Localization of the catalytic activity in restrictocin molecule by deletion mutagenesis.

Restrictocin, produced by the fungus Aspergillus restrictus, is a highly specific ribonucleolytic toxin which cleaves a single phosphodiester bond between G4325 and A4326 in the 28S rRNA. It is a nonglycosylated, single-chain, basic protein of 149 amino acids. The putative catalytic site of restrictocin includes Tyr47, His49, Glu95, Arg120 and His136. To map the catalytic activity in the restrictocin molecule, and to study the role of N- and C-terminus in its activity, we have systematically deleted amino-acid residues from both the termini. Three N-terminal deletions removing 8, 15 and 30 amino acids, and three C-terminal deletions lacking 4, 6, and 11 amino acids were constructed. The deletion mutants were expressed in Escherichia coli, purified to homogeneity and functionally characterized. Removal of eight N-terminal or four C-terminal amino acids rendered restrictocin partially inactive, whereas any further deletions from either end resulted in the complete inactivation of the toxin. The study demonstrates that intact N- and C-termini are required for the optimum functional activity of restrictocin.     

Dissection of functional domains in phage fd adsorption protein. Discrimination between attachment and penetration sites

We constructed a set of deletion mutants in the attachment protein of phage fd. These mutants lack sequences coding for sections in the amino-terminal half. All the mutants that comprise a leader sequence are incorporated into phage particles. Our data strongly suggest a bipartite organization of the amino-terminal domain with (1) a region for receptor recognition and (2) a region that is necessary for penetration of the DNA into the host cell. These regions were mapped. Some evidence suggesting different roles for gene 3 protein in penetration of the outer and inner membrane are discussed. We demonstrate that the phenotypes caused by gene 3 protein in host cells can be subdivided into two groups with different sequence requirements: (1) phenotypes related to outer membrane disturbance; and (2) phenotypes related to the tolQRA transport system.     

Identification of structural domains within the cauliflower mosaic virus movement protein by scanning deletion mutagenesis and epitope tagging.

Plant viruses encode proteins that mediate their movement from cell to cell through plasmodesmata. Currently, the mechanisms of action of these movement proteins (MPs) can be divided broadly into two types, requiring or not requiring the presence of viral capsid protein. Cauliflower mosaic virus encodes a multifunctional MP (P1) that modifies plasmodesmata through the formation of tubules that contain virus particles. To investigate the structure of P1, 26 small deletions (scanning deletions) were used to characterize regions of P1 essential for full biological activity. These deletions identified an N-terminal region and a region close to but not at the C terminus as domains that could tolerate manipulation, although gross deletions of either domain abolished infection. In sequence comparisons with other caulimovirus MPs, these regions coincided with the areas of least amino acid homology. Epitope tags inserted into either of these regions were stably maintained in systemic infections, and in extracts from infected plants, tagged P1 was detected on immunoblots. We predicted that, from the hypervariability of these regions, they would be located on the surface of the native P1 structure. Immunofluorescence of P1-specific tubules formed on the surface of infected protoplasts confirmed that the N-terminal and C terminus-proximal regions were exposed on the surface of the P1 tubule subunit. These findings establish a structure for P1 that is likely to be applicable to other tubule-forming MPs.     

Identification of a locus involved in meningococcal lipopolysaccharide biosynthesis by deletion mutagenesis

A novel method for insertion/deletion mutagenesis in meningococci was devised. This consisted of ligating a digest of total chromosomal DNA to a 1.1 kb restriction fragment containing an erythromycin-resistance marker ( ermC ), and subsequent transformation of the ligation mixture into the homologous meningococcal strain H44/76. Southern blotting of a number of the resulting erythromycin-resistant transformants demonstrated that all carried the ermC gene inserted at different positions in the chromosome. Mutants with a specific phenotype were identified by screening with the anti-lipopolysaccharide (LPS) monoclonal antibody MN4A8B2, which is specific for immunotype L3. In this way, two independent L3-negative mutant strains were isolated. In transformation experiments with chromosomal DNA from these mutants, erythromycin-resistance and lack of MN4A8B2 reactivity were always linked, showing that the insertion/deletion was in a locus involved in LPS biosynthesis. On SDS–PAGE, the mutant LPS displayed an electrophoretic mobility intermediate between that produced by the previously isolated galE and rfaF mutant strains. Chemical analysis of the mutant LPS revealed that the structure was probably lipid A–(KDO)₂–(Hep)₂. Chromosomal DNA flanking the ermC insertion in these two mutant strains was cloned, and used as probe for the isolation of the corresponding region of the wild-type strain. From hybridization and polymerase chain reaction (PCR) analysis, it could be concluded that both mutations map to the same locus. The affected gene probably encodes the glycosyltransferase necessary for adding N -acetylglucosamine to heptose.     

The properties of sequences involved in insertion and deletion mutagenesis in rodents

We analyzed nucleotide sequences that were inserted to or deleted from genomic DNA during the divergence from common ancestor. The median length of these sequences is 3 nucleotides; they are enriched with palindromes and often repeat in adjacent DNA.     

Defining the location and function of domains of McrB by deletion mutagenesis

The GTP-dependent restriction endonuclease McrBC of E. coli K12, which recognizes cytosine-methylated DNA, consists of two protein subunits, McrB and McrC. We have investigated the structural assignment and interdependence of the McrB subunit functions, namely (i) specific DNA recognition and (ii) GTP binding and hydrolysis. Extending earlier work, we have produced McrB variants comprising N- and C-terminal fragments. The variants McrB1-162 and McrB1-170 are still capable of specific DNA binding. McrB169-465 shows GTP binding and hydrolysis characteristics indistinguishable from full-length McrB as well as wild-type like interaction with McrC. Thus, DNA and GTP binding are spatially separated on the McrB molecule, and the respective domains function quite independently.     

Membrane-like protein involved in phage adsorption associated with phage-sensitivity in the bloom-forming cyanobacterium Microcystis aeruginosa

The cyanobacterium, Microcystis aeruginosa, contains a large number of defense genes (Makarova et al., 2011); thus, it is a good model to study the co-evolution of phage and bacteria. Here, we isolated and characterized two phage-resistant M. aeruginosa mutants that came from a phage intermediate-sensitive culture. To determine the mutation conferring resistance, a protein expression pattern analysis was performed comparing phage-sensitive and -resistant sub-strains using SDS-PAGE. There were no apparent differences in expression patterns in the soluble fraction; however, a ∼90 kDa protein in the hydrophobic fraction from the phage-sensitive sub-strain was observed. Using a successive thermal asymmetric interlaced-PCR, the entire sequence encoding the protein, assigned ISP90, as well as its neighboring regions (ca. 7.8 kb) was determined. ISP90 contained no conserved domains and was predicted to be a membrane-associated protein. No mutations were detected in the nucleotide sequences coding ISP90 and diversification of ISP90 regions within this species were observed. Diversification of ISP90 regions within this species suggests a possible genomic island that may be subjected to selective pressures from phages. The ISP90 sequence involving phage resistance/sensitivity contributes to the understanding of co-evolution between M. aeruginosa and phages.     

Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.     

Interchangeability of the adsorption proteins of bacteriophages Ff and IKe.

The wild-type adsorption protein (g3p) of filamentous phage IKe cannot be exchanged with its analogous protein in the related Ff (M13, fd, and f1) phage particles. Deletion mutants of the protein, however, are assembled into Ff phage particles. These hybrid Ff phage particles bearing deleted IKe g3p attach to N pili, thus conserving the host attachment property of the protein but not its infection-initiating function. This means that the attachment specificity is determined by IKe g3p independently of other phage components in contact with it. Infection initiation function, the process in which phage DNA is released into the host, in contrast seems to require either more complex structural features of the protein (for example, a certain oligomeric structure) provided only in the original particle, or a concerted action of g3p with another particle component, not replaceable by its homologous counterpart in the related phage.     

H protein of bacteriophage 16-3 and RkpM protein of Sinorhizobium meliloti 41 are involved in phage adsorption

The strain-specific capsular polysaccharide KR5 antigen of Sinorhizobium meliloti 41 is required both for invasion of the symbiotic nodule and for the adsorption of bacteriophage 16-3. In order to know more about the genes involved in these events, bacterial mutants carrying an altered phage receptor were identified by using host range phage mutants. A representative mutation was localized in the rkpM gene by complementation and DNA sequence analysis. A host range phage mutant isolated on these phage-resistant bacteria was used to identify the h gene, which is likely to encode the tail fiber protein of phage 16-3. The nucleotide sequences of the h gene as well as a host range mutant allele were also established. In both the bacterial and phage mutant alleles, a missense mutation was found, indicating a direct contact between the RkpM and H proteins in the course of phage adsorption. Some mutations could not be localized in these genes, suggesting that additional components are also important for bacteriophage receptor recognition.     

Cell-free expression of visual arrestin. Truncation mutagenesis identifies multiple domains involved in rhodopsin interaction.

Visual arrestin plays an important role in regulating light responsiveness via its ability to specifically bind to the phosphorylated and light-activated form of rhodopsin. To further characterize rhodopsin/arrestin interactions we have utilized a rabbit reticulocyte lysate translation system to synthesize bovine visual arrestin. The translated arrestin (404 amino acids) was demonstrated to be fully functional in terms of its ability to specifically recognize and bind to phosphorylated light-activated rhodopsin (P-Rh*). Competitive binding studies revealed that the in vitro synthesized arrestin and purified bovine visual arrestin had comparable affinities for P-Rh*. In an effort to assess the functional role of different regions of the arrestin molecule, two truncated arrestin mutants were produced by cutting within the open reading frame of the bovine arrestin cDNA with selective restriction enzymes. In vitro translation of the transcribed truncated mRNAs resulted in the production of arrestins truncated from the carboxyl terminus. The ability of each of the mutant arrestins to bind to dark (Rh), light-activated (Rh*), dark phosphorylated (P-Rh), and light-activated phosphorylated rhodopsin were then compared. Arrestin lacking 39 carboxyl-terminal residues binds specifically not only to P-Rh* but also to Rh* and P-Rh. This suggests that the carboxyl-terminal domain of arrestin plays an important regulatory role in ensuring strict arrestin binding selectivity to P-Rh*. Arrestin that has only the first 191 amino-terminal residues predominately discriminates the phosphorylation state of the rhodopsin; however, it also retains some binding specificity for the activation state. These results suggest that the amino-terminal half of arrestin contains key rhodopsin recognition sites responsible for interaction with both the phosphorylated and light-activated forms of rhodopsin.     

Clathrin domains involved in recognition by assembly protein AP-2

The domains on clathrin responsible for interaction with the plasma membrane-associated assembly protein AP-2 have been studied using a novel cage binding assay. AP-2 bound to pure clathrin cages but not to coat structures already containing AP that had been prepared by coassembly. Binding to preassembled cages also occurred in the presence of elevated Tris-HCl concentrations (greater than or equal to 200 mM) which block AP-2 interactions with free clathrin. AP-2 interactions with assembled cages could also be distinguished from AP-2 binding to clathrin trimers by sodium tripolyphosphate (NaPPPi), which binds to the alpha subunit of AP-2 (Beck, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4442-4447). At concentrations of 1-5 mM, NaPPPi blocked clathrin-triskelion binding; in contrast, interactions with cages persisted in the presence of 25 mM NaPPPi. To begin to identify the region(s) of the clathrin molecule important in recognition by AP-2, clathrin cages were proteolyzed to remove heavy chain terminal domains and portions of the distal leg as well as all of the light chains. AP-2 bound to these "clipped cages"; however, unlike the interaction with native cages, binding of AP-2 to clipped cages was sensitive to the lower concentrations of both Tris-HCl and NaPPPi which disrupt interactions of AP-2 with clathrin trimers. Reconstitution of the clipped cages with clathrin light chains did not restore resistance of AP-2 binding to Tris-HCl. We conclude that one binding site for AP-2 resides on the hub and/or proximal part of the clathrin triskelion whereas a second site is likely to involve the terminal domain and/or distal leg; the second site is manifested only in the assembled lattice structure. We suggest that these two distinct binding interactions may be mediated by the two unique large subunits within the AP-2 complex, acting sequentially during assembly.     

Binding of IKe gene 5 protein to polynucleotides. Fluorescence binding experiments of IKe gene 5 protein and mutual cooperativity of IKe and M13 gene 5 proteins

Fluorescence studies of the binding of IKe gene 5 protein to various polynucleotides were performed to obtain insight into the question as to what extent the binding characteristics of the gene 5 proteins of the IKe and M13 phages resemble and/or differ from each other. The fluorescence of IKe gene 5 protein is quenched 60% upon binding to most polynucleotides. At moderate salt concentrations, i.e., below 1 M salt, the binding stoichiometry is 4.0 +/- 0.5 nucleotides per IKe gene 5 protein monomer. The affinity of the protein for homopolynucleotides depends strongly on sugar and base type; in order of increasing affinities we find poly(rC) less than poly(dA) less than poly(rA) less than poly(dI) less than poly(rU) less than poly(dU) less than poly(dT). For most polynucleotides studied, the affinity depends linearly on the salt concentration: [d log (Kint omega)]/(d log [M+]) = -3. The binding is highly cooperative. The cooperativity parameter omega, as deduced from protein titration curves, is 300 +/- 150 and appears independent of the type of polynucleotide studied. Estimation of this binding parameter from salt titrations of gene 5 protein-polynucleotide complexes results in systematically higher values. A comparison of the binding data of the IKe and M13 gene 5 proteins shows that the fluorescence quenching, stoichiometry, order of binding affinities, and cooperativity in the binding are similar for both proteins. From this it is concluded that at least the DNA binding grooves of both proteins must show a close resemblance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Localization of the domains of fibrin involved in binding to platelets

The molecular basis of platelet-fibrin interactions has been investigated by using synthetic peptides as potential inhibitors of fibrin protofibril and fibrinogen binding to ADP-stimulated platelets, adhesion of fibrin fibers to the platelet surface, and platelet-mediated clot retraction. Synthetic peptides of sequence RGDS and HHLGGAKQAGDV, corresponding to regions of the fibrinogen alpha- and gamma-chains previously identified as platelet recognition sites, inhibited the binding of radiolabelled soluble fibrin oligomers to ADP-stimulated platelets with IC50 values of 10 and 40 microM, respectively. Synthetic GPRP and GHRP, corresponding to the N-terminal tripeptide sequence of the fibrin alpha-chains and the tetrapeptide sequence of the beta-chains, respectively, were minimally effective in blocking soluble fibrin polymer binding to ADP-stimulated platelets. Platelet functions which are unique to the three-dimensional fibrin network were examined by measurements of the extent of adhesion of fluorophore-labelled fibrin to platelets with a microfluorimetric technique and by light scattering measurements of the time course of clot retraction. Inhibition of fibrin-platelet adhesion by RGDS, HHLGGAKQAGDV and GHRP exhibited a similar, linear dependence reaching 1/2 maximum at about 200 microM, suggesting nonspecific effects. GPRP inhibited fibrin assembly but did not appear to have specific effects on fibrin-platelet adhesion. Only RGDS effected clot retraction, causing a 4-6-fold decrease in rate at 230 microM. These results indicate that fibrinogen and fibrin protofibrils, which are obligatory intermediates on the fibrin assembly pathway, share a set of common platelet recognition sites located at specific regions of the alpha- and gamma-chains of the multinodular fibrin(ogen) molecules. The RGDS site is also involved in mediating interactions between the three-dimensional fibrin network and stimulated platelets.     

Localization of a protein-DNA interface by random mutagenesis.

The type I restriction and modification enzymes do not possess obvious DNA-binding motifs within their target recognition domains (TRDs) of 150-180 amino acids. To identify residues involved in DNA recognition, changes were made in the amino-TRD of EcoKI by random mutagenesis. Most of the 101 substitutions affecting 79 residues had no effect on the phenotype. Changes at only seven positions caused the loss of restriction and modification activities. The seven residues identified by mutation are not randomly distributed throughout the primary sequence of the TRD: five are within the interval between residues 80 and 110. Sequence analyses have led to the suggestion that the TRDs of type I restriction enzymes include a tertiary structure similar to the TRD of the HhaI methyltransferase, and to a model for a DNA-protein interface in EcoKI. In this model, the residues within the interval identified by the five mutations are close to the protein-DNA interface. Three additional residues close to the DNA in the model were changed; each substitution impaired both activities. Proteins from twelve mutants were purified: six from mutants with partial or wild-type activity and six from mutants lacking activity. There is a strong correlation between phenotype and DNA-binding affinity, as determined by fluorescence anisotropy.     

Characterization of the DNA binding protein encoded by the N-specific filamentous Escherichia coli phage IKe. Binding properties of the protein and nucleotide sequence of the gene

A DNA binding protein encoded by the filamentous single-stranded DNA phage IKe has been isolated from IKe-infected Escherichia coli cells. Fluorescence and in vitro binding studies have shown that the protein binds co-operatively and with a high specificity to single-stranded but not to double-stranded DNA. From titration of the protein to poly(dA) it has been calculated that approximately four bases of the DNA are covered by one monomer of protein. These binding characteristics closely resemble those of gene V protein encoded by the F-specific filamentous phages M13 and fd. The nucleotide sequence of the gene specifying the IKe DNA binding protein has been established. When compared to the nucleotide sequence of gene V of phage M13 it shows an homology of 58%, indicating that these two phages are evolutionarily related. The IKe DNA binding protein is 88 amino acids long which is one amino acid residue larger than the gene V protein sequence. When the IKe DNA binding protein sequence is compared with that of gene V protein it was found that 39 amino acid residues have identical positions in both proteins. The positions of all five tyrosine residues, a number of which are known to be involved in DNA binding, are conserved. Secondary structure predictions indicate that the two proteins contain similar structural domains. It is proposed that the tyrosine residues which are involved in DNA binding are the ones in or next to a beta-turn, at positions 26, 41 and 56 in gene V protein and at positions 27, 42 and 57 in the IKe DNA binding protein.     

Localization and cloning of Xp21 deletion breakpoints involved in muscular dystrophy

Summary Twenty-nine deletion breakpoints were mapped in 220 kb of the DXS164 locus relative to potential exons of the Duchenne and Becker muscular dystrophy gene. Four deletion junction fragments were isolated to acquire outlying Xp21 loci on both the terminal and centromere side of the DXS164 locus. The junction loci were used for chromosome walking, searches for DNA polymorphisms, and mapping against deletion and translocation breakpoints. Forty-four unrelated deletions were analyzed using the junction loci as hybridization probes to map the endpoints between cloned Xp21 loci. DNA polymorphisms from the DXS164 and junction loci were used to follow the segregation of a mutation in a family that represents a recombinant. Both the physical and genetic data point to a very large size for this X-linked muscular dystrophy locus.     

Dominant-negative inhibition of prion formation diminished by deletion mutagenesis of the prion protein

Polymorphic basic residues near the C terminus of the prion protein (PrP) in humans and sheep appear to protect against prion disease. In heterozygotes, inhibition of prion formation appears to be dominant negative and has been simulated in cultured cells persistently infected with scrapie prions. The results of nuclear magnetic resonance and mutagenesis studies indicate that specific substitutions at the C-terminal residues 167, 171, 214, and 218 of PrP(C) act as dominant-negative, inhibitors of PrP(Sc) formation (K. Kaneko et al., Proc. Natl. Acad. Sci. USA 94:10069-10074, 1997). Trafficking of substituted PrP(C) to caveaola-like domains or rafts by the glycolipid anchor was required for the dominant-negative phenotype; interestingly, amino acid replacements at multiple sites were less effective than single-residue substitutions. To elucidate which domains of PrP(C) are responsible for dominant-negative inhibition of PrP(Sc) formation, we analyzed whether N-terminally truncated PrP(Q218K) molecules exhibited dominant-negative effects in the conversion of full-length PrP(C) to PrP(Sc). We found that the C-terminal domain of PrP is not sufficient to impede the conversion of the full-length PrP(C) molecule and that N-terminally truncated molecules (with residues 23 to 88 and 23 to 120 deleted) have reduced dominant-negative activity. Whether the N-terminal region of PrP acts by stabilizing the C-terminal domain of the molecule or by modulating the binding of PrP(C) to an auxiliary molecule that participates in PrP(Sc) formation remains to be established.