Gly Change Research Articles

Long-Range Transmission of Structural Instability due to the FHC Mutation E180G in Coiled-Coil Tm is inhibited by an Interchain Disulfide Bond

Previously we showed that the familial hypertrophic cardiomyopathy (FHC) mutation in Tm, E180G, increased the trypsin cleavage rate at R133 and introduced another cleavage site with a similar rate at K233 (Ly & Lehrer, 2012). To understand how this mutation can produce this dynamically unstable hot spot 53 residues away, we introduced a disulfide crosslink (XL) at C190 and studied its effect on limited trypsin digestion using MALDI MS and N-terminal sequencing.Trypsin digestions at 26° of native disulfide crosslinked (XL) ααTm, FHC mutant D175N and WT, produced a XL heterodimer intermediate for all, containing one cleaved chain at R133, XL to the full length chain (134-284/1-284) (50 kDa) as well as the doubly cleaved XL homodimer (134-284/134-284) (35 kDa). For XL E180G, however, another intermediate was also present (169-284/1-284) (46 kDa). For native XL ααTm, the cleavage site at K168 was only observed at 40° where the middle of Tm is unfolded. In contrast to uncrosslinked E180G, where cleavage occurred at residue 233, no cleavage was observed at residue 233 for crosslinked E180G.These results show: 1) that in addition to the increased destabilization near R133 due to the Glu to Gly change at residue 180 in XL E180G, further destabilization occurs in the region near residue 168; 2) the long-range destabilization near residue K233 caused by the E180G FHC mutation is inhibited by the intervening XL. This suggests that interchain dynamics is involved in propagating the local perturbation at 180G due to the mutation to the K233 region.Supported by NIH HL 91162.

Phenotypic and molecular characterization of a novel β-lactamase carried by Klebsiella pneumoniae, CTX-M-72, derived from CTX-M-3

This study reports phenotypic and molecular characterization of a novel CTX-M beta-lactamase carried by two Klebsiella pneumoniae isolates collected from two hospitals in China. Conjugation experiment, Southern hybridization, susceptibility testing, isoelectric focusing, PCR, and sequencing techniques as well as clone, expression, purification and kinetics were carried out to describe the characterization of the novel CTX-M-type enzyme. The analyses of plasmid profiling and pulsed-field gel electrophoresis of the novel enzyme were performed to investigate epidemiology. The PCR products had 967 nucleotides and a novel CTX-M enzyme with a pI of 8.5 was implicated in this resistance: CTX-M-72. Two strains exhibited a clavulanic acid-inhibited substrate profile that included extended-spectrum cephalosporins. The amino acid sequence of the CTX-M-72 beta-lactamase differed from that of the CTX-M-3 beta-lactamase by the Arg-->Gly change at position 164. The novel enzyme was susceptible to ceftazidime, the same response being observed for other CTX-M enzymes. The substrates of the beta-lactamase were also characterized. Furthermore, two resistant genes of clinical strains were closely related. The emergence of a novel CTX-M-type extended-spectrum beta-lactamase was rarely described in other areas. This study illustrated the importance of molecular surveillance in tracking CTX-M-producing strains in large teaching hospitals, suggested the horizontal transfer of plasmid-borne bla(CTX-M) genes contributed to the dissemination of CTX-M enzymes in hospital environments, and emphasized the need for epidemiological monitoring.

Molecular characterization of complement Factor I deficiency in two Spanish families

Complement Factor I (CFI) is a regulator of the classical and alternative pathways. CFI has enzymatic activity and is able to cleave C3b and C4b. Homozygous Factor I deficiency is associated with infectious and/or autoimmune diseases. Here we describe the biochemical and genetic characterization in two Spanish families with complete Factor I deficiency. In Family 1, the propositus suffered from several episodes of meningitis for more than a year. Biochemical complement studies showed undetectable Factor I levels in the propositus and in her sister, while their parents and a brother had partial Factor I deficiency and were healthy. In Family 2, three out of five children were homozygous for Factor I deficiency, two of whom suffered from meningitis and the third one from several infections. The parents and the other two siblings were healthy and heterozygous for Factor I deficiency. Molecular studies showed that the two families had different mutations at exon 5 of the Factor I gene, which codifies for module LDLr1. One mutation corresponds to a 772G>A change at the donor splice site that was originally found in a family from Northern England. The second is a new missense mutation 739T>G, that generates a Cys to Gly change.

Alternative Splicing Controls Neuronal Expression of v-ATPase Subunit a1 and Sorting to Nerve Terminals

Vacuolar proton ATPase accumulates protons inside various intracellular organelles such as synaptic vesicles; its membrane domain V0 could also be involved in membrane fusion. These different functions could require vacuolar proton ATPases possessing different V0 subunit a isoforms. In vertebrates, four genes encode isoforms a1-a4, and a1 variants are also generated by alternative splicing. We identified a novel a1 splice variant a1-IV and showed that the two a1 variants containing exon C are specifically expressed in neurons. Single neurons coexpress a2, a1-I, and a1-IV, and these subunit a isoforms are targeted to different membrane compartments. Recombinant a2 was accumulated in the trans-Golgi network, and a1-I was concentrated in axonal varicosities, whereas a1-IV was sorted to both distal dendrites and axons. Our results indicate that alternative splicing of exon N controls differential sorting of a1 variants to nerve terminals or distal dendrites, whereas exon C regulates their neuronal expression.

The R71G BRCA1 is a founder Spanish mutation and leads to aberrant splicing of the transcript.

In a BRCA1 screening in familial breast cancer carried out in different centres in Spain, France, and United Kingdom, a missense mutation 330A>G which results in a Arg to Gly change at codon 71 (R71G) was independently identified in 6 families, all of them with Spanish ancestors. This residue coincides with the -2 position of the exon 5 donor splice site. We further investigated the effect of this base substitution on the splicing of BRCA1 mRNA. The sequence analysis of the cDNA indicated that 22 bp of exon 5 were deleted, creating with the first bases of exon 6 a termination codon at position 64, which results in a truncated protein. The BRCA1 haplotype of the R71G carrier patients and Spanish controls was analysed by use of six microsatellites located within or near BRCA1. Our results are consistent with the possibility that these families shared a common ancestry with BRCA1 R71G being a founder mutation of Spanish origin.

Cell recognition by foot-and-mouth disease virus that lacks the RGD integrin-binding motif: flexibility in aphthovirus receptor usage.

Cell surface molecules that can act as virus receptors may exert an important selective pressure on RNA viral quasispecies. Large population passages of foot-and-mouth disease virus (FMDV) in cell culture select for mutant viruses that render dispensable a highly conserved Arg-Gly-Asp (RGD) motif responsible for integrin receptor recognition. Here, we provide evidence that viability of recombinant FMDVs including a Asp-143-->Gly change at the RGD motif was conditioned by a number of capsid substitutions selected upon FMDV evolution in cell culture. Multiply passaged FMDVs acquired the ability to infect human K-562 cells, which do not express integrin alpha(v)beta(3). In contrast to previously described cell culture-adapted FMDVs, the RGD-independent infection did not require binding to the surface glycosaminoglycan heparan sulfate (HS). Viruses which do not bind HS and lack the RGD integrin-binding motif replicate efficiently in BHK-21 cells. Interestingly, FMDV mutants selected from the quasispecies for the inability to bind heparin regained sensitivity to inhibition by a synthetic peptide that represents the G-H loop of VP1. Thus, a single amino acid replacement leading to loss of HS recognition can shift preferential receptor usage of FMDV from HS to integrin. These results indicate at least three different mechanisms for cell recognition by FMDV and suggest a potential for this virus to use multiple, alternative receptors for entry even into the same cell type.

Detection of grlA and gyrA mutations in 344 Staphylococcus aureus strains.

Mutations in the grlA and gyrA genes of 344 clinical strains of Staphylococcus aureus isolated in 1994 in Japan were identified by combinations of single-strand conformation polymorphism analysis, restriction fragment length analysis, and direct sequencing to identify possible relationships to fluoroquinolone resistance. Five types of single-point mutations and four types of double mutations were observed in the grlA genes of 204 strains (59.3%). Four types of single-point mutations and four types of double mutations were found in the gyrA genes of 188 strains (54.7%). Among them, the grlA mutation of TCC-->TTC or TAC (Ser-80-->Phe or Tyr) and the gyrA mutation of TCA-->TTA (Ser-84-->Leu) were principal, being detected in 137 (39.8%) and 121 (35.9%) isolates, respectively. The grlA point mutations of CAT-->CAC (His-77 [silent]), TCA-->CCA (Ser-81-->Pro), and ATA-->ATT (Ile-100 [silent]) were novel, as was the GAC-->GGC (Asp-73-->Gly) change in gyrA. A total of 15 types of mutation combinations within both genes were related to ciprofloxacin resistance (MIC > or = 3.13 microg/ml) and were present in 193 mutants (56.1%). Strains containing mutations in both genes were highly resistant to ciprofloxacin (MIC at which 50% of the isolates are inhibited [MIC50] = 50 microg/ml). Those with the Ser80-->Phe or Tyr alteration in grlA but wild-type gyrA showed a lower level of ciprofloxacin resistance (MIC50 < or = 12.5 microg/ml). Levofloxacin was active against 68 of 193 isolates (35.2%) with mutations at codon 80 of grlA in the presence or absence of a concomitant mutation at codon 73, 84, or 88 in gyrA (MIC < or = 6.25 microg/ml). The new fluoroquinolone DU-6859a showed good activity with 186 of 193 isolates (96.4%) for which the MIC was < or = 6.25 microg/ml.

Region 2.5 of the Escherichia coli RNA polymerase sigma70 subunit is responsible for the recognition of the 'extended-10' motif at promoters.

At some bacterial promoters, a 5'-TG-3' sequence element, located one base upstream of the -10 hexamer element, provides an essential motif necessary for transcription initiation. We have identified a mutant of the Escherichia coli RNA polymerase sigma70 subunit that has an altered preference for base sequences in this 'extended -10' region. We show that this mutant sigma70 subunit substantially increases transcription from promoters bearing 5'-TC-3' or 5'-TT-3' instead of a 5'-TG-3' motif, located one base upstream of the -10 hexamer. The mutant results from a single base pair substitution in the rpoD gene that causes a Glu to Gly change at position 458 of sigma70. This substitution identifies a functional region in sigma70 that is immediately adjacent to the well-characterized region 2.4 (positions 434-453, previously shown to contact the -10 hexamer). From these results, we conclude that this region (which we name region 2.5) is involved in contacting the 5'-TG-3' motif found at some bacterial promoters: thus, extended -10 regions are recognized by an extended region 2 of the RNA polymerase sigma70 subunit.

Perturbations in the surface structure of A22 Iraq foot-and-mouth disease virus accompanying coupled changes in host cell specificity and antigenicity.

Foot-and-mouth disease virus (FMDV) is an extremely infectious and antigenically diverse picornavirus of cloven-hoofed animals. Strains of the A22 subtype have been reported to change antigenically when adapted to different growth conditions. To investigate the structural basis of this phenomenon we have determined the structures of two variants of an A22 virus. The structures of monolayer- and suspension-cell-adapted A22 FMDV have been determined by X-ray crystallography. Picornaviruses comprise four capsid proteins, VP1-4. The major antigenic loop of the capsid protein VP1 is flexible in both variants of the A22 subtype but its overall disposition is distinct from that observed in other FMDV serotypes (O and C). A detailed structural comparison between A22 FMDV and a type O virus suggests that different conformations in a portion of the major antigenic loop of VP1 (the GH loop, which is also central to receptor attachment) result in distinct folds of the adjacent VP3 GH loop. Also, a single mutation (Glu82-->Gly) on the surface of VP2 in the suspension-cell-adapted virus appears to perturb the structure of the VP1 GH loop. The GH loop of VP1 is flexible in three serotypes of FMDV, suggesting that flexibility is important in both antigenic variability and structural communication with other regions of the virus capsid. Our results illustrate two instances of the propagation of structural perturbations across the virion surface: the change in the VP3 GH loop caused by the VP1 GH loop and the Glu82-->Gly change in VP2 which we believe perturbs the GH loop of VP1. In the latter case, the amplification of the sequence changes leads to differences, between the monolayer- and suspension-cell-adapted viruses, in host-cell interactions and antigenicity.

Mutational analysis of the human cyclin-dependent kinase inhibitor p27kip1 in primary breast carcinomas.

The human p27kip1 gene encodes a cyclin-dependent kinase inhibitor implicated in the negative regulation of the cell cycle. In order to elucidate the possible role of p27 mutations in the development or progression of human breast cancer, we have studied the occurrence of genetic abnormalities in this gene in a series of 30 primary breast carcinomas. Direct sequence analysis of the polymerase chain reaction amplified human p27 gene revealed the occurrence of two sequence variations with respect to the reported sequence; both variants were also present in the lymphocyte DNA from the same patients. First, a silent G to A change at codon 142 (Thr) was detected in a single case. Second, a T to G transversion at codon 109, resulting in a Val to Gly change, was observed in eight tumour DNA samples (26%) and in 31 out of 80 unrelated normal individuals (39%). This latter change creates a Bg/I restriction site that might be useful for genetic analysis of human tumours. Despite the occurrence of these polymorphisms, we did not however find any evidence of somatic mutations in the coding region of the p27 gene. On the basis of these results, we suggest that alterations in the integrity of the human p27 gene are not common events in human breast carcinomas.

Mutagenic Analysis and Localization of a Highly Conserved Epitope Near the Amino Terminal End of the Citrus Tristeza Closterovirus Capsid Protein

The monoclonal antibody (MAb) 3DF1 is the first commercially available citrus tristeza closterovirus (CTV)-specific MAb. It detects a broad spectrum of CTV isolates from various parts of the world. To precisely map the antigenic determinant recognized by 3DF1, the capsid protein (CP) genes of four 3DF1-nonreactive isolates were cloned as complementary DNA and their nucleotide sequences determined. Comparison of the deduced CP sequences of the four nonreactive isolates with those of previously sequenced 3DF1-reactive isolates revealed differences at three positions near their amino terminal ends. The amino acids Asp-2, Lys-13, and Phe-28 were conserved in all the 3DF1-reactive isolates, but they were replaced by Gly, Thr/Asp, and Tyr, respectively, in the CPs of the nonreactive isolates. Site-specific mutations were introduced into the cloned CP genes of the 3DF1-nonreactive isolate B215 and the 3DF1-reactive isolate T36. The serological reactivities of the wild-type and mutant CPs of B215 and T36 expressed as recombinant fusion proteins in Escherichia coli were evaluated by Western blot analysis. A point mutation (A→G) resulting in an Asp→Gly change at amino acid position 2 of the CP of isolate T36 abolished the reactivity with the MAb, whereas a reverse mutation resulting in a Gly→Asp change at the same position conferred reactivity on the CP of the nonreactive B215 isolate. The implications of the observed antigenic diversity on virus detection are discussed.

Ala--&gt;Gly mutation in the putative catalytic loop confers temperature sensitivity on Ros, insulin receptor, and insulin-like growth factor I receptor protein-tyrosine kinases.

Temperature-sensitive mutations in the avian sarcoma virus UR2 oncogene ros, encoding a receptor protein-tyrosine kinase (PTK), were identified. The Ala385-->Gly change mapping within the highly conserved RDLAARN motif in the Ros kinase domain was responsible for the temperature-sensitive phenotype. Based on the sequence homology of all known protein kinases and the crystalline structure of the cAMP-dependent protein kinase, this conserved region probably represents the PTK catalytic loop. The same mutation when introduced into the human insulin and insulin-like growth factor I receptors made these PTKs temperature sensitive in both biological function and kinase activity. Our results support the presumed catalytic role of this highly conserved sequence in PTKs. Due to its highly conserved nature, we predict that the same mutation would probably confer temperature sensitivity on other PTKs.

Characterization of HIV-1 neutralization escape mutants

Infection by molecularly cloned HIV-1, in the presence of a high-titre neutralizing monoclonal antibody (MAb), resulted in the selection of plaques in MT4 cells releasing HIV resistant to neutralization by the same MAb. The epitope recognized by the MAb was mapped to the V3 neutralization epitope at amino acids 305-321. The HIV-1 variants showed a reduced binding capacity for the selecting MAb as determined by immunofluorescence. Polymerase chain reaction (PCR) amplification of complementary DNA derived from viral RNA, cloning and sequencing identified a base pair (bp) change C----G at position 6663 in variant 110.5/1, predicting a change at amino acid 308 Arg----Gly. No other changes in the epitope were observed by sequencing three other variants. Differential hybridization of PCR amplified viral RNA and DNA, with oligonucleotides specific for the observed bp change or the 'wild type' sequence, indicated that the variants 110.5/1 and 110.5/7 were genotypically mixed for 308Gly/Arg. Subsequent screening of biologically 'recloned' variants 110.5/1 and 110.5/7 identified two subclones homozygous for the 308Gly change. The Arg----Gly change appears to affect the binding of the antibody to the epitope, since the linear peptide substituting 308Gly for 'wild type' 308Arg was 100 times less potent in blocking the neutralization of parental HIV. Amino-acid residue 308 thus appears to be crucial for antibody binding to the epitope. In addition, mutations distant from the monoclonal antibody binding site may also affect neutralization by antibodies recognizing the V3 loop.

Glu-111 is required for activation of the DNA cleavage center of EcoRI endonuclease

Gap repair in the presence of 2'-deoxycytosine 5'-O-(1-thiotriphosphate) has been utilized to mutagenize the amino-terminal one-half of the structural gene for EcoRI endonuclease. This approach has led to identification of over 200 mutants defective in endonuclease function. One mutant protein, which binds to the EcoRI sequence but displays greatly reduced cleavage activity, is the consequence of a Glu to Gly change at position 111. This protein has been purified to homogeneity and characterized in detail. Subunit interactions governing the tetramer to dimer transition of the mutant endonuclease are near normal as are parameters governing its interaction with specific and nonspecific DNA sequences. However, the rate constants for first and second strand cleavage steps are reduced by 60,000- and 30,000-fold, respectively, as a consequence of the Glu----Gly change. The defect in chemical cleavage steps can be partially overcome by elevating the pH of the reaction buffer from 7.6 to 8.5, conditions which enhance the rate of EcoRI* strand cleavage by wild type enzyme to a similar degree. We suggest that the Glu-111 mutation affects an interface between recognition and cleavage functions of the enzyme, an idea consistent with the suggestion that the cleavage center of the endonuclease is subject to activation upon specific recognition of the EcoRI sequence.

Chloroplast-coded atrazine resistance in Solanum nigrum: psbA loci from susceptible and resistant biotypes are isogenic except for a single codon change.

The 32-kDa photosystem II protein of the chloroplast is thought to be a target molecule for the herbicide atrazine. The psbA gene coding for this protein was cloned from Solanum nigrum atrazine-susceptible ('S') and atrazine-resistant ('R') biotypes. The 'S' and 'R' genes are identical in nucleotide sequence except for an A to G transition, predicting a Ser to Gly change at codon 264. The same predicted amino acid change in psbA was previously shown for an Amaranthus hybridus 'S' and 'R' biotypes which had, in addition, two silent nucleotide changes between the genes (Hirschberg, J. and McIntosh, L., Science 222, 1346-1349, 1983). Occurrence of the identical, non-silent change in psbA in different 'S' and 'R' weed biotype pairs suggests a functional, herbicide-related role for this codon position.

Isolation and characterization of φX174 mutants carrying lethal missense mutations in gene G

A previously constructed Escherichia coli transformant carrying a functional copy of bacteriophage phi X174 gene G on a plasmid, p phi XG, was used to isolate gene G mutants carrying temperature sensitive and lethal missense mutations. Two of the mutations have been characterized by sequencing: one carries a G --> A transition at residue 2821 producing a Gly --> Ser change in codon 143 of the G spike protein; the other carries an A --> G transition at residue 2678 producing Glu --> Gly change in codon 95. Sequencing DNA from 2 other mutants carrying lethal mutations that are rescued with p phi XG did not reveal any changes in the coding sequence. The lesion is believed to be in the intercistronic region between genes F and G. The adsorption kinetics for these mutants appear to be normal. Their burst size is about 25% that of wild type phi X174 on the host carrying p phi XG. These results along with previous results from the senior author's laboratory demonstrate that p phi XG can be used to rescue any gene G mutant of phi X174 regardless of the nature of the mutation involved.