Specific Mutagenesis Research Articles

Mutating aspartate in the calcium-binding site of alpha-lactalbumin: effects on the protein stability and cation binding.

The residue Asp87, which is in the calcium-binding loop of bovine alpha-lactalbumin (alpha-LA) and provides a side-chain carboxylate oxygen for ligand Ca(II) co-ordination, was substituted by either alanine or asparagine. The physical properties and calcium-binding affinities were monitored by intrinsic fluorescence and circular dichroism spectroscopy. D87A alpha-LA displayed a total loss of rigid tertiary structure, a dramatic loss in secondary structure and negligible calcium affinity [Anderson et al. (1997) Biochemistry, 36, 11648-11654]. On the contrary, D87N alpha-LA displayed native-like secondary structure with a somewhat de-stabilized tertiary structure. When the well-documented N-terminal methionine was enzymatically removed from D87N alpha-LA [Veprintsev et al. (1999) PROTEINS: Struct. Funct. Genet., 37, 65-72], the structure appeared to more closely resemble native alpha-LA. Remarkably, the thermal transition mid-temperature of apo-desMetD87N alpha-LA was approximately 31 degrees C versus native apo- alpha-LA (approximately 25 degrees C), probably due to negative charge 'compensation' in the calcium co-ordination site. On the other hand, the transition mid-temperature of Ca(II)-bound desMetD87N alpha-LA was approximately 57 degrees C versus native alpha-LA (approximately 66 degrees C), which was related to a decreased Ca(II) affinity (K = approximately 2.1 x 10(5) versus approximately 1.7 x 10(7)/M at 40 degrees C, respectively). These results reaffirm that alanine substitution in site specific mutagenesis is not always a prudent choice. Substitutions must be conservative with only minimal changes in functional groups and side-chain volume.

Abstracts Poster Presentations (Z5)

The structural basis for the redox properties of the tetrahaem Desulfovibrio vulgaris cytochrome c3, has been investigated by site specific mutagenesis. Thermodynamic and structural characterisation of mutated cytochromes revealed the functional role of Lys45 and Thr24 in the wild type protein. Mutation of Lys45, located in the vicinity of the propionates of haem I, by the uncharged residues Thr and Gin, allowed to conclude that Lys45 controls the pKa of such propionate groups and confirms the involvement of this residue in the redox-Bohr effect ~. More recently, the replacement of Thr24, located in the vicinity ofhaem III, by Val showed how crucial Thr24 is for the stabilisation of the oxidised form of the protein. This single mutation increases the microscopic redox potential ofhaem III by 106 mV. This mutation also destabilizes the concerted two-electron step between the intermediate oxidation stages, 1 and 3, which can occur in the wild type protein. Therefore, Thr24 is responsible for the balance of the global network of cooperativities tuned to control thermodynamically the directionality of the stepwise electron transfer 2.

Identification of a p53 Response Element in the Promoter Region of the hMSH2 Gene Required for Expression in A2780 Ovarian Cancer Cells

Defects in the human MSH2 mismatch repair system have been implicated in cellular mutagenesis, tumorigenesis, and chemotherapeutic resistance. The current studies characterized the 5' upstream proximal promoter region of the hMSH2 gene using transient transfection of A2780 ovarian cancer cells. Serial deletions of a 1.88-kb fragment of the proximal promoter region of the hMSH2 gene revealed that promoter activity was restricted to the first -281 bp. Targeted deletions within this -281 bp region coupled with specific sequence mutagenesis identified a response element for the p53 tumor suppressor protein located between -242 and -222 bp. The -242 hMSH2 p53 element is configured as a direct tandem repeat palindrome with 80% homology to the p53 consensus binding sequence. Co-transfection of an hMSH2 reporter and p53 expression vector into the p53-null cell line SK-OV-3 produced 10-fold enhanced transcription, which was lost when the -242 to -222 p53 binding site was mutated. These results clearly demonstrate the presence of a previously unidentified p53 response element in the hMSH2 proximal promoter. Its location at -242 bp upstream of the start site of transcription is distinct from two previously reported p53 sites at -447 and -416, which transactivate in Saos-2 cells (Scherer, S. J., Maier, S. M., Seifert, M., Hanselmann, R. G., Zang, K. D., Muller-Hermelink, H. K., Angel, P., Welter, C., and Schartl, M. (2000) J. Biol. Chem. 275, 37469-37473). Finally, in sharp contrast to their activity in Saos-2 cells, deletion of the -447 and -416 sites in A2780 cells had no effect on hMSH2 promoter activity. Thus, it appears that p53 regulates hMSH2 expression through multiple cell type-specific DNA response elements.

Interruption of coding sequences by heterologous introns can enhance the functional expression of recombinant genes.

Sustained expression of recombinant proteins is a critical factor for the effectiveness of numerous applications in the biomedical sciences including the treatment of human disease by gene therapy, the large scale production of therapeutic proteins, as well as the investigation of gene function by transgenesis or cell type specific mutagenesis. Although much attention has been paid to the optimisation of regulatory sequences such as promoters, untranslated regions and polyadenylation signals, effective and sustained expression of recombinant genes in vivo is often difficult to achieve. Here we report that the creation of artificial exons, by insertion of two short heterologous introns into open reading frames, is not only compatible with functional expression, but also leads to a 30-fold enhancement of mRNA production for both green fluorescent protein and the bacteriophage P1-derived Cre recombinase. The levels of green fluorescence were increased five-fold in cell lines and sustained long-term expression at increased levels was observed in rat brain after transduction with a herpes simplex virus-based vector. The data presented identify a means by which the expression of recombinant genes can be enhanced considerably, in addition to and independently from the surrounding regulatory sequences. The method should help obtain sustained and effective expression of recombinant proteins in vivo.

A role for Octamer binding protein motifs in the regulation of the proximal preprotachykinin-A promoter

We have recently developed cell line models that express the endogenous rat preprotachykinin-A (rPPT) gene and support reporter gene expression directed by the rPPT promoter. These are the neuronal derived cell line NF2C and the pancreatic cell lines RINm5F and a derivative RIN-1027-B2. Reporter gene activity in these cell lines is similar to that observed in primary cultures of rat dorsal root ganglion neurons. Analysis of reporter gene expression supported by various fragments of the rPPT promoter demonstrated that although –865 to +92 supported expression, addition of fragments between +92 and +500 led to repression of expression. Two previously defined octamer binding motifs, present both 5′ and 3′ of the major transcriptional start site, have been postulated to be potential enhancers of rPPT activity and we have now used these cell lines to determine the role of these regions in the rPPT promoter. Site specific mutagenesis of these elements has shown not only that both sites are potential enhancers of gene expression but also that the 3′ element binds multiple factors, of which at least one has repressor function. Binding of this repressor protein over or adjacent to the 3′ octamer binding protein site leads to the observed repression of promoter activity in the –865 to +500 construct relative to the to –865 to +92 the fragment.

An approach to the removal of yeast specific O-linked oligo-mannoses from human midkine expressed in Pichia pastoris using site-specific mutagenesis.

Human midkine is expressed and secreted in the medium under the control of an AOX1 gene promoter in Pichia pastoris using its own secretion signal. The midkine precursor is properly processed to yield the correct amino-terminus of mature midkine. However, more than half of the product receives yeast specific mannosylations. The sites for the mannosylations were determined to be the three threonine residues in the carboxy-terminal region of human midkine. In order to obtain non-mannosylated midkine, alanine residues were substituted for the three threonine residues by site specific mutagenesis. HPLC and mass spectrometry confirmed that the mutant midkine contained almost no mannose residues. Despite the amino acid substitutions in the carboxy-terminal region, mutant human midkine, promoted CHO cell proliferation as well as normal midkine.

Retroviral expression of the hepatitis B virus x gene promotes liver cell susceptibility to carcinogen-induced site specific mutagenesis

Mutational inactivation of the tumor suppressor gene p53 is common in hepatocellular carcinomas (HCC). AGG to AGT transversion in codon 249 of exon 7 of the p53 gene occurs in over 50% of HCC from endemic regions, where both chronic infection with the hepatitis B virus (HBV) and exposure to carcinogens such as aflatoxin B1 (AFB1) prevail. In this study, we report the effect of the HBV x protein (HBx) on carcinogen-induced cytotoxicity and AGG to AGT mutation in codon 249 of the p53 gene in the human liver cell line CCL13. Expression of HBx, as revealed by its transactivation function, results in enhanced cell susceptibility to cytotoxicity induced by the AFB1 active metabolite, AFB1-8,9-epoxide, and benzo(a)pyrene diol-epoxide. Under similar conditions, expression of HBx promotes apoptosis in a subset of cell population. Exposure to AFB1-8,9-epoxide alone induces a low frequency of AGG to AGT mutation in codon 249 of the p53 gene, as determined by an allele-specific polymerase chain reaction (AS-PCR) assay. However, expression of HBx enhances the frequency of AFB1-epoxide-induced AGG to AGT mutation compared to control cells. In summary, this study demonstrates that expression of HBx enhances liver cell susceptibility to carcinogen-induced mutagenesis, possibly through alteration of the balance between DNA repair and apoptosis, two cellular defense mechanisms against genotoxic stress.

Analysis of the DNA-binding domain of Escherichia coli DnaA protein.

The DNA-binding domain of the Escherichia coli DnaA protein is represented by the 94 C-terminal amino acids (domain 4, aa 374-467). The isolated DNA-binding domain acts as a functional repressor in vivo, as monitored with a mioC:lacZ translational fusion integrated into the chromosome of the indicator strain. In order to identify residues required for specific DNA binding, site-directed and random PCR mutagenesis were performed, using the mioC:lacZ construct for selection. Mutations defective in DNA binding were found all over the DNA-binding domain with some clustering in the basic loop region, within presumptive helix B and in a highly conserved region at the N-terminus of presumptive helix C. Surface plasmon resonance (SPR) analysis revealed different binding classes of mutant proteins. No or severely reduced binding activity was demonstrated for amino acid substitutions at positions R399, R407, Q408, H434, T435, T436 and A440. Altered binding specificity was found for mutations in a 12 residue region close to the N-terminus of helix C. The defects of the classical temperature sensitive mutants dnaA204, dnaA205 and dnaA211 result from instability of the proteins at higher temperatures. dnaX suppressors dnaA71 and dnaA721 map to the region close to helix C and bind DNA non-specifically.

活性酸素によるアポトーシスとレドックス制御系の役割

Apoptosis is induced by reactive oxygen species (ROS) and nitrogen species (RNS) which are produced under inflammatory conditions. Antioxidative enzymes such as superoxide dismutases and glutathione peroxidases (GPx) are known to prevent apoptosis through scavenging cytotoxic ROS. We found that nitric oxide (NO) specifically inhibits GPx activity, resulting in augmentation of peroxides. This suggests that dysfunction of GPx by binding of NO to the active site, selenocysteine, is at leaset in part responsible for the cellular damage. Since imbalance of a redox state caused by overexpression of aldose reductase also induces apoptosis, a significant role of redox system in controlling apoptosis is implicated. We have cloned and analyzed five members of the peroxiredoxin (Prx) gene family, which detoxify peroxides with thioredoxin and/or glutathione as electron donors. Analysis of the fourth member, Prx IV, indicates that it is a secretable form and exerts its protective role outside of the cells. We also demonstrated the essential role of selenocysteine residue in thioredoxin reductase by site specific mutagenesis as well as by deletion analysis of the selenocysteine insertion sequence element located at the 3' -untranslated region of the gene. Possible roles of these redox systems in protection against apoptotic stimuli are discussed.

10.2172/10116384

During the first phase of the project regarding specific mutagenesis of the chlorophyll-binding protein CP47 in photosystem II (PS II) most of the time has been devoted to (1) establishment of an optimal procedure for the reintroduction of psbB (the gene encoding CP47) carrying a site-directed mutation into the experimental organism, the cyanobacterium Synechocystis sp. PCC 6803, (2) preparations for site-directed mutagenesis, and (3) creation and analysis of chimaeric spinach/cyanobacterial CP47 mutants of Synechocystis. In the coming year, psbB constructs with site-directed mutations in potential chlorophyll-binding regions of CP47 will be introduced into the Synechocystis genome, and site-directed mutants will be characterized according to procedures described in the original project description. In addition, analysis of chimaeric CP47 mutants will be continued.

ChemInform Abstract: Inhibitors of p38 MAP Kinase: Therapeutic Intervention in Cytokine‐Mediated Diseases

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Inhibitors of p38 MAP Kinase: Therapeutic Intervention in Cytokine-Mediated Diseases

p38 MAP kinase is a member of the family of kinases which mediate intracellular transduction pathways. The activation of this particular MAP kinase pathway is in response to a broad variety of extracellular stimuli. Subsequent downstream events triggered by p38 activation result in the production of IL-1 and TNF-a, suggesting that inhibition of this enzyme may provide a useful therapeutic target for intervention in various diseases mediated by these cytokines. Understanding the biological consequences of p38 activation and inhibition has been the subject of intensive research over the past several years and there is now ample evidence to suggest that inhibition of this enzyme represents a valid approach for target intervention in various cytokine-mediated diseases. Crystal structures of both apo enzyme and enzyme bound to various ligands in conjunction with site specific mutagenesis studies have provided a wealth of information regarding the interactions necessary to result in potent inhibition and selectivity from other kinases. This information has proven useful towards the analysis of previously reported compounds and will provide additional insight towards the design of new compounds and building upon existing SAR.

Needle-in-a-haystack detection and identification of base substitution mutations in human tissues.

Background and induced germline mutagenesis and other genotoxicity studies have been hampered by the lack of a sufficiently sensitive technique for detecting mutations in a small cluster of cells or a single cell in a tissue sample composed of millions of cells. The most frequent type of genetic alteration is intragenic. The vast majority of oncogenic mutations in human and mammalian cancer involves only single base substitutions. We have developed universally applicable techniques that not only provide the necessary sensitivity and specificity for site specific mutagenesis studies, but also identify the point mutation. The exponential amplification procedures of polymerase chain reaction (PCR) and ligase chain reaction (LCR) have been combined with restriction endonuclease (RE) digestion to enable the selective enrichment and detection of single base substitution mutations in human oncogenic loci at a sensitivity of one mutant in more than 10(7) wild type alleles. These PCR/RE/LCR procedures have been successfully designed and used for codons 12 and 248 of the Ha-ras and p53 genes, respectively, both of which contain a natural MspI restriction endonuclease recognition sequence. These procedures have also been adapted for the detection and identification of mutations in oncogenic loci that do not contain a natural restriction endonuclease recognition sequence. Using PCR techniques, a HphI site was incorporated into the codons 12/13 region of the human N-ras gene, which was then used for the selective enrichment of mutants at this oncogenic locus. These PCR/RE/LCR procedures for base substitution mutations in codon 12 of the N-ras gene were found to have the sensitivity of detection of at least one mutant allele in the presence of the DNA equivalent of 10(6) wild type cells. Only one peripheral blood leukocyte DNA specimen out of nine normal individuals displayed an observable Ha-ras mutation that was present at frequency between 10(-5) and 10(-6). These PCR/RE/LCR techniques for detecting and identifying base substitution mutations are universally applicable to almost any locus or base site within the human or animal genome. With the added advantage of the adjustability of both the amount of DNA (number of genomes) to be tested and the sensitivity (10(-2) to 10(-7)) of the assay selection or enrichment procedures, these PCR/RE/LCR techniques will be useful in addressing a broad range of important questions in mutagenesis and carcinogenesis.

A novel means of self-protection, unrelated to toxin activation, confers immunity to the bactericidal effects of the Enterococcus faecalis cytolysin.

Enterococcus faecalis has become a pervasive clinical problem due to the emergence of resistance to most antibiotics. The cytolysin of E. faecalis is a novel bacterial toxin that contributes to the severity of disease. It consists of two structural subunits, which together possess both hemolytic and bactericidal activity. Both toxin subunits are encoded in a complex operon frequently harbored on pheromone-responsive plasmids. E. faecalis strains lacking such plasmids are susceptible to the bactericidal effects of the cytolysin. A novel cytolysin immunity determinant at the 3' end of the pAD1 cytolysin operon is described in the present study. Deletion analysis and specific mutagenesis isolated the immunity function to a single open reading frame. Specific mutagenesis experiments demonstrate that cytolysin immunity is unrelated to cytolysin activator (CylA) expression as previously proposed. Cytolysin immunity is, however, encoded on the same transcript as and 3' to CylA, and previous associations between immunity and CylA can be ascribed to the polar behavior of Tn917 insertion.

Mechanisms of mutation induction in germ cells of the mouse as assessed by the specific locus test

Mouse germ cell specific locus mutagenesis data and a molecular characterization of mutant alleles have been reviewed to arrive at an understanding of the mechanism of mutation induction in mammals. (a) The spermatogenic stage specificity for the sensitivity to mutation induction by 20 chemical mutagens is considered. (b) The effects of a saturable repair process and its recovery over time are examined for the mutagenic efficiency of ethylnitrosourea. (c) The mutagenic events following methylnitrosourea and chlorambucil are shown to be mainly deletions. In contrast the mutations recovered after ethylnitrosourea treatment are almost exclusively base pair substitutions. (d) It is emphasized that to date very few specific locus experiments have been designed to test for mutagenic events outside the interval stem cell spermatogonia—mature spermatozoa. A specific locus mutation has recently been shown to be due to loss of heterozygosity via mitotic recombination in an early zygote stage and suggests a broader range of possible mechanisms of mutation when these stages are considered. (e) With the cloning of all 7 marker loci mutation analysis at the molecular level will allow a more direct assessment of the mutation process in future studies.

Fine mapping of the antigen-antibody interaction of scFv215, a recombinant antibody inhibiting RNA polymerase II from Drosophila melanogaster.

A bacterially expressed single chain antibody (scFv215) directed against the largest subunit of drosophila RNA polymerase II was analysed. Structure and function of the antigen binding site in scFv215 were probed by chain shuffling and by site-specific mutagenesis. The entire variable region of either the heavy or light chain was replaced by an unrelated heavy or light chain. Both replacements resulted in a total loss of binding activity suggesting that the antigen binding site is contributed by both chains. The functional contributions of each complementarity determining region (CDR) were investigated by site specific mutagenesis of each CDR separately. Mutations in two of the CDRs, CDR1 of light chain and CDR2 of heavy chain, reduced the binding activity significantly. Each of the amino acids in these two CDRs was replaced individually by alanine (alanine walking). Seven amino acid substitutions in the two CDRs were found to reduce the binding activity by more than 50%. The data support a computer model of scFv215 which fits an epitope model based on a mutational analysis of the epitope suggesting an alpha-helical structure for the main contact area.

Essential role of interferon regulatory factor 3 in direct activation of RANTES chemokine transcription.

Localized and systemic cytokine production in virus-infected cells play an important role in the outcome of viral infection and pathogenicity. Activation of the interferon regulatory factors (IRF) in turn is a critical mediator of cytokine gene transcription. Recent studies have focused on the 55-kDa IRF-3 gene product as a direct transcriptional regulator of type 1 interferon (IFN-alpha and IFN-beta) activation in response to virus infection. Virus infection induces phosphorylation of IRF-3 on specific C-terminal serine residues and permits cytoplasmic-to-nuclear translocation of IRF-3, activation of DNA binding and transactivation potential, and association with the CBP/p300 coactivator. We previously generated constitutively active [IRF-3(5D)] and dominant-negative forms of IRF-3 that control IFN-beta and IFN-alpha gene expression. In an effort to characterize the range of immunoregulatory genes controlled by IRF-3, we now demonstrate that endogenous human RANTES gene transcription is directly induced in tetracycline-inducible IRF-3(5D)-expressing cells or paramyxovirus-infected cells. We also show that a dominant-negative IRF-3 mutant inhibits virus-induced expression of the RANTES promoter. Specific mutagenesis of overlapping ISRE-like sites located between nucleotides -123 and -96 in the RANTES promoter reduces virus-induced and IRF-3-dependent activation. These studies broaden the range of IRF-3 immunoregulatory target genes to include at least one member of the chemokine superfamily.

Cleavage of rhesus rotavirus VP4 after arginine 247 is essential for rotavirus-like particle-induced fusion from without.

We recently described our finding that recombinant baculovirus-produced virus-like particles (VLPs) can induce cell-cell fusion similar to that induced by intact rotavirus in our assay for viral entry into tissue culture cells (J. M. Gilbert and H. B. Greenberg, J. Virol. 71:4555-4563, 1997). The conditions required for syncytium formation are similar to those for viral penetration of the plasma membrane during the course of viral infection. This VLP-mediated fusion activity was dependent on the presence of the outer-layer proteins, viral protein 4 (VP4) and VP7, and on the trypsinization of VP4. Fusion activity occurred only with cells that are permissive for rotavirus infection. Here we begin to dissect the role of VP4 in rotavirus entry by examining the importance of the precise trypsin cleavage of VP4 and the activation of VP4 function related to viral entry. We present evidence that the elimination of the three trypsin-susceptible arginine residues of VP4 by specific site-directed mutagenesis prevents syncytium formation. Two of the three arginine residues in VP4 are dispensable for syncytium formation, and only the arginine residue at site 247 appears to be required for activation of VP4 functions and cell-cell fusion. Using the recombinant VLPs in our syncytium assay will aid in understanding the conformational changes that occur in VP4 involved in rotavirus penetration into host cells.

Mutagenesis and characterization of specific residues in fatty acid ethyl ester synthase: a gene for alcohol-induced cardiomyopathy.

Fatty acid ethyl ester synthase-III metabolizes both ethanol and carcinogens. Structure-function studies of the enzyme have not been performed in relation to site specific mutagenesis. In this study, three residues (Gly 32, Cys 39 and His 72) have been mutated to observe their role in enzyme activity. Gly to Gln, Cys to Trp and His to Ser mutations did not affect fatty acid ethyl ester synthase activity, but His to Ser mutant had less than 9% of control glutathione S-transferase activity. The apparent loss of transferase activity reflected a 28 fold weaker binding constant for glutathione. Thus, this study indicates that Gly and Cys may not be important for synthase or transferase activities however, histidine may play a role in glutathione binding, but it is not an essential catalytic residue of glutathione S-transferase or for fatty acid ethyl ester synthase activity.

Control of alternative pre-mRNA splicing by distributed pentameric repeats.

Multiple copies of the hexamer TGCATG have been shown to regulate fibronectin pre-mRNA alternative splicing. GCATG repeats also are clustered near the regulated calcitonin-specific 3' splice site in the rat calcitonin/CGRP gene. Specific mutagenesis of these repeats in calcitonin/CGRP pre-mRNA resulted in the loss of calcitonin-specific splicing, suggesting that the native repeats act to enhance alternative exon inclusion. Mutation of subsets of these elements implies that alternative splicing requires a minimum of two repeats, and that the combination of one intronic and one exonic repeat is necessary for optimal cell-specific splicing. However, multimerized intronic repeats inhibited calcitonin-specific splicing in both the wild-type context and in a transcript lacking endogenous repeats. These results suggest that both the number and distribution of repeats may be important features for the regulation of tissue-specific alternative splicing. Further, RNA containing a single repeat bound cell-specific protein complexes, but tissue-specific differences in protein binding were not detected by using multimerized repeats. Together, these data support a novel model for alternative splicing regulation that requires the cell-specific recognition of multiple, distributed sequence elements.