood lymphocyte cultures from 10 individuals, before and after metronidazole treatment . A significant increase in the percentage of cells with chromatid and isochromatid breaks was observed after metronidazole treatment (1500 mg per day for 10 days) . The percentages of cells with aberrations did not correlate with the levels of MTZ found in plasma . Individual variability was observed with respect to both the induction of aberrations and the concentration of MTZ in plasma . They could represent differences at the metabolic level, since metronidazole is known to be biotransformed by a polymorphic P450 cytochrome, and its metabolites have shown mutagenic activity.

Biochem Biophys Res Commun, 1996 Sep 13, 226(2), 498 - 505
Prokaryotic DNA ligases unwind superhelical DNA; Ivanchenko M et al.; We have studied the effect on DNA topology of binding of prokaryotic DNA ligases (T4 and E . coli) to superhelical or nicked circular DNA . Performing topoisomerase I-mediated relaxation in the presence of increasing amounts of T4 ligase led to a shift in the topoisomer distribution to increasingly more negative values . This result suggested that T4 ligase unwound the DNA and was further substantiated by ligation of nicked circular molecules by E . coli DNA ligase in the presence of increasing amounts of T4 ligase . Such an experiment was possible since the two DNA ligases require different cofactors for enzymatic activity . Performing a similar experiment with reverse partners, using E . coli DNA ligase as ligand, and T4 ligase as sealing agent, we observed that the E . coli enzyme also unwound the DNA . Thus, prokaryotic DNA ligases can be added to an ever-growing list of DNA-binding proteins that unwind the DNA upon binding.

J Theor Biol, 1996 Sep 7, 182(1), 45 - 58
A complementary circular code in the protein coding genes; Arques DG et al.; Recently, shifted periodicities 1 modulo 3 and 2 modulo 3 have been identified in protein (coding) genes of both prokaryotes and eukaryotes with autocorrelation functions analysing eight of 64 trinucleotides (Arques et al., 1995) . This observation suggests that the trinucleotides are associated with frames in protein genes . In order to verify this hypothesis, a distribution of the 64 trinucleotides AAA,..., TTT is studied in both gene populations by using a simple method based on the trinucleotide frequencies per frame . In protein genes, the trinucleotides can be read in three frames: the reading frame 0 established by the ATG start trinucleotide and frame 1 (resp . 2) which is the frame 0 shifted by 1 (resp . 2) nucleotide in the 5'-3' direction . Then, the occurrence frequencies of the 64 trinucleotides are computed in the three frames . By classifying each of the 64 trinucleotides in its preferential occurrence frame, i.e . the frame associated with its highest frequency, three subsets of trinucleotides can be identified in the three frames . This approach is applied in the two gene populations . Unexpectedly, the same three subsets of trinucleotides are identified in these two gene populations: Tzero = Xzero {symbol: see text} inverted question markAAA,TTT inverted question mark with Xzero = inverted question markAAC,AAT,ACC,ATC,ATT, CAG,CTC,CTG,GAA,GAC,GAG, GAT,GCC,GGC,GGT,GTA,GTC,GTT,TAC,TTC inverted question mark in frame 0, T1 = X1 {symbol: see text} inverted question markCCC inverted question mark in frame 1 and T2 = X2 {symbol: see text} inverted question markGGG inverted question mark in frame 2, each subset Xzero, X1 and X2 having 20 trinucleotides . Surprisingly, these three subsets have five important properties: (i) the property of maximal circular code for Xzero (resp . X1, X2) allowing the automatical retrieval of frame 0 (resp . 1, 2) in any region of a protein gene model (formed by a series of trinucleotides of Xzero) without using a start codon; (ii) the DNA complementarity property C (e.g . C(AAC) = GTT): C(T0) = T0, C(T1) = T2 and C(T2) = T1 allowing the two paired reading frames of a DNA double helix simultaneously to code for amino acids; (iii) the circular permutation property P (e.g . P(AAC) = ACA): P(Xzero) = X1 and P(X1) = X2 implying that the two subsets X1 and X2 can be deduced from Xzero; (iv) the rarity property with an occurrence probability of Xzero equal to 6 x 10(-8); and (v) the concatenation property with: a high frequency (27.5%) of misplaced trinucleotides in the shifted frames, a maximum (13 nucleotides) length of the minimal window to automatically retrieve the frame and an occurrence of the four types of nucleotides in the three trinucleotides sites, in favour of an evolutionary code . In the Discussion, the identified subsets Tzero, T1 and T2 replaced in the three two-letter genetic alphabets purine/pyrimidine, amino/ceto and strong/weak interaction, allow us to deduce that the RNY model (R = purine = A or G, Y = pyrimidine = C or T, N = R or Y) (Eigen & Schuster, 1978) is the closest two-letter codon model to the trinucleotides of Tzero . Then, these three subsets are related to the genetic code . The trinucleotides of Tzero code for 13 amino acids: Ala, Asn, Asp, Gln, Glu, Gly, Ile, Leu, Lys, Phe, Thr, Tyr and Val . Finally, a strong correlation between the usage of the trinucleotides of Tzero in protein genes and the amino acid frequencies in proteins is observed as six among seven amino acids not coded by Tzero, have as expected the lowest frequencies in proteins of both prokaryotes and eukaryotes.

J Mol Biol, 1996 Sep 6, 261(5), 407 - 13
A 5 S rRNA gene is present in the mitochondrial genome of the protist Reclinomonas americana but is absent from red algal mitochondrial DNA; Lang BF et al.; Except in the case of land plants, mitochondrial ribosomes apparently lack a 5 S rRNA species, even though this small RNA is a component of all prokaryotic, chloroplast and eukaryotic cytosol ribosomes . In plants, the mitochondrial 5 S rRNA is encoded by mtDNA and differs in sequence from the 5 S rRNA specified by plant nuclear and chloroplast genomes . A distinctive 5 S rRNA component has not been found in the mitochondrial ribosomes of non-plant eukaryotes and, with the notable exception of the chlorophycean alga, Prototheca wickerhamii, a 5 S rRNA gene has not been identified in those non-plant mtDNAs characterized to date . Here, we report the presence of a 5 S rRNA gene in the mtDNA of the heterotrophic flagellate Reclinomonas americana . This unicellular eukaryote is a member of the jakobid flagellates, an early-diverging group of protists that share ultrastructural characteristics with the retortamonads, primitive protists that lack mitochondria . We report sequence data from the mtDNAs of the red algae Porphyra purpurea and Gracilariopsis lemaneiformis, which we use to evaluate a recent claim that a 5 S rRNA gene exists in the mtDNA of a third rhodophyte alga, Chondrus crispus . Our results lead us to the opposite conclusion: that a 5 S rRNA gene is not encoded by red algal mtDNA . In view of the accumulating evidence favoring a monophyletic origin of the mitochondrial genome, it is likely that a 5 S rRNA gene was present in an ancestral proto-mitochondrial genome, and that contemporary mtDNA-encoded 5 S rRNA genes have all descended from this ancestral gene . Considering the highly restricted phylogenetic distribution of identified mtDNA-encoded 5 S rRNA genes, it follows that the mitochondrial 5 S rRNA gene must have been lost multiple times during evolutionary diversification of the eukaryotic lineage.

Proc Natl Acad Sci U S A, 1996 Sep 3, 93(18), 9384 - 8
Chromatin structure and gene expression; Felsenfeld G et al.; It is now well understood that chromatin structure is perturbed in the neighborhood of expressed genes . This is most obvious in the neighborhood of promoters and enhancers, where hypersensitivity to nucleases marks sites that no longer carry canonical nucleosomes, and to which transcription factors bind . To study the relationship between transcription factor binding and the generation of these hypersensitive regions, we mutated individual cis-acting regulatory elements within the enhancer that lies between the chicken beta- and epsilon-globin genes . Constructions carrying the mutant enhancer were introduced by stable transformation into an avian erythroid cell line . We observed that weakening the enhancer resulted in creation of two classes of site: those still completely accessible to nuclease attack and those that were completely blocked . This all-or-none behavior suggests a mechanism by which chromatin structure can act to sharpen the response of developmental systems to changing concentrations of regulatory factors . Another problem raised by chromatin structure concerns the establishment of boundaries between active and inactive chromatin domains . We have identified a DNA element at the 5' end of the chicken beta-globin locus, near such a boundary, that has the properties of an insulator; in test constructions, it blocks the action of an enhancer on a promoter when it is placed between them . We describe the properties and partial dissection of this sequence . A third problem is posed by the continued presence of nucleosomes on transcribed genes, which might prevent the passage of RNA polymerase . We show, however, that a prokaryotic polymerase can transcribe through a histone octamer on a simple chromatin template . The analysis of this process reveals that an octamer is capable of transferring from a position in front of the polymerase to one behind, without ever losing its attachment to the DNA.

EMBO J, 1996 Sep 2, 15(17), 4749 - 58
Structure of crystalline Escherichia coli methionyl-tRNA(f)Met formyltransferase: comparison with glycinamide ribonucleotide formyltransferase; Schmitt E et al.; Formylation of the methionyl moiety esterified to the 3' end of tRNA(f)Met is a key step in the targeting of initiator tRNA towards the translation start machinery in prokaryotes . Accordingly, the presence of methionyl-tRNA(f)Met formyltransferase (FMT), the enzyme responsible for this formylation, is necessary for the normal growth of Escherichia coli . The present work describes the structure of crystalline E.coli FMT at 2.0 A, resolution . The protein has an N-terminal domain containing a Rossmann fold . This domain closely resembles that of the glycinamide ribonucleotide formyltransferase (GARF), an enzyme which, like FMT, uses N-10 formyltetrahydrofolate as formyl donor . However, FMT can be distinguished from GARF by a flexible loop inserted within its Rossmann fold . In addition, FMT possesses a C-terminal domain with a beta-barrel reminiscent of an OB fold . This latter domain provides a positively charged side oriented towards the active site . Biochemical evidence is presented for the involvement of these two idiosyncratic regions (the flexible loop in the N-terminal domain, and the C-terminal domain) in the binding of the tRNA substrate.

J Photochem Photobiol B, 1996 Sep, 35(3), 141 - 8
Stress proteins in the cellular response to ultraviolet radiation; Trautinger F et al.; Virtually all cells-from prokaryotes to highly differentiated mammalian tissues-respond to a sudden increase in temperature with increased production of a limited set of proteins, called heat shock proteins or stress proteins (hsp) . Other stress factors such as alcohol, heavy metals, oxidants and agents leading to protein denaturation are equally able to induce a similar response . Induction of hsp is followed by a transient state of increased resistance to further stress . Many hsp function as "molecular chaperones" by binding to partially folded or misfolded proteins thus preventing their irreversible denaturation during stress exposure . The high evolutionary conservation of this reaction suggests its importance for the survival of cells and tissues under hostile environment conditions . Ultraviolet radiation (UV) exerts many potentially harmful effects on prokaryotic and eukaryotic cells and hsp may help the cell to cope with UV-induced damage . This review will focus on the role of hsp in the cellular response of mammalian skin to UV . Hsp have been detected in resting as well as stress exposed epidermal and dermal cells and experimental evidence points to the fact that these proteins mediate protection from UV induced cell death in vitro and in vivo . Experimental studies further indicate that UV itself might be able to induce the expression of specific hsp . Thus, hsp might provide an adaptive cellular response to increasing exposure to UV . Furthermore, UV-activation of hsp synthesis may provide a valuable model for investigation of the transcription regulation of UV-induced gene expression.

Plant Mol Biol, 1996 Sep, 31(6), 1185 - 94
Light-inducible gene HSP70B encodes a chloroplast-localized heat shock protein in Chlamydomonas reinhardtii; Drzymalla C et al.; The nuclear heat shock gene HSP70B of Chlamydomonas reinhardtii is inducible by heat stress and light . Induction by either environmental cue resulted in a transient elevation in HSP70B protein . Here we describe the organization and nucleotide sequence of the HSP70B gene . The deduced protein exhibits a distinctly higher homology to prokaryotic HSP70s than to those of eukaryotes, including the cytosolic HSP70A of Chlamydomonas reinhardtii . The HSP70B protein, as previously demonstrated by in vitro translation, is synthesized with a cleavable presequence . Using an HSP70B-specific antibody, this heat shock protein was localized to the chloroplast by cell fractionation experiments . A stromal location was suggested by the presence of a conserved sequence motif used for cleavage of presequences by a signal peptidase of the stroma . Amino acid alignments of HSP70 proteins from various organisms and different cellular compartments allowed the identification of sequence motifs, which are diagnostic for HSP70s of chloroplasts and cyanobacteria.

Med Hypotheses, 1996 Sep, 47(3), 199 - 213
Evolution of Hodgkin's disease; Okuyama S; Hodgkin's disease is an oncogenic core disorder characterized by both mitotic and amitotic neoplastic multiplication, and is associated with collateral disorders such as lacunar formation and leukocytic infiltration . Research has demonstrated that Hodgkin's disease progresses stepwise, beginning with a reversible, biological stage during which Hodgkin and Reed-Sternberg cells are formed, followed by constitutive, but reversible Hodgkinogenic medical stage that leads to an irreversible, systemic and fatal proto-oncogenic stage . This disease results from collateral activation of cytokine and archaic oncogenes, suppression of DNA repair genes in multiple chromosomes . The variability of Hodgkin's disease manifestations has required antisynthetic (antimetabolites, radiotherapy), anti-viral (acyclovir) and anti-mitotic (vincristine, vinblastine) for different loci minores of treatment . Continued molecular biological research of the ancestral and prokaryotic oncogenes is recommended.

Mol Biochem Parasitol, 1996 Sep, 80(1), 113 - 7
Ancylostoma caninum anticoagulant peptide: cloning by PCR and expression of soluble, active protein in E . coli; Cappello M et al.; Ancylostoma caninum Anticoagulant Peptide (AcAP) is the major anticoagulant activity present in extracts of adult Ancylostoma caninum hookworms . This 8.7 kDa protein is a potent and specific inhibitor of human coagulation factor Xa . Using PCR, we have isolated a cDNA encoding for AcAP from an adult A . caninum cDNA library . The 5' end of the AcAP cDNA was identified by reverse transcription PCR (RT-PCR) using A . caninum cDNA and a 5' primer corresponding to a nematode spliced leader sequence . The AcAP cDNA was expressed in E . coli using a prokaryotic expression vector, and the recombinant fusion protein (rAcAP) was purified to homogeneity using nickel resin affinity chromatography and reverse phase HPLC . Purified rAcAP is comparable to the native protein in inhibitor activity, with an apparent equilibrium inhibitory dissociation constant (Ki*) for the inhibition of factor Xa of 265 +/- 71 pM . The purified protein also prolongs the prothrombin and partial thromboplastic times of human plasma in a dose dependent manner.

Eur J Cell Biol, 1996 Sep, 71(1), 105 - 19
Internucleosomal DNA fragmentation in cultured cells under conditions reported to induce apoptosis may be caused by mycoplasma endonucleases; Paddenberg R et al.; DNA fragmentation is a common biochemical hallmark of apoptosis . It is catalyzed by endogenous Ca2+, Mg(2+)-dependent endonuclease(s) . Although the exact identity of the apoptotic endonuclease is still a matter of debate, a number of candidate nucleases have been proposed like NUC18, DNase II and DNase I . Relatively large amounts of nucleases are also expressed by mycoplasmas, cell wall-less bacteria of the class Mollicutes, which are found as contaminants in up to 45% of the continuous cell lines in current use . In order to clarify the effect of these pathogens on the investigation of apoptosis in cell culture systems, we looked for biochemical markers (DNA fragmentation, nuclease expression) and morphological changes characteristic of apoptosis (cell shrinkage, chromatin condensation, apoptotic bodies) in Mycoplasma hyorhinis-free and -infected cultures of the human pancreatic adenocarcinoma cell line PaTu 8902 and of mouse NIH 3T3 fibroblasts . For that purpose we employed cells cultured under standard conditions and cells exposed to the protein synthesis inhibitor cycloheximide, which is known to induce apoptosis in various cell systems . After exposure to cycloheximide only the mycoplasma-positive cells exhibited internucleosomal DNA degradation . In contrast, nuclease activities in the molecular range of 47 to 54 kDa were detected in cell homogenates and culture supernatants of infected cultures of both control and cycloheximide-treated cells, whereas mycoplasma-free cultures were nuclease-negative . The expression of the nucleases and the cycloheximide-induced DNA fragmentation were suppressed by the prokaryote-specific protein synthesis inhibitor chloramphenicol . Moreover, partially purified nucleases from supernatants of infected cells were able to cleave the DNA of isolated substrate nuclei at internucleosomal sites . These data indicate that DNA ladder formation in cell culture systems can also be caused by mycoplasmal nucleases which apparently penetrate the host cells after cycloheximide treatment or more generally after cellular stress . Therefore, internucleosomal DNA fragmentation in established cell lines has to be regarded with care, unless mycoplasmal infection can be excluded, or the existence of endogenous endonucleases can be proven . The presence of endonucleolytic activities of about 47 to 54 kDa molecular mass has now to be regarded as highly indicative of contaminations with M . hyorhinis . In contrast, the expression of an apoptotic morphology was not restricted to infected cells; in both mycoplasma-free and -contaminated cultures, cells with condensed chromatin were observed after staining with the DNA binding dye Hoechst 33342 . Electron microscopic studies revealed that most of the cells containing compacted DNA were phagocytosed by unaffected fellow cells . Presumably because of the relatively long exposure (72 h) to cycloheximide we also observed secondary necrosis as indicated by the parallel occurrence of morphological characteristics of apoptosis (chromatin condensation) and necrosis (loss of membrane integrity and organelle swelling).

Protein Sci, 1996 Sep, 5(9), 1939 - 41
Members of the immunoglobulin superfamily in bacteria; Bateman A et al.; We report a prediction that two prokaryotic proteins contain immunoglobulin superfamily domains . Immunoglobulin-like folds have been identified previously in prokaryotic proteins, but these share no recognizable sequence similarity with eukaryotic immunoglobulin superfamily (IgSF) folds, and may be the result of the physics and chemistry of proteins favoring certain common folds . In contrast, the prokaryotic proteins identified have sequences whose match to the immunoglobulin superfamily can be detected by hidden Markov modeling, BLASTP matches, key residue analysis, and secondary structure predictions . We propose that these prokaryotic immunoglobulin-like domains are almost certain to be related by divergence from a common ancestor to eukaryotic immunoglobulin superfamily domains.

Biotechnol Prog, 1996 Sep-Oct, 12(5), 645 - 9
A semicontinuous prokaryotic coupled transcription/translation system using a dialysis membrane; Kim DM et al.; This report describes a novel and simple cell-free protein synthesis system . In this paper, we prove that the short duration of protein synthesis in a conventional cell-free protein synthesis system of batch configuration can be attributed both to depletion of energy sources and deactivation of S30 extract by small-molecule byproducts produced during the protein synthesis . The reaction period of cell-free protein synthesis system could be extended through an operation of a continuous-flow cell-free protein synthesis system, which was originally developed by Spirin . However, inspite of the greatly extended reaction period, the final amount of cell-free produced protein was not significantly larger than that can be obtained from a batch system due to the reduced rate of protein synthesis . It was supposed that the reduced rate of protein synthesis in the continuous-flow system was attributed to leakage of translational components through the ultrafiltration membrane during the operation of the continuous-flow system . To solve such a problem of the continuous-flow system, we have developed and operated a novel reactor for cell-free protein synthesis . By use of this system, protein synthesis occurred for at least 14 h, yielding 1.2 mg/mL CAT protein . The present system is superior to the continuous-flow system as well as the conventional batch system in that it enables extremely high productivity without using any complex and hard-to-handle apparatus . As far as we know, the yield of cell-free protein synthesis given above is the best of the results reported to date.

Am J Surg, 1996 Sep, 172(3), 291 - 6
The pathogenesis of Staphylococcus aureus in the trauma patient and potential future therapies; Villavicencio RT et al.; BACKGROUND: Staphylococcus aureus is the most frequently isolated pathogen in the trauma patient and uses multiple virulent factors to cause infection . At the cellular level, infection begins with the prokaryotic bacterial cell manipulating the eukaryotic host cell through its virulent factors . Researching this cellular interaction by describing the mechanisms of actions of various virulent factors may lead to new preventive therapies which will make the trauma patient less susceptible to S aureus infections . METHODS: Surgical, medical, and microbial literature was reviewed to provide an update on S aureus pathogenesis . RESULTS: Novel future therapies, in addition to antibiotics, are being devised based on understanding the molecular nature of S aureus pathogenesis . CONCLUSION: The impact of S aureus on trauma will increase as S aureus develops more antibiotic resistance and as the trauma population becomes older and includes an increasing proportion of immunocompromised patients . To meet the challenge of increased virulence, trauma surgeons should be directly involved in the research of microbial pathogenesis.

Microbiol Rev, 1996 Sep, 60(3), 512 - 38
Strategies for achieving high-level expression of genes in Escherichia coli; Makrides SC; Progress in our understanding of several biological processes promises to broaden the usefulness of Escherichia coli as a tool for gene expression . There is an expanding choice of tightly regulated prokaryotic promoters suitable for achieving high-level gene expression . New host strains facilitate the formation of disulfide bonds in the reducing environment of the cytoplasm and offer higher protein yields by minimizing proteolytic degradation . Insights into the process of protein translocation across the bacterial membranes may eventually make it possible to achieve robust secretion of specific proteins into the culture medium . Studies involving molecular chaperones have shown that in specific cases, chaperones can be very effective for improved protein folding, solubility, and membrane transport . Negative results derived from such studies are also instructive in formulating different strategies . The remarkable increase in the availability of fusion partners offers a wide range of tools for improved protein folding, solubility, protection from proteases, yield, and secretion into the culture medium, as well as for detection and purification of recombinant proteins . Codon usage is known to present a potential impediment to high-level gene expression in E . coli . Although we still do not understand all the rules governing this phenomenon, it is apparent that "rare" codons, depending on their frequency and context, can have an adverse effect on protein levels . Usually, this problem can be alleviated by modification of the relevant codons or by coexpression of the cognate tRNA genes . Finally, the elucidation of specific determinants of protein degradation, a plethora of protease-deficient host strains, and methods to stabilize proteins afford new strategies to minimize proteolytic susceptibility of recombinant proteins in E . coli.

Protein Expr Purif, 1996 Sep, 8(2), 175 - 82
Expression and radiolabeling of recombinant proteins containing a phosphorylation motif; Mohanraj D et al.; Radiolabeled proteins are useful in basic and clinical research . Current methods available for radiolabeling proteins involve chemical derivatization, resulting in multiple additions of radionuclides at random sites . A method designed to specifically localize the radionuclide to a unique site will offer advantages of control and predictability in radiolabeling . We have studied the usefulness of a prokaryotic expression vector by incorporating the coding sequence of a consensus phosphorylation motif (Kemptide) for the cAMP-dependent protein kinase A immediately upstream to the multiple cloning site . This vector was used to express five different recombinant proteins with a phosphorylation site at the amino terminus . In addition, the phosphorylation motif was introduced into two other proteins and expressed in yeast . The genetically engineered proteins were purified to homogeneity by affinity chromatography and radiolabeled with {gamma32P}ATP in vitro . All seven proteins used in this study could be expressed with the phosphorylation sequence at their amino terminus and specifically labeled without loss of biological activity . This strategy allows the option of labeling proteins to high or low specific radioactivity and holds potential for in vitro binding and in vivo localization studies.

Arch Biochem Biophys, 1996 Sep 1, 333(1), 66 - 74
Functional expression of the human MDR1 gene in Escherichia coli; George AM et al.; In this preliminary study, we report the cloning of the human MDR1 cDNA into a prokaryotic expression vector and the consequent functional expression of heterologous P-glycoprotein in Escherichia coli . We demonstrate increased resistance to the P-glycoprotein substrates TPA+, TPP+, and puromycin; reduced accumulation of TPP+ and tetracycline by resistant cells; and the expression of a full-length immunoreactive P-glycoprotein molecule in the membrane fraction of resistant cells . The obvious structural and functional similarities of P-gp to prokaryotic ABC transporters and other efflux transporters argues for a more complete study of the consequences pertaining to the expression of human P-glycoprotein in E . coli.

J Bacteriol, 1996 Sep, 178(17), 5263 - 71
Isolation, purification, and in vitro characterization of recessive-lethal-mutant RNA polymerases from Escherichia coli; Tavormina PL et al.; The beta subunit of prokaryotic RNA polymerase shares significant sequence similarity with its eukaryotic and archaeal counterparts across most of the protein . Nine segments of particularly high similarity have been identified and are termed segments A through I . We have isolated severely defective Escherichia coli RNA polymerase mutants, most of which are unable to support bacterial growth . The majority of the substitutions affect residues in one of the conserved segments of beta, including invariant residues in segments D (amino acids 548 to 577), E (amino acids 660 to 678), and I (amino acids 1198 to 1296) . In addition, recessive-lethal mutations that affect residues highly conserved only among prokaryotes were identified . They include a substitution in the extreme amino terminus of beta, a region in which no substitutions have previously been identified, and one rpoB mutation that truncates the polypeptide without abolishing minimal polymerase function in vitro . To examine the recessive-lethal alleles in vitro, we devised a novel method to remove nonmutant enzyme from RNA polymerase preparations by affinity tagging the chromosomal rpoB gene . In vitro examination of a subset of purified recessive-lethal RNA polymerases revealed that several substitutions, including all of those altering conserved residues in segment I, severely decrease transcript elongation and increase termination . We discuss the insights these mutants lend to a structure-function analysis of RNA polymerase.

J Bacteriol, 1996 Sep, 178(17), 5065 - 70
A modular family 19 chitinase found in the prokaryotic organism Streptomyces griseus HUT 6037; Ohno T et al.; The specificity of chitinase C-1 of Streptomyces griseus HUT 6037 for the hydrolysis of the beta-1,4-glycosidic linkages in partially acetylated chitosan is different from that of other microbial chitinases . In order to study the primary structure of this unique chitinase, the chiC gene specifying chitinase C-1 was cloned and its nucleotide sequence was determined . The gene encodes a polypeptide of 294 amino acids with a calculated size of 31.4 kDa . Comparison of the amino acid sequence of the deduced polypeptide with that of other proteins revealed a C-terminal catalytic domain displaying considerable sequence similarity to the catalytic domain of plant class I, II, and IV chitinases which form glycosyl hydrolase family 19 . The N-terminal domain of the deduced polypeptide exhibits sequence similarity to substrate-binding domains of several microbial chitinases and cellulases but not to the chitin-binding domains of plant chitinases . The previously purified chitinase C-1 from S . griseus is suggested to be generated by proteolytic removal of the N-terminal chitin-binding domain and corresponds to the catalytic domain of the chitinase encoded by the chiC gene . High-performance liquid chromatography analysis of the hydrolysis products from N-acetyl chitotetraose revealed that chitinase C-1 catalyzes hydrolysis of the glycosidic bond with inversion of the anomeric configuration, in agreement with the previously reported inverting mechanism of plant class I chitinases . This is the first report of a family 19 chitinase found in an organism other than higher plants.

J Mol Evol, 1996 Sep, 43(3), 216 - 23
A relationship between GC content and coding-sequence length; Oliver JL et al.; Since base composition of translational stop codons (TAG, TAA, and TGA) is biased toward a low G+C content, a differential density for these termination signals is expected in random DNA sequences of different base compositions . The expected length of reading frames (DNA segments of sense codons flanked by in-phase stop codons) in random sequences is thus a function of GC content . The analysis of DNA sequences from several genome databases stratified according to GC content reveals that the longest coding sequences-exons in vertebrates and genes in prokaryotes-are GC-rich, while the shortest ones are GC-poor . Exon lengthening in GC-rich vertebrate regions does not result, however, in longer vertebrate proteins, perhaps because of the lower number of exons in the genes located in these regions . The effects on coding-sequence lengths constitute a new evolutionary meaning for compositional variations in DNA GC content.

J Mol Biol, 1996 Aug 30, 261(4), 568 - 85
Molecular evolution of the C-terminal cytoplasmic domain of a superfamily of bacterial receptors involved in taxis; Le Moual H et al.; Twenty-nine proteins from 16 different species of prokaryotes revealed an extensive sequence homology with the cytoplasmic domain of the Escherichia coli aspartate receptor . The high percentage of identity indicated that they constitute a superfamily of proteins . A consensus secondary structure consisting mostly of alpha-helices was predicted . The occurrence of a seven-residue repeat (a-b-c-d-e-f-g), in which both the a and d residues were hydrophobic with few exceptions, provided additional evidence for a conserved alpha-helical conformation . Sequence alignments, together with the predicted secondary structure, led to identification of the boundaries for the functional units constituting the cytoplasmic domain . Putative methylation sites were assigned for all the members of this superfamily . These proteins could be grouped into three classes based on the presence of 14-residue insertion/deletion regions found within both the signalling and the methylation functional units of the cytoplasmic domain . The gene coding for the C-terminal cytoplasmic domain of these proteins apparently evolved through gene duplication from a common ancestor in which the four original 14-residue insertion/deletion regions were deleted two by two during evolution.

J Biol Chem, 1996 Aug 30, 271(35), 21340 - 4
Primary folding of aspartylglucosaminidase . Significance of disulfide bridges and evidence of early multimerization; Riikonen A et al.; Aspartylglucosaminidase (AGA) is a lysosomal enzyme involved in the degradation of N-linked glycoproteins in lysosomes . AGA is synthesized as an inactive precursor molecule, which is rapidly activated in the endoplasmic reticulum by a proteolytic cleavage into alpha- and beta-subunits . We have recently determined the three-dimensional structure of AGA and shown that it is a globular molecule with a heterotetrameric (alphabeta)2 structure . On the basis of structural and functional analyses, AGA seems to be the first mammalian protein belonging to a newly described protein family, the N-terminal nucleophile hydrolases . Because the activation of the prokaryotic members of the N-terminal nucleophile hydrolase family seems to be triggered by the assembly of the subunits, we have studied the initial folding and oligomerization of AGA and provide evidence that dimerization of two precursor molecules in the endoplasmic reticulum is a prerequisite for the activation of AGA . To gain further information on the structural determinants influencing the early folding of AGA, we used site-specific mutagenesis of cysteine residues to define the role of intrachain disulfide bridges in the folding and activation of the enzyme . The N-terminal disulfide bridges in both the alpha- and beta-subunits seem to have only a stabilizing role, whereas the C-terminal disulfide bridge in both subunits evidently plays an important role in the early folding and activation of AGA.

J Biol Chem, 1996 Aug 23, 271(34), 20861 - 7
The amino-terminal module of the C4b-binding protein beta-chain contains the protein S-binding site; Hardig Y et al.; Human C4b-binding protein (C4BP) is composed of multiple alpha-chains associated with a single beta-chain . Each chain is composed of homologous, tandemly arranged repeats of so-called short consensus repeats (SCRs) . We have previously shown that the three SCR modules of the beta-chain contain a high affinity binding site for anticoagulant vitamin K-dependent protein S . On the basis of experiments using synthetic peptides, residues 31-45 of the amino-terminal SCR (SCR-1) in the beta-chain were suggested to be involved in protein S binding, but it is not known whether SCR-1 contains the entire protein S-binding site . To address this question, two different truncated forms of the beta-chain (beta1,2 and beta2, 3) were expressed in a prokaryotic expression system . The beta1,2 construct (SCR-1 + SCR-2) contained the high affinity binding site for protein S in contrast to beta2,3 (SCR-2 + SCR-3), which did not bind protein S . Unfortunately, it was not possible to express SCR-1 alone in this system . To further elucidate whether the protein S-binding site is fully contained in SCR-1 or whether SCR-2 is also required, recombinant alpha/beta-chain chimeras were constructed . These chimeras were composed of alpha-chains with one, two, or three of the amino-terminal SCR modules replaced by the beta-chain counterpart and were expressed in a eukaryotic expression system . All recombinant variants were retained within the cells and could be extracted in biologically active forms . The three alpha/beta-chain chimeras bound protein S equally well, with a Ka of approximately 2.3 x 10(8) +/- 0.2 M-1 as compared with 2.1 x 10(8) +/- 0.3 M-1 for plasma-purified C4BP . These results show that the entire protein S-binding site on C4BP is contained within beta-chain SCR-1.

J Biol Chem, 1996 Aug 23, 271(34), 20242 - 5
Two yeast homologs of ECA39, a target for c-Myc regulation, code for cytosolic and mitochondrial branched-chain amino acid aminotransferases; Eden A et al.; ECA39 was isolated as a target gene for c-Myc regulation in mice . We identified two homologs for the murine ECA39 in the yeast Saccharomyces cerevisiae, ECA39 and ECA40, as well as two human homologs . These genes show a significant homology to prokaryotic branched-chain amino acid aminotransferase (BCAT) (EC) . To understand the function of eukaryotic ECA39 and ECA40, we deleted either gene from the yeast genome . Activity of branched-chain amino acid aminotransferase was measured in the wild-type and mutants with either leucine, isoleucine, or valine as substrates . The results demonstrate that in S . cerevisiae ECA39 and ECA40 code for mitochondrial and cytosolic branched-chain amino acid aminotransferases, respectively . ECA39 is highly expressed during log phase and is down-regulated during the stationary phase of growth, while ECA40 shows an inverse pattern of gene expression . In agreement with these results, while we previously showed that deletion of ECA39 affected the cell cycle in proliferating cells, we do not observe a growth phenotype in eca40Delta cells . We suggest that BCAT is a target for c-Myc activity and discuss the evolutionary conservation of prokaryotic and eukaryotic BCATs and their possible involvement in regulation of cell proliferation.

Proc Natl Acad Sci U S A, 1996 Aug 20, 93(17), 9061 - 6
Gene recognition via spliced sequence alignment; Gelfand MS et al.; Gene recognition is one of the most important problems in computational molecular biology . Previous attempts to solve this problem were based on statistics, and applications of combinatorial methods for gene recognition were almost unexplored . Recent advances in large-scale cDNA sequencing open a way toward a new approach to gene recognition that uses previously sequenced genes as a clue for recognition of newly sequenced genes . This paper describes a spliced alignment algorithm and software tool that explores all possible exon assemblies in polynomial time and finds the multiexon structure with the best fit to a related protein . Unlike other existing methods, the algorithm successfully recognizes genes even in the case of short exons or exons with unusual codon usage; we also report correct assemblies for genes with more than 10 exons . On a test sample of human genes with known mammalian relatives, the average correlation between the predicted and actual proteins was 99% . The algorithm correctly reconstructed 87% of genes and the rare discrepancies between the predicted and real exon-intron structures were caused either by short (less than 5 amino acids) initial/terminal exons or by alternative splicing . Moreover, the algorithm predicts human genes reasonably well when the homologous protein is nonvertebrate or even prokaryotic . The surprisingly good performance of the method was confirmed by extensive simulations: in particular, with target proteins at 160 accepted point mutations (PAM) (25% similarity), the correlation between the predicted and actual genes was still as high as 95%.

EMBO J, 1996 Aug 15, 15(16), 4423 - 33
A conserved domain of the large subunit of replication factor C binds PCNA and acts like a dominant negative inhibitor of DNA replication in mammalian cells; Fotedar R et al.; Replication factor C (RF-C), a complex of five polypeptides, is essential for cell-free SV40 origin-dependent DNA replication and viability in yeast . The cDNA encoding the large subunit of human RF-C (RF-Cp145) was cloned in a Southwestern screen . Using deletion mutants of RF-Cp145 we have mapped the DNA binding domain of RF-Cp145 to amino acid residues 369-480 . This domain is conserved among both prokaryotic DNA ligases and eukaryotic poly(ADP-ribose) polymerases and is absent in other subunits of RF-C . The PCNA binding domain maps to amino acid residues 481-728 and is conserved in all five subunits of RF-C . The PCNA binding domain of RF-Cp145 inhibits several functions of RF-C, such as: (i) in vitro DNA replication of SV40 origin-containing DNA; (ii) RF-C-dependent loading of PCNA onto DNA; and (iii) RF-C-dependent DNA elongation . The PCNA binding domain of RF-Cp145 localizes to the nucleus and inhibits DNA synthesis in transfected mammalian cells . In contrast, the DNA binding domain of RF-Cp145 does not inhibit DNA synthesis in vitro or in vivo . We therefore conclude that amino acid residues 481-728 of human RF-Cp145 are critical and act as a dominant negative mutant of RF-C function in DNA replication in vivo.

Structure, 1996 Aug 15, 4(8), 943 - 55
Crystal structure of a eukaryotic (pea seedling) copper-containing amine oxidase at 2.2 A resolution; Kumar V et al.; BACKGROUND: Copper-containing amine oxidases catalyze the oxidative deamination of primary amines to aldehydes, in a reaction that requires free radicals . These enzymes are important in many biological processes, including cell differentiation and growth, would healing, detoxification and signalling . The catalytic reaction requires a redox cofactor, topa quinone (TPQ), which is derived by post-translational modification of an invariant tyrosine residue . Both the biogenesis of the TPQ cofactor and the reaction catalyzed by the enzyme require the presence of a copper atom at the active site . The crystal structure of a prokaryotic copper amine oxidase from E . coli (ECAO) has recently been reported . RESULTS: The first structure of a eukaryotic (pea seedling) amine oxidase (PSAO) has been solved and refined at 2.2 A resolution . The crystallographic phases were derived from a single phosphotungstic acid derivative . The positions of the tungsten atoms in the W12 clusters were obtained by molecular replacement using E . coli amine oxidase as a search model . The methodology avoided bias from the search model, and provides an essentially independent view of a eukaryotic amine oxidase . The PSAO molecule is a homodimer; each subunit has three domains . The active site of each subunit lies near an edge of the beta-sandwich of the largest domain, but is not accessible from the solvent . The essential active-site copper atom is coordinated by three histidine side chains and two water molecules in an approximately square-pyramidal arrangement . All the atoms of the TPQ cofactor are unambiguously defined, the shortest distance to the copper atom being approximately 6 A . CONCLUSIONS: There is considerable structural homology between PSAO and ECAO . A combination of evidence from both structures indicates that the TPQ side chain is sufficiently flexible to permit the aromatic grouf to rotate about the Cbeta-Cgamma bond, and to move between bonding and non-bonding positions with respect to the Cu atom . Conformational flexibility is also required at the surface of the molecule to allow the substrates access to the active site, which is inaccessible to solvent, as expected for an enzyme that uses radical chemistry.

Nucleic Acids Res, 1996 Aug 15, 24(16), 3267 - 75
Methylation inhibitors can increase the rate of cytosine deamination by (cytosine-5)-DNA methyltransferase; Zingg JM et al.; The target cytosines of (cytosine-5)-DNA methyltransferases in prokaryotic and eukaryotic DNA show increased rates of C-->T transition mutations compared to non-target cytosines . These mutations are induced either by the spontaneous deamination of 5-mC-->T generating inefficiently repaired G:T rather than G:U mismatches, or by the enzyme-induced C-->U deamination which occurs under conditions of reduced levels of S-adenosylmethionine (AdoMet) and S-adenosylhomocysteine (AdoHcy) . We tested whether various inhibitors of (cytosine-5)-DNA methyltransferases analogous to AdoMet and AdoHcy would affect the rate of enzyme-induced deamination of the target cytosine by M.HpaII and M.SssI . Interestingly, we found two compounds, sinefungin and 5'-amino-5'-deoxyadenosine, that increased the rate of deamination 10(3)-fold in the presence and 10(4)-fold in the absence of AdoMet and AdoHcy . We have therefore identified the first mutagenic compounds specific for the target sites of (cytosine-5)-DNA methyltransferases . A number of analogs of AdoMet and AdoHcy have been considered as possible antiviral, anticancer, antifungal and antiparasitic agents . Our findings show that chemotherapeutic agents with affinities to the cofactor binding pocket of (cytosine-5)-DNA methyltransferase should be tested for their potential mutagenic effects.

Biochem J, 1996 Aug 15, 318 ( Pt 1), 133 - 8
Towards a classification of glycosyltransferases based on amino acid sequence similarities: prokaryotic alpha-mannosyltransferases; Geremia RA et al.; A number of genes encoding bacterial glycosyltransferases have been sequenced during the last few years, but their low sequence similarity has prevented a straightforward grouping of these enzymes into families . The sequences of several bacterial alpha-mannosyltransferases have been compared using current alignment algorithms as well as hydrophobic cluster analysis (HCA) . These sequences show a similarity which is significant but too low to be reliably aligned using automatic alignment methods . However, a region spanning approx . 270 residues in these proteins could be aligned by HCA, and several invariant amino acid residues were identified . These features were also found in several other glycosyltransferases, as well as in proteins of unknown function present in sequence databases . This similarity most probably reflects the existence of a family of proteins with conserved structural and mechanistic features . It is argued that the present IUBMB classification of glycosyltransferases could be complemented by a classification of these enzymes based on sequence similarities analogous to that which we proposed for glycosyl hydrolases {Henrissat, B . (1991) Biochem . J . 280, 309-316}.

FEBS Lett, 1996 Aug 12, 391(3), 330 - 2
Role of the conserved aspartate and phenylalanine residues in prokaryotic and mitochondrial elongation factor Ts in guanine nucleotide exchange; Zhang Y et al.; The guanine nucleotide exchange reaction catalyzed by elongation factor Ts is proposed to arise from the intrusion of the side chains of D80 and F81 near the Mg2+ binding site in EF-Tu . D80A and F81A mutants of E . coli EF-Ts were 2-3-fold less active in promoting GDP exchange with E . coli EF-Tu while the D80AF81A mutant was nearly 10-fold less active . The D84 and F85 mutants of EF-Tsmt were 5-10-fold less active in stimulating the activity of EF-Tumt . The double mutation completely abolished the activity of EF-Tsmt.

J Biol Chem, 1996 Aug 9, 271(32), 19617 - 24
Regulation of the heat-shock protein 70 reaction cycle by the mammalian DnaJ homolog, Hsp40; Minami Y et al.; The effects of the human DnaJ homolog, Hsp40, on the ATPase and chaperone functions of the constitutively expressed Hsp70 homolog, Hsc70, were analyzed . Hsp40 stimulates the hydrolysis of ATP by Hsc70, causing a approximately 7-fold increase in its steady-state ATPase activity . In contrast to the prokaryotic Hsp70 system, ATP-hydrolysis and not the release of bound ADP is the rate-limiting step in the overall ATPase cycle of mammalian Hsc70 . The ability to activate the Hsc70 ATPase is partially preserved in a deletion mutant containing the J-domain and the G/F region of Hsp40 but not in a deletion mutant that contains the J-domain alone . As a result of its ATPase stimulating activity, addition of Hsp40 allows Hsc70 to bind peptide in the presence of ATP, whereas in the absence of Hsp40, peptide is efficiently released upon ATP binding to Hsc70 . The functional cooperation of Hsp40 with Hsc70 is essential to ensure the ATP hydrolysis-dependent binding of aggregation-sensitive denatured polypeptides, such as thermally denatured firefly luciferase and chemically denatured rhodanese . Binding of these proteins results in the formation of ternary complexes of Hsc70, Hsp40, and substrates . Hsc70 and Hsp40 cooperate with further factors in protein renaturation, as demonstrated by the finding that luciferase, thermally denatured in the presence of Hsc70, Hsp40, and ATP, refolds upon addition of rabbit reticulocyte cytosol . Our results indicate that Hsp40 has a critical regulatory function in the Hsc70 ATPase cycle that is required for the efficient loading of peptide substrate onto Hsc70.

Proc Natl Acad Sci U S A, 1996 Aug 6, 93(16), 8508 - 11
Simultaneous fluorescence-activated cell sorter analysis of two distinct transcriptional elements within a single cell using engineered green fluorescent proteins; Anderson MT et al.; Green fluorescent protein (GFP) is widely used as a reporter gene in both prokaryotes and eukaryotes . However, the fluorescence levels of wild-type GFP (wtGFP) are not bright enough for fluorescence-activated cell sorting or flow cytometry . Several GFP variants were generated that are brighter or have altered excitation spectra when expressed in prokaryotic cells . We engineered two GFP genes with different combinations of these mutations, GFP(S65T,V163A) termed GFP-Bex1, and GFP(S202F,T203I,V163A) termed GFP-Vex1 . Both show enhanced brightness and improved signal-to-noise ratios when expressed in mammalian cells and appropriately excited, compared with wtGFP . Each mutant retains only one of the two excitation peaks of the wild-type protein . GFP-Bex1 excites at 488 nm (blue) and GFP-Vex1 excites at 406 nm (violet), both of which are available laser lines . Excitation at these wavelengths allows for the independent analyses of these mutants by fluorescence-activated cell sorting, permitting simultaneous, quantitative detection of expression from two different genes within single mammalian cells.

Front Biosci, 1996 Aug 01, 1, e42 - 54
Mycoplasmas and HIV infection: from epidemiology to their interaction with immune cells; Brenner C et al.; Mycoplasmas are possible HIV cofactors, contributing to the evolution of AIDS . Our knowledge about mycoplasma prevalence in HIV-infected subjects has considerably increased due the development of specific detection assays . A new mycoplasma, Mycoplasma penetrans, has been identified and has been shown to be associated with HIV infection, at least among individuals with homosexual practices . We and others investigated the properties of M . fermentans and M . penetrans concerning cell colonization, cell invasion and cytopathogenicity . The molecular components which are involved in the interaction between these bacteria and immune cells are beginning to be identified and characterized . Membrane lipoproteins of these wall-less prokaryotes are key components in their interaction with B cells and surface capsular material may contribute to their defense from the host immune response.

Comput Appl Biosci, 1996 Aug, 12(4), 319 - 26
Constraining volume by matching the moments of a distance distribution; Chen CC et al.; The problem of computing a molecular structure from a set of distances arises in the interpretation of NMR data as well as other experimental methods that yield distance information . Techniques for computing structures must find conformations consistent with the distance data . There are often other constraints on the structure that must be satisfied as well . One of the most problematic constraints is the constraint on the total volume occupied by the atoms . In this paper, we use the first two moments (mean and variance) of an estimated distance distribution to constrain the volume of a computed structure . We show that a probabilistic algorithm for matching the first two moments of the estimated distance distribution significantly improves the quality of the solution, especially when the distance information alone is not sufficient to define the structure precisely . We also show that our method is not sensitive to small errors in the estimates of mean and variance of the distance distribution . Finally, we demonstrate the use of this constraint in computing a low-resolution structure of the 30S prokaryotic ribosomal subunit . Quantitative analysis of our results allows us to assess the information content contained in constraints on volume, and to show that in some cases addition of a volume constraint adds information roughly equivalent to doubling the number of input distances . Our results also demonstrate the flexibility of probabilistic representations of structural constraints, and the importance of including volume information to constrain structural computations-especially in the case of sparse data.

J Mol Med, 1996 Aug, 74(8), 423 - 39
Throwing a spanner in the works: antibiotics and the translation apparatus; Spahn CM et al.; The protein synthetic machinery is essential to all living cells and is one of the major targets for antibiotics . Knowledge of the structure and function of the ribosome and its associated factors is key to understanding the mechanism of drug action . Conversely, drugs have been used as tools to probe the translation cycle, thus providing a means to further our understanding of the steps that lead to protein synthesis . Our current understanding as to how antibiotics disrupt this process is reviewed here, with particular emphasis on the prokaryotic elongation cycle and those drugs that interact with ribosomal RNAs.

Mol Microbiol, 1996 Aug, 21(3), 543 - 56
The use of differential display-PCR to isolate and characterize a Legionella pneumophila locus induced during the intracellular infection of macrophages; Abu Kwaik Y et al.; The differential display (DD)-PCR technique has been modified to identify prokaryotic cDNA fragments that are differentially induced by facultative intracellular bacteria in response to the intracellular environment of eukaryotic cells . Several DD-PCR fragments identified from the intracellular bacterium Legionella pneumophila were induced at 4 h post-infection of the U937 macrophage-like cells . From these, a 700 bp fragment was cloned and sequenced . Neither the DNA sequence nor the predicted protein sequence from the open reading frame has similarity to other sequences in genetic databases . Transcription of the chromosomal locus containing the 700 bp fragment (eml, for early stage macrophage-induced locus) was induced by intracellular bacteria during the first few hours post-infection of macrophages but the expression was downregulated by 12 h post-infection . Transcription of eml was not growth phase-related in vitro, and was not affected by in vitro stress stimuli . A 3.7 kb EcoRI genomic fragment containing the 700 bp DD-PCR product was cloned . Six mini-Tn 10 insertions in the 3.7 kb EcoRI fragment were recombined into the L . pneumophila chromosome . Compared to the wild-type strain, five of the eml isogenic mutants had a similar phenotype of reduced cytopathicity to the U937 cells, showed a 100-fold increase in killing by macrophages during the first 5 h of the intracellular infection, and showed a 100-fold increase in killing during the first 24h of infection of the amoeba Hartmanella vermiformis . The 6th mutant had a phenotype indistinguishable from the wild-type strain . The cytopathicity defect of the mutants to the U937 cells was restored to wild-type levels by complementation of the mutants with a plasmid containing the 3.7 kb EcoRI fragment . These data showed that the 3.7 kb fragment containing eml is a novel L . pneumophila locus whose expression is uniquely induced by non-stress stimuli during early stages of the intracellular infection of phagocytic cells . Expression of this locus is required for survival of L . pneumophila within macrophages and within amoebae during early stages of the infection.

J Cell Biochem, 1996 Aug, 62(2), 210 - 22
Sequence and context effects on origin function in mammalian cells; Dijkwel PA et al.; Jacob and Brenner proposed a model for control of DNA replication in which a trans-acting initiator protein binds to a cis-acting replicator to effect initiation of nascent DNA chains at a fixed locus . Although replicators have been identified in prokaryotic and simple eukaryotic genomes, it has been much more difficult to demonstrate their presence in mammalian chromosomes . Owing to the lack of genetic approaches for identifying mammalian replicators, investigators have directed attention to localizing nascent strand start sites, which should lie close to replicators . Toward this end, a variety of clever techniques have been invented for analyzing replication intermediates, but only rarely have more than one of these techniques been applied to a single locus . However, virtually all have been used to analyze the dihydrofolate reductase locus in CHO cells . The picture that has developed in this locus is that initiation can occur at any of a large number of sites scattered throughout a broad zone, but somewhat more frequently near two sites that may correspond to true genetic replicators . Furthermore, it appears that local transcriptional activity, as well as appropriate torsional stress (as imparted by local attachment to the nuclear matrix), may have profound effects on origin activity.

Zentralbl Veterinarmed B, 1996 Aug, 43(6), 343 - 9
Investigations of the Japanese bovine tumour virus (BLV)--its ability to express structural and regulatory BLV proteins; Blankenstein P et al.; The mechanism of BLV-induced tumorigenesis has not been clear up to now . Changes of viral protein expression in infected cells may be involved in the molecular events leading to BLV-induced leukaemogenesis . In this study Western blot investigations of cells transfected with plasmid DNA containing the complete Japanese BLV tumour clone provirus demonstrate that this provirus is unable to express gag and env proteins . Following this an attempt was made to express the genes from this provirus in eukaryotic and prokaryotic cells using the phagemid pBK-RSV (Stratagene), but not as fusion proteins . The protein patterns expressed from the 5' and the 3' region of the BLV genome were compared with those of FLK/BLV cells . The results indicate that there is a defect in this provirus located in the genome region between the gag and env gene.

Nucleic Acids Res, 1996 Aug 1, 24(15), 2981 - 9
Sequence specific binding of chlamydial histone H1-like protein; Kaul R et al.; Chlamydia trachomatis is one of the few prokaryotic organisms known to contain proteins that bear homology to eukaryotic histone H1 . Changes in macromolecular conformation of DNA mediated by the histone H1-like protein (Hc1) appear to regulate stage specific differentiation . We have developed a cross-linking immunoprecipitation protocol to examine in vivo protein-DNA interaction by immune precipitating chlamydial Hc1 cross linked to DNA . Our results strongly support the presence of sequence specific binding sites on the chlamydial plasmid and hc1 gene upstream of its open reading frame . The preferential binding sites were mapped to 520 bp BamHI-XhoI and 547 bp BamHI-DraI DNA fragments on the plasmid and hc1 respectively . Comparison of these two DNA sequences using Bestfit program has identified a 24 bp region with >75% identity that is unique to the chlamydial genome . Double-stranded DNA prepared by annealing complementary oligonucleotides corresponding to the conserved 24 bp region bind Hc1, in contrast to control sequences with similar A+T ratios . Further, Hc1 binds to DNA in a strand specific fashion, with preferential binding for only one strand . The site specific affinity to plasmid DNA was also demonstrated by atomic force microscopy data images . Binding was always followed by coiling, shrinking and aggregation of the affected DNA . Very low protein-DNA ratio was required if incubations were carried out in solution . However, if DNA was partially immobilized on mica substrate individual strands with dark foci were still visible even after the addition of excess Hc1.

Bioessays, 1996 Aug, 18(8), 661 - 71
Molecular mechanisms of drug inhibition of DNA gyrase; Lewis RJ et al.; DNA gyrase, an enzyme unique to prokaryotes, has been implicated in almost all processes that involve DNA . Although efficient inhibitors of this protein have been known for more than 20 years, none of them have enjoyed prolonged pharmaceutical success . It is only recently that the mechanisms of inhibition for some of these classes of drugs have been established unequivocally by X-ray crystallography . It is hoped that this detailed structural information will assist the design of novel, effective inhibitors of DNA gyrase.

Oncogene, 1996 Aug 1, 13(3), 495 - 503
Isolation from a multigene family of the active human gene of the metastasis-associated multifunctional protein 37LRP/p40 at chromosome 3p21.3; Jackers P et al.; The 37 kD precursor of the 67 kD laminin receptor (37LRP) is a polypeptide whose expression is consistently upregulated in aggressive carcinoma . Interestingly, the 37LRP appears to be a multifunctional protein involved in the translational machinery and has also been identified as p40 ribosome-associated protein . Although highly conserved cDNAs corresponding to this polypeptide have been isolated from several species including vertebrates, invertebrates, plants and prokaryotes, characterization of any of the corresponding active genes has never been reported . In this study, we have cloned an intron-containing fragment which permitted us to isolate the active 37LRP/p40 human gene . This gene contains seven exons and six introns . Ribonuclease protection experiments suggest multiple transcription start sites . The promoter area does not bear a TATA box but contains four Sp1 sites . The first intron is also GC rich containing five Sp1 sites . Intron 4 contains the full sequence of the small nuclear RNA E2 and two Alu sequences are found in intron 3 . Fluorescent in situ hybridization localized the 37LRP/p40 active gene on chromosome 3 in the locus 3p21.3 which, interestingly, is a hot spot for genetic alterations in several cancers and particularly in small cell lung carcinoma.

J Bacteriol, 1996 Aug, 178(16), 4839 - 46
The heat shock protein ClpB mediates the development of thermotolerance in the cyanobacterium Synechococcus sp . strain PCC 7942; Eriksson MJ et al.; The heat shock protein CIpB (HSP100) is a member of the diverse group of Clp polypeptides that function as molecular chaperones and/or regulators of energy-dependent proteolysis . A single-copy gene coding for a ClpB homolog was cloned and sequenced from the unicellular cyanobacterium Synechococcus sp . strain PCC 7942 . The predicted polypeptide sequence was most similar to sequences of cytosolic ClpB from bacteria and higher plants (i.e., 70 to 75%) . Inactivation of clpB in Synechococcus sp . strain PCC 7942 resulted in no significant differences from the wild-type phenotype under optimal growth conditions . In the wild type, two forms of ClpB were induced during temperature shifts from 37 to 47.5 or 50 degrees C, one of 92 kDa, which matched the predicted size, and another smaller protein of 78 kDa . Both proteins were absent in the delta clpB strain . The level of induction of the two ClpB forms in the wild type increased with increasingly higher temperatures, while the level of the constitutive ClpC protein remained unchanged . In the delta clpB strain, however, the ClpC content almost doubled during the heating period, presumably to compensate for the loss of ClpB activity . Photosynthetic measurements at 47.5 and 50 degrees C showed that the null mutant was no more susceptible to thermal inactivation than the wild type . Using photosynthesis as a metabolic indicator, an assay was developed for Synechococcus spp . to determine the importance of ClpB for acquired thermotolerance . Complete inactivation of photosynthetic oxygen evolution occurred in both the wild type and the delta clpB strain when they were shifted from 37 directly to 55 degrees C for 10 min . By preexposing the cells at 50 degrees C for 1.5 h, however, a significant level of photosynthesis was retained in the wild type but not in the mutant after the treatment at 55 degrees C for 10 min . Cell survival determinations confirmed that the loss of ClpB synthesis caused a fivefold reduction in the ability of Synechococcus cells to develop thermotolerance . These results clearly show that induction of ClpB at high temperatures is vital for sustained thermotolerance in Synechococcus spp., the first such example for either a photosynthetic or a prokaryotic organism.

J Bacteriol, 1996 Aug, 178(16), 4759 - 64
Fancy meeting you here! A fresh look at "prokaryotic" protein phosphorylation; Kennelly PJ et al.; Bacteria play host to a wide range of protein phosphorylation-dephosphorylation systems (Fig . 1) . As little as five years ago the known systems were thought to be late-emerging and absolutely prokaryote specific . Today we know that most protein kinases and protein phosphatases are descended from a set of common, and possibly quite ancient, prototypes . Prokaryote- and eukaryote-specific protein kinases and protein phosphatases are rare and represent exceptions, not the rule as previously thought . Commonality suggests that a dynamic and versatile regulatory mechanism was first adapted to the modulation of protein function as early if not earlier than more "basic" mechanisms such as allosterism, etc . The existence of common molecular themes confirms that the microbial world offers a unique, largely untapped library and a powerful set of tools for the understanding of a regulatory mechanism which is crucial to all organisms, tools whose diversity and experimental malleability will provide new avenues for exploring and understanding key modes of cellular regulation.

Plant Physiol, 1996 Aug, 111(4), 1097 - 107
Cloning of a cDNA encoding cytosolic acetoacetyl-coenzyme A thiolase from radish by functional expression in Saccharomyces cerevisiae; Vollack KU et al.; A cDNA coding for radish (Raphanus sativus L.) acetoacetyl-coenzyme A thiolase (AACT) was cloned by complementation of the erg10 mutation affecting AACT in yeast (Saccharomyces cerevisiae) . The longest reading frame encodes a protein of 406 amino acids with a predicted relative molecular weight of 42,032, with significant similarities to eukaryotic and prokaryotic thiolases . There is no evidence for the presence of a leader peptide characteristic, e.g . of glyoxysomal thiolase . Yeast transformants expressing the radish AACT gene placed under the control of the GAL1 promoter exhibited a 10-fold higher enzyme activity than a wild-type yeast strain after induction by galactose . This enzyme activity is exclusively localized in the soluble fraction but not in membranes . These data indicate that we have cloned a gene encoding cytoplasmic (biosynthetic) AACT . Genomic DNA gel blot analysis suggests the presence of a single AACT gene, which is expressed in all parts of the seedling . Expression in cotyledons appears to be light-stimulated . We present preliminary evidence that a smaller transcript represents an antisense species being read from the same gene.

Genes Dev, 1996 Aug 1, 10(15), 1890 - 903
In vitro selection of preferred DNA pairing sequences by the Escherichia coli RecA protein; Tracy RB et al.; The RecA protein and other DNA strand exchange proteins are characterized by their ability to bind and pair DNA in a sequence-independent manner . In vitro selection experiments demonstrate, unexpectedly, that RecA protein has a preferential affinity for DNA sequences rich in GT composition . Such GT-rich sequences are present in loci that display increased recombinational activity in both eukaryotes and prokaryotes, including the Escherichia coli recombination hotspot, chi (5'-GCTGGTGG-3') . Interestingly, these selected sequences, or chi-containing substrates, display both an enhanced rate and extent of homologous pairing in RecA protein-dependent homologous pairing reactions . Thus, the binding and pairing of DNA by RecA protein is composition-dependent, suggesting that a component of the elevated recombinational activity of chi and increased genomic rearrangements at certain DNA sequences in eukaryotes is contributed by enhanced DNA pairing activity.

J Bacteriol, 1996 Aug, 178(15), 4563 - 70
The molecular architecture of the sar locus in Staphylococcus aureus; Bayer MG et al.; The global regulator sar in Staphylococcus aureus controls the synthesis of a variety of cell wall and extracellular proteins, many of which are putative virulence factors . The sar locus in strain RN6390 contains a 339-bp open reading frame (sarA) and an 860-bp upstream region . Transcriptional analyses of this locus revealed three different transcripts of 0.58, 0.84, and 1.15 kb (designated sarA, sarC, and sarB, respectively) . All three transcripts seemed to be under temporal, growth cycle-dependent regulation, with sarA and sarB being most abundant in early log phase and the sarC concentration being highest toward the late stationary phase . Mapping of the 5' ends of the sar transcripts by primer extension and modified S1 nuclease protection assays demonstrated that transcription is initiated from three separate, widely spaced promoters . The 3' ends of all three sar transcripts are identical, and transcriptional termination occurs upstream of a typical prokaryotic poly(T) termination signal . Northern (RNA) analysis of sar mutant clones containing plasmids that comprised various promoters and the termination signal revealed that individual transcripts can be generated from each of the three promoters, thus suggesting possible activation as independent promoters . The multipromoter system, from which transcription is initiated, bears conserved features for recognition by homologous sigma 70 transcription factors and also by those expressed in the general stress response . Downstream of the two distal promoters (P3 and P2) are two regions potentially encoding short peptides . It is conceivable that posttranslational cooperation between these short peptides and the sarA gene product occurs to modulate sar-related functions . Complementation studies of a sar mutant with a clone expressing all three sar transcripts showed that this clone was able to restore the sar wild-type phenotype to the sar mutant.

RNA, 1996 Aug, 2(8), 824 - 34
Identification of cis-acting signals in the giardiavirus (GLV) genome required for expression of firefly luciferase in Giardia lamblia; Yu DC et al.; Giardiavirus (GLV) is a 6,277-bp double-stranded RNA virus of Giardia lamblia, one of the earliest eukaryotic divergents from the prokaryotes . Our previous success in GLV-mediated transfection of G . lamblia has provided an effective way of monitoring the mechanisms underlining GLV gene replication and mRNA translation in this organism . Here we have investigated the cis-acting signals in the GLV genome that regulate replication, transcription, and translation of an inserted firefly luciferase gene in GLV-infected G . lamblia . By modifying the two terminal regions of a full-length GLV cDNA clone used to flank a luciferase gene, various in vitro chimeric transcripts were generated and introduced into GLV-infected G . lamblia via electroporation . Expression of luciferase (+) strand and (-) strand RNAs in the transfected cells was monitored and the luciferase activity assayed . The results indicated that the 5'-untranslated region (UTR) of 366 nt and the 3'-terminal 2,022 nt of the viral transcript are both needed for optimal expression of the two RNA strands . Although the entire 5'-UTR is needed for the chimeric mRNA synthesis, both the primary sequence and the secondary structure at the 3' end of GLV transcript are essential for the synthesis of (-) strand RNA . When the 5' end of GLV transcript was extended 265 nt into the capsid protein open reading frame and fused with that of luciferase, there was no change in the level of luciferase chimeric RNA, but a 5,000-fold increase of luciferase activity was observed that may be attributed to an enhanced translational efficiency of the chimeric mRNA in G . lamblia.

Biochim Biophys Acta, 1996 Jul 31, 1308(1), 31 - 40
Maternal and zygotic expression of mRNA for S-adenosylmethionine decarboxylase and its relevance to the unique polyamine composition in Xenopus oocytes and embryos; Shinga J et al.; From Xenopus tailbud cDNA library, we isolated the cDNA for S-adenosylmethionine decarboxylase (SAMDC), an enzyme which provides putrescine and spermidine with the aminopropyl group to form spermidine and spermine, respectively . The cDNA coded for 335 amino acids whose sequence had high homology (ca . 83%) to other vertebrate SAMDCs, preserving the sequences reportedly essential for enzyme activity, proenzyme processing, and putrescine stimulation of the enzyme activity . Northern blot analysis showed one major mRNA signal of ca . 3.5 kb, with a minor signal of ca 2.0 kb which may probably be due to cross-hybridization . In oocytes the SAMDC mRNA occurred from stage I, and its amount peaked at stage II, then gradually decreased from stage III to VI . The decreased level of the mRNA was maintained during oocyte maturation, further decreased from the cleavage to early neurula stage, and then increased greatly due to the zygotic expression during late neurula stages (stage 21-25), reaching a plateau level at the late tailbud stage (stage 28) . Enzyme assays showed that the changing level of the SAMDC mRNA was reflected in the level of the functional enzyme, suggesting strongly that the zygotic expression of the mRNA leads to a large increase in the amount of SAMDC, albeit in the pre-neurula embryo the amount of the enzyme is very small . We found that the relative composition of polyamines is the eukaryote-type (high-level spermine) at the beginning of oogenesis, but it changes to the prokaryote-type, or more appropriately Escherichia coli-type (high-level putrescine but background level spermine) during oocyte maturation, and remains E . coli-type throughout embryogenesis . We assume that the E . coli-type polyamine composition is a necessary factor for the normal embryogenic development in Xenopus and its maintenance, especially that in pre-neurula stages, can be explained by the low level of both SAMDC mRNA and SAMDC.

Biochim Biophys Acta, 1996 Jul 18, 1275(1-2), 61 - 9
A compilation of mutations located in the cytochrome b subunit of the bacterial and mitochondrial bc1 complex; Brasseur G et al.; In anticipation of the structure of the bc1 complex which is now imminent, we present here a preliminary compilation of all available cytochrome b mutants that have been isolated or constructed to date both in prokaryotic and eukaryotic species . We have briefly summarized their salient properties with respect to the structure and function of cytochrome b and to the Qo and Qi sites of the bc1 complex . In conjunction with the high resolution structure of the bc1 complex, this database is expected to serve as a useful reference point for the available data and help to focus and stimulate future experimental work in this field.

J Mol Biol, 1996 Jul 12, 260(2), 120 - 5
Localized DNA flexibility contributes to target site selection by DNA-bending proteins; Grove A et al.; Certain DNA-binding proteins function as architectural elements by bending DNA . We have studied the binding of three such proteins, the prokaryotic HU and integration host factor (IHF) and the eukaryotic HMG1, to DNA in which flexibility is enhanced by tandem mismatches and by substituting 5-hydroxymethyluracil (hmU) for thymine (T) . IHF and HU have higher affinity for DNA with two 4-nt loops than for perfect duplex DNA with a sequence that corresponds to a binding site for the phage-encoded homolog, TF1 . HU has a high affinity for DNA with 4-nt loops separated by 9 bp (Kd = 3.5 nM), with suboptimal binding for other loop separations . IHF-binding is optimal when 4-nt loops are 8 to 9 bp apart; optimal complex formation with DNA representing the specific IHF-binding site H' requires that loops do not disrupt the consensus sequence and that one 4-nt loop borders the dyad axis-proximal block of consensus sequence (Kd = 0.3 nM, approximately tenfold lower than for H' perfect duplex DNA) . HMG1 also binds preferentially to DNA with loops . All three proteins bind more tightly to DNA in which thymine is replaced with hmU . IHF has a tenfold higher affinity for hmU-DNA without a consensus IHF site (Kd = 7.6 nM) than for the corresponding T-DNA but does exhibit site-selectivity in hmU-DNA; Kd = 0.6 nM for the hmU-containing version of H' . Tighter binding to hmU-DNA is consistent with greater flexibility, and the distinct influence of loop position on complex formation suggests that sequence-dependent variations in flexibility of duplex DNA play a significant role in target-site selection by these DNA-bending proteins.

J Biol Chem, 1996 Jul 12, 271(28), 16460 - 5
The homologue of mammalian SPC12 is important for efficient signal peptidase activity in Saccharomyces cerevisiae; Fang H et al.; The multisubunit signal peptidase catalyzes the cleavage of signal peptides and the degradation of some membrane proteins within the endoplasmic reticulum (ER) . The only subunit of this enzyme functionally examined to date, yeast Sec11p, is related to signal peptidase I from bacteria . Since bacterial signal peptidase is capable of processing both prokaryotic and eukaryotic signal sequences as a monomer, it is unclear why the analogous enzyme in the ER contains proteins unrelated to signal peptidase I . To address this issue, the gene encoding Spc1p, the yeast homologue to mammalian SPC12, is isolated from the yeast Saccharomyces cerevisiae . Spc1p co-purifies and genetically interacts with Sec11p, but unlike Sec11p, Spc1p is not required for cell growth or the proteolytic processing of tested proteins in yeast . This indicates that only a subset of the ER signal peptidase subunits is required for signal peptidase and protein degradation activities in vivo . Through both genetic and biochemical criteria, Spc1p appears, however, to be important for efficient signal peptidase activity.

FEBS Lett, 1996 Jul 8, 389(3), 249 - 52
Fructose-1,6-bisphosphatase . Primary structure of the rabbit liver enzyme . 'Intermediate' variability of an oligomeric protein; Kaiser R et al.; The primary structure of rabbit liver fructose-1,6-bisphosphatase was determined by peptide analysis of digests with different proteases . The results establish the primary structure, complete data bank entries, and show that this enzyme variant is indeed homologous with other liver fructose-1,6-bisphosphatases . Residue differences with the enzymes from other mammals are 9-15%, with those from plants and yeasts about 50%, and with those from characterized prokaryotes up to 70%, showing an enzyme variability intermediate between those of 'variable' and 'constant' oligomeric dehydrogenases . Structural relationships, conformations and catalytic mechanisms are consistent within the family of fructose-1,6-bisphosphatases, and the rabbit protein is a typical rather than an aberrant form of the enzyme.

Genet Anal, 1996 Jul, 13(2), 25 - 31
The construction of novel mobilizable YAC plasmids and their behavior during trans-kingdom conjugation between bacteria and yeasts; Mahmood A et al.; Trans-kingdom conjugation is an easy and efficient method for gene transfer from prokaryotes to eukaryotes since it does not require DNA extraction and purification . We constructed novel mobilizable plasmids pAY-YAC-B and pAY-YAC-E . The origin of conjugal transfer (oriT) was inserted at two different positions, pAY-YAC-B contains oriT region in between two telomeres whereas pAY-YAC-E has oriT at the cloning site of pYAC4 . By conjugation, both plasmids were successfully transferred from E . coli to S . cerevisiae and S . kluyveri yeasts with the aid of helper plasmid pRH220 which harbors mob and tra genes . The plasmids were transferred more efficiently in S . cerevisiae compared to S . kluyveri . The analyses by restriction enzyme digestion and Southern hybridization indicated that both plasmids maintained their original structure and size in transconjugant yeasts, therefore, reflecting the faithful nicking and subsequent resealing of plasmids during conjugation . The comparison between conjugative transfer and transformation has also been performed and discussed.

Mol Microbiol, 1996 Jul, 21(1), 5 - 11
Circadian clocks in prokaryotes; Johnson CH et al.; Prokaryotes have long been thought incapable of expressing circadian (daily) rhythms . Recently, however, such biological 'clocks' have been discovered in several species of cyanobacteria . These endogenous timekeepers control gene expression on a global level in cyanobacteria . Even in cyanobacterial cultures that are growing with average doubling times more rapid than one per 24 h, the circadian clock controls gene expression and cell division . We have isolated mutants of the cyanobacterial circadian pacemaker and are currently characterizing the loci responsible for their altered period phenotypes.

Yeast, 1996 Jul, 12(9), 877 - 85
Sequence and analysis of a 33 kb fragment from the right arm of chromosome XV of the yeast Saccharomyces cerevisiae; Galisson F et al.; We have determined the nucleotide sequence of a cosmid (pEOA423) from chromosome XV of Saccharomyces cerevisiae . Analysis of the 33,173 bp sequence reveals the presence of 20 putative open reading frames (ORFs) . Five of them correspond to previously known genes (MGM1, STE4, CDC44, STE13, RPB8) . The previously published nucleotide sequences are in perfect agreement with our sequence except for STE4 and MGM1 . In the latter case, 59 amino acids were truncated from the published protein at its N-terminal end due to a frameshift . The putative translation products of six other ORFs exhibit significant homology with protein sequences in public databases: O50 03 and O50 17 products are homologs of the ANC1 and MIP1 proteins of S . cerevisiae, respectively; O50 05 product is similar to that of a protein of unknown function from Myxococcus xanthus; O50 12 product is probably a new ATP/ADP carrier; O50 13 product shows homology with group II tRNA synthetases; and the O50 16 product exhibits strong similarity with the N-terminal domain of the NifU proteins from several prokaryotes . The remaining nine ORFs show no significant similarity . Among these, two contiguous ORFs (O50 19 and O50 20) are very similar to each other, suggesting an ancient tandem duplication.

Protein Sci, 1996 Jul, 5(7), 1421 - 5
The protein phosphatase 2C (PP2C) superfamily: detection of bacterial homologues; Bork P et al.; A thorough sequence analysis of the various members of the eukaryotic protein serine/threonine phosphatase 2C (PP2C) family revealed the conservation of 11 motifs . These motifs could be identified in numerous other sequences, including fungal adenylate cyclases that are predicted to contain a functionally active PP2C domain, and a family of prokaryotic serine/threonine phosphatases including SpoIIE . Phylogenetic analysis of all the proteins indicates a widespread sequence family for which a considerable number of isoenzymes can be inferred.

Atherosclerosis, 1996 Jul, 124(1), 49 - 60
Adenovirus-assisted lipofection: efficient in vitro gene transfer of luciferase and cytosine deaminase to human smooth muscle cells; Kreuzer J et al.; Smooth muscle cells (SMC) are a central cell type involved in multiple processes of coronary artery diseases including restenosis and therefore are major target cells for different aspects of gene transfer . Previous attempts to transfect primary arterial cells using different techniques like liposomes, CaPO4 and electroporation resulted in only low transfection efficiency . The development of recombinant adenoviruses dramatically improved the delivery of foreign genes into different cell types including SMC . However, cloning and identification of recombinants remain difficult and time-consuming techniques . The present study demonstrates that a complex consisting of reporter plasmid encoding firefly luciferase (pLUC), polycationic liposomes and replication-deficient adenovirus was able to yield very high in vitro transfection of primary human smooth muscle cells under optimized conditions . The technique of adenovirus-assisted lipofection (AAL) increases transfer and expression of plasmid DNA in human smooth muscle cells in vitro up to 1000-fold compared to lipofection . To verify the applicability of AAL for gene transfer into human smooth muscle cells we studied a gene therapy approach to suppress proliferation of SMC in vitro, using the prokaryotic cytosine deaminase gene (CD) which enables transfected mammalian cells to deaminate 5-fluorocytosine (5-FC) to the highly toxic 5-fluorouracil (5-FU) . The effect of a transient CD expression on RNA synthesis was investigated by means of a cotransfection with a RSV-CD expression plasmid and the luciferase reporter plasmid . Western blot analysis demonstrated high expression of CD protein in transfected SMC . Cotransfected SMC demonstrated two-fold less luciferase activity in the presence of 5-FC (5 mmol/l) after 48 h compared to cells transfected with a non-CD coding plasmid . The data demonstrate that a transient expression of CD could be sufficient to reduce the capacity of protein synthesis in human SMC . This simple and effective in vitro transfection method may also be applicable to in vivo delivery of target genes to the vascular wall to inhibit SMC proliferation.

J Bacteriol, 1996 Jul, 178(14), 4084 - 8
Tyrosine phosphorylation in Myxococcus xanthus, a multicellular prokaryote; Frasch SC et al.; Tyrosine phosphorylation is an extremely rare event in prokaryotes, occurring almost exclusively in multicellular eukaryotes . We have identified, for the first time, by the use of antiphosphotyrosine monoclonal antibody and Western blot (immunoblot) analysis, two tyrosine-phosphorylated membrane proteins in the multicellular prokaryote Myxococcus xanthus . The pattern of tyrosine phosphorylation was shown to change during development, indicating a possible role for this regulatory modification during two stages of development, i.e., aggregation and sporulation . Furthermore, the altered pattern of tyrosine phosphorylation observed in a variety of signaling mutants was shown to differ from that observed in the wild type, suggesting further the possible involvement of tyrosine phosphorylation during the development program.

Comp Biochem Physiol B Biochem Mol Biol, 1996 Jul, 114(3), 287 - 93
Biochemical evidence for the presence of an unconventional actin protein in a prokaryotic organism; Labbe JP et al.; The ubiquity of actin, like the functional diversity of many associated proteins, raises a question concerning diversification of motility mechanisms and thus the emergence of an elementary functional system . Our aim was to investigate, in particular, mobiles prokaryotics cells as Synechocystis lacking cilia and flagella, search for actin essential properties and then locate the molecular behaviours . Here we report the presence and purification of a 56-kDa (apparent molecular weight) prokaryotic protein that polymerizes to form filaments, activates myosin Mg(++)-ATPase activity, inhibits DNase-1 activity and affords close antigenic homology to skeletal actin . This protein was found to be associated with thylakoid membranes and extracted in the presence of Triton X-100.

Eur J Biochem, 1996 Jul 1, 239(1), 67 - 73
Primary structure of omega-hordothionin, a member of a novel family of thionins from barley endosperm, and its inhibition of protein synthesis in eukaryotic and prokaryotic cell-free systems; Mendez E et al.; A new sulfur-rich basic polypeptide, so called omega-hordothionin, has been isolated from barley endosperm by extractions with NaCl and ammonium bicarbonate followed by reverse-phase high performance liquid chromatography . Purified omega-hordothionin was found to be homogeneous by SDS/polyacrylamide gel electrophoresis, N-terminal amino-acid sequencing and electrospray-ionization mass spectrometric analysis . The complete primary structure of omega-hordothionin was determined by automatic degradation of the intact molecule and peptides obtained by proteolytic cleavage . Omega-hordothionin consists of a single polypeptide chain of 48 amino acids with a molecular mass of 5508 Da deduced from its amino acid sequence, which fully coincides with the 5508.2 Da determined by electrospray-ionization mass spectrometry . The isolated polypeptide showed a characteristic composition with a high content of basic amino acids (five arginine residues, two lysine residues and six histidine residues) and eight cysteine residues, and has strong sequence identity (66%) with the sorghum SI alpha 1 alpha-amylase inhibitor . Omega-hordothionin, like gamma-hordothionin, exhibited translation inhibitory activity on both eukaryotic cell-free systems from mammalian (rat liver and rabbit reticulocyte lysates) and prokaryotic cell-free systems (Escherichia coli) . However, in contrast to gamma-hordothionin, omega-hordothionin did not inhibit plant systems such as Triticum aestivum, Cucumis sativus, Vicia sativa and Hordeum vulgare . Gamma-hordothionin also inhibited the alpha-amylase activity from human saliva, while omega-hordothionin and the other different genetic variants of thionins, alpha-hordothionin and beta-hordothionin, failed to show any inhibitory effect.

J Neurochem, 1996 Jul, 67(1), 105 - 10
Recoverin in cultured human retinoblastoma cells: enhanced expression during morphological differentiation; Wiechmann AF; Recoverin is a calcium-binding protein expressed in retinal photoreceptors . It appears to delay the termination of the phototransduction cascade by blocking the phosphorylation of photoexcited rhodopsin . The goal of this study was to determine if recoverin mRNA and protein are expressed in cultured human Y79 retinoblastoma cells, so that this cell line could be used as a model to study the mechanism of recoverin gene expression in the retina . A cDNA encoding human recoverin was PCR cloned and used for prokaryotic expression of recoverin protein . Polyclonal antibodies raised against pure recombinant recoverin were used for western blotting and immunocytochemistry of Y79 cells grown as attachment cultures in the presence of the differentiating agents dibutyryl cyclic AMP (dbcAMP) or butyrate . Northern blot analysis was performed on mRNA extracted from Y79 cells that were also treated with the differentiating agents . In Y79 cell monolayer cultures, recoverin was immunolocalized to the cell cytoplasm, and immunoreactivity was increased dramatically by the addition of 2 mM butyrate to the culture medium . Butyrate treatment also caused an increase in the development of neurite-like cellular processes . Addition of 4 mM dbcAMP resulted in a moderate increase in both recoverin immunoreactivity and number of cellular processes . Western and northern blots of butyrate and dbcAMP-treated Y79 cell cultures demonstrated an increase in recoverin protein and RNA expression, respectively, comparable with that observed with immunocytochemistry . These data suggest that, under the influence of the differentiating agent butyrate, Y79 cells exhibit an increase in expression of the photoreceptor protein recoverin and a concomitant morphological differentiation toward a neuronal phenotype.

Microb Ecol, 1996 Jul, 32(1), 81 - 99
Evaluation of Extracellular, High-affinity beta-N-acetylglucosaminidase Measurements from Freshwater Lakes: An Enzyme Assay to Estimate Protistan Grazing on Bacteria and Picocyanobacteria
Vrba J, Simek K, Pernthaler J, Psenner R.
Protistan community grazing rates upon both bacterioplankton and autotrophic picoplankton were estimated using fluorescently-labeled prey and by measurement of extracellular hydrolysis of 4-methylumbelliferyl (MUF) beta-N-acetylglucosaminide in a eutrophic reservoir and an oligo-mesotrophic lake during phytoplankton blooms . In addition, enzyme methods were optimized in bacterivorous flagellate cultures by two enzyme assays, based on fluorometric detection of protistan digestive activity, which were compared and calibrated independently against flagellate bacterivory . Enzymatic hydrolyses of MUF beta-N,N',N''-triacetylchitotriose and MUF beta-N-acetylglucosaminide were measured in cell-free (sonicated) and whole-cell (unsonicated) samples . The hydrolysis of both substrates, using the whole-cell enzyme assay at in situ pH, was correlated significantly with total grazing rate of Bodo saltans . Thus the whole-cell enzyme assay with MUF beta-N-acetylglucosaminide was used for freshwater samples . High-affinity (Km < 1 mumol l-1) and low-affinity (Km > 100mumol l-1) enzymes were distinguished kinetically in most samples from both systems studied . Activities (Vmax) of the high-affinity enzyme varied from 0.24 to 1.43 nmol l-1 h-1 . Protistan community grazing on bacterioplankton was in the range of 0.15-1.36 mug C l-1 h-1 both for lake and reservoir, the differences being observed in grazing on picocyanobacteria (lake, 0.03-0.22 mug C l-1 h-1; reservoir, 0.35-1.56 mug C l-1 h-1) . The enzyme activities were correlated significantly with the protistan grazing both on bacterioplakton (rs=0.62, P<0.001) and total procaryotic picoplankton (the sum of organic carbon grazed from bacteria and picocyanobacteria, rs=0.73, P<0.001) in the eutrophic reservoir . Weaker relationships (rs=0.42) with a lower slope were found for the oligo-mesotrophic lake . Ingestion rate studies are time-consuming and the digestive enzyme assay with MUF beta-N-acetylglucosaminide presents a rapid alternative for estimating total protistan prokaryotic picoplanktivory in freshwaters.

Cell, 1996 Jun 28, 85(7), 1101 - 12
Structure of bacteriophage T4 RNase H, a 5' to 3' RNA-DNA and DNA-DNA exonuclease with sequence similarity to the RAD2 family of eukaryotic proteins; Mueser TC et al.; Bacteriophage T4 RNase H is a 5' to 3' exonuclease that removes RNA primers from the lagging strand of the DNA replication fork and is a member of the RAD2 family of eukaryotic and prokaryotic replication and repair nucleases . The crystal structure of the full-length native form of T4 RNase H has been solved at 2.06 angstroms resolution in the presence of Mg2+ but in the absence of nucleic acids . The most conserved residues are clustered together in a large cleft with two Mg2+ in the proposed active site . This structure suggests the way in which the widely separated conserved regions in the larger nucleotide excision repair proteins, such as human XPG, could assemble into a structure like that of the smaller replication nucleases.

J Biol Chem, 1996 Jun 28, 271(26), 15782 - 6
Functional analysis of a p21WAF1,CIP1,SDI1 mutant (Arg94 --> Trp) identified in a human breast carcinoma . Evidence that the mutation impairs the ability of p21 to inhibit cyclin-dependent kinases; Balbin M et al.; Human p21 (also known as WAF1, CIP1, or SDI1) is a dual inhibitor of cyclin dependent kinases (CDKs) and the replication factor PCNA, which plays a role as a downstream mediator of the cell-cycle arrest induced by the tumor suppressor p53 . To determine whether inactivation of downstream targets of p53 might contribute to cellular transformation, we have examined the integrity of the p21 gene in 36 invasive ductal breast carcinomas . Direct sequence analysis of the polymerase chain reaction-amplified p21 gene revealed a C to T transition in codon 94 that caused the substitution of a tryptophan for an arginine in a tumor specimen . This mutation was not detected in normal DNA extracted from the same patient nor in a polymerase chain reaction-restriction fragment length polymorphism of 50 unrelated individuals, indicating that it corresponds to a tumor-specific alteration . Functional analysis of the p21(R94W) protein produced in different eukaryotic and prokaryotic expression systems revealed that this mutation impaired the ability of p21 to inhibit CDKs . By contrast, the R94W mutant was unaltered in its ability to promote cyclin-CDK association as well as in its ability to bind proliferating cell nuclear antigen, thus leaving its putative functions as kinase activator or as inhibitor of replicative DNA synthesis intact . On the basis of these functional analysis, we propose that the Arg residue at position 94 is important for the CDK inhibitory role of p21.

Gene, 1996 Jun 26, 172(2), 273 - 7
The cloning and characterization of a cDNA encoding Xenopus laevis DNA ligase I; Lepetit D et al.; A cDNA clone coding for DNA ligase I (LigI) was isolated from a Xenopus laevis oocyte cDNA library . The 3766-bp sequence showed a putative ORF capable of encoding a 1070-amino-acid protein whose overall identity with two mammalian sequences is 63% . This identity, however, rises to 72.5% in the C-terminal portion of the protein that contains the active site . Expression of the cDNA in a prokaryotic system produces a protein that is immunologically identical to LigI and can be adenylated . The 180-kDa size of the recombinant protein is similar to the LigI detected in oocyte . Northern blot analysis of ovary and embryo RNAs revealed the expression of two (4.1 and 6 kb) LigI transcripts.

Proc Natl Acad Sci U S A, 1996 Jun 25, 93(13), 6464 - 9
Complementation of an Escherichia coli adhE mutant by the Entamoeba histolytica EhADH2 gene provides a method for the identification of new antiamebic drugs; Yong TS et al.; The pathogenic protozoan parasite Entamoeba histolytica, the cause of amebic dysentery and amebic liver abscess, is an obligate anaerobe, and derives energy from the fermentation of glucose to ethanol with pyruvate and acetyl coenzyme A as intermediates . We have isolated EhADH2, a key enzyme in this pathway, that is a NAD+- and Fe2+-dependent bifunctional enzyme with acetaldehyde dehydrogenase and alcohol dehydrogenase activities . EhADH2 is the only known eukaryotic member of a newly defined family of prokaryotic multifunctional enzymes, which includes the Escherichia coli AdhE enzyme, an enzyme required for anaerobic growth of E . coli . Because of the critical role of EhADH2 in the amebic fermentation pathway and the lack of known eukaryotic homologues of the EhADH2 enzyme, EhADH2 represents a potential target for antiamebic chemotherapy . However, screening of compounds for antiamebic activity is hampered by the cost of large scale growth of Ent . histolytica, and difficulties in quantitating drug efficacy in vitro . To approach this problem, we expressed the EhADH2 gene in a mutant strain of E . coli carrying a deletion of the adhE gene . Expression of EhADH2 restored the ability of the mutant E . coli strain to grow under anaerobic conditions . By screening compounds for the ability to inhibit the anaerobic growth of the E . coli/EhADH2 strain, we have developed a rapid assay for identifying compounds with anti-EhADH2 activity . Using bacteria to bypass the need for parasite culture in the initial screening process for anti-parasitic agents could greatly simplify and reduce the cost of identifying new therapeutic agents effective against parasitic diseases.

Biochem Biophys Res Commun, 1996 Jun 25, 223(3), 514 - 9
Purification and characterization of recombinant human 5'-methylthioadenosine phosphorylase: definite identification of coding cDNA; Della Ragione F et al.; 5'-Methylthioadenosine phosphorylase gene maps on the 9p21 chromosome, strictly linked to the important tumor suppressor gene p16INK4A . Chromosomal deletions encompassing both the phosphorylase and p16INK4A genes cause the complete absence of the enzymatic activity in a large number of tumors, thus resulting in well-defined metabolic differences between malignant and normal cells . Recently, the cloning of the phosphorylase gene has been reported on the basis of indirect evidence . In order to demonstrate definitely the identification of 5'-methylthioadenosine phosphorylase gene, we have cloned the putative enzyme coding sequence in a prokaryotic expression vector and expressed the protein in bacteria . The recombinant phosphorylase has been purified to homogeneity and its physicochemical, immunological and kinetic features have been characterized . The results obtained allowed the conclusive demonstration of 5'-methylthioadenosine phosphorylase gene cloning and the use of recombinant protein for further characterization.

J Mol Biol, 1996 Jun 21, 259(4), 579 - 88
Short-range order in two eukaryotic genomes: relation to chromosome structure; Widom J; Fourier transform techniques have been used to analyze the distributions of all ten independent DNA dinucleotide steps in two eukaryotic genomes and one prokaryotic genome, for periodicities of approximately 2 to 500 bp . The results reveal systematic deviations from random expectation for certain dinucleotide steps over this entire range of periodicities, together with striking peaks at certain spatial periodicities for particular dinucleotide steps . Several dinucleotides yield peaks at a periodicity of approximately 10.2 bp that are unique to the eukaryotic genomes . Certain members of this set of dinucleotide signals were previously identified as involved in nucleosome positioning, while others were previously unrecognized . In real-space, these dinucleotides are uncorrelated or even anticorrelated (relative to random expectation) at distances of 10 and 11 bp, despite having greater than random spectral power at the corresponding periodicity . Real-space correlations of these dinucleotides at distances of 10 and 11 bp are suppressed by another spectral component, a 3 bp periodicity attributed to codons, which has a local minimum probability at approximately 10.5 bp . When the two eukaryotic genomes are encoded for the signal "AA or TT", the peak at approximately 10.2 bp periodicity is strengthened, whereas for the prokaryotic genome such a peak remains absent . For the Caenorhabditis elegans genome, this peak becomes the dominant feature in the transform, surpassing a peak owing to the existence of codons in both height and integrated intensity . These results suggest that the requirements of chromosome structure place significant constraints on eukaryotic genome organization; they reveal additional signals that may be related to nucleosome positioning; and they reveal a wealth of additional new non-random aspects of genome sequence organization.

EMBO J, 1996 Jun 17, 15(12), 2988 - 96
Calmodulin binding to glutamate decarboxylase is required for regulation of glutamate and GABA metabolism and normal development in plants; Baum G et al.; Glutamate decarboxylase (GAD) catalyzes the decarboxylation of glutamate to CO2 and gamma-aminobutyrate (GABA) . GAD is ubiquitous in prokaryotes and eukaryotes, but only plant GAD has been shown to bind calmodulin (CaM) . Here, we assess the role of the GAD CaM-binding domain in vivo . Transgenic tobacco plants expressing a mutant petunia GAD lacking the CaM-binding domain (GADdeltaC plants) exhibit severe morphological abnormalities, such as short stems, in which cortex parenchyma cells fail to elongate, associated with extremely high GABA and low glutamate levels . The morphology of transgenic plants expressing the full-length GAD (GAD plants) is indistinguishable from that of wild-type (WT) plants . In WT and GAD plant extracts, GAD activity is inhibited by EGTA and by the CaM antagonist trifluoperazine, and is associated with a CaM-containing protein complex of approximately 500 kDa . In contrast, GADdeltaC plants lack normal GAD complexes, and GAD activity in their extracts is not affected by EGTA and trifluoperazine . We conclude that CaM binding to GAD is essential for the regulation of GABA and glutamate metabolism, and that regulation of GAD activity is necessary for normal plant development . This study is the first to demonstrate an in vivo function for CaM binding to a target protein in plants.

Experientia, 1996 Jun 15, 52(6), 597 - 9
Antioxidant survey to assess antagonism to redox stress using a prokaryotic and an eukaryotic system; Baker H et al.; Using a prokaryote (Escherichia coli) and a metazoa-resembling eukaryote (Ochromonas danica), we surveyed antioxidants which might overcome redox stress imposed by menadione sodium bisulphite (MD) and buthionine sulphoximine (BSO) . BSO oxidant stress was evident only in O . danica; MD oxidant stress was evident in both organisms . Glutathione, its precursors, e.g . cysteine, homocysteine, and 2-oxo-4-thiazolidine carboxylic acid, and red blood cells, emerged as prime antioxidants for relieving BSO and MD oxidant stress . BSO and MD oxidant activity and antioxidant-annulling effect in O . danica were judged comparable to those found in animal cells whereas the results E . coli were not entirely equivalent . The O . danica system emerged as a practical, rapid, and useful system for pinpointing oxidant stressors and antioxidants, and shows promise for studies with mammalian systems.

Biochem J, 1996 Jun 15, 316 ( Pt 3), 853 - 8
Cloning and functional expression of glycosyltransferases from parasitic protozoans by heterologous complementation in yeast: the dolichol phosphate mannose synthase from Trypanosoma brucei brucei; Mazhari-Tabrizi R et al.; The gene for the enzyme dolichol phosphate mannose (Dol-P-Man) synthase from the parasitic protozoan Trypanosoma brucei brucei (T . brucei) was cloned by screening a T . brucei cDNA library and then sequenced . The library was constructed in a yeast expression vector and the positive clone was identified by complementation of a temperature-sensitive defect in the yeast strain DPM 1-6 {Orlean, Albright and Robbins (1988) J . Biol . Chem . 263, 17499-17507} . The insert of this clone displayed an open reading frame of 801 nucleotides coding for a putative protein of 267 amino acids . The deduced protein sequence showed an identity of 49% and a similarity of 69% with the published yeast sequence . Additional features of the T . brucei sequence are the presence of a putative signal sequence, a C-terminal transmembrane domain, a consensus sequence for phosphorylation by cAMP-dependent protein kinase and a stretch of five nucleotides immediately upstream from the putative initiation codon that could function as a prokaryotic ribosome binding site . A consensus sequence for dolichol binding (FI/VXF/YXXIPFXF/Y) found in the yeast protein could not be detected in the putative transmembrane domain of the T . brucei sequence . Biochemical characterization of the recombinant protein showed that it is functionally expressed in the yeast strain DPM 1-6 and Escherichia coli . In both constructs Dol-P-Man synthesis was shown in a cell-free system . Synthesis was stimulated by exogenous dolichol phosphate and inhibited by amphomycin . These results confirm that we have cloned the T . brucei Dol-P-Man synthase by heterologous complementation in yeast, an approach that might be applicable for other glycosyltransferases from various sources.

Virology, 1996 Jun 15, 220(2), 390 - 401
Conservation of DNA sequence in the predicted major late promoter regions of selected mastadenoviruses; Song B et al.; The major late promoter (MLP) of the subgroup C human adenoviruses is a preeminent model for the study of the mechanisms of basal and activated transcription, both in vivo and in vitro . However, while the structure and function of the human virus MLP has been the subject of extensive investigation, the conservation of the various promoter elements among the adenoviruses from different species has not been examined . Conservation of specific elements would strongly suggest the importance and universality of their function . To address this issue, sequences were obtained from cloned DNAs of several representative Mastadenoviridae, mouse adenovirus type 1 (MAV-1), Tupaia adenovirus type 1 (TAV-1), and two bovine adenoviruses of two distinct subgroups, BAV-3 and BAV-7 . The results of the sequencing studies showed that the TATA box and an upstream inverted CAAT box are conserved in all species and that the binding site for transcription factor USF is present in all except MAV-1, in which a sequence similar to an Sp1-binding site is present at a similar position . The initiator element (INR) sequence is not well conserved, and only one or other of the two downstream activating elements, DE1 and DE2, is predicted to be present in the nonprimate virus MLP regions . Ribonuclease protection assays on RNA isolated from MAV-1-infected cells late in infection indicated that the predicted MLP is functional, and transcription initiation and splice donor sites were identified . The human virus MLP is embedded in the essential DNA polymerase sequence on the opposite DNA strand . The primary amino acid sequences of the C-terminal regions of the predicted DNA polymerases show strong conservation of sequence motifs observed in replicative polymerases ranging from prokaryotes to mammals, and additional regions of strong conservation among the adenovirus polymerases . Pairwise comparisons between the newly sequenced regions of the polymerases and previously published sequences show that BAV-7 is most dissimilar to all others, while TAV-1 has a greater similarity to the primate sequences than to the others . The sequence data from both strands were also used to construct phylogenetic trees, based on BAV-7 as the outgroup . The trees constructed from the two sets of sequences are broadly similar, showing close relationships between primate viruses, but differing in the order of divergence of TAV-1 and MAV-1 branches.

Science, 1996 Jun 14, 272(5268), 1644 - 6
Arrested DNA replication in Xenopus and release by Escherichia coli mutagenesis proteins; Oda N et al.; Xenopus oocytes and oocyte nuclear extracts repair ultraviolet photoproducts on double-stranded (ds) DNA and replicate single-stranded (ss) to ds DNA . M13 ss DNA molecules containing cyclobutane pyrimidine dimers were maintained but not replicated in Xenopus oocytes yet were replicated in progesterone-matured oocytes . The replication arrest functioned only in cis . The replication arrest was alleviated by injection into oocytes of messenger RNAs encoding the prokaryotic mutagenesis proteins UmuD'C or MucA'B . These results may help explain how cells stabilize repair or replication events on DNA with unrepairable lesions.

Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 6059 - 63
An Escherichia coli chromosomal "addiction module" regulated by guanosine {corrected} 3',5'-bispyrophosphate: a model for programmed bacterial cell death; Aizenman E et al.; "Addiction modules" consist of two genes . In most of them the product of one is long lived and toxic while the product of the second is short lived and antagonizes the toxic effect; so far, they have been described mainly in a number of prokaryotic extrachromosomal elements responsible for the postsegregational killing effect . Here we show that the chromosomal genes mazE and mazF, located in the Escherichia coli rel operon, have all of the properties required for an addiction module . Furthermore, the expression of mazEF is regulated by the cellular level of guanosine {corrected} 3',5'-bispyrophosphate, the product of the RelA protein under amino acid starvation . These properties suggest that the mazEF system may be responsible for programmed cell death in E . coli and thus may have a role in the physiology of starvation.

Proc Natl Acad Sci U S A, 1996 Jun 11, 93(12), 5936 - 40
Structure and conformational changes of DNA topoisomerase II visualized by electron microscopy; Schultz P et al.; Type II DNA topoisomerases, which create a transient gate in duplex DNA and transfer a second duplex DNA through this gate, are essential for topological transformations of DNA in prokaryotic and eukaryotic cells and are of interest not only from a mechanistic perspective but also because they are targets of agents for anticancer and antimicrobial chemotherapy . Here we describe the structure of the molecule of human topoisomerase II {DNA topoisomerase (ATP-hydrolyzing), EC 5.99.1.3} as seen by scanning transmission electron microscopy . A globular approximately 90-angstrom diameter core is connected by linkers to two approximately 50-angstrom domains, which were shown by comparison with genetically truncated Saccharomyces cerevisiae topoisomerase II to contain the N-terminal region of the approximately 170-kDa subunits and that are seen in different orientations . When the ATP-binding site is occupied by a nonhydrolyzable ATP analog, a quite different structure is seen that results from a major conformational change and consists of two domains approximately 90 angstrom and approximately 60 angstrom in diameter connected by a linker, and in which the N-terminal domains have interacted . About two-thirds of the molecules show an approximately 25 A tunnel in the apical part of the large domain, and the remainder contain an internal cavity approximately 30 A wide in the large domain close to the linker region . We propose that structural rearrangements lead to this displacement of an internal tunnel . The tunnel is likely to represent the channel through which one DNA duplex, after capture in the clamp formed by the N-terminal domains, is transferred across the interface between the enzyme's subunits . These images are consistent with biochemical observations and provide a structural basis for understanding the reaction of topoisomerase II.

FEBS Lett, 1996 Jun 10, 388(1), 43 - 6
Two different respiratory Rieske proteins are expressed in the extreme thermoacidophilic crenarchaeon Sulfolobus acidocaldarius: cloning and sequencing of their genes; Schmidt CL et al.; We have isolated two genes encoding Rieske iron sulfur proteins from the genomic DNA of the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius (DSM 639) . One of the genes, named soxL, codes for the previously isolated novel Rieske-I protein . The second gene (soxF) 121 codes for the Rieske-II protein associated with the second terminal oxidase of Sulfolobus . Both proteins exhibit only 24% identical residues . The Rieske-I protein shows a number of unusual features . (i) The distance between the two cluster binding sites is significantly larger than in all known proteins . (ii) An unexpected Pro --> Asp exchange in one of the cluster binding sites . (iii) It shows some resemblance to the mitochondrial and plastidic Rieske proteins insofar as the soxL gene codes for a pre-sequence which is no longer present in the mature Rieske-I protein . Both proteins cluster together on a separate branch of the phylogenetic tree . To our knowledge this is the first proven case of two significantly different Rieske proteins in a prokaryote.

J Biol Chem, 1996 Jun 7, 271(23), 13746 - 53
Comparative topology studies in Saccharomyces cerevisiae and in Escherichia coli . The N-terminal half of the yeast ABC protein Ste6; Geller D et al.; Gene fusions have provided a strategy for determining the topology of polytopic membrane proteins in Escherichia coli . To evaluate whether this highly effective approach is applicable to heterologously expressed eukaryotic integral membrane proteins, we have carried out a comparative topological study of the eukaryotic membrane protein Ste6 both in bacteria and in yeast . Ste6, is an ATP binding cassette (ABC) protein, essential for export of the a-factor mating pheromone in Saccharomyces cerevisiae . The topogenic reporters, invertase in S . cerevisiae and alkaline phosphatase in E . coli, were fused to Ste6 at identical sites and the fusions were expressed in yeast and bacteria, respectively . The results obtained in both systems are similar, although more definitive in E . coli, and support the predicted six-transmembrane spans organization of the N-terminal half of Ste6 . Thus, the topological determinants for membrane insertion of polytopic proteins in prokaryotic and in eukaryotic systems appear to be highly similar . In this study we also demonstrate that Ste6 does not contain a cleaved signal sequence.

Biochem Biophys Res Commun, 1996 Jun 5, 223(1), 19 - 23
The basic fibroblast growth factor (FGF-2) antisense RNA (GFG) is translated into a MutT-related protein in vivo; Li AW et al.; The basic fibroblast growth factor (FGF-2) gene is transcribed bidirectionally to yield multiple sense (coding) transcripts and a unique 1.5 kb antisense transcript which may regulate sense RNA stability . The antisense RNA also contains a long open reading frame that predicts a hypothetical protein with homology to the prokaryotic MutT antimutator proteins . However, translation of this protein has not previously been demonstrated . We employed antibodies against the conserved MutT-domain of the deduced human FGF-2 antisense protein (GFG) to demonstrate expression of an immunoreactive 24 kDa protein in liver extracts from Xenopus laevis, and two proteins of 28 and 35 kDa in rat liver . In rats, GFG protein expression detected by western blot was tissue-specific and correlated with the level of FGF-2 antisense mRNA expression . These findings demonstrate that, in addition to its possible RNA regulatory function, the FGF-2 antisense transcript is translated into a conserved MutT-related protein.

Zhonghua Yi Xue Za Zhi, 1996 Jun, 76(6), 447 - 50
{The expression of pulmonary surfactant proteins B and surfactant protein C in E . coli}; Liu X et al.; OBJECTIVE: To express biological active surfactant protein B(SP-B) and surfactant protein C(SP-C) combined with synthetic phospholipids to make an ideal exogenous pulmonary surfactant (PS) for the treatment of respiratory distress syndrome (RDS) in future . METHOD: Prokaryote expression vector pIN-II-ompA2-B-C was established with the cDNA fragments of mature human SP-B and SP-C, then the fusion protein of mature SP-B with SP-C was expressed in E . coli BL21 induced by IPTG . The expression of fusion protein in E . coli was confirmed by Western blot anlaysis . The biological activity of the expressed product was measured by Wilhelmy's surface balance (MTP-2) . RESULTS: The fusion protein of SP-B with SP-C could be expressed in E . coli BL21, and their molecular weight (Mr) was 20000 on gel electrophoresis, but the amount was small . According to the measurement of Wilhelmy's surface balance (MTP-2), the minimal surface tension of expressed protein in E . coli BL21 could be reduced to 0.004 N/m2 and the area of hysteresis loop was relatively large . CONCLUSIONS: The results of the investigation will be a basis to obtain SP-B and SP-C with gene engineering technology and to create a new way for producing ideal exogenous PS.

Photodermatol Photoimmunol Photomed, 1996 Jun, 12(3), 122 - 30
Enzyme therapy of xeroderma pigmentosum: safety and efficacy testing of T4N5 liposome lotion containing a prokaryotic DNA repair enzyme; Yarosh D et al.; Xeroderma pigmentosum (XP) is a rare genetic disease in which patients are defective in DNA repair and are extremely sensitive to solar UV radiation exposure . A new treatment approach was tested in these patients, in which a prokaryotic DNA repair enzyme specific for UV-induced DNA damage was delivered into the skin by means of topically applied liposomes to supplement the deficient activity . Acute and chronic safety testing in both mice and humans showed neither adverse reactions nor significant changes in serum chemistry or in skin histology . The skin of XP patients treated with the DNA repair liposomes had fewer cyclobutylpyrimidine dimers in DNA and showed less erythema than did control sites . The results encourage further clinical testing of this new enzyme therapy approach.

Mol Biochem Parasitol, 1996 Jun, 78(1-2), 249 - 57
Isolation, characterization and expression of the gene encoding cytidine triphosphate synthetase from Giardia intestinalis; Lim RL et al.; The cytidine triphosphate synthetase gene from Giardia intestinalis was cloned using a PCR-based strategy . A 519 bp PCR product was obtained from the amplification of genomic DNA using two oligonucleotides derived from the CTP synthetase amino acid consensus sequences DPYINVDPG and KTKPTQ . This product was used to probe restriction endonuclease digested genomic DNA and the respective plasmid mini-libraries . Two genomic clones were obtained one with a 3.6 kb HindIII DNA fragment, containing approximately three-quarters of the 5'-end of the synthetase gene and subsequently, a 5.8 kb PstI DNA fragment which contained the whole gene . The intronless gene has a 1863 bp open reading frame encoding 620 amino acids (M(r) of 68.3 kDa) . A well conserved catalytic glutamine aminotransferase (GAT) domain was identified . In addition, three insert sequences were found which are not present in CTP synthetase from other species . Alignment and comparison of the deduced amino acid sequence relative to CTP synthetases from other species revealed a high degree of identity (34%) with a greater resemblance to prokaryotes than eukaryotes . The gene is located on chromosome 6 and the messenger RNA encoding it is estimated to be 1.9 kb . The coding region of G . intestinalis CTP synthetase was generated by PCR and subsequently cloned into the pQE30 vector for expression in E . coli . This construct yielded a soluble and enzymatically active recombinant protein which was purified by a Ni-NTA affinity column . The purified recombinant protein had a subunit molecular weight of 69.5 kDa and a native molecular weight of approximately 274 kDa . Kinetic studies of the partially purified recombinant G . intestinalis CTP synthetase gave apparent K(m) values of 0.1 mM and approximately 0.5 mM for the substrates UTP and L-glutamine respectively in accord with previously reported values for the native enzyme.

Trends Microbiol, 1996 Jun, 4(6), 237 - 42
Do bacteria need to communicate with each other for growth?
Kaprelyants AS, Kell DB.
It is usually assumed that most prokaryotes, when given appropriate nutrients, can grow and divide in the absence of other cells of the same species . However, recent studies have suggested that, for growth, prokaryotes need to communicate with each other using signalling molecules, and a variety of 'eukaryotic' hormones have been shown to stimulate bacterial growth . These observations have important implications for our understanding of bacterial pathogenicity.

Lipids, 1996 Jun, 31(6), 557 - 69
Uses of biotechnology in modifying plant lipids; Budziszewski GJ et al.; This review discusses fatty acid modification of oilseeds with additional emphasis on production of oxygenated derivatives . In a relatively short period, less than a decade, our understanding of the enzymes involved in plant fatty acid synthesis has increased to the point where we understand how they might be used in oilseed modification . Further, through modern molecular biological techniques, the actual genes for many of these important enzymes have been cloned . Use of genetic transformation systems has allowed us to fundamentally alter the normal biosynthetic pathways in highly specific ways, in manners that would be either difficult or impossible using traditional breeding techniques . Alteration of plant lipid biosynthesis is not restricted to using genes from the plants themselves, but interspecies transfer is possible, either from completely unrelated plant species (often of no commercial value but possessing unusual biochemical properties) or from animals, fungi, and prokaryotic organisms . In this way "designer" plants possessing altered metabolism, tailored to the interests or needs of certain industries, nutritionists, and the consumer can be created.

Biotechniques, 1996 Jun, 20(6), 1070 - 6, 1078, 1080-1
Phage display shot-gun cloning of ligand-binding domains of prokaryotic receptors approaches 100% correct clones; Jacobsson K et al.; We recently presented an application of the phage display technique enabling cloning of DNA encoding ligand-binding domain(s) of prokaryotic receptors directly from chromosomal DNA . Here we show that the use of a gene VIII-based, instead of a gene III-based, phagemid vector system results in a much more efficient selection for phage displaying a binding capacity . A phagemid library was made by insertion of randomly fragmented chromosomal DNA from Staphylococcus aureus strain 8325-4 into gene VIII in the constructed phagemid vector pG8H6 . The library, which in theory should express parts of all proteins encoded by the bacterial genome, was affinity panned against the ligands IgG, fibronectin and fibrinogen, respectively . After a second panning against the same ligand, a significant increase in the number of eluted phagemid particles was observed, and 75%-100% of randomly picked clones contained inserts derived from genes encoding proteins with a binding affinity for the respective ligand . The results show that this technique can be used for cloning prokaryotic receptor genes without any prior knowledge of the receptor, thus eliminating the need for probes in the identification of receptor genes.

Trends Biochem Sci, 1996 Jun, 21(6), 203 - 8
Knowing when not to stop: selenocysteine incorporation in eukaryotes; Low SC et al.; The regulation of translation frequently involves protein-RNA interactions . An intriguing example of this is the alternative decoding of UGA, typically a stop codon, as selenocysteine . Two RNA structures, the mRNA selenocysteine insertion sequence (SECIS element) and a unique selenocysteyl-tRNA, are required for this process . In prokaryotes, a single RNA-binding protein, a selenocysteine-specific elongation factor, interacts with both the tRNA and mRNA to confer decoding . Whether eukaryotes use a similar mechanism is currently the subject of intense investigation.

Microbiology, 1996 Jun, 142 ( Pt 6), 1429 - 35
Size and genomic location of the pMGA multigene family of Mycoplasma gallisepticum; Baseggio N et al.; The pMGA multigene family encodes variant copies of the cell surface haemagglutinin of Mycoplasma gallisepticum . Quantitative Southern blotting, using an oligonucleotide probe complementary to a region conserved in the leader sequence of all known pMGA genes, was used to estimate the number of members of the family in the genome of seven strains of M . gallisepticum . The number of copies estimated to be present in the genome varied from 32 in strain F to 70 in strain R, indicating that the pMGA gene family may be second in size only to the tRNA family among prokaryotes . If all members of the pMGA family are of similar length to those which have been characterized, a minimum of 79 kb (7.7%) of the genome of strain S6, 82 kb (8.2%) of PG31 and 168 kb (16%) of the genome of strain R is dedicated to encoding variants of the same haemagglutinin . The GAA repeat motif identified in the intergenic region between all characterized pMGA genes appeared to be a feature common to most, if not all, pMGA genes, and furthermore probably exclusive to them . The genomic locations of members of the pMGA family were determined by PFGE and Southern blot hybridization of M . gallisepticum strain S6 . The hybridizing regions were localized to four separate regions on the chromosome . The pMGA genes are likely to be predominantly arranged as tandem repeats within these regions, similar to the restricted regions for which the genomic sequence has been determined.

J Gen Virol, 1996 Jun, 77 ( Pt 6), 1259 - 63
Identification of the non-virion (NV) protein of fish rhabdoviruses viral haemorrhagic septicaemia virus and infectious haematopoietic necrosis virus; Schutze H et al.; Sequence analysis of a 795 nucleotide region of the fish rhabdovirus viral haemorrhagic septicaemia virus (VHSV) genome revealed one complete and one partial ORF of 369 and 153 nucleotides, respectively . The latter ORF probably encodes the amino-terminal part of the L (polymerase) protein . The former ORF potentially encodes a 122 amino acid protein . The location of this ORF as well as the size and deduced structure of the translation product indicate that it represents a homologue of the non-virion (NV) protein of the related infectious haematopoietic necrosis virus (IHNV) . Antisera raised against prokaryotically expressed NV protein of VHSV and IHNV were used to detect NV expression in VHSV- and IHNV-infected cells by Western Blot and immunofluorescence analyses . We present here the sequence of the VHSV NV gene and demonstrate the presence of IHNV and VHSV NV proteins in virus-infected cells.

J Gen Virol, 1996 Jun, 77 ( Pt 6), 1151 - 7
Human cytomegalovirus pp65 lower matrix phosphoprotein harbours two transplantable nuclear localization signals; Gallina A et al.; Human cytomegalovirus phosphoprotein pp65 is targeted to the cell nucleus immediately after infection . Deletion and point mutation analysis of the pp65 gene expressed in insect cells showed that two hydrophilic regions (HP1 and HP2) within the pp65 C-terminal 40% each harboured an independent nuclear localization signal (NLS); strong association to the nuclear stroma also requires the N-terminal domain . Either region, when fused to chloramphenicol acetyltransferase, localized the reporter protein to the nucleus in insect cells as well as in NIH 3T3 cells and human lung fibroblasts . In addition, HP1 was found to be the target of pp65 Ser/Thr phosphorylation in insect cells and a prokaryotically expressed HP1 was actively phosphorylated in vitro by casein kinase II, for which two site clusters map in HP1 . These findings indicate that pp65 includes two NLSs, one of which has the potential to be modulated by phosphorylation.

J Bacteriol, 1996 Jun, 178(12), 3564 - 71
Genetic analysis of the Mycobacterium smegmatis rpsL promoter; Kenney TJ et al.; The DNA sequence of the promoter region of the Mycobacterium smegmatis rpsL gene, which encodes the S12 ribosomal protein, was determined . Primer extension analysis and S1 nuclease protection experiments identified the 5' end of the rpsL mRNA to be 199 bp upstream of the translation initiation codon . The rpsL promoter contained sequences upstream of this start point for transcription that were similar to the canonical hexamers found at the -10 and -35 regions of promoters recognized by Esigma70, the major form of RNA polymerase in Escherichia coli . To define the promoter of the rpsL gene, DNA fragments containing progressive deletions of the upstream region of the rpsL gene were inserted into a plasmid vector containing a promoterless xylE gene . These insertions revealed that the 200 bp of DNA sequence immediately upstream from the translation initiation codon was not essential for promoter function . In addition, 5' deletions removing all but 34 bp upstream of the transcription start point retained greater than 90% promoter activity, suggesting that the -35 hexamer was not essential for promoter activity . To determine which nucleotides were critical for promoter function, oligonucleotide-directed mutagenesis and mutagenic PCR amplification were used to produce point mutations in the region upstream of the start point of transcription . Single base substitutions in the -10 hexamer, but not in the -35 hexamer, severely reduced rpsL promoter activity in vivo . Within the -10 hexamer, nucleotide substitutions causing divergence from the E . Coli sigma70 consensus reduced promoter activity . The DNA sequence immediately upstream from the - 10 hexamer contained the TGn motif described as an extended -10 region in prokaryotic promoters . Mutations in this motif, in combination with a transition at either the -38 or -37 position within the -35 hexamer, severely reduced promoter activity, indicating that in the absence of a functional -35 region, the rpsL promoter is dependent on the TGn sequence upstream from the -10 hexamer . Comparison of the nucleotide sequence of the rpsL promoter region of M . smegmatis with the homologous sequences from Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium tuberculosis showed the presence in these slowly growing mycobacterial species of conserved promoter elements a similar distance upstream of the translation initiation codon of the rpsL gene, but these other mycobacterial promoters did not contain the extended -10 motif.

J Bacteriol, 1996 Jun, 178(11), 3177 - 87
Characterization of the cytoplasmic filament protein gene (cfpA) of Treponema pallidum subsp . pallidum; You Y et al.; Treponema pallidum and other members of the genera Treponema, Spirochaeta, and Leptonema contain multiple cytoplasmic filaments that run the length of the organism just underneath the cytoplasmic membrane . These cytoplasmic filaments have a ribbon-like profile and consist of a major cytoplasmic filament protein subunit (CfpA, formerly called TpN83) with a relative molecular weight of approximately 80,000 . Degenerate DNA primers based on N-terminal and CNBr cleavage fragment amino acid sequences of T . pallidum subsp . pallidum (Nichols) CfpA were utilized to amplify a fragment of the encoding gene (cfpA) . A 6.8-kb EcoRI fragment containing all but the 5' end of cfpA was identified by hybridization with the resulting PCR product and cloned into Lambda ZAP II . The 5' region was obtained by inverse PCR, and the complete gene sequence was determined . The cfpA sequence contained a 2,034-nucleotide coding region, a putative promoter with consensus sequences (5'-TTTACA-3' for -35 and 5'-TACAAT-3' for -10) similar to the sigma70 recognition sequence of Escherichia coli and other organisms, and a putative ribosome-binding site (5'-AGGAG-3') . The deduced amino acid sequence of CfpA indicated a protein of 678 residues with a calculated molecular mass of 78.5 kDa and an estimated pI of 6.15 . No significant homology to known proteins or structural motifs was found among known prokaryotic or eukaryotic sequences . Expression of a LacZ-CfpA fusion protein in E . coli was detrimental to survival and growth of the host strain and resulted in the formation of short, irregular filaments suggestive of partial self-assembly of CfpA . The cytoplasmic filaments of T . pallidum and other spirochetes appear to represent a unique form of prokaryotic intracytoplasmic inclusions.

J Parasitol, 1996 Jun, 82(3), 423 - 7
The putative acetyl-CoA synthetase gene of Cryptosporidium parvum and a new conserved protein motif in acetyl-CoA synthetases; Khramtsov NV et al.; We determined the nucleotide (nt) sequence of the putative gene encoding acetyl-coenzyme A synthetase (ACS) from the parasitic protozoan Cryptosporidium parvum . The gene is single copy, located on a chromosome of approximately 1.08 mb, and has no introns . The gene is characterized by low codon usage bias and encodes a 694-amino acid (aa) protein with a predicted molecular size of 78 kDa, similar to other ACSs from different prokaryotic and eukaryotic species . Comparison of multiple protein alignments of ACSs revealed a new conserved sequence motif PKT(R/V/L)SGK(I/V/T)(T/M/V/K)R(R/N) near the C-terminus, which may be a signature for ACSs . This motif shares significant homology with sequences from other members of the AMP-binding family, has secondary structure similar to the purine-binding motif of ATP- and GTP-ases, and may play a role in the enzymatic activity of proteins from the AMP-binding family.

Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5443 - 8
Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis; Ito K et al.; Translation termination requires two codon-specific polypeptide release factors in prokaryotes and one omnipotent factor in eukaryotes . Sequences of 17 different polypeptide release factors from prokaryotes and eukaryotes were compared . The prokaryotic release factors share residues split into seven motifs . Conservation of many discrete, perhaps critical, amino acids is observed in eukaryotic release factors, as well as in the C-terminal portion of elongation factor (EF) G . Given that the C-terminal domains of EF-G interacts with ribosomes by mimicry of a tRNA structure, the pattern of conservation of residues in release factors may reflect requirements for a tRNA-mimicry for binding to the A site of the ribosome . This mimicry would explain why release factors recognize stop codons and suggests that all prokaryotic and eukaryotic release factors evolved from the progenitor of EF-G.

Gene, 1996 May 24, 171(1), 83 - 7
Phylogeny based on elongation factor Tu reflects the phenotypic features of mycoplasmas better than that based on 16S rRNA; Kamla V et al.; A universal phylogenetic tree of organisms from all kingdoms was constructed by the use of elongation factor Tu (EF-Tu) as the marker molecule . As in the 16S ribosomal RNA (16S rRNA)-based phylogeny, the EF-Tu tree divides eukaryotes, archaebacteria, and prokaryotes into three main branches . Furthermore, the EF-Tu-based tree shows, in contrast to the 16S rRNA tree, some interesting evolutionary relationships between mycoplasmas, better reflecting phenotypic features of these organisms.

FEBS Lett, 1996 May 20, 386(2-3), 95 - 8
A 6 kDa protein homologous to the N-terminus of the HMG1 protein promoting stimulation of murine erythroleukemia cell differentiation; Sparatore B et al.; Murine erythroleukemia (MEL) cells, in addition to an mRNA coding for a 30 kDa high mobility group (HMG)-1 protein, contain an mRNA coding for a 6 kDa HMG1 protein having the following structural properties: (1) its primary structure has 90% homology with the N-terminal sequence of the 30 kDa HMG1 protein; (2) it contains a consensus region of the HMG1 protein family; (3) it is deprived of the cluster of acidic amino acids that characterizes the C-terminal region of the 30 kDa HMG1 protein . This novel small Mr HMG1 protein has been expressed in prokaryotic cells and tested to establish similarities and differences in activity compared to the homologous higher Mr HMG1 protein . It has been found that the low Mr HMG1 form is not released from MEL cells following induction to erythroid differentiation, but is still effective, although with much less efficiency, when added to the external medium, in promoting acceleration in the rate of MEL cell differentiation as well as in activation of alpha-protein kinase C . Altogether these results provide evidence for the presence in MEL cells of a multigene family that encodes at least two different HMG1-type sequences most presumably involved, at distinct cellular sites, in different functions although commonly related to the promotion of cell differentiation . Additional information can be considered concerning the relationship between the characteristic N-terminal sequence of HMG1 protein and the extracellular activity on MEL cell differentiation.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 612 - 8
Synthesis of the bacteriophage lambda P protein in amino acid-starved Escherichia coli cells; Obuchowski M et al.; It was demonstrated previously that in isoleucine-starved Escherichia coli relA mutants harboring a plasmid derived from bacteriophage lambda the lambda O protein is not synthesized . However, a protein which coprecipited with the lambda O during immunoprecipitation with anti-lambda O serum was synthesized during the relaxed response . Here we found that this protein is the lambda P gene product . Despite significant inhibition of transcription from the pR promoter (which produces mRNA for the lambda P protein synthesis) during the stringent response, the lambda P protein was efficiently synthesized in relA- as well as relA+ strains starved for isoleucine, threonine and histidine, whereas the synthesis was negligible during starvation for arginine and leucine . The synthesis of the lambda P protein in amino acid-starved cells is sensitive to rifampicin . Thus we presume that this phenomenon is not caused by eventual increased stability of the lambda P mRNA but rather is an effect of preferential translation of this mRNA and incorporation of limited amount of amino acids arising in the starved cells as a result of intracellular proteolysis . One of possible explanations of the mechanism of this phenomenon may suggest that the same signals can be recognized in both prokaryotic and eukaryotic cells during initiation of translation at non-AUG codons.

Biochem Biophys Res Commun, 1996 May 15, 222(2), 512 - 8
Linker histones inhibit T4 and Escherichia coli DNA ligases; Ray E et al.; Based on some preliminary observations that linker histones strongly inhibit the activity of prokaryotic DNA ligases, we studied the effect of these histones on the ligation of short restriction DNA fragments by either T4 or E . coli DNA ligases . The inhibitory effect was strong, but it appeared only after two molecules of H1 bound to a approximately 200 bp-long DNA fragment . A similar pattern of inhibition (but at much higher concentration) was observed with the isolated globular domain of histone H5 . That the inhibition was specific to the linker histones became clear when other basic proteins, such as the core histone octamer or cytochrome C, were tested . They did not inhibit the ligases but rather significantly stimulated them . The other major linker DNA-binding protein in chromatin, the non-histone protein HMG1, showed no significant effect on the ligase activity.

EMBO J, 1996 May 15, 15(10), 2530 - 9
The relationship between sequence-specific termination of DNA replication and transcription; Mohanty BK et al.; In Escherichia coli and Bacillus subtilis replication fork arrest occurs in the terminus at sequence-specific sites by the binding of replication terminator proteins to the fork arrest sites . The protein-DNA complex causes polar arrest of the replication forks by inhibiting the activity of the replicative helicases in only one orientation of the terminus with respect to the replication origin . This activity has been named as polar contrahelicase . In this paper we report on a second novel activity of the terminator proteins of E.coli and B.subtilis, namely the ability of the proteins to block RNA chain elongation by several prokaryotic RNA polymerases in a polar mode . The replication terminator proteins ter and RTP of E.coli and B.subtilis respectively, impeded RNA chain elongation catalyzed by T7, SP6 and E.coli RNA polymerases in a polar mode at the replication arrest sites . The RNA chain anti-elongation and the contrahelicase activities were isopolar . Whereas one monomer of ter was necessary and sufficient to block RNA chain elongation, two interacting dimers of RTP were needed to effect the same blockage . The biological significance of the RNA chain anti-elongation activity is manifested in the functional inactivation of a replication arrest site by invasion of RNA chains from outside, and the consequent need to preserve replication arrest activity by restricting the passage of transcription through the terminus-terminator protein complex.

Nucleic Acids Res, 1996 May 15, 24(10), 1865 - 72
Interaction of the IciA protein with AT-rich regions in plasmid replication origins; Wei T et al.; A set of AT-rich repeats is a common motif in prokaryotic replication origins . We have screened for proteins binding to the AT-rich repeat region in plasmids F, R1 and pSC101 using an electrophoretic mobility shift assay with PCR-amplified DNA fragments from the origins . The IciA protein, which is known to bind to the AT-rich repeat region in the Escherichia coli origin of chromosome replication, oriC, was found to bind to the corresponding region from plasmids F (oriS) and R1, but not to pSC101 . DNase I footprint analysis showed that IciA interacted with the AT-rich region in both F and R1 . When the IciA gene was deleted, the copy number of plasmid F increased somewhat, whereas there was no major effect on the replication of pSC101 and R1, or on the E . coli chromosome.

Biochem J, 1996 May 15, 316 ( Pt 1), 57 - 63
Pyrophosphate-dependent phosphofructokinase of Entamoeba histolytica: molecular cloning, recombinant expression and inhibition by pyrophosphate analogues; Bruchhaus I et al.; By using oligonucleotide primers derived from regions highly conserved in prokaryotic and eukaryotic phosphofructokinase sequences, a genomic DNA fragment was amplified and used to isolate cDNA and genomic clones coding for PPi-dependent phosphofructokinase (PPi-PFK) of Entamoeba histolytica . The open reading frame consists of 1308 bp and the corresponding protein has a calculated molecular mass of 47.6 kDa . The N-terminal half of the protein shows 27-35% identity with PPi-PFKs or ATP-dependent phosphofructokinases (ATP-PFKs) of various eukaryotic and prokaryotic organisms . The amino acid residues that form the active site of the PPi-PFK from Propionibacterium freudenreichii and the allosteric ATP-PFK from Escherichia coli are conserved within the amoeba sequence . The PPi-PFK was recombinantly expressed by using a prokaryotic expression system . The purified recombinant protein was found to be enzymically active . The K(m) values for PPi and fructose 6-phosphate of the native and the recombinant PPi-PFKs were nearly identical . Various bisphosphonates (synthetic pyrophosphate analogues) were tested for their ability to inhibit PPi-PFK activity or amoebic growth . All bisphosphonates tested were competitive inhibitors for amoeba PPi-PFK activity . The best inhibitors were CGP 48048 and zoledronate, with Ki values of 50 microM . All bisphosphonates inhibited amoebic growth . One of them (risedronate) was inhibitory at a concentration of 10 microM . Bisphosphonates are therefore potential therapeutic agents for the treatment of amoebiasis.

Biochem J, 1996 May 15, 316 ( Pt 1), 251 - 7
The catalase-peroxidase of Synechococcus PCC 7942: purification, nucleotide sequence analysis and expression in Escherichia coli; Mutsuda M et al.; Synechococcus PCC 7942, a cyanobacterium, possesses catalaseperoxidase as the sole hydrogen peroxide-scavenging system . The enzyme has been purified to electrophoretic homogenenity from the cells . The native enzyme had a molecular mass of 150 kDa and was composed of two identical subunits of molecular mass 79 kDa . The apparent Km value of the catalase activity for H2O2 was 4.2 +/- 0.27 mM and the kcat value was 2.6 x 10(4) s-1 . The enzyme contained high catalase activity and an appreciable peroxidase activity with o-dianisidine and pyrogallol . The catalase activity was not inhibited by 3-amino-1,2,4-triazole but by KCN and NaN3 (apparent Ki values 19.3 +/- 0.84 and 20.2 +/- 0.95 microM respectively) . The enzyme showed an absorption spectrum of typical protohaem and contained one protohaem molecule per dimer . The gene encoding catalase-peroxidase was cloned from the chromosomal DNA of Synechococcus PCC 7942 . A 2160 bp open reading frame (ORF), coding a catalase-peroxidase of 720 amino acid residues (approx . 79.9 kDa), was observed . The deduced amino acid sequence coincided with that of the N-terminus of the purified enzyme and showed a remarkable similarity to those of a family of catalase-peroxidases of prokaryotic cells . Escherichia coli BL21 (DE3)plysS, harbouring a recombinant plasmid containing the catalase-peroxidase gene, produced a large amount of proteins that co-migrated on SDS/PAGE with the native enzyme . The recombinant enzyme showed the same ratio of catalase activity to peroxidase activity with o-dianisidine and the same Km for H2O2 as the native enzyme.

J Biol Chem, 1996 May 10, 271(19), 11468 - 76
A delayed-early response nuclear gene encoding MRPL12, the mitochondrial homologue to the bacterial translational regulator L7/L12 protein; Marty L et al.; We have characterized a new delayed-early response mRNA encoding a 21-kDa product (MRPL12) that accumulates during the G1 phase of growth-stimulated cells . MRPL12 is the mammalian homologue to chloroplastic and bacterial L12 ribosomal proteins . Immunofluorescence microscopy and cell fractionation indicate a predominant mitochondrial localization in various mammalian cell lines . The NH2-terminal 49 amino acids are necessary and sufficient to target the protein within the mitochondria and are probably cleaved off during import . MRPL12 proteins associated in vitro and cofractionate with ribosomal structures, as is the case for prokaryotic L12 proteins . Expression of a dominant inhibitory truncated protein leads to a severe reduction in cell growth by inhibiting mitochondrial ATP production . MRPL12 is the first mammalian mitochondrial ribosomal protein to be characterized.

Nature, 1996 May 9, 381(6578), 169 - 72
A DEAD-box RNA helicase in the Escherichia coli RNA degradosome; Py B et al.; The Escherichia coli RNA degradosome is a multi-enzyme complex that contains the exoribonuclease polynucleotide phosphorylase (PNPase) and the endoribonuclease RNase E . Both enzymes are important in RNA processing and messenger RNA degradation . Here we report that enolase and RhlB are two other major components of the degradosome . Enolase is a glycolytic enzyme with an unknown role in RNA metabolism . RhlB is a member of the DEAD-box family of ATP-dependent RNA helicases, which are found in both prokaryotes and eukaryotes . We show that the degradosome has an ATP-dependent activity that aids the degradation of structured RNA by PNPase . Incubation of the degradosome with affinity-purified antibody against RhlB inhibited the ATP-stimulated RNA degradation . These results suggest that RhlB acts by unwinding RNA structures that impede the processive activity of PNPase . RhlB is thus an important enzyme in mRNA turnover.

Protein Expr Purif, 1996 May, 7(3), 315 - 22
Characterization and partial purification of human monoamine oxidase-B expressed in Escherichia coli; Lu G et al.; Monoamine oxidases (MAO-A and MAO-B) are enzymes that play a key role in the degradation of endogenous and dietary monoamines . A full-length cDNA of the B-type of MAO, isolated from a human liver cDNA library, was cloned into a prokaryotic expression vector (pET11c) . Escherichia coli which was transfected with the recombinant plasmid expressed an insoluble protein product with the expected molecular weight (65 kDa) . However, in the inclusion body fraction, where most of the recombinant protein was present, no MAO activity was observed . In contrast, the membrane fraction of the bacterial lysates expressed catalytic activity as estimated by oxidative deamination of beta-phenylethylamine and tyramine . The active enzyme protein was solubilized with Triton X-100 and partly purified (80-fold) on a DEAE-Sepharose column . This enzyme activity showed properties very similar to those of human brain and platelet MAO-B . Moreover, a single band of the expected molecular size was observed on an immunoblot . The peak fraction from the DEAE-Sepharose separation was further purified on a tyramine-Sepharose column, yielding a highly purified enzyme (190-fold), visible as a band on a sodium dodecyl sulfate-containing polyacrylamide gel.

Semin Liver Dis, 1996 May, 16(2), 201 - 10
Hepatocellular transport: role of ATP-binding cassette proteins; Lomri N et al.; The interest of mammalian biologists in ATP-binding cassette (ABC) proteins is relatively recent . However, ABC proteins are widespread in distribution and have long been known to play an important transport role in prokaryotes . The review includes a brief overview of the structure, regulation, and varied functions of ABC proteins in different cell types as well as a synopsis of the emerging role of ABC proteins in human biology and disease . The review then focuses on the established (canalicular secretion of organic cations by the multidrug resistance, or MDR 1, gene product; ductular secretion of fluid and electrolytes mediated by CFTR), probable (biliary phospholipid secretion by the MDR 2 gene product; secretion of non-bile acid organic anions by the multidrug resistance protein, or MRP), and possible (bile acid secretion; biliary secretion of the signaling molecule, ATP; hormone transport) roles of known and novel ABC proteins in hepatobiliary secretion.

Plant Mol Biol, 1996 May, 31(2), 337 - 54
Function of 3' non-coding sequences and stop codon usage in expression of the chloroplast psaB gene in Chlamydomonas reinhardtii; Lee H et al.; The rate of mRNA decay is an important step in the control of gene expression in prokaryotes, eukaryotes and cellular organelles . Factors that determine the rate of mRNA decay in chloroplasts are not well understood . Chloroplast mRNAs typically contain an inverted repeat sequence within the 3' untranslated region that can potentially fold into a stem-loop structure . These stem-loop structures have been suggested to stabilize the mRNA by preventing degradation by exonuclease activity, although such a function in vivo has not been clearly established . Secondary structures within the translation reading frame may also determine the inherent stability of an mRNA . To test the function of the inverted repeat structures in chloroplast mRNA stability mutants were constructed in the psaB gene that eliminated the 3' flanking sequences of psaB or extended the open reading frame into the 3' inverted repeat . The mutant psaB genes were introduced into the chloroplast genome of Chlamydomonas reinhardtii . Mutants lacking the 3' stem-loop exhibited a 75% reduction in the level of psaB mRNA . The accumulation of photosystem I complexes was also decreased by a corresponding amount indicating that the mRNA level is limiting to PsaB protein synthesis . Pulse-chase labeling of the mRNA showed that the decay rate of the psaB mRNA was significantly increased demonstrating that the stem-loop structure is required for psaB mRNA stability . When the translation reading frame was extended into the 3' inverted repeat the mRNA level was reduced to only 2% of wild-type indicating that ribosome interaction with stem-loop structures destabilizes chloroplast mRNAs . The non-photosynthetic phenotype of the mutant with an extended reading frame allowed us to test whether infrequently used stop codons (UAG and UGA) can terminate translation in vivo . Both UAG and UGA are able to effectively terminate PsaB synthesis although UGA is never used in any of the Chlamydomonas chloroplast genes that have been sequenced.

Biochem Mol Biol Int, 1996 May, 38(6), 1211 - 21
Prokaryotic homolog of tubulin? Consideration of FtsZ and glyceraldehyde 3-phosphate dehydrogenase as probable candidates; Gupta RS et al.; The bacterial FtsZ protein has recently been suggested as a probable prokaryotic homolog of the tubulin family of proteins (Cell 80, 1995, 367-370) . We have compared the sequence similarity of tubulins to FtsZ and another protein glyceraldehyde 3-phosphate dehydrogenase (GAPDH) . Both these proteins exhibited similar levels of sequence identity to the tubulins, which in a few cases was indicated to be significant . We report that incubation of the GAPDH in microtubule assembly buffer causes its polymerization into filamentous structures . In eukaryotic cells, GAPDH is known to be associated with cytoskeletal structures and it binds specifically to both tubulins and colchicine . The latter is a distinctive characteristic of the tubulin family of proteins . These observations indicate that similar to the FtsZ proteins, GAPDH also exhibits a number of intriguing similarities to the tubulins . Whether any of these proteins truly represent the prokaryotic homolog of tubulin, however, is unclear at present.

Mol Microbiol, 1996 May, 20(3), 461 - 6
A flexible partnership: the CytR anti-activator and the cAMP-CRP activator protein, comrades in transcription control; Valentin-Hansen P et al.; A vital point in gene regulation is control at the level of transcription initiation . Recent research has established that this regulation can involve sophisticated networks of interacting proteins that modulate the activity of the transcription machinery by DNA looping, direct protein-protein interactions or changing DNA topology in the promoter region . This Micro-Review focuses on our investigations of a relatively simple prokaryotic gene regulatory system, the Escherichia coli CytR regulon, which exhibits a number of these features . This work has opened the door to the molecular understanding of how a prokaryotic repressor can be correctly positioned at specific DNA sequences with the help of a global activator, and how the repressor subsequently inhibits factor-dependent transcription initiation.

Antimicrob Agents Chemother, 1996 May, 40(5), 1126 - 33
Cloning and nucleotide sequence of the DNA gyrase gyrA gene from the fish pathogen Aeromonas salmonicida; Oppegaard H et al.; The DNA gyrase gyrA gene from the fish pathogen Aeromonas salmonicida 2148/89 was cloned, and the nucleotide sequence was determined . An open reading frame of 2,766 nucleotides was identified and was found to encode a protein of 922 amino acids with a calculated molecular mass of 101.1 kDa . The derived amino acid sequence shared a high degree of identity with other DNA gyrase A proteins, in particular, with other gram-negative GyrA sequences . When the amino acid sequence of A . salmonicida GyrA was compared with that of Escherichia coli GyrA, a number of conserved residues were present at identical coordinates, including the catalytic Tyr residue at position 122 (Tyr-122) and residues whose substitution confers quinolone resistance, notably, Ser-83, Ala-67, Gly-81, Asp-87, Ala-84, and Gln-106 . An intragenic region corresponding to 48 amino acids, which is not present in E . coli or other bacteria, was identified in the C-terminal part of A . salmonicida GyrA . This intragenic region shared sequence identity with various DNA-binding proteins of both prokaryotic and eukaryotic origins.

Microbiology, 1996 May, 142 ( Pt 5), 1265 - 72
Transcription of the glnB and glnA genes in the photosynthetic bacterium Rhodospirillum rubrum; Johansson M et al.; The PII protein, encoded by glnB, has a central role in the control of nitrogen metabolism in nitrogen-fixing prokaryotes . The glnB gene of Rhodospirillum rubrum was isolated and sequenced . The deduced amino acid sequence had very high sequence identity to other PII proteins . The glnA gene, encoding glutamine synthetase, was located 135 bp downstream of glnB and was partially sequenced . glnB is cotranscribed with glnA from a promoter with high similarity to the sigma 54-dependent promoter consensus sequence . A putative sigma 70 promoter was also identified further upstream of glnB . Northern blotting analyses showed that in addition glnA is either transcribed from an unidentified promoter or, more likely, that the glnBA transcript is processed to give the glnA mRNA . The total level of the two transcripts was much higher in nitrogen-fixing cells than in ammonia-grown cells.

J Mol Evol, 1996 May, 42(5), 570 - 9
Molecular evolution of maize catalases and their relationship to other eukaryotic and prokaryotic catalases; Guan L et al.; We have compared the nucleotide and protein sequences of the three maize catalase genes with other plant catalases to reconstruct the evolutionary relationship among these catalases . These sequences were also compared with other eukaryotic and prokaryotic catalases . Phylogenies based on distances and parsimony analysis show that all plant catalases derive from a common ancestral catalase gene and can be divided into three distinct groups . The first, and major, group includes maize Cat1, barley Cat1, rice CatB, and most of the dicot catalases . The second group is an apparent dicot-specific catalase group encompassing the tobacco Cat2 and tomato Cat . The third is a monocot-specific catalase class including the maize Cat3, barley Cat2, and rice CatA . The maize Cat2 gene is loosely related to the first group . The distinctive features of monocot-specific catalases are their extreme high codon bias at the third position and low degree of sequence similarity to other plant catalases . Similarities in the intron positions for several plant catalase genes support the conclusion of derivation from a common ancestral gene . The similar intron position between bean catalases and human catalase implies that the animal and plant catalases might have derived from a common progenitor gene sequence.

J Mol Evol, 1996 May, 42(5), 537 - 42
Phylogenetic analysis of the isopenicillin-N-synthetase horizontal gene transfer; Buades C et al.; A phylogenetic study of the isopenicillin-N-synthetase (IPNS) gene sequence from prokaryotic and lower eukaryotic producers of beta-lactam antibiotics by means of a maximum-likelihood approach has been carried out . After performing an extensive search, rather than invoking a global molecular clock, the results obtained are best explained by a model with three rates of evolution . Grouped in decreasing order, these correspond to A . nidulans and then to the rest of the eukaryotes and prokaryotes, respectively . The estimated branching date between prokaryotic and fungal IPNS sequences (852 +/- 106 MY) strongly supports the hypothesis that the IPNS gene was horizontally transferred from bacterial beta-lactam producers to filamentous fungi.

Nucleic Acids Res, 1996 May 1, 24(9), 1780 - 6
Examining the contribution of a dA+dT element to the conformation of Escherichia coli integration host factor-DNA complexes; Hales LM et al.; DNA binding proteins that induce structural changes in DNA are common in both prokaryotes and eukaryotes . Integration host factor (IHF) is a multi-functional DNA binding and bending protein of Escherichia coli that can mediate protein-protein and protein-DNA interactions by bending DNA . Previously we have shown that the presence of a dA+dT element 5'-proximal to an IHF consensus sequence can affect the binding of IHF to a particular site . In this study the contribution of various sequence elements to the formation of IHF-DNA complexes was examined . We show that IHF bends DNA more when it binds to a site containing a dA+dT element upstream of its core consensus element than to a site lacking a dA+dT element . We demonstrate that IHF can be specifically crosslinked to DNA with binding sites either containing or lacking this dA+dT element . These results indicate the importance of flanking DNA and a dA+dT element in the binding and bending of a site by IHF.

Biochem J, 1996 May 1, 315 ( Pt 3), 857 - 62
A protein targeting signal that functions in polarized epithelial cells in vivo; Ali S et al.; Eukaryotic membrane-associated polypeptides often contain a glycosylphosphatidylinositol (GPI) anchor that signals the attachment of GPI lipids to these proteins . The GPI anchor can function as a basolateral or apical targeting signal in mammalian cells cultured in vitro, although the function of the GPI anchor in vivo remains to be elucidated . In this study we have evaluated the effect of fusing a GPI anchor sequence to a prokaryotic reporter protein on the cellular location of the polypeptide in polarized epithelial cells of transgenic mice . The bacterial enzyme, when fused to a eukaryotic signal peptide, was secreted through the basolateral membrane of small-intestinal enterocytes; however, when the enzyme was lined to the GPI anchor sequence the polypeptide was redirected to the apical surface of the epithelial cells . These data provide the first direct evidence that the GPI anchor functions as an apical membrane protein sorting signal in polarized epithelial cells in vivo.

Am J Trop Med Hyg, 1996 May, 54(5), 471 - 4
Short report: regulation of inducible heat shock protein 70 genes in Leishmania chagasi; Andersen KA et al.; Eukaryotic and prokaryotic cells undergo an increase in heat shock proteins, including hsp70, during exposure to environmental stress and during some developmental changes . In trypanosomatid protozoa such as Leishmania sp . that cycle between poikilothermic vectors and mammalian hosts, this heat shock response occurs at programmed times in the parasite's life cycle . The increase in heat shock proteins in mammalian cells is initiated by an increased rate of transcription, resulting in greater amounts of total hsp70 RNA and protein . In contrast, we found a dramatic increase in hsp70 RNA during growth of Leishmania chagasi promastigotes from logarithmic to stationary phase in liquid culture, which was not accompanied by an increased amount of hsp70 protein . Furthermore, there was a 1.8-fold increase in hsp70 protein induced by exposure of L . chagasi to superoxide, but this was not associated with an increase in hsp70 RNA . We conclude that in contrast to higher eukaryotes, the amount of hsp70 protein produced by Leishmania sp . is not regulated by the steady state level of total hsp70 RNA.

Appl Environ Microbiol, 1996 May, 62(5), 1649 - 55
Application of the novel nucleic acid dyes YOYO-1, YO-PRO-1, and PicoGreen for flow cytometric analysis of marine prokaryotes; Marie D et al.; Novel blue light-excited fluorescent dyes for nucleic acids (YOYO-1, YO-PRO-1, and PicoGreen) were tested on cultures of Escherichia coli and of a variety of marine prokaryotes . Results of flow cytometric DNA analyses were compared with those obtained with the UV-excited dyes bis-benzimide Hoechst 33342 or 4', 6-diamidino-2-phenylindole (DAPI) . YOYO-1, YO-PRO-1, and PicoGreen can be used only on aldehyde-fixed cells and need to be supplemented with cofactors such as potassium, citrate, or EDTA . They are highly sensitive to ionic strength . Consequently, seawater culture samples cannot be stained directly with these dyes and require at least a 10-fold dilution with distilled water to obtain reliable fluorescence signals . After treatment with RNase, coefficients of variation for the G1 peak of the DNA distributions of the different strains tested with YOYO-1 or PicoGreen indicated in general an improvement over Hoechst 33342 staining . These novel dyes can be used to enumerate prokaryotic cells by flow cytometry, as demonstrated with E . coli . However, their sensitivity to ionic strength makes them unsuitable for cell cycle analysis in natural samples.

Appl Environ Microbiol, 1996 May, 62(5), 1636 - 41
Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzyme; Cheng TC et al.; Organophosphorus acid (OPA) anhydrolase enzymes have been found in a wide variety of prokaryotic and eukaryotic organisms . Interest in these enzymes has been prompted by their ability to catalyze the hydrolysis of toxic organophosphorus cholinesterase-inhibiting compounds, including pesticides and chemical nerve agents . The natural substrates for these enzymes are unknown . The gene (opaA) which encodes an OPA anhydrolase (OPAA-2) was isolated from an Alteromonas sp . strain JD6.5 EcoRI-lambda ZAPII chromosomal library expressed in Escherichia coli and identified by immunodetection with anti-OPAA-2 serum . OPA anhydrolase activity expressed by the immunopositive recombinant clones was demonstrated by using diisopropylfluorophosphate (DFP) as a substrate . A comparison of the recombinant enzyme with native, purified OPAA-2 showed they had the same apparent molecular mass (60 kDa), antigenic properties, and enzyme activity against DFP and the chemical nerve agents sarin, soman, and O-cyclohexyl methylphosphonofluoridate . The gene expressing this activity was found in a 1.74-kb PstI-HindIII fragment of the original 6.1-kb EcoRI DNA insert . The nucleotide sequence of this PstI-HindIII fragment revealed an open reading frame of 1,551 nucleotides, coding for a protein of 517 amino acid residues . Amino acid sequence comparison of OPAA-2 with the protein database showed that OPAA-2 is similar to a 647-amino-acid sequence produced by an open reading frame which appears to be the E . coli pepQ gene . Further comparison of OPAA-2, the E . coli PepQ protein sequence, E . coli aminopeptidase P, and human prolidase showed regions of different degrees of similarity or functionally conserved amino acid substitutions . These findings, along with preliminary data confirming the presence of prolidase activity expressed by OPAA-2, suggest that the OPAA-2 enzyme may, in nature, be used in peptide metabolism.

Mol Cell Biol, 1996 May, 16(5), 2314 - 24
Replication initiates at multiple dispersed sites in the ribosomal DNA plasmid of the protozoan parasite Entamoeba histolytica; Dhar SK et al.; In the protozoan parasite Entamoeba histolytica (which causes amoebiasis in humans), the rRNA genes (rDNA) in the nucleus are carried on an extrachromosomal circular plasmid . For strain HM-1:IMSS, the size of the rDNA plasmid is 24.5 kb, and 200 copies per genome are present . Each circle contains two rRNA transcription units as inverted repeats separated by upstream and downstream spacers . We have studied the replication of this molecule by neutral/neutral two-dimensional gel electrophoresis and by electron microscopy . All restriction fragments analyzed by two-dimensional gel electrophoresis gave signals corresponding to simple Y's and bubbles . This showed that replication initiated in this plasmid at multiple, dispersed locations spread throughout the plasmid . On the basis of the intensity of the bubble arcs, initiations from the rRNA transcription units seemed to occur more frequently than those from intergenic spacers . Multiple, dispersed initiation sites were also seen in the rDNA plasmid of strain HK-9 when it was analyzed by two-dimensional gel electrophoresis . Electron microscopic visualization of replicating plasmid molecules in strain HM-1:IMISS showed multiple replication bubbles in the same molecule . The location of bubbles on the rDNA circle was mapped by digesting with PvuI or BsaHI, which linearize the molecule, and with SacII, which cuts the circle twice . The distance of the bubbles from one end of the molecule was measured by electron microscopy . The data corroborated those from two-dimensional gels and showed that replication bubbles were distributed throughout the molecule and that they appeared more frequently in rRNA transcription units . The same interpretation was drawn from electron microscopic analysis of the HK-9 plasmid . Direct demonstration of more than one bubble in the same molecule is clear evidence that replication of this plasmid initiates at multiple sites . Potential replication origins are distributed throughout the plasmid . Such a mechanism is not known to operate in any naturally occurring prokaryotic or eukaryotic plasmid.

J Bacteriol, 1996 May, 178(9), 2498 - 506
Definition of the full extent of glycosylation of the 45-kilodalton glycoprotein of Mycobacterium tuberculosis; Dobos KM et al.; Chemical evidence for the true glycosylation of mycobacterial proteins was recently provided in the context of the 45-kDa MPT 32 secreted protein of Mycobacterium tuberculosis (K . Dobos, K . Swiderek, K.-H . Khoo, P . J . Brennan, and J . T . Belisle, Infect . Immun . 63:2846-2853, 1995) . However, the full extent and nature of glycosylation as well as the location of glycosylated amino acids remained undefined . First, to examine the nature of the covalently attached sugars, the 45-kDa protein was obtained from cells metabolically labeled with D-{U-14C} glucose and subjected to compositional analysis, which revealed mannose as the only covalently bound sugar . Digestion of the protein with the endoproteinase subtilisin and analysis of products by liquid chromatography-electrospray-mass spectrometry on the basis of fragments demonstrating neutral losses of hexose (m/z 162) or pentose (m/z 132) revealed five glycopeptides, S7, S18, S22, S29, and S41 among a total of 50 peptides, all of which produced only m/z 162 fragmentation ion deletions . Fast atom bombardment-mass spectrometry, N-terminal amino acid sequencing, and alpha-mannosidase digestion demonstrated universal O glycosylation of Thr residues with a single alpha-D-Man, mannobiose, or mannotriose unit . Linkages within the mannobiose and mannotriose were all alpha 1-2, as proven by gas chromatography-mass spectrometry of oligosaccharides released by beta-elimination . Total sequences of many of the glycosylated and nonglycosylated peptides combined with published information on the deduced amino acid sequence of the entire 45-kDa protein demonstrated that the sites of glycosylation were located in Pro-rich domains near the N terminus and C terminus of the polypeptide backbone . Specifically, the Thr residues at positions 10 and 18 were substituted with alpha-D-Manp(1-->2)alpha-D-Manp, the Thr residue at position 27 was substituted with a single alpha-D-Manp, and Thr-277 was substituted with either alpha-D-Manp, alpha-D-Manp(1-->2)alpha-D-Manp, or alpha-D-Manp(1--> 2)alpha-D-Manp(1-->2)alpha-D-Manp . This report further corroborates the existence of true prokaryotic glycoproteins, defines the complete structure of a mycobacterial mannoprotein and the first complete structure of a mannosylated mycobacterial protein, and establishes the principles for the study of other mycobacterial glycoproteins.

J Bacteriol, 1996 May, 178(9), 2489 - 97
Sequence analysis, expression, and binding activity of recombinant major outer sheath protein (Msp) of Treponema denticola; Fenno JC et al.; The gene encoding the major outer sheath protein (Msp) of the oral spirochete Treponema denticola ATCC 35405 was cloned, sequenced, and expressed in Escherichia coli . Preliminary sequence analysis showed that the 5' end of the msp gene was not present on the 5.5-kb cloned fragment described in a recent study (M . Haapasalo, K . H . Muller, V . J . Uitto, W . K . Leung, and B . C . McBride, Infect . Immun . 60:2058-2065,1992) . The 5' end of msp was obtained by PCR amplification from a T . denticola genomic library, and an open reading frame of 1,629 bp was identified as the coding region for Msp by combining overlapping sequences . The deduced peptide consisted of 543 amino acids and had a molecular mass of 58,233 Da . The peptide had a typical prokaryotic signal sequence with a potential cleavage site for signal peptidase 1 . Northern (RNA) blot analysis showing the msp transcript to be approximately 1.7 kb was consistent with the identification of a promoter consensus sequence located optimally upstream of msp and a transcription termination signal found downstream of the stop codon . The entire msp sequence was amplified from T . denticola genomic DNA and cloned in E . coli by using a tightly regulated T7 RNA polymerase vector system . Expression of Msp was toxic to E . coli when the entire msp gene was present . High levels of Msp were produced as inclusion bodies when the putative signal peptide sequence was deleted and replaced by a vector-encoded T7 peptide sequence . Recombinant Msp purified to homogeneity from a clone containing the full-length msp gene adhered to immobilized laminin and fibronectin but not to bovine serum albumin . Attachment of recombinant Msp was decreased in the presence of soluble substrate . Attachment of T . denticola to immobilized laminin and fibronectin was increased by pretreatment of the substrate with recombinant Msp . These studies lend further support to the hypothesis that Msp mediates the extracellular matrix binding activity of T . denticola.

Proc Natl Acad Sci U S A, 1996 Apr 30, 93(9), 4036 - 9
Posttranslational amino acid epimerization: enzyme-catalyzed isomerization of amino acid residues in peptide chains; Heck SD et al.; Since ribosomally mediated protein biosynthesis is confined to the L-amino acid pool, the presence of D-amino acids in peptides was considered for many years to be restricted to proteins of prokaryotic origin . Unicellular microorganisms have been responsible for the generation of a host of D-amino acid-containing peptide antibiotics (gramicidin, actinomycin, bacitracin, polymyxins) . Recently, a series of mu and delta opioid receptor agonists {dermorphins and deltorphins} and neuroactive tetrapeptides containing a D-amino acid residue have been isolated from amphibian (frog) skin and mollusks . Amino acid sequences obtained from the cDNA libraries coincide with the observed dermorphin and deltorphin sequences, suggesting a stereospecific posttranslational amino acid isomerization of unknown mechanism . A cofactor-independent serine isomerase found in the venom of the Agelenopsis aperta spider provides the first major clue to explain how multicellular organisms are capable of incorporating single D-amino acid residues into these and other eukaryotic peptides . The enzyme is capable of isomerizing serine, cysteine, O-methylserine, and alanine residues in the middle of peptide chains, thereby providing a biochemical capability that, until now, had not been observed . Both D- and L-amino acid residues are susceptible to isomerization . The substrates share a common Leu-Xaa-Phe-Ala recognition site . Early in the reaction sequence, solvent-derived deuterium resides solely with the epimerized product (not substrate) in isomerizations carried out in 2H2O . Significant deuterium isotope effects are obtained in these reactions in addition to isomerizations of isotopically labeled substrates (2H at the epimerizeable serine alpha-carbon atom) . The combined kinetic and structural data suggests a two-base mechanism in which abstraction of a proton from one face is concomitant with delivery from the opposite face by the conjugate acid of the second enzymic base.

Biochemistry, 1996 Apr 30, 35(17), 5441 - 50
Purification and characterization of a prokaryotic xanthine dehydrogenase from Comamonas acidovorans; Xiang Q et al.; Xanthine dehydrogenase (XDH) is induced in Comamonas acidovorans cells incubated in a limited medium with hypoxanthine as the only carbon and nitrogen source . The enzyme has been purified to homogeneity using standard techniques and characterized . It contains two subunits with M(r) values of 90 and 60 kDa . Gel filtration studies show the enzyme to have an alpha 2 beta 2 native structure . No precursor form of the enzyme is observed on Western blot analysis of cell extracts obtained at various stages of enzyme induction . Metal analysis of the purified enzyme shows 1.1 Mo, 4.0 Fe, and 3.6 phosphorus atoms per alpha beta protomer . Cofactor analysis shows the enzyme to contain a single molybdopterin mononucleotide and one FAD per alpha beta protomer . Electron spin resonance and circular dichroism spectral studies of the oxidized and reduced forms of the enzyme suggest the Fe centers to be two nonidentical {2Fe-2S} clusters . Electron spin resonance signals due to Mo(V) and neutral FAD radical are also observed in the reduced form of the enzyme . Purified enzyme preparations ranged from 70% to 100% functionality . The enzyme is irreversibly inactivated by CN- and is inhibited on incubation with allopurinol . With xanthine and NAD+ as substrates the enzyme has a specific activity of 50 units/mg, a kcat value of 120 s-1, an activity/flavin ratio of 1930, and respective Km values of 66 and 160 mM . Using 8-D-xanthine as substrate, a DV value of 1.8 is found with no change in Km . Thus, the Km and KD values of the enzyme for xanthine are equal . These data show Comamonas XDH to exhibit structural properties similar to bovine milk xanthine oxidase/dehydrogenase and to chicken liver xanthine dehydrogenase . Although the bacterial enzyme exhibits a 6-7-fold greater turnover rate than bovine or avian enzymes, the catalytic efficiencies (as measured by V/K) are similar for all three enzymes.

J Biol Chem, 1996 Apr 26, 271(17), 10137 - 42
Substitution of PIM1 protease in mitochondria by Escherichia coli Lon protease; Teichmann U et al.; PIM1 protease in mitochondria belongs to a conserved family of ATP-dependent proteases, which includes the Escherichia coli Lon protease . Yeast cells lacking PIM1 are largely defective in degrading misfolded proteins in the mitochondrial matrix, are respiratory deficient, and lose integrity of mitochondrial DNA . In order to analyze whether E . coli Lon protease is functionally equivalent to mitochondrial PIM1 protease, yeast cells lacking the PIM1 gene were transformed with a construct consisting of a mitochondrial targeting sequence fused onto the Lon protease . In these cells, the fusion protein was expressed and imported into mitochondria, and the targeting sequence was removed . In the absence of PIM1 protease, the E . coli Lon protease mediated the degradation of misfolded proteins in the matrix space in cooperation with the mitochondrial hsp70 system . These cells maintained the integrity of the mitochondrial genome and the respiratory function at 30 degrees C but not at 37 degrees C . Stabilization of mitochondrial DNA in Deltapim1 cells depended on protein degradation by the E . coli Lon protease, as a proteolytically inactive Lon variant was not capable of substituting for a loss of PIM1 protease . These results demonstrate functional conservation of Lon-like proteases from prokaryotes to eukaryotes and shed new light on the role of Lon-like proteases in mitochondrial biogenesis.

J Mol Biol, 1996 Apr 26, 258(1), 53 - 61
Antibiotic-induced oligomerisation of group I intron RNA; Wank H et al.; Antibiotics act as inhibitors of various biological processes . Here we demonstrate that some tuberactinomycins, hitherto known as inhibitors of prokaryotic protein synthesis and of group I intron self-splicing, have a modulatory effect on group I intron RNAs . The linear intron, which is excised during the self-splicing process, is still an active molecular capable of performing an intramolecular transesterification resulting in a circular molecule . However, in the presence of sub-inhibitory concentrations of tuberactinomycins, the intron reacts intermolecularly leading to the formation of linear head-to-tail intron-oligomers . The antibiotic stimulates the intron to react in trans instead of in cis . The phage T4-derived td intron uses the same sites for oligomerisation as for circularisation . Gel- retardation experiments demonstrate that the intron RNA forms non-covalent complexes in the presence of the antibiotic . It might be envisaged that the role of these peptide antibiotics is to bridge RNA molecules mediating RNA-RNA interactions and thus enabling their reaction . The tuberactinomycins are further able to induce the interaction of heterologous introns . The ligation of the T4 phage-derived td intron with the Tetrahymena rRNA intron is very efficient, resulting in molecules composed of two introns derived from different species . The td intron attacks the Tetrahymena intron at various sites, which are located within double-stranded regions . These observations suggest that small molecules like these basic peptide antibiotics could have mediated RNA-RNA interactions in a pre-protein era.

Biochemistry, 1996 Apr 23, 35(16), 5137 - 44
Molecular structure of the NADH/UDP-glucose abortive complex of UDP-galactose 4-epimerase from Escherichia coli: implications for the catalytic mechanism; Thoden JB et al.; UDP-galactose 4-epimerase is one of three enzymes in the metabolic pathway that converts galactose into glucose1-phosphate . Specifically this enzyme catalyzes the interconversion of UDP-galactose and UDP-glucose . The molecular structure of the NADH/UDP-glucose abortive complex of the enzyme from Escherichia coli has been determined by X-ray diffraction analysis to a nominal resolution of 1.8 A and refined to an R-factor of 18.2% for all measurement X-ray data . The nicotinamide ring of the dinucleotide adopts the syn conformation in relationship to the ribose . Both the NADH and UDP-glucose are in the proper orientation for a B-side specific transfer from C4 of the sugar to C4 of the dinucleotide . Those residues implicated in glucose binding include Ser 124, tyr 149, Asn 179, Asn199, Arg 231, and Tyr 299 . An amino acid sequence alignment of various prokaryotic and eukaryotic epimerases reveals a high degree of conservation with respect to those residues involved in both NADH and substrate binding . The nonstereospecificity displayed by epimerase was originally thought to occur through a simple rotation about the bond between the glycosyl C1 oxygen of the 4-ketose intermediate and the beta-phosphorous of the UDP moiety, thereby allowing the opposite side of the sugar to face the NADH . The present structure reveals that additional rotations about the phosphate backbone of UDP are necessary . Furthermore, the abortive complex model described here suggests that Ser 124 and Tyr 149 are likely to play important roles in the catalytic mechanism of the enzyme.

Proc Natl Acad Sci U S A, 1996 Apr 16, 93(8), 3416 - 21
Hyphal development in Neurospora crassa: involvement of a two-component histidine kinase; Alex LA et al.; Two-component signal transduction systems are most often found in prokaryotic organisms where they are responsible for mediating the cellular responses to many environmental stimuli . These systems are composed of an autophosphorylating histidine kinase and a response regulator . We have found evidence for the existence of two-component histidine kinases in the eukaryotic filamentous fungus Neurospora crassa based on screening with degenerate primers to conserved regions of these signaling proteins . Subsequent cloning and sequencing of one member of this newly discovered group, nik-1+, shows that the predicted protein sequence shares homology with both the kinase and response regulator modules of two-component signaling proteins . In addition, the N-terminal region of the protein has a novel repeating 90-amino acid motif . Deletion of the nik-1+ gene in N . crassa results in an organism that displays aberrant hyphal structure, which is enhanced under conditions of high osmostress . Increased osmotic pressure during growth on solid medium leads to restricted colonial growth, loss of aerial hyphae formation, and no subsequent conidiophore development . This finding may have implications for mechanisms of fungal colonization and pathogenicity.

EMBO J, 1996 Apr 15, 15(8), 1992 - 2002
p53 is linked directly to homologous recombination processes via RAD51/RecA protein interaction; Sturzbecher HW et al.; The tumour suppressor p53 prevents tumour formation after DNA damage by halting cell cycle progression to allow DNA repair or by inducing apoptotic cell death . Loss of wild-type p53 function renders cells resistant to DNA damage-induced cell cycle arrest and ultimately leads to genomic instabilities including gene amplifications, translocations and aneuploidy . Some of these chromosomal lesions are based on mechanisms that involve recombinatorial events . Here we report that p53 physically interacts with key factors of homologous recombination: the human RAD51 protein and its prokaryotic homologue RecA . In vitro, wild-type p53 inhibits defined biochemical activities of RecA protein, such as three-way DNA strand exchange and single strand DNA-dependent ATPase activity . In vivo, temperature-sensitive p53 forms complexes with RAD51 only in wild-type but not in mutant conformation . These observations suggest that functional wild-type p53 may select directly the appropriate pathway for DNA repair and control the extent and timing of the production of genetic variation via homologous recombination . Gene amplification an other types of chromosome rearrangements involved in tumour progression might occur not only as result of inappropriate cell proliferation but as a direct consequence of a defect in p53-mediated control of homologous recombination processes due to mutations in the p53 gene.

Biochem J, 1996 Apr 15, 315 ( Pt 2), 481 - 6
Functional domains of chlamydial histone H1-like protein; Remacha M et al.; Chlamydial trachomatis is one of the few prokaryotic organisms known to contain proteins that bear amino acid similarity to eukaryotic histone H1 . It is also appreciated that chlamydial histone-like proteins, designated Hc1 and Hc2, can bind DNA and are presumably involved in the condensation of infectious elementary bodies . However, there is no information on either the orientation of Hc1 and Hc2 or the mechanism of their DNA-protein and protein-protein interactions . Whereas the C-terminal domain of Hc1 between amino acids 63 and 125 shows best alignment with sea-urchin histone H1, and N-terminus between amino acids 1 and 62 is highly conserved among various chlamydial species, suggesting a bifunctional role for this unique protein . In order to delineate the regions responsible for the Hc1 characteristics, we have expressed these two fragments independently in Escherichia coli and studied the binding of double-stranded DNA to either whole Hc1 protein or its two termini . Our results support the role of the carboxyl portion in DNA-protein interaction, a function similar to its eukaryotic counterpart . Although this interaction initiates DNA condensation in the absence of the N-terminal domain, it is not sufficient to produce complete compaction . Intra- or inter-molecular protein-protein interactions may be necessary to achieve such an effect.

Biochim Biophys Acta, 1996 Apr 10, 1306(1), 23 - 6
Cloning and expression of an Entamoeba histolytica NAPD+(-)dependent alcohol dehydrogenase gene; Rodriguez MA et al.; In this paper we cloned, sequenced and expressed a novel Entamoeba histolytica alcohol dehydrogenase gene (Ehadh3) . Ehadh3 has a predicted 383 amino acids open reading frame, encoding for a 42.3 kDa protein . The deduced amino acid sequence showed 24 to 26% identity to other type III alcohol dehydrogenases found in prokaryotic and lower eukaryotic organisms, but not in mammalia . There are at least two Ehadh3 gene copies in the genome, but only a 1.2 kb transcript was detected . The EhADH3 fusion protein showed a NADP+(-)dependent ADH activity . Ehadh3 may be a good target for the developing of anti-E . histolytica drugs, without producing damage to the human.

Proc Natl Acad Sci U S A, 1996 Apr 2, 93(7), 3094 - 8
RecA protein stimulates homologous recombination in plants; Reiss B et al.; A number of RecA-like proteins have been found in eukaryotic organisms . We demonstrate that the prokaryotic recombination protein RecA itself is capable of interacting with genomic homologous DNA in somatic plant cells . Resistance to the DNA crosslinking agent mitomycin C requires homologous recombination as well as excision repair activity . Tobacco protoplasts expressing a nucleus-targeted RecA protein were at least three times as efficient as wild-type cells in repairing mitomycin C-induced damage . Moreover, homologous recombination at a defined locus carrying an endogenous nuclear marker gene was stimulated at least 10-fold in transgenic plant cells expressing nucleus-targeted RecA . The increase in resistance to mitomycin C and the stimulation of intrachromosomal recombination demonstrate that Escherichia coli RecA protein is functional in genomic homologous recombination in plants, especially when targeted to the plant nucleus.

Genes Cells, 1996 Apr, 1(4), 337 - 46
The major pathways of protein translocation across membranes; Ito K; The initial events in the localization of cell surface proteins include targeting of precursor molecules to the membrane and subsequent translocation across the membrane . The translocation reaction is mediated through a series of molecular interactions involving a number of protein factors . The trimeric SecY/Sec61 core complex, which is conserved throughout evolution, provides a proteinaceous pathway for translocation . The driving force for translocation is provided by the dynamic motion of the SecA ATPase in prokaryotes and by polypeptide elongation through the ribosome-Sec61 junction in eukaryotes.

Parasitology, 1996 Apr, 112 ( Pt 4), 363 - 9
Direct sequencing of the PCR amplified SSU rRNA gene of Entamoeba dispar and the design of primers for rapid differentiation from Entamoeba histolytica; Novati S et al.; Since 1993, strains of Entamoeba histolytica sensu lato have been assigned to 2 species on the basis of clinical, biochemical, immunological and genetic evidence: the pathogenic strains to E . histolytica sensu stricto, the non-pathogenic strains to Entamoeba dispar . Analysis of the gene encoding for the small subunit ribosomal RNA (SSU rDNA) supports the existence of 2 species . However, while 3 whole SSU rDNA sequences are available in the data bases for E . histolytica, only a partial sequence has been published for E . dispar . Here we report a SSU rDNA sequence for E . dispar . Compared to those of E . histolytica, this sequence shows 1.7% nucleotide substitutions . On the basis of our rDNA data, 2 primers were designed to produce polymerase chain reaction (PCR) amplification from both E . histolytica and E . dispar . Primer specificity for the 2 amoebae was assessed both theoretically against the data bases, and experimentally against a collection of eukaryotic and prokaryotic DNAs . The amplified stretch encompasses a polymorphic Dde I restriction site which allows, after cleavage of the fragment, E . histolytica and E . dispar to be distinguished . The reliability of this method of identification was assessed comparing the results with those based on classic isoenzyme analysis.

Comp Biochem Physiol B Biochem Mol Biol, 1996 Apr, 113(4), 707 - 9
Holotranscobalamins in B12 and non B12 requiring prokaryotes and eukaryotes; Baker H et al.; Transcobalamins, vitamin B12 binding proteins, deliver B12 to cell surface receptors which then permit B12 to cross cell membranes for metabolic use . There is little documentation concerning B12 binding proteins in bacteria and protists . We found that prokaryotes and eukaryotes requiring B12, as well as those protists synthesizing B12, also produce several transcobalamins for functionally transporting B12 similar to humans.

Mol Microbiol, 1996 Apr, 20(1), 9 - 15
Bacterial signalling involving eukaryotic-type protein kinases; Zhang CC; Protein Ser, Thr and Tyr kinases play essential roles in signal transduction in organisms ranging from yeast to mammals, where they regulate a variety of cellular activities . During the last few years, a number of genes that encode eukaryotic-type protein kinases have also been identified in four different bacterial species, suggesting that such enzymes are also widespread in prokaryotes . Although many of them have yet to be fully characterized, several studies indicate that eukaryotic-type protein kinases play important roles in regulating cellular activities of these bacteria, such as cell differentiation, pathogenicity and secondary metabolism . A model based on the possible coupling between two-component systems and eukaryotic-type protein kinases is proposed to explain the function of eukaryotic-type protein kinases in bacterial signalling in the light of studies in bacteria, as well as in plants and yeast . These two groups of eukaryotes possess signal-transduction pathways involving both two-component systems and eukaryotic protein kinases.

Virus Res, 1996 Apr, 41(2), 193 - 200
Prokaryotic expression of an immediate-early gene of human herpesvirus 6 and analysis of its viral antigen expression in human cells; Takeda K et al.; Segments of an immediate-early (1E) protein (1E03; 958 amino acids (aa)), encoded by clone pSTY03, of human herpesvirus 6 (HHV-6) variant B strain HST were expressed as beta-galactosidase fusion proteins in Escherichia coli . Using Western blot analysis, and the serum of a patient having high titer anti-HHV-6 antibodies, an antigenic region of the IE03 protein was mapped between residues 340 and 505 (pUE03IE-M) . The fusion protein expressed in E . coli harboring plasmid pUE03IE-M was purified after electrophoresis in SDS-PAGE, and then immunized in mice to obtain a monospecific antibody . Monospecific antibody raised against the fusion protein reacted with IE03 protein species with apparent molecular weights of 155 and 170 kDa, and was detected as granular fluorescence in nuclei of infected cells by an immunofluorescence antibody test . Furthermore, this antibody reacted only with HHV-6 variant B, but did not react with HHV-6 variant A . The IE03 protein was confirmed to be an IE protein, since the synthesis of this protein was observed in infected cells that were first treated with cycloheximide, which was then replaced with actinomycin D . Further, it was also detected as early as 4 h after infection.

Mol Microbiol, 1996 Apr, 20(2), 247 - 53
Mycoplasma pneumoniae cytadherence: unravelling the tie that binds; Krause DC; Mycoplasma pneumoniae is the leading cause of pneumonia in older children and young adults . Mycoplasma adherence to the respiratory epithelium (cytadherence) is required for colonization and pathogenesis . Although considered to be among the smallest and simplest known prokaryotes, this cell-wall-less bacterium possesses a highly differentiated terminal structure that is thought to be functional in mycoplasma cell division, gliding motility, and cytadherence . Mutant analysis has identified mycoplasma proteins associated with cytadherence, and revealed novel regulatory features . Ultrastructural and biochemical studies have established the subcellular location and interaction of key components, several of which are phosphorylated by ATP-dependent kinase(s) in a manner that is responsive to changing nutritional conditions . This review summarizes recent progress in defining the composition, organization and regulation of the attachment organelle . What emerges is a picture of M . pneumoniae cytadherence as a multifactorial process that extends well beyond adhesin-receptor recognition.

Curr Biol, 1996 Apr 1, 6(4), 484 - 6
MSH6, a Saccharomyces cerevisiae protein that binds to mismatches as a heterodimer with MSH2; Iaccarino I et al.; The process of post-replicative DNA-mismatch repair seems to be highly evolutionarily conserved . In Escherichia coli, DNA mismatches are recognized by the MutS protein . Homologues of the E . coli mutS and mutL mismatch-repair genes have been identified in other prokaryotes, as well as in yeast and mammals . Recombinant Saccharomyces cerevisiae MSH2 (MSH for MutS homologue) and human hMSH2 proteins have been shown to bind to mismatch-containing DNA in vitro . However, the physiological role of hMSH2 is unclear, as shown by the recent finding that the mismatch-binding factor hMutS alpha isolated from extracts of human cells is a heterodimer of hMSH2 and another member of the MSH family, GTBP . It has been reported that S . cerevisiae possesses a mismatch-binding activity, which most probably contains MSH2 . We show here that, as in human cells, the S . cerevisiae binding factor is composed of MSH2 and a new functional MutS homologue, MSH6, identified by its homology to GTBP.

Toxicol Appl Pharmacol, 1996 Apr, 137(2), 182 - 92
Toxicity of ochratoxin A, its opened lactone form and several of its analogs: structure-activity relationships; Xiao H et al.; Ochratoxin A (OA); its three natural analogs, ochratoxin C (OC), B (OB), and alpha (Oalpha); and its six synthetic analogs, the epimere of OA (d-OA), the ethylamide of OA (OE-OA), decarboxylated OA (DC-OA), O-methylated OA (OM-OA), lactone-opened OA (OP-OA), and the methyl ester of Oalpha (M-Oalpha) were assayed for their toxicities in prokaryotic (Bacillus brevis) and eukaryotic (HeLa cell) systems and in animals (mouse and rat) . The LC50S (mM) for HeLa cells, were 0.005 (OA), 0.009 (OC), 0.163 (d-OA), 10.1 (OE-OA), 7.6 (DC-OA), 0.83 (OM-OA), 0.054 (OB), and 0.56 Oalpha) . The minimum inhibitory doses (nmol/disc) for the growth of B . brevis (pH 6.5) were 8.7 (OA), 2.0 (OC), 5.5 (d-OA), 1.1 (OE-OA), 54 (OB), 390 (Oalpha), and 90 (M-Oalpha) while no inhibition of the bacterial growth was observed for OM-OA, DC-OA, and OP-OA at doses as high as 350 nmol/disc . The results indicate that the toxicities of OA were associated with its isocoumarin moiety but that neither the dissociation of the phenolic hydroxyl group nor the iron-chelating properties of OA were directly related to its toxicities . The lactone carbonyl group of OA, however, appears to be involved in OA toxicity as OP-OA is found in the bile of rats injected with OA and has similar toxicity to that of OA when administered intravenously to the rat . Overall, the structure-activity studies suggest that the toxicity of OA is attributable to its isocoumarin moiety and that the lactone carbonyl group may be involved in its toxicity.

J Mol Evol, 1996 Apr, 42(4), 469 - 71
Anomalous phylogenies based on bacterial catalase gene sequences; Mayfield JE et al.; Phylogenies based on nine prokaryotic catalase sequences demonstrate no relationship to phylogenies based on rDNA sequences or other known criteria . When this observation is considered together with the monophyletic relationship observed for eukaryotic catalase sequences, it seems likely that the catalase gene sequence has migrated repeatedly from eukaryotes to prokaryotes.

J Mol Evol, 1996 Apr, 42(4), 422 - 31
Intron position as an evolutionary marker of thioredoxins and thioredoxin domains; Sahrawy M et al.; In contrast to prokaryotes, which typically possess one thioredoxin gene per genome, three different thioredoxin types have been described in higher plants . All are encoded by nuclear genes, but thioredoxins m and f are chloroplastic while thioredoxins h have no transit peptide and are probably cytoplasmic . We have cloned and sequenced Arabidopsis thaliana genomic fragments encoding the five previously described thioredoxins h, as well as a sixth gene encoding a new thioredoxin h . In spite of the high divergence of the sequences, five of them possess two introns at positions identical to the previously sequenced tobacco thioredoxin h gene, while a single one has only the first intron . The recently published sequence of Chlamydomonas thioredoxin h shows three introns, two at the same positions as in higher plants . This strongly suggests a common origin for all cytoplasmic thioredoxins of plants and green algae . In addition, we have cloned and sequenced pea DNA genomic fragments encoding thioredoxins m and f . The thioredoxin m sequence shows only one intron between the regions encoding the transit peptide and the mature protein, supporting the prokaryotic origin of this sequence and suggesting that its association with the transit peptide has been facilitated by exon shuffling . In contrast, the thioredoxin f sequence shows two introns, one at the same position as an intron in various plant and animal thioredoxins and the second at the same position as an intron in thioredoxin domains of disulfide isomerases . This strongly supports the hypothesis of a eukaryotic origin for chloroplastic thioredoxin f.

Chromosoma, 1996 Apr, 104(7), 479 - 88
A recA-like gene in Drosophila melanogaster that is expressed at high levels in female but not male meiotic tissues; McKee BD et al.; The RecA protein is the central enzyme in prokaryotic recombination . It catalyzes pairing and strand exchange between homologous DNA molecules, and functions in both DNA repair and genetic recombination . The RecA-like proteins Rad51 and Dmc1 of yeast are both required for meiotic recombination and the former is also necessary for repair of double-strand breaks in vegetative cells . Genes encoding Rad51 homologs have been isolated recently from several higher eukaryotes . This paper describes the isolation and molecular characterization of a genomic DNA fragment from Drosophila melanogaster containing the coding sequence for a RecA-like protein . This protein exhibits strong sequence homology with the Rad51 proteins of budding yeast, fission yeast, chickens, mouse and humans, and slightly less (but still strong) homology with yeast Dmc1 . Both in situ hybridization and Southern analysis indicate that the Rad51 gene is present only once per genome in Drosophila (at 99D on chromosome arm 3R) . However, there are at least three other fragments that cross-hybridize strongly at low stringency . RNA blotting analysis detects a single transcript of about 1.35 kb that is present throughout development at low levels . Transcript levels are induced at least tenfold in ovaries, as measured by RNase protection analysis, suggestive of a role in female meiosis . Transcript levels are significantly lower in testes than in bulk RNA of adult males, however, indicating that Rad51 may be repressed in meiosis of Drosophila males.

Eur J Biochem, 1996 Apr 1, 237(1), 240 - 6
Protein purification, and cloning and characterization of the cDNA and gene for xylose isomerase of barley; Kristo P et al.; The first eukaryotic xylose isomerase protein was purified from barley Hordeum vulgare . The enzyme requires Mn2+ for its activity and is fairly thermostable, with the optimum temperature being 60 degrees C . It showed maximum activity over a broad pH range (7.0-9.0) . The molecular mass of the monomer was about 50,000 Da based on the SDS/PAGE, and the calculated value from the cDNA-deduced polypeptide sequence was 53,620 Da . A relative mass estimation of 100,000 Da was obtained from the Superose 12 chromatography, suggesting that the barley enzyme is a dimer . The cloned corresponding cDNA sequence of 1710 nucleotides encoded a polypeptide of 480 amino acids . The genomic sequence of 4473 nucleotides, revealed that the isomerase gene contained 20 introns, all starting with GT and ending with AG . One large intron was located in the 5'untranslated region . The barley isomerase has an insertion of about 40 residues at its amino terminus when compared to the prokaryotic cluster (family) II isomerases; cluster (family) I and cluster (family) II isomerases vary from the former in an insertion of around 50 residues at their amino termini . Comparison of the barley protein with the prokaryotic isomerases shows that the conserved catalytic and metal binding regions are also well conserved in barley.

Development, 1996 Apr, 122(4), 1291 - 302
A cell- and developmental stage-specific promoter drives the expression of a truncated c-kit protein during mouse spermatid elongation; Albanesi C et al.; In the postnatal testis, the c-kit transmembrane tyrosine-kinase receptor is expressed in type A spermatogonia, and its transcription ceases at the meiotic phase of spermatogenesis . Alternative, shorter c-kit transcripts are expressed in post-meiotic germ cells . These transcripts should encode a truncated version of the c-kit protein, lacking the extracellular, the transmembrane and part of the intracellular tyrosine-kinase domains . The 5' end of the alternative c-kit transcripts maps within an intron of the mouse c-kit gene . We now show that this intron contains a promoter active in nuclear extracts of round spermatids, and that two discrete sequences upstream of the transcriptional start site bind spermatid-specific nuclear factors . Deletion of both these sequences abolishes activity of the promoter in vitro . We have also established that this promoter is functional in vivo, in a tissue-and cell-specific fashion, since intronic sequences drive the expression of the E . coli lacZ reporter gene in transgenic mice specifically in the testis . Transgene expression is confined to haploid germ cells of seminiferous tubules, starting from spermatids at step 9, and disappearing at step 13, indicating that cryptic promoter within the 16th intron of the mouse c-kit gene is active in a short temporal window at the end of the transcriptional phase of spermiogenesis . In agreement with these data, western blot experiments using an antibody directed against the carboxy-terminal portion of the mouse c-kit protein showed that a polypeptide, of the size predicted by the open reading frame of the spermatid-specific c-kit cDNA, accumulates in the latest stages of spermatogenesis and in epididymal spermatozoa . An immunoreactive protein of the same size can be produced in both eukaryotic and prokaryotic artificial expression systems.

EMBO J, 1996 Apr 1, 15(7), 1726 - 33
The mismatch repair system contributes to meiotic sterility in an interspecific yeast hybrid; Hunter N et al.; The mismatch repair system is the major barrier to genetic recombination during interspecific sexual conjugation in prokaryotes . The existence of this anti-recombination activity has implications for theories of evolution and the isolation of species . To determine if this phenomenon occurs in eukaryotes, the effect of a deficiency of mismatch repair on the meiotic sterility of an interspecific hybrid of Saccharomyces cerevisiae and the closely related species Saccharomyces paradoxus was examined . The results demonstrate that the rare viable spores from these hybrids have high frequencies of aneuploidy and low frequencies of genetic exchange . Hybrids lacking mismatch repair genes PMS1 or MSH2 display increased meiotic recombination, decreased chromosome non-disjunction and improved spore viability . These observations are consistent with the proposal that the mismatch repair system is an element of the genetic barrier between eukaryotic species . We suggest that an anti-recombination activity during meiosis contributes towards the establishment of post-zygotic species barriers.

J Surg Res, 1996 Apr, 62(1), 130 - 4
Preservation of skin free-flap using trehalose; Kitahara AK et al.; In this study, we looked for a solution able to preserve traumatically amputated tissues such as nose tip, ear, and scalp for at least 48 hr that was easy to handle and low in price . Trehalose is a nonreducing disaccharide that can protect many prokaryotes, fungi, and yeasts against adverse conditions by stabilizing cell membranes . In order to study its protective effect, 60 skin free-flaps of the ears of rabbits were preserved in Euro-Collins (EC) solution or in modified Euro-Collins solution in which 7% trehalose replaced glucose (EC + 7%T) for 24, 48, and 72 hr at 4 degrees C . After completion of preservation, these flaps were transferred to the other ear with microvascular anastomosis . After 7 days, we obtained a survival rate of 100% of flaps after preservation for 24 hr in both solutions . But, after preservation for 48 hr or longer, this decreased to 60% (6 of 10 preserved flaps survived) in EC solution and 90% (9 of 10 flaps) in EC + 7%T solution . This difference became clearer after 72 hr of preservation, when the survival rate was higher and light microscopic observation showed less damage for flaps preserved in EC + 7%T solution (20% in EC and 70% in EC + 7%T) (P < 0.05) . Thus, EC + 7%T solution was superior to EC solution in the preservation of skin free-flaps of the ear of rabbits . Trehalose shows great promise in the preservation of many organs, including storage of traumatically amputated tissues such as nose tip, ear, and scalp.

J Bacteriol, 1996 Apr, 178(7), 2010 - 7
Promoters controlling expression of the alternative nitrogenase and the molybdenum uptake system in Rhodobacter capsulatus are activated by NtrC, independent of sigma54, and repressed by molybdenum; Kutsche M et al.; The alternative nitrogenase of Rhodobacter capsulatus is expressed only under conditions of nitrogen and molybdenum depletion . The analysis of anfA-lacZ fusions demonstrated that this dual control occurred at the level of transcription of anfA, which encodes a transcriptional activator specific for the alternative nitrogenase . The anfA promoter was found to be activated under nitrogen-limiting conditions by NtrC in a sigma54-independent manner . In addition, anfA transcription was repressed by traces of molybdenum . This molybdenum-dependent repression of anfA was released in R . capsulatus mutants carrying either lesions in the high-affinity molybdenum uptake system (modABCD) or a double deletion of mopA and mopB, two genes encoding molybdenum-pterin-binding proteins . The expression of the molybdenum transport system itself was shown to be negatively regulated by molybdenum and, unexpectedly, to be also regulated by NtrC . This finding is in line with the presence of two tandemly arranged DNA motifs located in front of the R . capsulatus mopA-modABCD operon, which are homologous to R . capsulatus NtrC binding sites . Mapping of the transcriptional initiation sites of mopA and anfA revealed promoter sequences exhibiting significant homology to each other but no homology to known prokaryotic promoters . In addition, a conserved DNA sequence of dyad symmetry overlapping the transcriptional initiation sites of mopA and anfA was found . Deletions within this element resulted in molybdenum-independent expression of anfA, indicating that this DNA sequence may be the target of MopA/MopB-mediated repression.

J Bacteriol, 1996 Apr, 178(7), 1829 - 41
Identification of a Caulobacter crescentus operon encoding hrcA, involved in negatively regulating heat-inducible transcription, and the chaperone gene grpE; Roberts RC et al.; In response to elevated temperature, both prokaryotic and eukaryotic cells increase expression of a small family of chaperones . The regulatory network that functions to control the transcription of the heat shock genes in bacteria includes unique structural motifs in the promoter region of these genes and the expression of alternate sigma factors . One of the conserved structural motifs, the inverted repeat CIRCE element, is found in the 5' region of many heat shock operons, including the Caulobacter crescentus groESL operon . We report the identification of another C . crescentus heat shock operon containing two genes, hrcA (hrc for heat shock regulation at CIRCE elements) and a grpE homolog . Disruption of the hrcA gene, homologs of which are also found upstream of grpE in other bacteria, increased transcription of the groESL operon, and this effect was dependent on the presence of an intact CIRCE element . This suggests a role for HrcA in negative regulation of heat shock gene expression . We identified a major promoter transcribing both hrcA and grpE and a minor promoter located within the hrcA coding sequence just upstream of grpE . Both promoters were heat shock inducible, with maximal expression 10 to 20 min after heat shock . Both promoters were also expressed constitutively throughout the cell cycle under physiological conditions . C . crescentus GrpE, shown to be essential for viability at low and high temperatures, complemented an Escherichia coli delta grpE strain in spite of significant differences in the N- and C-terminal regions of these two proteins, demonstrating functional conservation of this important stress protein.

J Biol Chem, 1996 Mar 29, 271(13), 7820 - 8
Biosynthesis of the linkage region of the mycobacterial cell wall; Mikusova K et al.; The "core" structure of the cell wall of Mycobacterium and related genera is unique among prokaryotes, consisting of a covalently linked complex of mycolic acids, D-arabinan and D-galactan (mycolylarabinogalactan, mAG), which, in turn, is linked to peptidoglycan via a special linkage unit, -alpha-L-Rhap(1-->3)-D-GlcNAc-P- . Little is known of the biosynthesis of this complex, although it is the site of action of several common anti-tuberculosis drugs . Isolated cell membranes of Mycobacterium smegmatis catalyzed the incorporation of {14C}GlcNAc from UDP-{14C}GlcNAc into two glycolipids (1 and 2) and of {14C}Rha from TDP-{14C}Rha into glycolipid 2 . These products were characterized as polyprenol-P-P-GlcNAc (glycolipid 1) and polyprenol-P-P-GlcNAc-Rha (glycolipid 2) based on sensitivity of synthesis to tunicamycin, chromatographic characterization of the products of mild acid hydrolysis, and mass spectral analysis of the glycosyl and polyprenyl units . Glycolipids 1 and 2 were shown to be precursors of the linkage unit in polymerized cell wall . The inclusion in the assays of UDP-{14C}Galp and a preparation of cell walls allowed the incorporation of {14C}Gal into two further glycolipids (3 and 4) . Preliminary evidence indicates a precursor-product relationship among glycolipids 1, 2, 3, and 4 . Thus, the first steps in the biosynthesis of the mycobacterial cell wall involve synthesis of the linkage disaccharide on a polyprenyl-P-P carrier followed by growth of the galactan unit . Assays are thus defined for the screening of new anti-tuberculosis drugs active against cell wall synthesis.

J Biol Chem, 1996 Mar 29, 271(13), 7465 - 72
Detection of a physical and functional interaction between Csk and Lck which involves the SH2 domain of Csk and is mediated by autophosphorylation of Lck on tyrosine 394; Bougeret C et al.; The COOH-terminal Src kinase (Csk) is responsible for the phosphorylation of the conserved, negative regulatory, carboxyl-terminal tyrosine of most of the Src family protein tyrosine kinases . Up to now, no stable binding of Csk to Src kinases has been detected . We therefore decided to analyze this interaction using two systems which allow detection of transient interaction . We produced and purified recombinant proteins in the glutathione S-transferase prokaryotic expression system . First, using real-time biospecific interaction analysis (BIAcore(TM)), we detected in vitro a specific interaction between Csk and one of its substrates Lck, a lymphocyte-specific member of the Src family . This interaction requires the autophosphorylation of Lck on tyrosine 394 (the phosphorylation of which is correlated with an increase of the kinase activity) and involves a functional Csk SH2 domain . Second, using the yeast two-hybrid system, we confirmed in vivo the physical interaction between Csk and Lck . Furthermore, in vitro we showed that autophosphorylation of Lck on tyrosine 394 enhances the phosphorylation of Lck by Csk on the negative regulatory site, tyrosine 505, suggesting that activated Lck serves preferentially as substrate for Csk . These findings might explain the mechanism(s) by which Csk interacts with most of Src kinases to down-regulate their kinase activity.

J Biol Chem, 1996 Mar 29, 271(13), 7368 - 74
Functional expression of Escherichia coli endonuclease IV in apurinic endonuclease-deficient yeast; Ramotar D et al.; Saccharomyces cerevisiae Apn1 and Escherichia coli endonuclease IV are homologous enzymes that initiate the repair of abasic (AP) sites or oxidative DNA strand breaks . Yeast lacking Apn1 (apn1-) are hypersensitive to simple alkylating agents (which produce many AP sites) and to oxidants and display an elevated spontaneous mutation rate due to endogenous damages . We explored whether the prokaryotic repair enzyme could substitute for its yeast counterpart . Plasmid constructs were generated that expressed endonuclease IV at 1/20 to 10-fold the AP endonuclease activity of wild-type yeast; some of these plasmids expressed hybrid forms of endonuclease IV equipped with the C-terminal nuclear localization signal of Apn1 . Although hybrid endonuclease IV-Apn1 (but not native endonuclease IV) was selectively localized to the yeast nucleus, expression of this chimeric protein at 25% of the normal Apn1 level did not restore alkylation or oxidant resistance to apn1- yeast, but it did partially counteract the mutator phenotype of apn1- yeast . Expression of either the hybrid protein or native endonuclease IV at approximately 10 times the wild-type Apn1 levels restored wild-type resistance to methyl methanesulfonate and near-wild-type H2O2 resistance . High level expression of native endonuclease IV also restored the normal spontaneous mutation rate to apn1- yeast . These data place limits on the amounts of AP endonuclease activity necessary for repair of DNA damages caused by both endogenous and environmental agents and point to a direct role of spontaneous AP sites as potentially mutagenic lesions.

J Biol Chem, 1996 Mar 22, 271(12), 6866 - 73
Mutations in the second largest subunit of RNA polymerase II cause 6-azauracil sensitivity in yeast and increased transcriptional arrest in vitro; Powell W et al.; Yeast RNA polymerase II enzymes containing single amino acid substitutions in the second largest subunit were analyzed in vitro for elongation-related defects . Mutants were chosen for analysis based on their ability to render yeast cells sensitive to growth on medium containing 6-azauracil . RNA polymerase II purified from three different 6-azauracil-sensitive yeast strains displayed increased arrest at well characterized arrest sites in vitro . The extent of this defect did not correlate with sensitivity to growth in the presence of 6-azauracil . The most severe effect resulted from mutation rpb2 10 (P1018S), which occurs in region H, a domain highly conserved between prokaryotic and eukaryotic RNA polymerases that is associated with nucleotide binding . The average elongation rate of this mutant enzyme is also slower than wild type . We suggest that the slowed elongation rate and an increase in dwell time of elongating pol II leads to rpb2 10's arrest-prone phenotype . This mutant enzyme can respond to SII for transcriptional read-through and carry out SII-activated nascent RNA cleavage.

Oncogene, 1996 Mar 21, 12(6), 1299 - 307
c-myc expression is activated by the immunoglobulin kappa-enhancers from a distance of at least 30 kb but not by elements located within 50 kb of the unaltered c-myc locus in vivo; Mautner J et al.; 50 kb of contiguous DNA sequences covering the human c-myc coding region and approximately 20 kb of flanking upstream and downstream sequences were cloned onto a prokaryotic F-factor derived plasmid, which also contains a selectable marker and the plasmid origin of DNA replication oriP of Epstein Barr virus (EBV) . Since these plasmids replicate extrachromosomally after stable transfection into EBV-positive B-cell lines, the gene regulation of c-myc can be analysed independent from chromosomal integration positions . Despite the presence of all known c-myc regulatory elements on these constructs, expression from the stably transfected c-myc gene was barely detectable in either cell line . Hypermethylation of these plasmids could be excluded as a mechanism for the lack of gene expression . Insertion of the immunoglobulin kappa-intron and 3' enhancers, however, activated c-myc transcription, when placed adjacent to or separated from the c-myc promoters by as far as 30 kb . These results indicate that transcription of c-myc in vivo requires additional and still unidentified control elements located outside this 50 kb fragment, and experimentally demonstrate long range enhancer function in vivo.

J Mol Biol, 1996 Mar 15, 256(5), 849 - 58
A replicational model for DNA recombination between direct repeats; Bi X et al.; DNA rearrangement (recombination) mediated by direct repeats is a major cause of genome instability . In Escherichia coli, direct repeats in close proximity can mediate efficient recA-independent intramolecular recombination, which produces multiple products . Using plasmid substrates, three basic forms of products have been revealed: the monomeric deletion product and two dimeric products . The frequency of recombination has been shown to be affected by structural factors such as the length of the repeat and the distance between the repeats . We show here that these factors also affect the relative abundance of each form of the product . Recombination between very short tandem repeats yields exclusively the monomeric product . Lengthening the repeats increases the abundance of the dimeric products . Increasing the distance separating the repeats sharply reduces the formation of the monomeric product . These results can be explained by a model for DNA rearrangement (recombination) involving DNA replication . We propose that misalignment of the repeats at the replication fork creates a recombinogenic intermediate that can be differentially processed to form the three basic products . The proposed sister-strand recombination mediated by direct repeats might be a general mechanism for deletion and/or amplification of repeated sequence in both prokaryotic and eukaryotic genomes.

J Natl Cancer Inst, 1996 Mar 6, 88(5), 240 - 51
Molecular mechanisms underlying hereditary nonpolyposis colorectal carcinoma; Rhyu MS; MutS and MutL are bacterial genes that have critical roles in DNA repair and recombination . Mutations in homologues of these genes cause hereditary nonpolyposis colorectal carcinoma and are implicated in some sporadic (nonhereditary) colorectal cancers . Molecular functions of these genes have been defined through extensive work in bacteria and yeast . This article reviews and explores molecular events that require MutS and MutL, including mismatch repair, homologous recombination, and gene conversion . The mechanisms of action of eukaryotic MutS and MutL homologues are compared with those of their prokaryotic counterparts, and the relevance of these mechanisms to tumorigenesis is discussed.

Biochemistry, 1996 Mar 5, 35(9), 2845 - 53
Solution structure of prokaryotic ribosomal protein S17 by high-resolution NMR spectroscopy; Jaishree TN et al.; The solution of a primary 16S rRNA-binding ribosomal protein, S17, was investigated by two- and three-dimensional homonuclear and heteronuclear magnetic resonance spectroscopy . Almost complete chemical shift assignments for the 1H, 15N, and 13C resonances have been obtained . The NMR data have been rigorously analyzed using a combination of distance geometry, back-calculation, and simulated annealing refinement techniques, and a high-resolution three-dimensional structure has been deduced . The protein consists of a single twisted antiparallel beta-pleated sheet with Greek-key topology . The five beta-strands are connected by extended loops that are flexible compared to the beta-sheet core structure and appear not to adopt one definite conformation in solution . Two of these loops contain many of the residues that have been implicated in binding ribosomal RNA . The location and distribution of these residues and other positively charged side chains on the protein surface suggest an interaction with two distinct regions of ribosomal RNA.

Front Biosci, 1996 Mar 01, 1, d48 - 58
DnaA- and PriA-dependent primosomes: two distinct replication complexes for replication of Escherichia coli chromosome; Masai H et al.; Enzymatic analyses of primosome assembly at chromosomal and plasmid origins as well as that at single-stranded replication origins revealed the presence of two distinct primosomes in Escherichia coli for primer RNA synthesis and duplex unwinding . A DnaA-dependent primosome is assembled at oriC, the chromosomal origin of Escherichia coli, as well as at the A site, a single-stranded DNA hairpin containing a dnaA box sequence within its stem . In contrast, PriA protein recognizes a hairpin, called n'-pas (primosome assembly site), and initiates assembly of the phiX174-type PriA-dependent primosome in conjunction with other prepriming proteins . Genetic analyses of the prepriming proteins required specifically for the latter primosome strongly suggested that it is responsible for RecA-dependent, DnaA/oriC-independent replication of the Escherichia coli chromosome . Furthermore, primosome assembly in replication of various plasmids may also be classified into either DnaA-dependent or PriA-dependent type . We propose that Escherichia coli possesses two distinct, mutually exclusive primosomes which are differentially utilized by the chromosome as well as by the plasmids . PriA protein appears to be conserved in a wide range of prokaryotic species, and we will also discuss possible biological function of the PriA-dependent primosome in the process of responses to DNA damages.

Pol J Pharmacol, 1996 Mar-Apr, 48(2), 209 - 13
Novel mechanism to enhance tPA-induced fibrinolysis: effect of limited proteolysis of PAI-1 by neutrophil elastase; Urano T et al.; The effect of the proteolytic cleavage of plasminogen activator inhibitor type 1 (PAI-1) by human neutrophil elastase (HNE) on fibrinolysis was investigated . HNE cleaved active recombinant prokaryotic PAI-1 (rpPAI-1) resulting in the formation of low molecular weight forms of rpPAI-1 as previously reported . The latent form of rpPAI-1 was resistant to HNE . NH2-terminal sequence analysis indicated that the cleavage site was Val355-Ser356 (P4-P3) . The fact that the strained loop of the latent form of PAI-1 is buried inside the molecule most likely accounts for its resistance to HNE . After the cleavage by HNE, active rpPAI-1 lost its specific activity toward plasminogen activators . The cleavage was both enzyme concentration and time dependent, and the almost complete inactivation of rpPAI-1 (2.9 microM) activity was obtained by a HNE (83 nM) treatment for 30 min at 37 degrees C . Vitroectin partially protected active rpPAI-1 from the HNE digestion . The effect of PAI-1 cleavage by HNE on tissue type PA (tPA) induced clot lysis was studied in a purified system . Clot lysis time without rpPAI-1 was 20.0 +/- 5.0 min, and was prolonged to 86.7 +/- 2.9 min by 68 nM of rpPAI-1 . It was shortened when HNE (from 0.6 nM to 80 nM) was added and returned to the value obtained without rpPAI-1 when 80 nM of HNE was present (20.0 +/- 5.8 min) . In the absence of PAI-1, however, HNE did not enhance clot lysis at all . The cleavage and inactivation of PAI-1 by HNE was shown to be a novel pathway to enhance fibrinolysis.

Microb Pathog, 1996 Mar, 20(3), 155 - 69
Correlations between Mycoplasma pneumoniae sensitivity to cyclosporin A and cyclophilin-mediated regulation of mycoplasma cytadherence; Reddy SP et al.; Adhesins and adhesin-related accessory proteins of the bacterial pathogen, Mycoplasma pneumoniae, are proline-rich in composition and mediate successful parasitism of host target cells . A specific class of peptidyl-proyl cis-trans isomerases (PPIs), called cyclophilins (Cyps), activate proline-rich proteins, and this enzymatic activity is inhibited by the drug, cyclosporin A (CsA) . This study builds upon the connection between the structural/functional properties of the proline-rich proteins of M . pneumoniae and the mode of action of CsA to demonstrate that CsA reduces cytadherence capabilities of mycoplasmas, affects colony morphology and can be mycoplasmacidal . As a consequence of CsA treatment early passage mycoplasmas lacked the major adhesin, P1, explaining their cytadherence-negative phenotype . Three mycoplasma proteins with molecular masses of 160, 84 and 80 kDa were identified by CsA-affinity chromatography . A PCR cloned partial cyp gene of M . pneumoniae, which exhibited sequence homologies with prokaryotic and eukaryotic cyclophilins, was present in multiple copies . These results implicate the role of PPIs as important regulators of cytadherence, virulence and growth cycle events in mycoplasmas.

Microbiology, 1996 Mar, 142 ( Pt 3), 657 - 65
Cloning, sequencing and disruption of a bromoperoxidase-catalase gene in Streptomyces venezuelae: evidence that it is not required for chlorination in chloramphenicol biosynthesis; Facey SJ et al.; Genomic DNA libraries of Streptomyces venezuelae ISP5230 and of a mutant blocked at the chlorination step of chloramphenicol biosynthesis were probed by hybridization with a synthetic oligonucleotide corresponding to the N-terminal amino acid sequence of a bromoperoxidase-catalase purified from the wild-type strain . Hybridizing fragments obtained from the two strains were cloned and sequenced . Analysis of the nucleotide sequences demonstrated that the fragments contained the same 1449 bp open reading frame with no differences in nucleotide sequence . The deduced polypeptide encoded 483 amino acids with a calculated M(r) of 54,200; the N-terminal sequence was identical to that of the bromoperoxidase-catalase purified from wild-type S . venezuelae . Comparison of the amino acid sequence predicted for the cloned bromoperoxidase-catalase gene (bca) with database protein sequences showed a significant similarity to a group of prokaryotic and eukaryotic catalases, but none to other peroxidases or haloperoxidases . Replacement of the bca gene in the wild-type strain of S . venezuelae with a copy disrupted by insertion of a DNA fragment encoding apramycin resistance did not prevent chloramphenicol production . The results suggest that the role of the enzyme in S . venezuelae is related to its activity as a catalase rather than as a halogenating agent.

J Cell Biochem, 1996 Mar 1, 60(3), 297 - 316
Common structural features of replication origins in all life forms; Boulikas T; Origins of replication (ORIs) among prokaryotes, viruses, and multicellular organisms appear to possess simple tri-, tetra-, or higher dispersed repetitions of nucleotides, AT tracts, inverted repeats, one to four binding sites of an initiator protein, intrinsically curved DNA, DNase I-hypersensitive sites, a distinct pattern of DNA methylation, and binding sites for transcription factors . Eukaryotic ORIs are sequestered on the nuclear matrix; this attachment is supposed to facilitate execution of their activation/deactivation programs during development . Furthermore, ORIs fall into various classes with respect to their sequence complexity: those enriched in AT tracts, those with GA- and CT-rich tracts, a smaller class of GC-rich ORIs, and a major class composed of mixed motifs yet containing distinct AT and polypurine or GC stretches . Multimers of an initiator protein in prokaryotes and viruses that might have evolved into a multiprotein replication initiation complex in multicellular organisms bind to the core ORI, causing a structural distortion to the DNA which is transferred to the AT tract flanking the initiator protein site; single-stranded DNA-binding proteins then interact with the melted AT tract as well as with the DNA polymerase alpha-primase complex in animal viruses and mammalian cells, causing initiation in DNA replication . ORIs in mammalian cells seem to colocalize with matrix-attached regions and are proposed to become DNase I-hypersensitive during their activation.

Trends Biotechnol, 1996 Mar, 14(3), 98 - 105
High cell-density culture of Escherichia coli; Lee SY; Escherichia coli is the most widely used prokaryotic system for the synthesis of heterologous proteins . Once an optimal expression system has been constructed, protein production can be enhanced by increasing the production of protein per cell per unit time (specific productivity), or by increasing the cell concentration per unit time (cell productivity) . Various high cell-density culture (HCDC) techniques have been developed for growing recombinant and non-recombinant E . coli strains in fed-batch cultures at concentrations greater than 100 grams (dry cell weight) per liter . This article reviews the problems encountered in HCDC of E . coli, and discusses various solutions . Feeding strategies for HCDC of E . coli, and the results obtained using them, are also described.

J Appl Bacteriol, 1996 Mar, 80(3), 333 - 7
Effect of water activity on production of beta-lactam antibiotics by Streptomyces clavuligerus in submerged culture; Cochet N et al.; The amount of available water in the environment of micro-organisms, defined as water activity (aw), has been shown to affect growth, respiration, enzyme synthesis, sporulation and other physiological functions . The aim of this study was to examine the impact of aw on production/excretion of a secondary metabolite . For this purpose, the production of beta-lactam antibiotics and biomass of Streptomyces clavuligerus was studied in relation to the aw-depressing agents glucose, sorbitol and NaCl . These were chosen because NaCl and sorbitol are often used to depress aw and glucose was not thought to be taken up by S . clavuligerus . The filamentous bacterium S . clavuligerus NRRL 3585 (ATCC 27064) is a prokaryotic producer of penicillin N, cephalosporins including cephamycin C and clavulanic acid . Under water stress conditions, a greater effect upon antibiotic biosynthesis than upon growth was consistently observed . When aw was decreased to below 0.997, antibiotic production began to decrease . For growth, inhibition was much more gradual and did not become intensive until an aw of 0.990 was reached.

Genetics, 1996 Mar, 142(3), 789 - 800
eth-1, the Neurospora crassa locus encoding S-adenosylmethionine synthetase: molecular cloning, sequence analysis and in vivo overexpression; Mautino MR et al.; Intense biochemical and genetic research on the eth-1r mutant of Neurospora crassa suggested that this locus might encode S-adenosylmethionine synthetase (S-Adomet synthetase) . We have used protoplast transformation and phenotypic rescue of a thermosensitive phenotype associated with the eth-1r mutation to clone the locus . Nucleotide sequence analysis demonstrated that it encodes S-Adomet synthetase . Homology analyses of prokaryotic, fungal and higher eukaryotic S-Adomet synthetase polypeptide sequences show a remarkable evolutionary conservation of the enzyme . N . crassa strains carrying S-Adomet synthetase coding sequences fused to a strong heterologous promoter were constructed to assess the phenotypic consequences of in vivo S-Adomet synthetase overexpression . Studies of growth rates and microscopic examination of vegetative development revealed that normal growth and morphogenesis take place in N . crassa even at abnormally high levels of cellular S-Adomet . The degree of cytosine methylation of a naturally methylated genomic region was dependent on the cellular levels of S-Adomet . We conclude that variation in S-Adomet levels in N . crassa cells, which in addition to the status of genomic DNA methylation could modify the flux of other S-Adomet-dependent metabolic pathways, does not affect growth rate or morphogenesis.

Mol Microbiol, 1996 Mar, 19(5), 933 - 9
The signal-transduction network for Pho regulation in Bacillus subtilis; Hulett FM; Depletion of nutrients, including phosphate, is a stress often encountered by a bacterial cell, and results in slowed growth, marking the cessation of exponential growth . Genes that are transcriptionally activated during phosphate starvation have been used to examine the signal-transduction mechanisms governing the Pho regulon in Bacillus subtilis . Alkaline phosphatase, the traditional reporter protein for Pho regulation in prokaryotes, is encoded by a multigene family in B . subtilis . Characterization of the alkaline phosphatase family was a breakthrough in the study of regulation of the Pho regulon, especially the discovery of promoter elements exclusively responsive to phosphate-starvation regulation . Current data suggest that at least three two-component signal-transduction systems interact, forming a regulatory network that controls the phosphate-deficiency response in B . subtilis . The interconnected pathways involve the PhoP-PhoR system, whose primary role is to mediate the phosphate-deficiency response; the SpoO phosphorelay required for the initiation of sporulation; and a newly discovered signal-transduction system, ResD-ResE, which also has a role in respiratory regulation during late growth . Parallel pathways positively regulate the Pho response via PhoP-PhoR . One pathway includes the ResD-ResE system, while the other involves a transition-state regulator, AbrB . The SpoO system represses the Pho response by negatively regulating both pathways . This review will discuss how the characterization of the APase multigene family made possible studies which show that the Pho regulon in B . subtilis is regulated by the integrated action of the Res, Pho and Spo signal-transduction systems.

J Endocrinol, 1996 Mar, 148(3), 531 - 43
Expression of alpha 2- and beta-adrenoceptor subtypes in human islets of Langerhans; Lacey RJ et al.; Sequences from cDNA molecules encoding alpha 2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human alpha 2C2-, alpha 2C4- and alpha 2C10-adrenoceptor subtype mRNA species . The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of alpha 2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic beta-cells . Using a ribonuclease protection assay protocol, expression of mRNA species encoding both alpha 2 C2 and alpha 2 C10 was demonstrated in preparations of isolated human islets of Langerhans . mRNA encoding alpha 2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction . In situ hybridisation was then employed to examine the distribution of each alpha 2-adrenoceptor subtype in sections of human pancreas . All three subtypes of alpha 2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffin-embedded human pancreas using riboprobes labelled with digoxigenin . Although some labelling of the three alpha 2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype . The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas . The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA . The results demonstrate that multiple alpha 2-adrenoceptor subtypes are expressed in human pancreas . Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue . The presence of alpha 2-adrenoceptor subtype mRNA species in pancreatic beta-cells was confirmed by Northern blotting of RNA extracted from the clonal beta-cell line, HIT-T15 . Transcripts encoding each of the three cloned alpha 2-adrenoceptor subtypes were detected in HIT-T15 cells . Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with beta 2-adrenoceptor mRNA revealed expression of this species in islet beta-cells but not in the exocrine tissue of the pancreas.

Mol Microbiol, 1996 Mar, 19(6), 1177 - 84
Regulatory noise in prokaryotic promoters: how bacteria learn to respond to novel environmental signals; de Lorenzo V et al.; Various features of the regulation of pathways for biodegradation of recalcitrant compounds by Pseudomonas provide insights into the mechanisms by which operons evolve to acquire conditionally active promoters that permit the corresponding genes to be transcribed only when required . The "regulatory noise hypothesis' proposes that transcriptional control systems develop responsiveness to new signals due to the leakiness and lack of specificity of preexisting promoters and regulators . When needed, these may become more specific through suppression of undesirable signals and further fine-tuning of the recruited proteins to interact with distinct chemicals . This hypothesis is supported by the sophisticated regulation of sigma 54-dependent promoters of the TOL (toluene biodegradation) operons, which can be activated to various degrees by heterologous proteins . Such "illegitimate' activation is suppressed by bent DNA structures, either static or protein induced, between promoter core elements . Therefore, not only the regulators but also the DNA sequences participate in the process that gives rise to novel specificities.

Plant Cell Physiol, 1996 Mar, 37(2), 117 - 22
Acetyl-CoA carboxylase in higher plants: most plants other than gramineae have both the prokaryotic and the eukaryotic forms of this enzyme; Konishi T et al.; The presence and the absence of a prokaryote type and a eukaryote type of acetyl-CoA carboxylase (EC 6.4.1.2; ACCase) were examined in members of 28 plant families by two distinct methods: the detection of biotinylated subunits of ACCase with a streptavidin probe, and the detection of the accD gene, which encodes a subunit of the prokaryotic ACCase, by Southern hybridization analysis . The protein extracts of all the plants studied contained a biotinylated polypeptide of 220 kDa, which was probably the eukaryotic ACCase . All the plants but those belonging to Gramineae also contained a biotinylated polypeptide of ca . 35 kDa, which is a putative subunit of the prokaryotic ACCase . In all plants but those in Gramineae, the ca . 35 kDa polypeptide was found in the protein extracts of plastids, while the 220 kDa polypeptide was absent from these plastid extracts . The plastid extracts of the plants in Gramineae contained the 220 kDa polypeptide, as did the homogenates of the leaves . Southern hybridization analysis demonstrated that all the plants but those in the Gramineae contained the accD gene . These findings suggest that most higher plants have the prokaryotic ACCase in the plastids and the eukaryotic ACCase in the cytosol . Only Gramineae plants might contain the eukaryotic ACCases both in the plastids and in the cytosol . The origin of the plastid-located eukaryotic ACCase in Gramineae is discussed as the first possible example of substitution of a plastid gene by a nuclear gene for a non-ribosomal component.

Infect Immun, 1996 Mar, 64(3), 1060 - 4
Molecular characterization of a ribonucleotide reductase (nrdF) gene fragment of Mycoplasma hyopneumoniae and assessment of the recombinant product as an experimental vaccine for enzootic pneumonia; Fagan PK et al.; A Mycoplasma hyopneumoniae clone bank was screened with hyperimmune pig serum . One clone exhibited sequence homology to the prokaryotic R2 subunit of ribonucleotide reductase and was expressed as an 11-kDa protein fused to beta-galactosidase . The vaccine potential of the fusion protein was assessed in pig trials . Following experimental challenge with a virulent isolate of M . hyopneumoniae, gross lung pathology (mean Goodwin lung score) of vaccinated animals, irrespective of adjuvant treatment, was significantly reduced compared with that of control unvaccinated pigs (P < 0.05).

J Bacteriol, 1996 Mar, 178(5), 1386 - 93
Characterization of a glutathione-dependent formaldehyde dehydrogenase from Rhodobacter sphaeroides; Barber RD et al.; Glutathione-dependent formaldehyde dehydrogenases (GSH-FDH) represent a ubiquitous class of enzymes, found in both prokaryotes and eukaryotes . During the course of studying energy-generating pathways in the photosynthetic bacterium Rhodobacter sphaeroides, a gene (adhI) encoding a GSH-FDH homolog has been identified as part of an operon (adhI-cycI) that also encodes an isoform of the cytochrome c2 family of electron transport proteins (isocytochrome c2) . Enzyme assays with crude Escherichia coli extracts expressing AdhI show that this protein has the characteristic substrate preference of a GSH-FDH . Ferguson plot analysis with zymograms suggests that the functional form of AdhI is a homodimer of approximately40-kDa subunits, analogous to other GSH-FDH enzymes . These properties of AdhI were used to show that mutations which increase or decrease adhI expression change the specific activity of GSH-FDH in R . sphaeroides extracts . In addition, expression of the presumed adhI-cycI operon appears to be transcriptionally regulated, since the abundance of the major adhI-specific primer extension product is increased by the trans-acting spd-7 mutation, which increases the level of both isocytochrome c2 and AdhI activity . While transcriptional linkage of adhI and cycI could suggest a function in a common metabolic pathway, isocytochrome c2 (periplasm) and AdhI (cytoplasm) are localized in separate compartments of R . sphaeroides . Potential roles for AdhI in carbon and energy generation and the possible relationship of GSH-FDH activity to isocytochrome c2 will be discussed based on the commonly accepted physiological functions of GSH-FDH enzymes in prokaryotes and eukaryotes.

Mol Cell Biol, 1996 Mar, 16(3), 1085 - 93
Mitotic crossovers between diverged sequences are regulated by mismatch repair proteins in Saccaromyces cerevisiae; Datta A et al.; Mismatch repair systems correct replication- and recombination-associated mispaired bases and influence the stability of simple repeats . These systems thus serve multiple roles in maintaining genetic stability in eukaryotes, and human mismatch repair defects have been associated with hereditary predisposition to cancer . In prokaryotes, mismatch repair systems also have been shown to limit recombination between diverged (homologous) sequences . We have developed a unique intron-based assay system to examine the effects of yeast mismatch repair genes (PMS1, MSH2, and MSH3) on crossovers between homologous sequences . We find that the apparent antirecombination effects of mismatch repair proteins in mitosis are related to the degree of substrate divergence . Defects in mismatch repair can elevate homologous recombination between 91% homologous substrates as much as 100-fold while having only modest effects on recombination between 77% homologous substrates . These observations have implications for genome stability and general mechanisms of recombination in eukaryotes.

J Biol Chem, 1996 Mar 1, 271(9), 5085 - 94
Association between 36- and 13.6-kDa alpha-like subunits of Arabidopsis thaliana RNA polymerase II; Ulmasov T et al.; Two subunits in RNA polymerase II (e.g . RPB3 and RPB11 in yeast) and two subunits common to RNA polymerases I and III (e.g . AC40 and AC19 in yeast) contain one or two motifs related to the alpha subunit in prokaryotic RNA polymerases . We have sequenced two different cDNAs (AtRPB36a and AtRPB36b), the two corresponding genes from Arabidopsis thaliana that are homologs of yeast RPB3, and an Arabidopsis cDNA (AtRPB13.6) that is a homolog of yeast RPB11 . The B36a subunit is the predominant B36 subunit associated with RNA polymerase II purified from Arabidopsis suspension culture cells, and this subunit has a stoichiometry of about 1 . Results from protein association assays showed that the B36a and B36b subunits did not associate, but each of these subunits did associate with the B13.6 subunit in vivo and in vitro . Two motifs in the B36b subunit related to the prokaryotic alpha subunit were shown to be required for the in vitro interactions with the B13.6 subunit . Our results suggest that the B36 and B13.6 subunits associate to form heterodimers in Arabidopsis RNA polymerase II like the AC40 and AC19 heterodimers reported for yeast RNA polymerases I and III but unlike the B44 homodimers reported for yeast RNA polymerase II.

J Biol Chem, 1996 Mar 1, 271(9), 5040 - 8
Identification and characterization of a thermostable MutS homolog from Thermus aquaticus; Biswas I et al.; Recognition of mispaired or unpaired bases during DNA mismatch repair is carried out by the MutS protein family . Here, we describe the isolation and characterization of a thermostable MutS homolog from Thermus aquaticus YT-1 . Sequencing of the mutS gene predicts an 89.3-kDa polypeptide sharing extensive amino acid sequence homology with MutS homologs from both prokaryotes and eukaryotes . Expression of the T . aquaticus mutS gene in Escherichia coli results in a dominant mutator phenotype . Initial biochemical characterization of the thermostable MutS protein, which was purified to apparent homogeneity, reveals two thermostable activities, an ATP hydrolysis activity in which ATP is hydrolyzed to ADP and Pi and a specific DNA mismatch binding activity with affinities for heteroduplex DNAs containing either an insertion/deletion of one base or a GT mismatch . The ATPase activity exhibits a temperature optimum of approximately 80 degrees C . Heteroduplex DNA binding by the T . aquaticus MutS protein requires Mg2+ and occurs over a broad temperature range from 0 degrees C to at least 70 degrees C . The thermostable MutS protein may be useful for further biochemical and structural studies of mismatch binding and for applications involving mutation detection.

Eur J Biochem, 1996 Mar 1, 236(2), 335 - 51
A nonribosomal system of peptide biosynthesis; Kleinkauf H et al.; This review covers peptide structures originating from the concerted action of enzyme systems without the direct participation of nucleic acids . Biosynthesis proceeds by formation of linear peptidyl intermediates which may be enzymatically modified as well as transformed into specific cyclic structures . The respective enzyme systems are constructed of biosynthetic modules integrated into multienzyme structures . Genetic and DNA-sequence analysis of biosynthetic gene clusters have revealed extensive similarities between prokaryotic and eukaryotic systems, conserved principles of organisation, and a unique mechanism of transport of intermediates during elongation and modification steps involving 4'-phospho-pantetheine . These similarities permit the identification of peptide synthetases and related aminoacyl-ligases and acyl-ligases from sequence data . Similarities to other biosynthetic systems involved in the assembly of polyketide metabolites are discussed.

EMBO J, 1996 Mar 1, 15(5), 1193 - 200
Phosphorylation of Okazaki-like DNA fragments in mammalian cells and role of polyamines in the processing of this DNA; Pohjanpelto P et al.; In mammalian cells DNA synthesis is more complicated than in prokaryotes and less well understood . Here we incubated intact mammalian cells (polyamine auxotrophic Chinese hamster ovary cells and primary human fibroblasts) with {32P}orthophosphate and found that, besides high molecular weight DNA, a species of low molecular weight DNA, approximately 450 bp in size, became efficiently labeled . The short DNA was labeled first, and in pulse-chase experiments the labeling was transient . The isolated small DNA fragments (RNase A-treated) were phosphorylated by T4 polynucleotide kinase specific for polynucleotides with 5'-OH ends . A polynucleotide kinase phosphorylating these DNA pieces was also detected in nuclear extracts of the cells . Treatment with alkaline phosphatase removed most of the 32P label incorporated into the small DNA in vivo . Labeling with deoxyribonucleosides did not reveal these fragments . We hypothesize that the low molecular weight DNA represents Okazaki fragments and that the mammalian DNA replication machinery includes a polynucleotide kinase phosphorylating the 5'-termini of Okazaki fragments . This would imply a novel step in DNA synthesis . We also show that depriving cells of polyamines reversibly blocks synthesis of high molecular weight DNA and leads to accumulation of the short DNA pieces, suggesting a role for polyamines in joining the Okazaki fragments.

Mutat Res, 1996 Mar 1, 367(3), 105 - 14
Genotoxicity and DNA adduct formation of incense smoke condensates: comparison with environmental tobacco smoke condensates; Chen CC et al.; Indoor air pollution has now been recognized as a potentially important problem for public health, since people spend most of their day in closed environments . Incense burning is possibly associated with elevated risks of leukemia and brain tumor in children from the epidemiological studies . Thus, evaluation of the genotoxicity of smoke condensates from incense burning is needed . We examined the genotoxicity of incense smoke condensates (ISC) using the Ames test in S . typhimurium strains with different mutagenic specificity and level of metabolic enzyme, the SOS chromotest in E . coli PQ37, and sister chromatid exchange assay in Chinese hamster ovary cells (SCE/CHO) . The genotoxicity of environmental tobacco smoke condensates (TSC) was also evaluated by the three assays to compare with the genotoxicity of ISC . ISC showed a positive response in TA98, but not in TA100 . It suggested that ISC only contained frame shift mutagens . The mutagenicity of ISC in both strains of TA98NR with deficient nitroreductase and TA98/1,8-DNP6 with deficient O-acetyltransferase was markedly decreased compared to that in TA98 strain . However, the mutagenicity was enhanced in YG1024 with overexpression of O-acetyltransferase activity . Thus, nitroarenes seemed to be responsible in part for the mutagenicity of ISC . Interestingly, all of the four ISC and two TSC samples showed a dose-dependent genotoxic response in the SOS chromotest with E . coli PQ37 but a low SCE induction of those samples were observed in CHO cells . When the genotoxicity was analyzed based on the condensates per one gram of original samples, the genotoxicity of two TSC condensates in prokaryotic cells was higher than that of four ISC samples except for the genotoxicity of TSC-2 in TA98 strain . However, the genotoxicity of certain ISC in eukaryotic cells based on the SCE/CHO assay was higher than that of TSC . To compare the covalent binding of DNA reactive intermediates of ISC and TSC to S . typhimurium TA98, the DNA adducts were evaluated by the 32P-postlabeling method with butanol extraction version . Similar diagonal radioactive zone (DRZ) was observed between ISC and CSC . However, DNA adduct levels induced by TSC were much greater than that of ISC.

Curr Genet, 1996 Mar, 29(4), 352 - 9
catA, a new Aspergillus nidulans gene encoding a developmentally regulated catalase; Navarro RE et al.; Aspergillus nidulans asexual sporulation (conidiation) is a model system for studying gene regulation and development . The CAN5 cDNA is one of several clones isolated based on transcript induction during conidiation . Here we present the molecular characterization of its corresponding gene, demonstrating that it encodes a developmentally regulated catalase, designated catA . The catA 744-amino-acid-residue polypeptide shows significant identity to other catalases . Its similarity to prokaryotic catalases is greater than to other fungal catalases . catA mRNA is barely detectable in growing mycelia, highly induced during sporulation, and present in isolated spores . However, catA expression is not dependent on the developmental regulatory genes brlA, abaA and wetA . Direct catalase activity determination in native gels revealed the existence of two bands of activity . One of these bands represented the major activity during vegetative growth and was induced during sporulation . The second catalase activity appeared after the induction of sporulation and was the predominant activity in spores . Disruption of catA abolished the major spore catalase without eliminating the vegetative activity, indicating the existence of at least two catalase genes in A . nidulans . catA-disrupted mutants produced spores that were sensitive to H2O2, as compared to wild-type spores . The increase in the activity of the vegetative catalase and the appearance of a second catalase during asexual sporulation is consistent with the occurrence of an oxidative stress during development.

Curr Genet, 1996 Mar, 29(4), 327 - 34
The response regulator-like protein Pos9/Skn7 of Saccharomyces cerevisiae is involved in oxidative stress resistance; Krems B et al.; We have isolated mutants of Saccharomyces cerevisiae with an increased sensitivity to oxidative stress . All pos9 mutants (pos for peroxide sensitivity) were hypersensitive to methylviologene, hyperbaric oxygen or hydrogen peroxide, but grew similarly to the wild-type under all other conditions tested . Isolation and sequencing of the respective POS9 gene revealed that it was identical to SKN7 . The predicted Skn7/Pos9 protein possesses a domain with high homology to prokaryotic response regulators . These regulatory proteins are part of a simple signalling cascade termed a "two-component system", where a phosphorylation signal of a histidine kinase is transferred to a conserved aspartate residue of the response regulator . To test the functional role of the respective aspartate residue of Skn7/Pos9 protein in oxidative stress, we mutagenized this residue in vitro to alanine, arginine and glutamate . Only the glutamate allele (D427 to E) was able to rescue the hydrogen peroxide-sensitivity of pos9 mutants . By fusion experiments with the Gal4 DNA-binding domain we identified the isolated response regulator-like domain as a novel eukaryotic domain sufficient for gene activation . Whereas this hybrid protein activated transcription of a lacZ reporter gene under aerobic conditions, no activation was observed under anaerobic conditions, indicating that the response regulator domain is involved in a signalling reaction . Two-hybrid investigations also suggest an oligomerization of the Pos9 protein . Our results indicate that a two-component system is involved in the oxidative-stress response of yeast.

DNA Res, 1996 Feb 29, 3(1), 25 - 30
Conservation and periodicity of DNA bend sites in eukaryotic genomes; Wada-Kiyama Y et al.; DNA bend sites appear every 680 bp on average in the human epsilon- and beta-globin gene regions . Although most of their molecular nature has not been unraveled, a potential bend core sequence A2N8A2N8A2 (A/A/A) and its complementary T2N8T2N8T2 (T/T/T) appeared preferentially either in or very close to most of the bend sites, whereas other combinations of A2 and T2 dinucleotides, A/T/T + A/A/T, T/T/A + T/A/A and A/T/A + T/A/T, did not . The distances between any two of the core sequences in the entire beta-globin locus showed a strong bias to a length of 701-800 bp and multiples thereof, suggesting that there is periodicity throughout the locus . This bias was not found for other combinations of A2 and T2 . Again, this periodicity was identified in many eukaryotic genes, whereas the tendency was absent in mRNAs and prokaryotic as well as viral genomes.

Biochemistry, 1996 Feb 27, 35(8), 2699 - 704
Function of the {2FE-2S} cluster in mammalian ferrochelatase: a possible role as a nitric oxide sensor; Sellers VM et al.; Ferrochelatase (E.C . 4.99.1.1) is the terminal enzyme of the heme biosynthetic pathway, catalyzing the insertion of ferrous iron into protoporphyrin . In mammals the enzyme contains a labile {2Fe-2S} center . Although this cluster is absent in all prokaryotic, plant, and yeast ferrochelatases, its destruction or elimination from the mammalian enzyme results in loss of enzyme activity . In the current study we present data which clearly demonstrate that mammalian ferrochelatase is strongly inhibited by nitric oxide and that this effect is mediated via destruction of the {2Fe-2S} cluster . Carbon monoxide has no inhibitory effect, and yeast ferrochelatase, which lacks the {2Fe-2S} cluster, is not affected by NO (or CO) . EPR and UV-visible absorption of purified recombinant human ferrochelatase provides evidence that NO is targeting the {2Fe-2S} center . UV-visible absorption spectroscopy of both human and murine recombinant ferrochelatase incubated with NO or the NO donor, S-nitroso-N-acetylpenicillamine (SNAP), indicate a rapid loss of the visible absorption spectrum of the {2Fe-2S} cluster . EPR studies of the resulting samples reveal the characteristic axial S = 1/2 resonance, g perpendicular = 2.033, and g parallel = 2.014 of a cysteinyl-coordinated monomeric iron-dinitrosyl cluster degradation product . Parallel spectroscopic studies of spinach ferredoxin, which also contains a {2Fe-2S} cluster, gave no indication of NO-induced cluster degradation under the same experimental conditions . Exposure of DMSO-induced murine erythroleukemia cells exposed to SNAP results in an initial decrease in heme production, suggesting that in vivo the cluster is rapidly destroyed . The potential physiological relevance of these data to the anemias that are found in individuals with chronic infections is discussed.

Biochemistry, 1996 Feb 27, 35(8), 2505 - 11
Purification of a mammalian homologue of Escherichia coli endonuclease III: identification of a bovine pyrimidine hydrate-thymine glycol DNAse/AP lyase by irreversible cross linking to a thymine glycol-containing oligoxynucleotide; Hilbert TP et al.; We purified a homologue of the Escherichia coli DNA repair enzyme endo nuclease III 5000-fold from calf thymus which, like endonuclease III, demonstrates DNA-glycosylase activity against pyrimidine hydrates and thymine glycol and AP lyase activity (DNA strand cleavage at AP sites via beta-elimination) . The functional similarity between the enzymes suggested a strategy for definitive identification of the bovine protein based on the nature of its enzyme-substrate (ES) intermediate . Prokaryotic DNA glycosylase/AP lyases function through N-acylimine (Schiff's base) ES intermediates which, upon chemical reduction to stable secondary amines, irreversibly cross link the enzyme to oligodeoxynucleotides containing substrate modified bases . We incubated endonuclease III with a 32P- labeled thymine glycol-containing oligodeoxynucleotide in the presence of NaCNBH3 . This resulted in an increase in the apparent molecular weight of the enzyme by SDS-PAGE . Phosphorimaging confirmed irreversible cross linking between enzyme and DNA . Identical treatment of the most purified bovine enzyme fraction resulted in irreversible cross linking of the oligodeoxynucleotide to a predominant 31 kDa species . Amino acid analysis of the 31 kDa species revealed homology to the predicted amino acid sequence of a Caenorhabditis elegans 27.8 kDa protein which, in turn, has homology to endonuclease III . The translated amino acid sequences of two partial 3' cDNAs, from Homo sapiens and Rattus sp., also demonstrate homology to the C . elegans and bovine sequences suggesting a homologous family of endonuclease III-like DNA repair enzymes is present throughout phylogeny.

Proc R Soc Lond B Biol Sci, 1996 Feb 22, 263(1367), 209 - 15
Long, polymorphic microsatellites in simple organisms; Field D et al.; We have examined the phylogenetic distribution of the longest, perfect microsatellites in GenBank . Despite the large contributions of model higher-eukaryotic organisms to GenBank, the selective cloning of long microsatellites from these organisms as genetic markers, and the relative lack of concentration on the microsatellites in lower eukaryotes and prokaryotes, we found that simple organisms, defined here as slime molds, fungi, protists, prokaryotes, viruses, organelles and plasmids, contributed 78 of the 375 examined sequences . These 78 simple-organism microsatellites are characterized predominantly by trinucleotide repeats, nearly half of which lie in exons, and in general show a bias towards A+T rich motifs . Simple-organism microsatellites represented more than once in GenBank displayed length polymorphisms when independent clones were compared . These facts collectively raise speculation as to the role of these 'junk' sequences in such highly economical genomes, especially when precise changes in long microsatellites are known to regulate critical virulence factors in several prokaryotes . Regardless of their biological significance, simple-organism microsatellites may provide a general source of molecular markers to track disease outbreaks and the evolution of microorganisms in unprecedented detail.

J Theor Biol, 1996 Feb 21, 178(4), 375 - 80
Tumour progression: random mutations or an integrated survival response to cellular stress conserved from unicellular organisms?
Israel L.
The current paradigm states that cancer progression is caused by random independent mutations, each selected for its survival advantages . The accelerated rates of phenotypic changes, the pleiotropic effect of several genes involved in progression--which need not be necessarily mutated for inducing the observed changes in cancer cell behaviour--lead us to propose an alternative hypothesis . Malignant progression might be a result of the unveiling of a cell-survival program, induced by various aggressions in the same way as the SOS system is induced and regulated in bacteria . This hypothesis depends on the homology between several genes involved in cancer progression (such as bcl2, mdm2, the mismatch repair genes, the heat shock protein genes, the pleiotropic resistance genes, the telomerase gene ...) and several genes involved in the survival of prokaryotes and eukaryotes under stress . The development of multicellular organisms could not take place without the building of a control program, exemplified by the so-called anti-oncogenes . However, this control program had to integrate some weaknesses, in order to allow for embryogenesis, growth, and wound healing . These weaknesses, neutral from an evolutionary point of view--since most cancers are sporadic and kill their hosts long after the birth of the offspring--are exploited by the survival program of individual cells, inherited from the genome of prokaryotes and unicellular eukaryotes, and repressed but not suppressed in animals . If this theory is true, it is probable that (i) no anti-oncogenes will be found in unicellular organisms, (ii) the sensitivity to mutations will be higher in genes involved in proliferation and in anti-oncogenes such as p53 and Rb, than in genes not involved in the cancer process, (iii) a process of transfer of genetic information exists in cancer cells as it exists in bacteria . The identification of the genes governing the survival program could lead to new therapeutic approaches.

Mutat Res, 1996 Feb 19, 350(1), 143 - 52
Natural antimutagenic agents; Mitscher LA et al.; Following a brief review of recent discoveries in the field of natural antimutagenic and tumor chemopreventive agents, contemporary findings in the author's laboratories employing the direct acting mutagen, ethyl methanesulfonate, in modified Ames tests and eukaryotic murine FM3A mammary tumor cells modified to be subject to thymidine-less death are described to illustrate the underlying principles . The EMS studies are illustrated with the isolation of the novel antimutagen, plicatin B, from the medicinal plants, Psoralea juncaea and P . plicata . The FM3A studies are carried out with extracts of Styrax asiatica, a plant previously studied extensively with the EMS system . The FM3A findings closely parallel the earlier work with EMS showing that the responsible agents, cinnamic acid, cinnamoyl ricinoleate and cinnamoyl cinnamate are effective both in prokaryotic and eukaryotic tests and that the new FM3A assay system has useful properties for screening and assay of novel antimutagenic agents.

Biochem J, 1996 Feb 15, 314 ( Pt 1), 241 - 8
Molecular cloning and functional identification of a plant ornithine decarboxylase cDNA; Michael AJ et al.; A cDNA for a plant ornithine decarboxylase (ODC), a key enzyme in putrescine and polyamine biosynthesis, has been isolated from root cultures of the solanaceous plant Datura stramonium . Reverse transcription-PCR employing degenerate oligonucleotide primers representing conserved motifs from other eukaryotic ODCs was used to isolate the cDNA . The longest open reading frame potentially encodes a peptide of 431 amino acids and exhibits similarity to other eukaryotic ODCs, prokaryotic and eukaryotic arginine decarboxylases (ADCs), prokaryotic meso-diaminopimelate decarboxylases and the product of the tabA gene of Pseudomonas syringae cv . tabaci . Residues involved at the active site of the mouse ODC are conserved in the plant enzyme . The plant ODC does not possess the C-terminal extension found in the mammalian enzyme, implicated in rapid turnover of the protein, suggesting that the plant ODC may have a longer half-life . Expression of the plant ODC in Escherichia coli and demonstration of ODC activity confirmed that the cDNA encodes an active ODC enzyme . This is the first description of the primary structure of a eukaryotic ODC isolated from an organism where the alternative ADC routine to putrescine is present.

Cancer Res, 1996 Feb 15, 56(4), 728 - 32
The t(6;16)(p21;q22) chromosome translocation in the LNCaP prostate carcinoma cell line results in a tpc/hpr fusion gene; Veronese ML et al.; Very little is known about the molecular and genetic mechanisms involved in prostate cancer . Previous studies have shown frequent loss of heterozygosity (40%) at chromosomal regions 8p, 10q, and 16q, suggesting the presence of tumor suppressor genes in these regions . The LNCaP cell line, established from a metastatic lesion of human prostatic adenocarcinoma, carries a t(6;16)(p21;q22) translocation . To determine whether this translocation involved genes important in the process of malignant transformation, we cloned and sequenced the t(6;16) breakpoint of this cell line . Sequence analysis showed that the breakpoint is within the haptoglobin gene cluster on chromosome 16, and that, on chromosome 6, the break occurs within a novel gene, tpc, similar to the prokaryotic S10 ribosomal protein gene . The translocation results in the production of a fusion transcript, tpc/hpr.

Eur J Biochem, 1996 Feb 15, 236(1), 240 - 8
Cloning, sequence analysis and overexpression of the rhodanese gene of Azotobacter vinelandii; Colnaghi R et al.; A gene encoding rhodanese (rhdA) was cloned from Azotobacter vinelandii on a 2.3-kb SphI fragment . This fragment was identified by its hybridization to a PCR product obtained by amplification of genomic DNA using degenerate primers encoding the N-terminal sequence of rhodanese purified from A . vinelandii . The sequence of a 1.2-kb region revealed an 813-bp open reading frame that encoded a polypeptide of 271 amino acids, the N-terminal sequence of which was identical to that of A . vinelandii rhodanese . In a search of database entries, eukaryotic rhodaneses and rhodanese-like proteins from bacteria gave the highest scores of identity (27-30%) with the predicted product of the 813-bp open reading frame . A . vinelandii RhdA shows less sequence similarity to vertebrate rhodaneses than it does to prokaryotic rhodanese-like proteins which did not show typical rhodanese activity . Basic residues thought to be catalytically important in bovine rhodanese are not conserved in A . vinelandii rhodanese . The sequence similarity between the two structurally similar domains of rhodanese is more pronounced for the A . vinelandii enzyme than the bovine enzyme, and supports the hypothesis that the complete structure was originally generated by gene duplication . When rhdA was overexpressed in Escherichia coli, rhodanese represented 30% of total cell protein and thiosulfate:cyanide sulfurtransferase activity increased >600 fold in cell-free extracts . A . vinelandii rhdA insertion/deletion mutants had no discernible phenotype distinct from the wild-type strain with respect to growth on various sulfur sources or nitrogenase activity . Mutants retained 20% of wild-type rhodanese thiosulfate:cyanide sulfurtransferase activity suggesting the presence of redundant sulfurtransferase enzymes in A . vinelandii.

Biochim Biophys Acta, 1996 Feb 15, 1273(2), 84 - 6
The coxD gene for heme O synthase in Synechocystis; Malakhov M et al.; The cyanobacterial coxD gene for heme O synthase was cloned from Synechocystis sp . PCC 6803 and its nucleotide sequence was determined . The deduced amino-acid sequence of the gene was homologous to the amino-acid sequences of bacterial heme O synthesis . In contrast to the genes for heme O synthases in other prokaryotes, which are clustered together with genes for the structural subunit(s) of cytochrome oxidase, the coxD gene is not linked to such genes on the chromosome of Synechocystis.

Gene, 1996 Feb 12, 168(2), 257 - 60
Prokaryotic production of the human T cell receptor Vbeta2 chain and binding to toxic-shock syndrome toxin-1; Ericson ML et al.; The human T-cell receptor Vbeta2-, D- and J-encoding domains were PCR-amplified from MOLT-4 total cDNA and subcloned in Escherichia coli . The V/D/J fragment was subsequently transferred to a prokaryotic expression vector in frame with a polyhistidine-encoding prosequence which enabled us to affinity-purify the fusion protein with IMAC (immobilized metal-ion affinity chromatography {correction of chromatorgraphy}) . Since the recombinant (re-) human T-cell receptor Vbeta2 fusion protein (Vbeta2 sol) produced in E.coli was found to be insoluble, purification was carried out under denaturating conditions . The purified and renatured re-protein, Vbeta2 sol, was immunoreactive with an anti-Vbeta2 monoclonal antibody in an ELISA assay . The specificity of Vbeta2 sol was shown by its binding in vitro to the staphylococcal superantigen TSST-1, but not to the Staphylococcus aureus exotoxin-1 (SEA).

J Biol Chem, 1996 Feb 9, 271(6), 2995 - 3004
The COQ7 gene encodes a protein in saccharomyces cerevisiae necessary for ubiquinone biosynthesis; Marbois BN et al.; Ubiquinone (coenzyme Q) is a lipid that transports electrons in the respiratory chains of both prokaryotes and eukaryotes . Mutants of Saccharomyces cerevisiae deficient in ubiquinone biosynthesis fail to grow on nonfermentable carbon sources and have been classified into eight complementation groups (coq1 coq8; Tzagoloff, A., and Dieckmann, C . L.(1990) Microbiol . Rev . 54, 211-225) . In this study we show that although yeast coq7 mutants lack detectable ubiquinone, the coq7 1 mutant does synthesize demethoxyubiquinone (2-hexaprenyl-3-methyl-6-methoxy-1,4-benzoquinone), a ubiquinone biosynthetic intermediate . The corresponding wild-type COQ7 gene was isolated, sequenced, and found to restore growth on nonfermentable carbon sources and the synthesis of ubiquinone . The sequence predicts a polypeptide of 272 amino acids which is 40% identical to a previously reported Caenorhabditis elegans open reading frame . Deletion of the chromosomal COQ7 gene generates respiration defective yeast mutants deficient in ubiquinone . Analysis of several coq7 deletion strains indicates that, unlike the coq7 1 mutant, demethoxyubiquinone is not produced . Both coq7 1 and coq7 deletion mutants, like other coq mutants, accumulate an early intermediate in the ubiquinone biosynthetic pathway, 3-hexaprenyl-4-hydroxybenzoate . The data suggest that the yeast COQ7 gene may encode a protein involved in one or more monoxygenase or hydroxylase steps of ubiquinone biosynthesis.

Proc Natl Acad Sci U S A, 1996 Feb 6, 93(3), 1210 - 4
A cell cycle-regulated bacterial DNA methyltransferase is essential for viability; Stephens C et al.; The CcrM adenine DNA methyltransferase, which specifically modifies GANTC sequences, is necessary for viability in Caulobacter crescentus . To our knowledge, this is the first example of an essential prokaryotic DNA methyltransferase that is not part of a DNA restriction/modification system . Homologs of CcrM are widespread in the alpha subdivision of the Proteobacteria, suggesting that methylation at GANTC sites may have important functions in other members of this diverse group as well . Temporal control of DNA methylation state has an important role in Caulobacter development, and we show that this organism utilizes an unusual mechanism for control of remethylation of newly replicated DNA . CcrM is synthesized de novo late in the cell cycle, coincident with full methylation of the chromosome, and is then subjected to proteolysis prior to cell division.

J Biochem Biophys Methods, 1996 Feb 5, 31(3-4), 189 - 93
Microcalorimetric study of mitochondria isolated from fish liver tissue; Tan AM et al.; We determined the thermogenesis curves of mitochondria isolated from fish liver tissue by using an LKB 2277 Bioactivity Monitor . After isolation from the fish liver, mitochondria still have activity and can live for a long time by using the stored nutrients . We calculated the recovery rate constants of mitochondria . We found that the thermogenesis curves of mitochondria are similar to those obtained from prokaryotic cells, but not similar to those obtained from eukaryotic cells . We determined the metabolic thermogenesis curves of mitochondria isolated from two kinds of carp liver tissue, scattered-scaled mirror carp and harvest carp . There are some important similarities and some important differences between these thermogenesis curves.

Gene, 1996 Feb 2, 168(1), 109 - 12
Two different macronuclear EF-1 alpha-encoding genes of the ciliate Euplotes crassus are very dissimilar in their sequences, copy numbers and transcriptional activities; Bergemann J et al.; Genes (EFA) encoding the translation elongation factor EF-1 alpha (EFA) or the prokaryotic homolog EFTu frequently occur in multiple copies in the same organism . This has been interpreted either in terms of a potential of differential gene expression during different phases of development, or as gene dosage adaptation to the need of high-level production of the gene products . Since ciliates can differentially amplify their genes, the latter argument would lead to the expectation of only one EFA gene in the macronucleus . However, we have found two such genes which strongly differ in both copy number and codon usage . Both transcripts are detectable at very different levels . The expression of the genes takes place both in the vegetative and sexual phases, i.e.,during conjugation.

J Biol Chem, 1996 Feb 2, 271(5), 2455 - 61
The C-terminal extension of yeast seryl-tRNA synthetase affects stability of the enzyme and its substrate affinity; Weygand-Durasevic I et al.; Saccharomyces cerevisiae seryl-tRNA synthetase (SerRS) contains a 20-amino acid C-terminal extension, which is not found in prokaryotic SerRS enzymes . A truncated yeast SES1 gene, lacking the 60 base pairs that encode this C-terminal domain, is able to complement a yeast SES1 null allele strain; thus, the C-terminal extension in SerRS is dispensable for the viability of the cell . However, the removal of the C-terminal peptide affects both stability of the enzyme and its affinity for the substrates . The truncation mutant binds tRNA with 3.6-fold higher affinity, while the Km for serine is 4-fold increased relative to the wild-type SerRS . This indicates the importance of the C-terminal extension in maintaining the overall structure of SerRS.

Mol Biochem Parasitol, 1996 Feb-Mar, 76(1-2), 159 - 73
Cloning and characterization of the NAD-linked glycerol-3-phosphate dehydrogenases of Trypanosoma brucei brucei and Leishmania mexicana mexicana and expression of the trypanosome enzyme in Escherichia coli; Kohl L et al.; A polyclonal antiserum raised against the purified glycosomal glycerol-3-phosphate dehydrogenase of Trypanosoma brucei brucei has been used to identify the corresponding cDNA clone in a T.b . brucei expression library . This cDNA was subsequently used to obtain genomic clones containing glycerol-3-phosphate dehydrogenase genes . Two tandemly arranged genes were detected in these clones . Characterization of one of the genes showed that it codes for a polypeptide of 353 amino acids, with a molecular mass of 37,651 Da and a calculated net charge of +8 . Using the T.b . brucei gene as a probe, a corresponding glycerol-3-phosphate dehydrogenase gene was also identified in a genomic library of Leishmania mexicana mexicana . The L.m . mexicana gene codes for a polypeptide of 365 amino acids, with a molecular mass of 39,140 Da and a calculated net charge of +8 . The amino-acid sequences of both polypeptides are 63% identical and carry a type-1 peroxisomal targeting signal (PTS1) SKM and -SKL at their respective C-termini . Moreover, the L.m . mexicana polypeptide also carries a short N-terminal extension reminiscent of a mitochondrial transit sequence . Subcellular localisation analysis showed that in L.m . mexicana the glycerol-3-phosphate dehydrogenase activity co-fractionated both with mitochondria and with glycosomes . This is not the case in T . brucei, where the enzyme is predominantly glycosomal . The two trypanosomatid sequences resemble their prokaryotic homologues (32-36%) more than their eukaryotic counterparts (25-31%) and carry typical prokaryotic signatures . The possible reason for this prokaryotic nature of a trypanosomatid glycerol-3-phosphate dehydrogenase is discussed.

Mol Biochem Parasitol, 1996 Feb-Mar, 76(1-2), 145 - 58
Molecular characterization of glycosomal NAD(+)-dependent glycerol 3-phosphate dehydrogenase from Trypanosoma brucei rhodesiense; Stebeck CE et al.; The primary structure of a 38-kDa protein isolated from membrane preparations of African trypanosomes was determined by protein and DNA sequencing . Searching of the protein database with the trypanosome translated amino acid sequence identified glycerol 3-phosphate dehydrogenase (EC 1.1.1.8) from various prokaryotic and eukaryotic organisms as the optimal scoring protein . Surprisingly, the eukaryotic trypanosome enzyme showed the highest degree of sequence identity with the corresponding enzyme from the prokaryote Escherichia coli . The trypanosome molecule was expressed in Escherichia coli and found to be enzymatically active, thus confirming the identity of the molecule as an NAD(+)-dependent glycerol 3-phosphate dehydrogenase . A monoclonal antibody specific for the 38-kDa protein was used to localize the enzyme to glycosomes . Immunoblotting showed that the monoclonal antibody bound to a 38-kDa protein in African trypanosomes but not in T . cruzi, Leishmania or Crithidia . The enzyme has a pI of 9.1, a net charge of +17 and contains the peroxisome-like targeting tripeptide SKM at its C-terminus, all characteristic of glycosomal enzymes . Amino acids predicted to be involved in the NAD(+)-dependent glycerol 3-phosphate dehydrogenase active site have diverged from those of the mammalian enzyme . Kinetic analyses of the trypanosome GPD and GPD from rabbit muscle showed that the Km values of the two enzymes are different . The data suggest that the trypanosome protein may be a candidate target for rational drug design.

J Virol Methods, 1996 Feb, 56(2), 179 - 89
Dengue virus envelope glycoprotein can be secreted from insect cells as a fusion with the maltose-binding protein; Staropoli I et al.; The maltose-binding protein (MalE) contains a signal sequence which allows its translocation in the periplasm of prokaryotic microorganisms . In this study, MalE was produced in Spodoptera frugiperda (Sf9) lepidopterian cells using the baculovirus expression system . The secretion of MalE, following cleavage of its signal sequence, to the supernatant fluid of recombinant baculovirus-infected Sf9 cells and its affinity for maltodextrin polymers allowed recovery of significant amounts (> or = 10 micrograms per 10(6) cells) of highly purified protein . The gene encoding the envelope glycoprotein E of the dengue (DEN) type 2 virus deleted of its C-terminal 102 amino acids (D2E delta 102) was fused to the MalE gene . The resulting hybrid MalE-D2E delta 102 glycoprotein was processed through the Golgi network of Sf9 cells and was secreted . It was retained on a maltodextrin column and was eluted with maltose . Antigenic and immunogenic properties dependent on the three-dimensional structure in the native E protein were preserved in the recombinant MalE-D2E delta 102 protein . Thus MalE with its signal sequence may be used as a carrier protein for production in the baculovirus system and purification of proteins which require transportation through intracellular compartments for correct folding and processing.

Pharmacogenetics, 1996 Feb, 6(1), 1 - 42
P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature; Nelson DR et al.; We provide here a list of 481 P450 genes and 22 pseudogenes, plus all accession numbers that have been reported as of October 18, 1995 . These genes have been described in 85 eukaryote (including vertebrates, invertebrates, fungi, and plants) and 20 prokaryote species . Of 74 gene families so far described, 14 families exist in all mammals examined to date . These 14 families comprise 26 mammalian subfamilies, of which 20 and 15 have been mapped in the human genome and the mouse genome, respectively . Each subfamily usually represents a cluster of tightly linked genes widely scattered throughout the genome, but there are exceptions . Interestingly, the CYP51 family has been found in mammals, filamentous fungi and yeast, and plants-attesting to the fact that this P450 gene family is very ancient . One functional CYP51 gene and two processed pseudogenes, which are the first examples of intronless pseudogenes within the P450 superfamily, have been mapped to three different human chromosomes . This revision supersedes the four previous updates in which a nomenclature system, based on divergent evolution of the superfamily, has been described . For the gene, we recommend that the italicized root symbol "CYP' for human ("Cyp' for mouse and Drosophila), 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 is no longer recommended in mouse gene nomenclature . "P' ("ps' in mouse and Drosophila) after the gene number denotes a pseudogene; "X' after the gene number means its use has been discontinued . If a gene is the sole member of a family, the subfamily letter and gene number would be helpful but need not be included . The human nomenclature system should be used for all species other than mouse and Drosophila . The cDNAs, mRNAs and enzymes in all species (including mouse) should include all capital letters, and without italics or hyphens . This nomenclature system is similar to that proposed in our previous updates.

Mol Microbiol, 1996 Feb, 19(4), 827 - 39
Two different mechanisms are involved in the heat-shock regulation of chaperonin gene expression in Bradyrhizobium japonicum; Babst M et al.; Heat-shock regulation was detected for three out of the five members of the groESL multigene family in Bradyrhizobium japonicum . The results uncovered the simultaneous presence of two distinct heat-shock control systems which so far have not been reported to co-exist in a single prokaryotic organism . The first system concerns groESL1 whose transcription is controlled in a sigma32-dependent manner similar to that known from work done with Escherichia coli . Heat-shock control of groESL4 is mediated by the second system, which is characterized by an inverted-repeat DNA structure originally described as a heat-shock regulatory element (CIRCE) in Bacillus subtilis . This element represses expression of groESL4 under non-stress conditions, as inferred from the increased expression of a groESL4'-'lacZ fusion suffering a 4 bp deletion within the CIRCE element . The two control systems clearly differ with respect to the temperature dependence and the kinetics of the heat-shock response, and they also respond differently to the stress signal elicited by incorporation of the amino acid analogue p-F-phenylalanine into cellular protein . Knock-out mutations in groEL4 resulted in an increased expression of groESL4, suggesting that repression via CIRCE depends, itself, upon the cellular level of GroEL4 protein.

Trends Microbiol, 1996 Feb, 4(2), 73 - 6
Lymphocyte activation by CpG dinucleotide motifs in prokaryotic DNA; Krieg AM; CpG dinucleotides are present at the expected frequency in prokaryotic DNA, but are underrepresented ('CpG suppression') and methylated in vertebrate DNA . The vertebrate immune system has apparently evolved the ability to recognize these unmethylated CpG motifs and respond with a rapid and coordinated cytokine response leading to the induction of humoral and cell-mediated immunity.

J Membr Biol, 1996 Feb, 149(3), 161 - 8
Phylogenetic, structural and functional characteristics of the Na-K-Cl cotransporter family; Park JH et al.; Bumetanide-sensitive Na-K-Cl cotransporters and thiazide-sensitive Na-Cl cotransporters comprise a family of integral membrane transport proteins, the Na-K-Cl cotransporter (NKCC) family . Each of the members of this family is over 1,000 amino acids in length . We have multiply aligned the ten currently sequenced members of this family from human, rabbit, rodent, shark, flounder, moth, worm and yeast sources . Phylogenetic analyses suggest the presence of at least six isoforms of these full length proteins in eukaryotes . Average hydropathy and average similarity plots have been derived revealing that each of these proteins possesses a central, well conserved, hydrophobic domain of almost invariant length, possibly consisting of twelve transmembrane alpha-helical spanners, an N-terminal, poorly conserved, hydrophilic domain of variable length, and a C-terminal, moderately conserved, hydrophilic domain of moderately constant length . A functionally uncharacterized homologue of this family occurs in the cyanobacterium Synechococcus sp . Limited sequence similarity of these proteins with members of a family of basic amino acid transporters suggest that the NKCC family may be distantly related to the previously characterized, ubiquitous, amino acid-polyamine-choline (APC) family of facilitators . These observations suggest that the NKCC family is an old family that has its roots in the prokaryotic kingdom.

Indian J Med Res, 1996 Feb, 103, 103 - 11
Immunohistochemical study of the expression of human groEL-stress protein in human nervous tissue; Khanna N et al.; Monoclonal antibody (ML-30) directed against 65 kDa stress protein of mycobacteria, is shown to identify human cellular protein homologous with the groEL heat shock protein in many prokaryotes . Immunohistochemical survey of nervous tissue, both central and peripheral, from patients dying of various inflammatory, degenerative and neoplastic conditions and from experimental animals, using this antibody showed punctate granular staining of the cells to a variable degree . The astrocytes showed strong immunolabelling . The normal neurons and oligodendroglia stained variably, while abnormal neurons were darkly labelled . Ependymal cells showed apical granular positivity . The ubiquitinated inclusion bodies in amyotrophic lateral sclerosis, Alzheimer's disease and Parkinson's disease were not recognised by the ML-30 antibody . In diseased and stressed nervous tissue from experimental animals, the expression of the ML-30 recognisable stress protein was variable . The epitope recognised by ML-30 was found stable in postmortem tissues collected up to 36 h after death and processed for paraffin sectioning, after fixation in formalin for many years . Enhanced expression of the human groEL stress protein homologue in mammalian nervous tissue following various forms of stress may play a role in modulating the extent of tissue damage by autoimmune mechanism because of its high immunogenic nature and constitutive presence in the cells.

Mol Cell Probes, 1996 Feb, 10(1), 7 - 14
Development of an amplification and hybridization assay for the specific and sensitive detection of Mycoplasma fermentans DNA; Berg S et al.; A polymerase-chain-reaction-based detection system for Mycoplasma fermentans was established . The highly conserved tuf gene, which encodes elongation factor Tu of prokaryotes, served as target sequence for the PCR . With two PCR oligodeoxynucleotides, which were selected from M . fermentans specific sequences of the tuf gene, we amplified a 850 base pair DNA fragment . Via the biotin-moiety of one primer the PCR fragments were immobilized on streptavidin-coated microtitre plates . After alkaline denaturation a digoxigenin-labelled M . fermentans specific DNA probe was hybridized to the single stranded immobilized PCR fragment . Detection was performed by addition of an alkaline phosphatase conjugated anti-digoxigenin antibody . 4-methyl-umbelliferyl-phosphate was used as a fluorogenic substrate . Amplification of 10 fg chromosomal target DNA was detected by this 'DNA enzyme immuno assay (DEIA)' technique, corresponding to seven genome copies . Our study supports the presumption that the tuf gene proves to be a suitable target sequence for the PCR based detection of any bacterial species . Furthermore, hybridization of PCR fragments with radio-labelled DNA probes should no longer be necessary, because a very sensitive non-radioactive test system can easily be established with the 'DEIA' technique.

Curr Biol, 1996 Feb 1, 6(2), 134 - 6
Immunoglobulin diversity: rearranging by cutting and repairing; Lieber M; The recombination process that assembles antigen-receptor genes is now understood in some biochemical detail . The initial steps reflect a common theme seen in retroviral integration and prokaryotic transposition, and the later steps involve the enzymatic machinery for double-strand break DNA repair.

J Chem Technol Biotechnol, 1996 Feb, 65(2), 123 - 30
Apparent heterogeneity of recombinant interferon gamma receptors produced in prokaryotic and eukaryotic expression systems; Fountoulakis M; Recombinant proteins show several types of heterogeneity and post-translational modifications which are usually related to their production system . The apparent heterogeneity of recombinant interferon gamma receptors and interferon gamma receptor-immunoglobulin G fusion proteins expressed in Escherichia coli, baculovirus-infected insect cells and Chinese hamster ovary cells have been studied . In general, all proteins tested showed some type of heterogeneity which was detectable by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The E . coli-derived receptor included non-native conformations involving mis-paired or non-formed disulfides . This type of heterogeneity affected the biological activity of the protein . In addition, the prokaryotic protein had trapped phosphoric acid during downstream processing . The phosphoric acid entrapment did not affect ligand binding capacity . The eukaryotic proteins showed heterogeneity because of the unequal cleavage of the signal peptide and because of differences in glycosylation . The latter types of heterogeneity did not affect activity . Glycosylation-related heterogeneity was partially derived from the unequal utilization of the potential N-glycosylation sites and differently affected the apparent molecular masses and migrations of the proteins on polyacrylamide gels . The results may be useful in characterization studies of recombinant proteins.

RNA, 1996 Feb, 2(2), 171 - 82
Selenocysteine incorporation in eukaryotes: insights into mechanism and efficiency from sequence, structure, and spacing proximity studies of the type 1 deiodinase SECIS element; Martin GW 3rd et al.; SECIS elements are stem-loop structures located in the 3' untranslated regions (UTRs) of eukaryotic selenoprotein mRNAs that are required for directing cotranslational selenocysteine incorporation at UGA codons . In prokaryotes, stem-loops mediating selenocysteine incorporation are located immediately downstream of the UGA selenocysteine codon, in the coding region . Previous characterization studies of the mammalian SECIS elements of type 1 deiodinase, glutathione peroxidase, and selenoprotein P showed that conserved nucleotides in the loops and unpaired bulges, and base pairing in the stems are required for SECIS function . These initial studies utilized approximately 175-230-nt segments of the 3'UTRs of the selenoprotein mRNAs . Here we define the minimal functional rat type 1 deiodinase SECIS element, a 45-nt segment, the 5' boundary of which corresponds precisely to the 5'-most critical conserved nucleotide identified previously . We also define base pairing requirements in the stem of this element . In view of the presence of SECIS elements in the open reading frames (ORFs) of bacterial selenoproteins, we examine the effects in the type 1 deiodinase of extending the ORF into the SECIS element, and find that this dramatically inhibits SECIS function . Finally, we define a minimal spacing requirement of 51-111 nt between a eukaryotic UGA selenocysteine codon and SECIS element.

Genes Dev, 1996 Feb 1, 10(3), 351 - 64
Intron mobility in phage T4 occurs in the context of recombination-dependent DNA replication by way of multiple pathways; Mueller JE et al.; Numerous group I introns in both prokaryotes and eukaryotes behave as mobile genetic elements . The functional requirements for intron mobility were determined in the T4 phage system using an in vivo assay to measure intron homing with wild-type and mutant derivatives . Thus, it was demonstrated that intron mobility occurs in the context of phage recombination-dependent replication, a pathway that uses overlapping subsets of replication and recombination functions . The functional requirements for intron homing and the nature of recombinant products are only partially consistent with the accepted double-strand-break repair (DSBR) model for intron inheritance, and implicate additional homing pathways . Whereas ambiguities in resolvase requirements and underrepresentation of crossover recombination products are difficult to rationalize strictly by DSBR, these properties are most readily consistent with a synthesis-dependent strand annealing (SDSA) pathway . These pathways share common features in the strand invasion steps, but differ in subsequent repair synthesis and resolution steps, influencing the genetic consequences of the intron transfer event.

Appl Environ Microbiol, 1996 Feb, 62(2), 332 - 9
The endosymbiont (Buchnera sp.) of the aphid Diuraphis noxia contains plasmids consisting of trpEG and tandem repeats of trpEG pseudogenes; Lai CY et al.; Most aphids are dependent for their survival on prokaryotic endosymbionts assigned to the genus Buchnera . Among the functions of Buchnera species is the synthesis of tryptophan, which is required by the aphid host . In Buchnera species from the aphid Diuraphis noxia, the genes for anthranilate synthase (trpEG) were found on a plasmid which consisted of seven tandem repeats of a 3.2-kb unit and one 2.6-kb unit which differed in containing a 0.6-kb deletion . One of the 3.2-kb units contained open reading frames corresponding to trpEG; the remaining units contained trpEG pseudogenes (psi) . The nucleotide sequence upstream of trpE contained a region that has characteristics of an origin of replication (ori) . Relative to trpB (a chromosomal gene), there were about two copies of the trpEG-containing plasmid . Comparisons of the nucleotide sequences of the 3.2-kb units containing trpEG and psi trpEG indicated that most changes occurred in a 700-nucleotide segment that included the region upstream of trpE and the portion of this gene coding for the N terminus . The consequence of these changes was the silencing of trpEG by inactivation of the putative promoter region and premature termination of the TrpE peptide . In contrast, the nucleotide sequence of the segment corresponding to ori was conserved in the units containing trpEG and psi trpEG . We offer a number of speculations on the evolutionary pressure in this lineage which resulted in the silencing of most of trpEG while still retaining the regions resembling ori.

Curr Microbiol, 1996 Feb, 32(2), 89 - 94
Ribosomal protein S1 (RpsA) of Buchnera aphidicola, the endosymbiont of aphids: characterization of the gene and detection of the product; Clark MA et al.; Buchnera aphidicola is a prokaryotic endosymbiont found in specialized cells of the aphid Schizaphis graminum . Many of the previously cloned B . aphidicola genes are preceded by a poor ribosome-binding site . Ribosomal protein S1 (RpsA) allows the translation of messenger RNAs that lack or have a poor ribosome binding site . We have cloned and sequenced a 4.5-kilobase (kb) B . aphidicola DNA fragment containing four open reading frames corresponding to aroA-rpsA-himD-tpiA . The deduced amino acid sequence of B . aphidicola RpsA was 75% identical to that of the Escherichia coli protein . The major difference was in the number of basic amino acids, which were present in higher numbers in B . aphidicola RpsA . Antiserum to E . coli RpsA was prepared and used to detect B . aphidicola RpsA in cell-free extracts of aphids . During the first 12 days of aphid growth there is a slight decrease in the amount of RpsA per unit of aphid weight . The three additional genes found on the 4.5-kb DNA fragment encoded for proteins involved in aromatic amino acid biosynthesis (aroA), DNA bending (himD), and carbohydrate metabolism (tpiA) . The presence of these genes in B . aphidicola is additional evidence of its similarity to free-living bacteria.

Cancer Res, 1996 Feb 1, 56(3), 616 - 22
Expression of prokaryotic HhaI DNA methyltransferase is transforming and lethal to NIH 3T3 cells; Wu J et al.; In neoplastic cells, levels of DNA methyltransferase activity are often increased, and evidence is accruing to suggest an important role for this event in tumorigenesis . To evaluate this possibility further, and to investigate the contribution of increasing de novo, as opposed to maintenance, DNA methylation in mammalian cells, we expressed the bacterial HhaI methyltransferase in cultured murine fibroblasts . This enzyme is a pure de novo DNA methyltransferase that methylates the internal C in the sequence GCGC . We find that both constitutive and induced expression of the wild-type HhaI results, primarily, in lethality to the cells . However, surviving cell clones that express low levels of M . HhaI demonstrate increased tumorigenicity as assessed by soft agar cloning efficiency (8.6% for sense HhaI-transduced PA 317 cells versus 0.4% for antisense controls; 1.7% for sense HhaI-transfected NIH 3T3 cells versus 0% for a mutant HhaI control) and tumorigenicity in nude mouse heterotransplants (75% for sense HhaI-transduced PA 317 cells versus 18.5% for antisense controls) . DNA isolated from the clonogenic sense HhaI clones, versus clones expressing the mutant HhaI gene, has no increase in overall CpG methylation but an average of 27% (range, 16.7-38.9) increase in methylcytosine content at GCGC sites . These findings suggest that eukaryotic cells tolerate a narrow window of increase de novo DNA methylating capacity, above which cell death occurs and within cell transformation results . Our results further emphasize the potential role of increased DNA methyltransferase activity in the evolution of cancer.

J Bacteriol, 1996 Feb, 178(3), 591 - 9
Characterization of uncultivated prokaryotes: isolation and analysis of a 40-kilobase-pair genome fragment from a planktonic marine archaeon; Stein JL et al.; One potential approach for characterizing uncultivated prokaryotes from natural assemblages involves genomic analysis of DNA fragments retrieved directly from naturally occurring microbial biomass . In this study, we sought to isolate large genomic fragments from a widely distributed and relatively abundant but as yet uncultivated group of prokaryotes, the planktonic marine Archaea . A fosmid DNA library was prepared from a marine picoplankton assemblage collected at a depth of 200 m in the eastern North Pacific . We identified a 38.5-kbp recombinant fosmid clone which contained an archaeal small subunit ribosomal DNA gene . Phylogenetic analyses of the small subunit rRNA sequence demonstrated it close relationship to that of previously described planktonic archaea, which form a coherent group rooted deeply within the Crenarchaeota branch of the domain Archaea . Random shotgun sequencing of subcloned fragments of the archaeal fosmid clone revealed several genes which bore highest similarity to archaeal homologs, including large subunit ribosomal DNA and translation elongation factor 2 (EF2) . Analyses of the inferred amino acid sequence of archaeoplankton EF2 supported its affiliation with the Crenarchaeote subdivision of Archaea . Two gene fragments encoding proteins not previously found in Archaea were also identified: RNA helicase, responsible for the ATP-dependent alteration of RNA secondary structure, and glutamate semialdehyde aminotransferase, an enzyme involved in initial steps of heme biosynthesis . In total, our results indicate that genomic analysis of large DNA fragments retrieved from mixed microbial assemblages can provide useful perspective on the physiological potential of abundant but as yet uncultivated prokaryotes.

Infect Immun, 1996 Feb, 64(2), 472 - 9
Sequence analysis of the chromosomal region around and within the V-1-encoding gene of Mycoplasma pulmonis: evidence for DNA inversion as a mechanism for V-1 variation; Simmons WL et al.; Although the variation of V-1 antigens of Mycoplasma pulmonis has been correlated with variable expression of the cytadherence properties of this organism and has been implicated as a virulence determining factor in M . pulmonis-induced murine respiratory disease, the precise function of these antigens remains unknown . We have cloned and characterized genes encoding V-1 from two M . pulmonis UAB CT V-1 variants that differ in hemadsorption properties . A comparison of the nucleotide sequences revealed that these two variant genes were identical in the 5'-most 724 nucleotides . Regions of extensive divergence that contained repeated sequences were found 3' to this conserved region . On the basis of their deduced amino acid sequences, one variant expressed a V-1 protein of 94.2 kDa presumptively containing 40 repeats of 17 amino acids and the other expressed a protein of 27.4 kDa consisting 2 direct, noncontiguous 9-amino-acid repeats . These general properties, as well as the presence of a prokaryotic lipoprotein acylation sequence (L-X-Y-C), indicated that the genes encoding V-1 were similar in structure to genes encoding other mycoplasma surface lipoproteins . Further analysis of sequences flanking these genes revealed that these variants arose via an inversion event which provided an interchange of the two variable regions as well as for the conserved region of these genes and immunoblot analyses using rabbit polyclonal antibodies specific for synthetic peptides derived from the sequences of the different variable regions indicated that DNA inversion acted as a switch which allowed only one of the two different genes to be expressed at any given time . This inversion model clearly provides a mechanism by which M . pulmonis can alter its surface architecture and also strongly suggests that the as-yet-undefined function of V-1 residues in the variable carboxy region of these proteins.

J Biol Chem, 1996 Jan 26, 271(4), 2225 - 33
The human purH gene product, 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase . Cloning, sequencing, expression, purification, kinetic analysis, and domain mapping; Rayl EA et al.; We report here the cloning and sequencing of the cDNA, purification, steady state kinetic analysis, and truncation mapping studies of the human 5-aminoimidazole- 4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase (AICARFT/IMPCHase) . These steps of de novo purine biosynthesis, respectively . In all species of both prokaryotes and eukaryotes studied, these two activities are present on a single bifunctional polypeptide encoded on the purH gene . The human purH cDNA is 1776 base pairs in length encoding for a 591-amino acid polypeptic (Mr = 64,425) . The human and avian purH cDNAs are 75 and 81% similar on the nucleotide and amino acid sequence level, respectively . The Km values for AICAR and (6R,6S)10-formyltetrahydrofolate are 16.8 microM +/- 1.5 and 60.2 microM +/- 5.0, respectively, for the cloned, purified human enzyme . A 10-amino acid sequence within the COOH-terminal portion of human AICARFT/IMPCHase has some degree of homology to a previously noted "folate binding site." Site directed mutagenesis studies indicate that this sequence plays no role in enzymatic activity . We have constructed truncation mutants which demonstrate that each of the two enzyme activities can be expressed independent of the other . IMPCHase and AICARFT activities are located within the NH2-terminal 223 and COOH-terminal 406 amino acids, respectively . The truncation mutant possessing AICARFT activity displays steady state kinetic parameters identical to those of the holoenzyme.

Nature, 1996 Jan 25, 379(6563), 364 - 8
Calcium oscillations in mammalian eggs triggered by a soluble sperm protein; Parrington J et al.; At fertilization in mammals, the sperm induces a characteristic series of Ca2+ oscillations in the egg which serve as the essential trigger for egg activation and early development of the embryo . It is not known how the sperm initiates this fundamental process, however, nor has any pathway linking sperm-egg membrane-receptor binding with intracellular Ca2+ release been demonstrated . Microinjection of sperm extracts into mammalian eggs elicits Ca2+ oscillations identical to those occurring at fertilization, which suggests that sperm may introduce a Ca2+ oscillation-inducing factor into the egg on gamete membrane fusion . Here we identify a soluble sperm protein that exhibits Ca2+ oscillation-inducing ('oscillogen') activity in eggs . Sperm oscillogen exists as an oligomer with a subunit of M(r) 33K and a specific intracellular localization at the equatorial segment of the sperm head . Cloning of the 33K oscillogen complementary DNA indicates similarity with a hexose phosphate isomerase found in prokaryotes . This sperm-derived oscillogen, termed oscillin, may represent the physiological trigger for development in mammals.

Proc Natl Acad Sci U S A, 1996 Jan 23, 93(2), 819 - 23
DNA rearrangement mediated by inverted repeats; Bi X et al.; Inverted repeats of DNA are widespread in the genomes of eukaryotes and prokaryotes and can mediate genome rearrangement . We studied rearrangement mediated by plasmid-borne inverted repeats in Escherichia coli . We show that inverted repeats can mediate an efficient and recA-independent recombination event . Surprisingly, the product of this recombination is not that of simple inversion between the inverted repeats, but almost exclusively an unusual head-to-head dimer with complex DNA rearrangement . Moreover, this recombination is dramatically reduced by increasing the distance separating the repeats . These results can be readily explained by a model involving reciprocal switching of the leading and lagging strands of DNA replication within the inverted repeats, which leads to the formation of a Holliday junction . Reciprocal strand switching during DNA replication might be a common mechanism for genome rearrangement associated with inverted duplication.

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