Drastic Amino Acid Research Articles

Correction: Within-patient mutation frequencies reveal fitness costs of CpG dinucleotides and drastic amino acid changes in HIV

[This corrects the article DOI: 10.1371/journal.pgen.1007420.].

Within-patient mutation frequencies reveal fitness costs of CpG dinucleotides and drastic amino acid changes in HIV.

HIV has a high mutation rate, which contributes to its ability to evolve quickly. However, we know little about the fitness costs of individual HIV mutations in vivo, their distribution and the different factors shaping the viral fitness landscape. We calculated the mean frequency of transition mutations at 870 sites of the pol gene in 160 patients, allowing us to determine the cost of these mutations. As expected, we found high costs for non-synonymous and nonsense mutations as compared to synonymous mutations. In addition, we found that non-synonymous mutations that lead to drastic amino acid changes are twice as costly as those that do not and mutations that create new CpG dinucleotides are also twice as costly as those that do not. We also found that G→A and C→T mutations are more costly than A→G mutations. We anticipate that our new in vivo frequency-based approach will provide insights into the fitness landscape and evolvability of not only HIV, but a variety of microbes.

Mutation analysis of GLDC, AMT and GCSH in cataract captive-bred vervet monkeys (Chlorocebus aethiops).

Non-ketotic hyperglycinaemia (NKH) is an autosomal recessive inborn error of glycine metabolism characterized by accumulation of glycine in body fluids and various neurological symptoms. This study describes the first screening of NKH in cataract captive-bred vervet monkeys (Chlorocebus aethiops). Glycine dehydrogenase (GLDC), aminomethyltransferase (AMT) and glycine cleavage system H protein (GCSH) were prioritized. Mutation analysis of the complete coding sequence of GLDC and AMT revealed six novel single-base substitutions, of which three were non-synonymous missense and three were silent nucleotide changes. Although deleterious effects of the three amino acid substitutions were not evaluated, one substitution of GLDC gene (S44R) could be disease-causing because of its drastic amino acid change, affecting amino acids conserved in different primate species. This study confirms the diagnosis of NKH for the first time in vervet monkeys with cataracts.

RNA editing differently affects protein-coding genes in D. melanogaster and H. sapiens.

When an RNA editing event occurs within a coding sequence it can lead to a different encoded amino acid. The biological significance of these events remains an open question: they can modulate protein functionality, increase the complexity of transcriptomes or arise from a loose specificity of the involved enzymes. We analysed the editing events in coding regions that produce or not a change in the encoded amino acid (nonsynonymous and synonymous events, respectively) in D. melanogaster and in H. sapiens and compared them with the appropriate random models. Interestingly, our results show that the phenomenon has rather different characteristics in the two organisms. For example, we confirm the observation that editing events occur more frequently in non-coding than in coding regions, and report that this effect is much more evident in H. sapiens. Additionally, in this latter organism, editing events tend to affect less conserved residues. The less frequently occurring editing events in Drosophila tend to avoid drastic amino acid changes. Interestingly, we find that, in Drosophila, changes from less frequently used codons to more frequently used ones are favoured, while this is not the case in H. sapiens.

WS13.1 Microevolution of Pseudomonas aeruginosa in cystic fibrosis lungs

Objectives Serial P. aeruginosa isolates in half year intervals from 35 CF patients at our local CF clinic who became colonized with P. aeruginosa in the 1980s were selected. The microevolution of P. aeruginosa in CF lungs was investigated in the six patients with the mildest and the six patients with the most severe course of their chronic P. aeruginosa infection. Methods Serial isolates were genotyped with a customized microarray. Sequential isolates of the initially colonizing clone were then subjected to whole genome sequencing by SOLiD5500 technology. Nucleotide variations compared to the PA14 genome were extracted, filtered, annotated and used for the reconstruction of clades. The 250 sequenced bacterial isolates were characterized in mutation rates, morphology, motility and secretion of virulence effectors. Results Exopolysaccharide biosynthesis, antimicrobial resistance and global regulators of lifestyle and metabolism are the most common functional categories whose genes were hit by mutations in the CF lungs. Microevolution was not uniform. The P. aeruginosa clone inhabiting severely affected lungs repetitively generated descendants with stop mutations or drastic amino acid changes in key genes of lifestyle, but these loss-of-function mutants were not recovered later. In contrast, P. aeruginosa clones predominantly acquired benign amino acid substitutions in patients who maintained a normal function of their chronically colonized lungs for up to 30 years. Conclusion Modes of microevolution of P. aeruginosa in CF lungs are associated with the severity of the chronic lung infection. Supported by DFG (SFB900) and Christiane Herzog Stiftung.

Microevolution of Pseudomonas aeruginosa in cystic fibrosis lungs

The chronic airway infections with Pseudomonas aeruginosa determine disease outcome in most individuals with cystic fibrosis (CF). We have collected serial P. aeruginosa isolates in half year intervals from all 35 CF patients at our local CF clinic who became colonized with P. aeruginosa in the 1980s. The microevolution of P. aeruginosa in CF lungs was investigated in the six patients with the mildest and most severe course of their chronic P. aeruginosa infection. Serial isolates were genotyped with a customized microarray. Isolates of the initially colonizing clone were then subjected to whole genome sequencing. Nucleotide variations compared to the PA14 genome were extracted, filtered, annotated and used for the reconstruction of clades. The 250 sequenced bacterial isolates were characterized in mutation rates, morphology, motility and secretion of virulence effectors. Exopolysaccharide biosynthesis, antimicrobial resistance and global regulators of lifestyle and metabolism are the most common functional categories whose genes were hit by mutations in the CF lungs. Microevolution was not uniform. The P. aeruginosa clone inhabiting severely affected lungs repetitively generated descendants with stop mutations or drastic amino acid changes in key genes of lifestyle, but these loss-of-function mutants were not recovered at later time points. In contrast, P. aeruginosa clones predominantly acquired benign amino acid substitutions in patients who maintained a normal function of their chronically colonized lungs for up to 30 years. Modes of microevolution of P. aeruginosa in CF lungs are associated with the severity of the chronic lung infection.

Exploring the Structural Basis of Substrate Preferences in Baeyer-Villiger Monooxygenases: INSIGHT FROM STEROID MONOOXYGENASE

Steroid monooxygenase (STMO) from Rhodococcus rhodochrous catalyzes the Baeyer-Villiger conversion of progesterone into progesterone acetate using FAD as prosthetic group and NADPH as reducing cofactor. The enzyme shares high sequence similarity with well characterized Baeyer-Villiger monooxygenases, including phenylacetone monooxygenase and cyclohexanone monooxygenase. The comparative biochemical and structural analysis of STMO can be particularly insightful with regard to the understanding of the substrate-specificity properties of Baeyer-Villiger monooxygenases that are emerging as promising tools in biocatalytic applications and as targets for prodrug activation. The crystal structures of STMO in the native, NADP(+)-bound, and two mutant forms reveal structural details on this microbial steroid-degrading enzyme. The binding of the nicotinamide ring of NADP(+) is shifted with respect to the flavin compared with that observed in other monooxygenases of the same class. This finding fully supports the idea that NADP(H) adopts various positions during the catalytic cycle to perform its multiple functions in catalysis. The active site closely resembles that of phenylacetone monooxygenase. This observation led us to discover that STMO is capable of acting also on phenylacetone, which implies an impressive level of substrate promiscuity. The investigation of six mutants that target residues on the surface of the substrate-binding site reveals that enzymatic conversions of both progesterone and phenylacetone are largely insensitive to relatively drastic amino acid changes, with some mutants even displaying enhanced activity on progesterone. These features possibly reflect the fact that these enzymes are continuously evolving to acquire new activities, depending on the emerging availabilities of new compounds in the living environment.

BMP15 and premature ovarian failure: causal mutations, variants, polymorphisms?

BMP15 and premature ovarian failure: causal mutations, variants, polymorphisms?

Evolutionary patterns at the RNase based gametophytic self - incompatibility system in two divergent Rosaceae groups (Maloideae and Prunus)

BackgroundWithin Rosaceae, the RNase based gametophytic self-incompatibility (GSI) system has been studied at the molecular level in Maloideae and Prunus species that have been diverging for, at least, 32 million years. In order to understand RNase based GSI evolution within this family, comparative studies must be performed, using similar methodologies.ResultIt is here shown that many features are shared between the two species groups such as levels of recombination at the S-RNase (the S-pistil component) gene, and the rate at which new specificities arise. Nevertheless, important differences are found regarding the number of ancestral lineages and the degree of specificity sharing between closely related species. In Maloideae, about 17% of the amino acid positions at the S-RNase protein are found to be positively selected, and they occupy about 30% of the exposed protein surface. Positively selected amino acid sites are shown to be located on either side of the active site cleft, an observation that is compatible with current models of specificity determination. At positively selected amino acid sites, non-conservative changes are almost as frequent as conservative changes. There is no evidence that at these sites the most drastic amino acid changes may be more strongly selected.ConclusionsMany similarities are found between the GSI system of Prunus and Maloideae that are compatible with the single origin hypothesis for RNase based GSI. The presence of common features such as the location of positively selected amino acid sites and lysine residues that may be important for ubiquitylation, raise a number of issues that, in principle, can be experimentally addressed in Maloideae. Nevertheless, there are also many important differences between the two Rosaceae GSI systems. How such features changed during evolution remains a puzzling issue.

DNA polymorphism in the SUPERWOMAN1 (SPW1) locus of the wild rice Oryza rufipogon and its related species

The level and pattern of nucleotide variation in the SUPERWOMAN1(SPW1) locus of the wild rice Oriza rufipogon and its related species were analyzed. SPW1 is orthologous to APETELA3(AP3) in Arabidopsis thaliana. The estimated level of nucleotide variation for the entire region (pi = 0.0046) was intermediate among those for other genes of O. rufipogon, although the estimates varied considerably among the SPW1 domains. Complicated haplotype structure was detected, resulting in a high proportion of significant linkage disequilibrium. Deviation from the neutrality was not detected. However, purifying selection was suggested by lack of replacement variation in the MADS-box and I-domain regions, which function as DNA-binding domain. On the other hand, an excess of drastic amino acid changes was detected in the C-domain, as in the AP3 region of A. thaliana. Taken together, these results imply that different types of natural selection are acting among different domains of a single protein. In the phylogenetic tree, O. sativa strains were included in the same cluster of O. rufipogon, supporting the hypothesis that O. rufipogon is the wild ancestor of O. sativa.

Molecular characterization of Erwinia amylovora strains from different host plants through RFLP analysis and sequencing of hrpN and dspA/E genes

A total of 73 Erwinia amylovora strains obtained from 13 Maloideae host species and from Rubus spp., and isolated from different geographic areas, were assessed using RFLP and DNA sequencing analysis of the 3′hrpN gene and/or of a fragment of 1341 bp of the dspA/E region. An Erwinia pyrifoliae strain, used as outgroup, was checked in the same way. For the three strains isolated from Rubus spp. and for one strain from Amelanchier sp., RFLP analysis of the hrpN gene using the RsaI enzyme yielded a PCR product 60 bp smaller than that of all the other strains. Sequence analysis of the gene revealed this was due to the absence of a 60 bp fragment in the noncoding region downstream of the gene. The strain PD 2915, isolated from Amelanchier sp. grown in Canada, showed five same‐sense substitutions and one missense substitution at position 868 of the hrpN gene, converting aspartic acid into asparagine. Also, restriction analysis of a fragment of 613 bp of the dspA/E region with CfoI revealed an RFLP pattern suitable for differentiating the E. amylovora strains isolated from Rubus spp. and Amelanchier sp. from all the others. In the dspA/E coding region, the four strains showed 13–14 missense point mutations, in some cases yielding drastic amino acid substitutions. In addition, partial sequencing of the dspA/E region of PD 2915 from Amelanchier sp. indicated a higher similarity to E. amylovora strains isolated from Rubus spp. than towards strains from other Maloideae hosts. The E. pyrifoliae strain showed 23 single nucleotide substitutions along the hrpN gene and 88% of nucleotide identity with E. amylovora strains in the portion of dspA/E region. Artificial inoculations on immature pear fruits and young shoots of Maloideae and Ruboideae showed a restricted pathogenicity for the strains from Rubus and Amelanchier, with the latter inciting blight symptoms only on Amelanchier.

A Point Mutation within the Replicase Gene Differentially Affects Coronavirus Genome versus Minigenome Replication

During the construction of the transmissible gastroenteritis virus (TGEV) full-length cDNA clone, a point mutation at position 637 that was present in the defective minigenome DI-C was maintained as a genetic marker. Sequence analysis of the recovered viruses showed a reversion at this position to the original virus sequence. The effect of point mutations at nucleotide 637 was analyzed by reverse genetics using a TGEV full-length cDNA clone and cDNAs from TGEV-derived minigenomes. The replacement of nucleotide 637 of TGEV genome by a T, as in the DI-C sequence, or an A severely affected virus recovery from the cDNA, yielding mutant viruses with low titers and small plaques compared to those of the wild type. In contrast, T or A at position 637 was required for minigenome rescue in trans by the helper virus. No relationship between these observations and RNA secondary-structure predictions was found, indicating that mutations at nucleotide 637 most likely had an effect at the protein level. Nucleotide 637 occupies the second codon position at amino acid 108 of the pp1a polyprotein. This position is predicted to map in the N-terminal polyprotein papain-like proteinase (PLP-1) cleavage site at the p9/p87 junction. Replacement of G-637 by A, which causes a drastic amino acid change (Gly to Asp) at position 108, affected PLP-1-mediated cleavage in vitro. A correlation was found between predicted cleaving and noncleaving mutations and efficient virus rescue from cDNA and minigenome amplification, respectively.

Nucleotide sequence analysis of rotavirus gene 11 from two tissue culture-adapted ATCC strains, RRV and Wa.

We report here nucleotide sequence and characterization of gene 11 from two tissue culture-adapted ATCC rhesus (RRV) and human (Wa) strains of rotavirus. Gene 11 sequence encodes a nonstructural protein, NSP5 and also encodes NSP6, from an out of phase open reading frame. Sequence of RRV(ATCC) gene 11 represents the first report from a rhesus rotavirus which has more than 90% homology at the nucleotide and deduced amino acid sequence level with that of its closely related simian SA11 strain. The WaATCC gene sequence differed from that of published Wa (WaPub) at three nucleotide positions, one at 264 (G(Wa-Pub) to A(ATCC-Wa)), another a nucleotide insertion (A) at position 388 and the third, a deletion (A) at 416. The latter two changes in WaATCC NSP5 resulted in drastic amino acid changes within a 10-residue region (123-132) from VHVYQFQLTN in WaPub to DSCVSISTNH in WaATCC NSP5 protein. In this region, WaATCC NSP5 is closer to published sequences from other strains, suggesting the authenticity of the present sequence. The nucleotide difference between WaPub and WaATCC NSP5 sequences, however, did not affect the NSP6 deduced amino acid sequence, which is overall highly conserved among all the strains compared. Sequence-based phylogenetic analysis of gene 11 identified a high degree of conservation within the Group A rotaviruses. In addition, it also separated RRV(ATCC) and WaATCC, suggesting rotavirus segregation by genogroup. An anti-NSP5 monoclonal antibody of SA11 recognized RRV NSP5 protein but not WaATCC NSP5 from the infected cells, further supporting the phylogenetic segregation of RRV(ATCC) and WaATCC strains based on their NSP5 coding sequence.

Creation of a functional S-nitrosylation site in vitro by single point mutation

Here we show that in extrahepatic methionine adenosyltransferase replacement of a single amino acid (glycine 120) by cysteine is sufficient to create a functional nitric oxide binding site without affecting the kinetic properties of the enzyme. When wild-type and mutant methionine adenosyltransferase were incubated with S-nitrosoglutathione the activity of the wild-type remained unchanged whereas the activity of the mutant enzyme decreased markedly. The mutant enzyme was found to be S-nitrosylated upon incubation with the nitric oxide donor. Treatment of the S-nitrosylated mutant enzyme with glutathione removed most of the S-nitrosothiol groups and restored the activity to control values. In conclusion, our results suggest that functional S-nitrosylation sites can develop from existing structures without drastic or large-scale amino acid replacements.

Emerging foot-and-mouth disease virus variants with antigenically critical amino acid substitutions predicted by model studies using reference viruses

One of the major obstacles to the design of effective antiviral vaccines is the frequent generation of antigenic viral variants in the field. The types of variants that will become dominant during disease outbreaks is often unpredictable. However, here we report the genetic and antigenic characterization of emerging foot-and-mouth disease virus (FMDV) variants with antigenically critical amino acid substitutions predicted by model studies using reference viruses and monoclonal antibodies. The new variants belong to serotype C and have caused a number of recent disease outbreaks in Argentina. The variants harbor antigenically drastic amino acid substitutions in each of the antigenic sites identified in FMDV. In particular, a substitution found at a major antigenic site (site A, the G-H loop of VP1) had been repeatedly selected in viruses resistant to neutralization by monoclonal and polyclonal antibodies. The association of critical amino acid replacements at predicted positions with new FMD outbreaks has a number of implications for FMD epidemiology and for the design of vaccines intended to control diseases caused by highly variable RNA viruses.

Molecular Genetic Aspects of Tetraploidy in the Common Carp Cyprinus carpio

Genome duplications are believed to have occurred on multiple occasions in vertebrate evolution. Studies of duplicate gene loci in tetraploid animals may reveal important general aspects of gene duplication, an important mode of gene evolution in metazoans. The common carp Cyprinus carpio has twice as many chromosomes as most other cyprinid fishes due to tetraploidization previously estimated to have occurred 50 Myr ago. Our sequence analyses of duplicate carp loci suggest that the tetraploidization took place less than 16 Myr ago. This is further supported by sequence comparisons with the diploid grass carp, which seems to have diverged from the common carp approximately 19 Myr ago. Duplicate loci appear to remain expressed for millions of years and may accumulate mutations leading to drastic amino acid replacements as shown here for somatotropin. Therefore, both loci should always be characterized in molecular studies of tetraploid animals such as goldfish, salmonid fishes, and Xenopus laevis. The long life of duplicate genes may explain the occurrence of numerous large multigene families in higher metazoans.

JunD mutants with spontaneously acquired transforming potential have enhanced transactivating activity in combination with Fra-2.

Although a replication-competent retrovirus that carries junD has no transforming activity in chicken embryo fibroblasts, we have isolated mutant viruses that have spontaneously acquired transforming activity. The molecularly cloned junD genes of three such mutant viruses (T1, T2, and T3) were shown to be responsible for the cellular transformation. DNA sequence analysis indicated that a specific polynucleotide in the junD sequence was tandemly multiplied three times of five times in T1 and T2, respectively. The repeated polynucleotide encodes 16 amino acid residues that are located in a highly conserved region among Jun family proteins. The junD mutation in T3 involved an inversion, a translocation, and nucleotide substitutions that caused drastic amino acid exchanges in another well-conserved region among Jun family proteins. The transcriptional activity of these mutants was analyzed by means of transient expression experiments in F9 cells using a reporter gene containing a single AP-1 binding site. Compared with the wild-type JunD, none of them showed enhanced transactivating activity in the forms of homodimers or of heterodimers with c-Fos or Fra-1. However, they did exhibit much higher transactivating activity than the wild type when they formed heterodimers with Fra-2, indicating that the mutated regions function as transactivation domains in a partner-specific manner. Since we have previously reported that there is a basal level of Fra-2 expression in chicken embryo fibroblasts, the results may indicate that protein complexes between JunD mutants and Fra-2 play a crucial role in the cellular transforming activity.

The G protein of human respiratory syncytial virus: significance of carbohydrate side-chains and the C-terminal end to its antigenicity

The reactivities of eighteen monoclonal antibodies with different glycosylated forms of the human respiratory syncytial (RS) virus G protein were tested in Western blots. Only five antibodies recognized the unglycosylated precursor. The majority of antibodies, however, reacted with the O-glycosylated form of the G protein, emphasizing the importance of this type of modification for the antigenicity of the mature molecule. Human antisera, which recognized the RS virus G protein in Western blots, failed to inhibit the binding of anti-G antibodies to the virus but inhibited the binding of anti-F antibodies in the same type of assay. The human antibodies, however, did not recognize the G protein of some neutralization-resistant mutants selected with one anti-G monoclonal antibody. These mutants contain drastic amino acid sequence changes in the C-terminal end of the G molecule. The results are discussed in terms of the G protein antigenic structure.