A single amino acid converts a repressor to an activator of flowering

Homologous proteins occurring through gene duplication may give rise to novel functions through mutations affecting protein sequence or expression. Comparison of such homologues allows insight into how morphological traits evolve. However, it is often unclear which changes are key to determining new functions. To address these ideas, we have studied a system where two homologues have evolved clear and opposite functions in controlling a major developmental switch. In plants, flowering is a major developmental transition that is critical to reproductive success. Arabidopsis phosphatidylethanolamine-binding protein homologues TERMINAL FLOWER 1 (TFL1) and FLOWERING LOCUS T (FT) are key controllers of flowering, determining when and where flowers are made, but as opposing functions: TFL1 is a repressor, FT is an activator. We have uncovered a striking molecular basis for how these homologous proteins have diverged. Although <60% identical, we have shown that swapping a single amino acid is sufficient to convert TFL1 to FT function and vice versa. Therefore, these key residues may have strongly contributed to the selection of these important functions over plant evolution. Further, our results suggest that TFL1 and FT are highly conserved in biochemical function and that they act as repressors or activators of flowering through discrimination of structurally related interactors by a single residue.     

Genomic analysis of para-Bombay individuals in south-eastern China: the possibility of linkage and disequilibrium between FUT1 and FUT2

BackgroundThe para-Bombay phenotype results from a variety of mutations in the α-(1,2)-fucosyltransferase gene (FUT1). We investigated samples from seven Chinese probands serologically typed as having the para-Bombay phenotype.ResultsThree FUT1 genotypes, h1/h1 (5 individuals), h1/h6 (1 individual) and h3/h2 (1 individual), and three FUT2 genotypes, Se³⁵⁷/Se³⁵⁷ (5 individuals), Se³⁵⁷/Se^{357, 385} (1 individual) and Se³⁵⁷/Se^{357, 716} (1 individual) were observed in seven para-Bombay probands. Among 331 donors, only one individual carried the G716A and 880delTT mutations in heterozygosity; this subjects FUT1 and FUT2 genotypes were H/h2 and Se³⁵⁷/Se^{357, 716}, respectively.ConclusionThe review of all para-Bombay probands identified in the Fujian Blood Centre showed that h1 and h2 are the predominant non-functional FUT1 alleles in Fujian para-Bombay individuals. Our data confirm the hypothesis that the h2 allele is linked to Se^{357, 716}, and the concurrence of unique FUT1 and FUT2 mutations is geographically specific.     

Genetic suppression of the circadian Clock mutation by the melatonin biosynthesis pathway

Most laboratory mouse strains including C57BL/6J do not produce detectable levels of pineal melatonin owing to deficits in enzymatic activity of arylalkylamine N-acetyltransferase (AANAT) and N-acetylserotonin O-methyl transferase (ASMT), two enzymes necessary for melatonin biosynthesis. Here we report that alleles segregating at these two loci in C3H/HeJ mice, an inbred strain producing melatonin, suppress the circadian period-lengthening effect of the Clock mutation. Through a functional mapping approach, we localize mouse Asmt to chromosome X and show that it, and the Aanat locus on chromosome 11, are significantly associated with pineal melatonin levels. Treatment of suprachiasmatic nucleus (SCN) explant cultures from Period2^Luciferase (Per2^Luc) Clock/+ reporter mice with melatonin, or the melatonin agonist, ramelteon, phenocopies the genetic suppression of the Clock mutant phenotype observed in living animals. These results demonstrate that melatonin suppresses the Clock/+ mutant phenotype and interacts with Clock to affect the mammalian circadian system.     

Polymorphisms of MDR1, CYP2C19 and P2Y12 genes in Indian population: Effects on clopidogrel response

Aims/objectiveInfluence of genetic variations on the response of clopidogrel, an antiplatelet drug is implicated. In the present study, the prevalence of single nucleotide polymorphisms of MDR1 (C3435T), CYP2C19 [CYP2C19*2 CYP2C19*3, CYP2C19*17] and P2Y₁₂ (i-T744C) in Indian population and their effects on clopidogrel response was analyzed.Methods and resultsTo analyze the prevalence of polymorphisms, 102 healthy individuals were recruited. Clopidogrel response was assessed by ADP induced platelet aggregation in clopidogrel naïve acute myocardial infarction (AMI) patients (n = 26) screened from 100 AMI cases, before loading dose of 300 mg, at 24 h before next dose and 6 days after on 75 mg per day and platelet aggregation inhibition (PAI) was calculated between these time intervals. Genotyping was carried out by PCR-based restriction enzyme digestion method for C3435T of MDR1 and i-T744C of P2Y₁₂, by multiplex PCR for CYP2C19*2 (G681A) and CYP2C19*3 (G636A) and by nested PCR for CYP2C19*17 (C806T). The effect of the above mentioned genetic variations on PAI was analyzed. Variant allele of CYP2C19*3 was not observed while the prevalence of 3435T of MDR1 (0.524), CYP2C19*2 (681A, 0.352); i-744C of P2Y₁₂ (0.088), as well as wild type allele CYP2C19*17 (C806, 0.897) associated with decrease clopidogrel response were observed. Trend toward poor response to clopidogrel was observed at 24 h with the variant genotypes of CYP2C19*2 and i-T744C of P2Y₁₂ as compared to wild type.ConclusionThe present study did show a trend toward impaired response of clopidogrel to inhibit platelet aggregation with variant genotypes of CYP2C19*2 and iT744C of P2Y₁₂ compared to respective wild type genotype at 24 h.     

The role of the JAK2 GGCC haplotype and the TET2 gene in familial myeloproliferative neoplasms

Background

Myeloproliferative neoplasms constitute a group of diverse chronic myeloid malignancies that share pathogenic features such as acquired mutations in the JAK2, TET2, CBL and MPL genes. There are recent reports that a JAK2 gene haplotype (GGCC or 46/1) confers susceptibility to JAK2 mutation-positive myeloproliferative neoplasms. The aim of this study was to examine the role of the JAK2 GGCC haplotype and germline mutations of TET2, CBL and MPL in familial myeloproliferative neoplasms.

Design and Methods

We investigated patients with familial (n=88) or sporadic (n=684) myeloproliferative neoplasms, and a control population (n=203) from the same demographic area in Italy. Association analysis was performed using tagged single nucleotide polymorphisms (rs10974944 and rs12343867) of the JAK2 haplotype. Sequence analysis of TET2, CBL and MPL was conducted in the 88 patients with familial myeloproliferative neoplasms.

Results

Association analysis revealed no difference in haplotype frequency between familial and sporadic cases of myeloproliferative neoplasms (P=0.6529). No germline mutations in TET2, CBL or MPL that segregate with the disease phenotype were identified. As we observed variability in somatic mutations in the affected members of a pedigree with myeloproliferative neoplasms, we postulated that somatic mutagenesis is increased in familial myeloproliferative neoplasms. Accordingly, we compared the incidence of malignant disorders between sporadic and familial patients. Although the overall incidence of malignant disorders did not differ significantly between cases of familial and sporadic myeloproliferative neoplasms, malignancies were more frequent in patients with familial disease aged between 50 to 70 years (P=0.0198) than in patients in the same age range with sporadic myeloproliferative neoplasms.

Conclusions

We conclude that the JAK2 GGCC haplotype and germline mutations of TET2, CBL or MPL do not explain familial clustering of myeloproliferative neoplasms. As we observed an increased frequency of malignant disorders in patients with familial myeloproliferative neoplasms, we hypothesize that the germline genetic lesions that underlie familial clustering of myeloproliferative neoplasms predispose to somatic mutagenesis that is not restricted to myeloid hematopoietic cells but cause an increase in overall carcinogenesis.     

Nonsense suppression in archaea

Bacterial strains carrying nonsense suppressor tRNA genes played a crucial role in early work on bacterial and bacterial viral genetics. In eukaryotes as well, suppressor tRNAs have played important roles in the genetic analysis of yeast and worms. Surprisingly, little is known about genetic suppression in archaea, and there has been no characterization of suppressor tRNAs or identification of nonsense mutations in any of the archaeal genes. Here, we show, using the β-gal gene as a reporter, that amber, ochre, and opal suppressors derived from the serine and tyrosine tRNAs of the archaeon Haloferax volcanii are active in suppression of their corresponding stop codons. Using a promoter for tRNA expression regulated by tryptophan, we also show inducible and regulatable suppression of all three stop codons in H. volcanii. Additionally, transformation of a ΔpyrE2 H. volcanii strain with plasmids carrying the genes for a pyrE2 amber mutant and the serine amber suppressor tRNA yielded transformants that grow on agar plates lacking uracil. Thus, an auxotrophic amber mutation in the pyrE2 gene can be complemented by expression of the amber suppressor tRNA. These results pave the way for generating archaeal strains carrying inducible suppressor tRNA genes on the chromosome and their use in archaeal and archaeviral genetics. We also provide possible explanations for why suppressor tRNAs have not been identified in archaea.The availability of Escherichia coli strains carrying nonsense suppressor tRNA genes played a crucial role in much of the early work on bacterial (1) and bacterial viral genetics (2–4). For example, use of strains carrying amber suppressors enabled the isolation and propagation of T4 bacteriophage mutants defective in phage assembly and morphogenesis. Infection of E. coli strains not carrying an amber suppressor by the mutant T4 phages and biochemical and EM analyses of the phage lysates led to identification of the step at which phage morphogenesis was blocked in each of the mutants and provided a picture of the T4 phage genes involved in morphogenesis (5, 6). Similar approaches were used to identify genes involved in morphogenesis of phages P22 and λ and dissect their morphogenetic pathways (7, 8).In eukaryotes as well, nonsense suppressor tRNAs have played important roles in the genetic analysis of yeast (9, 10) and worms (11, 12). Suppressor tRNAs have not been identified in flies and mammals. However, ectopic expression of suppressor tRNA genes has been used to identify and suppress nonsense (stop codon) mutations in Drosophila (13, 14) and mammalian cell lines or viruses (15). A mammalian cell line carrying an inducible amber suppressor tRNA gene has been used to propagate a Polio virus mutant carrying an amber mutation in the RNA replicase gene (16).The availability of archaeal strains carrying suppressor tRNA genes would greatly facilitate archaeal and archaeviral genetics. Surprisingly, in contrast to bacteria and eukaryotes, little is known about genetic suppression in archaea (17), and there has been no characterization of suppressor tRNAs and identification of nonsense mutations in any of the archaeal genes. A few of the methanogenic archaea contain a tRNA that can read the stop codon UGA and insert the noncanonical amino acid selenocysteine at specific sites in a protein. These cases are, however, specialized, in that read through of the UGA codon requires a cis-acting structural element elsewhere in the mRNA (18). Some archaea, including Methanosarcinaceae, use the stop codon UAG to insert the noncanonical amino acid pyrrolysine into a few proteins (19, 20). It is possible, however, that, in this case, UAG has been or is being usurped as a sense codon (21) in much the same way as UGA as a sense codon for tryptophan in mitochondria and Mycoplasma and UAA and UAG as sense codons for glutamine in Tetrahymena (22, 23).There could be several reasons why suppressor tRNAs have not been identified in archaea. First, suppression could be weak and difficult to detect. Second, suppressor tRNAs, particularly those expressed constitutively, may be inherently toxic to archaea. Third, in contrast to bacteria and eukaryotes, archaea contain very few tRNA genes that are redundant (24). Therefore, mutation of any archaeal tRNA gene to produce a suppressor could abrogate the normal function of the tRNA and therefore, be lethal. This scenario is reminiscent of the situation in E. coli, where isolation of an amber or ochre suppressor derived from the tryptophan tRNA (tRNA^Trp) gene, of which there is only a single copy in WT strains, required the generation of merodiploid strains carrying an additional copy of the tRNA^Trp gene (25, 26).To distinguish between the possibilities mentioned above and with the eventual goal of generating archaeal strains carrying suppressor tRNA genes on the chromosome, we mutated the serine and tyrosine tRNA genes of the archaeon Haloferax volcanii to amber, ochre, and opal suppressors and studied their activities in suppression of amber, ochre, and opal stop codons using β-galactosidase (β-gal) genes carrying the corresponding mutations as reporters. We show that the suppressor tRNAs are active in suppression of the corresponding codons in H. volcanii. Using a promoter for tRNA expression regulated by concentrations of tryptophan in the medium, we have also demonstrated inducible and regulatable suppression of stop codons. Furthermore, we show that transformation of a ΔpyrE2 H. volcanii strain with plasmids carrying the genes for a pyrE2 amber mutant and the serine amber suppressor tRNA produced transformants that could grow on plates in minimal medium lacking uracil. In addition to providing the only example of suppression of amber, ochre, and opal stop codons in archaea, these results pave the way for generation of archaeal strains carrying inducible suppressor tRNA genes on the chromosome and their use in genetic analyses of archaea and archaeal viruses.     

Comparative study of mutations in SNP loci of K-RAS,hMLH1 and hMSH2 genes in neoplastic intestinal polyps and colorectal cancer

AIM: To clarify the molecular mechanism involved in pathogenesis of colorectal cancer as well as clinical significance of genetic analysis of histological samples.METHODS: A total of 480 blood and tissue specimens were collected in our hospital from January 2011 to October 2012. In the observation group, there were 120 blood specimens and 120 intestinal tract tissue specimens collected from patients with neoplastic intestinal polyps. In the control group I there were 80 blood specimens and 80 intestinal tract tissue specimens collected from patients with colorectal cancer. In the control group II there were 40 blood specimens and 40 intestinal tract tissue specimens collected from healthy individuals. The gene segments were amplified using PCR and DNA gel electrophoresis along with DNA sequence analysis were employed for the detection of the following single nucleotide polymorphisms (SNPs): K-RAS codons 12 and 13; hMLH1 (human mutS homolog 1) gene missense mutation at Va1384Asp; hMSH2 (human mutS homolog 2) gene missense mutation at 2783C/A.RESULTS: The mutation rate of the SNP at Va1384Asp locus of the hMLH1 gene from blood and tissue specimens in the observation group showed no statistical difference from those in the control group I. The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene were significantly lower in the observation group than in the control group I (χ² = 15.476, 29.670, 10.811, 16.618, 33.538, 7.898, P < 0.05). The mutation rate of SNP at Va1384Asp locus of the hMLH1 gene was significantly higher in the observation group when compared to the control group II (χ² = 10.486, 4.876, P < 0.05). The mutation rates of SNPs in codons 12 and 13 of K-RAS and at 2783C/A locus of the hMSH2 gene did not show any statistical difference from those in the control group II.CONCLUSION: There may be important clinical significance and relevance between neoplastic intestinal polyps and colorectal cancer in terms of the mechanisms involved in the pathogenesis.     

Synergistic effect of Bcl2, Myc and Ccnd1 transforms mouse primary B cells into malignant cells

Background

A synergistic effect resulting from a combination of BCL2 and MYC or MYC and CCND1 has been implicated in human B-cell lymphomas. Although the identification of other cooperative genes involved is important, our present understanding of such genes remains scant. The objective of this study was to identify the additional cooperative gene(s) associated with BCL2 and MYC or MYC and CCND1. First, we assessed whether Bcl2, Myc and Ccnd1 could cooperate. Next, we developed a synergism-based functional screening method for the identification of other oncogene(s) that act with Bcl2 and Myc.

Design and Methods

Growth in culture, colony formation and oncogenicity in vivo were assessed in mouse primary B cells exogenously expressing various combinations of Bcl2, Myc and Ccnd1. For the functional screening, Bcl2- and Myc-expressing primary B cells were infected with a retroviral cDNA library. Inserted cDNA of transformed cells in culture were then identified.

Results

Primary B cells exogenously expressing Bcl2, Myc and Ccnd1 showed factor-independent growth ability, enhanced colony-forming capability and aggressive oncogenicity, unlike the cases observed with the expression of any combination of only two of the genes. We identified CCND3 and NRAS as cooperative genes with Bcl2 and Myc through the functional screening.

Conclusions

Bcl2, Myc and Ccnd1 or Bcl2, Myc and CCND3 synergistically transformed mouse primary B cells into aggressive malignant cells. Our new synergism-based method is useful for the identification of synergistic gene combinations in tumor development, and may expand our systemic understanding of a wide range of cancer-causing elements.     

Antagonistic pleiotropic effects reduce the potential adaptive value of the FRIGIDA locus

Although the occurrence of epistasis and pleiotropy is widely accepted at the molecular level, its effect on the adaptive value of fitness-related genes is rarely investigated in plants. Knowledge of these features of a gene is critical to understand the molecular basis of adaptive evolution. Here we investigate the importance of pleiotropy and epistasis in determining the adaptive value of a candidate gene using the gene FRI (FRIGIDA), which is thought to be the major gene controlling flowering time variation in Arabidopsis thaliana. The effect of FRI on flowering time was analyzed in an outbred population created by randomly mating 19 natural accessions of A. thaliana. This unique population allows the estimation of FRI effects independent of any linkage association with other loci due to demographic processes or to coadapted genes. It also allows for the estimation of pleiotropic effects of FRI on fitness and inflorescence architecture. We found that FRI explains less variation in flowering time than previously observed among natural accessions, and interacts epistatically with the FLC locus. Although early flowering plants produce more fruits under spring conditions, and nonfunctional alleles of FRI were associated with early flowering, variation at FRI was not associated with fitness. We show that nonfunctional FRI alleles have negative pleiotropic effects on fitness by reducing the numbers of nodes and branches on the inflorescence. We propose that these antagonistic pleiotropic effects reduce the adaptive value of FRI, and helps explain the maintenance of alternative life history strategies across natural populations of A. thaliana.     

Schizosaccharomyces pombe Rtf2 mediates site-specific replication termination by inhibiting replication restart

Here, we identify a phylogenetically conserved Schizosaccharomyces pombe factor, named Rtf2, as a key requirement for efficient replication termination at the site-specific replication barrier RTS1. We show that Rtf2, a proliferating cell nuclear antigen-interacting protein, promotes termination at RTS1 by preventing replication restart; in the absence of Rtf2, we observe the establishment of “slow-moving” Srs2-dependent replication forks. Analysis of the pmt3 (SUMO) and rtf2 mutants establishes that pmt3 causes a reduction in RTS1 barrier activity, that rtf2 and pmt3 are nonadditive, and that pmt3 (SUMO) partly suppresses the rtf2-dependent replication restart. Our results are consistent with a model in which Rtf2 stabilizes the replication fork stalled at RTS1 until completion of DNA synthesis by a converging replication fork initiated at a flanking origin.     

Arabidopsis semidwarfs evolved from independent mutations in GA20ox1, ortholog to green revolution dwarf alleles in rice and barley

Understanding the genetic bases of natural variation for developmental and stress-related traits is a major goal of current plant biology. Variation in plant hormone levels and signaling might underlie such phenotypic variation occurring even within the same species. Here we report the genetic and molecular basis of semidwarf individuals found in natural Arabidopsis thaliana populations. Allelism tests demonstrate that independent loss-of-function mutations at GA locus 5 (GA5), which encodes gibberellin 20-oxidase 1 (GA20ox1) involved in the last steps of gibberellin biosynthesis, are found in different populations from southern, western, and northern Europe; central Asia; and Japan. Sequencing of GA5 identified 21 different loss-of-function alleles causing semidwarfness without any obvious general tradeoff affecting plant performance traits. GA5 shows signatures of purifying selection, whereas GA5 loss-of-function alleles can also exhibit patterns of positive selection in specific populations as shown by Fay and Wu’s H statistics. These results suggest that antagonistic pleiotropy might underlie the occurrence of GA5 loss-of-function mutations in nature. Furthermore, because GA5 is the ortholog of rice SD1 and barley Sdw1/Denso green revolution genes, this study illustrates the occurrence of conserved adaptive evolution between wild A.thaliana and domesticated plants.Bioactive gibberellins (GAs) are plant growth regulators involved in important traits such as seed germination, flowering time, flower development, and elongation growth (1). GA biosynthesis and signaling pathways are well defined (1, 2) and have been targeted in crop breeding. Modification of GA pathways was crucial in the green revolution because it conferred semidwarfness, thus reducing lodging and increasing crop yields (3–6). Green revolution semidwarf varieties in wheat are due to mutations in DELLA genes, whereas many short straw rice varieties carry a mutation in the Semi-Dwarf-1 (SD1) locus. This locus codes for GA 20-oxidase-2, a GA biosynthesis gene that is also mutated in most modern barley varieties in which the gene was called Denso or Semi-dwarf 1 (Sdw1) (7).GA 20-oxidases are involved in the later steps of GA biosynthesis and belong to the group of 2-oxoglutarate–dependent dioxygenases that, together with GA 3-oxidases, form biologically active GA (8). Arabidopsis thaliana has five GA20ox paralogous genes. AtGA20ox-1, AtGA20ox-2, AtGA20ox-3, and AtGA20ox-4 can catalyze the in vitro conversion of GA₁₂ to GA₉. Therefore, GA20ox paralogs might have partial redundant functions (9). However, among paralog genes, only AtGA20ox-1 (GA5), which was cloned on the basis of the ga5 mutant (10), affected plant height (8).Natural variation for GA biosynthesis has been previously described in A. thaliana because the Bur-0 accession carries a loss-of-function allele at GA20ox4 (9), which does not result in a semidwarf phenotype. In addition, genetic variation in GA1 has been associated with variation in floral morphology (11). Furthermore, the semidwarf phenotype (here defined as a plant height shorter than half the size of genetically related individuals) observed in the Kas-2 accession is due to a recessive allele at the GA5 locus (12). The latter finding led to the questions of whether green revolution alleles, artificially selected in cereals, could also occur in natural populations of the wild species A. thaliana, and if so, how many different GA5 loss-of-function alleles exist, how they are distributed, and why they occur in some populations.     

Major flowering time gene,FLOWERING LOCUS C,regulates seed germination in Arabidopsis thaliana

FLOWERING LOCUS C (FLC) is a major regulator of flowering responses to seasonal environmental factors. Here, we document that FLC also regulates another major life-history transition-seed germination, and that natural variation at the FLC locus and in FLC expression is associated with natural variation in temperature-dependent germination. FLC-mediated germination acts through additional genes in the flowering pathway (FT, SOC1, and AP1) before involving the abscisic acid catabolic pathway (via CYP707A2) and gibberellins biosynthetic pathway (via GA20ox1) in seeds. Also, FLC regulation of germination is largely maternally controlled, with FLC peaking and FT, SOC1, and AP1 levels declining at late stages of seed maturation. High FLC expression during seed maturation is associated with altered expression of hormonal genes (CYP707A2 and GA20ox1) in germinating seeds, indicating that gene expression before the physiological independence of seeds can influence gene expression well after any physical connection between maternal plants and seeds exists. The major role of FLC in temperature-dependent germination documented here reveals a much broader adaptive significance of natural variation in FLC. Therefore, pleiotropy between these major life stages likely influences patterns of natural selection on this important gene, making FLC a promising case for examining how pleiotropy influences adaptive evolution.