Sequence Mutagenesis Research Articles

Potent cross-reactive neutralization of SARS coronavirus isolates by human monoclonal antibodies

The severe acute respiratory syndrome coronavirus (SARS-CoV) caused a worldwide epidemic in late 2002/early 2003 and a second outbreak in the winter of 2003/2004 by an independent animal-to-human transmission. The GD03 strain, which was isolated from an index patient of the second outbreak, was reported to resist neutralization by the human monoclonal antibodies (hmAbs) 80R and S3.1, which can potently neutralize isolates from the first outbreak. Here we report that two hmAbs, m396 and S230.15, potently neutralized GD03 and representative isolates from the first SARS outbreak (Urbani, Tor2) and from palm civets (SZ3, SZ16). These antibodies also protected mice challenged with the Urbani or recombinant viruses bearing the GD03 and SZ16 spike (S) glycoproteins. Both antibodies competed with the SARS-CoV receptor, ACE2, for binding to the receptor-binding domain (RBD), suggesting a mechanism of neutralization that involves interference with the SARS-CoV-ACE2 interaction. Two putative hot-spot residues in the RBD (Ile-489 and Tyr-491) were identified within the SARS-CoV spike that likely contribute to most of the m396-binding energy. Residues Ile-489 and Tyr-491 are highly conserved within the SARS-CoV spike, indicating a possible mechanism of the m396 cross-reactivity. Sequence analysis and mutagenesis data show that m396 might neutralize all zoonotic and epidemic SARS-CoV isolates with known sequences, except strains derived from bats. These antibodies exhibit cross-reactivity against isolates from the two SARS outbreaks and palm civets and could have potential applications for diagnosis, prophylaxis, and treatment of SARS-CoV infections.

Controlled shedding of platelet glycoprotein (GP)VI and GPIb-IX-V by ADAM family metalloproteinases.

Platelet glycoprotein (GP)VI that binds collagen, and GPIb-IX-V that binds von Willebrand factor, initiate thrombus formation. In this study, we investigated the mechanisms of metalloproteinase-mediated ectodomain shedding that regulate the surface expression of GPVI, GPIbalpha (the major ligand-binding subunit) and GPV (that regulates thrombin-dependent activation via GPIbalpha). Immunoblotting human platelet lysates using affinity-purified antibodies against cytoplasmic domains of GPVI, GPIbalpha or GPV allowed simultaneous analysis of intact and cleaved receptor, and revealed (i) that a significant fraction of GPIbalpha, but not GPVI, exists in a cleaved state on platelets, even when isolated in the presence of metalloproteinase inhibitor (GM6001) or EDTA; (ii) the same-sized membrane-associated fragments of GPVI or GPIbalpha are generated by phorbol-ester (PMA), the mitochondrial-targeting reagent CCCP, the calmodulin inhibitor W7, or the thiol-modifying reagent, N-ethylmaleimide, that directly activates ADAM10/ADAM17; and (iii) GPV is shed by both metalloproteinase- and thrombin-dependent mechanisms, depending on the concentration of thrombin. Based on the predicted cleavage area defined by these studies, ADAM10, but not ADAM17, cleaved a GPVI-based synthetic peptide within the extracellular membrane-proximal sequence (PAR;Q(243)YY) as analyzed by MALDI-TOF-MS. In contrast, ADAM17, but not ADAM10, cleaved within the GPIbalpha-based peptide (LRG;V(465)LQ). Both ADAM10 and ADAM17 cleaved within a GPV-based peptide (AQP;V(494)TT). Metalloproteinase-mediated shedding of GPIbalpha from GPIb-IX-transfected or GPVI-transfected cells induced by W7 or N-ethylmaleimide was inhibited by mutagenesis of sequences identified from peptide analysis. These findings suggest surface levels of GPVI, GPIbalpha and GPV may be controlled by distinct mechanisms involving ADAM10 and/or ADAM17.

Hypoxia-inducible Factor 1α (HIF-1α)-mediated Hypoxia Increases BACE1 Expression and β-Amyloid Generation

The incidence of Alzheimer disease (AD) and vascular dementia is greatly increased following cerebral ischemia and stroke in which hypoxic conditions occur in affected brain areas. beta-Amyloid peptide (Abeta), which is derived from the beta-amyloid precursor protein (APP) by sequential proteolytic cleavages from beta-secretase (BACE1) and presenilin-1 (PS1)/gamma-secretase, is widely believed to trigger a cascade of pathological events culminating in AD and vascular dementia. However, a direct molecular link between hypoxic insults and APP processing has yet to be established. Here, we demonstrate that acute hypoxia increases the expression and the enzymatic activity of BACE1 by up-regulating the level of BACE1 mRNA, resulting in increases in the APP C-terminal fragment-beta (betaCTF) and Abeta. Hypoxia has no effect on the level of PS1, APP, and tumor necrosis factor-alpha-converting enzyme (TACE, an enzyme known to cleave APP at the alpha-secretase cleavage site). Sequence analysis, mutagenesis, and gel shift studies revealed binding of HIF-1 to the BACE1 promoter. Overexpression of HIF-1alpha increases BACE1 mRNA and protein level, whereas down-regulation of HIF-1alpha reduced the level of BACE1. Hypoxic treatment fails to further potentiate the stimulatory effect of HIF-1alpha overexpression on BACE1 expression, suggesting that hypoxic induction of BACE1 expression is primarily mediated by HIF-1alpha. Finally, we observed significant reduction in BACE1 protein levels in the hippocampus and the cortex of HIF-1alpha conditional knock-out mice. Our results demonstrate an important role for hypoxia/HIF-1alpha in modulating the amyloidogenic processing of APP and provide a molecular mechanism for increased incidence of AD following cerebral ischemic and stroke injuries.

Mycoplasma hyopneumoniae mhp379 is a Ca2+-dependent, sugar-nonspecific exonuclease exposed on the cell surface.

Mycoplasma hyopneumoniae mhp379 is a putative lipoprotein that shares significant amino acid sequence similarity with a family of bacterial thermostable nucleases. To examine the nuclease activity of mhp379, the gene was cloned and expressed in Escherichia coli following the deletion of the amino-terminal signal sequence and prokaryotic lipoprotein cleavage site and mutagenesis of the mycoplasma TGA tryptophan codons to TGG. The recombinant fusion protein yielded a 33-kDa thrombin cleavage product, corresponding in size to the mature mhp379 protein. Exonuclease activity was indicated by agarose gel electrophoresis analysis of the reaction products that were released when different nucleic acid substrates were used. Endonuclease activity was also indicated by the digestion of closed circular plasmid DNA. The recombinant mhp379 fusion protein completely digested single-stranded DNA, double-stranded DNA (dsDNA), and RNA. The optimal reaction conditions were determined with a novel nuclease assay based on the enhancement of fluorescence of SYBR green I bound to dsDNA. Optimal activity was observed in the presence of calcium ions at a concentration of 15 mM and a pH of 9.5. No nuclease activity was observed in the absence of calcium ions. Mycoplasmas do not have the ability to synthesize nucleic acid precursors, and thus, nucleases are likely to be important in the acquisition of precursors for the synthesis of nucleic acids. Homologs of an ATP-binding cassette (ABC) transport system were identified immediately downstream of the gene encoding mhp379, and two homologs of M. pneumoniae lipoprotein multigene family 2 were also identified immediately upstream. Homologs of mhp379 were identified in the sequenced genomes of a number of mycoplasma species, and in most cases the homologous ABC transport system was identified immediately downstream of the homologous gene; in several cases a homolog of M. pneumoniae lipoprotein multigene family 2 was also identified immediately upstream. These observations suggest that mhp379 comprises part of a conserved ABC transport operon in mycoplasmas and that the exonuclease activity of mhp379 may be associated with the conserved function of the ABC transport system in the import of nucleic acid precursors. This is the first study to identify the gene and characterize the activity of a mycoplasma exonuclease.

In vitro 'sexual' evolution through the PCR-based staggered extension process (StEP)

This protocol describes a directed evolution method for in vitro mutagenesis and recombination of polynucleotide sequences. The staggered extension process (StEP) is essentially a modified PCR that uses highly abbreviated annealing and extension steps to generate staggered DNA fragments and promote crossover events along the full length of the template sequence(s). The resulting library of chimeric polynucleotide sequence(s) is subjected to subsequent high-throughput functional analysis. The recombination efficiency of the StEP method is comparable to that of the most widely used in vitro DNA recombination method, DNA shuffling. However, the StEP method does not require DNA fragmentation and can be carried out in a single tube. This protocol can be completed in 4-6 h.

The structure of the endoribonuclease XendoU: From small nucleolar RNA processing to severe acute respiratory syndrome coronavirus replication.

Small nucleolar RNAs (snoRNAs) play a key role in eukaryotic ribosome biogenesis. In most cases, snoRNAs are encoded in introns and are released through the splicing reaction. Some snoRNAs are, instead, produced by an alternative pathway consisting of endonucleolytic processing of pre-mRNA. XendoU, the endoribonuclease responsible for this activity, is a U-specific, metal-dependent enzyme that releases products with 2'-3' cyclic phosphate termini. XendoU is broadly conserved among eukaryotes, and it is a genetic marker of nidoviruses, including the severe acute respiratory syndrome coronavirus, where it is essential for replication and transcription. We have determined by crystallography the structure of XendoU that, by refined search methodologies, appears to display a unique fold. Based on sequence conservation, mutagenesis, and docking simulations, we have identified the active site. The conserved structural determinants of this site may provide a framework for attempting to design antiviral drugs to interfere with the infectious nidovirus life cycle.

Transcriptional regulation of APH‐1A and increased γ‐secretase cleavage of APP and Notch by HIF‐1 and hypoxia

The proteolytic cleavage of Alzheimer beta-amyloid precursor protein (APP) and signaling receptor Notch is mediated by the PS/gamma-secretase complex, which consists of presenilins, nicastrin, APH-1, and PEN-2. Although the four components are known to coordinately regulate each other at the protein level, information regarding their transcription regulation is scarce. Here we characterized the 5'-flanking region of the human APH-1A gene and identified a 271-bp fragment containing the transcription initiation site for the promoter activity. Sequence analysis, mutagenesis, and gel shift studies revealed a binding of AP4 and HIF-1 to the promoter, which affects the promoter activity. Activation of HIF-1 by short-term NiCl2 treatments (a condition of chemical hypoxia) dramatically increased APH-1A mRNA and protein expression, accompanied by increased secretion of Abeta and decreased APP CTFs formation, indicative of an increase in gamma-secretase activity. NiCl2 treatments had little effect on APP and the other three components of the gamma-secretase complex. The cellular concentration of Notch intracellular domain (NICD) was also increased by the hypoxic treatment. Our results demonstrate that APH-1A expression and the gamma-secretase mediated Abeta and Notch NICD generation are regulated by HIF-1, and the specific control of APH-1A expression may imply physiological functions uniquely assigned to APH-1A.

Identification of active site residues of the inverting glycosyltransferase Cgs required for the synthesis of cyclic β-1,2-glucan, a Brucella abortus virulence factor

Brucella abortus cyclic glucan synthase (Cgs) is a 320-kDa (2868-amino acid) polytopic integral inner membrane protein responsible for the synthesis of the virulence factor cyclic beta-1,2-glucan by a novel mechanism in which the enzyme itself acts as a protein intermediate. Cgs functions as an inverting processive beta-1,2-autoglucosyltransferase and has the three enzymatic activities required for the synthesis of the cyclic glucan: initiation, elongation, and cyclization. To gain further insight into the protein domains that are essential for the enzymatic activity, we have compared the Cgs sequence with other glycosyltransferases (GTs). This procedure allowed us to identify in the Cgs region (475-818) the widely spaced D, DxD, E/D, (Q/R)xxRW motif that is highly conserved in the active site of numerous GTs. By site-directed mutagenesis and in vitro and in vivo activity assays, we have demonstrated that most of the amino acid residues of this motif are essential for Cgs activity. These sequence and site-directed mutagenesis analyses also indicate that Cgs should be considered a bi-functional modular GT, with an N-terminal GT domain belonging to a new GT family related to GT-2 (GT-84) followed by a GH-94 glycoside hydrolase C-terminal domain. Furthermore, over-expression of inactive mutants results in wild-type (WT) production of cyclic glucan when bacteria co-express the mutant and the WT form, indicating that Cgs may function in the membrane as a monomeric enzyme. Together, these results are compatible with a single addition model by which Cgs acts in the membrane as a monomer and uses the identified motif to form a single center for substrate binding and glycosyl-transfer reaction.

Identification of a Virulence-Associated Determinant, Dihydrolipoamide Dehydrogenase ( lpd ), in Mycoplasma gallisepticum through In Vivo Screening of Transposon Mutants

To effectively analyze Mycoplasma gallisepticum for virulence-associated determinants, the ability to create stable genetic mutations is essential. Global M. gallisepticum mutagenesis is currently limited to the use of transposons. Using the gram-positive transposon Tn4001mod, a mutant library of 110 transformants was constructed and all insertion sites were mapped. To identify transposon insertion points, a unique primer directed outward from the end of Tn4001mod was used to sequence flanking genomic regions. By comparing sequences obtained in this manner to the annotated M. gallisepticum genome, the precise locations of transposon insertions were discerned. After determining the transposon insertion site for each mutant, unique reverse primers were synthesized based on the specific sequences, and PCR was performed. The resultant amplicons were used as unique Tn4001mod mutant identifiers. This procedure is referred to as signature sequence mutagenesis (SSM). SSM permits the comprehensive screening of the M. gallisepticum genome for the identification of novel virulence-associated determinants from a mixed mutant population. To this end, chickens were challenged with a pool of 27 unique Tn4001mod mutants. Two weeks postinfection, the birds were sacrificed, and organisms were recovered from respiratory tract tissues and screened for the presence or absence of various mutants. SSM is a negative-selection screening technique whereby those mutants possessing transposon insertions in genes essential for in vivo survival are not recovered from the host. We have identified a virulence-associated gene encoding dihydrolipoamide dehydrogenase (lpd). A transposon insertion in the middle of the coding sequence resulted in diminished biologic function and reduced virulence of the mutant designated Mg 7.

Localization of human T-cell lymphotropic virus type II Tax protein is dependent upon a nuclear localization determinant in the N-terminal region

Human T-cell lymphotropic viruses (HTLV) types I and II are closely related oncogenic retroviruses that have been associated with lymphoproliferative and neurological disorders. The proviral genome encodes a trans-regulatory Tax protein that activates viral genes and upregulates various cellular genes involved in both cell growth and transformation. Tax proteins of HTLV-I (Tax-I) and HTLV-II (Tax-II) exhibit more than 77% aa homology and expression of either Tax-I or Tax-II is sufficient for immortalization of cultured T lymphocytes. Tax-I shuttles from the nucleus to the cytoplasm and accumulates within the nucleus, whereas Tax-II is found mainly in the cytoplasm. In the present study we have used recombinant vectors to analyze the size and structure of the nuclear localization domain within the Tax-II protein sequence. The Tax-II protein was expressed in HeLa cells either as the complete protein, or regions thereof, that were individually fused to the green fluorescent protein (GFP). Immunoblot analysis of the fused Tax-II products confirmed their expression and size. Fluorescence microscopy studies indicated that the complete Tax-II as well as N-truncated forms presented a punctuate cytoplasmic distribution and that a nuclear localization determinant is confined to within the first 60 aa of Tax-II. Accordingly, site directed mutagenesis and deletion of specific sequences within the first 60 aa showed that the nuclear determinant lies within the first 41 residues of Tax-II. These results point to a direct involvement of the amino-terminal residues of Tax-II protein in determining its nuclear functionality.

Insilico.mutagenesis: A Primer Selection Tool Designed for Sequence Scanning Applications Used in Directed Evolution Experiments

Several primer prediction programs have been developed for a variety of applications. However none of these tools allows the prediction of a large set of primers for whole gene site-directed mutagenesis experiments using the megaprimer method. We report a novel primer prediction tool (insilico.mutagenesis), accessible at www.insilico.uni-duesseldorf.de, developed for the application to high-throughput mutagenesis used in directed evolution or structure-function dependency projects, which involve the subsequent mutagenesis of a large number of amino acid positions (e.g., in whole gene saturation or gene scanning mutagenesis experiments). Furthermore, the program is suitable for all site-directed (saturation) mutagenesis approaches, such as saturation mutagenesis of promoter sequences and other types of untranslated intergenic regions. In anticipation of downstream cloning steps, the primer design tool also includes a restriction site control feature alerting the user if unwanted restriction sites have been introduced within the mutagenesis primer. The use of our tool promises to speed up the process of site-directed mutagenesis, as it instantly allows predicting a large set of primers.

Diverse Plasmid DNA Vectors by Directed Molecular Evolution of Cytomegalovirus Promoters

Genetic vaccinations, gene therapy, and manufacturing of therapeutic proteins would benefit from promoter sequences that provide improved or prolonged expression levels. The cytomegalovirus (CMV) promoter is one of the most potent promoters known to date, and no previous examples of improved activity of this promoter by sequence mutagenesis have been reported. This study describes directed molecular evolution of CMV promoters derived from two human and two nonhuman primate strains of CMV by DNA shuffling and screening. Libraries of chimeric promoters were screened and analyzed for expression levels and immune responses, using plasmid DNA vectors encoding luciferase and beta-galactosidase. The results indicate that high functional diversity among CMV promoters can be generated, and the chimeric promoters selected after two rounds of DNA shuffling and particularly designed screening assays provided approximately 2-fold increased luciferase reporter gene expression and anti-beta-galactoside antibody response in vivo when compared with wild-type promoters. Sequence analysis of the shuffled promoters identified several mutations potentially contributing to the observed enhanced or reduced promoter activities and identified a 42-nucleotide region that appears obsolete for the functioning of the CMV promoter. Taken together, these data demonstrate the feasibility of generating diverse promoter sequences by DNA shuffling and screening methods, and provide novel structure- function information about CMV promoters. DNA shuffling and screening technologies provide a new approach to promoter optimization and development of optimal expression vectors for genetic vaccinations, gene therapy, and protein expression.

Probing the Catalytically Essential Residues of the α-l-Fucosidase from the Hyperthermophilic Archaeon Sulfolobus solfataricus

Retaining glycosidases promote the hydrolysis of the substrate by following a double-displacement mechanism involving a covalent intermediate. The catalytic residues are a general acid/base catalyst and the nucleophile. Experimental identification of these residues in a specific glycosidase allows for the assigning of the corresponding residues in all of the other enzymes belonging to the same family. By means of sequence alignment, mutagenesis, and detailed kinetic studies of the alpha-fucosidase from Sulfolobus solfataricus (Ssalpha-fuc) (family 29), we show here that the residues, invariant in this family, have the function inferred from the analysis of the 3D structure of the enzyme from Thermotoga maritima (Tmalpha-fuc). These include in Ssalpha-fuc the substrate-binding residues His46 and His123 and the nucleophile of the reaction, previously described. The acid/base catalyst could be assigned less easily. The k(cat) of the Ssalpha-fucGlu292Gly mutant, corresponding to the acid/base catalyst of Tmalpha-fuc, is reduced by 154-fold but could not be chemically rescued. Instead, the Ssalpha-fucGlu58Gly mutant revealed a 4000-fold reduction of k(cat)/K(M) if compared to the wild-type and showed the rescue of the k(cat) by sodium azide at wild-type levels. Thus, our data suggest that a catalytic triad, namely, Glu58, Glu292, and Asp242, is involved in catalysis. Glu58 and Glu292 cooperate in the role of acid/base catalyst, while Asp242 is the nucleophile of the reaction. Our data suggest that in glycosidase family 29 alpha-fucosidases promoting the retaining mechanism with slightly different catalytic machineries coexist.

Regulation of Hepatitis C Virus Polyprotein Processing by Signal Peptidase Involves Structural Determinants at the p7 Sequence Junctions

The hepatitis C virus genome encodes a polyprotein precursor that is co- and post-translationally processed by cellular and viral proteases to yield 10 mature protein products (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Although most cleavages in hepatitis C virus polyprotein precursor proceed to completion during or immediately after translation, the cleavages mediated by a host cell signal peptidase are partial at the E2/p7 and p7/NS2 sites, leading to the production of an E2p7NS2 precursor. The sequences located immediately N-terminally of E2/p7 and p7/NS2 cleavage sites can function as signal peptides. When fused to a reporter protein, the signal peptides of p7 and NS2 were efficiently cleaved. However, when full-length p7 was fused to the reporter protein, partial cleavage was observed, indicating that a sequence located N-terminally of the signal peptide reduces the efficiency of p7/NS2 cleavage. Sequence analyses and mutagenesis studies have also identified structural determinants responsible for the partial cleavage at both the E2/p7 and p7/NS2 sites. Finally, the short distance between the cleavage site of E2/p7 or p7/NS2 and the predicted transmembrane alpha-helix within the P' region might impose additional structural constraints to the cleavage sites. The insertion of a linker polypeptide sequence between P-3' and P-4' of the cleavage site released these constraints and led to improved cleavage efficiency. Such constraints in the processing of a polyprotein precursor are likely essential for hepatitis C virus to post-translationally regulate the kinetics and/or the level of expression of p7 as well as NS2 and E2 mature proteins.

Architecture of P2Y nucleotide receptors: structural comparison based on sequence analysis, mutagenesis, and homology modeling.

Human P2Y receptors encompass at least eight subtypes of Class A G protein-coupled receptors (GPCRs), responding to adenine and/or uracil nucleotides. Using a BLAST search against the Homo sapiens subset of the SWISS-PROT and TrEMBL databases, we identified 68 proteins showing high similarity to P2Y receptors. To address the problem of low sequence identity between rhodopsin and the P2Y receptors, we performed a multiple-sequence alignment of the retrieved proteins and the template bovine rhodopsin, combining manual identification of the transmembrane domains (TMs) with automatic techniques. The resulting phylogenetic tree delineated two distinct subgroups of P2Y receptors: Gq-coupled subtypes (e.g., P2Y1) and those coupled to Gi (e.g., P2Y12). On the basis of sequence comparison we mutated three Tyr residues of the putative P2Y1 binding pocket to Ala and Phe and characterized pharmacologically the mutant receptors expressed in COS-7 cells. The mutation of Y306 (7.35, site of a cationic residue in P2Y12) or Y203 in the second extracellular loop selectively decreased the affinity of the agonist 2-MeSADP, and the Y306F mutation also reduced antagonist (MRS2179) affinity by 5-fold. The Y273A (6.48) mutation precluded the receptor activation without a major effect on the ligand-binding affinities, but the Y273F mutant receptor still activated G proteins with full agonist affinity. Thus, we have identified new recognition elements to further define the P2Y1 binding site and related these to other P2Y receptor subtypes. Following sequence-based secondary-structure prediction, we constructed complete models of all the human P2Y receptors by homology to rhodopsin. Ligand docking on P2Y1 and P2Y12 receptor models was guided by mutagenesis results, to identify the residues implicated in the binding process. Different sets of cationic residues in the two subgroups appeared to coordinate phosphate-bearing ligands. Within the P2Y1 subgroup these residues are R3.29, K/R6.55, and R7.39. Within the P2Y12 subgroup, the only residue in common with P2Y1 is R6.55, and the role of R3.29 in TM3 seems to be fulfilled by a Lys residue in EL2, whereas the R7.39 in TM7 seems to be substituted by K7.35. Thus, we have identified common and distinguishing features of P2Y receptor structure and have proposed modes of ligand binding for the two representative subtypes that already have well-developed ligands.

An algorithm for the study of DNA sequence evolution based on the genetic code

Recent studies of the quantum-mechanical processes in the DNA molecule have seriously challenged the principle that mutations occur randomly. The proton tunneling mechanism causes tautomeric transitions in base pairs resulting in mutations during DNA replication. The meticulous study of the quantum-mechanical phenomena in DNA may reveal that the process of mutagenesis is not completely random. We are still far away from a complete quantum-mechanical model of DNA sequence mutagenesis because of the complexity of the processes and the complex three-dimensional structure of the molecule. In this paper we have developed a quantum-mechanical description of DNA evolution and, following its outline, we have constructed a classical model for DNA evolution assuming that some aspects of the quantum-mechanical processes have influenced the determination of the genetic code. Conversely, our model assumes that the genetic code provides information about the quantum-mechanical mechanisms of mutagenesis, as the current code is the product of an evolutionary process that tries to minimize the spurious consequences of mutagenesis. Based on this model we develop an algorithm that can be used to study the accumulation of mutations in a DNA sequence. The algorithm has a user-friendly interface and the user can change key parameters in order to study relevant hypotheses.

Protein tolerance to random amino acid change.

Mutagenesis of protein-encoding sequences occurs ubiquitously; it enables evolution, accumulates during aging, and is associated with disease. Many biotechnological methods exploit random mutations to evolve novel proteins. To quantitate protein tolerance to random change, it is vital to understand the probability that a random amino acid replacement will lead to a protein's functional inactivation. We define this probability as the "x factor." Here, we develop a broadly applicable approach to calculate x factors and demonstrate this method using the human DNA repair enzyme 3-methyladenine DNA glycosylase (AAG). Three gene-wide mutagenesis libraries were created, each with 10(5) diversity and averaging 2.2, 4.6, and 6.2 random amino acid changes per mutant. After determining the percentage of functional mutants in each library using high-stringency selection (>19,000-fold), the x factor was found to be 34% +/- 6%. Remarkably, reanalysis of data from studies of diverse proteins reveals similar inactivation probabilities. To delineate the nature of tolerated amino acid substitutions, we sequenced 244 surviving AAG mutants. The 920 tolerated substitutions were characterized by substitutability index and mapped onto the AAG primary, secondary, and known tertiary structures. Evolutionarily conserved residues show low substitutability indices. In AAG, beta strands are on average less substitutable than alpha helices; and surface loops that are not involved in DNA binding are the most substitutable. Our results are relevant to such diverse topics as applied molecular evolution, the rate of introduction of deleterious alleles into genomes in evolutionary history, and organisms' tolerance of mutational burden.

Identification and Kinetics Analysis of a Novel Heparin-binding Site (KEDK) in Human Tenascin-C

The interaction between tenascin-C (TN-C), a multi-subunit extracellular matrix protein, and heparin was examined using a surface plasmon resonance-based technique on a Biacore system. The aims of the present study were to examine the affinity of fibronectin type III repeats of TN-C fragments (TNIII) for heparin, to investigate the role of the TNIII4 domains in the binding of TN-C to heparin, and to delineate a sequence of amino acids within the TNIII4 domain, which mediates cooperative heparin binding. At a physiological salt concentration, and pH 7.4, TNIII3-5 binds to heparin with high affinity (K(D) = 30 nm). However, a major heparin-binding site in TNIII5 produces a modest affinity binding at a K(D) near 4 microm, and a second site in TNIII4 enhances the binding by several orders of magnitude, although it was far too weak to produce an observable binding of TNIII4 by itself. Moreover, mutagenesis of the KEDK sequence in the TNIII4 domain resulted in the significant reduction of heparin-binding affinity. In addition, residues in the KEDK sequences are conserved in TN-C throughout mammalian evolution. Thus the structure-based sequence alignment, mutagenesis, and sequence conservation data together reveal a KEDK sequence in TNIII4 suggestive of a minor heparin-binding site. Finally, we demonstrate that TNIII4 contains binding sites for heparin sulfate proteoglycan and enhances the heparin sulfate proteoglycan-dependent human gingival fibroblast adhesion to TNIII5, thus providing the biological significance of heparin-binding site of TNIII4. These results suggest that the heparin-binding sites may traverse TNIII4-5 and thus require KEDK in TNIII4 for optimal heparin-binding.

Discovery of mammalian genes that participate in virus infection

BackgroundViruses are obligate intracellular parasites that rely upon the host cell for different steps in their life cycles. The characterization of cellular genes required for virus infection and/or cell killing will be essential for understanding viral life cycles, and may provide cellular targets for new antiviral therapies.ResultsCandidate genes required for lytic reovirus infection were identified by tagged sequence mutagenesis, a process that permits rapid identification of genes disrupted by gene entrapment. One hundred fifty-one reovirus resistant clones were selected from cell libraries containing 2 × 105 independently disrupted genes, of which 111 contained mutations in previously characterized genes and functionally anonymous transcription units. Collectively, the genes associated with reovirus resistance differed from genes targeted by random gene entrapment in that known mutational hot spots were under represented, and a number of mutations appeared to cluster around specific cellular processes, including: IGF-II expression/signalling, vesicular transport/cytoskeletal trafficking and apoptosis. Notably, several of the genes have been directly implicated in the replication of reovirus and other viruses at different steps in the viral lifecycle.ConclusionsTagged sequence mutagenesis provides a rapid, genome-wide strategy to identify candidate cellular genes required for virus infection. The candidate genes provide a starting point for mechanistic studies of cellular processes that participate in the virus lifecycle and may provide targets for novel anti-viral therapies.

Transcriptional control of the mouse Col7a1 gene in keratinocytes: basal and transforming growth factor-beta regulated expression.

Anchoring fibrils at the cutaneous basement membrane zone of the stratified squamous epithelia are essential to maintaining skin integrity, as absence of these structures leads to the chronic blistering disease, dystrophic epidermolysis bullosa. Type VII collagen, the major component of anchoring fibrils, is synthesized primarily by basal keratinocytes and to a lesser degree by dermal fibroblasts. To elucidate the transcriptional control elements of the type VII collagen gene (Col7a1), 3 kb of 5' flanking sequence of the mouse gene was cloned, sequenced, and fused to the chloramphenicol acetyltransferase reporter gene. Promoter deletion analyses revealed that 560 bp of Col7a1 5' flanking sequence was sufficient and necessary for basal level of transcription in cultured murine keratinocytes. Mutagenesis of DNA sequences with similarity to consensus binding sites for transcription factors, including Sp1/Sp3, AP2, AP1, and Smads, within the p-560Col7a1 promoter/chloramphenicol acetyltransferase construct, coupled with DNA binding assays, revealed the importance of these sites for basal Col7a1 expression. The effect of transforming growth factor beta, an activator of Col7a1 expression in keratinocytes and dermal fibroblasts, was examined using the same Col7a1 promoter/chloramphenicol acetyltransferase constructs. These analyses demonstrated that transforming growth factor beta1 stimulation of Col7a1 transcription is dependent on a putative interaction between Smads and AP1. Interestingly, the Smad-like binding site was essential for both basal and transforming growth factor beta1 stimulated Col7a1 transcription. Collectively, these findings attest to the complex regulation of Col7a1 transcription in epidermal keratinocytes.