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