Characterization of in-frame proteins encoded by cvaA, an essential gene in the colicin V secretion system: CvaA* stabilizes CvaA to enhance secretion.

Colicin V (ColV), an antibacterial peptide toxin, uses a dedicated signal sequence-independent export system for its extracellular secretion in Escherichia coli. The products of at least three genes (a chromosomal tolC gene and two plasmid-born cvaA and cvaB genes) are involved in this process. To characterize the gene products, the cvaA gene was subcloned and expressed under the control of T7 RNA polymerase promoter. Two in-frame proteins, CvaA and CvaA*, were expressed and identified. DNA sequences predicted that both proteins have two potential translational initiation sites. N-terminal peptide sequencing showed that the translation of CvaA starts from a TTG, 11 amino acids upstream of the previously proposed ATG initiation site. CvaA* is translated from an upstream ATG. Expression of both CvaA and CvaA* was induced by the iron chelator 2,2'-dipyridyl, indicating that cvaA is negatively regulated at least partially by Fur. CvaA*-depleted cells were found to secrete less ColV, based on reduced activity in the supernatant, than did wild type, which was recovered by the addition of a plasmid producing CvaA*. Interestingly, CvaA*-depleted and wild-type cells had similar levels of intracellular ColV activity. Translational fusions showed that the syntheses of ColV and CvaA are not affected by CvaA* depletion. However, CvaA in CvaA*-depleted cells was less stable than that in wild-type cells, indicating that CvaA* may directly or indirectly affect the stability of CvaA. We conclude that CvaA* is not essential for ColV secretion but that it enhances the ColV secretion by stabilizing the CvaA protein.     

Functional complementation between bacterial MDR-like export systems: colicin V, alpha-hemolysin, and Erwinia protease.

The antibacterial protein Colicin V (ColV) is secreted from gram-negative bacteria by a signal sequence-independent pathway. The proteins that mediate the export of ColV share sequence similarities with components from other signal sequence-independent export systems such as those for alpha-hemolysin (Hly) and Erwinia protease (Prt). We report here that the intact HlyBD export system can export active ColV from Escherichia coli strains lacking the ColV export proteins CvaA and CvaB. The individual Hly export genes complement mutations in their respective ColV homologs, but do so at a lower efficiency. When CvaA or CvaB is expressed along with the intact HlyBD exporter, the Cva export protein interferes with export of ColV through the HlyBD system. Gene fusions and point mutations in the ColV structural gene were used to define signals in ColV recognized by the Hly exporter. An export signal in ColV recognized by HlyBD is localized to the amino-terminal 57 amino acids of the protein. In addition, mutations in the ColV export signal differentially affect export through CvaAB and HlyBD, suggesting differences in signal specificity between the Cva and Hly systems. The three Erwinia protease export proteins can also export active ColV, and interference is seen when CvaA or CvaB is expressed along with the intact Prt exporter. Functional complementation is not reciprocal; alpha-hemolysin is not exported through either the ColV system or the Prt system.     

Genetic Analysis of the Colicin V Secretion Pathway

Colicin V (ColV) is peptide antibiotic secreted by Escherichia coli through a dedicated exporter composed of three proteins, CvaA, CvaB, and TolC. ColV secretion is independent of the E. coli general secretory pathway (Sec) but requires an N-terminal export signal specific for the CvaAB/TolC exporter. ColV secretion was characterized using genetic and biochemical methods. When the ColV N-terminal extension is replaced with the OmpA signal sequence, the Sec system can localize ColV to the periplasm. Periplasmic ColV is lethal to cells lacking the ColV immunity protein, Cvi. Based on this result, a genetic assay was designed to monitor for the presence of periplasmic ColV during normal CvaAB/TolC mediated secretion. Results indicate that low levels of ColV may be present in the periplasm during secretion. Precursor and mature ColV were also characterized from the wild-type system and in various exporter mutant backgrounds using immunoprecipitation. ColV processing is rapid in wild-type cells, and CvaA and CvaB are critical for processing to occur. In contrast, processing occurs normally, albeit more slowly, in a TolC mutant.     

Positive and negative mutant selection in the human histone hairpin-binding protein using the yeast three-hybrid system

We have used the yeast three-hybrid system in a positive selection for mutants of the human histone hairpin-binding protein (HBP) capable of interacting with non-canonical hairpins and in a negative selection for loss-of-binding mutants. Interestingly, all mutations from the positive selection are located in the N- and C-terminal regions flanking a minimal RNA-binding domain (RBD) previously defined between amino acids 126 and 198. Further, in vitro binding studies demonstrate that the RBD, which shows no obvious similarity to other RNA-binding motifs, has a relaxed sequence specificity compared to full-length HBP, allowing it to bind to mutant hairpin RNAs not normally found in histone genes. These findings indicate that the sequences flanking the RBD are important for restricting binding to the highly conserved histone hairpin structure. Among the loss-of-binding mutations, about half are nonsense mutations distributed throughout the N-terminal part and the RBD whereas the other half are missense mutations restricted to the RBD. Whereas the nonsense mutations permit a more precise definition of the C-terminal border of the RBD, the missense mutations identify critical residues for RNA binding within the RBD.     

Sequence variation in two lignin biosynthesis genes,cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase 2 (CAD2)

Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase 2 (CAD2) are genes which may influence variation in lignin content and composition within plants. Sequence variation within these genes may be responsible for changes in enzyme activity and/or specificity, which could cause variation in lignin content or composition. This study examines sequence variation within these two genes in Eucalyptus globulus, an important species used in pulp and paper-making. Twenty-one single nucleotide polymorphisms (SNPs) were identified in the exons of CCR, of which nine were neutral mutations and 12 were missense mutations. Six of the missense mutations affected highly conserved amino acids within the protein sequence of CCR. Eight SNPs were identified in the CAD2 exons, six of which were neutral mutations and two which were missense mutations. One of the missense mutations affected a highly conserved amino acid within the protein sequence. In addition, 32 SNPs were identified in the CCR introns along with four insertion/deletions and two polyA length variation regions. Polymorphism affecting highly conserved amino acids may alter enzyme function and this molecular variation may be linked to variation in lignin profiles. Selecting positive alleles which produce favourable lignin profiles would be advantageous in tree breeding programs.     

Domain wise docking analyses of the modular chitin binding protein CBP50 from Bacillus thuringiensis serovar konkukian S4

This paper presents an in silico characterization of the chitin binding protein CBP50 from B. thuringiensis serovar konkukian S4 through homology modeling and molecular docking. The CBP50 has shown a modular structure containing an N-terminal CBM33 domain, two consecutive fibronectin-III (Fn-III) like domains and a C-terminal CBM5 domain. The protein presented a unique modular structure which could not be modeled using ordinary procedures. So, domain wise modeling using MODELLER and docking analyses using Autodock Vina were performed. The best conformation for each domain was selected using standard procedure. It was revealed that four amino acid residues Glu-71, Ser-74, Glu-76 and Gln-90 from N-terminal domain are involved in protein-substrate interaction. Similarly, amino acid residues Trp-20, Asn-21, Ser-23 and Val-30 of Fn-III like domains and Glu-15, Ala-17, Ser-18 and Leu-35 of C-terminal domain were involved in substrate binding. Site-directed mutagenesis of these proposed amino acid residues in future will elucidate the key amino acids involved in chitin binding activity of CBP50 protein.     

Molecular analysis of type I-A (tyrosinase negative) oculocutaneous albinism

Type I oculocutaneous albinism (OCA) is caused by the reduction in or absence of activity of tyrosinase in melanocytes in skin, hair, and the eyes, the result of mutations of the tyrosinase gene. To date, a total of 22 unique mutations in the coding region of tyrosinase have been described in the literature. In this report we present 5 additional mutations of the tyrosinase gene associated with type I-A OCA in four individuals, including 2 missense, 1 frameshift and 2 nonsense mutations, and review the relevant literature on all published mutations. Analysis of the distribution of all identified missense mutations (n = 17) shows that most cluster in three areas of the gene and involve amino acids conserved between humans and the mouse. Two clusters involve the copper A and copper B binding sites and may disrupt the metal ion-protein interaction necessary for enzyme function. The third cluster in exon I could represent a functional domain important in enzyme function such as the tyrosine or the dihydroxyphenylalanine (DOPA) binding site of the enzyme. Small deletions or insertions resulting in frameshift mutations and nonsense mutations are distributed throughout the coding region and do not appear to cluster.     

A comparison of the mutation spectra of Menkes disease and Wilson disease

The genes for two copper-transporting ATPases, ATP7A and ATP7B, are defective in the heritable disorders of copper imbalance, Menkes disease (MNK) and Wilson disease (WND), respectively. A comparison of the two proteins shows extensive conservation in the signature domains, with amino acid identities outside of the conserved domains being limited. The mutation spectra of MNK and WND were compared to confirm and refine further regions critical for normal function. Mutations were found to be relatively widespread; however, the majority was concentrated within defined functional domains and membrane-spanning segments, reinforcing the importance of these regions for protein function. Of the total published point mutations in ATP7A, 23.0% are splice-site, 20.7% nonsense, 17.2% missense, and 39.1% small insertions/deletions. There is a high prevalence (58.2%) of missense mutations in ATP7B. For the other mutations in ATP7B, 7.4% are splice-site, 7.4% nonsense, and 27.0% small insertions/deletions. A region of possible importance is the intervening sequence between the last copper-binding domain and the first transmembrane helix, as this region has a high percentage of MNK mutations. Similarly, the region containing the ATP-binding domain has 24.6% of all WND mutations. The study of mutation locations is useful for defining critical regions or residues and for efficient molecular diagnosis.Electronic Supplementary Material Supplementary material is available for this article if you access the article at .     

Mutations in IMPG2, Encoding Interphotoreceptor Matrix Proteoglycan 2, Cause Autosomal-Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a heterogeneous group of inherited retinal diseases caused by progressive degeneration of the photoreceptor cells. Using autozygosity mapping, we identified two families, each with three affected siblings sharing large overlapping homozygous regions that harbored the IMPG2 gene on chromosome 3. Sequence analysis of IMPG2 in the two index cases revealed homozygous mutations cosegregating with the disease in the respective families: three affected siblings of Iraqi Jewish ancestry displayed a nonsense mutation, and a Dutch family displayed a 1.8 kb genomic deletion that removes exon 9 and results in the absence of seven amino acids in a conserved SEA domain of the IMPG2 protein. Transient transfection of COS-1 cells showed that a construct expressing the wild-type SEA domain is properly targeted to the plasma membrane, whereas the mutant lacking the seven amino acids appears to be retained in the endoplasmic reticulum. Mutation analysis in ten additional index cases that were of Dutch, Israeli, Italian, and Pakistani origin and had homozygous regions encompassing IMPG2 revealed five additional mutations; four nonsense mutations and one missense mutation affecting a highly conserved phenylalanine residue. Most patients with IMPG2 mutations showed an early-onset form of RP with progressive visual-field loss and deterioration of visual acuity. The patient with the missense mutation, however, was diagnosed with maculopathy. The IMPG2 gene encodes the interphotoreceptor matrix proteoglycan IMPG2, which is a constituent of the interphotoreceptor matrix. Our data therefore show that mutations in a structural component of the interphotoreceptor matrix can cause arRP.     

Identification of Glutamate Residues Important for Catalytic Activity or Thermostability of a Truncated Bacillus sp. Strain TS-23 α-amylase by Site-directed Mutagenesis

The importance of 17 glutamate residues of a truncated Bacillus sp. strain TS-23 α-amylase (BACΔNC) was investigated by site-directed mutagenesis. The Ala- and Asp-substituted variants were overexpressed in the recombinant E. coli cells and the 54-kDa proteins were purified to nearly homologous by nickel-chelate chromatography. Glu-295, which locates in the conserved region III of amylolytic enzymes, mutations resulted in a complete loss of enzyme activity. The specific activity for E151A was decreased by more than 30%, while other variants showed activity comparable to that of BACΔNC. A decreased half-life at 70°C was observed for Glu-219 variants with respective to the wild-type enzyme, suggesting that replacement of Glu-219 by either Ala or Asp might have a significant destabilizing effect on the protein structure.     

Identification of novel alleles induced by EMS-mutagenesis in key genes of kernel hardness and starch biosynthesis in wheat by TILLING

To identify novel allelic variations in key genes of wheat quality, the present study used the targeting induced local lesions in genomes platform to detect point mutations in target genes. The wheat variety Longfumai 17 was treated by the mutagen ethyl methanesulfonate to produce a bulk M₂ generation, and the population included 1122 plants. A total length of 3906.80 kb nucleotides was analyzed, and the average mutation density was 1/244.17 kb. The identified mutations included G>A substitutions (43.75%), C>T substitutions (31.25%), A insertions (12.50%), T insertions (6.25%), and deletions (6.25%). These point mutations led to changes in amino acids and thus the encoded protein sequences, ultimately producing 18.75% of missense mutations, 12.50% of frame shift mutations, 6.25% of nonsense mutations, 25.00% of silent mutations and 37.50% of non-coding region mutations. In the kernel hardness gene Pinb and 3 starch synthesis genes waxy, Agp2 and SSIIa-A, we detected 16 different point mutations in 25 mutant lines. The Pinb gene harbored two missense mutations and a nonsense mutation; the C>T missense mutation resulted in a novel allele, this novel allele and the nonsense mutation alerted protein 3D structure; the waxy gene presented missense and frame shift mutations; the Agp2 gene carried a missense mutation; the SSIIa-A incurred a missense mutation and a frame shift mutation that resulted in premature protein termination. All the frame shift mutations, nonsense mutations and the Pinb novel allele resulted in allelic variation of their corresponding genes, which in turn affected their gene functions. The identified mutant lines can be used as intermediate materials in wheat quality improvement schemes.     

Spectrum of MECP2 gene mutations in a cohort of Indian patients with Rett syndrome: Report of two novel mutations

Rett syndrome (RTT) is an X-linked neurodevelopmental disorder, primarily affecting females and characterized by developmental regression, epilepsy, stereotypical hand movements, and motor abnormalities. Its prevalence is about 1 in 10,000 female births. Rett syndrome is caused by mutations within methyl CpG-binding protein 2 (MECP2) gene. Over 270 individual nucleotide changes which cause pathogenic mutations have been reported. However, eight most commonly occurring missense and nonsense mutations account for almost 70% of all patients. We screened 90 individuals with Rett syndrome phenotype. A total of 19 different MECP2 mutations and polymorphisms were identified in 27 patients. Of the 19 mutations, we identified 7 (37%) frameshift, 6 (31%) nonsense, 14 (74%) missense mutations and one duplication (5%). The most frequent pathogenic changes were: missense p.T158M (11%), p.R133C (7.4%), and p.R306C (7.4%) and nonsense p.R168X (11%), p.R255X (7.4%) mutations. We have identified two novel mutations namely p.385-388delPLPP present in atypical patients and p.Glu290AlafsX38 present in a classical patient of Rett syndrome. Sequence homology for p.385-388delPLPP mutation revealed that these 4 amino acids were conserved across mammalian species. This indicated the importance of these 4 amino acids in structure and function of the protein. A novel variant p.T479T has also been identified in a patient with atypical Rett syndrome.     

Identification of residues of the Caenorhabditis elegans LIN-1 ETS domain that are necessary for DNA binding and regulation of vulval cell fates

LIN-1 is an ETS domain protein. A receptor tyrosine kinase/Ras/mitogen-activated protein kinase signaling pathway regulates LIN-1 in the P6.p cell to induce the primary vulval cell fate during Caenorhabditis elegans development. We identified 23 lin-1 loss-of-function mutations by conducting several genetic screens. We characterized the molecular lesions in these lin-1 alleles and in several previously identified lin-1 alleles. Nine missense mutations and 10 nonsense mutations were identified. All of these lin-1 missense mutations affect highly conserved residues in the ETS domain. These missense mutations can be arranged in an allelic series; the strongest mutations eliminate most or all lin-1 functions, and the weakest mutation partially reduces lin-1 function. An electrophoretic mobility shift assay was used to demonstrate that purified LIN-1 protein has sequence-specific DNA-binding activity that required the core sequence GGAA. LIN-1 mutant proteins containing the missense substitutions had dramatically reduced DNA binding. These experiments identify eight highly conserved residues of the ETS domain that are necessary for DNA binding. The identification of multiple mutations that reduce the function of lin-1 as an inhibitor of the primary vulval cell fate and also reduce DNA binding suggest that DNA binding is essential for LIN-1 function in an animal.     

Asparagine 26, glutamic acid 31, valine 45, and tyrosine 64 of Ras proteins are required for their oncogenicity.

Ras and Rap1 proteins are related GTP-dependent signal transducers which require Gly-12, the effector domain (residues 32-40), and Ala-59 for stimulation of their GTPase activities by GAP1 and GAP3, respectively. The replacement of Gly-12 by Val or Ala-59 by Thr potentiates the Ras oncogenicity and Rap1A antioncogenicity. However, the mutations in the effector domain, in particular the replacement of Thr-35 by Ala, abolish both Ras oncogenicity and Rap1A antioncogenicity, indicating that the effector domain is involved in interactions of these signal transducers with their targets as well as the GAPs. In this paper, we demonstrate that (i) replacement of Tyr-64 of the Ha-Ras protein or Phe-64 of the Rap1A protein by Glu or other non-hydrophobic amino acids reduces their intrinsic GTPase activities and abolishes their stimulation by GAP1 or GAP3, respectively, (ii) replacement of Tyr-64 by Gly and other non-hydrophobic amino acids results in complete loss of the oncogenicity of the v-Ha-Ras protein, indicating that the hydrophobic residue 64, in addition to the known effector domain, is essential for the Ras protein to interact with its target as well as GAP1. In addition we have found that Asn-26, Glu-31, and Val-45 of the v-Ha-Ras protein are required for its oncogenicity. Replacement of the Ras residues at either positions 26, 31, or 45 by the corresponding Rap1A residues abolishes the Ras oncogenicity.     

Effect of precisely identified mutations in the spoIIAC gene of Bacillus subtilis on the toxicity of the sigma-like gene product to Escherichia coli

Summary Yudkin (1986) has shown that the spoIIAC gene of Bacillus subtilis cannot be cloned in Escherichia coli in such an orientation that it is expressed. This toxicity of the gene product has been attributed to its close homology with the sigma subunit of the E. coli RNA polymerase. The effect of six individual mutations in spoIIAC has now been studied. All six mutant genes could be cloned in E. coli in an orientation that does not allow expression. When in the orientation that permits expression, one mutant gene could not be cloned, and a second substantially hampered growth; both mutations lie in the region that is believed to encode the DNA-binding domain of the protein. By contrast, two missense mutations in the region of the gene thought to encode the domain that binds to the core RNA polymerase rendered the protein harmless in E. coli, as did two nonsense mutations.     

Characterization of a germline mosaicism in families with Lowe syndrome, and identification of seven novel mutations in the OCRL1 gene.

The oculocerebrorenal syndrome of Lowe (OCRL) is an X-linked disorder characterized by major abnormalities of eyes, nervous system, and kidneys. Mutations in the OCRL1 gene have been associated with the disease. OCRL1 encodes a phosphatidylinositol 4, 5-biphosphate (PtdIns[4,5]P2) 5-phosphatase. We have examined the OCRL1 gene in eight unrelated patients with OCRL and have found seven new mutations and one recurrent in-frame deletion. Among the new mutations, two nonsense mutations (R317X and E558X) and three other frameshift mutations caused premature termination of the protein. A missense mutation, R483G, was located in the highly conserved PtdIns(4,5)P2 5-phosphatase domain. Finally, one frameshift mutation, 2799delC, modifies the C-terminal part of OCRL1, with an extension of six amino acids. Altogether, 70% of missense mutations are located in exon 15, and 52% of all mutations cluster in exons 11-15. We also identified two new microsatellite markers for the OCRL1 locus, and we detected a germline mosaicism in one family. This observation has direct implications for genetic counseling of Lowe syndrome families.     

Genetic analysis of the LexA repressor: Isolation and characterization of LexA(Def) mutant proteins

Summary We report the isolation of LexA mutant proteins with impaired repressor function. These mutant proteins were obtained by transforming a LexA-deficient recA-lacZ indicator strain with a randomly mutagenized plasmid harbouring the lexA gene and subsequent selection on MacConkey-lactose indicator plates. A total of 24 different lexA(Def) missense mutations were identified. All except three mutant proteins are produced in near-normal amounts suggesting that they are fairly resistant to intracellular proteases. All lexA(Def) missense mutations are situated within the first 67 amino acids of the amino-terminal DNA binding domain. The properties of an intragenic deletion mutant suggest that the part of the amino-terminal domain important for DNA recognition or domain folding should extent at least to amino acids 69 or 70. A recent 2D-NMR study (Lamerichs et al. 1989) has identified three a helices in the DNA binding domain of LexA. The relative orientation of two of them (helices 2 and 3) is reminiscent of, but not identical to, the canonical helix-turn-helix motif suggesting nevertheless that helix 3 might be involved in DNA recognition. The distribution of the lexA(Def) missense mutations along the first 67 amino-terminal amino acids indeed shows some clustering within helix 3, since 8 out of the 24 different missense mutations are found in this helix. However one mutation in front of helix 1 and five mutations between amino acids 61 and 67 suggest that elements other than helices 2 and 3 may be important for DNA binding.     

Probing the structure-activity relationship of Escherichia coli LT-A by site-directed mutagenesis

Computer analysis of the crystallographic structure of the A subunit of Escherichia coil heat-labile toxin (LT) was used to predict residues involved in NAD binding, catalysis and toxicity. Following site-directed mutagenesis, the mutants obtained could be divided into three groups. The first group contained fully assembled, non-toxic new molecules containing mutations of single amino acids such as Val-53 → Glu or Asp, Ser-63 → Lys, Val-97 → Lys, Tyr-104 → Lys or Asp, and Ser-14 → Lys or Glu. This group also included mutations in amino acids such as Arg-7, Glu-110 and Glu-112 that were already known to be important for enzymatic activity. The second group was formed by mutations that caused the collapse or prevented the assembly of the A subunit: Leu-41 → Phe, Ala-45 → Tyr or Glu, Val-53 → Tyr, Val-60 → Gly, Ser-68 → Pro, His-70 → Pro, Val-97 → Tyr and Ser-114 → Tyr. The third group contained those molecules that maintained a wild-type level of toxicity in spite of the mutations introduced: Arg-54 → Lys or Ala, Tyr-59 → Met, Ser-68 → Lys, Ala-72 → Arg, His or Asp and Arg-192 → Asn. The results provide a further understanding of the structure–function of the active site and new, non-toxic mutants that may be useful for the development of vaccines against diarrhoeal diseases.     

Cloning,mutagenesis, and expression of the acetylcholinesterase gene from a strain of Musca domestica; the change from a drug-resistant to a sensitive enzyme

Insect acetylcholinesterase (AChE) is known to be a primary target of organophosphorus and carbamate insecticides. However chronic exposure to these chemicals has led to resistance to applied insecticides, due usually to mutation of the AChE gene. Analysis of the AChE gene (hm) of Musca domestica (the housefly), which is cloned in this report, reveals the relationship between mutation and insecticide resistance. The 2,076 bp hm encodes a mature protein of 612 amino acids (67 kDa), and an 80 residue signal peptide. Unlike the enzyme of 'sensitive' strains, the AChE used in this study was resistant to the organophosphorus insecticide, trichlorphon. DNA sequencing showed that this AChE is identical to that of the sensitive strains with the exception of three amino acids Met-82, Ala-262, and Tyr-327. Site-directed mutagenesis of the Ala-262 and Tyr-327 residues largely restored sensitivity to the insecticide, suggesting that these two residues are the key structural elements controlling sensitivity. In addition to these residues, Glu-234 and Ala-236 in the conserved sequence FGESAG are thought to play a role in modulating sensitivity to organophosphorus insecticides.