Prokaryotes

Prep Biochem Biotechnol, 1999 Feb, 29(1), 77 - 90
Prokaryotic gene fusion expression systems and their use in structural and functional studies of proteins; Sheibani N; The ability to express and purify large quantity of proteins in bacteria has greatly impacted many aspects of biological research . These include their use as a source of reagent for biochemical and biophysical studies as well as a source of antigen for antibody production . Currently many different expression systems are available and new ones are being developed . These systems allow inducible expression of a desired protein as a fusion with an affinity tag for simple purification . The affinity tags can generally be removed by specific proteases which recognize cleavage sites engineered between the affinity tag and the desired protein . Presence of tags that encode epitopes of specific antibodies provide additional means for identification of recombinant proteins . This review provides an overview of some of the most commonly utilized expression systems and examples of the use of these proteins in biochemical and biophysical studies . I will also describe other available systems which may provide suitable alternative for expression of recombinant proteins.

Curr Opin Microbiol, 1998 Oct, 1(5), 530 - 4
New antibiotic discovery, novel screens, novel targets and impact of microbial genomics; Allsop AE; The clinical need for new classes of antibiotic continues to grow, as drug resistance erodes the efficacy of current therapies . Historically, most antibiotics were discovered by random screening campaigns, but over the past 20 years, this strategy has largely failed to deliver a sufficient range of chemical diversity to keep pace with changing clinical profiles . A more rational approach to drug hunting has been greatly potentiated by the availability of bacterial genomic information . The rapid progress in sequencing and analysis of these small, prokaryotic genomes has enabled the concomitant development of powerful new technologies that are already enhancing the potential utility of genomic information . The future promises versatile and precise tools to understand what makes a successful antibiotic and moreover the means to identify and evaluate novel classes of drug.

J Invertebr Pathol, 1999 Mar, 73(2), 162 - 72
Studies on rickettsia-like organism disease of the tropical marine pearl oyster I: the fine structure and morphogenesis of pinctada maxima pathogen rickettsia-like organism
Wu X, Pan J.
The present paper reports for the first time the discovery of a rickettsia-like organism (RLO) in the cultured tropical marine pearl oyster Pinctada maxima with mass mortality in the Hainan Province of China . This organism parasitizes the cytoplasm of host cells and forms intracytoplasmic eosinophilic inclusions . These organisms are extremely pleiomorphic in shape and average 967 x 551 nm in size, as measured in cross sections of transmission electron micrographs . The organisms exhibit clearly recognizable ultrastructural characteristics of prokaryotic bacteria-like cells, including two trilaminar membranes, an increasing electron-dense periplasmic ribosome zone, and a thread-like DNA nucleoidal structure . In addition to the above prokaryotic characteristics, the following unique biological characteristics were confirmed by TEM: (i) These organisms are usually located in host cells in two ways, namely, free in the cell cytoplasm and involved within membrane-limited phagolysosomes; (ii) The organisms exist in two morphological cell types, namely a small cell variant (SCV) and a large cell variant (LCV) . The most important morphological difference between two cell types is that the SCV is obviously ribosome-rich in the periphery of the body, which makes SCV more electron-dense in the cytoplasm and narrower in the central nucleoid area than the LCV; (iii) Two propagative modes of the organisms, transverse binary fission and budding, are observed in cytoplasm and phagolysosomes of host cells under TEM, in which the budding is more often seen in phagolysosomes . These characteristics indicate that the organism is a separate species in the family Rickettsiaceae and should be classified into the genus Rickettsia . On the basis of the existence of the two propagative modes and two cell types, and intracellular location, we propose a developmental cycle for this organism which includes a vegetative differentiation stage to develop LCV by transverse binary fisson and a budding differentiation stage to develop resistant SCV .

Microbiol Mol Biol Rev, 1999 Mar, 63(1), 106 - 27
Prochlorococcus, a marine photosynthetic prokaryote of global significance; Partensky F et al.; The minute photosynthetic prokaryote Prochlorococcus, which was discovered about 10 years ago, has proven exceptional from several standpoints . Its tiny size (0.5 to 0.7 microm in diameter) makes it the smallest known photosynthetic organism . Its ubiquity within the 40 degrees S to 40 degrees N latitudinal band of oceans and its occurrence at high density from the surface down to depths of 200 m make it presumably the most abundant photosynthetic organism on Earth . Prochlorococcus typically divides once a day in the subsurface layer of oligotrophic areas, where it dominates the photosynthetic biomass . It also possesses a remarkable pigment complement which includes divinyl derivatives of chlorophyll a (Chl a) and Chl b, the so-called Chl a2 and Chl b2, and, in some strains, small amounts of a new type of phycoerythrin . Phylogenetically, Prochlorococcus has also proven fascinating . Recent studies suggest that it evolved from an ancestral cyanobacterium by reducing its cell and genome sizes and by recruiting a protein originally synthesized under conditions of iron depletion to build a reduced antenna system as a replacement for large phycobilisomes . Environmental constraints clearly played a predominant role in Prochlorococcus evolution . Its tiny size is an advantage for its adaptation to nutrient-deprived environments . Furthermore, genetically distinct ecotypes, with different antenna systems and ecophysiological characteristics, are present at depth and in surface waters . This vertical species variation has allowed Prochlorococcus to adapt to the natural light gradient occurring in the upper layer of oceans . The present review critically assesses the basic knowledge acquired about Prochlorococcus both in the ocean and in the laboratory.

Curr Opin Plant Biol, 1998 Dec, 1(6), 475 - 9
Plastid division: evidence for a prokaryotically derived mechanism; Osteryoung KW et al.; Plastid division is a critical process in plant cell biology but it is poorly understood . Recent studies combining mutant analysis, gene cloning, and exploitation of genomic resources have revealed that the molecular machinery associated with plastid division is derived evolutionarily from the bacterial cell division apparatus . Comparison of the two processes provides a basis for identifying new components of the plastid division mechanism, but also serves to highlight the differences, not least of which is the nuclear control of the plastid division process.

Curr Opin Microbiol, 1998 Jun, 1(3), 271 - 7
A natural species concept for prokaryotes; Ward DM; Direct molecular analyses of natural microbial populations reveal patterns that should compel microbiologists to adopt a more natural species concept that has been known to biologists for decades . The species debate can be exploited to address a larger issue - microbiologists need, in general, to take a more natural view of the organisms they study.

Curr Opin Microbiol, 1998 Apr, 1(2), 175 - 82
Signal transduction by MAP kinase cascades in budding yeast; Posas F et al.; Budding yeast contain at least four distinct MAPK (mitogen activated protein kinase) cascades that transduce a variety of intracellular signals: mating-pheromone response, pseudohyphal/invasive growth, cell wall integrity, and high osmolarity adaptation . Although each MAPK cascade contains a conserved set of three protein kinases, the upstream activation mechanisms for these cascades are diverse, including a trimeric G protein, monomeric small G proteins, and a prokaryotic-like two-component system . Recently, it became apparent that there is extensive sharing of signaling elements among the MAPK pathways; however, little undesirable cross-talk occurs between various cascades . The formation of multi-protein signaling complexes is probably centrally important for this insulation of individual MAPK cascades.

Biochem Biophys Res Commun, 1999 Mar 5, 256(1), 63 - 7
Cloned prokaryotic iron superoxide dismutase protects yeast cells against oxidative stress depending on mitochondrial location; Balzan R et al.; Superoxide dismutase (SOD) is considered to be the first line of defense against oxygen toxicity . It exists as a family of three metalloproteins with copper,zinc (Cu,ZnSOD), manganese (MnSOD), and iron (FeSOD) forms . In this work, we have targeted Escherichia coli FeSOD to the mitochondrial intermembrane space (IMS) of yeast cells deficient in mitochondrial MnSOD . Our results show that FeSOD in the IMS increases the growth rate of the cells growing in minimal medium in air but does not protect the MnSOD-deficient yeast cells when exposed to induced oxidative stress . Cloned FeSOD must be targeted to the mitochondrial matrix to protect the cells from both physiological and induced oxidative stress . This confirms that the superoxide radical is mainly generated on the matrix side of the inner mitochondrial membrane of yeast cells, without excluding its potential appearance in the mitochondrial IMS where its elimination by SOD is beneficial to the cells .

Int J Neural Syst, 1997 Oct-Dec, 8(5-6), 581 - 99
A neural network method for identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites; Nielsen H et al.; We have developed a new method for the identification of signal peptides and their cleavage sites based on neural networks trained on separate sets of prokaryotic and eukaryotic sequences . The method performs significantly better than previous prediction schemes, and can easily be applied to genome-wide data sets . Discrimination between cleaved signal peptides and uncleaved N-terminal signal-anchor sequences is also possible, though with lower precision . Predictions can be made on a publicly available WWW server: http://www.cbs.dtu.dk/services/SignalP/.

J Struct Biol, 1998 Dec 15, 124(2-3), 303 - 10
Nucleotide-dependent interaction of the N-terminal domain of MukB with microtubules; Lockhart A et al.; The MukB protein from Escherichia coli has a domain structure that is reminiscent of the eukaryotic motor proteins kinesin and myosin: N-terminal globular domains, a region of coiled-coil, and a specialised C-terminal domain . Sequence alignment of the N-terminal domain of MukB with the kinesin motor domain indicated an approximately 22% sequence identity . These observations raised the possibility that MukB might be a prokaryotic motor protein and, due to the sequence homology shared with kinesin, might bind to microtubules (Mts) . We found that a construct encoding the first 342 residues of MukB (Muk342) binds specifically to Mts and shares a number of properties with the motor domain of kinesin . Visualisation of the Muk342 decorated Mt complexes using negative stain electron microscopy indicated that the Muk342 smoothly decorates the outside of Mts . Biochemical data demonstrate that Muk342 decorates Mts with a binding stoichiometry of one Muk342 monomer per tubulin monomer . These findings strongly suggest that MukB has a role in force generation and that it is a prokaryotic homologue of kinesin and myosin .

J Struct Biol, 1998 Dec 15, 124(2-3), 276 - 302
Structural research on surface layers: a focus on stability, surface layer homology domains, and surface layer-cell wall interactions; Engelhardt H et al.; Surface layers (S-layers) from Bacteria and Archaea are built from protein molecules arrayed in a two-dimensional lattice, forming the outermost cell wall layer in many prokaryotes . In almost half a century of S-layer research a wealth of structural, biochemical, and genetic data have accumulated, but it has not been possible to correlate sequence data with the tertiary structure of S-layer proteins to date . In this paper, some highlights of structural aspects of archaeal and bacterial S-layers that allow us to draw some conclusions on molecular properties are reviewed . We focus on the structural requirements for the extraordinary stability of many S-layer proteins, the structural and functional aspects of the S-layer homology domain found in S-layers, extracellular enzymes and related functional proteins, and outer membrane proteins, and the molecular interactions of S-layer proteins with other cell wall components . Finally, the perspectives and requirements for structural research on S-layers, which indicate that the investigation of isolated protein domains will be a prerequisite for solving S-layer structures at atomic resolution, are discussed .

Biochem Biophys Res Commun, 1999 Feb 24, 255(3), 735 - 9
Antiangiogenic activity of restin, NC10 domain of human collagen XV: comparison to endostatin; Ramchandran R et al.; Based on a homology search with endostatin, the C-terminus 185 aa of collagen XVIII, we report the cloning, expression, and antiangiogenic activity of a 22 kDa human collagen XV fragment, that we have named restin . Restin was expressed in the prokaryotic pET expression system . We have shown that restin inhibits the migration of endothelial cells in vitro but has no effect on the proliferation of these cells . A polyclonal antibody raised against endostatin cross-reacted with restin . Systemic administration of restin suppressed the growth of tumors in a xenograft renal carcinoma model .

J Bacteriol, 1999 Mar, 181(5), 1562 - 8
Characterization of an insertion sequence element associated with genetically diverse plant pathogenic Streptomyces spp; Healy FG et al.; Streptomycetes are common soil inhabitants, yet few described species are plant pathogens . While the pathogenicity mechanisms remain unclear, previous work identified a gene, nec1, which encodes a putative pathogenicity or virulence factor . nec1 and a neighboring transposase pseudogene, ORFtnp, are conserved among unrelated plant pathogens and absent from nonpathogens . The atypical GC content of nec1 suggests that it was acquired through horizontal transfer events . Our investigation of the genetic organization of regions adjacent to the 3' end of nec1 in Streptomyces scabies 84.34 identified a new insertion sequence (IS) element, IS1629, with homology to other IS elements from prokaryotic animal pathogens . IS1629 is 1,462 bp with 26-bp terminal inverted repeats and encodes a putative 431-amino-acid (aa) transposase . Transposition of IS1629 generates a 10-bp target site duplication . A 77-nucleotide (nt) sequence encompassing the start codon and upstream region of the transposase was identified which could function in the posttranscritpional regulation of transposase synthesis . A functional copy of IS1629 from S . turgidiscabies 94.09 (Hi-C-13) was selected in the transposon trap pCZA126, through its insertion into the lambda cI857 repressor . IS1629 is present in multiple copies in some S . scabies strains and is present in all S . acidiscabies and S . turgidiscabies strains examined . A second copy of IS1629 was identified between ORFtnp and nec1 in S . acidiscabies strains . The diversity of IS1629 hybridization profiles was greatest within S . scabies . IS1629 was absent from the 27 nonpathogenic Streptomyces strains tested . The genetic organization and nucleotide sequence of the nec1-IS1629 region was conserved and identical among representatives of S . acidiscabies and S . turgidiscabies . These findings support our current model for the unidirectional transfer of the ORFtnp-nec1-IS1629 locus from IS1629-containing S . scabies (type II) to S . acidiscabies and S . turgidiscabies.

J Bacteriol, 1999 Mar, 181(5), 1474 - 80
An Lrp-like protein of the hyperthermophilic archaeon Sulfolobus solfataricus which binds to its own promoter; Napoli A et al.; Regulation of gene expression in the domain Archaea, and specifically hyperthermophiles, has been poorly investigated so far . Biochemical experiments and genome sequencing have shown that, despite the prokaryotic cell and genome organization, basal transcriptional elements of members of the domain Archaea (i.e., TATA box-like sequences, RNA polymerase, and transcription factors TBP, TFIIB, and TFIIS) are of the eukaryotic type . However, open reading frames potentially coding for bacterium-type transcription regulation factors have been recognized in different archaeal strains . This finding raises the question of how bacterial and eukaryotic elements interact in regulating gene expression in Archaea . We have identified a gene coding for a bacterium-type transcription factor in the hyperthermophilic archaeon Sulfolobus solfataricus . The protein, named Lrs14, contains a potential helix-turn-helix motif and is related to the Lrp-AsnC family of regulators of gene expression in the class Bacteria . We show that Lrs14, expressed in Escherichia coli, is a highly thermostable DNA-binding protein . Bandshift and DNase I footprint analyses show that Lrs14 specifically binds to multiple sequences in its own promoter and that the region of binding overlaps the TATA box, suggesting that, like the E . coli Lrp, Lrs14 is autoregulated . We also show that the lrs14 transcript is accumulated in the late growth stages of S . solfataricus.

J Biol Chem, 1999 Mar 5, 274(10), 6634 - 40
The prokaryotic beta-recombinase catalyzes site-specific recombination in mammalian cells; Diaz V et al.; The development of new strategies for the in vivo modification of eukaryotic genomes has become an important objective of current research . Site-specific recombination has proven useful, as it allows controlled manipulation of murine, plant, and yeast genomes . Here we provide the first evidence that the prokaryotic site-specific recombinase (beta-recombinase), which catalyzes only intramolecular recombination, is active in eukaryotic environments . beta-Recombinase, encoded by the beta gene of the Gram-positive broad host range plasmid pSM19035, has been functionally expressed in eukaryotic cell lines, demonstrating high avidity for the nuclear compartment and forming a clear speckled pattern when assayed by indirect immunofluorescence . In simian COS-1 cells, transient beta-recombinase expression promoted deletion of a DNA fragment lying between two directly oriented specific recognition/crossing over sequences (six sites) located as an extrachromosomal DNA substrate . The same result was obtained in a recombination-dependent lacZ activation system tested in a cell line that stably expresses the beta-recombinase protein . In stable NIH/3T3 clones bearing different number of copies of the target sequences integrated at distinct chromosomal locations, transient beta-recombinase expression also promoted deletion of the intervening DNA, independently of the insertion position of the target sequences . The utility of this new recombination tool for the manipulation of eukaryotic genomes, used either alone or in combination with the other recombination systems currently in use, is discussed.

J Biol Chem, 1999 Mar 5, 274(10), 6366 - 73
Identification and functional characterization of a novel mitochondrial thioredoxin system in Saccharomyces cerevisiae; Pedrajas JR et al.; The so-called thioredoxin system, thioredoxin (Trx), thioredoxin reductase (Trr), and NADPH, acts as a disulfide reductase system and can protect cells against oxidative stress . In Saccharomyces cerevisiae, two thioredoxins (Trx1 and Trx2) and one thioredoxin reductase (Trr1) have been characterized, all of them located in the cytoplasm . We have identified and characterized a novel thioredoxin system in S . cerevisiae . The TRX3 gene codes for a 14-kDa protein containing the characteristic thioredoxin active site (WCGPC) . The TRR2 gene codes for a protein of 37 kDa with the active-site motif (CAVC) present in prokaryotic thioredoxin reductases and binding sites for NADPH and FAD . We cloned and expressed both proteins in Escherichia coli, and the recombinant Trx3 and Trr2 proteins were active in the insulin reduction assay . Trx3 and Trr2 proteins have N-terminal domain extensions with characteristics of signals for import into mitochondria . By immunoblotting analysis of Saccharomyces subcellular fractions, we provide evidence that these proteins are located in mitochondria . We have also constructed S . cerevisiae strains null in Trx3 and Trr2 proteins and tested them for sensitivity to hydrogen peroxide . The Deltatrr2 mutant was more sensitive to H2O2, whereas the Deltatrx3 mutant was as sensitive as the wild type . These results suggest an important role of the mitochondrial thioredoxin reductase in protection against oxidative stress in S . cerevisiae.

Int J Mol Med, 1999 Mar, 3(3), 291 - 5
M161Ag is a potent cytokine inducer with complement activating function (review); Matsumoto M et al.; We discovered a membrane-associated novel gene product expressed on some malignant human cells/cell lines undergoing apoptosis . This protein, named M161Ag, activated human complement and efficiently induced the pro-inflammatory cytokines IL-1 Beta , TNF-alpha and IL-6, and also IL-10 and IL-12 in human peripheral blood monocytes . M161Ag was a 43 kDa palmitoylated protein containing five amino acids encoded by TGA codons . These TGA codons were found to be translated into Trp, consistent with expression in prokaryotes including mitochondria and mycoplasma . The amino-terminal lipid was characteristic of prokaryote proteins participating in membrane anchoring . The M161Ag genomic clone contained a Pribnow box at the -35 and -10 promoter portions and the Shine-Dalgarno ribosomal binding site approximately 10 bp upstream of the translational start codon . The Mycoplasma fermentans origin of this protein was then confirmed by genomic Southern analysis; cells only infected with M . fermentans were positive for M161Ag . Thus, latent infection with M . fermentans allows tumor cells to produce M161Ag leading to activation of the host immune system . Here, we summarize bacterial proteins resembling M161Ag which may be candidates for therapeutic use if they exert immuno-regulatory functions.

Mol Biochem Parasitol, 1999 Jan 5, 98(1), 67 - 79
L-myo-Inositol 1-phosphate synthase from Entamoeba histolytica; Lohia A et al.; L-myo-Inositol 1-phosphate synthase (I-1-P synthase) catalyses the primary reaction for the synthesis of inositol in a variety of prokaryotes, eukaryotes and in the chloroplasts of algae and higher plants . Inositol is a precursor of essential macromolecules like membrane phospholipids, GPI anchor proteins and lipophosphoglycans, which play a determinant role in the pathogenesis of protozoan parasites such as Leishmania and Entamoeba . However, there is no report of I-1-P synthase or its gene from these organisms . The gene INO1 coding for this enzyme was first cloned from Saccharomyces cerevisiae and subsequently from several plants . Using molecular cloning techniques we have isolated and characterised the INO1 gene coding for the enzyme I-1-P synthase from Entamoeba histolytica . Simultaneously, we have purified and characterised the native enzyme from E . histolytica trophozoites and the cloned gene product from Escherichia coli . The gene product and the purified enzyme were both shown to be recognised by a heterologous anti-I-1-P synthase antibody from the phytoflagellate Euglena gracilis . Phylogenetic analysis of I-1-P synthase sequences from different eukaryotes suggest that it is highly conserved across species and the origin of this enzyme precedes the evolutionary divergence of modern eukaryotes.

Microb Comp Genomics, 1998, 3(4), 219 - 35
Microbial genescapes: a prokaryotic view of the yeast genome; Ragan MA et al.; We examine the translated open reading frames (ORFs) of the yeast Saccharomyces cerevisiae, focusing on those that have FASTA matches in phyletically defined sets of completely sequenced genomes . On this basis, we identify archaeal yeast, bacterial yeast, universal yeast, and yeast ORFs that do not have a match in any of nine prokaryote genomes . Similarly, we examine the yeast mitochondrial genome and the subset of the yeast nuclear ORFs identified as being involved in mitochondrial biogenesis . For the yeast ORFs that match one or more ORFs in these prokaryote genomes, we examine the phyletic and functional distributions of these matches as a function of match strength . These results provide genome level insights into the origin of the eukaryotic cell and the origin of mitochondria . More generally, they exemplify how the growing database of prokaryote genome sequences can help us understand eukaryote genomes.

Microb Comp Genomics, 1998, 3(4), 199 - 217
Microbial genescapes: phyletic and functional patterns of ORF distribution among prokaryotes; Gaasterland T et al.; We have implemented a statistically based approach to comparative genomics that allows us to define and characterize distributional patterns of conceptually translated open reading frames (ORFs) at different confidence levels based on pairwise FASTA matches . In this report, we apply this methodology to nine microbial genomes, focusing particularly on phyletic and functional patterns of ORF distribution within and between the two prokaryotic domains of life, Bacteria and Archaea . We examine patterns of presence and absence of matches, determine the universal ORF set, analyze features of genome specialization between closely related organisms, and present genomic evidence for the monophyly of Archaea . These analyses illustrate how a quantitative approach to comparative genomics can illuminate questions of fundamental biological significance.

Adv Exp Med Biol, 1998, 449, 39 - 53
POU domain factors in neural development; Schonemann MD et al.; Transcription factors serve critical roles in the progressive development of general body plan, organ commitment, and finally, specific cell types . Comparison of the biological roles of a series of individual members within a family permits some generalizations to be made regarding the developmental events that are likely to be regulated by a particular class of transcription factors . Here, we evidence that the developmental functions of the family of transcription factors characterized by the POU DNA binding motif exerts roles in mammalian development . The POU domain family of transcription factors was defined following the observation that the products of three mammalian genes, Pit-1, Oct-1, and Oct-2, and the protein encoded by the C . elegans gene unc-86, shared a region of homology, known as the POU domain . The POU domain is a bipartite DNA binding domain, consisting of two highly conserved regions, tethered by a variable linker . The approximately 75 amino acid N-terminal region was called the POU-specific domain and the C-terminal 60 amino acid region, the POU-homeodomain . High-affinity site-specific DNA binding by POU domain transcription factors requires both the POU-specific and the POU-homeodomain . Resolution of the crystal structures of Oct-1 and Pit-1 POU domains bound to DNA as a monomer and homodimer, respectively, confirmed several of the in vitro findings regarding interactions of this bipartite DNA binding domain with DNA and has provided important information regarding the flexibility and versatility of POU domain proteins . Overall the crystal structure of a monomer of the Oct-1 POU domain bound to the octamer element was similar to that predicted by the NMR solution structures of the POU-specific domain and the POU-homeodomain in isolation, with the POU-specific domain consists of four alpha helices, with the second and third helices forming a structure similar to the helix-turn-helix motif of the lambda and 434 repressors; several of the DNA base contacts are also conserved . A homodimer of the Pit-1 POU domain was crystallized bound to a Pit-1 dimer DNA element that is closely related to a site in the proximal promoter of the prolactin gene . The structure of the Pit-1 POU domain on DNA is very similar to that of Oct-1, and the Pit-1 POU-homeodomain/DNA structure is strikingly similar to that of other homeodomains, including the Oct-1 POU-homeodomain . The DNA contacts made by the Pit-1 POU-specific domain are also similar to those of Oct-1 and conserved with many made by the prokaryotic repressors . In the Oct-1 crystal, the POU-specific domain recognizes a GCAT half-site, while the corresponding sequence recognized by the Pit-1 POU-specific domain, GTAT, is on the opposing strand . As a result, the orientation of the Pit-1 POU-specific domain relative to the POU-homeodomain is flipped, as compared to the Oct-1 crystal structure, indicating the remarkable flexibility of the POU-specific domain in adapting to variations in sequence within the site . Also in contrast to the Oct-1 monomer structure is the observation that the POU-specific and POU-homeodomain of each Pit-1 molecule make major groove contacts on the same face of the DNA, consistent with the constraints imposed by its 15 amino acid linker . As a result, the Pit-1 POU domain homodimer essentially surrounds its DNA binding site . In the Pit-1 POU domain homodimer the dimerization interface is formed between the C-terminal end of helix 3 of the POU-homeodomain of one Pit-1 molecule and the N-terminus of helix 1 and the loop between helices 3 and 4 of the POU-specific domain of the other Pit-1 molecule . In contrast to other homeodomain crystal structures, the C-terminus of helix 3 in the Pit-1 POU-homeo-domain has an extended structure . (ABSTRACT TRUNCATED)

Biochemistry, 1999 Feb 9, 38(6), 1884 - 92
Purification, cloning, and characterization of the 16S RNA m5C967 methyltransferase from Escherichia coli; Tscherne JS et al.; The methyltransferase that forms m5C967 in Escherichia coli small subunit ribosomal RNA has been purified, cloned, and characterized . The gene was identified from the N-terminal sequence of the purified enzyme . The gene is a fusion of two open reading frames, fmu and fmv, previously believed to be distinct due to a DNA sequencing error . The gene, here named rsmB, encodes a 429-amino acid protein that has a number of homologues in prokaryotes, Archaea, and eukaryotes . C-Terminal sequencing of the overexpressed and affinity-purified protein by mass spectrometry methods verified the sequence expected for the gene product . The recombinant protein exhibited the same specificity as the previously described native enzyme; that is, it formed only m5C and only at position 967 . C1407, which is also m5C in natural 16S RNA, was not methylated . In vitro, the enzyme only recognized free 16S RNA . 30S ribosomal subunits were not a substrate . There was no requirement for added magnesium, suggesting that extensive secondary or tertiary structure in the RNA substrate may not be a requirement for recognition.

J Biol Chem, 1999 Feb 26, 274(9), 5948 - 52
Deletion mutation analysis of the mutS gene in Escherichia coli; Wu TH et al.; The MutS protein is part of the dam-directed MutHLS mismatch repair pathway in Escherichia coli . We have constructed deletion derivatives in the mutS gene, which retain the P-loop coding region for ATP binding . The mutant proteins were assayed for ATP hydrolysis, heteroduplex DNA binding, heterodimer MutS formation, and the ability to interact with MutL . Dimerization was assayed by expressing His6-tagged wild-type and non-tagged deletion mutant proteins in the same cell and isolating the His6-tagged protein followed by MutS immunoblotting after SDS-polyacrylamide gel electrophoresis . MutS-MutL interaction was measured using the same technique except that the MutL protein carried the His6 tag . Our results indicate that DNA binding ability resides in the N-terminal end of MutS, and dimerization and MutL interactions are located in the C-terminal end . Given the extensive amino acid homology in the MutS family our results with E . coli should be applicable to MutS homologues in other prokaryotes and eukaryotes.

J Nutr, 1999 Feb, 129(2), 325 - 7
Update on interconversions of vitamin B-6 with its coenzyme; McCormick DB et al.; Biosynthesis of pyridoxal 5'-phosphate (PLP) depends upon the relatively specific action of two consecutive enzymes, viz . pyridoxal (pyridoxine, pyridoxamine) kinase and pyridoxine (pyridoxamine) phosphate oxidase . Less specific phosphatases catalyze hydrolyses of the 5'-phosphates of the vitamers pyridoxal, pyridoxamine, and pyridoxine . From the recognition a generation ago of these processes by which the three forms of vitamin B-6 and their 5'-phosphates are interconverted, more recent studies have provided a fairly sophisticated understanding of the molecular characteristics of the enzymes involved . The evolutionary retention of homologous portions of pyridoxal kinase in humans as well as bacteria and the most recent finding of a highly conserved region of the pyridoxine (pyridoxamine) phosphate oxidase, also from both prokaryotic and eukaryotic organisms, emphasize the importance of these catalysts in the formation of a coenzyme that is essential for most organisms . Both kinase and oxidase involved in B-6 metabolism are potential targets for pharmacologic agents.

Protein Expr Purif, 1999 Feb, 15(1), 99 - 104
The refolding, purification, and activity analysis of a rice Bowman-Birk inhibitor expressed in Escherichia coli; Li N et al.; A putative rice trypsin/chymotrypsin inhibitor of the Bowman-Birk family, RBBI-8 of about 20 kDa, was expressed in Escherichia coli as a fusion protein bearing an N-terminal (His)6 purification tag . The expressed recombinant protein, rRBBI-8, is insoluble and accumulates as inclusion bodies . The insoluble protein was solubilized in 8 M urea under reducing environment and then refolded into its active conformation under optimized redox conditions . Strategies used to optimize yield and efficiency include selecting the redox system, increasing protein concentration during refolding by adding the denatured protein in a stepwise way, utilizing additives to prevent aggregation, and selecting buffer-exchanging conditions . A Ni-chelate affinity column was then employed to purify the renatured protein . rRBBI-8 shows strong inhibitory activity against trypsin and it can slightly inhibit chymotrypsin . In this study, a refolding and purification system was set up for this cysteine-rich recombinant protein expressed in a prokaryotic system .

Biotechniques, 1999 Feb, 26(2), 276 - 80, 282
Modified miniprep method for the rapid recovery of episomes from transfected breast epithelial cells; Bowers MT et al.; Episomal vectors such as pCEP4 are useful in expression cloning because they can replicate in both prokaryotes and eukaryotic cells . We have found a rapid and efficient means of extracting them from transfected MCF-10A nonmalignant human breast epithelial cells . We show that a plasmid miniprep protocol, modified by the addition of an extraction that eliminates a DNase activity, can consistently harvest pCEP4 episomes from the transfected cells (516 +/- 112 pg/harvest, mean +/- standard deviation; n = 11) . The quality of the episomal DNA obtained in this manner was verified by PCR, Southern blot and the retransformation of Escherichia coli . This simple method enables the efficient recovery of episomes and is applicable in the expression cloning of potential oncogenes using host MCF-10A cells.

Biochim Biophys Acta, 1999 Jan 4, 1436(3), 437 - 50
Phosphatidylinositol synthesis in mycobacteria; Salman M et al.; The metabolism and synthesis of an important mycobacterial lipid component, phosphatidylinositol (PI), and its metabolites, was studied in Mycobacterium smegmatis and M . smegmatis subcellular fractions . Little is known about the synthesis of PI in prokaryotic cells . Only a cell wall fraction (P60) in M . smegmatis was shown to possess PI synthase activity . Product was identified as PI by migration on TLC, treatment with phospholipase C and ion exchange chromatography . PI was the only major product (92.3%) when both cells and P60 fraction were labeled with {3H}inositol . Also, a neutral lipid inositol-containing product (4.1% of the total label) was identified in the P60 preparations . Strangely, PI synthase substrates, CDP-dipalmitoyl-DAG and CDP-NBD-DAG, added to the assay did not stimulate {3H}PI and NBD-PI yield by M . smegmatis . At the same time, addition of both substrates to rat liver and Saccharomyces cerevisiae PI synthase assays resulted in an increase in the product yield . Upon addition of CHAPS to the mycobacterial PI synthase assay, both substrates were utilized in a dose-dependent manner for the synthesis of NBD-PI and {3H}PI . These results demonstrate a strict substrate specificity of mycobacterial PI synthase toward endogenous substrates . K(m) of the enzyme toward inositol was shown to be 25 microM; Mg2+ stimulated the enzyme to a greater degree than Mn2+ . Structural analogs of myo-inositol, epi-inositol and scyllo-inositol and Zn2+ were shown to be more potent inhibitors of mycobacterial PI synthase than of mammalian analogs . Lack of sequence homology with mammalian PI synthases, different kinetic characteristics, existence of selective inhibitors and an important physiological role in mycobacteria, suggest that PI synthase may be a good potential target for antituberculosis therapy.

J Biol Chem, 1999 Feb 19, 274(8), 4684 - 92
Detailed architecture of the barley chloroplast psbD-psbC blue light-responsive promoter; Kim M et al.; The photosystem II reaction center chlorophyll protein D2, is encoded by the chloroplast gene psbD . PsbD is transcribed from at least three different promoters, one which is activated by high fluence blue light . Sequences within 130 base pairs (bp) of the psbD blue light-responsive promoter (BLRP) are highly conserved in higher plants . In this study, the structure of the psbD BLRP was analyzed in detail using deletion and site-directed mutagenesis and in vitro transcription . Deletion analysis showed that a 53-bp DNA region of the psbD BLRP, from -57 to -5, was sufficient for transcription in vitro . Mutation of a putative prokaryotic -10 element (TATTCT) located from -7 to -12 inhibited transcription from the psbD BLRP . In contrast, mutation of a putative prokaryotic -35 element, had no influence on transcription . Mutation of a TATATA sequence located between the barley psbA -10 and -35 elements significantly reduced transcription from this promoter . However, site-directed mutation of sequences located between -35 and -10 had no effect on transcription from the psbD BLRP . Transcription from the psbD BLRP was previously shown to require a 22-bp sequence, termed the AAG-box, located between -36 and -57 . The AAG-box specifically binds the protein complex AGF . Site-directed mutagenesis identified two different sequence motifs in the AAG-box that are important for transcription in vitro . Based on these results, we propose that positive factors bind to the AAG-box and interact with the chloroplast-encoded RNA polymerase to promote transcription from the psbD BLRP . Transcription from the psbD BLRP is thus similar to type II bacterial promoters that use activating proteins to stimulate transcription . Transcription of the psbD BLRP was approximately 6 . 5-fold greater in plastid extracts from illuminated versus dark-grown plants . This suggests that light-induced activation of this promoter in vivo involves factors interacting with the 53-bp psbD BLRP in vitro.

J Biol Chem, 1999 Feb 19, 274(8), 4537 - 44
A new antioxidant with alkyl hydroperoxide defense properties in yeast; Lee J et al.; To isolate new antioxidant genes, we have searched for activities that would rescue the tert-butyl hydroperoxide (t-BOOH)-hypersensitive phenotype of a Saccharomyces cerevisiae strain deleted for the gene encoding the oxidative stress response regulator Skn7 . We report the characterization of AHP1, which encodes a 19-kDa protein similar to the AhpC/TSA protein family within a small region encompassing Cys-62 of Ahp1p and the highly conserved N-terminal catalytic AhpC/TSA cysteine . Ahp1p contains a peroxisomal sorting signal, suggesting a peroxisomal localization . AHP1 exerts strong antioxidant protective functions, as demonstrated both by gene overexpression and deletion analyses, and is inducible by peroxides in an Yap1- and Skn7-dependent manner . Similar to yeast Tsa1p, Ahp1p forms a disulfide-linked homodimer upon oxidation and in vivo requires the presence of the thioredoxin system but not of glutathione to perform its antioxidant protective function . Furthermore, in contrast to Tsa1p, which is specific for H2O2, Ahp1p is specific for organic peroxides . Therefore, with respect to substrate specificity, Ahp1p differs from Tsa1p and is similar to prokaryotic alkyl hydroperoxide reductase AhpC . These data suggest that Ahp1p is a yeast orthologue of prokaryotic AhpC and justifies its name of yeast alkyl hydroperoxide reductase.

Mol Microbiol, 1998 Dec, 30(5), 1101 - 12
Nitrogen control of the glnN gene that codes for GS type III, the only glutamine synthetase in the cyanobacterium Pseudanabaena sp . PCC 6903; Crespo JL et al.; Pseudanabaena sp . strain PCC 6903 is the first cyanobacteria lacking the typical prokaryotic glutamine synthetase type I encoded by the glnA gene . The glnN gene product, glutamine synthetase type III, is the only glutamine synthetase activity present in this cyanobacterium . Analysis of glnN expression clearly indicated a nitrogen-dependent regulation . Pseudanabaena glnN gene expression and GSIII activity were upregulated under nitrogen starvation or using nitrate as a nitrogen source, while low levels of transcript and activity were found in ammonium-containing medium . Primer extension analysis showed that the glnN gene promoter structure resembled that of the NtcA-related promoters . Mobility shift assays demonstrated that Synechocystis sp . PCC 6803 NtcA protein, expressed and purified from Escherichia coli, bound to the promoter of the Pseudanabaena 6903 glnN gene . The NtcA control of the glnN gene in this cyanobacterium suggested that, in the absence of a glnA gene, NtcA took control of the only glutamine synthetase gene in a fashion similar to the way the glnA gene is governed in those cyanobacteria harbouring a glnA gene.

Biochemistry (Mosc), 1999 Jan, 64(1), 8 - 16
Termination of translation in eukaryotes: new results and new hypotheses; Kisselev LL et al.; Important new results obtained in studies of prokaryotic and eukaryotic translation termination during 1994-1998 are reviewed . Properties of the newly discovered factors RF3, eRF1, and eRF3 are described . Similarity and difference between prokaryotic and eukaryotic systems of translation termination and recent models of molecular mechanisms of protein synthesis at the termination stage are discussed . Hypotheses concerning the biological role of eRF3 are formulated and discussed.

J Clin Microbiol, 1999 Mar, 37(3), 518 - 23
Detection of viable Mycobacterium tuberculosis by reverse transcriptase-strand displacement amplification of mRNA; Hellyer TJ et al.; Numerous assays have been described for the detection of DNA and rRNA sequences that are specific for the Mycobacterium tuberculosis complex . Although beneficial to initial diagnosis, such assays have proven unsuitable for monitoring therapeutic efficacy owing to the persistence of these nucleic acid targets long after conversion of smears and cultures to negative . However, prokaryotic mRNA has a typical half-life of only a few minutes and we have previously shown that the presence of mRNA is a good indicator of bacterial viability . The purpose of the present study was to develop a novel reverse transcriptase-strand displacement amplification system for the detection of M . tuberculosis alpha-antigen (85B protein) mRNA and to demonstrate the use of this assay in assessing chemotherapeutic efficacy in patients with pulmonary tuberculosis . The assay was applied to sequential, noninduced sputum specimens collected from four patients: 10 of 11 samples (91%) collected prior to the start of therapy were positive for alpha-antigen mRNA, compared with 1 of 8 (13%), 2 of 8 (25%), 2 of 8 (25%), and 0 of 8 collected on days 2, 4, 7, and 14 of treatment, respectively . In contrast, 39 of 44 samples (89%) collected on or before day 14 were positive for alpha-antigen DNA . The loss of detectable mRNA corresponded to a rapid drop over the first 4 days of treatment in the number of viable organisms present in each sputum sample, equivalent to a mean fall of 0.43 log10 CFU/ml/day . Analysis of mRNA is a potentially useful method for monitoring therapeutic efficacy and for rapid in vitro determination of drug susceptibility.

Nucleic Acids Res, 1999 Mar 1, 27(5), 1283 - 8
The exosome subunit Rrp43p is required for the efficient maturation of 5.8S, 18S and 25S rRNA; Zanchin NI et al.; The Saccharomyces cerevisiae protein Rrp43p co-purifies with four other 3'-->5' exoribonucleases in a complex that has been termed the exosome . Rrp43p itself is similar to prokaryotic RNase PH . Individual exosome subunits have been implicated in the 3' maturation of the 5.8S rRNA found in 60S ribosomes and the 3' degradation of mRNAs . However, instead of being deficient in 60S ribosomes, Rrp43p-depleted cells were deficient in 40S ribosomes . Pulse-chase and steady-state northern analyses of pre-RNA and rRNA levels revealed a significant delay in the synthesis of both 25S and 18S rRNAs, accompanied by the stable accumulation of 35S and 27S pre-rRNAs and the under-accumulation of 20S pre-rRNA . In addition, Rrp43p-depleted cells accumulated a 23S aberrant pre-rRNA and a fragment excised from the 5' ETS . Therefore, in addition to the maturation of 5.8S rRNA, Rrp43p is required for the maturation 18S and 25S rRNA.

J Mol Biol, 1999 Feb 19, 286(2), 475 - 87
Characterization of the interactions between human cdc25C, cdks, cyclins and cdk-cyclin complexes; Morris MC et al.; We have overexpressed and purified human dual-specificity phosphatase cdc25C from a prokaryotic expression system at high levels and in a soluble, active form, and have studied and quantified its potential to interact with cdks, cyclins and preformed cdk-cyclin complexes by fluorescence spectroscopy and size-exclusion chromatography . Our data indicate that human cdc25C forms stable complexes, through hydrophobic contacts, with cdk and cyclin monomers, as well as with preformed cdk-cyclin complexes . In vitro, cdc25C interacts with cyclin monomers with high affinity, with tenfold less affinity with cdks, and with intermediate affinity with cdk-cyclin complexes . Moreover, changes observed in the intrinsic fluorescence of cdks, cyclins and cdk-cyclin complexes upon interaction with cdc25C are indicative of concomitant conformational changes within cdks and cyclins . From our results, we propose that in vitro, in the presence of monomeric cdks and cyclins, cdc25C forms stable ternary complexes, first through a high affinity interaction with a cyclin, which may then help target cdc25C towards a cdk . We discuss the biological relevance of our results and propose that a similar, two-step mechanism of interaction between cdc25C and cdk-cyclin complexes may occur in vivo .

Prenat Diagn, 1998 Dec, 18(13), 1366 - 73
The expression of genes in human preimplantation embryos; Pergament E et al.; The study of gene expression in human preimplantation embryos is establishing itself as a necessary dimension of developmental biology and medical genetics . Transcripts identified in human preimplantation embryos include housekeeping genes, transcription and growth factor genes, sex-determining genes, tissue-specific genes and novel genes, as well as genes of unknown function . Strategies are being developed which will eventually permit the most sophisticated gene expression studies on single human embryos of co-ordinated transcription and translational regulation . There is both a need for international co-operation for the systematic construction of expression maps and a need to establish databases of expression patterns during different stages of human development . Understanding how genes are regulated in humans is essential for understanding both normal development and disease . Until recently, studies of gene expression and regulation during embryogenesis were almost exclusively limited to prokaryotes and to eukaryotes other than man . The introduction of artificial reproductive technologies in conjunction with the development of recombinant molecular technologies applicable to single cells has made possible the study of human development at its earliest stages (Pergament and Bonnicksen, 1994) . Although there are still enormous technical challenges, robust strategies have been, and continue to be, developed for connecting DNA sequence to such endophenotypes as timing and level of genes expression at the single cell level . Questions currently being asked in human developmental genetic studies concern the pronucleus, the zygote and the preimplantation embryo: what genes are expressed? When are they expressed? What functions do they perform and how, in sequence or in combination? And, what elements control and regulate their expression? This review provides an overview of current knowledge about the expression of different embryonic genes during early human development and discusses future prospects, which includes a need for international co-operation similar to the Human Genome Project.

Novartis Found Symp, 1998, 217, 178 - 90; discussion 190-4
Bacterial genetics and strain variation; van Helden PD; An entire genome sequence will provide valuable information, but the genome of only one individual will limit interpretation of that information . Knowledge concerning genome variation in both eukaryotic and prokaryotic organisms such as Mycobacterium tuberculosis is likely to yield information of equal value and provide fundamental insights concerning the function of the genome . The variability in the genome between individual strains may be small and well defined, but it may cause large phenotypic changes (e.g . point mutations causing drug resistance) . Clinical and epidemiological observations have led to the development of hypotheses, assumptions and models concerning disease dynamics . However, genome variation studied by molecular epidemiology has made new insights possible, which have allowed us to examine prevailing dogmas concerning tuberculosis . Recent results suggest that historical dogmas may well hold true in some communities, but not all . The information gathered from studying strain variation can be used for modelling disease dynamics, prediction of epidemics, policy planning and for monitoring the outcome of new interventions, as well as for gaining insight into the life processes of the organism . However, molecular epidemiology has its own limitations, some of which result from our lack of understanding of genome variation . We need further information in order to understand clonality and evolution of this organism so that our use of molecular tools in epidemiology and drug development may become more relevant and accurate.

J Gen Virol, 1999 Jan, 80 ( Pt 1), 39 - 46
The major capsid protein, VP1, of human JC virus expressed in Escherichia coli is able to self-assemble into a capsid-like particle and deliver exogenous DNA into human kidney cells; Ou WC et al.; The full-length major capsid protein, VP1, of the human polyomavirus JC virus was cloned and expressed in Escherichia coli . VP1 protein expressed in E . coli self-assembled into capsid-like particles and caused haemagglutination of human O-type red blood cells . Caesium chloride density-gradient centrifugation analysis revealed that the capsid-like particles consisted of virion-like pseudovirion and empty capsid-like pseudocapsid populations . The morphology of pseudo-virion and pseudocapsid particles was observed under the electron microscope . The pseudovirions contained DNA and RNA molecules but the pseudocapsids did not contain any nucleic acid, as analysed by DNA extraction . DNA-binding activity of VP1 was also demonstrated by the South-Western probing method in vitro . Furthermore, pseudocapsids were able to deliver exogenous DNA into human foetal kidney epithelial cells . These results indicate that recombinant JC virus VP1 is able to self-assemble into capsid-like particles and to package DNA in the absence of the minor capsid proteins, VP2 and VP3 . This prokaryotic assembly system may facilitate the investigation of maturation mechanism(s) of polyomaviruses . Furthermore, capsid-like particles of JC virus VP1 generated in E . coli potentially could be used as a human gene transfer vector.

Prog Nucleic Acid Res Mol Biol, 1999, 62, 155 - 75
The initiation of DNA base excision repair of dipyrimidine photoproducts; Lloyd RS; One of the major DNA repair pathways is base excision repair, in which DNA bases that have been damaged by endogenous or exogenous agents are removed by the action of a class of enzymes known as DNA glycosylases . One subset of the known DNA glycosylases has an associated abasic lyase activity that generates a phosphodiester bond scission . The base excision pathway is completed by the sequential action of abasic endonucleases, DNA polymerases, and DNA ligases . Base excision repair of ultraviolet (UV) light-induced dipyrimidine photoproducts has been described in a variety of prokaryotic and eukaryotic organisms and phages . These enzymes vary significantly in their exact substrate specificity and in the catalytic mechanism by which repair is initiated . The prototype enzyme within this class of UV-specific DNA glycosylases is T4 endonuclease V . Endonuclease V holds the distinction of being the first glycosylase (1) to have its structure solved by X-ray diffraction of the enzyme alone as well as in complex with pyrimidine dimer-containing DNA, (2) to have its key catalytic active site residues identified, and (3) to have its mechanism of target DNA site location determined and the biological relevance of this process established . Thus, the study of endonuclease V has been critical in gaining a better understanding of the mechanisms of all DNA glycosylases.

Prog Nucleic Acid Res Mol Biol, 1999, 62, 109 - 54
Regulation of mammalian ribosomal gene transcription by RNA polymerase I; Grummt I; All cells, from prokaryotes to vertebrates, synthesize vast amounts of ribosomal RNA to produce the several million new ribosomes per generation that are required to maintain the protein synthetic capacity of the daughter cells . Ribosomal gene (rDNA) transcription is governed by RNA polymerase I (Pol I) assisted by a dedicated set of transcription factors that mediate the specificity of transcription and are the targets of the pleiotrophic pathways the cell uses to adapt rRNA synthesis to cell growth . In the past few years we have begun to understand the specific functions of individual factors involved in rDNA transcription and to elucidate on a molecular level how transcriptional regulation is achieved . This article reviews our present knowledge of the molecular mechanism of rDNA transcriptional regulation.

J Mol Biol, 1999 Feb 12, 286(1), 105 - 20
The preprotein translocase of the mitochondrial inner membrane: function and evolution; Rassow J et al.; Growing mitochondria acquire most of their proteins by the uptake of mitochondrial preproteins from the cytosol . To mediate this protein import, both mitochondrial membranes contain independent protein transport systems: the Tom machinery in the outer membrane and the Tim machinery in the inner membrane . Transport of proteins across the inner membrane and sorting to the different inner mitochondrial compartments is mediated by several protein complexes which have been identified in the past years . A complex containing the integral membrane proteins Tim17 and Tim23 constitutes the import channel for preproteins containing amino-terminal hydrophilic presequences . This complex is associated with Tim44 which serves as an adaptor protein for the binding of mtHsp70 to the membrane . mtHsp70, a 70 kDa heat shock protein of the mitochondrial matrix, drives the ATP-dependent import reaction of the processed preprotein after cleavage of the presequence . Preproteins containing internal targeting information are imported by a separate import machinery, which consists of the intermembrane-space proteins Tim9, Tim10, and Tim12, and the inner membrane proteins Tim22 and Tim54 . The proteins Tim17, Tim22, and Tim23 have in common a similar topology in the membrane and a homologous amino acid sequence . Moreover, they show a sequence similarity to OEP16, a channel-forming amino acid transporter in the outer envelope of chloroplasts, and to LivH, a component of a prokaryotic amino acid permease, defining a new PRAT-family of preprotein and amino acid transporters .

Arch Virol, 1998, 143(12), 2413 - 9
Prokaryotic expression of human cytomegalovirus pUS22 and its reactivity with human antibody; Dal Monte P et al.; This work demonstrates that antibodies to the product of the recombinant pUS22 of human cytomegalovirus (HCMV) are present in human sera during natural infection . US22 gene product has been identified as a member of the US22 family which may be secreted from infected cells . It is an early protein of 593 amino acids, 76 Kd in molecular weight . US22 seems to be an antigen which stimulates a good IgG response . In fact specific IgGs were found in approximately 40% of the CMV positive sera irrespective of their anti-CMV IgG titer . Specific IgM antibodies to pUS22 were observed exclusively during primary infection and in the sera with a high anti-CMV IgM titer . pUS22 could be considered for inclusion in a cocktail of CMV recombinant proteins to determine seropositivity to CMV and also to diagnose an active CMV infection.

C R Acad Sci III, 1998 Dec, 321(12), 961 - 78
{Is the replicon model applicable to higher eukaryotes?}; de Recondo AM; Thirty-five years ago, the Replicon model was proposed by Jacob, Brenner and Cuzin to explain the regulation of the Escherichia coli DNA replication . In this model, a genetic element, the replicator, would function as a target for a positive-acting initiator protein to drive the initiation of replication . This simple idea has been extremely useful in providing a framework to explain how the initiation of DNA replication occurs in all organisms . The identification of autonomously replicating sequences (ARSs) in budding yeast was the first extension of the Replicon model to eukaryotic chromosomes . In the higher eukaryotes, many biochemically defined replication start sites have been identified; nevertheless there is little genetic data indicating that these sites contain DNA sequences that are essential for replication . Moreover, in early Xenopus or Drosophila embryos, specific DNA sequences are not required either for initiating DNA replication or for preventing rereplication within a single cell cycle . This apparently fundamental difference between replicators in yeast and metazoan embryos may be more superficial than initially thought . In fact, during the past several years, an eukaryotic initiator conserved from yeast to man and also present in embryonic cells, the origin recognition complex (ORC), has been characterized, suggesting that the initiation mechanism should be essentially the same in prokaryotes and eukaryotes . In addition, the efficient once-per-cell-cycle replication of DNA is ensured in eukaryotes by a simple two-step mechanism in which the assembly of stable prereplicative complexes (PreRCs) at origins precedes and is temporally separated from the firing of these origins . Regulation of this process by cyclin-dependent kinases ensures that when origins fire, the cell is no longer competent to form new PreRCs . Now, it is important to understand how these complexes are remodeled or disassembled during replication initiation to trigger the transition from a stable origin-bound complex to a mobile replication machine.

J Mol Evol, 1999 Feb, 48(2), 213 - 7
On negative selection against ATG triplets near start codons in eukaryotic and prokaryotic genomes; Saito R et al.; The frequencies of ATG triplets in the genomes of various species were systematically analyzed, and the frequency of ATG triplets was significantly low around start codons in both prokaryotic and eukaryotic genomes . In eukaryotes, however, the frequency decrease before the start codon is much more evident than that after the start codon . In prokaryotes, on the other hand, the ATG frequency pattern around the start codon is less evident, and-more importantly-symmetric . We also computed average distances between a start codon and its nearest upstream-located ATG triplet and found a general tendency for the average distances to be longer in higher organisms.

J Mol Evol, 1999 Feb, 48(2), 209 - 12
Synonymous and nonsynonymous substitution rates in diatoms: a comparison between chloroplast and nuclear genes; Sorhannus U et al.; Rates of synonymous and nonsynonymous nucleotide substitutions and codon usage bias (ENC) were estimated for a number of nuclear and chloroplast genes in a sample of centric and pennate diatoms . The results suggest that DNA evolution has taken place, on an average, at a slower rate in the chloroplast genes than in the nuclear genes: a rate variation pattern similar to that observed in land plants . Synonymous substitution rates in the chloroplast genes show a negative association with the degree of codon usage bias, suggesting that genes with a higher degree of codon usage bias have evolved at a slower rate . While this relationship has been shown in both prokaryotes and multicellular eukaryotes, it has not been demonstrated before in diatoms.

J Mol Evol, 1999 Feb, 48(2), 178 - 86
Horizontal gene transfer involved in the convergent evolution of the plasmid-encoded enantioselective 6-hydroxynicotine oxidases; Schenk S et al.; The D- and L-specific nicotine oxidases are flavoproteins involved in the oxidative degradation of nicotine by the Gram-positive soil bacterium Arthrobacter nicotinovorans . Their structural genes are located on a 160-kbp plasmid together with those of other nicotine-degrading enzymes . They are structurally unrelated at the DNA as well as at the protein level . Each of these oxidases possesses a high degree of substrate specificity; their catalytic stereoselectivity is absolute, although they are able to bind both enantiomeric substrates with a similar affinity . It appears that the existence of these enzymes is the result of convergent evolution . The amino acid sequence of 6-hydroxy-l-nicotine oxidase (EC 1.5.3.6) as derived from the respective structural gene shows considerable structural similarity with eukaryotic monoamine oxidases (EC 1.4.3.4) but not with monoamine oxidases from prokaryotic bacteria including those of the genus Arthrobacter . These similarities are not confined to the nucleotide-binding sites . A 100-amino acid stretch at the N-terminal regions of 6-hydroxy-l-nicotine oxidase and human monoamine oxidases A possess a 35% homology . Overall, 27.0, 26.9, and 25.8% of the amino acid positions of the monoamine oxidases of Aspergillus niger (N), humans (A), and rainbow trout (Salmo gairdneri) are identical to those of 6-hydroxy-l-nicotine oxidase (Smith-Waterman algorithm) . In addition, the G+C content of the latter enzyme is in the range of that of eukaryotic monoamine oxidases and definitely lower than that of the A . nicotinovorans DNA and even that of the pAO1 DNA . The primary structure of 6-hydroxy-d-nicotine oxidase (EC 1.5.3.5) does not reveal its evolutionary history as easily . Significant similarities are found with a mitomycin radical oxidase from Streptomyces lavendulae (23.3%) and a "hypothetical protein" from Mycobacterium tuberculosis (26.0%) . It is proposed that the plasmid-encoded gene of 6-hydroxy-l-nicotine oxidase evolved after horizontal transfer from an eukaryotic source.

Annu Rev Genet, 1998, 32, 185 - 225
Comparative DNA analysis across diverse genomes; Karlin S et al.; We review concepts and methods for comparative analysis of complete genomes including assessments of genomic compositional contrasts based on dinucleotide and tetranucleotide relative abundance values, identifications of rare and frequent oligonucleotides, evaluations and interpretations of codon biases in several large prokaryotic genomes, and characterizations of compositional asymmetry between the two DNA strands in certain bacterial genomes . The discussion also covers means for identifying alien (e.g . laterally transferred) genes and detecting potential specialization islands in bacterial genomes.

Bioinformatics, 1998, 14(10), 884 - 5
GeneDn: for high-level expression design of heterologous genes in a prokaryotic system; Ju LW et al.; RESULTS: Based on the mathematical model of high-level expression of heterologous genes in prokaryotic vector pBV220, we developed a program GeneDn for high-level expression design of natural and synthetic genes . AVAILIBILITY: The program is written in Turbo Pascal 7.0 . The source code and related material are available upon request . CONTACT: wujj@nic.bmi.ac.cn

Genome Res, 1999 Jan, 9(1), 27 - 43
AAA+: A class of chaperone-like ATPases associated with the assembly, operation, and disassembly of protein complexes; Neuwald AF et al.; Using a combination of computer methods for iterative database searches and multiple sequence alignment, we show that protein sequences related to the AAA family of ATPases are far more prevalent than reported previously . Among these are regulatory components of Lon and Clp proteases, proteins involved in DNA replication, recombination, and restriction (including subunits of the origin recognition complex, replication factor C proteins, MCM DNA-licensing factors and the bacterial DnaA, RuvB, and McrB proteins), prokaryotic NtrC-related transcription regulators, the Bacillus sporulation protein SpoVJ, Mg2+, and Co2+ chelatases, the Halobacterium GvpN gas vesicle synthesis protein, dynein motor proteins, TorsinA, and Rubisco activase . Alignment of these sequences, in light of the structures of the clamp loader delta' subunit of Escherichia coli DNA polymerase III and the hexamerization component of N-ethylmaleimide-sensitive fusion protein, provides structural and mechanistic insights into these proteins, collectively designated the AAA+ class . Whole-genome analysis indicates that this class is ancient and has undergone considerable functional divergence prior to the emergence of the major divisions of life . These proteins often perform chaperone-like functions that assist in the assembly, operation, or disassembly of protein complexes . The hexameric architecture often associated with this class can provide a hole through which DNA or RNA can be thread; this may be important for assembly or remodeling of DNA-protein complexes.

EMBO J, 1999 Feb 1, 18(3), 727 - 32
Indirect regulation of translational termination efficiency at highly expressed genes and recoding sites by the factor recycling function of Escherichia coli release factor RF3; Crawford DJ et al.; Prokaryotic release factor RF3 is a stimulatory protein that increases the rate of translational termination by the decoding release factors RF1 and RF2 . The favoured model for RF3 function is the recycling of RF1 and RF2 after polypeptide release by displacing the factors from the ribosome . In this study, we have demonstrated that RF3 also plays an indirect role in the decoding of stop signals of highly expressed genes and recoding sites by accentuating the influence of the base following the stop codon (+4 base) on termination signal strength . The efficiency of decoding strong stop signals (e.g . UAAU and UAAG) in vivo is markedly improved with increased RF3 activity, while weak signals (UGAC and UAGC) are only modestly affected . However, RF3 is not responsible for the +4 base influence on termination signal strength, since prfC- strains lacking the protein still exhibit the same qualitative effect . The differential effect of RF3 at stop signals can be mimicked by modest overexpression of decoding RF . These findings can be interpreted according to current views of RF3 as a recycling factor, which functions to maintain the concentration of free decoding RF at stop signals, some of which are highly responsive to changes in RF levels.

J Mol Biol, 1999 Feb 5, 285(5), 1965 - 75
Integration of the colicin A pore-forming domain into the cytoplasmic membrane of Escherichia coli; Duche D et al.; The pore-forming domain of colicin A (pfColA) fused to a prokaryotic signal peptide (sp-pfColA) inserted into the inner membrane of Escherichia coli and apparently formed a functional channel, when generated in vivo . We investigated pfColA functional activity in vivo by the PhoA gene fusion approach, combined with cell fractionation and protease susceptibility experiments . Alkaline phosphatase was fused to the carboxy-terminal end of each of the ten alpha-helices of sp-pfColA to form a series of differently sized fusion proteins . We suggest that the alpha-helices anchoring pfColA in the membrane are first translocated into the periplasm . We identify two domains that anchor pfColA to the membrane in vivo: domain 1, extending from helix 1 to helix 8, which contains the voltage-responsive segment and domain 2 consisting of the hydrophobic helices 8 and 9 . These two domains function independently . Fusion proteins with a mutation inactivating the voltage-responsive segment or with a domain 1 lacking helix 8 were peripherally associated with the outside of the inner membrane, and were therefore digested by proteases added to spheroplasts . In contrast, fusion proteins with a functional domain 1 were protected from proteases, suggesting as expected that most of domain 1 is inserted into the membrane or is indeed translocated to the cytoplasm during pfColA channel opening .

Biochimie, 1998 Dec, 80(12), 1069 - 76
Effect of N-terminal deletions on the activity of pokeweed antiviral protein expressed in E . coli; Xu J et al.; Pokeweed antiviral protein (PAP) from Phytolacca americana is a highly specific N-glycosidase removing adenine residues (A4324 in 28S rRNA and A2660 in 23S rRNA) from intact ribosomes of both eukaryotes and prokaryotes . Due to the ribosome impairing activity the gene coding for mature PAP has not been expressed so far in bacteria whereas the full-length gene (coding for the mature 262 amino acids plus two signal peptides of 22 and 29 amino acids at both N- and C-termini, respectively) has been expressed in Escherichia coli . In order to determine: 1) the size of the N-terminal region of PAP which is required for toxicity to E . coli; and 2) the location of the putative enzymatic active site of PAP, 5'-terminal progressive deletion of the PAP full-length gene was carried out and the truncated forms of the gene were cloned in a vector containing a strong constitutive promoter and a consensus Shine-Dalgarno ribosome binding site . The ribosome inactivation or toxicity of the PAP is used as a phenotype characterized by the absence of E . coli colonies, while the mutation of PAP open reading frames in the small number of survived clones is used as an indicator of the toxicity to E . coli cells . Results showed that the native full-length PAP gene was highly expressed and was not toxic to E . coli cells although in vitro ribosome inactivating activity assay indicated it was active . However, all of the N-terminal truncated forms (removal of seven to 107 codons) of the PAP gene were toxic to E . coli cells and were mutated into either out of frame, early termination codon or inactive form of PAP (i.e., clone PAP delta107) . Deletion of more than 123 codons restored the correct gene sequence but resulted in the loss of the antiviral and ribosome inactivating activities and by the formation of a large number of clones . These results suggest that full-length PAP (with N- and C-terminal extensions) might be an inactive form of the enzyme in vivo presumably by inclusion body formation or other unknown mechanisms and is not toxic to E . coli cells . However, it is activated by at least seven codon deletions at the N-terminus . Deletions from seven through to 107 amino acids were lethal to the cells and only mutated forms (inactive) of the gene were obtained . But deletion of more than 123 amino acids resulted in the loss of enzymatic activity and made it possible to express the correct PAP gene in E . coli . Because deletion of Tyr94 and Val95, which are involved in the binding of the target adenine base, did not abolish the activity of PAP, it is concluded that the location previously proposed for PAP enzymatic active site should be reassessed.

FEBS Lett, 1999 Jan 8, 442(1), 1 - 6
Hepatitis B core particles as a universal display model: a structure-function basis for development; Pumpens P et al.; Because it exhibits a remarkable capability to accept mutational intervention and undergo correct folding and self-assembly in all viable prokaryotic and eukaryotic expression systems, hepatitis B core (HBc) protein has been favored over other proposed particulate carriers . Structurally, the unusual alpha-helical organization of HBc dimeric units allows introduction of foreign peptide sequences into several areas of HBc shells, including their most protruding spikes . Progress toward full resolution of the spatial structure as well as accumulation of chimeric HBc-based structures has brought closer the knowledge-based design of future vaccines, gene therapy tools and other artificial particulate objects.

Res Microbiol, 1998 Nov-Dec, 149(10), 689 - 701
Orientation of the peptidoglycan chains in the sacculus of Escherichia coli; Koch AL; The organization of chains of oligopeptidoglycan in the saccular wall is of critical importance in the study of the mechanism and physiology of prokaryotic wall growth . The electron microphotographs of De Pedro et al . present new findings and can be used to negate or at least raise questions about the previously accepted conclusion that the glycan chains are oriented transversely to the axis of rod-shaped Escherichia coli . This suggests caution in assuming that the glycan chains in the murein structure are parallel to each other and are perpendicular to the axis of the cell . These results should reopen the question of not only the orientation of the peptidoglycan chains, but the possibility of variability in orientation . Three classes of hypotheses about wall growth are reconsidered and problems with them are presented . The new results from De Pedro's laboratory and the experimental glycan chain length distribution argue against proposed systematic models . These include models that postulate belts or hoops stretched around the circumference of the cell and mechanisms that insert new chains of the length of presumptive "docking" strands in the stress-bearing wall . They are consistent, however, with the surface stress theory that proposes that random enzyme action together with physical forces are involved in the elongation of the rod-shaped Gram-negative wall.

Biochim Biophys Acta, 1998 Dec 10, 1448(2), 212 - 26
Calcium signalling in Bacillus subtilis; Herbaud ML et al.; Few systematic studies have been devoted to investigating the role of Ca2+ as an intracellular messenger in prokaryotes . Here we report an investigation on the potential involvement of Ca2+ in signalling in Bacillus subtilis, a Gram-positive bacterium . Using aequorin, it is shown that B . subtilis cells tightly regulate intracellular Ca2+ levels . This homeostasis can be changed by an external stimulus such as hydrogen peroxide, pointing to a relationship between oxidative stress and Ca2+ signalling . Also, B . subtilis growth appears to be intimately linked to the presence of Ca2+, as normal growth can be immediately restored by adding Ca2+ to an almost non-growing culture in EGTA containing Luria broth medium . Addition of Fe2+ or Mn2+ also restores growth, but with 5-6 h delay, whereas Mg2+ did not have any effect . In addition, the expression of alkyl hydroperoxide reductase C (AhpC), which is strongly enhanced in bacteria grown in the presence of EGTA, also appears to be regulated by Ca2+ . Finally, using 45Ca2+ overlay on membrane electrotransferred two-dimensional gels of B . subtilis, four putative Ca2+ binding proteins were found, including AhpC . Our results provide strong evidence for a regulatory role for Ca2+ in bacterial cells.

Virology, 1999 Jan 20, 253(2), 208 - 18
The ORF, regulated synthesis, and persistence-specific variation of influenza C viral NS1 protein; Marschall M et al.; The open reading frame (ORF) and the regulated synthesis of the influenza C viral NS1 protein were analyzed in view of viruses possessing different biological activities . We provide evidence for a 246-amino-acid NS1-ORF, encoded by five viral strains and variants . Prokaryotic expression of the prototype NS1-ORF resulted in a product of 27 kDa, confirming the predicted molecular weight . Using an antiserum raised against recombinant NS1 protein, nonstructural proteins of wild-type virus were detected in infected cells for a limited course of time, whereas a persistent virus variant was characterized by a long-term nonstructural gene expression . As examined by infection experiments, the intracellular distribution of nonstructural protein was nuclear and cytoplasmic, whereas in NS1 gene-transfected cells, the cytoplasmic localization occurred in a fine-grained structure, suggesting an analogy to influenza A viral NS1 protein . Concerning persistent infection, NS1 protein species differing in sizes and posttranslational modifications were observed for a persistent virus variant, as particularly illustrated by a high degree of NS1 phosphorylation . Virus reassortant analyses proved the importance of the NS-coding genomic segment: the minimal viral properties required for the establishment of persistence were transferred with this segment to a monoreassortant virus . Thus the influenza C viral NS1 protein is a 246-amino-acid nuclear-cytoplasmic phosphoprotein that can be subject to specific variations being functionally linked to a persistent virus phenotype .

Acta Biochim Pol, 1998, 45(3), 645 - 52
Overexpression of the yeast HAM1 gene prevents 6-N-hydroxylaminopurine mutagenesis in Escherichia coli; Kozmin SG et al.; The base analogue 6-N-hydroxylaminopurine (HAP) is a potent mutagen in a variety of prokaryotic and eukaryotic organisms . Mutations in the yeast ham1 gene render the cells hypersensitive to the mutagenic effect of HAP . We have found that this gene has homologues in a variety of organisms from bacteria to man . We have overexpressed yeast Ham1p in E . coli . We demonstrate that under conditions when this protein constitutes approximately 30% of cellular protein, the host strain is protected both from toxic and mutagenic effects of HAP . This result indicates that sole Ham1p activity might be sufficient for destruction of HAP or its metabolites in bacterial cells.

J Mol Biol, 1999 Jan 29, 285(4), 1401 - 15
Toxin-antitoxin systems homologous with relBE of Escherichia coli plasmid P307 are ubiquitous in prokaryotes; Gronlund H et al.; Toxin-antitoxin systems encoded by bacterial plasmids and chromosomes specify two proteins, a cytotoxin and an antitoxin . The antitoxins neutralize the cognate toxins by forming tight complexes with them . The antitoxins are unstable due to degradation by cellular proteases (Lon or Clp), whereas the toxins are stable . Here we show that orf7 (denoted relBP307) and orf6 (denoted relEP307) of Escherichia coli plasmid P307 are homologous to the relBE genes of E . coli and constitute a two-component toxin-antitoxin system: (i) relEP307 encodes a cytotoxin lethal or inhibitory to host cells; (ii) relBP307 encodes an antitoxin that prevents the lethal action of the relE-encoded toxin; (iii) RelBP307 antitoxin is degraded by Lon protease; (iv) RelBP307 antitoxin autoregulates the relBE operon of P307 at the level of transcription; (v) RelEP307 toxin acts as a co-repressor of transcription; and (vi) the relBE system stabilizes a mini-P307 replicon by the killing of plasmid-free cells . Using database searching, we found relBE homologues on the chromosomes of many Gram-negative and Gram-positive bacteria . Even more surprising, numerous relBE-homologous gene systems are present on the chromosomes of Archae . Thus, toxin-antitoxin systems homologous with relBE of E . coli are ubiquitous in prokaryotic organisms .

Arch Biochem Biophys, 1999 Feb 1, 362(1), 123 - 30
Rhodobacter capsulatus DNA topoisomerase I purification and characterization; Alkorta I et al.; A 30-kDa DNA topoisomerase has been purified to near homogeneity from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus . The enzyme is recognized by an antibody against a 16-mer peptide sequence from human DNA topoisomerase I . The purified enzyme is a type I topoisomerase . Consistent with the properties of other prokaryotic type I DNA topoisomerases, the isolated enzyme is unable to relax positively supercoiled DNA and absolutely requires divalent cations for its relaxation activity . However, regardless of the Mg+2 concentrations, ATP concentrations above 5 mM completely inhibit the relaxing activity . The enzyme is sensitive to high salt concentrations and the optimal activity occurs at salt concentrations between 3 and 30 mM for monovalent cations . Single-stranded M13 DNA is a strong inhibitor of this relaxing activity . The enzyme is inhibited by ethidium bromide, confirming that this DNA topoisomerase is incapable of relaxing positive supercoils . Topoisomerase I-specific inhibitors like Hoechst 32258 and actinomycin D inhibit the enzymatic activity while the enzyme is resistant to type II topoisomerase inhibitors such as norfloxacin, nalidixic acid, and novobiocin . From these enzymatic characteristics, we conclude that the R . capsulatus DNA topoisomerase is a prokaryotic type I DNA topoisomerase .

RNA, 1999 Jan, 5(1), 66 - 81
The yeast Saccharomyces cerevisiae YDL112w ORF encodes the putative 2'-O-ribose methyltransferase catalyzing the formation of Gm18 in tRNAs; Cavaille J et al.; The protein sequences of three known RNA 2'-O-ribose methylases were used as probes for detecting putative homologs through iterative searches of genomic databases . We have identified 45 new positive Open Reading Frames (ORFs), mostly in prokaryotic genomes . Five complete eukaryotic ORFs were also detected, among which was a single ORF (YDL112w) in the yeast Saccharomyces cerevisiae genome . After genetic depletion of YDL112w, we observed a specific defect in tRNA ribose methylation, with the complete disappearance of Gm18 in all tRNAs that naturally contain this modification, whereas other tRNA ribose methylations and the complex pattern of rRNA ribose methylations were not affected . The tRNA G18 methylation defect was suppressed by transformation of the disrupted strain with a plasmid allowing expression of YDL112wp . The formation of Gm18 on an in vitro transcript of a yeast tRNASer naturally containing this methylation, which was efficiently catalyzed by cell-free extracts from the wild-type yeast strain, did not occur with extracts from the disrupted strain . The protein encoded by the YDL112w ORF, termed Trm3 (tRNA methylation), is therefore likely to be the tRNA (Gm18) ribose methylase . In in vitro assays, its activity is strongly dependent on tRNA architecture . Trm3p, the first putative tRNA ribose methylase identified in an eukaryotic organism, is considerably larger than its Escherichia coli functional homolog spoU (1,436 amino acids vs . 229 amino acids), or any known or putative prokaryotic RNA ribose methyltransferase . Homologs found in human (TRP-185 protein), Caenorhabditis elegans and Arabidopsis thaliana also exhibit a very long N-terminal extension not related to any protein sequence in databases.

J Biol Chem, 1999 Jan 29, 274(5), 2907 - 15
The DNA binding properties of Saccharomyces cerevisiae Rad51 protein; Zaitseva EM et al.; Saccharomyces cerevisiae Rad51 protein is the paradigm for eukaryotic ATP-dependent DNA strand exchange proteins . To explain some of the unique characteristics of DNA strand exchange promoted by Rad51 protein, when compared with its prokaryotic homologue the Escherichia coli RecA protein, we analyzed the DNA binding properties of the Rad51 protein . Rad51 protein binds both single-stranded DNA (ssDNA) and double-stranded DNA (dsDNA) in an ATP- and Mg2+-dependent manner, over a wide range of pH, with an apparent binding stoichiometry of approximately 1 protein monomer per 4 (+/-1) nucleotides or base pairs, respectively . Only dATP and adenosine 5'-gamma-(thiotriphosphate) (ATPgammaS) can substitute for ATP, but binding in the presence of ATPgammaS requires more than a 5-fold stoichiometric excess of protein . Without nucleotide cofactor, Rad51 protein binds both ssDNA and dsDNA but only at pH values lower than 6.8; in this case, the apparent binding stoichiometry covers the range of 1 protein monomer per 6-9 nucleotides or base pairs . Therefore, Rad51 protein displays two distinct modes of DNA binding . These binding modes are not inter-convertible; however, their initial selection is governed by ATP binding . On the basis of these DNA binding properties, we conclude that the main reason for the low efficiency of the DNA strand exchange promoted by Rad51 protein in vitro is its enhanced dsDNA-binding ability, which inhibits both the presynaptic and synaptic phases of the DNA strand exchange reaction as follows: during presynapsis, Rad51 protein interacts with and stabilizes secondary structures in ssDNA thereby inhibiting formation of a contiguous nucleoprotein filament; during synapsis, Rad51 protein inactivates the homologous dsDNA partner by directly binding to it.

Curr Opin Genet Dev, 1998 Dec, 8(6), 649 - 54
Everything in moderation: archaea as 'non-extremophiles'; DeLong EF; Well characterized and cultivated archaea are prokaryotic specialists that thrive in habitats of elevated temperature, low pH, high salinity, or strict anoxia . Recently, however, new groups of abundant, uncultivated archaea have been found to be widespread in more pedestrian biotopes, including marine plankton, terrestrial soils, lakes, marine and freshwater sediments, and in association with metazoa . Research efforts are presently focused on characterizing the physiology, biochemistry and genetics of these abundant and cosmopolitan but poorly understood archaea.

Curr Opin Cell Biol, 1998 Dec, 10(6), 742 - 8
Emerging mechanisms of eukaryotic DNA replication initiation; Leatherwood J; Recent research has focused on proteins important for early steps in replication in eukaryotes, and particularly on Cdc6/Cdc18, the MCMs, and Cdc45 . Although it is still unclear exactly what role these proteins play, it is possible that they are analogous to initiation proteins in prokaryotes . One specific model is that MCMs form a hexameric helicase at replication forks, and Cdc6/Cdc18 acts as a 'clamp-loader' required to lock the MCMs around DNA . The MCMs appear to be the target of Cdc7-Dbf4 kinase acting at individual replication origins . Finally, Cdc45 interacts with MCMs and may shed light on how cyclin-dependent kinases activate DNA replication.

Biol Chem, 1998 Dec, 379(12), 1407 - 12
Cloning, subcellular localization and functional expression of human RNase HII; Frank P et al.; Recently we showed that the major mammalian RNase H, RNase HI, is evolutionarily related to prokaryotic RNase HII (Frank et al., FEBS-Lett . 421, 23-26, 1998), an enzyme described to be a minor activity in E . coli . As a consequence we addressed the question of whether a human RNase H exists, sharing homology with the main E . coli enzyme, RNase HI . Employing sequence analysis of expressed sequence tags, followed by specific PCR amplification of human cDNA, we cloned, sequenced and expressed a human open reading frame, coding for a 32 kDa protein . Purification of the recombinant His(6)-tagged protein from E . coli extracts using Ni(2+)-chelating chromatography and subsequent renaturation gel assay proved that it is an active RNase H . The properties of this enzyme suggest that it is identical with the human RNase HII, previously purified by one of us (Frank et al., Nucleic Acids Res . 22, 5247-5254, 1994) . Studies using a green fluorescent protein-fusion construct reveal that this protein is located in the nucleus.

Biochimie, 1998 Nov, 80(11), 923 - 31
The metabolism of 6-deoxyhexoses in bacterial and animal cells; Tonetti M et al.; L-fucose and L-rhamnose are two 6-deoxyhexoses naturally occurring in several complex carbohydrates . In prokaryotes both of them are found in polysaccharides of the cell wall, while in animals only L-fucose has been described, which mainly participates to the structure of glycoconjugates, either in the cell membrane or secreted in biological fluids, such as ABH blood groups and Lewis system antigens . L-fucose and L-rhamnose are synthesized by two de novo biosynthetic pathways starting from GDP-D-mannose and dTDP-D-glucose, respectively, which share several common features . The first step for both pathways is a dehydration reaction catalyzed by specific nucleotide-sugar dehydratases . This leads to the formation of unstable 4-keto-6-deoxy intermediates, which undergo a subsequent epimerization reaction responsible for the change from D- to L-conformation, and then a NADPH-dependent reduction of the 4-keto group, with the consequent formation of either GDP-L-fucose or dTDP-L-rhamnose . These compounds are then the substrates of specific glycosyltransferases which are responsible for insertion of either L-fucose or L-rhamnose in the corresponding glycoconjugates . The enzyme involved in the first step of GDP-L-fucose biosynthesis in E . coli, i.e., GDP-D-mannose 4,6 dehydratase, has been recently expressed as recombinant protein and characterized in our laboratory . We have also cloned and fully characterized a human protein, formerly named FX, and an E . coli protein, WcaG, which display both the epimerase and the reductase activities, thus indicating that only two enzymes are required for GDP-L-fucose production . Fucosylated complex glycoconjugates at the cell surface can then be recognized by specific counter-receptors in interacting cells, these mechanisms initiating important processes including inflammation and metastasis . The second pathway starting from dTDP-D-glucose leads to the synthesis of antibiotic glycosides or, alternatively, to the production of dTDP-L-rhamnose . While several sets of data are available on the first enzyme of the pathway, i.e., dTDP-D-glucose dehydratase, the enzymes involved in the following steps still need to be identified and characterized.

Cell Mol Life Sci, 1998 Dec, 54(12), 1350 - 64
Diversity of prokaryotic chromosomal proteins and the origin of the nucleosome; Sandman K et al.; All cells employ architectural proteins to confine and organize their chromosomes, and to prevent the otherwise thermodynamically favored collapse of concentrated DNA into compact structures . To accomplish this, prokaryotes have evolved a variety of phylogenetically unrelated, small, basic, sequence-independent DNA-binding proteins that include histones in Euryarchaeota, and members of the HU family in many Bacteria . In contrast, virtually, all Eukarya employ histones, and recently a metabolism-based hypothesis proposed that the eukaryal nucleus originated from a hydrogen-consuming, histone-containing Archaeon . Histones may have prevailed during the evolution of the Eukarya because of their extended interactions with DNA and, as noted, the histone fold now exists not only in histones but also as a structural motif in eukaryal transcription factors.

J Cell Biochem Suppl, 1998, 30-31, 18 - 29
DNA replication machinery of the mammalian cell; Malkas LH; The process of DNA replication in mammalian cells is highly complex and has several unique features that distinguish it from simpler prokaryotic systems . The study of mammalian DNA replication lagged behind that of prokaryotes for many years . This was because of the lack of a reliable and efficient mammalian cell-based in vitro DNA replication system . In 1984, the first mammalian-based DNA replication system that initiated DNA synthesis successfully in vitro was developed . The employment of the mammalian in vitro DNA replication system has led to the identification of several DNA replication proteins . This article describes the current knowledge regarding the proteins mediating mammalian DNA replication, as well as how they are proposed to function during DNA synthesis . There is also a discussion of the role the mammalian cell nuclear architecture plays in DNA replication . The evidence for the existence of an organized DNA replication machine in mammalian cells is also presented.

Proc Natl Acad Sci U S A, 1999 Jan 19, 96(2), 784 - 9
The evolutionary origin of the protein-translocating channel of chloroplastic envelope membranes: identification of a cyanobacterial homolog; Reumann S et al.; The known envelope membrane proteins of the chloroplastic protein import apparatus lack sequence similarity to proteins of other eukaryotic or prokaryotic protein transport systems . However, we detected a putative homolog of the gene encoding Toc75, the protein-translocating channel from the outer envelope membrane of pea chloroplasts, in the genome of the cyanobacterium Synechocystis sp . PCC 6803 . We investigated whether the low sequence identity of 21% reflects a structural and functional relationship between the two proteins . We provide evidence that the cyanobacterial protein is also localized in the outer membrane . From this information and the similarity of the predicted secondary structures, we conclude that Toc75 and the cyanobacterial protein, referred to as SynToc75, are structural homologs . synToc75 is essential, as homozygous null mutants were not recovered after directed mutagenesis . Sequence analysis indicates that SynToc75 belongs to a family of outer membrane proteins from Gram-negative bacteria whose function is not yet known . However, we demonstrate that these proteins are related to a specific group of prokaryotic secretion channels that transfer virulence factors, such as hemolysins and adhesins, across the outer membrane.

Cancer Res, 1999 Jan 1, 59(1), 189 - 97
Endostatin: yeast production, mutants, and antitumor effect in renal cell carcinoma; Dhanabal M et al.; Endostatin is a Mr 20,000 COOH-terminal fragment of collagen XVIII that inhibits the growth of several primary tumors . We report here the cloning and expression of mouse endostatin in both prokaryotic and eukaryotic expression systems . Soluble recombinant protein expressed in yeast (15-20 mg/L) inhibited the proliferation and migration of endothelial cells in response to stimulation by basic fibroblast growth factor . A rabbit polyclonal antibody was raised that showed positive immunoreactivity to the recombinant protein expressed from both systems . Importantly, the biological activity of the mouse recombinant protein could be neutralized by this antiserum in both endothelial proliferation and chorioallantoic membrane assays . Systemic administration of endostatin at 10 mg/kg suppressed the growth of renal cell cancer in a nude mouse model . The inhibition of tumor growth with soluble yeast-produced protein was comparable to that obtained with non-refolded precipitated protein expressed from bacteria . In addition, two closely related COOH-terminal deletion mutants of endostatin were also tested and showed strikingly differing activity . Collectively, these findings demonstrate the expression of a biologically active form of mouse endostatin in yeast, define a role for the molecule in inhibiting endothelial cell migration, extend its antitumor effects to renal cell carcinoma, and provide a formal proof (via the neutralizing antiserum experiments and the mutant data) that endostatin (and not a possible contaminant) acts as an antiangiogenic agent . Finally, the high level expression of mouse endostatin in yeast serves as an endotoxin free, soluble source of protein for fundamental studies on the mechanisms of tumor growth suppression by angiogenesis inhibitors.

Biochem Mol Biol Int, 1998 Dec, 46(6), 1093 - 100
Use of prokaryotically expressed nucleocapsid protein as positive antigen in ELISA; Kumar SS et al.; A cDNA library of Rinderpest vaccine virus was prepared in Zap Express vector (Stratagene) . The Rinderpest 'N' gene specific clones were selected, characterized and thereafter expressed in E . coli XLOLR strain . The expressed protein was found to be immunogenic in western blot with hyperimmune sera . It reacted with rinderpest and 'N' protein specific monoclonal antibodies in Enzyme Linked Immunosorbent Assay (ELISA) . Prokaryotically expressed 'N' protein also gave precipitin band in counter immunoelectrophoresis test (CIE) . The expression of N protein was sufficient for its utility as positive antigen in CIE and ELISA used for rinderpest diagnosis.

Annu Rev Microbiol, 1998, 52, 591 - 625
Thymine metabolism and thymineless death in prokaryotes and eukaryotes; Ahmad SI et al.; For many years it has been known that thymine auxotrophic microorganisms undergo cell death in response to thymine starvation {thymineless death (TLD)} . This effect is unusual in that deprivation of many other nutritional requirements has a biostatic, but not lethal, effect . Studies of numerous microbes have indicated that thymine starvation has both direct and indirect effects . The direct effects involve both single- and double-strand DNA breaks . The former may be repaired effectively, but the latter lead to cell death . DNA damaged by thymine starvation is a substrate for DNA repair processes, in particular recombinational repair . Mutations in recBCD recombinational repair genes increase sensitivity to thymineless death, whereas mutations in RecF repair protein genes enhance the recovery process . This suggests that the RecF repair pathway may be critical to cell death, perhaps because it increases the occurrence of double-strand DNA breaks with unique DNA configurations at lesion sites . Indirect effects in bacteria include elimination of plasmids, loss of transforming ability, filamentation, changes in the pool sizes of various nucleotides and nucleosides and in their excretion, and phage induction . Yeast cells show effects similar to those of bacteria upon thymine starvation, although there are some unique features . The mode of action of certain anticancer drugs and antibiotics is based on the interruption of thymidylate metabolism and provides a major impetus for further studies on TLD . There are similarities between TLD of bacteria and death of eukaryotic cells . Also, bacteria have "survival" genes other than thy (thymidylate synthetase), and this raises the question of whether there is a relationship between the two . A model is presented for a molecular basis of TLD.

J Biochem Mol Toxicol, 1999, 13(2), 93 - 106
Molecular mechanisms of copper metabolism and the role of the Menkes disease protein; Harrison MD et al.; Menkes disease is an X-linked, recessive disorder of copper metabolism that occurs in approximately 1 in 200,000 live births . The condition is characterized by skeletal abnormalities, severe mental retardation, neurologic degeneration, and patient mortality in early childhood . The symptoms of Menkes disease result from a deficiency of serum copper and copper-dependent enzymes . A candidate gene for the disease has been isolated and designated MNK . The MNK gene codes for a P-type cation transporting ATPase, based on homology to known P-type ATPases and in vitro experimentation . cDNA clones of MNK in Menkes patients show diminished or absented hybridization in northern blot experiments . The Menkes protein functions to export excess intracellular copper and activates upon Cu(I) binding to the six metal-binding repeats in the amino-terminal domain . The loss of Menkes protein activity blocks the export of dietary copper from the gastrointestinal tract and causes the copper deficiency associated with Menkes disease . Each of the Menkes protein amino-terminal repeats contains a conserved -X-Met-X-Cys-X-X-Cys- motif (where X is any amino acid) . These metal-binding repeats are conserved in other cation exporting ATPases involved in metal metabolism and in proteins involved in cellular defense against heavy metals in both prokaryotes and eukaryotes . An overview of copper metabolism in humans and a discussion of our understanding of the molecular basis of cellular copper homeostasis is presented . This forms the basis for a discussion of Menkes disease and the protein deficit in this disease.

J Biol Chem, 1999 Jan 22, 274(4), 2014 - 20
Cloning and characterization of a eukaryotic pantothenate kinase gene (panK) from Aspergillus nidulans; Calder RB et al.; Pantothenate kinase (PanK) is the key regulatory enzyme in the CoA biosynthetic pathway . The PanK gene from Escherichia coli (coaA) has been previously cloned and the enzyme biochemically characterized; highly related genes exist in other prokaryotes . We isolated a PanK cDNA clone from the eukaryotic fungus Aspergillus nidulans by functional complementation of a temperature-sensitive E . coli PanK mutant . The cDNA clone allowed the isolation of the genomic clone and the characterization of the A . nidulans gene designated panK . The panK gene is located on chromosome 3 (linkage group III), is interrupted by three small introns, and is expressed constitutively . The amino acid sequence of A . nidulans PanK (aPanK) predicted a subunit size of 46.9 kDa and bore little resemblance to its bacterial counterpart, whereas a highly related protein was detected in the genome of Saccharomyces cerevisiae . In contrast to E . coli PanK (bPanK), which is regulated by CoA and to a lesser extent by its thioesters, aPanK activity was selectively and potently inhibited by acetyl-CoA . Acetyl-CoA inhibition of aPanK was competitive with respect to ATP . Thus, the eukaryotic PanK has a distinct primary structure and unique regulatory properties that clearly distinguish it from its prokaryotic counterpart.

Adv Microb Physiol, 1998, 40, 401 - 38
Energetics of alkaliphilic Bacillus species: physiology and molecules; Krulwich TA et al.; The challenge of maintaining a cytoplasmic pH that is much lower than the external pH is central to the adaptation of extremely alkaliphilic Bacillus species to growth at pH values above 10 . The success with which this challenge is met may set the upper limit of pH for growth in these bacteria, all of which also exhibit a low content of basic amino acids in proteins or protein segments that are exposed to the outside bulk phase liquid . The requirement for an active Na(+)-dependent cycle and possible roles of acidic cell wall components in alkaliphile pH homeostasis are reviewed . The gene loci that encode Na+/H+ antiporters that function in the active cycle are described and compared with the less Na(+)-specific homologues thus far found in non-alkaliphilic Gram-positive prokaryotes . Alkaliphilic Bacillus species carry out oxidative phosphorylation using an exclusively H(+)-coupled ATPase (synthase) . Nonetheless, ATP synthesis is more rapid and reaches a higher phosphorylation potential at highly alkaline pH than at near-neutral pH even though the bulk electrochemical proton gradient across the coupling membrane is lower at highly alkaline pH . It is possible that some of the protons extruded by the respiratory chain are conveyed to the ATP synthase without first equilibrating with the external bulk phase . Mechanisms that might apply to oxidative phosphorylation in this type of extensively studied alkaliphile are reviewed, and note is made of the possibility of different kinds of solutions to the problem that may be found in new alkaliphilic bacteria that are yet to be isolated or characterized.

Hum Mutat, 1999, 13(1), 44 - 53
Systematic analysis of coproporphyrinogen oxidase gene defects in hereditary coproporphyria and mutation update; Rosipal R et al.; Hereditary coproporphyria (HC) is an acute hepatic porphyria with autosomal dominant inheritance caused by deficient activity of coproporphyrinogen III oxidase (CPO) . Clinical manifestations of the disease are characterized by acute attacks of neurological dysfunction often precipitated by drugs, fasting, cyclical hormonal changes, or infectious diseases . Skin photosensitivity may also be present . The seven exons, the exon/intron boundaries and part of 3' noncoding sequence of the CPO gene were systematically analyzed by an exon-by-exon denaturing gradient gel electrophoresis (DGGE) strategy followed by direct sequencing in seven unrelated heterozygous HC patients from France, Holland, and Czech Republic . Seven novel mutations and two new polymorphisms were detected . Among these mutations: two are missense (G197W, W427R), two are nonsense (Q306X, Q385X), two are small deletions (662de14bp; 1168del3bp removing a glycine at position 390), and one is a splicing mutation (IVS1-15c-->g) which creates a new acceptor splice site . The pathological significance of the point mutations G197W, W427R, and the in-frame deletion 390delGly were assessed by their respective expression in a prokaryotic system using site-directed mutagenesis . These mutations resulted in the absence or a dramatic decrease of CPO activity . The two polymorphisms were localized in noncoding part of the gene: 1) a C/G polymorphism in the promotor region, 142 bp upstream from the transcriptional initiation site (-142C/G), and 2) a 6 bp deletion polymorphism in the 3' noncoding part of the CPO gene, 574 bp downstream of the last base of the normal termination codon (+574 delATTCTT) . Five intragenic dimorphisms are now well characterized and the high degree of allelic heterogeneity in HC is demonstrated with seven new different mutations making a total of nineteen CPO gene defects reported so far.

Endocr Res, 1998 Aug-Nov, 24(3-4), 541 - 7
The use of computational chemistry in the study of sex steroid biosynthesis; Auchus RJ; Many of the steroidogenic enzymes and cofactor proteins are bound to intracellular membranes, frustrating standard methods of structure determination . Structural models of steroidogenic P450 enzymes, however, may be predicted from the x-ray crystal structures of prokaryotic P450s . Using P450-BMP as primary structural template, models of hepatic and steroidogenic P450s have been generated using computational chemistry and graphics techniques . We have developed an analogous model of human P450c17 using an approach that relies heavily on energy minimization and molecular dynamics to yield the final structure . The final model predicts the known activities of the enzyme and explains why all reported mutations disrupt one or more activities . Although the term "computational chemistry" suggests that modeling is an operator-independent, fully automated process, modeling exercises are fraught with pitfalls, choices, and practical dilemmas which make each attempt a unique endeavor . This paper describes the procedure in detail, using P450c17 as an example, and highlights the opportunities that computational chemistry offers for the study of sex steroid biosynthesis.

Mol Neurobiol, 1998 Winter, 17(1-3), 157 - 74
Nitric oxide in invertebrates; Colasanti M et al.; Nitric oxide (NO) is considered an important signaling molecule implied in different physiological processes, including nervous transmission, vascular regulation, immune defense, and in the pathogenesis of several diseases . The presence of NO is well demonstrated in all vertebrates . The recent data on the presence and roles of NO in the main invertebrate groups are reviewed here, showing the widespread diffusion of this signaling molecule throughout the animal kingdom, from higher invertebrates down to coelenterates and even to prokaryotic cells . In invertebrates, the main functional roles described for mammals have been demonstrated, whereas experimental evidence suggests the presence of new NOS isoforms different from those known for higher organisms . Noteworthy is the early appearance of NO throughout evolution and striking is the role played by the nitrergic pathway in the sensorial functions, from coelenterates up to mammals, mainly in olfactory-like systems . All literature data here reported suggest that future research on the biological roles of early signaling molecules in lower living forms could be important for the understanding of the nervous-system evolution.

Am J Physiol, 1999 Jan, 276(1 Pt 1), G7 - G13
Genetic Disorders of Membrane Transport III . Congenital chloride diarrhea; Kere J et al.; Congenital chloride diarrhea (CLD) is a recessively inherited disorder of intestinal electrolyte absorption that involves, specifically, Cl-/HCO-3 exchange . CLD is caused by mutations in a chromosome 7 gene, first known as DRA (for downregulated in adenoma) . The disease occurs in all parts of the world but is more common in some populations with genetic founder effects . More than 20 mutations in the gene are known to date . The CLD (or DRA) gene encodes a transmembrane protein belonging to the sulfate transporter family with three known members in humans, all associated with a distinct genetic disease . Members of the gene family can transport other anions as well that may turn out to be physiologically more important than sulfate transport . The gene family is well conserved in many prokaryotic and eukaryotic species and is expected to be much larger than presently known.

Microbiology, 1998 Dec, 144 ( Pt 12), 3351 - 8
Site-specific integration of bacteriophage VWB genome into Streptomyces venezuelae and construction of a VWB-based integrative vector; Van Mellaert L et al.; The temperate bacteriophage VWB integrates into the chromosome of Streptomyces venezuelae ETH14630 via site-specific integration . Following recombination of the VWB attP region with the chromosomal attB sequence, the host-phage junctions attL and attR are formed . Nucleotide sequence analysis of attP, attB, attL and attR revealed a 45 bp common core sequence . In attB this 45 bp sequence consists of the 3' end of a putative tRNA Arg(AGG) gene with a 3'-terminal CCA sequence which is typical for prokaryotic tRNAs . Phage DNA integration restores the putative tRNA Arg(AGG) gene in attL . However, following recombination the CCA sequence is missing as is the case for most Streptomyces tRNA genes described so far . Adjacent to VWB attP, an ORF encoding a 427 aa protein was detected . The C-terminal region of this protein shows high similarity to the conserved C-terminal domain of site-specific recombinases belonging to the integrase family . To prove the functionality of this putative integrase gene (int), an integrative vector pKT02 was constructed . This vector consists of a 2.3 kb HindIII-SphI restriction fragment of VWB DNA containing attP and int cloned in a non-replicative Escherichia coli vector carrying a thiostrepton-resistance (tsr) gene . Integration of pKT02 was obtained after transformation of Streptomyces venezuelae ETH14630 and Streptomyces lividans TK24 protoplasts . This vector will thus be useful for a number of additional Streptomyces species in which a suitable tRNA gene can be functional as integration site.

Protein Expr Purif, 1998 Dec, 14(3), 403 - 8
Large-scale preparation of biologically active recombinant chicken obese protein (leptin); Raver N et al.; Prokaryotic expression vector pMON3401 encoding full size A(-1) chicken leptin (AF012727) was prepared by PCR of previously described cDNA . Escherichia coli cells transformed with this vector overexpressed large amounts of chicken leptin upon induction with nalidixic acid . The expressed protein found in the inclusion bodies was refolded and purified to homogeneity on a Q-Sepharose column, yielding two electrophoretically pure fractions (leptin-1 and leptin-2), eluted from the column by 100 and 125 mM NaCl . Both fractions showed a single band of the expected molecular mass of 16 kDa and were composed of over 95% of monomeric protein . The biological activity of both fractions, resulting from proper renaturation, was further evidenced by their ability to stimulate proliferation of leptin-sensitive BAF/3 cells transfected with a long form of human leptin-receptor construct and by lowering the food intake of starved chicken following intravenous or intraperitoneal injections .

Gen Comp Endocrinol, 1999 Jan, 113(1), 155 - 64
Preparation of recombinant gilthead seabream (Sparus aurata) growth hormone and its use for stimulation of larvae growth by oral administration; Ben-Atia I et al.; Gilthead seabream (Sparus aurata) growth hormone (gsGH) cDNA coding for the mature protein was cloned in a pGEM-T vector and then transferred into prokaryotic expression vector pET-8 and expressed in E . coli BL21 (DE3) cells upon induction with IPTG . The expressed protein, contained within the inclusion-body pellet, was solubilized in 4.5 M urea, refolded at pH 11.3 in the presence of catalytic amounts of cysteine, and purified to over 98% purity, as evidenced by SDS-PAGE . Gel-filtration on a Superdex column under nondenaturing conditions and partial amino acid N-terminal sequence showed the purified protein to be a monomeric alanyl-gsGH . Over 90% pure bacterial beta-lactamase was copurified as a by-product . Binding assays of the {125I}gsGH to gs liver microsomal fraction resulted in high specific binding characterized by a Kd = 1.93 nM . Recombinant gsGH, like ovine placental lactogen, exhibited growth-stimulating activity when applied orally to S . aurata larvae or intraperitoneally to juvenile fish .

Med Hypotheses, 1998 Jul, 51(1), 5 - 9
Natural killer cell reactivity: activation and cytolysis mechanism models, involving heat shock protein, haemopoietic histocompatibility, major histocompatibility complex and complement molecules; Manzo G; The close association of heat shock protein (HSP), haemopoietic histocompatibility (Hh), major histocompatibility complex (MHC), and complement genes on the same chromosomal region, and the fact that all these genes are inherited on the whole in each haplotype of an individual, might indicate some evolutionary and functional correlations among them . Several data suggest for HSP70 molecules a possible role as a molecular target recognizable by natural killer (NK) cells . HSP70 sequences from both prokaryotic and eukaryotic organisms reveal that about half of the amino acid residues are identical and many of the remaining residues are similar . I here assume that NK reactivity might start, early in the immunogenesis process, as a effect of the interaction between HSP70 molecules and a hypothetical HSP receptor of yet immature non-cytolytic NK cells . To this receptor, an HSP molecule might act as an activator or an inhibitor depending on whether its amino acid residues are reactive or not with it, respectively . Later in the immunogenesis process, murine Hh or human equivalent molecules, dominantly expressed in bone marrow target cells, might select the non-reactive NK clones of an individual, inducing them to mature and express a lytic machinery . As a consequence of the NK maturation, proliferating hemopoietic target cells expressing only or mainly activator HSPs on their surface might undergo NK cytolysis . This might explain the NK lysis of apparently normal cells found in human foetal marrow; moreover, this might explain in some way the F1 hybrid resistance phenomenon . The NK reactivity of an individual would be further modulated by the expression on the NK surface of particular receptors (CD94, p58) specific for defined MHC molecules (Cw1, Cw3, Bw6, B7) on the target cells . Such a specific interaction would induce an 'NK effector inhibition' . The NK reactivity mechanism might have been further evolutionarily modified and adapted by the involvement of other NK receptors, such as CD11b (specific for the C3b factor of the complement) and CD16 (specific for the IgG Fc piece).Cooperation among HSP, MHC, CD11b, CD16, C3b and Fc allows us to propose original models of the activation and cytolysis mechanisms in the NK cytotoxicity and antibody-dependent cell cytotoxicity phenomena.

J Biochem (Tokyo), 1999 Jan, 125(1), 210 - 6
Identification of carboxyl residues in pepstatin-insensitive carboxyl proteinase from Pseudomonas sp . 101 that participate in catalysis and substrate binding; Ito M et al.; Pseudomonas carboxyl proteinase (PCP), isolated from Pseudomonas sp . 101, is the first example from a prokaryote of unique carboxyl proteinases {EC 3.4.23.33} which are insensitive to aspartic proteinase inhibitors, such as pepstatin, diazoacetyl-DL-norleucine methylester, and 1,2-epoxy-3(p-nitrophenoxy)propane . To identify the catalytic residue(s) of PCP, chemical modification was carried out using carboxyl residue-specific reagents, carbodiimides . PCP was inactivated effectively by N,N'-dicyclohexylcarbodiimide (DCCD) with pseudo-first-order kinetics . For the inactivation, 0.7 mol DCCD was involved per 1 mol PCP . The effects of pH and methanol on the inactivation showed that two carboxyl residues (Asp and/or Glu) were involved in the reaction.The inactivation by DCCD was prevented by a competitive inhibitor, tyrostatin, or a synthetic substrate in a concentration-dependent manner . Based on these data, differential labeling of PCP with DCCD was carried out: Firstly, PCP was treated with cold DCCD in the presence of tyrostatin . After removal of the tyrostatin, which covered the substrate binding site, by dialysis, the PCP was treated with {14C}DCCD to label carboxyl residue(s) essential for its function . Two labeled peptides were isolated by HPLC from a trypsin digest of cold- and {14C}DCCD modified PCP . On analysis of their amino acid sequences, it was revealed that the {14C}DCCD was bound to Asp140 and Glu222 of PCP, respectively . Based on these data, it was strongly suggested that Asp140 and Glu222 of PCP were involved in its catalytic function or substrate binding.

Clin Microbiol Rev, 1999 Jan, 12(1), 19 - 39
Role of heat shock proteins in protection from and pathogenesis of infectious diseases; Zugel U et al.; Increased synthesis of heat shock proteins (hsp) occurs in prokaryotic and eukaryotic cells when they are exposed to stress . By increasing their hsp content, cells protect themselves from lethal assaults, primarily because hsp interfere with the uncontrolled protein unfolding that occurs under stress . However, hsp are not produced only by stressed cells; some hsp are synthesized constitutively and perform important housekeeping functions . Accordingly, hsp are involved in the assembly of molecules which play important roles in the immune system . It is not surprising that due to their wide distribution and their homology among different species, hsp represent target antigens of the immune response . Frequent confrontation of the immune system with conserved regions of hsp which are shared by various microbial pathogens can potentiate antimicrobial immunity . However, long-term confrontation of the immune system with hsp antigens which are similar in the host and invaders may convert the immune response against these host antigens and promote autoimmune disease . This review provides an overview of the role of hsp in immunity with a focus on infectious and autoimmune diseases.

Plant Cell, 1999 Jan, 11(1), 87 - 99
A chromodomain protein encoded by the arabidopsis CAO gene is a plant-specific component of the chloroplast signal recognition particle pathway that is involved in LHCP targeting; Klimyuk VI et al.; A recessive mutation in Arabidopsis, named chaos (for chlorophyll a/b binding protein harvesting-organelle specific; designated gene symbol CAO), was isolated by using transposon tagging . Characterization of the phenotype of the chaos mutant revealed a specific reduction of pigment binding antenna proteins in the thylakoid membrane . These nuclear-encoded proteins utilize a chloroplast signal recognition particle (cpSRP) system to reach the thylakoid membrane . Both prokaryotes and eukaryotes possess a cytoplasmic SRP containing a 54-kD protein (SRP54) and an RNA . In chloroplasts, the homolog of SRP54 was found to bind a 43-kD protein (cpSRP43) rather than to an RNA . We cloned the CAO gene, which encodes a protein identified as Arabidopsis cpSRP43 . The product of the CAO gene does not resemble any protein in the databases, although it contains motifs that are known to mediate protein-protein interactions . These motifs include ankyrin repeats and chromodomains . Therefore, CAO encodes an SRP component that is unique to plants . Surprisingly, the phenotype of the cpSRP43 mutant (i.e., chaos) differs from that of the Arabidopsis cpSRP54 mutant, suggesting that the functions of the two proteins do not strictly overlap . This difference also suggests that the function of cpSRP43 is most likely restricted to protein targeting into the thylakoid membrane, whereas cpSRP54 may be involved in an additional process(es), such as chloroplast biogenesis, perhaps through chloroplast-ribosomal association with chloroplast ribosomes.

Science, 1999 Jan 8, 283(5399), 220 - 1
A nonhyperthermophilic common ancestor to extant life forms; Galtier N et al.; The G+C nucleotide content of ribosomal RNA (rRNA) sequences is strongly correlated with the optimal growth temperature of prokaryotes . This property allows inference of the environmental temperature of the common ancestor to all life forms from knowledge of the G+C content of its rRNA sequences . A model of sequence evolution, assuming varying G+C content among lineages and unequal substitution rates among sites, was devised to estimate ancestral base compositions . This method was applied to rRNA sequences of various species representing the major lineages of life . The inferred G+C content of the common ancestor to extant life forms appears incompatible with survival at high temperature . This finding challenges a widely accepted hypothesis about the origin of life.

Mol Biochem Parasitol, 1998 Nov 30, 97(1-2), 149 - 60
A Plasmodium falciparum aminopeptidase gene belonging to the M1 family of zinc-metallopeptidases is expressed in erythrocytic stages; Florent I et al.; A new single copy gene has been isolated from Plasmodium falciparum, by immunoscreening a genomic DNA expression library . The gene appears devoid of introns, displays the classical A + T richness and codon usage of P . falciparum genes, and is transcribed into a 4 kb mRNA in erythrocytic stages . The deduced amino acid sequence corresponds to a 1056 residue protein (122 kDa) containing the canonical HExxHx18E signature of zinc-metallopeptidase active sites of the M1 family at position 467-490, a downstream conserved tyrosine residue involved in catalysis in position 551, and the GAMEN conserved motif characteristic of aminopeptidases in the M1 family, at position 431-435 . The greatest similarities were found with aminopeptidases N of Escherichia coli and Haemophilius influenza (more than 80% identical residues in the canonical signature of the active site) but significant similarities centred on the active site region exist with all other members of the M1 family such as other prokaryotic aminopeptidases, eukaryotic aminopeptidases A and N and leukotriene A4 hydrolases (40-50% identical residues in the canonical signature of the active site) . A polyclonal serum raised to a synthetic peptide deduced from the gene labelled schizont proteins of 96 and 68 kDa purified to homogeneity and both displaying aminopeptidase activity, as well as cytoplasmic structures in schizont stages.

Mutat Res, 1999 Jan, 436(1), 11 - 9
Spontaneous mutations in the Big Blue transgenic system are primarily mouse derived; Hill KA et al.; The Big Blue transgenic mouse mutation detection system provides a powerful approach for measuring spontaneous and induced mutations in vivo . The observed mutations may contain a fraction of ex vivo or prokaryotic mutational events . Indeed, a modified, selectable form of the Big Blue assay seem to generate artifactual mutants under certain circumstances . Herein we review the evidence that circular mutants (i.e., the plaque circumference is at least 50% blue) collected in the standard Big Blue assay are derived primarily from the mouse . The most direct evidence is the similarity in the types of mutations found in jackpot and nonjackpot mutations . In addition, about half of the spontaneous mutations in the lacI transgene are transitions and transversions at CpG dinucleotides, a mammalian-specific feature . The mutation pattern observed at lacI is consistent with AT mutation pressure operating in a GC rich DNA and approaches that reported for observed germline human factor IX mutations . Furthermore, the spontaneous mutation pattern of circular Big Blue mutants differs significantly from that of an endogenous lacI gene in E . coli . Pinpoint mutants (a dot of blue color peripherally located in a wild type plaque), which a priori were not expected to be mouse-derived, have a mutation pattern consistent with the mutation pattern of an endogenous E . coli lacI gene . Analysis of induced mutagenesis studies reveals mutation frequencies and patterns for the Big Blue circular mutants which are comparable to endogenous genes . In reconstruction experiments, blue plaques derived from a superinfection with wild type and mutant phage produced approximately 50% blue and 50% clear plaques on replating . This phenomenon has not been seen when plaques derived from mouse were replated in the Big Blue assay . Collectively, the evidence strongly supports a murine origin for circular mutants recovered in the standard Big Blue assay . Validation of current assays is an essential step in determining the frequency and pattern of spontaneous murine-specific mutations . Defining this benchmark will be helpful in evaluating the next generation of transgenic mutation detection systems .

J Struct Biol, 1998 Nov, 123(3), 269 - 71
Crystallization and preliminary X-ray analysis of FlhD from Escherichia coli; Campos A et al.; The flhD gene from Escherichia coli codes for one of the transcriptional activators of flagellar genes and is a putative global regulator in the cell . FlhD together with FlhC forms a complex that positively regulates the expression of flagellar genes . However, FlhD is able to change its DNA binding specificity, probably by forming another complex with some unknown factor or by binding to other targets by itself . FlhD is a good model to study the DNA binding specificity by complex formation . The FlhD three-dimensional structure will certainly improve our understanding of this protein, which is the first case of combinatory specificity in prokaryotes . The flhD gene was over-expressed, and the resulting protein was purified to homogeneity . FlhD crystals were obtained from 15-25% PEG 5000, 0.05-0.1 M lithium sulfate, 0.1 M Tris, pH 8.5, or 20-30% PEG 5000, 0.05-0.2 M sodium acetate, and 0.1 M Tris-HCl, pH 8.5, as precipitant solutions, and belong to the space group I4 with unit cell parameters of a = b = 90 A, c = 42.6 A, and alpha = beta = gamma = 90 degrees and contain two molecules per asymmetric unit . A complete native data set has been collected to 2.3-A resolution .

Eur J Biochem, 1998 Dec 1, 258(2), 485 - 90
Reactivity studies of the tyrosyl radical in ribonucleotide reductase from Mycobacterium tuberculosis and Arabidopsis thaliana--comparison with Escherichia coli and mouse; Elleingand E et al.; Ribonucleotide reductase (RNR) is a key enzyme for DNA synthesis since it provides cells with deoxyribonucleotides, the DNA precursors . Class I alpha2beta2 RNRs contain a dinuclear iron center and an essential tyrosyl radical in the beta2 component (protein R2) . This is also true for the purified protein R2 of Mycobacterium tuberculosis RNR, as shown by iron analysis, light absorption and EPR spectroscopy . EPR spectroscopy at 286 GHz revealed a high g(x) value, suggesting that the radical is not hydrogen bonded, as in other prokaryotic R2s and in contrast with eukaryotic R2s (from Arabidopsis thaliana and mouse) . Furthermore, it proved to be very resistant to scavenging by a variety of phenols and thiols and by hydroxyurea, similar to the Escherichia coli radical . By comparison, the plant and mouse radicals are very sensitive to drugs such as resveratrol and 2-thiophenthiol . The radical from M . tuberculosis RNR does not seem to be an appropriate target for new antituberculous agents.

Acta Microbiol Immunol Hung, 1998, 45(3-4), 349 - 90
The origin and evolution of viruses (a review); Sinkovics J et al.; Viroids and prions might have existed early at the border of inanimate and living worlds . Most extant viruses can be characterized as derivatives of ancestors originating from episomal elements of prokaryotes (DNA phages) and later from eukaryotes . Retroviruses very likely originated from cellular retrotransposons . Retrograde evolution of some large viruses from obligatory intracellular bacteria is possible but the ontogenesis of extant bacteria does not include a viral form of existence (the filterable L forms are not viruses) and well-defined viruses do not regenerate back into vegetative bacterial forms . Biologists experimenting with the evolution of prokaryotic and eukaryotic ancient cells cannot ignore the earliest appearance of viruses within or outside the living matter . Viruses participated in and gave direction to the evolution and natural selection by coexisting with uni- and multicellular organisms for billions of years . The coevolution of viruses and their host cells is characterized by incessant attacks and counterattacks through gene rearrangements and mutations (induced in the virus by an immunological counterattack of the host or by transgression of species barriers by the virus) and recombinations . Recombinations occurred between viral and viral or viral and host genes . Acts of "molecular piracy" as practiced by ancient viruses endowed the virus with the expression of several host genes for the advantage of the virus in its replicative cycle and host-to-host spread . Probably the first immortalized and malignantly transformed cells were induced by viruses as viruses evolved anti-apoptotic measures . While infected cells resort to apoptotic death before the assembly of a new viral progeny, prominent are the anti-apoptotic measures viruses evolved in order to assure the completion of their full replicative cycle . Further, viruses may escape neutralization by host antibodies and may survive a counterattack by the host's T cells directed at virally infected cells of its own . Viruses may induce a form of tolerance and coexist with their host without inducing disease . Persistent and apparently or deceivingly apathogenic or even attenuated viral "quasi-species" populations may contain individual particles that regain virulence due to recombinations and/or gene rearrangements, especially when transgressing species barriers . Xenotropic viruses of animals may replicate in human cells and vice versa confounding experiments with xenotransplants or with use of veterinary viral vaccines for the treatment of human diseases.

Exp Mol Med, 1998 Jun 30, 30(2), 65 - 71
Expression of a recombinant branched chain alpha-oxo acid dehydrogenase complex E2 (BCOADC-E2) in insect cells and its immunoreactivity to autoimmune sera; Lee SM et al.; Preparation of a pure autoantigen by way of recombinant DNA technology has an important value in an accurate diagnosis or prognosis of an autoimmune disease . BCOADC-E2 subunit, a mitochondrial protein, has been known to be the autoantigen of primary biliary cirrhosis (PBC), a chronic autoimmune liver disease, as well as idiopathic dilated cardiomypathy (IDCM), a chronic autoimmune heart disease . Recombinant form of this molecule had been expressed in E . coli but with low yield and severe degradation . Furthermore, sera from IDCM patients failed to recognized BCOADC-E2 molecule produced in prokaryotic expression system . In this study, a recombinant bovine BCOADC-E2 fusion protein has been expressed in insect cells using baculovirus expression system and analyzed anti-BCOADC-E2 reactivity in sera from patients with PBC or with IDCM . Optimal production of the recombinant fusion protein has been achieved at 20 multiplicity of infection (MOI), and the protein was affinity-purified using metal-binding resins . The affinity-purified BCOADC-E2 protein was successfully recognized by sera from PBC patients, but not by sera from IDCM patients suggesting that the different auto-immune response against BCOADC-E2 is needed to be elucidated in terms of epitope recognition.

Bioorg Med Chem Lett, 1998 Sep 22, 8(18), 2479 - 82
H-phosphonate derivatives as novel peptide deformylase inhibitors; Hu YJ et al.; Peptide deformylase catalyzes the removal of the N-terminal formyl group from nascent polypeptides during prokaryotic protein maturation and is essential for bacterial survival . Its absence from eukaryotic organisms makes it an attractive target for designing novel antibacterial agents . Peptidyl H-phosphonates were synthesized and shown to be competitive inhibitors of the deformylase.

J Mol Evol, 1999 Jan, 48(1), 77 - 85
Genetic characterization of plasmids containing genes encoding enzymes of leucine biosynthesis in endosymbionts (Buchnera) of aphids; Baumann L et al.; The prokaryotic endosymbionts (Buchnera) of aphids are known to provision their hosts with amino acids that are limiting in the aphid diet . Buchnera from the aphids Schizaphis graminum and Diuraphis noxia have plasmids containing leuABCD, genes that encode enzymes of the leucine biosynthetic pathway, as well as genes encoding proteins probably involved in plasmid replication (repA1 and repA2) and an open reading frame (ORF1) of unknown function . The newly reported plasmids closely resemble a plasmid previously described in Buchnera of the aphid Rhopalosiphum padi {Bracho AM, Martinez-Torres D, Moya A, Latorre A (1995) J Mol Evol 41:67-73} . Nucleotide sequence comparisons indicate conserved regions which may correspond to an origin of replication and two promoters, as well as inverted repeats, one of which resembles a rho-independent terminator . Phylogenetic analyses based on amino acid sequences of leu gene products and ORF1 resulted in trees identical to those obtained from endosymbiont chromosomal genes and the plasmid-borne trpEG . These results are consistent with a single evolutionary origin of the leuABCD-containing plasmid in a common ancestor of Aphididae and the lack of plasmid exchange between endosymbionts of different aphid species . Trees for ORF1 and repA (based on both nucleotides and amino acids) are used to examine the basis for leu plasmid differences between Buchnera of Thelaxes suberi and Aphididae . The most plausible explanation is that a single transfer of the leu genes to an ancestral replicon was followed by rearrangements . The related replicon in Buchnera of Pemphigidae, which lacks leuABCD, appears to represent the ancestral condition, implying that the plasmid location of the leu genes arose after the Pemphigidae diverged from other aphid families . This conclusion parallels previously published observations for the unrelated trpEG plasmid, which is present in Aphididae and absent in Pemphigidae . Recruitment of amino acid biosynthetic genes to plasmids has been ongoing in Buchnera lineages after the infection of aphid hosts.

J Mol Evol, 1999 Jan, 48(1), 22 - 41
Getting in or out: early segregation between importers and exporters in the evolution of ATP-binding cassette (ABC) transporters; Saurin W et al.; ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate in the transport of a wide variety of molecules . ABC systems are characterized by a highly conserved ATPase module called here the ABC module, involved in coupling transport to ATP hydrolysis . We have used the sequence of one of the first representatives of bacterial ABC transporters, the MalK protein, to collect 250 closely related sequences from a nonredundant protein sequence database . The sequences collected by this objective method are all known or putative ABC transporters . After having eliminated short protein sequences and duplicates, the 197 remaining sequences were subjected to a phylogenetic analysis based on a mutational similarity matrix . An unrooted tree for these modules was found to display two major branches, one grouping all collected uptake systems and the other all collected export systems . This remarkable disposition strongly suggests that the divergence between these two functionally different types of ABC systems occurred once in the history of these systems and probably before the differentiation of prokaryotes and eukaryotes . We discuss the implications of this finding and we propose a model accounting for the generation and the diversification of ABC systems.

J Biol Chem, 1999 Jan 8, 274(2), 924 - 9
Purification, cloning, and characterization of the 16 S RNA m2G1207 methyltransferase from Escherichia coli; Tscherne JS et al.; The methyltransferase that forms m2G1207 in Escherichia coli small subunit rRNA has been purified, cloned, and characterized . The gene was identified from the N-terminal sequence of the purified enzyme as the open reading frame yjjT (SWISS-PROT accession number ) . The gene, here renamed rsmC in view of its newly established function, codes for a 343-amino acid protein that has homologs in prokaryotes, Archaea, and possibly also in lower eukaryotes . The enzyme reacted well with 30 S subunits reconstituted from 16 S RNA transcripts and 30 S proteins but was almost inactive with the corresponding free RNA . By hybridization and protection of appropriate segments of 16 S RNA that had been extracted from 30 S subunits methylated by the enzyme, it was shown that of the three naturally occurring m2G residues, only m2G1207 was formed . Whereas close to unit stoichiometry of methylation could be achieved at 0.9 mM Mg2+, both 2 mM EDTA and 6 mM Mg2+ markedly reduced the level of methylation, suggesting that the optimal substrate may be a ribonucleoprotein particle less structured than a 30 S ribosome but more so than free RNA.

J Biol Chem, 1999 Jan 8, 274(2), 748 - 54
The flavohemoglobin of Escherichia coli confers resistance to a nitrosating agent, a "Nitric oxide Releaser," and paraquat and is essential for transcriptional responses to oxidative stress; Membrillo-Hernandez J et al.; Escherichia coli possesses a flavohemoglobin (Hmp), product of hmp, the first microbial globin gene to be sequenced and characterized at the molecular level . Although related proteins occur in numerous prokaryotes and eukaryotic microorganisms, the function(s) of these proteins have been elusive . Here we report construction of a defined hmp mutation and its use to probe Hmp function . As anticipated from up-regulation of hmp expression by nitric oxide (NO), S-nitrosoglutathione (GSNO) or sodium nitroprusside (SNP), the hmp mutant is hypersensitive to these agents . The hmp promoter is more sensitive to SNP and S-nitroso-N-penicillamine (SNAP) than is the soxS promoter, consistent with the role of Hmp in protection from reactive nitrogen species . Additional functions for Hmp are indicated by (a) parallel sensitivity of the hmp mutant to the redox-cycling agent, paraquat, (b) inability of the mutant to up-regulate fully the soxS and sodA promoters in response to oxidative stress caused by paraquat, GSNO and SNP, and (c) failure of the mutant to accumulate reduced paraquat radical after anoxic growth . We conclude that Hmp plays a role in protection from nitrosating agents and NO-related species and oxidative stress . This protective role probably involves direct detoxification of those species and sensing of NO-related and oxidative stress.

Genetics, 1999 Jan, 151(1), 57 - 75
Saccharomyces cerevisiae Mod5p-II contains sequences antagonistic for nuclear and cytosolic locations; Tolerico LH et al.; MOD5 encodes a tRNA modification activity located in three subcellular compartments . Alternative translation initiation generates Mod5p-I, located in the mitochondria and the cytosol, and Mod5p-II, located in the cytosol and nucleus . Here we study the nucleus/cytosol distribution of overexpressed Mod5p-II . Nuclear Mod5p-II appears concentrated in the nucleolus, perhaps indicating that the nuclear pool may have a different biological role than the cytoplasmic and mitochondrial pools . Mod5p contains three motifs resembling bipartite-like nuclear localization sequences (NLSs), but only one is sufficient to locate a passenger protein to the nucleus . Mutations of basic residues of this motif cumulatively contribute to a cytosolic location for the fusion proteins . These alterations also cause decreased nuclear pools of endogenous Mod5p-II . Depletion of nuclear Mod5p-II does not affect tRNATyr function . Despite the NLS, most Mod5p is cytosolic . We assessed whether Mod5p sequences cause a karyophilic reporter to be located in the cytosol . By this assay, Mod5p may contain more than one region that functions as cytoplasmic retention and/or nuclear export sequences . Thus, distribution of Mod5p results from the presence/absence of mitochondrial targeting information and sequences antagonistic for nuclear and cytosolic locations . Mod5p is highly conserved; sequences responsible for subcellular distribution appear to reside in "accessory" motifs missing from prokaryotic counterparts.

Genetics, 1999 Jan, 151(1), 45 - 55
The MSN1 and NHP6A genes suppress SWI6 defects in Saccharomyces cerevisiae; Sidorova J et al.; Ankyrin (ANK) repeats were first found in the Swi6 transcription factor of Saccharomyces cerevisiae and since then were identified in many proteins of eukaryotes and prokaryotes . These repeats are thought to serve as protein association domains . In Swi6, ANK repeats affect DNA binding of both the Swi4/Swi6 and Mbp1/Swi6 complexes . We have previously described generation of random mutations within the ANK repeats of Swi6 that render the protein temperature sensitive in its ability to activate HO transcription . Two of these SWI6 mutants were used in a screen for high copy suppressors of this phenotype . We found that MSN1, which encodes a transcriptional activator, and NHP6A, which encodes an HMG-like protein, are able to suppress defective Swi6 function . Both of these gene products are involved in HO transcription, and Nhp6A may also be involved in CLN1 transcription . Moreover, because overexpression of NHP6A can suppress caffeine sensitivity of one of the SWI6 ANK mutants, swi6-405, other SWI6-dependent genes may also be affected by Nhp6A . We hypothesize that Nhp6A and Msn1 modulate Swi6-dependent gene transcription indirectly, through effects on chromatin structure or other transcription factors, because we have not been able to demonstrate that either Msn1 or Nhp6A interact with the Swi4/Swi6 complex.

Bioorg Med Chem Lett, 1998 Jan 6, 8(1), 87 - 92
Antibiotic inhibitors of the peptidyl transferase center . 1 . Clindamycin as a composite analogue of the transfer RNA fragments L-Pro-Met and the D-ribosyl ring of adenosine; Fitzhugh AL; Clindamycin's three-dimensional structure is shown via a computer rendered stereomodel to be strikingly similar to L-Pro-Met and the D-ribosyl ring of adenosine . This discovery has important implications for the rational design of new licosamides and for efforts to understand how this and related classes of agents selectively inhibit protein biosynthesis on the prokaryotic ribosome.

Curr Microbiol, 1999 Feb, 38(2), 135 - 6
Detection of messenger RNA transcribed from genes encoding enzymes of amino acid biosynthesis in Buchnera aphidicola (endosymbiont of aphids); Baumann L et al.; The aphid Schizaphis graminum is dependent on Buchnera aphidicola, a prokaryotic endosymbiont . One of the functions of the endosymbiont is the synthesis of essential amino acids for the aphid host . Previously we have found that B . aphidicola has many of the genes that encode enzymes of amino acid biosynthesis . Using reverse transcriptase and the polymerase chain reaction, we have detected messenger RNA corresponding to genes involved in the synthesis of tryptophan, isoleucine, valine, leucine, and histidine.

Mol Gen Genet, 1998 Nov, 260(4), 346 - 56
A second gene for type I signal peptidase in Bradyrhizobium japonicum, sipF, is located near genes involved in RNA processing and cell division; Bairl A et al.; The TnphoA-induced Bradyrhizobium japonicum mutant 184 shows slow growth and aberrant colonization of soybean nodules . Using a DNA fragment adjacent to the transposon insertion site as a probe, a 3.4-kb BglII fragment of B . japonicum 110spc4 DNA was identified and cloned . Sequence analysis indicated that two truncated ORFs and three complete ORFs were encoded on this fragment . A database search revealed homologies to several other prokaryotic proteins: PdxJ (an enzyme involved in vitamin B6 biosynthesis), AcpS (acyl carrier protein synthase), Lep or Sip (prokaryotic type I signal peptidase), RNase III (an endoribonuclease which processes double-stranded rRNA precursors and mRNA) and Era (a GTP-binding protein required for cell division) . The mutation in strain 184 was found to lie within the signal peptidase gene, which was designated sipF . Therefore, sipF is located in a region that encodes gene products involved in posttranscriptional and posttranslational processing processes . By complementation of the lep(ts) E . coli mutant strain IT41 it was demonstrated that sipF indeed encodes a functional signal peptidase, and genetic complementation of B . japonicum mutant 184 by a 2.8-kb SalI fragment indicated that sipF is expressed from a promoter located directly upstream of sipF . Using a non-polar kanamycin resistance cassette, a specific sipF mutant was constructed which exhibited defects in symbiosis similar to those of the original mutant 184.

Free Radic Biol Med, 1998 Dec, 25(9), 1106 - 11
Oxidative stress causes a general, calcium-dependent degradation of mitochondrial polynucleotides; Crawford DR et al.; Oxidative stress has many effects on biological cells, including the modulation of gene expression . Reactive oxygen species are known to up-regulate and down-regulate RNA expression in prokaryotic and eukaryotic cells . We have previously reported that a preferential and calcium-dependent down-regulation of mitochondrial RNAs occurs when HA-1 hamster fibroblasts are exposed to hydrogen peroxide . Here we extend these studies to determine whether this down-regulation is specific to mitochondria RNA or involves general polynucleotide degradation . Degradation and associated decreases in the levels of 16S mitochondrial rRNA following exposure of cells to 400 microM hydrogen peroxide were found to be dependent on calcium at 2 and 5 h . Degradation of mitochondrial genomic DNA was also observed following peroxide exposure, and occurred at similar time points as for mitochondrial RNA degradation . As with mitochondrial RNA degradation, this mitochondrial genomic DNA degradation was dependent on calcium . These results indicate that there is a general, calcium-dependent degradation of mitochondrial polynucleotides following exposure of HA-1 fibroblasts to oxidative stress, and suggest that a dramatic shut-down in mitochondrial biosynthesis is an early-stage response to oxidative stress.

J Biol Chem, 1999 Jan 1, 274(1), 86 - 92
A mechanism for Tn5 inhibition . carboxyl-terminal dimerization; Braam LA et al.; Tn5 is unique among prokaryotic transposable elements in that it encodes a special inhibitor protein identical to the Tn5 transposase except lacking a short NH2-terminal DNA binding sequence . This protein regulates transposition through nonproductive protein-protein interactions with transposase . We have studied the mechanism of Tn5 inhibition in vitro and find that a heterodimeric complex between the inhibitor and transposase is critical for inhibition, probably via a DNA-bound form of transposase . Two dimerization domains are known in the inhibitor/transposase shared sequence, and we show that the COOH-terminal domain is necessary for inhibition, correlating with the ability of the inhibitor protein to homodimerize via this domain . This regulatory complex may provide clues to the structures of functional synaptic complexes . Additionally, we find that NH2- and COOH-terminal regions of transposase or inhibitor are in functional contact . The NH2 terminus appears to occlude transposase homodimerization (hypothetically mediated by the COOH terminus), an effect that might contribute to productive transposition . Conversely, a deletion of the COOH terminus uncovers a secondary DNA binding region in the inhibitor protein which is probably located near the NH2 terminus.

Protein Sci, 1998 Dec, 7(12), 2684 - 7
Yeast methionine aminopeptidase I can utilize either Zn2+ or Co2+ as a cofactor: a case of mistaken identity?
Walker KW, Bradshaw RA.
Yeast methionine aminopeptidase I (MetAP I) is one of two enzymes in Saccharomyces cerevisiae that is responsible for cotranslational cleavage of initiator methionines . It has previously been classified as a Co2+ metalloprotease in all prokaryotic and eukaryotic forms studied . However, treatment of recombinant apo-MetAP I with 12.5 microM Zn2+ produces an enzyme that is as active as that reconstituted with 200 microM Co2+ . In the presence of physiological concentrations of reduced glutathione (GSH), Co-MetAP I is inactive, while the activity of Zn-MetAP I is increased more than 1.7-fold over Zn-MetAP I assayed in the absence of GSH . Given that the in vivo concentration of Zn2+ is at least 1,000-fold higher than that of Co2+, and that Co2+ is insoluble in physiological concentrations of GSH, it is probable that yeast MetAP I is actually a Zn2+ metalloprotease . Furthermore, unless there are extraordinary conditions that insulate or sequester them from this reducing milieu, that have yet to be identified, there are not likely to be any cytoplasmic enzymes that use free Co2+.

Biol Chem, 1998 Nov, 379(11), 1355 - 8
RNA splicing of bacterial genes in eukaryotes; Lorbach E et al.; The presence of intervening sequences or introns in eukaryotic genes has been known for more than 20 years, and the mechanisms underlying RNA splicing have been studied in depth both genetically and biochemically . In recent years, however, an increasing number of bacterial genes have been introduced into higher eukaryotes as important tools for genetic studies . Their gene products are frequently used as an indirect measure for cell type-specific promoter activity, as, for example, in the case of chloramphenicol acetyl transferase (CAT assay) or beta-galactosidase . Here we show that RNA splicing of two prokaryotic genes encoding site-specific DNA recombinases occurs in eukaryotic cells . In one case, splicing is only observed after treatment of cells with the cytokine alpha interferon . We further demonstrate that mutating an intragenic donor splice site in a bacterial gene apparently activates a second, alternative splicing pathway . In conjunction with previous reports, our findings should also be regarded as a warning that splicing of bacterial genes in higher eukaryotes is a more common phenomenon than presently recognized, which may be difficult to overcome and may cause problems in the interpretation of experimental results.

J Bacteriol, 1999 Jan, 181(1), 167 - 76
Recruitment of ZipA to the septal ring of Escherichia coli is dependent on FtsZ and independent of FtsA; Hale CA et al.; Cell division in prokaryotes is mediated by the septal ring . In Escherichia coli, this organelle consists of several essential division proteins, including FtsZ, FtsA, and ZipA . To gain more insight into how the structure is assembled, we studied the interdependence of FtsZ, FtsA, and ZipA localization using both immunofluorescence and Gfp tagging techniques . To this end, we constructed a set of strains allowing us to determine the cellular location of each of these three proteins in cells from which one of the other two had been specifically depleted . Our results show that ZipA fails to accumulate in a ring shape in the absence of FtsZ . Conversely, depletion of ZipA does not abolish formation of FtsZ rings but leads to a significant reduction in the number of rings per unit of cell mass . In addition, ZipA does not appear to require FtsA for assembly into the septal ring and vice versa . It is suggested that septal ring formation starts by assembly of the FtsZ ring, after which ZipA and FtsA join this structure in a mutually independent fashion through direct interactions with the FtsZ protein.

Nucleic Acids Res, 1999 Jan 15, 27(2), 616 - 23
Nucleotide excision repair affects the stability of long transcribed (CTG*CAG) tracts in an orientation-dependent manner in Escherichia coli; Parniewski P et al.; The influence of nucleotide excision repair (NER), the principal in vivo repair system for DNA damages, was investigated in Escherichia coli with uvrA, uvrB and uvrAuvrB mutants with the triplet repeat sequences (TRS) involved in myotonic dystrophy, the fragile X syndrome and Friedreich's ataxia . (CTG*CAG)175was more stable when the (CTG) strand was transcribed than when the (CAG) strand was transcribed in the alternate orientation . A lack of the UvrA protein dramatically increases the instability of this TRS in vivo as compared with the stability of the same sequence in uvrB mutant, which produces an intact UvrA protein . We propose that transcription transiently dissociates the triplet repeat complementary strands enabling the non-transcribed strand to fold into a hairpin conformation which is then sufficiently stable that replication bypasses the hairpin to give large deletions . If the TRS was not transcribed, fewer deletions were observed . Alternatively, in the uvrA-mutant, the hairpins existing on the lagging strand will suffer bypass DNA synthesis to generate deleted molecules . Hence, NER, functionally similar in both prokaryotes and eukaryotes, is an important factor in the genetic instabilities of long transcribed TRS implicated in human hereditary neuro-logical diseases.

Mol Gen Genet, 1998 Nov, 260(2-3), 289 - 94
The Caenorhabditis elegans RAD51 homolog is transcribed into two alternative mRNAs potentially encoding proteins of different sizes; Rinaldo C et al.; In prokaryotes, the RecA protein plays a pivotal role in homologous recombination, catalyzing the transfer of a single DNA strand into an homologous molecule . Structural homologs of the bacterial RecA protein, called Rad51, have been found in different eukaryotes (from yeast to man), suggesting a certain level of conservation in recombination pathways among living organisms . We have cloned the homolog of RAD51 in Caenorhabditis elegans . The CeRAD51 gene is transcribed into two alternative mRNAs and potentially codes for two proteins of 395 and 357 amino acids in length, respectively . We discuss the evolutionary implications of these findings.

FEBS Lett, 1998 Nov 27, 440(1-2), 172 - 4
Differential inhibition of transcription from sigma70- and sigma32-dependent promoters by rifampicin; Wegrzyn A et al.; Rifampicin is an antibiotic which binds to the beta subunit of prokaryotic RNA polymerases and prevents initiation of transcription . It was found previously that production of heat shock proteins in Escherichia coli cells after a shift from 30 degrees C to 43 degrees C is not completely inhibited by this antibiotic . Here we demonstrate that while activity of a pL-lacZ fusion (pL is a sigma70-dependent promoter) in E . coli cells is strongly inhibited by rifampicin, a p(groE)-lacZ fusion, whose activity is dependent on the sigam32 factor, retains significant residual activity even at relatively high rifampicin concentrations . Differential sensitivity to this antibiotic of RNA polymerase holoenzymes containing either the sigma70 or the sigma32 subunit was confirmed in vitro . Since the effects of an antibiotic that binds to the beta subunit can be modulated by the presence of either the sigma70 or the sigma32 subunit in the holoenzyme, it is tempting to speculate that binding of various sigma factors to the core of RNA polymerase results in different conformations of particular holoenzymes, including changes in the core enzyme.

Proc Natl Acad Sci U S A, 1998 Dec 22, 95(26), 15831 - 6
Origin of a chloroplast protein importer; Bolter B et al.; During evolution, chloroplasts have relinquished the majority of their genes to the nucleus . The products of transferred genes are imported into the organelle with the help of an import machinery that is distributed across the inner and outer plastid membranes . The evolutionary origin of this machinery is puzzling because, in the putative predecessors, the cyanobacteria, the outer two membranes, the plasma membrane, and the lipopolysaccharide layer lack a functionally similar protein import system . A 75-kDa protein-conducting channel in the outer envelope of pea chloroplasts, Toc75, shares approximately 22% amino acid identity to a similarly sized protein, designated SynToc75, encoded in the Synechocystis PCC6803 genome . Here we show that SynToc75 is located in the outer membrane (lipopolysaccharide layer) of Synechocystis PCC6803 and that SynToc75 forms a voltage-gated, high conductance channel with a high affinity for polyamines and peptides in reconstituted liposomes . These findings suggest that a component of the chloroplast protein import system, Toc75, was recruited from a preexisting channel-forming protein of the cyanobacterial outer membrane . Furthermore, the presence of a protein in the chloroplastic outer envelope homologous to a cyanobacterial protein provides support for the prokaryotic nature of this chloroplastic membrane.

Drug Metab Dispos, 1998 Dec, 26(12), 1199 - 201
Lanosterol 14alpha-demethylase (CYP51) and spermatogenesis; Rozman D et al.; CYP51 is the only gene of the cytochrome P450 (P450, or CYP) superfamily that is expressed in prokaryotes and eukaryotes . In animals, the gene product, P45014DM, catalyzes the lanosterol 14alpha-demethylase reaction, an essential step in cholesterol biosynthesis . P45014DM serves a housekeeping role, and it was surprising to find the highest level of CYP51 expression in the testes . This is a result of very high-level CYP51 expression in postmeiotic, haploid spermatids and results in elevated P45014DM activity in these cells . It is proposed that the elevated level of 14alpha-demethylase activity leads to production of signaling sterols by haploid germ cells, although the function of such sterols in males is unknown.

Biochim Biophys Acta, 1998 Nov 10, 1388(2), 465 - 77
In situ characterization of Helicobacter pylori arginase; Mendz GL et al.; The properties of Helicobacter pylori arginase activity in metabolically competent cells and lysates were investigated with the aim of obtaining a better understanding of the nitrogen metabolism of the bacterium . One-dimensional 1H- and 13C-nuclear magnetic resonance spectroscopy, spectrophotometry, radio tracer analysis and protein purification techniques were employed to characterize in situ the first step in the utilization of l-arginine by the bacterium . Arginase activity was associated with the cell-envelope fraction obtained by centrifugation of lysates . A Km of 22+/-3 mM was determined for the enzyme activity, and differences of Vmax were observed between strains . Divalent cations stimulated arginase activity, and the most potent activators were Co2+>Ni2+>Mn2+ . The activity was highly specific for l-arginine and did not catabolize analogs recognized by other arginases of prokaryote and eukaryote origin . The Ki of several inhibitors was measured and served also to characterize the enzyme activity . The presence of bicarbonate enhanced the hydrolysis of l-arginine in cell suspensions, but not in lysates or semi-purified enzyme preparations . Amino acid sequence analyses revealed important differences between the deduced structures of H . pylori arginase and those of other organisms . This finding was consistent with experimental data which showed that H . pylori arginase has unique properties.

Biochim Biophys Acta, 1998 Nov 10, 1388(2), 405 - 18
Lipoamide dehydrogenase from streptomyces seoulensis: biochemical and genetic properties; Youn H et al.; Lipoamide dehydrogenase was purified around 22-fold relative to the crude extracts of Streptomyces seoulensis with an overall yield of 9 . 5% . The enzyme was composed of two identical subunits with a molecular mass of 54 kDa and contained 1 mol of FAD per mol of subunit . The absorption spectra of the enzyme revealed the absorption maxima of flavoprotein at 272, 349, and 457 nm . Catalytically active two-electron reduced lipoamide dehydrogenase was produced by anaerobic reduction with one equivalent of NADH . Addition of excess amount of NADH led to the four-electron reduced lipoamide dehydrogenase . The reaction of the enzyme in the reduction reaction of lipoamide or lipoic acid could be explained by a ping-pong mechanism like many other lipoamide dehydrogenases reported earlier . The enzyme also catalysed the reduction of various quinone compounds with NADH as electron donor via a ping-pong mechanism . The enzyme can catalyse a single electron transfer in case of quinone-reducing process, evidenced by the production of 1, 4-naphthosemiquinone radical anion . The quinone-reducing activity of the enzyme was dramatically inhibited by NAD+, indicating the involvement of four-electron reduced form . The structural gene for the enzyme was cloned using a DNA fragment PCR-amplified with the primers designed from N-terminal and internal amino acid sequences . The deduced amino acid sequence shared striking similarity with those of lipoamide dehydrogenases from prokaryotes and eukaryotes . The gene was named lpd . All tested Streptomyces contained one homologue of the lpd gene, which is consistent with the fact that most organisms contain only one lipoamide dehydrogenase.

Mol Cell Biol, 1999 Jan, 19(1), 471 - 83
The yeast RER2 gene, identified by endoplasmic reticulum protein localization mutations, encodes cis-prenyltransferase, a key enzyme in dolichol synthesis; Sato M et al.; As an approach to understand the molecular mechanisms of endoplasmic reticulum (ER) protein sorting, we have isolated yeast rer mutants that mislocalize a Sec12-Mfalpha1p fusion protein from the ER to later compartments of the secretory pathway (S . Nishikawa and A . Nakano, Proc . Natl . Acad . Sci . USA 90:8179-8183, 1993) . The temperature-sensitive rer2 mutant mislocalizes different types of ER membrane proteins, suggesting that RER2 is involved in correct localization of ER proteins in general . The rer2 mutant shows several other characteristic phenotypes: slow growth, defects in N and O glycosylation, sensitivity to hygromycin B, and abnormal accumulation of membranes, including the ER and the Golgi membranes . RER2 and SRT1, a gene whose overexpression suppresses rer2, encode novel proteins similar to each other, and their double disruption is lethal . RER2 homologues are found not only in eukaryotes but also in many prokaryote species and thus constitute a large gene family which has been well conserved during evolution . Taking a hint from the phenotype of newly established mutants of the Rer2p homologue of Escherichia coli, we discovered that the rer2 mutant is deficient in the activity of cis-prenyltransferase, a key enzyme of dolichol synthesis . This and other lines of evidence let us conclude that members of the RER2 family of genes encode cis-prenyltransferase itself . The difference in phenotypes between the rer2 mutant and previously obtained glycosylation mutants suggests a novel, as-yet-unknown role of dolichol.

Science, 1998 Dec 18, 282(5397), 2220 - 6
Structure of the MscL homolog from Mycobacterium tuberculosis: a gated mechanosensitive ion channel; Chang G et al.; Mechanosensitive ion channels play a critical role in transducing physical stresses at the cell membrane into an electrochemical response . The MscL family of large-conductance mechanosensitive channels is widely distributed among prokaryotes and may participate in the regulation of osmotic pressure changes within the cell . In an effort to better understand the structural basis for the function of these channels, the structure of the MscL homolog from Mycobacterium tuberculosis was determined by x-ray crystallography to 3.5 angstroms resolution . This channel is organized as a homopentamer, with each subunit containing two transmembrane alpha helices and a third cytoplasmic alpha helix . From the extracellular side, a water-filled opening approximately 18 angstroms in diameter leads into a pore lined with hydrophilic residues which narrows at the cytoplasmic side to an occluded hydrophobic apex that may act as the channel gate . This structure may serve as a model for other mechanosensitive channels, as well as the broader class of pentameric ligand-gated ion channels exemplified by the nicotinic acetylcholine receptor.

Arch Virol, 1998, 143(11), 2203 - 14
Chaperone protein GrpE and the GroEL/GroES complex promote the correct folding of tobacco mosaic virus coat protein for ribonucleocapsid assembly in vivo; Hwang DJ et al.; Several prokaryotic chaperone proteins were shown to promote the correct folding and in vivo assembly of tobacco mosaic virus coat protein (TMV CP) using a chimaeric RNA packaging system in control or chaperone-deficient mutant strains of Escherichia coli . Mutations in groEL or dnaK reduced the amount of both total and soluble TMV CP, and the yield of assembled TMV-like particles, several-fold . Thus both GroEL and DnaK have significant direct or indirect effects on the overall expression, stability, folding and assembly of TMV CP in vivo . In contrast, while cells carrying a mutation in grpE expressed TMV CP to a higher overall level than control E . coli, the amounts of both soluble CP and assembled TMV-like particles were below control levels, suggesting a negative effect of GrpE on overall CP accumulation, but positive role(s) in CP folding and assembly . Curiously, cells with mutations in groES and, to a lesser extent, dnaJ expressed total, soluble and assembled forms of TMV CP significantly above control values, suggesting some form of negative control by these chaperone proteins . To avoid pleiotropic effects or artefacts in chaperone-null mutants, selected chaperone proteins were also over-expressed in control E . coli cells . Overproduction of GroEL or GroES alone had little effect . However, co-overexpression of GroEL and GroES resulted in a two-fold increase in soluble TMV CP and a four-fold rise in assembled TMV-like (pseudovirus) particles in vivo . Moreover, TMV CP was shown to interact directly with GroEL in vivo . Together, these results suggest that GrpE and the GroEL/GroES chaperone complex promote the correct folding and assembly of TMV CP into ribonucleocapsids in vivo.

Comp Biochem Physiol B Biochem Mol Biol, 1998 Aug, 120(4), 693 - 700
Coevolution of actin and associated proteins: an alpha-actinin-like protein in a cyanobacterium (Spirulina platensis); Usmanova A et al.; Actin, together with associated proteins, such as myosin, cross-linking or capping proteins, has been observed in all eukaryotic cells . Presence of actin or actin-like proteins has also been reported in prokaryotic organisms belonging to the cyanobacteria . Our aim was first to extend the characterization of an actin-like protein to another prokaryotic cell, i.e . Spirulina, then to compare the antigenic reactivity of this new protein with that of Synechocystis and skeletal actins . We observed that some of the conserved antigenic epitopes corresponded to actin regions known to interact with cross-linking proteins . We also report for the first time that alpha-actinin and filamin purified from chicken gizzard both interact with a prokaryotic actin-like protein . Finally, we searched for the occurrence of a cross-linking protein in these cyanobacteria and identified a 105-kDa protein as an alpha-actinin-like protein using specific antibodies.

Trends Cell Biol, 1998 Nov, 8(11), 454 - 9
SMC proteins and chromosome structure; Strunnikov AV; The structure of chromosomes is largely determined by chromosome-associated proteins . Members of the SMC (structural maintenance of chromosomes) family play an important role in both prokaryotic and eukaryotic chromosome structure and dynamics . SMC proteins are involved in chromosome condensation, sister-chromatid cohesion, sex-chromosome dosage compensation, genetic recombination and DNA repair . There have been major advances recently in understanding the function of SMC proteins--including the identification of biochemical activities of SMC-containing protein complexes and the realization that individual SMC proteins might link seemingly unrelated aspects of chromosomal metabolism.

Gene, 1998 Oct 9, 221(1), 117 - 25
Isolation of an Arabidopsis thaliana cDNA by complementation of a yeast abc1 deletion mutant deficient in complex III respiratory activity; Cardazzo B et al.; The yeast Abc1 protein acts as a chaperone-like protein essential for the proper conformation and efficient functioning of the respiratory complex III . By functional complementation of a yeast abc1 mutant, we have identified an Arabidopsis thaliana cDNA that corresponds to a single copy gene and encodes a protein sharing 45% similarity with the yeast Abc1p protein . Cytochrome spectra and respiratory activity measurements have shown that the plant protein allows a partial restoration of the complex III activity . No major difference in the steady-state level of ABC1At mRNA was observed in various plant tissues, suggesting that ABC1At is constitutively expressed in A . thaliana . Phylogenetic analysis revealed that the Abc1At protein belongs to a large family of proteins composed of two eukaryotic and one prokaryotic subgroups differing by their degree of similarity and probably by their function.

Trends Biochem Sci, 1998 Nov, 23(11), 434 - 8
Contrasting lifestyles of rolling-circle phages and plasmids; Novick RP; The rolling-circle mechanism of DNA replication is used by small prokaryotic genomes, such as single-stranded phages and plasmids . However, phages and plasmids have adapted the rolling-circle mechanism differently to suit their contrasting biological needs . The phi X174 phage uses a monomeric initiator protein catalytically, displays incomplete termination and recycles the initiator protein, in order to mass-produce phage progeny . By contrast, to control replication precisely, the pT181 plasmid uses a dimeric initiator protein stochiometrically, completes termination and inactivates the initiator after each replication cycle . The phi X174 phage and the pT181 plasmid represent paradigmatic adaptations of the rolling-circle mechanism and could provide models for other replicons.

Int J Mol Med, 1998 Jan, 1(1), 147 - 56
Stalling of DNA methyltransferase in chromosome stability and chromosome remodelling (Review); Smith SS; As a consequence of their mechanism of action, DNA (cytosine-5) methyltransferases from both prokaryotes and eukaryotes necessarily recognize mispaired bases in unusual DNA structures as catalytic transition-state analogs . A review of the available data suggests that the enzymes are designed to stall at these sites because they are unable to release substrates or products that are fixed in a conformation resembling the transition state . The enzymes can operate by a two-step process in which they first methylate extrahelical cytosines satisfying their recognition requirements and subsequently stall at the site of methylation . On RNA and DNA RNA hybrids they may operate by a similar one-step process in which they stall at transition-state analogs without methylating cytosine moieties . These natural capacities suggest that the enzymes may physically participate in stable nucleoprotein assemblies formed as components of normal chromatin structure or as intermediates in the repair of unusual structures . The methyltransferases, themselves, may physically participate in chromosome remodelling as part of a mechanism of inactivation or imprinting by stabilizing RNA DNA hybrids or RNA RNA secondary structure involving cis-acting untranslated RNAs like the product of the Xist gene . Methyl-transferase may physically participate in the repair of certain unusual structures by serving as a nucleation point . The affinity for secondary structure in nucleic acids may account for the spreading of DNA methylation patterns . Titration of host methyltransferase by RNA DNA hybrids and RNA secondary structure formed during retroviral replication in certain tumorigenic retroviruses, like MMTV, may account for global hypomethylation observed in retrovirally transformed cells . In a similar fashion, titration of methyltransferase by secondary structures associated with chromosome instability may account for global hypomethylation observed in association with local hypermethylation in tumorigenesis.

Int J Mol Med, 1998 Jan, 1(1), 83 - 90
A 5'-monophosphate form of bredinin selectively inhibits the activities of mammalian DNA polymerases in vitro; Horie T et al.; Bredinin is an immunosuppressive drug which is used clinically in Japan . In this study, we investigated bredinin's molecular mode of action to clarify its immunosuppressive effects . We focused on the DNA polymerases in the somatic DNA synthesis which may be required in the process of lymphocyte differentiation . We found that bredinin-5'-monophosphate (breMP) could be a potent inhibitor of mammalian DNA polymerase alpha(pol.alpha) and (pol.beta) in vitro, although bredinin itself has no such effects . BreMP inhibited the pol . alpha activity at less than 7 micrograms/ml and the pol . activity at 7 micrograms/ml . Neither breMP nor bredinin influenced the activities of a plant DNA polymerase, prokaryotic DNA polymerases such as E . coli DNA polymerase I and Taq DNA polymerase, or DNA-metabolic enzymes such as DNase I, indicating that breMP selectively suppressed the activities of the mammalian DNA polymerases . For pol., beta breMP acted by competing with both the substrate and template-primer . For pol . alpha, it acted by competing only with the substrate, and non-competitively with the template-primer . The ribose of bredinin is quickly and quantitatively converted to its ribose-5'-phosphate form in vivo as soon as it is incorporated into cells . The action mode of bredinin and its use as an immunosuppressive drug are discussed based on these results.

J Biol Chem, 1998 Dec 18, 273(51), 34255 - 62
Escherichia coli DnaA protein . The N-terminal domain and loading of DnaB helicase at the E . coli chromosomal origin; Sutton MD et al.; Initiation of DNA replication at the Escherichia coli chromosomal origin occurs through an ordered series of events that depends first on the binding of DnaA protein, the replication initiator, to DnaA box sequences followed by unwinding of an AT-rich region . A step that follows is the binding of DnaB helicase at oriC so that it is properly positioned at each replication fork . We show that DnaA protein actively mediates the entry of DnaB at oriC . One region (amino acids 111-148) transiently binds to DnaB as determined by surface plasmon resonance . A second functional domain, possibly involving formation of a unique nucleoprotein structure, promotes the stable binding of DnaB during the initiation process and is inactivated in forming an intermediate termed the prepriming complex by removal of the N-terminal 62 residues . Based on similarities in the replication process between prokaryotes and eukaryotes, these results suggest that a similar mechanism may load the eukaryotic replicative helicase.

J Biol Chem, 1998 Dec 18, 273(51), 34247 - 54
1.85-A resolution crystal structure of human ornithine transcarbamoylase complexed with N-phosphonacetyl-L-ornithine . Catalytic mechanism and correlation with inherited deficiency; Shi D et al.; The crystal structure of human ornithine transcarbamoylase complexed with the bisubstrate analog N-phosphonacetyl-L-ornithine has been solved at 1.85-A resolution by molecular replacement . Deleterious mutations produce clinical hyperammonia that, if untreated, results in neurological symptoms or death (ornithine transcarbamylase deficiency) . The holoenzyme is trimeric, and as in other transcarbamoylases, each subunit contains an N-terminal domain that binds carbamoyl phosphate and a C-terminal domain that binds L-ornithine . The active site is located in the cleft between domains and contains additional residues from an adjacent subunit . Binding of N-phosphonacetyl-L-ornithine promotes domain closure . The resolution of the structure enables the role of active site residues in the catalytic mechanism to be critically examined . The side chain of Cys-303 is positioned so as to be able to interact with the delta-amino group of L-ornithine which attacks the carbonyl carbon of carbamoyl phosphate in the enzyme-catalyzed reaction . This sulfhydryl group forms a charge relay system with Asp-263 and the alpha-amino group of L-ornithine, instead of with His-302 and Glu-310, as previously proposed . In common with other ureotelic ornithine transcarbamoylases, the human enzyme lacks a loop of approximately 20 residues between helix H10 and beta-strand B10 which is present in prokaryotic ornithine transcarbamoylases but has a C-terminal extension of 10 residues that interacts with the body of the protein but is exposed . The sequence of this C-terminal extension is homologous to an interhelical loop found in several membrane proteins, including mitochondrial transport proteins, suggesting a possible mode of interaction with the inner mitochondrial membrane.

J Bacteriol, 1998 Dec, 180(24), 6776 - 9
Sucrose-phosphate synthase from Synechocystis sp . strain PCC 6803: identification of the spsA gene and characterization of the enzyme expressed in Escherichia coli; Curatti L et al.; The first identification and characterization of a prokaryotic gene (spsA) encoding sucrose-phosphate synthase (SPS) is reported for Synechocystis sp . strain PCC 6803, a unicellular non-nitrogen-fixing cyanobacterium . Comparisons of the deduced amino acid sequence and some relevant biochemical properties of the enzyme with those of plant SPSs revealed important differences in the N-terminal and UDP-glucose binding site regions, substrate specificities, molecular masses, subunit compositions, and regulatory properties.

Blood Cells Mol Dis, 1998 Dec, 24(4), 401 - 11
Expression, purification, and characterization of a recombinant erythroid-specific hexokinase isozyme; Bianchi M et al.; Hexokinase type I (HK I; ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1), the predominant glucose-phosphorylating enzyme in red blood cells, exists in human erythrocytes in multiple molecular forms that differ in isoelectric point and are separable by ion-exchange chromatography . The major forms, designated HK Ia, Ib and Ic, have similar kinetic properties but are characterized by different age-dependent decay and different intracellular distribution in reticulocytes . HK Ib, which elutes between HK I and HK II in the DEAE ion-exchange chromatography, appears to be unique to RBCs and different from any other hexokinase isozyme previously described . Indeed, Murakami and Piomelli recently reported the presence of a specific HK isozyme (named HKr) expressed in K562 cells and in human reticulocytes and, moreover, the resolution of the human HK I gene structure provided the direct evidence of an erythroid-specific exon 1 . To further investigate the microheterogeneity of HK I in human RBCs we established a prokaryotic expression system for the HKr isozyme, using the pET plasmid, inducible with IPTG . The recombinant HKr, expressed in bacterial cells as a catalytically active enzyme, was purified to homogeneity by a combination of DEAE ionexchange chromatography followed by hydrophobic interaction chromatography and dye-ligand affinity chromatography . The kinetic and chromatographic properties of the homogeneous recombinant HKr suggest that this erythroid-specific HK isozyme in fact corresponds to the HK isoform previously described in human RBCs and referred to as HK Ib .

Mol Biochem Parasitol, 1998 Oct 30, 96(1-2), 151 - 65
Protein synthesis in Giardia lamblia may involve interaction between a downstream box (DB) in mRNA and an anti-DB in the 16S-like ribosomal RNA; Yu DC et al.; Giardia lamblia, a parasitic protozoan, has been regarded as one of the most conserved eukaryotes evolved from the prokaryotes . One of its unique features appears to be the unusually short 5'-untranslated regions (UTR) (1-6 nucleotides (nts)) and the apparent absence of 5'-cap structures from its mRNAs . Transfection of the Giardia trophozoites with luciferase-encoding chimeric transcripts, flanked by the 5'- and 3'-ends of giardiavirus (GLV) (+)-strand RNA, indicated that the translational efficiency was enhanced by 5000-fold when the 5'-viral sequence extended 264 nts into the capsid coding region and fused with the luciferase open reading frame (ORF) . A 13-nt downstream box (DB) was identified within this region which complements a 15-nt sequence between nts # 1382 and 1396 near the 3'-end of the Giardia 16S-like ribosomal RNA (the anti-DB) . Deletion or scrambling of this DB in the mRNA leads to a significant loss of the translational efficiency in Giardia . A Shine-Dalgarno (SD)-like element was also identified at 9-14 nts upstream from the initiation codon in the viral (+)-strand RNA, but alteration of its sequence led to no change in translation . Using the sequence complementary to ribosomal anti-DB to probe the Giardia mRNAs available in the databases, each mRNA was found to contain a putative DB with an average length from 8 to 13 nts . It is thus possible that initiation of translation in Giardia may involve a DB in the coding region of mRNA that may bind to a putative anti-DB in the small ribosomal RNA through base pairing . This mechanism of ribosome recruitment, which finds a potential parallel in Escherichia coli, could illustrate a relatively close distance between Giardia and prokaryotes in terms of translation initiation, and may provide a model for studying the evolution of translation machinery.

Proteins, 1998, Suppl 2, 74 - 89
Mass spectrometric identification and microcharacterization of proteins from electrophoretic gels: strategies and applications; Jensen ON et al.; The entire genomic DNA sequences of a number of prokaryotic and eukaryotic species are now available and many more, including the human genome, will be completed in the near future . The state-of-life of a cell at any given time, however, is defined by its protein composition, i.e., its proteome . Gel electrophoresis, mass spectrometry, and bioinformatics will be important tools for protein and proteome analysis in the post-genome era . Protein identification from electrophoretic gels by mass spectrometric peptide mapping or peptide sequencing combined with sequence database searching is established and has been applied to numerous biological systems . We describe current strategies and selected applications in molecular and cell biology . The next challenges are detailed structure/function analyses, which include studying the molecular composition of multiprotein complexes and characterization of secondary modifications of proteins . The advantages and limitations of a number of mass spectrometry-based strategies designed for microcharacterization of low amounts of protein from electrophoretic gels are discussed and illustrated by examples.

RNA, 1998 Dec, 4(12), 1653 - 63
Neomycin B inhibits splicing of the td intron indirectly by interfering with translation and enhances missplicing in vivo; Waldsich C et al.; The aminoglycoside antibiotic neomycin B inhibits translation in prokaryotes and interferes with RNA-protein interactions in HIV both in vivo and in vitro . Hitherto, inhibition of ribozyme catalysis has only been observed in vitro . We therefore monitored the activity of neomycin B and several other aminoglycoside antibiotics on splicing of the T4 phage thymidylate synthase (td) intron in vivo . All antibiotics tested inhibited splicing, even chloramphenicol, which does not inhibit splicing in vitro . Splicing of the td intron in vivo requires translation for proper folding of the pre-mRNA . In the absence of translation, two interactions between sequences in the upstream exon and the 5' and 3' splice sites trap the pre-mRNA in splicing-incompetent conformations . Their disruption by mutations rendered splicing less dependent on translation and also less sensitive to neomycin B . Intron splicing was affected by neither neomycin B nor gentamicin in Escherichia coli strains carrying antibiotic-resistance genes that modify the ribosomal RNA . Taken together, this demonstrates that in vivo splicing of td intron is not directly inhibited by aminoglycosides, but rather indirectly by their interference with translation . This was further confirmed by assaying splicing of the Tetrahymena group I intron, which is inserted in the E . coli 23 S rRNA and, thus, not translated . Furthermore, neomycin B, paromomycin, and streptomycin enhanced missplicing in antibiotic-sensitive strains . Missplicing is caused by an alternative structural element containing a cryptic 5' splice site, which serves as a substrate for the ribozyme . Our results demonstrate that aminoglycoside antibiotics display different effects on ribozymes in vivo and in vitro.

RNA, 1998 Dec, 4(12), 1549 - 68
Cbf5p, a potential pseudouridine synthase, and Nhp2p, a putative RNA-binding protein, are present together with Gar1p in all H BOX/ACA-motif snoRNPs and constitute a common bipartite structure; Watkins NJ et al.; The eukaryotic nucleolus contains a large number of small nucleolar RNAs (snoRNAs) that are involved in preribosomal RNA (pre-rRNA) processing . The H box/ACA-motif (H/ACA) class of snoRNAs has recently been demonstrated to function as guide RNAs targeting specific uridines in the pre-rRNA for pseudouridine (psi) synthesis . To characterize the protein components of this class of snoRNPs, we have purified the snR42 and snR30 snoRNP complexes by anti-m3G-immunoaffinity and Mono-Q chromatography of Saccharomyces cerevisiae extracts . Sequence analysis of the individual polypeptides demonstrated that the three proteins Gar1p, Nhp2p, and Cbf5p are common to both the snR30 and snR42 complexes . Nhp2p is a highly basic protein that belongs to a family of putative RNA-binding proteins . Cbf5p has recently been demonstrated to be involved in ribosome biogenesis and also shows striking homology with known prokaryotic psi synthases . The presence of Cbf5p, a putative psi synthase in each H/ACA snoRNP suggests that this class of RNPs functions as individual modification enzymes . Immunoprecipitation studies using either anti-Cbf5p antibodies or a hemagglutinin-tagged Nhp2p demonstrated that both proteins are associated with all H/ACA-motif snoRNPs . In vivo depletion of Nhp2p results in a reduction in the steady-state levels of all H/ACA snoRNAs . Electron microscopy of purified snR42 and snR30 particles revealed that these two snoRNPs possess a similar bipartite structure that we propose to be a major structural determining principle for all H/ACA snoRNPs.

Nucleic Acids Res, 1999 Jan 1, 27(1), 293 - 4
TransTerm, the translational signal database, extended to include full coding sequences and untranslated regions; Dalphin ME et al.; TransTerm is a database of mRNA sequences and parameters useful for detecting translational control signals in general . TransTerm-98 has been expanded beyond previous years to include full coding sequences and UTRs, while retaining the original small contexts about the coding sequence start- and stop-codons . The database contains more than 130 000 non-redundant coding sequences with associated untranslated regions (UTRs) from over 450 species . This includes the complete genomes of 12 prokaryotic and one eukaryotic organism . Several coding sequence parameters are available: coding sequence length, Nc, GC3 and, when it is computable, Codon Adaptation Index (CAI) . Codon usage tables and summaries of start- and stop-codon contexts are also included . TransTerm-98 has both a relational database form with a WWW interface and a flatfile format, also available by Internet browser . TransTerm is available at: tml

Microbiology, 1998 Nov, 144 ( Pt 11), 3229 - 37
Malate synthase from Streptomyces clavuligerus NRRL3585: cloning, molecular characterization and its control by acetate; Chan M et al.; Malate synthase is a key enzyme of the glyoxylate cycle, which is an anaplerotic pathway essential for growth on acetate as the sole carbon source . The aceB gene, encoding malate synthase from Streptomyces clavuligerus NRRL 3585, was cloned using PCR and fully sequenced . The ORF obtained encodes 541 amino acids with a deduced Mr of 60000, consistent with the observed Mr (62000-64000) of most malate synthase enzymes reported so far . The aceB gene has a high G+C content (71.5 mol%), especially in the third codon position . A 50 bp region upstream of the malate synthase ORF was predicted to be a prokaryotic promoter region . The relationship between carbon source, antibiotic (cephalosporin) biosynthesis and malate synthase activity was investigated . Growth of S . clavuligerus on acetate as the major carbon source was delayed, compared to that on glycerol . Furthermore, high levels of malate synthase activity were associated with the presence of acetate in the growth medium . Growth on acetate also resulted in lower levels of cephalosporin production, compared to that on glycerol . The cloned S . clavuligerus aceB gene was expressed in Escherichia coli BL21(DE3) . Transformants exhibited an approximately 71-fold increase in malate synthase activity, compared to the control, thereby demonstrating high-level expression of soluble and enzymically active malate synthase in the heterologous host.

Kidney Int, 1998 Nov, 54(5), 1444 - 54
Molecular cloning of KS, a novel rat gene expressed exclusively in the kidney; Hilgers KF et al.; BACKGROUND: We aimed to identify genes with kidney specific, developmentally regulated expression . Here we report the cDNA sequence and expression pattern of KS, a novel kidney-specific rat gene . METHODS: A partial cDNA was identified by differential display polymerase chain reaction (PCR) of a renal cell fraction enriched for proximal tubular and renin-expressing cells . Using the partial cDNA as a probe, a rat kidney cDNA library was screened . The full-length KS sequence was obtained by PCR amplification of cDNA ends . The expression pattern of KS was investigated by Northern blot . RNA was extracted from several organs of newborn and adult rats, as well as from the kidneys of rats with altered tubular function, that is, rats that had undergone unilateral nephrectomy, unilateral ureteral obstruction, neonatal losartan treatment, and the appropriate control animals . The expression of KS was also investigated in the kidneys of rats with spontaneous or renovascular hypertension . RESULTS: The KS cDNA (2426 bp) contained one open reading frame encoding a predicted 572 amino acid protein . The derived peptide sequence displayed approximately 70% similarity to the hypertension-related SA gene product and approximately 50% similarity to prokaryotic and eukaryotic acetyl-CoA synthases (EC 6 . 2.1.1) . KS was expressed in the kidney and not in any other organ assayed . KS RNA was not detected in fetal and newborn rat kidney but became apparent after one week of postnatal life . Gene expression was downregulated in rat models of altered tubular function . KS expression was decreased in spontaneously hypertensive rats but not in renovascular hypertension . CONCLUSION: KS, a novel rat gene, exhibits a unique tissue-specific expression exclusively in mature kidneys . The data suggest KS may encode an adenosine monophosphate binding enzyme.

EMBO J, 1998 Dec 1, 17(23), 6952 - 62
The yeast histidine protein kinase, Sln1p, mediates phosphotransfer to two response regulators, Ssk1p and Skn7p; Li S et al.; The Saccharomyces cerevisiae Sln1 protein is a 'two-component' regulator involved in osmotolerance . Two-component regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes . Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p . SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p . We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway . Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p . The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427 . In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427 . The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p . The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.

Bioessays, 1998 Aug, 20(8), 652 - 9
Transposons in filamentous fungi--facts and perspectives; Kempken F et al.; Transposons are ubiquitous genetic elements discovered so far in all investigated prokaryotes and eukaryotes . In remarkable contrast to all other genes, transposable elements are able to move to new locations within their host genomes . Transposition of transposons into coding sequences and their initiation of chromosome rearrangements have tremendous impact on gene expression and genome evolution . While transposons have long been known in bacteria, plants, and animals, only in recent years has there been a significant increase in the number of transposable elements discovered in filamentous fungi . Like those of other eukaryotes, each fungal transposable element is either of class or of class II . While class I elements transpose by a RNA intermediate and employ reverse transcriptases, class II elements transpose directly at the DNA level . We present structural and functional features for such transposons that have been identified so far in filamentous fungi . Emphasis is given to specific advantages or unique features when fungal systems are used to study transposable elements, e.g., the evolutionary impact of transposons in coenocytic organisms and possible experimental approaches toward horizontal gene transfer . Finally, we focus on the potential of transposons for tagging and identifying fungal genes.

Am J Hum Genet, 1998 Dec, 63(6), 1598 - 608
The first nuclear-encoded complex I mutation in a patient with Leigh syndrome; Loeffen J et al.; Nicotinamide adenine dinucleotide (NADH):ubiquinone oxidoreductase (complex I) is the largest multiprotein enzyme complex of the respiratory chain . The nuclear-encoded NDUFS8 (TYKY) subunit of complex I is highly conserved among eukaryotes and prokaryotes and contains two 4Fe4S ferredoxin consensus patterns, which have long been thought to provide the binding site for the iron-sulfur cluster N-2 . The NDUFS8 cDNA contains an open reading frame of 633 bp, coding for 210 amino acids . Cycle sequencing of amplified NDUFS8 cDNA of 20 patients with isolated enzymatic complex I deficiency revealed two compound heterozygous transitions in a patient with neuropathologically proven Leigh syndrome . The first mutation was a C236T (P79L), and the second mutation was a G305A (R102H) . Both mutations were absent in 70 control alleles and cosegregated within the family . A progressive clinical phenotype proceeding to death in the first months of life was expressed in the patient . In the 19 other patients with enzymatic complex I deficiency, no mutations were found in the NDUFS8 cDNA . This article describes the first molecular genetic link between a nuclear-encoded subunit of complex I and Leigh syndrome.

Microbiol Mol Biol Rev, 1998 Dec, 62(4), 1492 - 553
Posttranscriptional control of gene expression in yeast; McCarthy JE; Studies of the budding yeast Saccharomyces cerevisiae have greatly advanced our understanding of the posttranscriptional steps of eukaryotic gene expression . Given the wide range of experimental tools applicable to S . cerevisiae and the recent determination of its complete genomic sequence, many of the key challenges of the posttranscriptional control field can be tackled particularly effectively by using this organism . This article reviews the current knowledge of the cellular components and mechanisms related to translation and mRNA decay, with the emphasis on the molecular basis for rate control and gene regulation . Recent progress in characterizing translation factors and their protein-protein and RNA-protein interactions has been rapid . Against the background of a growing body of structural information, the review discusses the thermodynamic and kinetic principles that govern the translation process . As in prokaryotic systems, translational initiation is a key point of control . Modulation of the activities of translational initiation factors imposes global regulation in the cell, while structural features of particular 5' untranslated regions, such as upstream open reading frames and effector binding sites, allow for gene-specific regulation . Recent data have revealed many new details of the molecular mechanisms involved while providing insight into the functional overlaps and molecular networking that are apparently a key feature of evolving cellular systems . An overall picture of the mechanisms governing mRNA decay has only very recently begun to develop . The latest work has revealed new information about the mRNA decay pathways, the components of the mRNA degradation machinery, and the way in which these might relate to the translation apparatus . Overall, major challenges still to be addressed include the task of relating principles of posttranscriptional control to cellular compartmentalization and polysome structure and the role of molecular channelling in these highly complex expression systems.

Microbiol Mol Biol Rev, 1998 Dec, 62(4), 1353 - 70
A natural view of microbial biodiversity within hot spring cyanobacterial mat communities; Ward DM et al.; This review summarizes a decade of research in which we have used molecular methods, in conjunction with more traditional approaches, to study hot spring cyanobacterial mats as models for understanding principles of microbial community ecology . Molecular methods reveal that the composition of these communities is grossly oversimplified by microscopic and cultivation methods . For example, none of 31 unique 16S rRNA sequences detected in the Octopus Spring mat, Yellowstone National Park, matches that of any prokaryote previously cultivated from geothermal systems; 11 are contributed by genetically diverse cyanobacteria, even though a single cyanobacterial species was suspected based on morphologic and culture analysis . By studying the basis for the incongruity between culture and molecular samplings of community composition, we are beginning to cultivate isolates whose 16S rRNA sequences are readily detected . By placing the genetic diversity detected in context with the well-defined natural environmental gradients typical of hot spring mat systems, the relationship between gene and species diversity is clarified and ecological patterns of species occurrence emerge . By combining these ecological patterns with the evolutionary patterns inherently revealed by phylogenetic analysis of gene sequence data, we find that it may be possible to understand microbial biodiversity within these systems by using principles similar to those developed by evolutionary ecologists to understand biodiversity of larger species . We hope that such an approach guides microbial ecologists to a more realistic and predictive understanding of microbial species occurrence and responsiveness in both natural and disturbed habitats.

Pharmacol Ther, 1998 Nov, 80(2), 183 - 201
Heat shock protein 70 kDa: molecular biology, biochemistry, and physiology; Kiang JG et al.; Heat shock proteins (HSPs) are detected in all cells, prokaryotic and eukaryotic . In vivo and in vitro studies have shown that various stressors transiently increase production of HSPs as protection against harmful insults . Increased levels of HSPs occur after environmental stresses, infection, normal physiological processes, and gene transfer . Although the mechanisms by which HSPs protect cells are not clearly understood, their expression can be modulated by cell signal transducers, such as changes in intracellular pH, cyclic AMP, Ca2+, Na+, inositol trisphosphate, protein kinase C, and protein phosphatases . Most of the HSPs interact with other proteins in cells and alter their function . These and other protein-protein interactions may mediate the little understood effects of HSPs on various cell functions . In this review, we focus on the structure of the HSP-70 family (HSP-70s), regulation of HSP-70 gene expression, their cytoprotective effects, and the possibility of regulating HSP-70 expression through modulation of signal transduction pathways . The clinical importance and therapeutic potential of HSPs are discussed.

J Biol Chem, 1998 Dec 11, 273(50), 33305 - 10
GroEL traps dimeric and monomeric unfolding intermediates of citrate synthase; Grallert H et al.; The prokaryotic molecular chaperone GroE is increasingly expressed under heat shock conditions . GroE protects cells by preventing the irreversible aggregation of thermally unfolding proteins . Here, the interaction of GroE with thermally unfolding citrate synthase (CS) was dissected into several steps that occur before irreversible aggregation, and the conformational states of the unfolding protein recognized by GroEL were determined . The kinetic analysis of CS unfolding revealed the formation of inactive dimeric and monomeric intermediates . GroEL binds both intermediates without affecting the unfolding pathway . Furthermore, the dimeric intermediates are not protected against dissociation in the presence of GroEL . Monomeric CS is stably associated with GroEL, thus preventing further irreversible unfolding steps and subsequent aggregation . During refolding, monomeric CS is encapsulated inside the cavity of GroEL . GroES complexes . Taken together our results suggest that for protection of cells against heat stress both the ability of GroEL to interact with a large variety of nonnative conformations of proteins and the active, GroES-dependent refolding of highly unfolded species are important.

Virology, 1998 Nov 25, 251(2), 393 - 401
Expression of a uracil DNA glycosylase (UNG) inhibitor in mammalian cells: varicella-zoster virus can replicate in vitro in the absence of detectable UNG activity; Reddy SM et al.; Uracil DNA glycosylase (UNG) functions as a DNA repair or proofreading enzyme . The UNG gene is present in nearly all prokaryotes and eukaryotes screened to date and is found in herpesviruses and poxviruses . Prior studies showed that viral UNG is essential for poxvirus replication . Although viral UNG is not required for herpesvirus replication, cellular UNG was thought to be essential for virus replication . To study the role of UNG in herpesvirus replication, we first showed that varicella-zoster virus (VZV) ORF59 encodes a functional UNG . We then constructed a VZV mutant with a deletion in the UNG gene and showed that the mutant was unimpaired for replication in vitro . Because cultured cells express their own endogenous UNG, we next inserted a bacteriophage UNG inhibitor UGI gene into the VZV genome . Infection of cells with VZV lacking viral UNG and expressing UGI completely abrogated detectable cellular UNG activity in vitro . Parental VZV, VZV lacking viral UNG, and VZV expressing UGI all grew to similar titers in cell culture, indicating that VZV can replicate in vitro in the absence of detectable viral or cellular UNG activity .

Virology, 1998 Nov 25, 251(2), 370 - 82
Genetic analysis of the internal ribosome entry segment of bovine viral diarrhea virus; Chon SK et al.; Pestiviruses and hepaciviruses are atypical members of the Flaviviridae due to their unique biological properties, including the utilization of internal ribosome entry for translation initiation . In contrast to internal initiation in picornaviruses, which depends on numerous canonical initiation factors, the mode of internal ribosome entry in pestiviruses and hepaciviruses resembles prokaryotic translation initiation . To identify functionally important elements within the bovine viral diarrhea virus (BVDV) internal ribosome entry segment (IRES), we carried out a mutational analysis of the 5' untranslated region (5' UTR) of BVDV cloned in the intercistronic region of a bicistronic reporter plasmid . IRES function was assessed in a bicistronic transcript by inserting the 5' 901 nucleotides of BVDV genome, which correspond to the 385 nucleotides of the 5' UTR and 515 nucleotides of the open reading frame (ORF) encoding for Npro and 4 amino acids from the capsid protein . The resulting Npro-luciferase fusion encoded by the 3' cistron was cleaved efficiently to release the luciferase reporter . In vivo translation analyses showed that stem-loops Ia and Ib in the 5' UTR were completely dispensable for efficient translation, whereas stem-loop IIIe and the hairpin end of IIIb were only partially required . In contrast, deletions or insertions in any of other four stem-loop structures, including domains II, IIIa, IIIc, and IIId, caused nearly 10-fold reductions of in vivo IRES activity . The tolerance of structural modifications within the distal portion of domain IIIb and IIIe correlated with a low level of sequence conservation in these regions among pestiviruses . The 5' boundary of the IRES resides at the 5' end of stem-loop II near nucleotide 75 . The 3' of the IRES extends into the 5' end of the polyprotein ORF because removal of the Npro coding region reduced translation by 21% .

Plant Cell, 1998 Dec, 10(12), 1991 - 2004
Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ; Osteryoung KW et al.; The division of plastids is critical for viability in photosynthetic eukaryotes, but the mechanisms associated with this process are still poorly understood . We previously identified a nuclear gene from Arabidopsis encoding a chloroplast-localized homolog of the bacterial cell division protein FtsZ, an essential cytoskeletal component of the prokaryotic cell division apparatus . Here, we report the identification of a second nuclear-encoded FtsZ-type protein from Arabidopsis that does not contain a chloroplast targeting sequence or other obvious sorting signals and is not imported into isolated chloroplasts, which strongly suggests that it is localized in the cytosol . We further demonstrate using antisense technology that inhibiting expression of either Arabidopsis FtsZ gene (AtFtsZ1-1 or AtFtsZ2-1) in transgenic plants reduces the number of chloroplasts in mature leaf cells from 100 to one, indicating that both genes are essential for division of higher plant chloroplasts but that each plays a distinct role in the process . Analysis of currently available plant FtsZ sequences further suggests that two functionally divergent FtsZ gene families encoding differentially localized products participate in chloroplast division . Our results provide evidence that both chloroplastic and cytosolic forms of FtsZ are involved in chloroplast division in higher plants and imply that important differences exist between chloroplasts and prokaryotes with regard to the roles played by FtsZ proteins in the division process.

FEMS Microbiol Lett, 1998 Nov 15, 168(2), 187 - 94
Transcript analysis of the pcbABC genes encoding the antenna apoproteins in the photosynthetic prokaryote, Prochlorothrix hollandica; Nikolaitchik OA et al.; The tightly linked pcbABC genes encode the chlorophyll a/b-binding apoproteins in the oxygenic photosynthetic prokaryote Prochlorothrix hollandica . Northern blotting experiments employing gene-specific DNA probes have identified a complex pattern of transcription from the pcb region . A large 4.4-kb transcript detected in cultures maintained in high light, low light and in darkness results from the cotranscription of all three genes, whereas pcbAB, pcbBC and individual pcbA, B, and C mRNAs are similarly detected in all light regimes . The half lives of the RNAs vary from 15 min for the pcbABC transcript, to over 60 min for the pcbA and pcbC mRNAs . The lack of identifiable promoter sequences other than the region upstream from pcbA, plus the enhanced stability of the individual single gene transcripts, suggest that the smaller RNA species arise from processing of larger transcripts . Transcription and mRNA turnover occurs largely independent of light intensity, in contrast to what is seen in most other phototrophs, in which light influences the accumulation of antenna apoprotein gene mRNAs.

J Endocrinol, 1998 Dec, 159(3), 509 - 18
Cloning, preparation and characterization of biologically active recombinant caprine placental lactogen; Sakal E et al.; Caprine placental lactogen (cPL) cDNA was cloned by reverse transcription (RT)-PCR from total RNA of goat placenta . The PCR product encoding for the mature protein was gel purified, ligated to pGEM-T and finally subcloned into a pET8c prokaryotic expression vector . E . coli cells (BL-21) transformed with this vector overexpressed large amounts of cPL upon induction with Isopropyl-1-thio-beta-D-galactopyranoside . The expressed protein, found in the inclusion bodies, was refolded and purified to homogeneity on Q-Sepharose and SP-Sepharose columns, yielding two electrophoretically pure fractions (cPL-Q and cPL-S), composed of over 98% of monomeric protein of the expected molecular mass of approximately 23 kDa . Binding of cPL to the extracellular domain (ECD) of prolactin receptors (PRLR) from rat (r), rabbit (rb), and bovine (b), growth hormone receptors (GHR) from human (h) and rabbit, and binding to rabbit mammary gland membranes revealed similar binding profiles for cPL-Q, cPL-S and ovine (o)PL . Caprine PL was capable of forming 1:2 complexes with hGHR-ECD, rbGHR-ECD, rPRLR-ECD and rbPRLR-ECD whereas with bPRLR-ECD only a 1:1 complex was detected . The biological activity of both cPL fractions resulting from proper renaturation was further evidenced by their ability to stimulate proliferation of Nb2 cells, FDC-P1 cells transfected with rabbit or human GHRs and by stimulation of beta-casein synthesis in rabbit and ovine mammary gland acini cultures.

J Biol Chem, 1998 Dec 4, 273(49), 32857 - 63
Characterization of the syringomycin synthetase gene cluster . A link between prokaryotic and eukaryotic peptide synthetases; Guenzi E et al.; With this work we have completed the characterization of the syringomycin synthetase gene cluster . In particular, by sequencing additional 28.5 kilobase pairs we show that the nine modules involved in the binding of the nine amino acids of syringomycin are localized on SyrB and SyrE, with SyrE carrying eight modules . The recombinant SyrB and the first and second modules of SyrE (SyrE1 and SyrE2) have been expressed in Escherichia coli and purified . The biochemical data indicate that SyrB binds threonine, the putative precursor of the last amino acid of syringomycin, whereas SyrE1 and SyrE2 bind serine, the first and the second amino acids of syringomycin, respectively . On the basis of the sequence analysis and the biochemical data presented here, it appears that syringomycin synthetase is unique among peptide synthetases in that its genetic organization does not respect the "colinearity rule" according to which the order of the amino acid binding modules along the chromosome parallels the order of the amino acids on the peptide . This feature, together with the absence of a single transcription unit and the absence of epimerase-like domains make syringomycin synthetase more related to the eukaryotic peptide synthetases than to the bacterial counterparts.

Mol Gen Genet, 1998 Oct, 260(1), 20 - 9
The Nop60B gene of Drosophila encodes an essential nucleolar protein that functions in yeast; Phillips B et al.; The Cbf5 protein of Saccharomyces cerevisiae was originally identified as a low-affinity centromeric DNA-binding protein, and chf5 mutants have a defect in rRNA synthesis . A closely related protein from mammals, NAP57, is a nucleolar protein that coimmunoprecipitates with the nucleolar phosphoprotein Nopp140 . To study the function of this protein family in a higher eukaryote that is amenable to genetic approaches, the gene encoding a Drosophila melanogaster homolog, Nop60B, was identified . The predicted Drosophila protein shares a high degree of sequence identity over a 380-residue region with both the mammalian and yeast proteins, and shares several conserved motifs with the prokaryotic tRNA pseudouridine 55 synthases . Nop60B RNA is found at high levels in nurse cells and in the oocyte, and is present throughout development . Nop60B protein is localized primarily to the nucleolus of interphase cells, and is absent from the chromosomes during mitosis . Nop60B mutants were generated and shown to be homozygous lethal . The Drosophila gene can rescue the lethal phenotype of yeast chf5 mutations, showing that the function of this protein has been conserved from yeast to Drosophila.

Biosens Bioelectron, 1998 Sep 15, 13(6), 637 - 50
Dissecting the molecular details of prokaryotic transcriptional control by surface plasmon resonance: the methionine and arginine repressor proteins; Stockley PG et al.; The commercial surface plasmon resonance (SPR) biosensors, BIACORE, have been used to investigate the molecular details of macromolecular interactions at prokaryotic promoter-operators . For the Escherichia coli methionine repressor, MetJ, we have quantitated the interaction of the protein with synthetic and natural operator sites and shown that the SPR response is directly related to the stoichiometry of the complexes being formed . The utility of a continuous flow system has also been exploited to investigate transcription from an immobilised promoter-operator fragment; with transcripts collected and subsequently characterised by RT-PCR . This technique has enabled us to investigate how repressor binding affects (i) the interaction of the RNA polymerase (RNAP) with the promoter and (ii) the ability of RNAP to initiate transcription . Remarkably, the repression complex appears to stabilise binding of RNAP, whilst having the expected effects on the levels of transcripts produced . This may well be a general mechanism allowing rapid transcription initiation to occur as soon as the repression complex dissociates . These techniques have also been used to examine protein-DNA interactions in the E . coli and Bacillus subtilis arginine repressor systems . The repressors are the products of the argR and ahrC genes, respectively . Both proteins form hexamers in rapid equilibrium with smaller subunits believed to be trimers . There are three types of operator in these systems, autoregulatory, biosynthetic and catabolic (B . subtilis only) . Sensorgrams show that each protein recognises the three types of immobilised operator differently and that binding is stimulated over 100-fold by the presence of L-arginine.

Mol Cell Biochem, 1998 Nov, 188(1-2), 57 - 62
Genes regulating copper metabolism; Harris ED et al.; The metabolism of Cu is intimately linked with its nutrition . From gut to enzymes, Cu bioavailability to key enzymes and other components operates through a complex mechanism that uses transport proteins as well as small molecular weight ligands . Steps in Cu transport through the blood, absorption by cells, and incorporation into enzymes are slowly being understood . Cloning and sequencing of the genes for Menkes disease and Wilson disease has shown that membrane-bound enzymes analogous to Cu-ATPases in prokaryotes are equally important to Cu transport and homeostasis in mammalian cells . The primary structure of the mammalian Cu-ATPases has been deduced from cDNAs from tissues and organs . It now appears that mammalian Cu-ATPase have tissue and developmental specificity . In this review, we will focus on the Cu-ATPase that has been identified with Menkes disease . An emphasis will be placed on the existence of multiple forms of the ATPase and some indication as to how the different isoforms befit their role in the normal physiology of copper, specifically transmembrane transport and maintenance of a favorable internal cellular environment.

J Theor Biol, 1998 Nov 21, 195(2), 167 - 86
On the relationship between genomic regulatory element organization and gene regulatory dynamics; Wolf DM et al.; In this project we study the relationship between genomic regulatory element organization and gene regulatory dynamics . This paper illustrates an approach to investigating this relationship based on the application of classical nonlinear system analysis techniques to a transcription level, statistical thermodynamical model like that used in Shea & Ackers (1985) . Preliminary ideas presented at the ICMCM conference (Wolf & Eeckman, 1998) are developed in this manuscript . We show that, for prokaryotic gene circuits dominated by local promoter control, dynamical system behavior descriptors like the number and stability of equilibrium point steady states and their bifurcation potential can be largely determined from genomic organization (e.g . the number, type, and placement of regulatory protein binding sites) . Concepts are illustrated on hypothetical gene regulation systems with one or two genes and varying numbers of regulatory protein binding sites (operators) . Gene regulatory systems with a single gene and an arbitrary number of operator sites are shown to be globally stable, with the potential for having multiple equilibrium points and capable of bifurcating . A monomer-controlled gene regulation system with n operator sites is proven to have a maximum of 1+n/2 stable equilibria for even n, and (n+1)/2 for odd n, while a multimer-controlled, n operator site system is shown to have a maximum of 2+n/2 stable equilibria for even n, and (n+3)/2 for odd n . These results are applied to the design of a two-state switch using a gene regulation system with two operator sites . The question "what is the simplest possible gene regulation system capable of acting like a switch?" is answered . The paper ends with an analysis of a two-gene regulation system, the results of which point to the existence of a "soft-switching" mechanism that may account for the "on-off" hypothesized behavior of some gene networks .

Folia Microbiol (Praha), 1998, 43(4), 339 - 52
The role of GTP-binding proteins in mechanochemical movements of microorganisms and their potential to form filamentous structures; Mikulik K; Prokaryotic cells contain proteins which form extended chains or multimers that oscillate between monomers and oligomers of varying length . Hydrolysis of nucleoside triphosphates combined with site-specific disposition of substrates and products to monomers and multimers is the driving force of dynamic instability of these molecules . Polymeric structures are connected in some manner to a variety of signaling systems that adhere to the polymeric matrix, including the GTP-binding protein(s), protein kinases and phosphatases, and other proteins or systems that communicate between the cytoplasmic membrane and the cytosol . Flexible organization allowing regulated dynamic movement is one of the key elements in all living cells . In eukaryotic cells actin and tubulin are the two main components of dynamically controlled spatial system . These proteins are noteworthy for their ability to polymerize, reversibly, into filaments or microtubules in association with hydrolysis of ATP or GTP, respectively . As such, they regulate most of the mechanics of cell movement including cell division, cell differentiation, phagocytosis and other dynamic phenomena . Recent evidence revealed that microbial cells create functional domains at specific sites of the cells and can form cytoplasmic tubules and fibers.

Curr Opin Biotechnol, 1998 Oct, 9(5), 534 - 48
Recent advances in producing and selecting functional proteins by using cell-free translation; Jermutus L et al.; Prokaryotic and eukaryotic in vitro translation systems have recently become the focus of increasing interest for tackling fundamental problems in biochemistry . Cell-free systems can now be used to study the in vitro assembly of membrane proteins and viral particles, rapidly produce and analyze protein mutants, and enlarge the genetic code by incorporating unnatural amino acids . Using in vitro translation systems, display techniques of great potential have been developed for protein selection and evolution . Furthermore, progress has been made to efficiently produce proteins in batch or continuous cell-free translation systems and to elucidate the molecular causes of low yield and find possible solutions for this problem.

Curr Opin Biotechnol, 1998 Oct, 9(5), 506 - 9
Use of cell wall-less bacteria (L-forms) for efficient expression and secretion of heterologous gene products; Gumpert J et al.; In spite of many efforts and achievements to optimize the prokaryotic expression systems, there are still general and specific problems in obtaining sufficient yields of the functionally active gene products . The main problems concern the formation of inclusion bodies, incorrect folding, toxicity for the producer cells and degradation by proteases . One way to overcome these problems is with expression systems alternative to those of Escherichia coli . For the first time, cell wall-less L-form bacteria were used to establish such an alternative expression system and test its practicability . The results showed that various recombinant proteins can be synthesized in considerable amounts as soluble, functionally active products with these cell wall-less strains.

Mol Cell Biol, 1998 Dec, 18(12), 7225 - 34
Identification and characterization of the fourth single-stranded-DNA binding domain of replication protein A; Brill SJ et al.; Replication protein A (RPA), the heterotrimeric single-stranded-DNA (ssDNA) binding protein (SSB) of eukaryotes, contains two homologous ssDNA binding domains (A and B) in its largest subunit, RPA1, and a third domain in its second-largest subunit, RPA2 . Here we report that Saccharomyces cerevisiae RPA1 contains a previously undetected ssDNA binding domain (domain C) lying in tandem with domains A and B . The carboxy-terminal portion of domain C shows sequence similarity to domains A and B and to the region of RPA2 that binds ssDNA (domain D) . The aromatic residues in domains A and B that are known to stack with the ssDNA bases are conserved in domain C, and as in domain A, one of these is required for viability in yeast . Interestingly, the amino-terminal portion of domain C contains a putative Cys4-type zinc-binding motif similar to that of another prokaryotic SSB, T4 gp32 . We demonstrate that the ssDNA binding activity of domain C is uniquely sensitive to cysteine modification but that, as with gp32, ssDNA binding is not strictly dependent on zinc . The RPA heterotrimer is thus composed of at least four ssDNA binding domains and exhibits features of both bacterial and phage SSBs.

Yeast, 1998 Jun 30, 14(9), 853 - 60
The deletion of six ORFs of unknown function from Saccharomyces cerevisiae chromosome VII reveals two essential genes: YGR195w and YGR198w; Rodriguez-Pena JM et al.; We have deleted six different ORFs of unknown function located on the right arm of Saccharomyces cerevisiae chromosome VII; namely, YGR187c/HGH1, YGR189c, YGR194c, YGR195w, YGR196c and YGR198w . No basic phenotypes could be attributed to the strains deleted in any of genes YGR187c/HGH1, YGR189c, YGR194c and YGR196c . These deletants did not show mating, sporulation or growth defects under any of the conditions tested . However, spores bearing deletions in either the YGR195w or YGR198w genes were unable to develop into macroscopical colonies . The YGR195w gene product shows significant homology with bacterial ribonuclease PH, an enzyme hitherto undescribed in yeasts, and its deletion causes a loss of viability after one to three rounds of cell division . Overexpression of this gene, using a tetracycline-regulatable promoter system, did not cause any effect on the cells . Contrary to what has been reported for prokaryotic homologs, this enzyme could play an essential role in yeast cell biology . The product encoded by the other essential ORF, YGR198w, shows no significant homology with any protein of known function in the databases . Spores bearing the deletion usually germinate and give rise to microcolonies of 50-100 non-viable cells.

Arch Microbiol, 1998 Oct, 170(5), 385 - 8
The atpIBEXF operon coding for the F0 sector of the ATP synthase from the purple nonsulfur photosynthetic bacterium Rhodobacter capsulatus; Borghese R et al.; The atpIBEXF operon coding for the F0 sector of the ATP synthase from Rhodobacter capsulatus was cloned and sequenced . The genes for the five subunits were present in the order: atpI (subunit I), atpB (subunit a), atpE (subunit c), atpX (subunit b'), and atpF (subunit b) . The transcription initiation site was defined by primer-extension analysis . A duplicated and divergent copy of the b subunit gene (subunit b') was present . This duplication is found only in photosynthetic prokaryotes and in plant chloroplasts . F0 deletion mutants formed tiny colonies during anaerobic growth in the dark but could not sustain continuous growth . Based on the results of the present work, we conclude that a functioning ATP synthase is essential for normal growth under all conditions tested.

Biochem Mol Biol Int, 1998 Oct, 46(3), 607 - 17
The use of a chimera HIV-1/HIV-2 envelope protein for immunodiagnosis of HIV infection: its expression and purification in E . coli by use of a translation initiation site within HIV-1 env gene; Han B et al.; A chimera HIV-1/HIV-2 envelope sequence composed of multiple conserved immunodominant epitopes of HIV-1 envelope protein (HIV-1 IIIB: env482-518 + env548-675) and the HIV-2 gp36 immunodominant epitope (env592-603), was constructed and directly over-expressed in E . coli by using a prokaryotic translation initiation sequence contained within the gene of HIV-1 envelope . The recombinant product was purified and applied in antibody-screening assay . The purified chimera antigen reacted with all the thirty-eight HIV-1 positive serum samples, the two HIV-2 serum samples, and had no cross-reaction with all the eighty-eight normal healthy serum sample . The results indicated that this recombinant chimera HIV-1/HIV-2 envelope protein could be useful for diagnostic purposes of HIV infection.

Structure, 1998 Nov 15, 6(11), 1453 - 65
GDP-4-keto-6-deoxy-D-mannose epimerase/reductase from Escherichia coli, a key enzyme in the biosynthesis of GDP-L-fucose, displays the structural characteristics of the RED protein homology superfamily; Rizzi M et al.; BACKGROUND: The process of guanosine 5'-diphosphate L-fucose (GDP-L-fucose) biosynthesis is conserved throughout evolution from prokaryotes to man . In animals, GDP-L-fucose is the substrate of fucosyltransferases that participate in the biosynthesis and remodeling of glycoconjugates, including ABH blood group and Lewis-system antigens . The 'de novo' pathway of GDP-L-fucose biosynthesis from GDP-D-mannose involves a GDP-D-mannose 4,6 dehydratase (GMD) and a GDP-4-keto-6-deoxy-D-mannose epimerase/reductase (GMER) . Neither of the catalytic mechanisms nor the three-dimensional structures of the two enzymes has been elucidated yet . The severe leukocyte adhesion deficiency (LAD) type II genetic syndrome is known to result from deficiencies in this de novo pathway . RESULTS: The crystal structures of apo- and holo-GMER have been determined at 2.1 A and 2.2 A resolution, respectively . Each subunit of the homodimeric (2 x 34 kDa) enzyme is composed of two domains . The N-terminal domain, a six-stranded Rossmann fold, binds NADP+; the C-terminal domain (about 100 residues) displays an alpha/beta topology . NADP+ interacts with residues Arg12 and Arg36 at the adenylic ribose phosphate; moreover, a protein loop based on the Gly-X-X-Gly-X-X-Gly motif (where X is any amino acid) stabilizes binding of the coenzyme diphosphate bridge . The nicotinamide and the connected ribose ring are located close to residues Ser107, Tyr136 and Lys140, the putative GMER active-site center . CONCLUSIONS: The GMER fold is reminiscent of that observed for UDP-galactose epimerase (UGE) from Escherichia coli . Consideration of the enzyme fold and of its main structural features allows assignment of GMER to the reductase-epimerase-dehydrogenase (RED) enzyme homology superfamily, to which short-chain dehydrogenase/reductases (SDRs) also belong . The location of the NADP+ nicotinamide ring at an interdomain cleft is compatible with substrate binding in the C-terminal domain.

Nature, 1998 Nov 5, 396(6706), 88 - 92
NMR structure of the histidine kinase domain of the E . coli osmosensor EnvZ; Tanaka T et al.; Bacteria live in capricious environments, in which they must continuously sense external conditions in order to adjust their shape, motility and physiology . The histidine-aspartate phosphorelay signal-transduction system (also known as the two-component system) is important in cellular adaptation to environmental changes in both prokaryotes and lower eukaryotes . In this system, protein histidine kinases function as sensors and signal transducers . The Escherichia coli osmosensor, EnvZ, is a transmembrane protein with histidine kinase activity in its cytoplasmic region . The cytoplasmic region contains two functional domains: domain A (residues 223-289) contains the conserved histidine residue (H243), a site of autophosphorylation as well as transphosphorylation to the conserved D55 residue of response regulator OmpR, whereas domain B (residues 290-450) encloses several highly conserved regions (G1, G2, F and N boxes) and is able to phosphorylate H243 . Here we present the solution structure of domain B, the catalytic core of EnvZ . This core has a novel protein kinase structure, distinct from the serine/threonine/tyrosine kinase fold, with unanticipated similarities to both heatshock protein 90 and DNA gyrase B.

Gene, 1998 Nov 5, 222(1), 91 - 7
Effect of short 5' UTRs on protein synthesis in two biological kingdoms; Teilhet M et al.; Efficient ribosomal protein synthesis is dependent on cis-acting elements in the 5' untranslated region (UTR) of mRNAs . Between prokaryotes and eukaryotes, the sequence and location of these elements differ to the extent of not being functionally interchangeable . We explored the possibility of constructing bifunctional UTRs that could direct translation in both prokaryotes and eukaryotes . A variant of a UTR from ner of phage Mu (ner-ACC) enhanced protein synthesis in a rabbit reticulocyte lysate, and it was compared to a lacZ-CTA, containing the lambda cro RBS and the Escherichia coli lacZ spacer . Several mutants in the -3 to -1 regions of both lacZ-CTA and ner-ACC were tested in rabbit reticulocyte lysate and E . coli to select UTRs that were optimized simultaneously for both biological kingdoms . The lacZ-ATC proved 217-fold more effective than ner-ACC in this cross-species ability to enhance translation . The lacZ-ACC and ner-ATC were 83- and 78-fold, respectively, better than ner-ACC . We conclude that short UTRs (12-15 nt in length) can be fine-tuned in the -9 to -1 regions to enhance protein synthesis concurrently in prokaryotes and eukaryotes . In related studies, we show that nt at the -3 to -1 region of mRNAs exert an enormous impact on synthesis of proteins in E . coli.

Biochem Biophys Res Commun, 1998 Nov 9, 252(1), 63 - 8
Using discriminant function for prediction of subcellular location of prokaryotic proteins; Chou KC et al.; The discriminant function algorithm was introduced to predict the subcellular location of proteins in prokaryotic organisms from their amino-acid composition . The rate of correct prediction for the three possible subcellular locations of prokaryotic proteins studied by Reinhardt and Hubbard (Nucleic Acid Research, 1998, 26:2230-2236) was 90% by the self-consistency test, and 87% by the jackknife test . These rates are considerably higher than the results recently reported by them using the neural network method . Furthermore, the test procedure adopted here is also more rigorous . The core of the current algorithm is the covariance matrix, through which the collective interactions among different amino-acid components of a protein can be reflected . It is anticipated that, owing to the intimate correlation of the function of a protein with its subcellular location, the current algorithm will become a useful tool for the systematic analysis of genome data .

Science, 1998 Nov 13, 282(5392), 1321 - 4
Dual requirement for gephyrin in glycine receptor clustering and molybdoenzyme activity; Feng G et al.; Glycine receptors are anchored at inhibitory chemical synapses by a cytoplasmic protein, gephyrin . Molecular cloning revealed the similarity of gephyrin to prokaryotic and invertebrate proteins essential for synthesizing a cofactor required for activity of molybdoenzymes . Gene targeting in mice showed that gephyrin is required both for synaptic clustering of glycine receptors in spinal cord and for molybdoenzyme activity in nonneural tissues . The mutant phenotype resembled that of humans with hereditary molybdenum cofactor deficiency and hyperekplexia (a failure of inhibitory neurotransmission), suggesting that gephyrin function may be impaired in both diseases.

Hum Mol Genet, 1998 Nov, 7(12), 1921 - 5
Molecular characterization of homozygous variegate porphyria; Roberts AG et al.; Variegate porphyria (VP) is a low penetrance, autosomal dominant disorder that results from partial deficiency of protoporphyrinogen oxidase (PPOX) activity caused by mutation in the PPOX gene . The rare homozygous variant of VP is characterized by severe PPOX deficiency, onset of photosensitization by porphyrins in early childhood, skeletal abnormalities of the hand and, less constantly, short stature, mental retardation and convulsions . We have identified PPOX mutations on both alleles of five of the 11 unrelated patients with homozygous VP reported to date . Two patients were homoallelic for missense mutations (D349A and A433P), while three were heteroallelic . Functional analysis by prokaryotic expression showed that the D349A and A433P and one missense mutation in each of the three heteroallelic patients (G358R in two patients and A219KANA) preserved some PPOX activity (9.5-25% of wild-type) . Mutations on the other allele of the heteroallelic patients abolished or markedly decreased activity . There was no relation between genotype assessed by functional analysis and the presence or severity of non-cutaneous manifestations . The mutations were absent from 104 unrelated patients with autosomal dominant VP . Our findings define the molecular pathology of homozygous VP and suggest that mild PPOX mutations occur in the general population but have very low or no clinical penetrance in heterozygotes.

Proc Natl Acad Sci U S A, 1998 Nov 10, 95(23), 13976 - 81
Eukaryotic phytochromes: light-regulated serine/threonine protein kinases with histidine kinase ancestry; Yeh KC et al.; The discovery of cyanobacterial phytochrome histidine kinases, together with the evidence that phytochromes from higher plants display protein kinase activity, bind ATP analogs, and possess C-terminal domains similar to bacterial histidine kinases, has fueled the controversial hypothesis that the eukaryotic phytochrome family of photoreceptors are light-regulated enzymes . Here we demonstrate that purified recombinant phytochromes from a higher plant and a green alga exhibit serine/threonine kinase activity similar to that of phytochrome isolated from dark grown seedlings . Phosphorylation of recombinant oat phytochrome is a light- and chromophore-regulated intramolecular process . Based on comparative protein sequence alignments and biochemical cross-talk experiments with the response regulator substrate of the cyanobacterial phytochrome Cph1, we propose that eukaryotic phytochromes are histidine kinase paralogs with serine/threonine specificity whose enzymatic activity diverged from that of a prokaryotic ancestor after duplication of the transmitter module.

J Mol Biol, 1998 Nov 20, 284(1), 101 - 24
Crystal structure of methionine aminopeptidase from hyperthermophile, Pyrococcus furiosus; Tahirov TH et al.; The structure of methionine aminopeptidase from hyperthermophile Pyrococcus furiosus (PfMAP) with an optimal growth temperature of 100 degreesC was determined by the multiple isomorphous replacement method and refined in three different crystal forms, one monoclinic and two hexagonal, at resolutions of 2.8, 2.9, and 3.5 A . The resolution of the monoclinic crystal form was extended to 1.75 A by water-mediated transformation to a low-humidity form, and the obtained diffraction data used for high-resolution structure refinement . This is the first description of a eukaryotic type methionine aminopeptidase structure . The PfMAP molecule is composed of two domains, a catalytic domain and an insertion domain, connected via two antiparallel beta-strands . The catalytic domain, which possesses an internal 2-fold symmetry and contains two cobalt ions in the active site, resembles the structure of a prokaryotic type MAP from Escherichia coli (EcMAP), while the structure of the insertion domain containing three helices has a novel fold and accounts for a major difference between the eukaryotic and prokaryotic types of methionine aminopeptidase . Analysis of the PfMAP structure in comparison with EcMAP and other mesophile proteins reveals several factors which may contribute to the hyperthermostability of PfMAP: (1) a significantly high number of hydrogen bonds and ion-pairs between side-chains of oppositely charged residues involved in the stabilization of helices; (2) an increased number of hydrogen bonds between the positively charged side-chain and neutral oxygen; (3) a larger number of buried water molecules involved in crosslinking the backbone atoms of sequentially separate segments; (4) stabilization of two antiparallel beta-strands connecting the two domains of the molecule by proline residues; (5) shortening of N and C-terminal tails and stabilization of the loop c3E by deletion of three residues .

Trends Microbiol, 1998 Oct, 6(10), 407 - 10
Adaptive significance of circadian programs in cyanobacteria; Johnson CH et al.; Prokaryotic cyanobacteria express robust circadian (daily) rhythms, even when growing with doubling times that are considerably faster than once every 24 h . This biological clock orchestrates cellular events to occur in an optimal temporal program . Competition experiments demonstrate that fitness is enhanced when the circadian period resonates with the period of the environmental cycle.

Biochem J, 1998 Nov 15, 336 ( Pt 1), 69 - 76
Membrane topology of the Escherichia coli gamma-aminobutyrate transporter: implications on the topography and mechanism of prokaryotic and eukaryotic transporters from the APC superfamily; Hu LA et al.; The Escherichia coli gamma-aminobutyric acid permease (GabP) is a plasma membrane protein from the amine-polyamine-choline (APC) superfamily . On the basis of hydropathy analysis, transporters from this family are thought to contain 12, 13 or 14 transmembrane domains . We have experimentally analysed the topography of GabP by using the cytoplasmically active LacZ (beta-galactosidase) and the periplasmically active PhoA (alkaline phosphatase) as complementary topological sensors . The enzymic activities of 32 GabP-LacZ hybrids and 43 GabP-PhoA hybrids provide mutually reinforcing lines of evidence that the E . coli GabP contains 12 transmembrane segments that traverse the membrane in a zig-zag fashion with both N- and C-termini facing the cytoplasm . Interestingly, the resulting model predicts that the functionally important 'consensus amphipathic region' (CAR) {Hu and King (1998) Biochem . J . 330, 771-776} is at least partly membrane-embedded in many amino acid transporters from bacteria and fungi, in contrast with the apparent situation in mouse cationic amino acid transporters (MCATs), in which this kinetically significant region is thought to be fully cytoplasmic {Sophianopoulou and Diallinas (1995) FEMS Microbiol . Rev . 16, 53-75} . To the extent that conserved domains serve similar functions, the resolution of this topological disparity stands to have family-wide implications on the mechanistic role of the CAR . The consensus transmembrane structure derived from this analysis of GabP provides a foundation for predicting the topological disposition of the CAR and other functionally important domains that are conserved throughout the APC transporter superfamily.

Biochim Biophys Acta, 1998 Nov 8, 1442(2-3), 326 - 8
The secondary structure of Nosema apis large subunit ribosomal RNA; De Rijk P et al.; The microsporidia are a group of obligate intracellular eukaryotic parasites, that lack mitochondria . Their ribosomes show several prokaryote-like features . This paper presents the secondary structure of the large subunit ribosomal RNA (LSU rRNA) of the microsporidium Nosema apis . With its 2481 bases, it is the shortest known non-mitochondrial LSU rRNA . The seemingly prokaryote-like features of the molecule cannot be used as evidence for the ancient origin of the microsporidia . The reduction in size can be attributed to changes in the regions of the LSU rRNA that are known to show great variability in length and sequence within the eukaryotes . The lack of fragmentation commonly seen in other eukaryotes may also be a derived feature.

J Biol Chem, 1998 Nov 13, 273(46), 30704 - 12
Human Hsp70 and Hsp40 chaperone proteins facilitate human papillomavirus-11 E1 protein binding to the origin and stimulate cell-free DNA replication; Liu JS et al.; Human papillomavirus replication initiator, the E1 helicase, binds weakly to the origin of DNA replication . Purified human chaperone proteins Hsp70 and Hsp40 (HDJ-1 and HDJ-2) independently and additively enhanced E1 binding to the origin . The interaction between E1 and Hsp70 was transient and required ATP hydrolysis, whereas Hsp40 bound to E1 directly and remained in the complex . A peptide of 20 residues spanning the HPD loop and helix II of the J domain of YDJ-1 also stimulated E1 binding to the origin, alone or in combination with Hsp70 or Hsp40 . A mutated peptide (H34Q) had a reduced activity, while an adjacent or an overlapping peptide had no effect . Neither Hsp70 nor the J peptide altered the E1/DNA ratio in the complex . Electron microscopy showed that E1 mainly bound to DNA as a hexamer . In the presence of Hsp40, E1 primarily bound to DNA as a dihexamer . Preincubation of chaperones with viral E1 and template shortened the lag time and increased replication in a cell-free system . Since two helicases are essential for bidirectional replication of human papillomavirus DNA, these results demonstrate that, as in prokaryotes, chaperones play an important role in the assembly of preinitiation complexes on the origin.

FEBS Lett, 1998 Oct 16, 437(1-2), 70 - 4
Properties of a mutant form of the prokaryotic enhancer binding protein, NTRC, which hydrolyses ATP in the absence of effectors; Widdick D et al.; The mutation S170A in the proposed nucleotide binding site of the transcriptional activator protein NTRC abolishes its ability to catalyse open promoter complex formation by the sigma(N)-RNA polymerase holoenzyme . NTRC(S170A) has significant ATPase activity, which, in contrast to the wild-type protein, is unaffected by phosphorylation or binding to enhancer sites on DNA . The mutant protein appears to oligomerise normally on DNA in response to phosphorylation but the ATPase activity is apparently not responsive to changes in oligomerisation state . The defect in transcriptional activation is discussed in relation to mutations in other sigma(N)-dependent activators.

Philos Trans R Soc Lond B Biol Sci, 1998 Sep 29, 353(1374), 1425 - 30
Plant hormone perception and action: a role for G-protein signal transduction?
Hooley R.
Plants perceive and respond to a profusion of environmental and endogenous signals that influence their growth and development . The G-protein signalling pathway is a mechanism for transducing extracellular signals that is highly conserved in a range of eukaryotes and prokaryotes . Evidence for the existence of G-protein signalling pathways in higher plants is reviewed, and their potential involvement in plant hormone signal transduction evaluated . A range of biochemical and molecular studies have identified potential components of G-protein signalling in plants, most notably a homologue of the G-protein coupled receptor superfamily (GCR1) and the G alpha and G beta subunits of heterotrimeric G-proteins . G-protein agonists and antagonists are known to influence a variety of signalling events in plants and have been used to implicate heterotrimeric G-proteins in gibberellin and possibly auxin signalling . Antisense suppression of GCR1 in Arabidopsis leads to a phenotype which supports a role for this receptor in cytokinin signalling . These observations suggest that higher plants have at least some of the components of G-protein signalling pathways and that these might be involved in the action of certain plant hormones.

Curr Biol, 1998 Oct 22, 8(21), 1161 - 8
The junctional pore complex, a prokaryotic secretion organelle, is the molecular motor underlying gliding motility in cyanobacteria; Hoiczyk E et al.; BACKGROUND: Whereas most bacteria move by means of flagella, some prokaryotes move by gliding . In cyanobacteria, gliding motility is a slow uniform motion which is invariably accompanied by a continuous secretion of slime . On the basis of these characteristics, a model has been proposed in which the gliding motility of cyanobacteria depends on the steady secretion of slime using specific pores, as well as the interaction of the slime with the filament surface and the underlying substrate . RESULTS: The structures of the pore apparatus of two different filamentous cyanobacteria have been characterized . In both species, pores are formed by a hitherto uncharacterized type of prokaryotic organelle that spans the entire multilayered cell wall and possesses structural properties expected for an organelle that is involved in the rapid secretion of extracellular carbohydrates . Light microscopic observations of the secretion process provided direct evidence that the pore complexes are the actual sites of slime secretion, that the secreted slime fibrils are elongated at about the same rate as the filament glides (up to 3 micrometer s-1), and that gliding movements are caused directly by the secretion of slime . CONCLUSIONS: It has been known for a long time that carbohydrate secretion has an important role in the gliding motility of various prokaryotes . Our results strongly suggest that slime secretion is not only a prerequisite for this peculiar type of motility in cyanobacteria, but also directly generates the necessary thrust for locomotion.

J Mol Biol, 1998 Nov 13, 283(5), 963 - 76
Sequence-specific DNA binding by EcoKI, a type IA DNA restriction enzyme; Powell LM et al.; The type I DNA restriction and modification enzymes of prokaryotes are multimeric enzymes that cleave unmethylated, foreign DNA in a complex process involving recognition of the methylation status of a DNA target sequence, extensive translocation of DNA in both directions towards the enzyme bound at the target sequence, ATP hydrolysis, which is believed to drive the translocation possibly via a helicase mechanism, and eventual endonucleolytic cleavage of the DNA . We have examined the DNA binding affinity and exonuclease III footprint of the EcoKI type IA restriction enzyme on oligonucleotide duplexes that either contain or lack the target sequence . The influence of the cofactors, S-adenosyl methionine and ATP, on binding to DNA of different methylation states has been assessed . EcoKI in the absence of ATP, with or without S-adenosyl methionine, binds tightly even to DNA lacking the target site and the exonuclease footprint is large, approximately 45 base-pairs . The protection is weaker on DNA lacking the target site . Partially assembled EcoKI lacking one or both of the subunits essential for DNA cleavage, is unable to bind tightly to DNA lacking the target site but can bind tightly to the recognition site . The addition of ATP to EcoKI, in the presence of AdoMet, allows tight binding only to the target site and the footprint shrinks to 30 base-pairs, almost identical to that of the modification enzyme which makes up the core of EcoKI . The same effect occurs when S-adenosyl homocysteine or sinefungin are substituted for S-adenosyl methionine, and ADP or ATPgammaS are substituted for ATP . It is proposed that the DNA binding surface of EcoKI comprises three regions: a "core" region which recognises the target sequence and which is present on the modification enzyme, and a region on each DNA cleavage subunit . The cleavage subunits make tight contacts to any DNA molecule in the absence of cofactors, but this contact is weakened in the presence of cofactors to allow the protein conformational changes required for DNA translocation when a target site is recognised by the core modification enzyme . This weakening of the interaction between the DNA cleavage subunits and the DNA could allow more access of exonuclease III to the DNA and account for the shorter footprint .

Arch Biochem Biophys, 1998 Nov 1, 359(1), 17 - 23
Expression of the psbA gene in the marine oxyphotobacteria Prochlorococcus spp; Garcia-Fernandez JM et al.; The oxygenic photosynthetic prokaryotes Prochlorococcus marinus SS120 (CCMP1375) and Prochlorococcus sp . MED4 (CCMP 1378) were previously shown to exhibit different pigmentation and ecophysiological characteristics . The former strain has a much lower divinyl-Chl a to b ratio and is adapted to lower photon flux densities than the latter . In contrast to the cyanobacteria examined so far, both strains possess only one copy of the psbA gene, encoding the D1 protein of photosystem II core . In acclimated steady-state cultures, psbA transcript levels were always higher at high irradiances in both strains . Upon a shift from low to high light, the psbA transcript levels increased in both strains but more quickly in MED4 than in SS120 . They decreased during the opposite shift . Iron-starved MED4 cells overexpressed psbA at all assayed irradiances, suggesting that this species, representative of populations from naturally iron-depleted oceanic areas, may have developed a particular compensation mechanism . The similar effects of DCMU and DBMIB on the expression of psbA suggest that light regulation of psbA in Prochlorococcus may be mediated by the electron transport chain . The energy state of cells could, however, also be involved in this regulation, since cultures of both strains subjected to darkness showed psbA levels significantly lower when glucose was added .

Biochemistry, 1998 Nov 3, 37(44), 15336 - 44
Characterization of the nucleotide binding properties of SV40 T antigen using fluorescent 3'(2')-O-(2,4,6-trinitrophenyl)adenine nucleotide analogues; Huang SG et al.; ATP binding to the large tumor (T) antigen encoded by the simian virus 40 (SV40) genome plays an essential role in the replication of viral DNA {Fanning, E., and Knippers, R . (1992) Annu . Rev . Biochem . 61, 55-85} . To better explore the functions of T antigen during the replication process, we have studied the interactions of T antigen with fluorescent 3'(2')-O-(2,4,6-trinitrophenyl) (TNP) adenine nucleotide analogues . Binding of TNP-ATP and TNP-ADP was accompanied by an 8-fold fluorescence enhancement and a concomitant blue shift (11 nm) of the maximal emission wavelength; the intrinsic protein tryptophan fluorescence was quenched maximally by 50% . Both signals were utilized to characterize the nucleotide binding activity of T antigen . TNP-ATP and TNP-ADP bound to the ATP binding site with dissociation constants of 0.35 microM and 2.6 microM . TNP substitution enhanced the affinity of ADP for T antigen by approximately 11-fold . The binding stoichiometry was 1 mol of TNP nucleotide per mole of monomer T antigen . The binding of TNP-ATP was more temperature dependent than that of TNP-ADP . The enthalpy change contributed nearly half of the energy for TNP-ATP binding, whereas binding of TNP-ADP was primarily entropy driven . Both TNP-ATP and TNP-ADP were strong inhibitors of the T antigen ATPase activity, confirming the high affinities of the TNP nucleotides for the ATP binding site . Like the parent nucleotides, they also induced T antigen hexamer formation . Using the TNP nucleotides as fluorescent probes, we have measured the affinity of various nucleotides and analogues for T antigen . The results indicate that the nucleotide binding specificity of T antigen was similar to that of the prokaryotic helicases Dna B and Rep, suggesting closely related ATP binding sites in the three DNA helicases.

Curr Genet, 1998 Oct, 34(4), 336 - 41
Phylogenetic analysis of plastid origins based on secA sequences; Barbrook AC et al.; We have generated secA sequence data from a number of photosynthetic prokaryotes and carried out a phylogenetic analysis using secA sequences from prokaryotes, green plants, and red and brown algae . We have studied the substitution patterns that give rise to the apparent phylogenetic structure . We show that the high AT content of the plastid sequences significantly affects the amino-acid composition . We also show that most of the apparent evidence for an edge separating red and brown plastids from green plants within the phylogenetic tree is due to differences in nucleotide composition . The remaining apparent evidence is likely to be due, at least in part, to differences in the distribution of sites free to vary . We discuss the implications of this study for hypotheses of plastid origins.

Eur J Biochem, 1998 Oct 1, 257(1), 255 - 62
Involvement of the amino acids outside the active-site cleft in the catalysis of ricin A chain; Kitaoka Y; The A chain of ricin (RA) is a cytotoxic RNA N-glycosidase that inactivates ribosomes by depurination of the adenosine residue at position 4324 in 28S rRNA . Of the 267 amino acids in the protein, 231 could be deleted in one or another of 83 mutants, without the loss of the capacity to catalyze hydrolysis of a single specific nucleotide in rRNA {Morris, K . N . & Wool, I . G . (1992) Proc . Natl Acad . Sci . USA 89, 4869-4873} . Expression of 29 selected deletion mutants of RA in prokaryotic cell-free coupled transcription-translation reactions was carried out and the activities of the mutants were assessed by monitoring depurination of reticulocyte ribosomes . Kinetic analysis of five deletion mutants which retained detectable activity was performed . Deletion of amino acids outside the putative active-site cleft in these mutants significantly affected the catalytic rate rather than the interaction with ribosomes . From these data, the amino acids far from the active-site cleft appeared to be involved in alignment of the key residues for catalysis and interaction with the target tetraloop structure of 28S rRNA.

Biochim Biophys Acta, 1998 Oct 23, 1425(2), 348 - 60
Production and characterization of a bivalent single chain Fv/alkaline phosphatase conjugate specific for the hemocyanin of the scorpion Androctonus australis; Mousli M et al.; A102 is a monoclonal antibody raised against the hemocyanin of the Tunisian scorpion Androctonus australis . It is directed against the subunit Aa6 and does not cross-react when tested against a variety of similar scorpion hemocyanins . Here, we report the construction of a plasmid encoding a recombinant enzyme-linked antigen-binding protein with the antigen-binding specificity of antibody A102 . The DNA fragments encoding the variable domains of A102 were inserted into a prokaryotic expression vector so as to produce a single chain antibody variable fragment (scFv) fused to the bacterial alkaline phosphatase . The fusion protein preserved the IgG binding and alkaline phosphatase activities . Immunoelectron microscopic analysis showed that the recombinant protein bound antigen bivalently as is the case for natural antibodies . Crude preparations containing the conjugate were used in a rapid visual immunoassay for the specific detection of A . australis hemocyanin, using a droplet of hemolymph removed from live animals by puncture . The simplicity of the test made it suitable for the direct identification of animals belonging to this species . It could be useful in areas where A . australis, the most dangerous African scorpion, is found with other species from which it is not easy to distinguish using morphological criteria.

Gene, 1998 Sep 14, 217(1-2), 57 - 67
Skewed oligomers and origins of replication; Salzberg SL et al.; The putative origin of replication in prokaryotic genomes can be located by a new method that finds short oligomers whose orientation is preferentially skewed around the origin . The skewed oligomer method is shown to work for all bacterial genomes and one of three archaeal genomes sequences to date, confirming known or predicted origins in most cases and in three cases (H . pylori, M . thermoautotrophicum, and Synechocystis sp.), suggesting origins that were previously unknown . In many cases, the presence of conserved genes and nucleotide motifs confirms the predictions . An algorithm for finding these skewed seven-base and eight-base sequences is described, along with a method for combining evidence from multiple skewed oligomers to accurately locate the replication origin . Possible explanations for the phenomenon of skewed oligomers are discussed . Explanations are presented for why some bacterial genomes contain hundreds of highly skewed oligomers, whereas others contain only a handful.

J Bacteriol, 1998 Nov, 180(21), 5799 - 802
CHR, a novel family of prokaryotic proton motive force-driven transporters probably containing chromate/sulfate antiporters; Nies DH et al.; We describe a small family of proteins, CHR, which contains members that function in chromate and/or sulfate transport . CHR proteins occur in bacteria and archaea . They consist of about 400 amino acyl residues, appear to have 10 transmembrane alpha-helical segments in an unusual 4+6 arrangement, and arose by an intragenic duplication event.

J Biol Chem, 1998 Nov 6, 273(45), 29727 - 37
Loss of Hsp70-Hsp40 chaperone activity causes abnormal nuclear distribution and aberrant microtubule formation in M-phase of Saccharomyces cerevisiae; Oka M et al.; The 70-kDa heat shock proteins, hsp70, are highly conserved among both prokaryotes and eukaryotes, and function as chaperones in diverse cellular processes . To elucidate the function of the yeast cytosolic hsp70 Ssa1p in vivo, we characterized a Saccharomyces cerevisiae ssa1 temperature-sensitive mutant (ssa1-134) . After shifting to the restrictive temperature (37 degreesC), ssa1-134 mutant cells showed abnormal distribution of nuclei and accumulated as large-budded cells with a 2 N DNA content . We observed more prominent mutant phenotypes using nocodazole-synchronized cells: when cells were incubated at the restrictive temperature following nocodazole treatment, viability was rapidly lost and abnormal arrays of bent microtubules were formed . Chemical cross-linking and immunoprecipitation analyses revealed that the interaction of mutant Ssa1p with Ydj1p (cytosolic DnaJ homologue in yeast) was much weaker compared with wild-type Ssa1p . These results suggest that Ssa1p and Ydj1p chaperone activities play important roles in the regulation of microtubule formation in M phase . In support of this idea, a ydj1 null mutant at the restrictive temperature was found to exhibit more prominent phenotypes than ssa1-134 . Furthermore, both ssa1-134 and ydj1 null mutant cells exhibited greater sensitivity to anti-microtubule drugs . Finally, the observation that SSA1 and YDJ1 interact genetically with a gamma-tubulin, TUB4, supports the idea that they play a role in the regulation of microtubule formation.

Biol Chem, 1998 Aug-Sep, 379(8-9), 1019 - 23
S-phase DNA damage checkpoint in budding yeast; Foiani M et al.; Eukaryotic cells must be able to coordinate DNA repair, replication and cell cycle progression in response to DNA damage . A failure to activate the checkpoints which delay the cell cycle in response to internal and external cues and to repair the DNA lesions results in an increase in genetic instability and cancer predisposition . The use of the yeast Saccharomyces cerevisiae has been invaluable in isolating many of the genes required for the DNA damage response, although the molecular mechanisms which couple this regulatory pathway to different DNA transactions are still largely unknown . In analogy with prokaryotes, we propose that DNA strand breaks, caused by genotoxic agents or by replication-related lesions, trigger a replication coupled repair mechanism, dependent upon recombination, which is induced by the checkpoint acting during S-phase.

Mol Microbiol, 1998 Oct, 30(2), 431 - 41
Maximal transcriptional activation by the IHF protein of Escherichia coli depends on optimal DNA bending by the activator; Engelhorn M et al.; Transcriptional activation in prokaryotes can be mediated by at least two different mechanisms: direct contacts between the activator and RNA polymerase or modulation of the overall geometry of DNA . In the latter case, an activator protein that bends DNA favours contacts between the DNA upstream of the activator binding site and the back of RNA polymerase . The architectural protein integration host factor (IHF) of Escherichia coli bends DNA and activates transcription at several promoters . We have isolated mutants of IHF that maximize transcriptional activation by adjusting the bending angle of the DNA . The amino acid residues of IHF that adjust the bending angle are close to the DNA and probably make electrostatic interactions with the DNA . We show that transcriptional activation is maintained when the IHF binding site is moved further upstream or when its orientation is inverted, and we conclude from these data that direct interactions between IHF and RNA polymerase do not participate in activation . IHF acts merely by bending DNA; weaker bending leading to stronger activation . We propose that wild-type IHF induces too strong a DNA bend (180 degrees) for optimal interactions between DNA upstream of the IHF binding site and the back of RNA polymerase.

Mol Microbiol, 1998 Oct, 30(2), 365 - 79
An experimental chain of infection reveals that distinct Borrelia burgdorferi populations are selected in arthropod and mammalian hosts; Ryan JR et al.; The prokaryotic, spirochaetal microorganism Borrelia burgdorferi is the causative agent of Lyme disease, an arthropod-borne disease of a variety of vertebrates and the most prevalent arthropod-borne disease of humans in the United States . In order to understand better the normal life cycle of B . burgdorferi, an experimental chain of infection was devised that involved multiple sequential arthropod and mammalian passages . By examining populations of B . burgdorferi emerging from different points in this infectious chain, we demonstrate that selection of B . burgdorferi populations peculiar to arthropod or vertebrate hosts is a property of at least one of the two ecologically distinct strains we examined . Distinct B . burgdorferi populations were identified using an antigenic profile, defined by a set of monoclonal antibodies to eight B . burgdorferi antigens, and a plasmid profile, defined by the naturally occurring plasmids in the starting clonal populations . These two profiles constituted the phenotypical signature of the population . In the strain exhibiting selection in the different hosts, transition from one host to another produced a striking series of alternating phenotypical signatures down the chain of infection . At the molecular level, the alternating signatures were manifested as a reciprocal relationship between the expression of certain antigenic forms of outer surface protein (Osp) B and OspC . In the case of OspC, the antigenic changes could be correlated to the presence of one of two distinctly different alleles of the ospC gene in a full-length and presumably transcriptionally active state . In the case of OspB, two alleles were again identified . However, their differences were minor and their relationship to OspB antigenic variation more complicated . In addition to the reciprocating changes in the antigenic profile, a reciprocating change in the size (probably the multimeric state) of a 9.0 kbp supercoiled plasmid was also noted . Selection of distinct populations in the tick may be responsible for the microorganism's ability to infect a wide range of vertebrate hosts efficiently, in that the tick might provide selective pressure for the elimination of the population selected in the previous host.

Protein Expr Purif, 1998 Nov, 14(2), 229 - 36
Prokaryotic expression of bovine lactoferrin deletion mutants that bind to the Ca2+-dependent lactoferrin receptor on isolated rat hepatocytes; Sitaram MP et al.; We generated a series of recombinant variants of bovine lactoferrin (Lf) as fusion proteins using two prokaryotic expression vectors and examined the ability of the expressed proteins to compete with native Lf for binding to the Ca2+-dependent Lf receptor on isolated rat hepatocytes . A near-full-length bovine Lf cDNA (pN16b) was expressed in pGEMEX-2 as a gene 10 fusion protein (r-bLf10/-70) . Deletions of pN16b were cloned into the HindIII/NotI and BamHI/NotI restriction sites of expression vector pET 32 and expressed as thioredoxin fusion proteins, r-bLfT/-271 and r-bLfT/-310, respectively . r-bLf10/-70, r-bLfT/-271, and r-bLfT/-310 lacked, respectively, the NH2-terminal 70, 271, and 310 amino acids of Lf . Expression of recombinant proteins in Escherichia coli BL21-DE3 strain was monitored by denaturing gel electrophoresis or by immunoblot with anti-Lf antibodies . The yield of each of the soluble recombinant proteins was approximately 10 mg/L of BL21-DE3 suspension . r-bLf10/-70 and r-bLfT/-271 competed strongly with 125I-Lf for binding to hepatocytes but r-bLfT/-310 did not . Our findings are consistent with the conclusion that Lf binds to its Ca2+-dependent receptor on hepatocytes via noncarbohydrate determinants contained within its C-lobe .

Proc Natl Acad Sci U S A, 1998 Oct 27, 95(22), 13319 - 23
A photosystem I reaction center driven by chlorophyll d in oxygenic photosynthesis
Hu Q, Miyashita H, Iwasaki I I, Kurano N, Miyachi S, Iwaki M, Itoh S.
A far-red type of oxygenic photosynthesis was discovered in Acaryochloris marina, a recently found marine prokaryote that produces an atypical pigment chlorophyll d (Chl d) . The purified photosystem I reaction center complex of A . marina contained 180 Chl d per 1 Chl a with PsaA-F, -L, -K, and two extra polypeptides . Laser excitation induced absorption changes of reaction center Chl d that was named P740 after its peak wavelength . A midpoint oxidation reduction potential of P740 was determined to be +335 mV . P740 uses light of significantly low quantum energy (740 nm = 1.68 eV) but generates a reducing power almost equivalent to that produced by a special pair of Chl a (P700) that absorbs red light at 700 nm (1.77 eV) in photosystem I of plants and cyanobacteria . The oxygenic photosynthesis based on Chl d might either be an acclimation to the far-red light environments or an evolutionary intermediate between the red-absorbing oxygenic and the far-red absorbing anoxygenic photosynthesis that uses bacteriochlorophylls.

Proc Natl Acad Sci U S A, 1998 Oct 27, 95(22), 13227 - 32
Identification of differentially expressed mRNA in prokaryotic organisms by customized amplification libraries (DECAL): the effect of isoniazid on gene expression in Mycobacterium tuberculosis; Alland D et al.; Understanding the effects of the external environment on bacterial gene expression can provide valuable insights into an array of cellular mechanisms including pathogenesis, drug resistance, and, in the case of Mycobacterium tuberculosis, latency . Because of the absence of poly(A)+ mRNA in prokaryotic organisms, studies of differential gene expression currently must be performed either with large amounts of total RNA or rely on amplification techniques that can alter the proportional representation of individual mRNA sequences . We have developed an approach to study differences in bacterial mRNA expression that enables amplification by the PCR of a complex mixture of cDNA sequences in a reproducible manner that obviates the confounding effects of selected highly expressed sequences, e.g., ribosomal RNA . Differential expression using customized amplification libraries (DECAL) uses a library of amplifiable genomic sequences to convert total cellular RNA into an amplified probe for gene expression screens . DECAL can detect 4-fold differences in the mRNA levels of rare sequences and can be performed on as little as 10 ng of total RNA . DECAL was used to investigate the in vitro effect of the antibiotic isoniazid on M . tuberculosis, and three previously uncharacterized isoniazid-induced genes, iniA, iniB, and iniC, were identified . The iniB gene has homology to cell wall proteins, and iniA contains a phosphopantetheine attachment site motif suggestive of an acyl carrier protein . The iniA gene is also induced by the antibiotic ethambutol, an agent that inhibits cell wall biosynthesis by a mechanism that is distinct from isoniazid . The DECAL method offers a powerful new tool for the study of differential gene expression.

Comp Biochem Physiol B Biochem Mol Biol, 1998 Apr, 119(4), 677 - 90
The comparative amino acid sequences, substrate specificities and gene or cDNA nucleotide sequences of some prokaryote and eukaryote amidinotransferases: implications for evolution; Bedekar A et al.; The amino acid sequences of the amidinotransferases and the nucleotide sequences of their genes or cDNA from four Streptomyces species (seven genes) and from the kidneys of rat, pig, human and human pancreas were compared . The overall amino acid and nucleotide sequences of the prokaryotes and eukaryotes were very similar and further, three regions were identified that were highly identical . Evidence is presented that there is virtually zero chance that the overall and high identity regions of the amino acid sequence similarities and the overall nucleotide sequence similarities between Streptomyces and mammals represent random match . Both rat and lamprey amidinotransferases were able to use inosamine phosphate, the amidine group acceptor of Streptomyces . We have concluded that the structure and function of the amidinotransferases and their genes has been highly conserved through evolution from prokaryotes to eukaryotes . The evolution has occurred with: (1) a high degree of retention of nucleotide and amino acid sequences; (2) a high degree of retention of the primitive Streptomyces guanine + cytosine (G + C) third codon position composition in certain high identity regions of the eukaryote cDNA; (3) a decrease in the specificities for the amidine group acceptors; and (4) most of the mutations silent in the regions suggested to code for active sites in the enzymes.

Trends Biochem Sci, 1998 Sep, 23(9), 324 - 8
Conservation of gene order: a fingerprint of proteins that physically interact; Dandekar T et al.; A systematic comparison of nine bacterial and archaeal genomes reveals a low level of gene-order (and operon architecture) conservation . Nevertheless, a number of gene pairs are conserved . The proteins encoded by conserved gene pairs appear to interact physically . This observation can therefore be used to predict functions of, and interactions between, prokaryotic gene products.

J Biol Chem, 1998 Oct 30, 273(44), 28557 - 9
The polysialic acid units of the neural cell adhesion molecule N-CAM form filament bundle networks; Toikka J et al.; Polysialic acid is a developmentally regulated component in the neural cell adhesion molecule N-CAM which also occurs as the capsular polysaccharide of bacteria causing meningitis . Polysialic acid has been considered as a repulsive element that regulates intermolecular and intercellular adhesion . Using atomic force microscopy we unexpectedly find that oligomers of polysialic acid assemble with each other into filament bundle networks . Filaments were formed from oligomers containing 12 or more N-acetylneuraminic acid residues, and they were sensitive to sialidase digestion . The networks were also formed by the polysialic acid-containing carbohydrate units of N-CAM . The formation of filament bundles is a novel and unexpected property of polysialic acid and of short carbohydrate oligomers in general and represents a previously unrecognized molecular interaction mechanism which impacts both eukaryotic and prokaryotic cell-cell adhesions.

Mol Microbiol, 1998 Oct, 30(1), 121 - 34
Functional analysis of the Helicobacter pylori principal sigma subunit of RNA polymerase reveals that the spacer region is important for efficient transcription; Beier D et al.; We have cloned the rpoD gene encoding the principal sigma (sigma) factor of Helicobacter pylori . The deduced amino acid sequence reveals a predicted polypeptide of 676 residues that has amino acid homology with the principal sigma factors of a number of divergent prokaryotes . We have designated this factor sigma80 . Amino acid sequence analysis suggests that region 1.1 is missing in sigma80 and that a region with homology to a regulatory protein from Bacillus subtilis phage SPO1 is present . Genetic studies have indicated that sigma80 is not compatible with the transcriptional machinery of Escherichia coli . However, in vitro sigma80 could be assembled into the E . coli RNA polymerase and could bind to E . coli and H . pylori promoters, suggesting that the sigma80-containing RNA polymerase has the same stoichiometry as the native complex . By exchanging protein domains between E . coli and H . pylori sigma factors, we demonstrate that the sigma80 domain inhibiting transcription from E . coli promoters is confined within the non-conserved spacer region, implying that the spacer region of prokaryotic primary sigma factors plays an important role in the process of transcription . Consistent with its restricted niche and with the availability of a very restricted number of transcriptional regulators, H . pylori may have evolved a spacer region of the sigma factor to modulate total transcription and to quickly respond to microenvironmental changes.

J Mol Biol, 1998 Oct 30, 283(3), 537 - 47
Binding of the fur (ferric uptake regulator) repressor of Escherichia coli to arrays of the GATAAT sequence; Escolar L et al.; The mode of DNA binding of the Fur (ferric uptake regulator) repressor which controls transcription of iron-responsive genes in Escherichia coli, has been re-examined . Using as a reference the known sites at the promoter of the aerobactin operon of Escherichia coli, we have compared in detail the patterns of interaction between the purified Fur protein and natural or synthetic DNA targets . DNase I and hydroxyl radical footprinting, as well as missing-T assays, consistently revealed that functional Fur sites are composed of a minimum of three repeats of the hexameric motif GATAAT rather than by a palindromic 19 bp target sequence . Extended binding sites, constructed by stepwise addition of one or two direct repeats of the same sequence, were occupied co-operatively by Fur with the same pattern of interactions as those observed with the core of three repeats . This indicated that functional sites with a range of affinities can be formed by the addition of discrete GATAAT extensions to a minimal recognition sequence . The fashion in which Fur binds its target, virtually unknown in prokaryotic transcriptional regulators, accounts for the observed helical wrapping of the protein around the DNA helix .

Biochem Pharmacol, 1998 Sep 1, 56(5), 583 - 90
4-Hydroxy-17-methylincisterol, an inhibitor of DNA polymerase-alpha activity and the growth of human cancer cells in vitro; Togashi H et al.; An ergosterol derivative, 4-hydroxy-17-methylincisterol (HMI), was found to be an inhibitor of mammalian DNA polymerases in vitro . HMI inhibited the activity of calf thymus DNA polymerase alpha (pol . alpha) . Among the polymerases tested, pol . alpha was the most sensitive to inhibition by HMI, and the inhibition was concentration dependent . The inhibitory effect of HMI on pol . alpha was almost the same as that shown by aphidicolin, a well-known potent pol . alpha inhibitor . HMI had relatively less effect on rat DNA pol . beta, human immunodeficiency virus type 1 reverse transcriptase (HIV-RT), and calf thymus terminal deoxynucleotidyl transferase (TdT) in vitro, and did not influence the activities of prokaryotic DNA polymerases such as Klenow Fragment of DNA polymerase I, or the DNA-metabolic enzyme DNase I . HMI was found to be able to prevent the growth of human cancer cell lines originating from patients with leukemia or various solid tumors; its IC50 values ranged from 7.5 to 12 microM . We also synthesized other ergosterol derivatives and tested them, and found that two compounds, 17-methylincisterol and 4-acetyl-17-methylincisterol, have similar inhibitory effects.

Proc Int Conf Intell Syst Mol Biol, 1998, 6, 131 - 9
Bayesian protein family classifier; Qu K et al.; A Bayesian procedure for the simultaneous alignment and classification of sequences into subclasses is described . This Gibbs sampling algorithm iterates between an alignment step and a classification step . It employs Bayesian inference for the identification of the number of conserved columns, the number of motifs in each class, their size, and the size of the classes . Using Bayesian prediction, inter-class differences in all these variables are brought to bare on the classification . Application to a superfamily of cyclic nucleotide-binding proteins identifies both similarities and differences in the sequence characteristics of the five subclasses identified by the procedure: 1) cNMP-dependent kinases, 2) prokaryotic cAMP-dependent regulatory proteins, CRP-type, 3) prokaryotic regulatory proteins, FNR-type, 4) cAMP gated ion channel proteins of animals, and 5) cAMP gated ion channels of plants.

Extremophiles, 1998 Aug, 2(3), 191 - 200
Microbial diversity of soda lakes; Jones BE et al.; Soda lakes are highly alkaline extreme environments that form in closed drainage basins exposed to high evaporation rates . Because of the scarcity of Mg2+ and Ca2+ in the water chemistry, the lakes become enriched in CO3(2-) and Cl-, with pHs in the range 8 to > 12 . Although there is a clear difference in prokaryotic communities between the hypersaline lakes where NaCl concentrations are > 15% w/v and more dilute waters, i.e., NaCl concentrations about 5% w/v, photosynthetic primary production appears to be the basis of all nutrient recycling . In both the aerobic and anaerobic microbial communities the major trophic groups responsible for cycling of carbon and sulfur have in general been identified . Systematic studies have shown that the microbes are alkaliphilic and many represent separate lineages within accepted taxa, while others show no strong relationship to known prokaryotes . Although alkaliphiles are widespread it seems probable that these organisms, especially those unique to the hypersaline lakes, evolved separately within an alkaline environment . Although present-day soda lakes are geologically quite recent, they have probably existed since archaean times, permitting the evolution of independent communities of alkaliphiles since an early period in the Earth's history.

Adv Exp Med Biol, 1998, 440, 775 - 80
Prokaryotic expression of porcine epidemic diarrhoea virus ORF3; Schmitz A et al.; Wild type (wt) and cell culture adapted (ca) strains of the coronavirus PEDV differ in their ability to cause diarrhea in neonate piglets: the wt strains are virulent; the ca strains are attenuated . Comparison of the available nucleotide sequences obtained from the different viral isolates revealed almost complete sequence identity with the exception of variations and truncations in open reading frame 3 (ORF3) observed exclusively in ca-PEDV isolates . In order to study the biological function(s) of the putative ORF3 product, the molecule was expressed as a heterodimeric fusion protein in E . coli . ORF3 was fused in frame to the alkaline phosphatase gene . Simultaneously, the construct was designed to form specific heterodimers by inclusion of the well known leucine zipper motiv of Jun and Fos . The heterodimerization partner contained the E . coli heat-labile enterotoxin subunit B (LTB) to allow specific binding to the eukaryotic cell receptor GM1 . Our results indicate that heterodimeric fusion protein containing a truncated form of ORF3 was produced in high amounts, carried the expected ORF3 epitope, showed phosphatase activity, and was able to bind to the GM1 receptor . In contrast, a fusion protein containing the entire sequence of the ORF3 product was produced in minute amounts, indicating that it may have biological activity in prokaryotes, which led to the reduction of the amounts of proteins expressed.

Mol Biotechnol, 1998 Aug, 10(1), 83 - 5
A simple method to enrich mRNA from total prokaryotic RNA; Su C et al.; Isolation of prokaryotic mRNA by the poly(dT) method has been difficult, primarily due to the great instability of the poly(A) sequence in its mRNA . We developed a simple method to remove rRNA from total RNA of Staphylococcus aureus by cloning a PCR-amplified S . aureus rRNA gene fragment into a plasmid, and then synthesizing biotin-labeled antisense rRNA to subtract rRNA . By using this method, S . aureus rRNA is significantly reduced and mRNA is enriched . This method may be used to prepare prokaryotic mRNA for many molecular biology applications.

Curr Biol, 1998 Oct 8, 8(20), 1102 - 9
Chromosome arrangement within a bacterium; Teleman AA et al.; BACKGROUND: The contour length of the circular chromosome of bacteria is greater than a millimeter but must be accommodated within a cell that is only a few micrometers in length . Bacteria do not have nucleosomes and little is known about the arrangement of the chromosome inside a prokaryotic cell . RESULTS: We have investigated the arrangement of chromosomal DNA within the bacterium Bacillus subtilis by using fluorescence microscopy to visualize two sites on the chromosome simultaneously in the same cell . Indirect immunofluorescence with antibodies against the chromosome partition protein Spo0J were used to visualize the replication origin region of the chromosome . Green fluorescent protein fused to the lactose operon repressor Lacl was used to decorate tandem copies of the lactose operon operator lacO . A cassette of tandem operators was separately inserted into the chromosome near the origin (359 degrees), near the replication terminus (181 degrees), or at two points in between (90 degrees and 270 degrees) . The results show that the layout of the chromosome is dynamic but is principally arranged with the origin and terminus maximally apart and the quarter points of the chromosome in between . CONCLUSIONS: The use of cytological methods to visualize two chromosomal sites in the same cell has provided a glimpse of the arrangement of a bacterial chromosome . We conclude that, to a first approximation, the folding of the bacterial chromosome is consistent with, and may preserve, the linear order of genes on the DNA.

Nucleic Acids Res, 1998 Nov 1, 26(21), 4880 - 7
X-ray analysis of a complex of Escherichia coli uracil DNA glycosylase (EcUDG) with a proteinaceous inhibitor . The structure elucidation of a prokaryotic UDG; Ravishankar R et al.; Uracil-DNA glycosylase (UDG), a key highly conserved DNA repair enzyme involved in uracil excision repair, was discovered in Escherichia coli . The Bacillus subtilis bacteriophage, PBS-1 and PBS-2, which contain dUMP residues in their DNA, express a UDG inhibitor protein, Ugi which binds to UDG very tightly to form a physiologically irreversible complex . The X-ray analysis of the E . coli UDG ( Ec UDG)-Ugi complex at 3.2 A resolution, leads to the first structure elucidation of a bacterial UDG molecule . This structure is similar to the enzymes from human and viral sources . A comparison of the available structures involving UDG permits the delineation of the constant and the variable regions of the molecule . Structural comparison and mutational analysis also indicate that the mode of action of the enzyme from these sources are the same . The crystal structure shows a remarkable spatial conservation of the active site residues involved in DNA binding in spite of significant differences in the structure of the enzyme-inhibitor complex, in comparison with those from the mammalian and viral sources . Ec UDG could serve as a prototype for UDGs from pathogenic prokaryotes, and provide a framework for possible drug development against such pathogens with emphasis on features of the molecule that differ from those in the human enzyme.

Mol Cell Biol, 1998 Nov, 18(11), 6525 - 37
Mismatch repair proteins regulate heteroduplex formation during mitotic recombination in yeast; Chen W et al.; Mismatch repair (MMR) proteins actively inhibit recombination between diverged sequences in both prokaryotes and eukaryotes . Although the molecular basis of the antirecombination activity exerted by MMR proteins is unclear, it presumably involves the recognition of mismatches present in heteroduplex recombination intermediates . This recognition could be exerted during the initial stage of strand exchange, during the extension of heteroduplex DNA, or during the resolution of recombination intermediates . We previously used an assay system based on 350-bp inverted-repeat substrates to demonstrate that MMR proteins strongly inhibit mitotic recombination between diverged sequences in Saccharomyces cerevisiae . The assay system detects only those events that reverse the orientation of the region between the recombination substrates, which can occur as a result of either intrachromatid crossover or sister chromatid conversion . In the present study we sequenced the products of mitotic recombination between 94%-identical substrates in order to map gene conversion tracts in wild-type versus MMR-defective yeast strains . The sequence data indicate that (i) most recombination occurs via sister chromatid conversion and (ii) gene conversion tracts in an MMR-defective strain are significantly longer than those in an isogenic wild-type strain . The shortening of conversion tracts observed in a wild-type strain relative to an MMR-defective strain suggests that at least part of the antirecombination activity of MMR proteins derives from the blockage of heteroduplex extension in the presence of mismatches.

J Histochem Cytochem, 1998 Nov, 46(11), 1321 - 8
Immunoelectron microscopic study for polyamines; Fujiwara K et al.; The polyamines (PAs) are ubiquitous polycationic metabolites in eukaryotic and prokaryotic cells and are believed to be intimately involved in the regulation of DNA, RNA, and protein biosynthesis, the exact function of which remains unclear, mainly because of a lack of knowledge of PA subcellular localization . In this study, using immunoelectron microscopy, we have demonstrated that PAs are predominantly located on free and attached ribosomes of the rough endoplasmic reticulum in the neurons of the lateral reticular nucleus of rat medulla oblongata . The nuclei, axons, and nerve endings were devoid of PA . This suggests that PAs are one of the components of biologically active ribosomes, being closely involved in the translation processes of protein biosynthesis.

FEBS Lett, 1998 Sep 25, 436(1), 76 - 80
An Arabidopsis protein that interacts with the cytokinin-inducible response regulator, ARR4, implicated in the His-Asp phosphorylay signal transduction; Yamada H et al.; Previously, Arabidopsis thaliana was shown to possess a set of response regulators (ARR-series), which are implicated in the prokaryotic type of signal transduction mechanism, generally referred to as the His-Asp phosphorylay . Among them, ARR4 is a typical phospho-accepting response regulator, whose expression was recently demonstrated to be rapidly induced by a cytokinin-treatment of the plant . To gain insight into the presumed His-Asp phosphotransfer signaling mechanism as well as the role of ARR4 in this higher plant, in this study we adopt the widely used yeast two-hybrid system, and report the identification of an Arabidopsis protein that has an ability to interact physically with the cytokinin-inducible ARR4 response regulator.

Proc Natl Acad Sci U S A, 1998 Oct 13, 95(21), 12631 - 6
Sequence motifs in adenoviral DNA block immune activation by stimulatory CpG motifs; Krieg AM et al.; Unmethylated CpG dinucleotides in particular base contexts (CpG-S motifs) are relatively common in bacterial DNA but are rare in vertebrate DNA . B cells and monocytes have the ability to detect such CpG-S motifs that trigger innate immune defenses with production of Th1-like cytokines . Despite comparable levels of unmethylated CpG dinucleotides, DNA from serotype 12 adenovirus is immune-stimulatory, but serotype 2 is nonstimulatory and can even inhibit activation by bacterial DNA . In type 12 genomes, the distribution of CpG-flanking bases is similar to that predicted by chance . However, in type 2 adenoviral DNA the immune stimulatory CpG-S motifs are outnumbered by a 15- to 30-fold excess of CpG dinucleotides in clusters of direct repeats or with a C on the 5' side or a G on the 3' side . Synthetic oligodeoxynucleotides containing these putative neutralizing (CpG-N) motifs block immune activation by CpG-S motifs in vitro and in vivo . Eliminating 52 of the 134 CpG-N motifs present in a DNA vaccine markedly enhanced its Th1-like function in vivo, which was increased further by the addition of CpG-S motifs . Thus, depending on the CpG motif, prokaryotic DNA can be either immune-stimulatory or neutralizing . These results have important implications for understanding microbial pathogenesis and molecular evolution and for the clinical development of DNA vaccines and gene therapy vectors.

RNA, 1998 Oct, 4(10), 1203 - 15
Structure and stability of variants of the sarcin-ricin loop of 28S rRNA: NMR studies of the prokaryotic SRL and a functional mutant; Seggerson K et al.; NMR has been used to examine the conformational properties of two variants of the sarcin-ricin loop (SRL) from eukaryotic 28S rRNA, which is essential for elongation factor interactions with the ribosome: (1) its bacterial homologue, which lacks two of the bases that flank the conserved 12-nt sequence in the middle of the SRL, but which is functionally equivalent, and (2) a functionally active variant of the eukaryotic SRL in which the bulged G within the conserved sequence is replaced by an A . The data indicate that, although the bacterial SRL is less stable than the eukaryotic SRL, its conformation is closely similar . Furthermore, even though replacement of the bulged G in the SRL with an A seriously destabilizes the center of the loop, its effect on the overall conformation of the SRL appears to be modest . In the course of this work, it was serendipitously discovered that at neutral pH, the C8 proton of the bulged G, in both PRO-SRL and E73, exchanges about 10 times faster than it does in GMP.

Curr Biol, 1998 Sep 24, 8(19), 1079 - 82
The Antirrhinum ERG gene encodes a protein related to bacterial small GTPases and is required for embryonic viability; Ingram GC et al.; Small GTPases have diverse roles in animals and yeast, including signal transduction, regulation of secretion, organisation of the cytoskeleton, and control of cell division . Similar GTPases have also been found in bacteria, such as the Escherichia coli GTPase ERA, which is involved in regulating metabolism and cell division {1,2} . Many small GTPases have been cloned from plants but their functional analysis has largely been limited to complementation of mutations in corresponding yeast genes, and antisense experiments which have implicated these proteins in processes such as root nodulation {3,4} . No mutations in plant GTPases have been reported, and thus their true importance in plant growth and development is unknown . Here we report the isolation of a gene from Antirrhinum majus encoding a protein from an entirely novel class of eukaryotic GTPases showing strongest similarity to the prokaryotic protein ERA . We have named this gene ERG (for ERA-related GTPase) . The ERG gene is expressed in dividing or metabolically active cells . We generated a deletion allele of ERG by site-selected transposon mutagenesis and have shown that seeds containing embryos and endosperm homozygous for this deletion arrest soon after fertilisation . We conclude that ERG has a crucial role in plant growth and development, possibly by influencing mitochondrial division.

Gene, 1998 Oct 5, 220(1-2), 61 - 70
Genomic organization of four beta-1,4-endoglucanase genes in plant-parasitic cyst nematodes and its evolutionary implications; Yan Y et al.; The genomic organization of genes encoding beta-1,4-endoglucanases (cellulases) from the plant-parasitic cyst nematodes Heterodera glycines and Globodera rostochiensis (HG-eng1, Hg-eng2, GR-eng1, and GR-eng2) was investigated . HG-eng1 and GR-eng1 both contained eight introns and structural domains of 2151 and 2492bp, respectively . HG-eng2 and GR-eng2 both contained seven introns and structural domains of 2324 and 2388bp, respectively . No significant similarity in intron sequence or size was observed between HG-eng1 and HG-eng2, whereas the opposite was true between GR-eng1 and GR-eng2 . Intron positions among all four cyst nematode cellulase genes were conserved identically in relation to the predicted amino acid sequence . HG-eng1, GR-eng1, and GR-eng2 had several introns demarcated by 5'-GCellipsisAG-3' in the splice sites, and all four nematode cellulase genes had the polyadenylation and cleavage signal sequence 5'-GAUAAA-3'-both rare occurences in eukaryotic genes . The 5'- flanking regions of each nematode cellulase gene, however, had signature sequences typical of eukaryotic promoter regions, including a TATA box, bHLH-type binding sites, and putative silencer, repressor, and enhancer elements . Database searches and subsequent phylogenetic comparison of the catalytic domain of the nematode cellulases placed the nematode genes in one group, with Family 5, subfamily 2, glycosyl hydrolases from Scotobacteria and Bacilliaceae as the most homologous groups . The overall amino acid sequence identity among the four nematode cellulases was from 71 to 83%, and the amino acid sequence identity to bacterial Family 5 cellulases ranged from 33 to 44% . The eukaryotic organization of the four cyst nematode cellulases suggests that they share a common ancestor, and their strong homology to prokaryotic glycosyl hydrolases may be indicative of an ancient horizontal gene transfer.

Gene, 1998 Oct 5, 220(1-2), 1 - 12
A new member of YER057c family in Trypanosoma cruzi is adjacent to an ABC-transporter; Robello C et al.; Tcp17 is a Trypanosoma cruzi gene located contiguous to the ABC-transporter tcpgp2 . The protein contains 160 amino acid residues with a predicted molecular mass of 16.5kDa . Western blot analysis using a polyclonal antiserum against recombinant TCP17 revealed that the protein is only expressed in the epimastigote form of the parasite; we did not detect the protein either in the amastigote or trypomastigote forms . A sequence comparison of TCP17 showed a remarkable homology with a conserved family of prokaryotic and eukaryotic proteins called YER057c whose function has not yet been characterized . Here, we propose a new signature of this family considering the N-terminal: {IV}-X(4)-{AV}-{AP}-X-{AP}-X(3)-Y-X(9)-{LIVF}-X(2)-{SA}-G-{QS}, and the C-terminal: {AT}-R-X(2)-{IVFY}-X-{VC}-X(2)-L-P-X(4)-{LIVM}-E-{IVM} -{DE} motifs . Immunofluorescence and immunoelectron microscopy studies suggest that the protein has a wide distribution in the cell, with a higher concentration in the external side of the plasma membrane, on the Golgi complex and on cytoplasmic vacuoles . Although the physiological function of TCP17 is unknown, its conservation in evolution suggests biological relevance in the parasite.

Res Microbiol, 1998 Jan, 149(1), 55 - 64
Genetic and serological analysis of the immunogenic 67-kDa lipoprotein of Mycoplasma sp . bovine group 7; Frey J et al.; The gene encoding a lipoprotein of 67 kDa, named P67, was cloned from Mycoplasma sp . bovine group 7 strain PG50 and expressed in Escherichia coli K12 . Analysis of the amino acid sequence derived from the DNA sequence of the P67 gene revealed a typical prokaryotic signal peptidase II membrane lipoprotein lipid attachment site and a transmembrane structure domain in the leader sequence at the amino-terminal end of the protein . Protein P67 showed 91% identical amino acid residues to the lipoprotein P72 of Mycoplasma mycoides subsp . mycoides small colony type (SC) and 53% identical amino acid residues to a peptide of an unassigned gene on the genome of Mycoplasma capricolum subsp . capricolum . Antibodies made against recombinant P67 reacted with a 67-kDa protein in all Mycoplasma sp . bovine group 7 strains tested and also, to some extent, with P72 of Mycoplasma mycoides subsp . mycoides SC . The gene encoding P67 was present in all strains of Mycoplasma sp . bovine group 7 analysed, but not in other Mycoplasma sp . of the "mycoides cluster" and not in the phylogenetically related Mycoplasma putrefaciens . PCR and restriction fragment analysis revealed that the gene of P67 is conserved in all strains of Mycoplasma sp . bovine group 7 . A specific PCR reaction based on the P67 gene sequence enabled rapid identification of strains belonging to Mycoplasma sp . bovine group 7.

Res Microbiol, 1997 May, 148(4), 345 - 54
Prochlorothrix hollandica PCC 9006: genomic properties of an axenic representative of the chlorophyll a/b-containing oxyphotobacteria; Schyns G et al.; Prochlorothrix hollandica is an oxygenic photosynthetic prokaryote that differs from the cyanobacteria in having chlorophyll a/b-protein complexes instead of phycobilisomes as major light-harvesting antennae . We report the isolation and culturing of an axenic strain of P . hollandica, available from the Pasteur Culture Collection of Cyanobacteria as strain PCC 9006 . The strain has a mean DNA base composition of 51.6 +/- 0.1 mol% G+C and a genomic complexity of 3.37 +/- 0.17 x 10(9) daltons (5,505 kb) . A reiterated DNA sequence represents approximately 4.4% of the genome . Restriction enzyme isoschizomers with different sensitivities to base methylation were used to demonstrate that most A residues in the sequence GATC are methylated in P . hollandica DNA and that this methylation increases with culture age . Furthermore, some C residues are methylated, although the specificity of the C methylation system does not match that of well-characterized C methylases . Nucleotide analysis showed that up to approximately 3.5% of both dA and dC residues are methylated in P . hollandica DNA.

Biochim Biophys Acta, 1998 Aug 20, 1399(2-3), 141 - 53
A putative lichenysin A synthetase operon in Bacillus licheniformis: initial characterization; Yakimov MM et al.; Certain Bacillus licheniformis strains isolated from oil wells have been shown to produce a very effective biosurfactant, lichenysin A, which is structurally similar to another less active lipopeptide, surfactin . Surfactin, like many small peptides in prokaryotes and lower eukaryotes, is synthesized non-ribosomally by multi-enzyme peptide synthetase complex . Analysis of several peptide synthetases of bacterial and fungal origin has revealed a high degree of sequence conservation . Two 35-mer oligonucleotides derived from highly conserved motifs ('core I' and 'core II') of surfactin synthetase were used to identify the cloned putative operon of lichenysin A synthetase lchA from B . licheniformis BNP29, a strain not amenable to genetic manipulation in a BAC system (F-plasmid-based bacterial artificial chromosome) based on Escherichia coli and its single-copy plasmid F-factor . A 32.4 kb fragment containing lichenysin A biosynthesis locus was sequenced and analysed . The structural architecture of putative lichenysin A synthetase protein containing seven amino acid (aa) activation-thiolation, two epimerization and one thioesterase domains is discussed in terms of its similarity to surfactin and other peptide synthetases . The 100 aa peptide chain situated between the highly conserved signature sequences FDXX and NXYGPTE(IV)X within amino acid binding domains of peptide synthetases is proposed to be a minimal block dictating the substrate specificity of the enzymes . A new operon-type structure has been localized directly upstream from the lichenysin A synthetase genes which, on the basis of sequence determination, potentially encode a four-member ABC-type transport system involved in product secretion.

Plant Physiol, 1998 Oct, 118(2), 661 - 74
Comparative analysis of the regulation of expression and structures of two evolutionarily divergent genes for Delta1-pyrroline-5-carboxylate synthetase from tomato; Fujita T et al.; We isolated two tomato (Lycopersicon esculentum) cDNA clones, tomPRO1 and tomPRO2, specifying Delta1-pyrroline-5-carboxylate synthetase (P5CS), the first enzyme of proline (Pro) biosynthesis . tomPRO1 is unusual because it resembles prokaryotic polycistronic operons (M.G . Garcia-Rios, T . Fujita, P.C . LaRosa, R.D . Locy, J.M . Clithero, R.A . Bressan, L.N . Csonka {1997} Proc Natl Acad Sci USA 94: 8249-8254), whereas tomPRO2 encodes a full-length P5CS . We analyzed the accumulation of Pro and the tomPRO1 and tomPRO2 messages in response to NaCl stress and developmental signals . Treatment with 200 mM NaCl resulted in a >60-fold increase in Pro levels in roots and leaves . However, there was a <3-fold increase in the accumulation of the tomPRO2 message and no detectable induction in the level of the tomPRO1 message in response to NaCl stress . Although pollen contained approximately 100-fold higher levels of Pro than other plant tissues, there was no detectable increase in the level of either message in pollen . We conclude that transcriptional regulation of these genes for P5CS is probably not important for the osmotic or pollen-specific regulation of Pro synthesis in tomato . Using restriction fragment-length polymorphism mapping, we determined the locations of tomPRO1 and tomPRO2 loci in the tomato nuclear genome . Sequence comparison suggested that tomPRO1 is similar to prokaryotic P5CS loci, whereas tomPRO2 is closely related to other eukaryotic P5CS genes.

Farmaco, 1998 Jun 30, 53(6), 431 - 7
Synthesis and biological properties of a new series of N-pyrido substituted tetrahydrocarbazoles; Ferlin MG et al.; A series of methyl and ethyl quaternary pyridiniumtetrahydrocarbazoles was synthesized and studied in comparison with ellipticine, chosen as a reference . In general, their antiproliferative activity, tested in different biological substrates, appeared to be higher than that of the corresponding non-quaternarized compounds . This fact could be attributed to the introduction of a positive charge in the molecule, which can stabilize the molecular complex they form with DNA . In a prokaryotic system, the T2 bacteriophage, both quaternarized and non-quaternarized compounds inhibited its infectivity moderately, in a similar way to ellipticine . This effect seemed to be connected to a direct activity on the virions rather than on the indicator bacteria . In mammalian cells, the pyridiniumtetrahydrocarbazoles were more effective . In particular, they appeared to be very active in inhibiting DNA synthesis in Ehrlich ascites cells; some of them were as effective as ellipticine . However, pyridiniumtetrahydrocarbazoles were less active in comparison with ellipticine when their capacity for inhibiting the clonal growth in Chinese hamster ovary (CHO) cells was tested . A similar picture was obtained studying the formation of chromosome aberrations and of sister chromatid exchanges in the same cells . These different responses can be explained considering that the data on DNA synthesis reflect effects only on DNA replication within a short time, without considering any later consequences; on the contrary, in the long-term tests, other events, which lead to cell killing or genotoxicity, can take place . Pyridiniumtetrahydrocarbazoles damage DNA, inducing double-strand breaks efficiently . These observations, together with the data already obtained on unsubstituted derivatives, suggest the pyridiniumtetrahydrocarbazoles induce antiproliferative and genotoxic effects, very probably by inhibiting topoisomerase II.

J Mol Biol, 1998, 283(1), 43 - 58
Sequence-dependent extrusion of a small DNA hairpin at the N4 virion RNA polymerase promoters; Dai X et al.; Bacteriophage N4 virion RNA polymerase promoters contain five to seven-base inverted repeats separated by three bases and centered at position -12 from the site of transcription initiation . We have previously shown that these inverted repeats extrude as hairpins at physiological superhelical densities in a Mg(II)-dependent manner . Mg(II)-dependent hairpin extrusion at promoters P1 and P2 displays quantitative differences in reactivity to structural probes at different DNA superhelical densities, with extrusion at P2 being more favored at low superhelical density . Analyses of mutant promoters using structure-specific probes revealed that specific sequences, at the closing base-pair of the hairpin and at the loop (i.e . 5'-C-GXA-G-3' where X=G, A, T), are required for extrusion of the small promoter hairpins at physiological superhelical density . The sequence-dependent requirements for extrusion of the small N4 promoter hairpins may be generally applicable for other such sequences found both in prokaryotic and eukaryotic genomes .

Biochemistry, 1998 Oct 6, 37(40), 13941 - 6
Residues critical for formylglycine formation and/or catalytic activity of arylsulfatase A; Knaust A et al.; Sulfatases contain a unique posttranslational modification in their active site, a formylglycine residue generated from a cysteine or a serine residue . The formylglycine residue is part of a sequence that is highly conserved among sulfatases, suggesting that it might direct the generation of this unique amino acid derivative . In the present study residues 68-86 flanking formylglycine 69 in arylsulfatase A were subjected to an alanine/glycine scanning mutagenesis . The mutants were analyzed for the conversion of cysteine 69 to formylglycine and their kinetic properties . Only cysteine 69 turned out to be essential for formation of the formylglycine residue, while substitution of leucine 68, proline 71, and alanine 74 within the heptapeptide LCTPSRA reduced the formylglycine formation to about 30-50% . Several residues that are part of or directly adjacent to an alpha-helix presenting the formylglycine 69 at the bottom of the active site pocket were found to be critical for catalysis . A surprising outcome of this study was that a number of residues fully or highly conserved between all known eukaryotic and prokaryotic sulfatases turned out to be essential neither for generation of formylglycine nor for catalysis.

Clin Exp Allergy, 1998 Aug, 28(8), 984 - 91
Expression of two isoforms of Lep d 2, the major allergen of Lepidoglyphus destructor, in both prokaryotic and eukaryotic systems; Olsson S et al.; BACKGROUND: The dust mite Lepidoglyphus destructor is a major cause of allergic diseases among farmers . We have previously cloned and sequenced two isoforms of the major allergen Lep d 2 (formerly designated Lep d 1) and found significant homology to group 2 allergens of the house dust mite species Dermatophagoides . We now report on the production and characterization of recombinant Lep d 2 . OBJECTIVE: We have expressed both isoforms in two different expression systems; a eukaryotic system, baculovirus in insect cells and a prokaryotic system, E . coli . We have compared the two systems in regard to production yields and immunoreactivity of the recombinant allergens . METHODS: The complete cDNA including the natural leader sequence was cloned into the pBlueBacIII transfer vector, and the rLep d 2 was produced as a secreted protein in baculovirus . For the expression in E . coli, the cDNA was cloned into the pET vector, and the rLep d 2 was produced with six C-terminal histidine residues . The purified recombinant allergens were tested for immunoreactivity with 10 sera from subjects allergic to Lepidoglyphus destructor and were compared with native Lep d 2 using inhibition immunoblotting . The ability of the recombinant allergens to release histamine from basophils was evaluated using a histamine release assay . RESULTS: Both expression systems produced immunoreactive recombinant allergens . They inhibited the binding of human sera to native Lep d 2 confirming their retained IgE binding properties . The yield of pure recombinant protein from the prokaryotic system was approximately 1 mg/L compared to the eukaryotic system which produced up to 4 mg/L in an adherent cell culture system . CONCLUSIONS: We have produced recombinant Lep d 2 in prokaryotic and eukaryotic expression systems which are comparable to the native allergen . Recombinant Lep d 2 might now be included in more extensive clinical studies to confirm its usefulness in the in vitro and the in vivo diagnosis of Lepidoglyphus destructor.

EMBO J, 1998 Oct 1, 17(19), 5563 - 76
A plasma membrane-bound putative endo-1,4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis; Nicol F et al.; Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes . In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall . Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion . Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells . KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall . The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants . KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface . KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation . Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels . However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids . Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.

Indian J Exp Biol, 1998 Feb, 36(2), 136 - 47
Calcium-dependent metabolic regulations in prokaryotes indicate conserved nature of calmodulin gene; Sengupta LK et al.; Role of free calcium and calcium binding protein calmodulin as signal molecule in cellular regulation is well established in eukaryotes . However, reports on Ca(2+)-dependent processes and their inhibition by calcium and/or calmodulin antagonists indicate towards the presence of calmodulin in prokaryotes as well . The common evolutionary origin of pro- and eukaryotes and many examples of evolutionary conservation of structure and functions support the contention of such conservation of the role of Ca2+ and calmodulin . Eukaryotic calmodulin (CaM) contains four structurally and functionally similar Ca2+ domains named I, II, III and IV . Each Ca2+ binding loop consists of 12 amino acid residues with ligands arranged spatially to satisfy the octahedral symmetry of Ca2+ binding . Plant calmodulin differ from vertebrate ones in 13 to 14 amino acid positions of which nine occur at -COOH- terminal half . Differences between protozoan and mammalian CaM also occur mostly in the same half . Isolation and characterization, although to a little extent, of CaM-like proteins from bacteria and cyanobacteria and their comparison with CaMs from diverse origin suggest high degree of conservation . Non-bulky amino acids like glycine, alanine and serine with low specific rotation are present in greater number in the primitive form of calmodulin and have been significantly reduced in highly evolved form of calmodulin, suggesting that their requirement was insignificant and were eliminated from EF hand structure during evolution . However, amino acids like glutamate/glutamine and aspartate/asparagine were highly conserved and did not show any major change in their frequency since their positions are too significant in calcium binding domain . While the number of positively charged amino acids like arginine and leucine was increased, histidine containing weakly ionized group and having a significant buffering capacity was reduced to a major extent, further suggesting that the acidic nature of calmodulin protein has been maintained during evolution . Thus it is now clear that the entire superfamily of Ca2+ binding proteins have arisen from a common genetic ancestry . Two successive tandem duplications of gene encoding a single domain containing protein of 30-40 residues gave rise to a four domain molecule from which this family was then derived.

Nucleic Acids Res, 1998 Oct 15, 26(20), 4626 - 34
Regulated processive transcription of chromatin by T7 RNA polymerase in Trypanosoma brucei; Wirtz E et al.; Inability of T7 RNA polymerase to processively transcribe higher eukaryotic chromatin is interpreted as a correlate of its reported inhibition by nucleosomes on reconstituted templates in vitro . We used chromosomally integrated reporter cassettes to examine features of T7 transcription in a lower eukaryotic system . Luciferase reporters were targeted to rDNA in transgenic Trypanosoma brucei stably expressing the phage polymerase . Because trypanosome mRNAs are capped by RNA splicing in trans , T7 transcription could be gauged by luciferase activity . In contrast to findings from higher eukaryotes, T7 transcription is vigorous and processive on chromatin templates in T.brucei , surpassing levels achieved with endogenous promoters, including those recruiting RNA polymerase I . This may be a reflection of intrinsic differences in chromatin structure between differently evolved eukaryotes or of an integration site that is exceptionally permissive for T7 transcription due to a local accessible chromatin conformation . T7 transcription could be manipulated to achieve different levels of constitutive expression, through the use of promoter mutations . Moreover, T7 initiation could be regulated by the prokaryotic Tet repressor and elongation halted by T7 terminator sequences . We have exploited these features to construct a robust inducible expression system, whose utility potentially extends to other trans -splicing organisms.

Structure, 1998 Sep 15, 6(9), 1105 - 16
The crystal structure of human cytosolic serine hydroxymethyltransferase: a target for cancer chemotherapy; Renwick SB et al.; BACKGROUND: Serine hydroxymethyltransferase (SHMT) is a ubiquitous enzyme found in all prokaryotes and eukaryotes . As an enzyme of the thymidylate synthase metabolic cycle, SHMT catalyses the retro-aldol cleavage of serine to glycine, with the resulting hydroxymethyl group being transferred to tetrahydrofolate to form 5, 10-methylene-tetrahydrofolate . The latter is the major source of one-carbon units in metabolism . Elevated SHMT activity has been shown to be coupled to the increased demand for DNA synthesis in rapidly proliferating cells, particularly tumour cells . Consequently, the central role of SHMT in nucleotide biosynthesis makes it an attractive target for cancer chemotherapy . RESULTS: We have solved the crystal structure of human cytosolic SHMT by multiple isomorphous replacement to 2.65 A resolution . The monomer has a fold typical for alpha class pyridoxal 5'-phosphate (PLP) dependent enzymes . The tetramer association is best described as a 'dimer of dimers' where residues from both subunits of one 'tight' dimer contribute to the active site . CONCLUSIONS: The crystal structure shows the evolutionary relationship between SHMT and other alpha class PLP-dependent enzymes, as the fold is highly conserved . Many of the results of site-directed mutagenesis studies can easily be rationalised or re-interpreted in light of the structure presented here . For example, His 151 is not the catalytic base, contrary to the findings of others . A mechanism for the cleavage of serine to glycine and formaldehyde is proposed.

J Mol Biol, 1998 Oct 9, 282(5), 969 - 90
Complete sequence of the IncPbeta plasmid R751: implications for evolution and organisation of the IncP backbone; Thorsted PB et al.; The broad host range IncP plasmids are of particular interest because of their ability to promote gene spread between diverse bacterial species . To facilitate study of these plasmids we have compiled the complete sequence of the IncPbeta plasmid R751 . Comparison with the sequence of the IncPalpha plasmids confirms the conservation of the IncP backbone of replication, conjugative transfer and stable inheritance functions between the two branches of this family . As in the IncPalpha genome the DNA of this backbone appears to have been enriched for the GCCG/CGGC motifs characteristic of the genome of organisms with a high G+C content, such as P . aeruginosa, suggesting that IncPbeta plasmids have been subjected during their evolution to similar mutational and selective forces as IncPalpha plasmids and may have evolved in pseudomonad hosts . The IncP genome is consistently interrupted by insertion of phenotypic markers and/or transposable elements between oriV and trfA and between the tra and trb operons . The R751 genome reveals a family of repeated sequences in these regions which may form the basis of a hot spot for insertion of foreign DNA . Sequence analysis of the cryptic transposon Tn4321 revealed that it is not a member of the Tn21 family as we had proposed previously from an inspection of its ends . Rather it is a composite transposon defined by inverted repeats of a 1347 bp IS element belonging to a recently discovered family which is distributed throughout the prokaryotes . The central unique region of Tn4321 encodes two predicted proteins, one of which is a regulatory protein while the other is presumably responsible for an as yet unidentified phenotype . The most striking feature of the IncPalpha plasmids, the global regulation of replication and transfer by the KorA and KorB proteins encoded in the central control operon, is conserved between the two plasmids although there appear to be significant differences in the specificity of repressor-operator interactions . The importance of these global regulatory circuits is emphasised by the observation that the operator sequences for KorB are highly conserved even in contexts where the surrounding region, either a protein coding or intergenic sequence, has diverged considerably . There appears to be no equivalent of the parABCDE region which in the IncPalpha plasmids provides multimer resolution, lethality to plasmid-free segregants and active partitioning functions . However, we found that the continuous sector from co-ordinate 0 to 9100 bp, encoding the co-regulated klc and kle operons as well as the central control region, could confer a high degree of segregational stability on a low copy number test vector . Thus R751 appears to exhibit very clearly what was first revealed by study of the IncPalpha plasmids, namely a fully functional co-ordinately regulated set of replication, transfer and stable inheritance functions .

Cell, 1998 Sep 18, 94(6), 819 - 27
The structure of supercoiled intermediates in DNA replication; Peter BJ et al.; We studied the structure of replication intermediates accumulated by Tus-induced arrest of plasmid DNA replication at termination sites . For intermediates generated both in vitro with purified components and in vivo, superhelical stress is distributed throughout the entire partially replicated molecule; daughter DNA segments are wound around each other, and the unreplicated region is supercoiled . Thus, unlinking of parental DNA strands by topoisomerases can be carried out both behind and in front of the replication fork . We explain why previous studies with prokaryotic and eukaryotic replication intermediates discerned only supercoiling in the unreplicated portion.

Proc Natl Acad Sci U S A, 1998 Sep 29, 95(20), 11655 - 60
Promoter opening via a DNA fork junction binding activity; Guo Y et al.; The rate-limiting step in transcriptional initiation typically is opening the promoter DNA to expose the template strand . Opening is tightly regulated, but how it occurs is not known . These experiments identify an activity, recognition of specific DNA fork junctions, and suggest that it is critical to bacterial promoter opening . This activity is both sequence and structure specific; it recognizes the bases that constitute the upstream double-stranded/single-stranded boundary of the open complex . Promoter mutations known to reduce opening rates lead to comparable reductions in fork junction binding affinity . The activity acts to establish the upstream boundary of melted DNA and works in conjunction with two single-stranded DNA binding activities that recognize separately the two melted strands . The junction binding activity is contained within the sigma factor component of the holoenzyme . The activity occurs in both a typical prokaryotic transcription system and in a eukaryotic-like bacterial system that responds to enhancers and needs ATP . Thus DNA opening catalyzed by fork junction binding may occur in a variety of systems in which DNA must be opened to be copied.

Curr Opin Biotechnol, 1998 Aug, 9(4), 344 - 349
Membrane protein structures: the known world expands; Garavito RM; The structure determinations of the cytochrome bc1 complex and the prokaryotic potassium channel demonstrate that a wider range of membrane proteins are now amenable to study by X-ray crystallography . Furthermore, the structures of porins and interfacial membrane proteins show that membrane structural biology is becoming a mature and productive field.

Mol Cells, 1998 Aug 31, 8(4), 452 - 8
Expressed sequence tags of radish flower buds and characterization of a CONSTANS LIKE 1 gene; Moon YH et al.; Expressed sequence tag (EST) analysis was conducted for young flower buds of radish plants . Among a total of 66 ESTs examined, 40 showed a significant similarity to previously identified genes . Twenty-eight ESTs were similar to proteins identified in other plants, 11 were similar to eukaryotic proteins other than plants, and one was similar to a prokaryotic protein . Four clones were selected for further studies . EST clone 81, which showed a homology to germin-like proteins was expressed more abundantly in leaves and roots as compared to flower buds . Clone 105 was highly homologous to the translation inhibitor protein and was expressed in all three organs, but the expression level was higher in flower buds and roots . Another EST clone, 133, which shared a significant similarity with the Ran-binding protein, hybridized to two different size transcripts that were detectable only in flower buds . Clone 39 was a homolog of CONSTANS, which is a gene involved in controlling the flowering time in Arabidopsis . The cDNA clone of EST clone 39 containing the entire open reading frame was obtained and designated as RsCOL1 (Raphanus sativus CONSTANS LIKE 1) . It was 1049 bp long and contained an open reading frame of 307 amino acid residues (calculated molecular mass = 33.1 kDa) . The RsCOL1 protein contained two putative zinc finger motifs in the amino terminal region which were 59% identical to the corresponding region of the Arabidopsis CO protein . The radish protein also contained a predicted nuclear localization domain in the carboxyl terminal region which was 87% identical to the corresponding region of CO . DNA blot analysis revealed that the radish genome contained several genes similar to RsCOL1 . RNA blot analysis showed that RsCOL1 was strongly expressed in flower buds at the early bolting stage, and the expression level declined as the flower bud matured . The transcript was also detectable in leaves and roots . In mature flowers, the RsCOL1 transcript was present primarily in carpels.

J Bacteriol, 1998 Oct, 180(19), 5003 - 9
Genomic analysis reveals chromosomal variation in natural populations of the uncultured psychrophilic archaeon Cenarchaeum symbiosum; Schleper C et al.; Molecular phylogenetic surveys have recently revealed an ecologically widespread crenarchaeal group that inhabits cold and temperate terrestrial and marine environments . To date these organisms have resisted isolation in pure culture, and so their phenotypic and genotypic characteristics remain largely unknown . To characterize these archaea, and to extend methodological approaches for characterizing uncultivated microorganisms, we initiated genomic analyses of the nonthermophilic crenarchaeote Cenarchaeum symbiosum found living in association with a marine sponge, Axinella mexicana . Complex DNA libraries derived from the host-symbiont population yielded several large clones containing the ribosomal operon from C . symbiosum . Unexpectedly, cloning and sequence analysis revealed the presence of two closely related variants that were consistently found in the majority of host individuals analyzed . Homologous regions from the two variants were sequenced and compared in detail . The variants exhibit >99.2% sequence identity in both small- and large-subunit rRNA genes and they contain homologous protein-encoding genes in identical order and orientation over a 28-kbp overlapping region . Our study not only indicates the potential for characterizing uncultivated prokaryotes by genome sequencing but also identifies the primary complication inherent in the approach: the widespread genomic microheterogeneity in naturally occurring prokaryotic populations.

Plant Mol Biol, 1998 Oct, 38(3), 497 - 502
Maize mitochondrial seryl-tRNA synthetase recognizes Escherichia coli tRNA(Ser) in vivo and in vitro; Rokov J et al.; In our studies to analyze the structure/function relationships among cytoplasmic and organellar seryl-tRNA synthetases (SerRS), we have characterized a Zea mays cDNA (SerZMm) encoding a protein with significant similarity to prokaryotic SerRS enzymes . To demonstrate the functional identity of SerZMm, the gene sequence encoding the putative mature protein was cloned . This construct complemented in vivo a temperature-sensitive Escherichia coli serS mutant strain . The mature SerZMm protein overexpressed in Escherichia coli efficiently aminoacylated bacterial tRNA(Ser) in vitro, while yeast tRNA was a poor substrate . These data identify SerZMm as an organellar maize seryl-tRNA synthetase, the first plant organellar SerRS to be cloned . The analysis of its N-terminal targeting signal suggests a mitochondrial function for the SerZMm protein in maize.

Curr Eye Res, 1998 Sep, 17(9), 870 - 4
Cloning and expression of human corneal calgranulin C (CO-Ag); Gottsch JD et al.; PURPOSE: A host-parasite interaction is thought to be involved in the pathogenesis of Mooren's ulcer . We have identified a cornea-associated antigen (CO-Ag), which may be a target for the autoimmune process resulting in Mooren's ulcer . This study presents the cloning, expression, and identification of a cDNA encoding human CO-Ag . METHODS: Reverse transcription-polymerase chain reaction (RT-PCR) was performed to amplify a cDNA encoding CO-Ag in the human cornea . The cDNA fragment was cloned into a prokaryotic expression vector and the resulting plasmid was transformed into DH5 E . coli cells . Autoantibody reactivity to the CO-Ag fusion protein in patient sera was tested by Western blots . RESULTS: A cDNA encoding human CO-Ag was amplified by RT-PCR . The entire mRNA coding region was 273 nucleotides in length, predicting a 91-amino acid protein with a molecular weight of 10,683 daltons . The cDNA sequence was identical to human neutrophil calgranulin C (CaGC) . Human CO-Ag was expressed in E . coli carrying a plasmid in which the CO-Ag cDNA was under control of the E . coli trc promoter . The CO-Ag fusion protein, which comprised as much as 15% of the total bacterial protein, was purified to 90% homogeneity by affinity chromatography on an immobilized metal column . The recombinant CO-Ag protein produced was recognized by autoantibodies in the sera of 6 of 15 patients with Mooren's ulcer and none of 14 normal control sera by Western blots . CONCLUSION: CO-Ag is identical to calgranulin C, a neutrophil protein found on the surface of filarial nematodes . A host-parasite interaction may cause autoimmunity to CO-Ag (CaGC) in the cornea resulting in a Mooren's ulcer.

Curr Genet, 1998 Sep, 34(3), 212 - 5
The rbcL gene from the non-photosynthetic parasite Lathraea clandestina is not transcribed by a plastid-encoded RNA polymerase; Lusson NA et al.; In the plastome of the obligate root-parasitic plant, Lathraea clandestina, the rbcL gene has been maintained and is expressed, despite the reduced size and gene content of the plastid genome . Some of the plastid genes involved in translation (e . g . transfer RNAs, ribosomal RNAs and ribosomal proteins) have been sequenced and still appear to code for functional ribosomal components . Indeed, the 16S rRNA and rpl20 genes are expressed whilst other necessary tRNA and ribosomal protein-encoding genes have probably been deleted or truncated . Although obtained by PCR, the four rpo genes for Escherichia coli-like plastid encoded RNA polymerase appear to be pseudogenes . Nevertheless, the rbcL gene, with a "-10, -35" prokaryotic-like promoter, is still transcribed . In contrast to photosynthetic plants, rbcL transcripts in Lathraea are larger in their 5' region and cover the prokaryotic-like promoter . The transcription initiation site is located near the ATG start codon of the atpB pseudogene . Similarity to non-consensus E . coli-like plastid promoters suggests that rbcL transcription is driven by a nuclear-encoded RNA polymerase.

J Mol Biol, 1998 Oct 2, 282(4), 761 - 74
Cloning and characterization of a mouse homologue (mNthl1) of Escherichia coli endonuclease III; Sarker AH et al.; Endonuclease III (endoIII; nth gene product) of Escherichia coli is known to be a DNA repair enzyme having a relatively broad specificity for damaged pyrimidine bases of DNA . Here, we describe the cloning and characterization of the cDNA and the gene for a mouse homologue (mNthl1/mNth1) of endoIII . The cDNA was cloned from a mouse T-cell cDNA library with a probe prepared by PCR using the library and specific PCR primers synthesized based on the reported information of partial amino acid sequences of bovine NTHL1/NTH1 and of EST Data Bases . The cDNA is 1025 nucleotides long and encodes a protein consisting of 300 amino acids with a predicted molecular mass of 33.6 kDa . The amino acid sequence exhibits significant homologies to those of endoIII and its prokaryotic and eukaryotic homologues . The recombinant mNthl1 with a hexahistidine tag was overexpressed in a nth::cmr nei::Kmr double mutant of E . coli, and purified to apparent homogeneity . The enzyme showed thymine glycol DNA glycosylase, urea DNA glycosylase and AP lyase activities . Northern blot analysis indicated that mNthl1 mRNA is about 1 kb and is expressed ubiquitously . A 15 kb DNA fragment containing the mNthl1 gene was cloned from a mouse genomic library and sequenced . The gene consists of six exons and five introns spanning 6.09 kb . The sequenced 5' flanking region lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors such as Ets, Sp1, AP-1 and AP-2 . The mNthl1 gene was shown to lie immediately adjacent to the tuberous sclerosis 2 (Tsc2) gene in a 5'-to-5' orientation by sequence analysis and was assigned to chromosome 17A3 by in situ hybridization .

Orig Life Evol Biosph, 1998 Oct, 28(4-6), 583 - 96
A search for extraterrestrial eukaryotes: physical and paleontological aspects; Chela-Flores J; Physical and biochemical aspects of a proposed search for extraterrestrial eukaryotes (SETE) are considered . Such a program should approach the distinction between a primitive eukaryote and an archaebacteria . The emphasis on gene silencing suggests a possible assay suitable for a robotic investigation of eukaryoticity, so as to be able to decide whether the first steps towards eukaryogenesis have been taken in an extraterrestrial planet, or satellite . The experiment would consist of searching for cellular division and the systematic related delay in replication of heterochromatic chromosome segments . It should be noticed that the direct search for a membrane-bounded set of chromosomes does not necessarily determine eukaryotic identity, as there are prokaryotes that have membrane-bounded nucleoids . A closer look at the protein fraction of chromatin (mainly histones) does not help either, as there are some eukaryotes that may lack histones; there are also some bacteria as well as archaebacteria with histone-like proteins in their nucleoids . Comments on the recent suggestion of possible environments for a SETE program are discussed: the deep crust of Mars, and the Jovian satellite Europa, provided the existence of an ocean under its ice-covered surface is confirmed by the current Galileo mission.

Nucleic Acids Res, 1998 Oct 1, 26(19), 4374 - 81
Cross-species aminoacylation of tRNA with a long variable arm between Escherichia coli and Saccharomyces cerevisiae; Soma A et al.; Prokaryotes have three amino acid-specific class II tRNAs that possess a characteristic long variable arm, tRNASer, tRNALeuand tRNATyr, while eukaryotes have only two, tRNASerand tRNALeu . Because of such a phylogenetic divergence in the composition of tRNA, the class II tRNA system is a good candidate for studying how the tRNA recognition manner has evolved in association with the evolution of tRNA . We report here a cross-species aminoacylation study of the class II tRNAs, showing the unilateral aminoacylation specificity between Escherichia coli and a yeast, Saccharomyces cerevisiae . Both SerRS and LeuRS from E.coli were unable to aminoacylate yeast class II tRNAs; in contrast, the yeast counterparts were able to aminoacylate E.coli class II tRNAs . Yeast seryl-tRNA synthetase was able to aminoacylate not only E.coli tRNASerbut also tRNALeuand tRNATyr, and yeast LeuRS was able to aminoacylate not only E.coli tRNALeubut also tRNATyr . These results indicate that the recognition manner of class II tRNA, especially the discrimination strategy of each aminoacyl-tRNA synthetase against noncognate class II tRNAs, is significantly divergent between E.coli and yeast . This difference is thought to be due mainly to the different composition of class II tRNAs in E.coli and yeast.

Biochem J, 1998 Oct 1, 335 ( Pt 1), 67 - 77
Synthesis in Escherichia coli of two smaller enzymically active analogues of Coxiella burnetii macrophage infectivity potentiator (CbMip) protein utilizing a single open reading frame from the cbmip gene; Mo YY et al.; FK506-binding proteins (FKBPs) have been identified in a variety of eukaryotic and prokaryotic organisms . Macrophage infectivity potentiator (CbMip, 23.5 kDa) protein of the obligate intracellular bacterium, Coxiella burnetii, was shown previously to belong to the family of FKBPs based on sequence homology and peptidyl-prolyl cis/trans isomerase (PPIase) activity . Further characterization of the cbmip gene has identified two additional proteins with molecular masses of 15.5 and 15.0 kDa that are synthesized, in addition to the 23.5 kDa CbMip, when expressed in Escherichia coli . Amino acid sequencing at the N-terminus combined with transcription and translation fusion expression revealed that the two proteins were synthesized from the same open reading frame of the cbmip gene, but starting at different internal translation start codons, probably by translational reinitiation . When the internal methionines serving as start sites were replaced with lysine by site-directed mutagenesis, the synthesis of 15.5 and 15.0 kDa proteins was abolished even though the synthesis of 23.5 kDa CbMip was intact . This confirmed that the 15.5 and 15.0 kDa proteins are indeed generated by translational reinitiation and are not degradation products of the 23.5 kDa protein . Like other FKBPs, both 15.5 and 15.0 kDa proteins exhibit PPIase activity . Because they share significant sequence homology with FKBPs and have a similar PPIase activity, 15.5 and 15 . 0 kDa proteins are designated as C . burnetii FKBP (Cb-FKBP) analogues I and II, respectively . TnphoA mutagenesis demonstrated that whereas the large protein (CbMip) is secreted, Cb-FKBP analogues I and II are cytoplasmic, indicating that structural variations could allow for different subcellular compartmentalization of similar proteins . Western-blot analysis of lysates of purified C . burnetii using a CbMip-specific monoclonal antibody revealed the presence of a protein migrating at approximately 15 kDa, indicating the presence of smaller Cb-FKBP analogue(s) in C . burnetii, although at much lower levels compared with 23.5 kDa CbMip . This unique gene organization seen with cbmip may provide the organism with a mechanism of efficient use of its limited genetic information to synthesize proteins that are structurally different yet functionally similar.

Cell, 1998 Sep 4, 94(5), 595 - 605
The DNA replication and damage checkpoint pathways induce transcription by inhibition of the Crt1 repressor; Huang M et al.; We have identified the yeast CRT1 gene as an effector of the DNA damage and replication checkpoint pathway . CRT1 encodes a DNA-binding protein that recruits the general repressors Ssn6 and Tup1 to the promoters of damage-inducible genes . Derepression of the Crt1 regulon suppresses the lethality of mec1 and rad53 null alleles and is essential for cell viability during replicative stress . In response to DNA damage and replication blocks, Crt1 becomes hyperphosphorylated and no longer binds DNA, resulting in transcriptional induction . CRT1 is autoregulated and is itself induced by DNA damage, indicating the existence of a negative feedback pathway that facilitates return to the repressed state after elimination of damage . The inhibition of an autoregulatory repressor in response to DNA damage is a strategy conserved throughout prokaryotic and eukaryotic evolution.

Curr Biol, 1998 Sep 10, 8(18), R662 - 5
Phosphorelay signalling: new tricks for an ancient pathway; Brown JM et al.; 'Two-component' phosphorelay systems, once thought to be used exclusively by prokaryotes, have in the past few years been shown to exist in eukaryotes . A two-component system in Dictyostelium has now been shown to modulate protein kinase A, a key regulator of multicellular development in this organism.

Plant Mol Biol, 1998 Sep, 38(1-2), 247 - 63
Protein import into cyanelles and complex chloroplasts; Schwartzbach SD et al.; Higher-plant, green and red algal chloroplasts are surrounded by a double membrane envelope . The glaucocystophyte plastid (cyanelle) has retained a prokaryotic cell wall between the two envelope membranes . The complex chloroplasts of Euglena and dinoflagellates are surrounded by three membranes while the complex chloroplasts of chlorarachniophytes, cryptomonads, brown algae, diatoms and other chromophytes, are surrounded by 4 membranes . The peptidoglycan layer of the cyanelle envelope and the additional membranes of complex chloroplasts provide barriers to chloroplast protein import not present in the simpler double membrane chloroplast envelope . Analysis of presequence structure and in vitro import experiments indicate that proteins are imported directly from the cytoplasm across the two envelope membranes and peptidoglycan layer into cyanelles . Protein import into complex chloroplasts is however fundamentally different . Analysis of presequence structure and in vitro import into microsomal membranes has shown that translocation into the ER is the first step for protein import into complex chloroplasts enclosed by three or four membranes . In vivo pulse chase experiments and immunoelectronmicroscopy have shown that in Euglena, proteins are transported from the ER to the Golgi apparatus prior to import across the three chloroplast membranes . Ultrastructural studies and the presence of ribosomes on the outermost of the four envelope membranes suggests protein import into 4 membrane-bounded complex chloroplasts is directly from the ER like outermost membrane into the chloroplast . The fundamental difference in import mechanisms, posttranslational direct chloroplast import or co-translational translocation into the ER prior to chloroplast import, appears to reflect the evolutionary origin of the different chloroplast types . Chloroplasts with a two-membrane envelope are thought to have evolved through the primary endosymbiotic association between a eukaryotic host and a photosynthetic prokaryote while complex chloroplasts are believed to have evolved through a secondary endosymbiotic association between a heterotrophic or possibly phototrophic eukaryotic host and a photosynthetic eukaryote.

Plant Mol Biol, 1998 Sep, 38(1-2), 209 - 21
Multiple pathways for the targeting of thylakoid proteins in chloroplasts; Robinson C et al.; The assembly of the photosynthetic apparatus requires the import of numerous cytosolically synthesised proteins and their correct targeting into or across the thylakoid membrane . Biochemical and genetic studies have revealed the operation of several targeting pathways for these proteins, some of which are used for thylakoid lumen proteins whereas others are utilised by membrane proteins . Some pathways can be traced back to the prokaryotic ancestors of chloroplasts but at least one pathway appears to have arisen in response to the transfer of genes from the organelle to the nucleus . In this article we review recent findings in this field that point to the operation of a mechanistically unique protein translocase in both plastids and bacteria, and we discuss emerging data that reconcile the remarkable variety of targeting pathways with the natures of the substrate precursor proteins.

FEBS Lett, 1998 Aug 28, 434(1-2), 93 - 6
The relationship between synonymous codon usage and protein structure; Xie T et al.; The hypothesis that synonymous codon usage is related to protein three-dimensional structure is examined by investigating the correlation between synonymous codon usage and protein secondary structure . All except two codons in E . coli show the same secondary structural preference for alpha-helix, beta-strand or coil as that of amino acids to be encoded by the respective codons, while 17 codons show secondary structural bias in mammalian proteins . The results indicate that there is no significant correlation between synonymous codon usage and protein secondary structure in E . coli, but there is a correlation in mammals . It could be deduced that synonymous codons carry much less structural information in prokaryotes than in eukaryotes due to their divergent evolutionary mechanism.

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11268 - 73
Structure elucidation and chemical synthesis of stigmolone, a novel type of prokaryotic pheromone; Hull WE et al.; Approximately 2 micromol of a novel prokaryotic pheromone, involved in starvation-induced aggregation and formation of fruiting bodies by the myxobacterium Stigmatella aurantiaca, were isolated by a large-scale elution procedure . The pheromone was purified by HPLC, and high-resolution MS, IR, 1H-NMR, and 13C-NMR were used to identify the active substance as the hydroxy ketone 2,5, 8-trimethyl-8-hydroxy-nonan-4-one, which has been named stigmolone . The analysis was complicated by a solvent-dependent equilibrium between stigmolone and the cyclic enol-ether 3,4-dihydro-2,2, 5-trimethyl-6-(2-methylpropyl)-2H-pyran formed by intramolecular nucleophilic attack of the 8-OH group at the ketone C4 followed by loss of H2O . Both compounds were synthesized chemically, and their structures were confirmed by NMR analysis . Natural and synthetic stigmolone have the same biological activity at ca . 1 nM concentration.

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11263 - 7
Intercellular signaling in Stigmatella aurantiaca: purification and characterization of stigmolone, a myxobacterial pheromone; Plaga W et al.; The myxobacterium Stigmatella aurantiaca passes through a life cycle that involves formation of a multicellular fruiting body as the most complex stage . An early step in this differentiation process depends on a signal factor secreted by the cells when nutrients become limited . The formation of a fruiting body from a small cell population can be accelerated by addition of this secreted material . The bioactive compound was found to be steam volatile . It was purified to homogeneity by steam distillation followed by reversed-phase and normal-phase HPLC . The pheromone was named stigmolone, in accordance with the structure 2,5, 8-trimethyl-8-hydroxy-nonan-4-one, as determined by NMR and mass spectrometry . Stigmolone represents a structurally unique and highly bioactive prokaryotic pheromone that is effective in the bioassay at 1 nM concentration.

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11071 - 6
Base pair switching by interconversion of sugar puckers in DNA extended by proteins of RecA-family: a model for homology search in homologous genetic recombination; Nishinaka T et al.; Escherichia coli RecA is a representative of proteins from the RecA family, which promote homologous pairing and strand exchange between double-stranded DNA and single-stranded DNA . These reactions are essential for homologous genetic recombination in various organisms . From NMR studies, we previously reported a novel deoxyribose-base stacking interaction between adjacent residues on the extended single-stranded DNA bound to RecA protein . In this study, we found that the same DNA structure was induced by the binding to Saccharomyces cerevisiae Rad51 protein, indicating that the unique DNA structure induced by the binding to RecA-homologs was conserved from prokaryotes to eukaryotes . On the basis of this structure, we have formulated the structure of duplex DNA within filaments formed by RecA protein and its homologs . Two types of molecular structures are presented . One is the duplex structure that has the N-type sugar pucker . Its helical pitch is approximately 95 A (18.6 bp/turn), corresponding to that of an active, or ATP-form of the RecA filament . The other is one that has the S-type sugar pucker . Its helical pitch is approximately 64 A (12.5 bp/turn), corresponding to that of an inactive, or ADP-form of the RecA filament . During this modeling, we found that the interconversion of sugar puckers between the N-type and the S-type rotates bases horizontally, while maintaining the deoxyribose-base stacking interaction . We propose that this base rotation enables base pair switching between double-stranded DNA and single-stranded DNA to take place, facilitating homologous pairing and strand exchange . A possible mechanism for strand exchange involving DNA rotation also is discussed.

Proc Natl Acad Sci U S A, 1998 Sep 15, 95(19), 11043 - 6
Default taxonomy: Ernst Mayr's view of the microbial world; Woese CR; This perspective is a response to a taxonomic proposal by E . Mayr {"Two empires or three?" (1998) Proc . Natl . Acad . Sci . USA 95, 9720-9723} . Mayr has suggested that the now accepted classification of life into three primary domains, Archaea, Bacteria, and Eucarya-originally proposed by myself and others--be abandoned in favor of the earlier Prokaryote-Eukaryote classification . Although the matter appears a taxonomic quibble, it is not that simple . At issue here are differing views as to the nature of biological classification, which are underlain by differing views as to what biology is and will be--matters of concern to all biologists.

EMBO J, 1998 Sep 15, 17(18), 5286 - 97
A nuclear-encoded protein of prokaryotic origin is essential for the stability of photosystem II in Arabidopsis thaliana; Meurer J et al.; To understand the regulatory mechanisms underlying the biogenesis of photosystem II (PSII) we have characterized the nuclear mutant hcf136 of Arabidopsis thaliana and isolated the affected gene . The mutant is devoid of any photosystem II activity, and none of the nuclear- and plastome-encoded subunits of this photosystem accumulate to significant levels . Protein labelling studies in the presence of cycloheximide showed that the plastome-encoded PSII subunits are synthesized but are not stable . The HCF136 gene was isolated by virtue of its T-DNA tag, and its identity was confirmed by complementation of homozygous hcf136 seedlings . Immunoblot analysis of fractionated chloroplasts showed that the HCF136 protein is a lumenal protein, found only in stromal thylakoid lamellae . The HCF136 protein is produced already in dark-grown seedlings and its levels do not increase dramatically during light-induced greening . This accumulation profile confirms the mutational data by showing that the HCF136 protein must be present when PSII complexes are made . HCF136 homologues are found in the cyanobacterium Synechocystis species PCC6803 (slr2034) and the cyanelle genome of Cyanophora paradoxa (ORF333), but are lacking in the plastomes of chlorophytes and metaphytes as well as from those of rhodo- and chromophytes . We conclude that HCF136 encodes a stability and/or assembly factor of PSII which dates back to the cyanobacterial-like endosymbiont that led to the plastids of the present photosynthetic eukaryotes.

Plasmid, 1998 Sep, 40(2), 164 - 8
A versatile prokaryotic cloning vector with six dual restriction enzyme sites in the polylinker facilitates efficient subcloning into vectors with unique cloning sites; Sage DR et al.; In large and complex vectors a single restriction enzyme recognition site may be available for introduction of additional DNA requiring the development of linker fragments to create compatible insertion sites . This technology can be time consuming and costly . We describe the construction of a simple phagemid, pSFI, with a polylinker that contains six pairs of dual, rare-cutting, restriction enzyme recognition sites (NotI, SpeI, EcoRV, PstI, SacII, EagI) with multiple unique sites between each pair . This has permitted rapid subcloning of DNA with creation of single flanking restriction enzyme sites . pSFI was used to expedite transfer of viral genes to a LacZ-inducible expression vector and to an adenovirus expression cassette for production of replication-defective virus . The use of this phagemid has facilitated complex vector manipulations and is a valuable adjunct to the family of multifunctional cloning vectors.

J Biol Chem, 1998 Sep 18, 273(38), 24760 - 9
Transcription factor MSY-1 regulates expression of the murine growth hormone receptor gene; Schwartzbauer G et al.; Previous studies identified and partially characterized a 42-base pair regulatory element in the 5'-flanking region of the L1 transcript of the murine growth hormone (GH) receptor gene that interacted with both double- and single-stranded DNA-binding proteins . We present evidence that the double-stranded DNA-binding protein is NF-Y, a CCAAT box-binding protein . Experiments with a dominant negative form of NF-Y indicate that NF-Y does not play a direct role in regulating the activity of the FP42 element . A cDNA clone that specifically interacts with the upper (coding) strand of the regulatory element was isolated by screening a cDNA expression library using the Southwestern technique . DNA sequencing, electrophoretic mobility shift assay, Southwestern blot analysis, and supershift EMSA confirm the identity of the single-stranded binding protein to be MSY-1, a DNA-binding protein that is evolutionary conserved from prokaryotes to eukaryotes . Mapping of single-stranded DNA configurations reveals that MSY-1 can facilitate the formation of single-stranded DNA regions in the GH receptor 5'-flanking region . Transient transfection experiments support the role of MSY-1 as a repressor of GH receptor gene activation . Southwestern blot analysis indicates that the levels of nuclear MSY-1 are decreased in the livers of pregnant mice, suggesting a role for MSY-1 in the increased expression of the GH receptor during pregnancy.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 457 - 61
Spiroplasma turonicum sp . nov . from Haematopota horse flies (Diptera: Tabanidae) in France; Helias C et al.; Strain Tab4cT, a helical prokaryote that was isolated from the body of a Haematopota sp . fly collected in Champchevrier, Indre-et-Loire, Touraine, France, was found to be a member of the class Mollicutes . The cells of strain Tab4cT were small, motile helices that were devoid of a cell wall . The organism passed through filters with mean pore diameters as small as 0.20 mm . Strain Tab4cT grew rapidly in liquid SP-4 medium at both 30 and 37 degrees C . The organism fermented glucose but did not hydrolyse arginine or urea, and did not require serum for growth . In preliminary electrophoretic analyses, the cell protein patterns of strain Tab4cT were distinct from those of 14 other spiroplasmas found in mosquitoes, deer flies and horse flies from Europe and the Far-East . In reciprocal metabolism inhibition and deformation serological tests, employing antigens and antisera representative of spiroplasma groups I-XXXIII (including all sub-groups), plus ungrouped strains BARC 1901 and BARC 2649, no serological relationship with Tab4cT was found . The G + C content of the DNA of strain Tab4cT was about 25 +/- 1 mol% and its genome size was 1.305 kbp . It is proposed that spiroplasma strain Tab4cT be assigned to group XVII (presently vacant) and that strain (ATCC 700271T) is the type strain of a new species, Spiroplasma turonicum.

Int J Syst Bacteriol, 1998 Apr, 48 Pt 2, 389 - 94
An approach to characterizing uncultivated prokaryotes: the Grey Lung agent and proposal of a Candidatus taxon for the organism, 'Candidatus Mycoplasma ravipulmonis'; Neimark H et al.; An approach to characterizing uncultivated bacteria which combines a PFGE procedure for obtaining purified full-length chromosomes with PCR amplification is described . Isolated chromosomes from uncultivated organisms provide a specifically identifiable source material for hybridization, amplification and cloning . The availability of purified chromosomes for DNA amplification should aid in examining the diversity of microbial populations and should also reduce the possibility of forming hybrid DNA artifact molecules . The approach is illustrated by isolating the chromosome of the uncultivated agent of rodent Grey Lung disease and using the purified chromosomes to amplify and directly sequence the evolutionarily conserved 16S rRNA gene . The Grey Lung agent (GLA) contains a 650 kb chromosome and is shown to be a Mycoplasma sp . located phylogenetically in the hominis group of mycoplasmas . If a simple genomic lesion(s) is responsible for the unculturability of GLA, it is conceivable that complementation with DNA from a close relative could permit growth on artificial media.

Biochem Biophys Res Commun, 1998 Aug 28, 249(3), 579 - 82
On the coordination and oxidation states of the active-site copper ion in prokaryotic Cu,Zn superoxide dismutases; Stroppolo ME et al.; The active-site copper ion of the prokaryotic Cu,Zn superoxide dismutase from P . leiognathi is found to undergo reversible reduction upon irradiation of the protein solution with a high-intensity X-ray beam from a third-generation synchrotron source . The same phenomenon is observed for the enzyme crystals, whose diffraction pattern has been obtained from synchrotron sources . In this case the active-site copper-ligand coordination bond lengths and in particular the Cu-NE2(His61) distance are consistent with a copper ion in the reduced state . These results are in line with previous studies on the eukaryotic Cu,Zn superoxide dismutases and suggest the conservation of an identical catalytic mechanism in both the prokaryotic and eukaryotic enzymes.

Mol Biol Evol, 1998 Sep, 15(9), 1224 - 31
Optimally recovering rate variation information from genomes and sequences: pattern filtering; Lake JA; Nucleotide substitution rates vary at different positions within genes and genomes, but rates are difficult to estimate, because they are masked by the stochastic nature of substitutions . In this paper, a linear method, pattern filtering, is described which can optimally separate the signals (related to substitution rates or to other measures of sequence change) from stochastic noise . Pattern filtering promises to be useful in both genomic and molecular evolution studies . In an example using mitochondrial genomes, it is shown that pattern filtering can reveal coding and non-coding regions without the need for prior identification of reading frames or other knowledge of the sequence and promises to be an important tool for genomic analysis . In a second example, it is shown that pattern filtering allows one to classify sites on the basis of an estimator of substitution rates . Using elongation factor EF-1 alpha sequences, it is shown that the fastest sites favor archaea as the sister taxon of eukaryotes, whereas the slower sites support the eocyte prokaryotes as the sister taxon of eukaryotes, suggesting that the former result is an artifact of "long branch attraction."

Mol Biol Evol, 1998 Sep, 15(9), 1183 - 8
A covariotide model explains apparent phylogenetic structure of oxygenic photosynthetic lineages; Lockhart PJ et al.; The aims of the work were (1) to develop statistical tests to identify whether substitution takes place under a covariotide model in sequences used for phylogenetic inference and (2) to determine the influence of covariotide substitution on phylogenetic trees inferred for photosynthetic and other organisms . (Covariotide and covarion models are ones in which sites that are variable in some parts of the underlying tree are invariable in others and vice versa.) Two tests were developed . The first was a contingency test, and the second was an inequality test comparing the expected number of variable sites in two groups with the observed number . Application of these tests to 16S rDNA and tufA sequences from a range of nonphotosynthetic prokaryotes and oxygenic photosynthetic prokaryotes and eukaryotes suggests the occurrence of a covariotide mechanism . The degree of support for partitioning of taxa in reconstructed trees involving these organisms was determined in the presence or absence of sites showing particular substitution patterns . This analysis showed that the support for splits between (1) photosynthetic eukaryotes and prokaryotes and (2) photosynthetic and nonphotosynthetic organisms could be accounted for by patterns arising from covariotide substitution . We show that the additional problem of compositional bias in sequence data needs to be considered in the context of patterns of covariotide/covarion substitution . We argue that while covariotide or covarion substitution may give rise to phylogenetically informative patterns in sequence data, this may not always be so.

FEMS Microbiol Rev, 1998 Jun, 22(2), 65 - 78
The intracellular life of Chlamydia psittaci: how do the bacteria interact with the host cell?
Escalante-Ochoa C, Ducatelle R, Haesebrouck F.
Throughout the life of any organism interactions with the surrounding environment are always taking place, a process that leads to evolution . Chlamydia psittaci is an obligate intracellular parasite, but it must also be capable of extracellular survival in order to search for new host cells . Therefore, these peculiar prokaryotes have evolved two different particles and a unique developmental cycle that, together with a series of not yet fully understood interactions with their host cells, allow them to fulfil the requirements for their permanence in nature . These interactions are the subject of this paper . Particular attention is paid to the attachment and internalization of the bacteria, the chlamydial vacuole, and the avoidance of lysosomal degradation.

Curr Opin Genet Dev, 1998 Aug, 8(4), 386 - 91
Microbial asymmetric cell division: localization of cell fate determinants; Jacobs C et al.; The genetic mechanisms that control asymmetric cell divisions--yielding progeny cells that differ from one another--have been conserved among prokaryotes, eukaryotic microbes, and higher organisms . All use the paradigm of regulatory protein localization as a way of translating genetic information into three-dimensional spacePublication Types:

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