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Allelic diversification at the C (OsC1) locus of wild and cultivated rice: nucleotide changes associated with phenotypes.

Divergent phenotypes are often detected in domesticated plants despite the existence of invariant phenotypes in their wild forms. One such example in rice is the occurrence of varying degrees of apiculus coloration due to anthocyanin pigmentation, which was previously reported to be caused by a series of alleles at the C locus. The present study reveals, on the basis of comparison of its maps, that the C gene appears to be the rice homolog (OsC1) of maize C1, which belongs to the group of R2R3-Myb factors. Two different types of deletions causing a frameshift were detected in the third exon, and both of the deleted nucleotides corresponded to the positions of putative base-contacting residues, suggesting that the Indica and Japonica types carry loss-of-function mutations with independent origins. In addition, replacement substitutions were frequently detected in OsC1 of strains carrying the previously defined C alleles. Molecular population analysis revealed that 17 haplotypes were found in 39 wild and cultivated rices, and the haplotypes of most cultivated forms could be classified into one of three distinct groups, with few shared haplotypes among taxa, including Indica and Japonica types. The genealogy of the OsC1 gene suggests that allelic diversification causing phenotypic change might have resulted from mutations in the coding region rather than from recombination between preexisting alleles. The McDonald and Kreitman test revealed that the changes in amino acids might be associated with selective forces acting on the lineage of group A whose haplotypes were carried by most Asian cultivated forms. The results regarding a significant implication for genetic diversity in landraces of rice are also discussed.

Alleles↗

The evolutionary ecology of dominance-recessivity.

An "adaptive dynamics" modelling approach to the evolution of dominance-recessivity is presented. In this approach, fitness derives from an explicit ecological scenario, and both evolutionary attractivity and invasibility of resident populations are examined. The ecology consists of a within-individual part representing a locus with regulated activity and a between-individual part that is a two-patch soft selection model. Evolutionary freedom is allowed at a single locus. The evolutionary analysis considers directed random walks on trait space, generated by repeated invasions of mutants. The phenotype of an individual is determined by allelic parameters. Mutations can have two effects: they either affect the affinity of the promoter sequence for transcription factors, or they affect the gene product. The dominance interaction between alleles derives from their promoter affinities. Additive genetics is evolutionarily unstable when selection and evolution maintain two alleles in the population. In such a situation, dominance interactions can become stationary and close to additive genetics or they continue to evolve at a very slow pace towards dominance-recessivity. The probability that a specific dominance interaction will evolve depends on the relative mutation rate of promoter compared to gene product and the distribution of mutational effect sizes. Either allele in the dimorphism can become dominant, and dominance-recessivity is always most likely to evolve. Evolution then approaches a population state where every phenotype has maximum viability in one of the two patches. When the within-individual part is replaced by a housekeeping locus that codes for a metabolic enzyme, evolution favours a population of two alleles under the same conditions as for a regulated locus. In the case of a housekeeping gene, however, the evolutionary dynamical system approaches a population state where the heterozygote and only one homozygote phenotype are equivalent to the optimum phenotypes in the two patches.

Alleles↗

A common thrombomodulin amino acid dimorphism is associated with myocardial infarction.

Endothelial dysfunction and haemostatic imbalance are believed to be important aetiological factors in the development of acute coronary syndromes. Thrombomodulin (TM) is an integral membrane protein crucial for normal endothelial function and activation of the protein C anticoagulant pathway. We have investigated the importance of a common C/T dimorphism in the TM gene (nucleotide 1418) for development of premature myocardial infarction (MI). The C/T dimorphism predicts an Ala455 to Val replacement in the sixth EGF-like domain of TM. The dimorphism was investigated in 97 MI survivors and 159 healthy controls. The C allele was significantly more frequent among patients than controls (p = 0.035). The allele frequency for the C allele was 0.82 in the patients and 0.72 in the control group. The plasma concentration of TM was investigated among healthy controls but was not related to the C/T dimorphism. In conclusion, the association of the C allele with premature MI, suggests that the TM gene and the C/T dimorphism may be aetiological factors involved in the pathogenesis of MI. Possibly, the Ala455 to Val replacement may affect the function of the TM molecule and the activation of the protein C anticoagulant pathway.

Adult↗

Allelic expression analysis suggests that cis-acting polymorphism of FRZB expression does not contribute to osteoarthritis susceptibility.

OBJECTIVE: The FRZB gene codes for secreted frizzled-related protein 3 (sFRP3), a soluble antagonist of wnt signalling that is required for maintaining cartilage integrity. Two common single nucleotide polymorphisms (SNPs) that code for the substitution of conserved arginine residues have previously been identified in FRZB and found to be associated with osteoarthritis (OA). Functional studies revealed that the arginine substitutions lead to a loss-of-function of sFRP3 activity. We set out to assess whether cis-acting polymorphism in the regulation of FRZB expression exists, as this may be an additional mechanism through which sFRP3 activity could be modulated. METHODS: RNA was extracted from the articular cartilage chondrocytes of 25 individuals who had undergone joint replacement for OA and who were heterozygous for one of the two FRZB SNPs. Allelic output was measured by single base extension in to the SNPs and deviations from the expected 1:1 pattern were assessed using the Mann-Whitney U test. RESULTS: Differential allelic expression was observed in six of the 25 individuals. However, the average fold difference in allelic expression in the six was only 1.19. CONCLUSIONS: The presence of a small degree of differential allelic expression in a low proportion (24%) of the individuals studied suggests that polymorphism in FRZB cis-acting regulatory elements can be discounted as a major factor that could influence the development of OA.

Aged↗

Genetic instability in Drosophila melanogaster: P-element mutagenesis by gene conversion.

We report the molecular characterization of several P element-induced mutations and their revertants at the yellow (y) locus of Drosophila melanogaster. One of the mutants analyzed, y76d28, results from the insertion of a P element into the 5'-transcribed, untranslated portion of the y gene. Sequence analysis of several revertants of y76d28 shows that P excision occurs imprecisely. These events result in insertion of additional ATG codons in the y locus mRNA but are without phenotypic effect. In addition, we describe the molecular structure of P-associated mutations induced in a near wild-type revertant of y76d28 that carries an internally deleted 0.4-kilobase P element in the 5' noncoding region. Sequence analysis of two of these mutants demonstrates that they arose as a result of the integration of a larger P element at the exact location as in the parental stock without the 8-base-pair additional duplication associated with P insertions. The phenotype of these y alleles is dependent on the size and orientation of the integrated P element. We infer that P-element replacement in these mutants has occurred by a recombination/gene conversion mechanism.

Alleles↗

Molecular and mutational analysis of a gelsolin-family member encoded by the flightless I gene of Drosophila melanogaster.

The flightless locus of Drosophila melanogaster has been analyzed at the genetic, molecular, ultrastructural and comparative crystallographic levels. The gene encodes a single transcript encoding a protein consisting of a leucine-rich amino terminal half and a carboxyterminal half with high sequence similarity to gelsolin. We determined the genomic sequence of the flightless landscape, the breakpoints of four chromosomal rearrangements, and the molecular lesions in two lethal and two viable alleles of the gene. The two alleles that lead to flight muscle abnormalities encode mutant proteins exhibiting amino acid replacements within the S1-like domain of their gelsolin-like region. Furthermore, the deduced intron-exon structure of the D. melanogaster gene has been compared with that of the Caenorhabditis elegans homologue. Furthermore, the sequence similarities of the flightless protein with gelsolin allow it to be evaluated in the context of the published crystallographic structure of the S1 domain of gelsolin. Amino acids considered essential for the structural integrity of the core are found to be highly conserved in the predicted flightless protein. Some of the residues considered essential for actin and calcium binding in gelsolin S1 and villin V1 are also well conserved. These data are discussed in light of the phenotypic characteristics of the mutants and the putative functions of the protein.

Actins↗

An improved system for gene replacement and xylE fusion analysis in Pseudomonas aeruginosa.

A novel pUC19-based gene replacement vector has been developed. This vector incorporates (i) the counterselectable sacB marker, (ii) a lacZ alpha allele for blue-white screening, (iii) an oriT for conjugation-mediated plasmid transfer and (iv) unique cloning sites for SmaI and the rare-cutting meganuclease I-SceI. These rare restriction sites are also present on the helper plasmid pUC19Sce. The replacement vector is engineered to contain few restriction sites to gain greater access to restriction sites within cloned DNA fragments, thus facilitating their genetic manipulation. The usefulness of the system was demonstrated by chromosomal integration of a newly constructed xylE::GmR fusion cassette into the glpD gene of Pseudomonas aeruginosa.

Amino Acid Sequence↗

Serine-proline replacement at residue 127 of NADH-cytochrome b5 reductase causes hereditary methemoglobinemia, generalized type.

Hereditary methemoglobinemia is an autosomal recessive disorder characterized by NADH-cytochrome b5 reductase (b5R) deficiency. In an attempt to clarify the molecular mechanisms involved in the enzyme deficiency, we isolated the b5R gene from a patient homozygous for hereditary methemoglobinemia, generalized type, and compared its nucleotide sequence with that of the normal NADH-cytochrome b5R gene. Only one difference was observed; a thymidine at the first position of codon 127 (TCT) was altered to a cytidine in the b5R gene of the patient, resulting in replacement of serine with proline. Dot blot hybridization of the amplified DNA samples with allele-specific oligonucleotide probes showed that the proband and her brothers were homozygous for this mutation and that their father was heterozygous. Although the activity of b5R in lymphoblastoid cells from homozygotes was reduced to 10% of the normal level, RNA blot and protein blot analyses of the lymphoblastoid cells showed that synthesis of b5R messenger RNA and the b5R polypeptide were normal. Serine at residue 127 is presumed to be in an alpha-helix structure that is part of a nucleotide-binding domain. These observations suggest that replacement of Pro-127 causes a significant conformation change in the nucleotide-binding domain that affects electron transport from NADH to cytochrome b5. Functional enzyme deficiency results in a generalized type of hereditary methemoglobinemia.

Adult↗

Mutational analysis of bli-4/kpc-4 reveals critical residues required for proprotein convertase function in C. elegans.

Kex2/subtilisin-like proteinase activity is required for the production of the adult cuticle in the nematode Caenorhabditis elegans. Deletion of the carboxy termini of four of the bli-4/kpc-4 convertase isoforms results in blistering of the adult cuticle. The blisters vary in severity (expressivity) and are not evident in all individuals (reduced penetrance). We have isolated 13 bli-4/kpc-4 mutants that arrest development in late embryogenesis. Using a PCR-based heteroduplex technique, we have identified nucleotide changes responsible for eight of these lethal mutations. The lesions reside within the first 12 exons that are shared by all of the bli-4/kpc-4 gene products, with the majority of mutations clustered within the protease domain. This finding suggests that the protease domain represents a large mutable target. Among these mutations, allele h384 represents a molecular null mutant in which the catalytically essential serine residue (Ser415) is replaced by phenylalanine. Novel missense mutations that change the identity of amino acids evolutionary conserved in all kex2/subtilisin-convertases highlight critical residues essential for activity. We examined the functional activity of BLI-4/KPC-4 products expressed from several lethal mutants by testing their effect on the variable penetrance of blistering exhibited by the e937 allele. We found that the combination of a bli-4/kpc-4 lethal mutation in trans to the bli-4(e937) mutation was sufficient to cause severe blistering in heteroallelic progeny, even in the presence of a known dominant suppressor.

Alleles↗

Molecular basis of HLA polymorphism: implications in clinical transplantation.

Polymorphism of the human leukocyte antigens (HLA) represents a major barrier to organ and hematopoietic stem cell (HSC) transplantation. The cloning and sequencing of HLA class I and II genes has not only provided a clear picture of the molecular basis of allelic polymorphism, but also allowed the development of a variety of PCR-based DNA typing techniques. Such methods are now progressively replacing serological typing for assessing donor/recipient HLA compatibility in clinical transplantation. The 100 serological HLA-A,B,Cw,DR,DQ,DP specificities now comprise more than 1300 alleles defined at the DNA sequence level. Most of the serotypes are subdivided into numerous allelic subtypes in worldwide populations (up to 50 alleles in some cases), although a limited number of alleles are detected in a given population group. In organ transplantation application of HLA molecular typing allowed to improve typing quality, leading to a more precise matching assessment with better clinical results. Knowledge of the molecular basis of class I gene polymorphisms also led to the development of new matching algorithms such as HLA-Matchmaker, based on immunogenic amino acid triplets localized on antibody-accessible external domains of class I antigens. The most impressive impact of novel DNA typing methods concerns matching for allogeneic HSC transplantation because subtle serologically silent sequence differences between allelic subtypes are efficiently recognized by alloreactive T-cells with potentially serious consequences for graft outcome. High resolution HLA class I and II matching has contributed to improve patients survival after unrelated HSC transplantation, although the relative importance of individual loci remains to be elucidated. Donor matching criteria should take into account parameters such as the time frame allowed by the patient's disease and the probability to identify a well matched donor based on the patient's HLA phenotype.

Algorithms↗

An improved method for rapid generation of unmarked Pseudomonas aeruginosa deletion mutants.

BACKGROUND: Traditional gene replacement procedures are still time-consuming. They usually necessitate cloning of the gene to be mutated, insertional inactivation of the gene with an antibiotic resistance cassette and exchange of the plasmid-borne mutant allele with the bacterial chromosome. PCR and recombinational technologies can be exploited to substantially accelerate virtually all steps involved in the gene replacement process. RESULTS: We describe a method for rapid generation of unmarked P. aeruginosa deletion mutants. Three partially overlapping DNA fragments are amplified and then spliced together in vitro by overlap extension PCR. The resulting DNA fragment is cloned in vitro into the Gateway vector pDONR221 and then recombined into the Gateway-compatible gene replacement vector pEX18ApGW. The plasmid-borne deletions are next transferred to the P. aeruginosa chromosome by homologous recombination. Unmarked deletion mutants are finally obtained by Flp-mediated excision of the antibiotic resistance marker. The method was applied to deletion of 25 P. aeruginosa genes encoding transcriptional regulators of the GntR family. CONCLUSION: While maintaining the key features of traditional gene replacement procedures, for example, suicide delivery vectors, antibiotic resistance selection and sucrose counterselection, the method described here is considerably faster due to streamlining of some of the key steps involved in the process, especially plasmid-borne mutant allele construction and its transfer into the target host. With appropriate modifications, the method should be applicable to other bacteria.

Bacterial Proteins↗

Identification of a missense mutation in a Friedreich's ataxia patient: implications for diagnosis and carrier studies.

Approximately 95% of all Friedreich's ataxia (FA) patients are homozygous for a large GAA triplet-repeat expansion in the first intron of the Friedreich's ataxia gene (FRDA). The remaining cases are expected to be compound heterozygous with a GAA expansion on one allele and a point mutation on the other. Generally, the clinical diagnostic profile in this group of patients is indistinguishable from that in classic FA patients with homozygous expansions. This study describes a mildly affected patient who presents with only one expanded allele by Southern blot analysis. Point mutation screening shows a single base change in FRDA exon 3 resulting in a nonconservative amino acid replacement in the N-terminal portion of the frataxin protein. Extended family studies show that two of the patient's sibs are carriers of the expanded allele and one is a carrier of the missense mutation. This case study demonstrates the benefits of implementing a combined Southern blot and point mutation diagnostic protocol for compound heterozygous patients. By identifying both mutations, this procedure confirms the diagnosis of FA in patients with an atypical disease course and allows for more complete family studies.

Adult↗

Influence of HLA-DR genes on the production of rheumatoid arthritis-specific autoantibodies to citrullinated fibrinogen.

OBJECTIVE: Antibodies directed against citrullinated fibrinogen are highly specific for rheumatoid arthritis (RA). This study was undertaken to test whether RA-associated HLA-DR alleles are associated with anti-citrullinated fibrinogen in RA patient sera and whether replacement of arginyl by citrullyl residues on fibrinogen peptides modifies their binding to HLA-DR molecules and their recognition by T cells. METHODS: Antikeratin, antifilaggrin, and anti-citrullinated fibrinogen antibodies were assayed in RA patients who had undergone HLA-DR typing. Direct assays were performed to investigate binding of citrullinated or native fibrinogen peptides (encompassing the entire alpha- and beta-chains of fibrinogen) to purified HLA-DR molecules. T cell proliferative responses to citrullinated or native fibrinogen peptides were measured in RA patients and controls. RESULTS: HLA-DRB1*0404 was associated with anti-citrullinated fibrinogen in RA sera (P = 0.002). For the RA-associated alleles HLA-DRB1*0401 and HLA-DR1, there was a nonsignificant trend toward association (P = 0.07). Multiple peptides from the alpha- and beta-chains of fibrinogen bound many HLA-DR alleles; DRB1*0404 was the best fibrinogen peptide binder. Citrullination did not influence fibrinogen peptide binding to HLA-DR or fibrinogen peptide recognition by T cells. Peripheral blood T cells that recognized native or citrullinated fibrinogen peptides were common in RA patients but not in healthy controls. CONCLUSION: The RA-associated HLA-DRB1*0404 allele is also associated with production of antibodies to citrullinated fibrinogen. DRB1*0401 and DRB1*01 tend to be associated with anti-citrullinated fibrinogen, but this is not statistically significant. Citrullination of fibrinogen peptide does not influence peptide-DR-T cell interaction. Finally, T cell proliferation in response to citrullinated or uncitrullinated fibrinogen peptides is frequent in RA patients and very infrequent in controls.

Alleles↗

Extensive allelic variation in Cryptococcus neoformans.

The orotidine monophosphate pyrophosphorylase (OMPPase) gene locus of the DNA of 13 Cryptococcus neoformans var. neoformans strains, including 10 recent clinical isolates, was studied by using restriction fragment length polymorphisms and nucleotide sequence analysis. The OMPPase locus (URA5) is highly polymorphic, and at least six alleles were identified. The nucleotide sequences of some alleles differed by up to 5%. The majority of the nucleotide polymorphisms in the protein-coding region occurred at the third codon position and were silent. The low frequency of replacement nucleotide substitutions relative to silent nucleotide substitutions implied that there is strong selection against amino acid changes in OMPPase. The allelic variation suggested that there is extensive genomic diversity among C. neoformans clinical isolates from one geographic area. The various alleles are potentially useful markers in the study of the population structure, epidemiology, and pathogenesis of C. neoformans strains.

Alleles↗

Incremental increase in fitness cost with increased beta -lactam resistance in pneumococci evaluated by competition in an infant rat nasal colonization model.

BACKGROUND: We evaluated the impact of resistant penicillin-binding protein (PBP) allele acquisition on the ability of penicillin-resistant (PEN-R) pneumococcal strains to compete with penicillin-susceptible (PEN-S) ancestors for upper-respiratory-tract (URT) colonization. METHODS: PEN-S serotype 2, 6B, and 9V strains were transformed into derivatives expressing an increasing number of PEN-R PBP forms (2X, 2X-1A, and 2X-1A-2B for serotype 2 and 2X, 2X-2B, and 2X-2B-1A for 6B and 9V). Infant rats were inoculated intranasally with a mix of a PEN-R and PEN-S strains. For consecutive days, samples were collected for assessment of the ratio of PEN-S to PEN-R cells colonizing the URT. The selective index (SI), defined as the change in the natural logarithm of the ratio of PEN-S to PEN-R strains from the inoculum to the nasal-wash samples, quantified differences in fitness. RESULTS: SIs significantly > 0 (indicating a cost of resistant allele acquisition) were observed 4-5 days after colonization in all but serotype 6B pbp2x transfomants. Additional replacements with low-affinity forms of pbp2b and pbp1a genes reduced further ability to compete in all strains. CONCLUSIONS: The cost of penicillin-resistance acquisition for the Streptococcus pneumoniae strain competing with its susceptible ancestor to colonize the URT increases with the number of resistant pbp alleles acquired.

Amino Acid Sequence↗

[Genetic nature of atrophic rhinitis in swine. I. The results of a test cross and bacteriological study in atrophic rhinitis in swine].

It is well established that the swine atrophic rhinitis (AR) is controlled by the only gene with two alleles, R and r, the former being a dominant. Our study was designed to determine the role of these factors in appearance of AR of swine among the offspring. Normal and diseased pairs of animals were mated. The heterozygotic (Rr) phenotypically normal but potentially ill hogs and sows were revealed at the farm where AR was spread. The phenotypically normal hogs and sows (RR, Rr) were mated with diseased (rr) or potentially diseased ones, and phenotypically normal heterozygotic (Rr) hogs and sows (control). The diseased sucking-pigs revealed in the litter were removed. It has been established that the sucking-pigs were normal when mating normal homozygotic animals. These sucking-pigs were used to replace the live-stock of sows. When mating normal homozygotic animals with recessive ones, the sucking-pigs were also normal, the normal alleles being dominant. In matings of heterozygotic animals 27.3% of the litter were wry-snouted. However, even healthy heterozygotic animals should not be used for replacing the live-stock, because they have both the normal and recessive alleles. When recessive animals were mated, all sucking-pigs proved to have the symptoms of AR. Such sucking-pigs, together with their parents were necessarily removed.

Animals↗

Allelic variants of human TCR BV17S1 defined by restriction fragment length polymorphism, single strand conformation polymorphism, and amplification refractory mutation system analyses.

Several human TCR BV gene subfamilies, including BV3, BV14, and BV17S1, are single member genes but are overutilized among activated CD4+ synovial T cells in the rheumatoid arthritis (RA). To define the role of these TCR BV genes in the pathogenesis of disease, it is critical to characterize the genomic organization and the allelic variations of these genes. In this study we describe allelic variations of BV17S1 defined by restriction fragment length polymorphism (RFLP), single strand conformation polymorphism (SSCP), and amplification refractory mutation system (ARMS) analyses. A single nucleotide replacement (C/T) results in an amino acid substitution (F/L) in the leader and distinguishes BV17S1*1 from BV17S1*2. This nucleotide substitution was found to create a BsmAI restriction enzyme recognition site in BV17S1*2. Therefore genotypic analyses can be performed either by the SSCP or RFLP method. The analyses of 75 unrelated individuals show that the frequency for allele BV17S1*1 is 52.7% and for allele BV17S1*2 is 47.3%. Both alleles are functionally expressed and are distributed within CD4+/CD8+ T cell subsets. Another point mutation in the CDR2 region of BV17S1, which results in the amino acid replacement of Gln by His, originally identified form a cDNA clone, has now been confirmed as an allele by ARMS analysis using genomic DNA preparations and designated to as BV17S1*3. Screening of this CDR2 related variant among normal populations indicates that this is a rare allele (1 of 75). Although this variant may be of functional significance, the genotypic analysis and functional studies are difficult due to the low frequency of BV17S1*3. In an attempt to define a correlation between BV17S1 allelic usage and susceptibility to RA, the germline distribution of BV17S1 alleles *1 and *2 has been examined in a small number of RA patients and no skewed usage has been identified.

Alleles↗

Thermoconditional modulation of the pleiotropic sensitivity phenotype by the Saccharomyces cerevisiae PRP19 mutant allele pso4-1.

The conditionally-lethal pso4-1 mutant allele of the spliceosomal-associated PRP19 gene allowed us to study this gene's influence on pre-mRNA processing, DNA repair and sporulation. Phenotypes related to intron-containing genes were correlated to temperature. Splicing reporter systems and RT-PCR showed splicing efficiency in pso4-1 to be inversely correlated to growth temperature. A single amino acid substitution, replacing leucine with serine, was identified within the N-terminal region of the pso4-1 allele and was shown to affect the interacting properties of Pso4-1p. Amongst 24 interacting clones isolated in a two-hybrid screening, seven could be identified as parts of the RAD2, RLF2 and DBR1 genes. RAD2 encodes an endonuclease indispensable for nucleotide excision repair (NER), RLF2 encodes the major subunit of the chromatin assembly factor I, whose deletion results in sensitivity to UVC radiation, while DBR1 encodes the lariat RNA splicing debranching enzyme, which degrades intron lariat structures during splicing. Characterization of mutagen-sensitive phenotypes of rad2Delta, rlf2Delta and pso4-1 single and double mutant strains showed enhanced sensitivity for the rad2Delta pso4-1 and rlf2Delta pso4-1 double mutants, suggesting a functional interference of these proteins in DNA repair processes in Saccharomyces cerevisiae.

Alleles↗