The derived allele of ASPM is associated with lexical tone perception

The ASPM and MCPH1 genes have been implicated in the adaptive evolution of the human brain [Mekel-Bobrov N. et al., 2005. Ongoing adaptive evolution of ASPM, a brain size determinant in homo sapiens. Science 309; Evans P.D. et al., 2005. Microcephalin, a gene regulating brain size, continues to evolve adaptively in humans. Science 309]. Curiously, experimental attempts have failed to connect the implicated SNPs in these genes with higher-level brain functions. These results stand in contrast with a population-level study linking the population frequency of their alleles with the tendency to use lexical tones in a language [Dediu D., Ladd D.R., 2007. Linguistic tone is related to the population frequency of the adaptive haplogroups of two brain size genes, ASPM and microcephalin. Proc. Natl. Acad. Sci. U.S.A. 104]. In the present study, we found a significant correlation between the load of the derived alleles of ASPM and tone perception in a group of European Americans who did not speak a tone language. Moreover, preliminary results showed a significant correlation between ASPM load and hemodynamic responses to lexical tones in the auditory cortex, and such correlation remained after phonemic awareness, auditory working memory, and non-verbal IQ were controlled. As in previous studies, no significant correlation between ASPM and cognitive measures were found. MCPH1 did not correlate with any measures. These results suggest that the association between the recently derived allele of ASPM is likely to be specific and is tied to higher level brain functions in the temporal cortex related to human communication.     

Mimicking natural polymorphism in eIF4E by CRISPR‐Cas9 base editing is associated with resistance to potyviruses

In many crop species, natural variation in eIF4E proteins confers resistance to potyviruses. Gene editing offers new opportunities to transfer genetic resistance to crops that seem to lack natural eIF4E alleles. However, because eIF4E are physiologically important proteins, any introduced modification for virus resistance must not bring adverse phenotype effects. In this study, we assessed the role of amino acid substitutions encoded by a Pisum sativum eIF4E virus‐resistance allele (W69L, T80D S81D, S84A, G114R and N176K) by introducing them independently into the Arabidopsis thaliana eIF4E1 gene, a susceptibility factor to the Clover yellow vein virus (ClYVV). Results show that most mutations were sufficient to prevent ClYVV accumulation in plants without affecting plant growth. In addition, two of these engineered resistance alleles can be combined with a loss‐of‐function eIFiso4E to expand the resistance spectrum to other potyviruses. Finally, we use CRISPR‐nCas9‐cytidine deaminase technology to convert the Arabidopsis eIF4E1 susceptibility allele into a resistance allele by introducing the N176K mutation with a single‐point mutation through C‐to‐G base editing to generate resistant plants. This study shows how combining knowledge on pathogen susceptibility factors with precise genome‐editing technologies offers a feasible solution for engineering transgene‐free genetic resistance in plants, even across species barriers.     

Salicylic acid-, but not cytokinin-induced, resistance to WClMV is associated with increased expression of SA-dependent resistance genes in Phaseolus vulgaris

Two-week-old Phaseolus vulgaris plants, wick-fed with 1 mmol/L salicylic acid (SA) or 50 nmol/L dihydrozeatin (DHZ), showed partial inhibition of the accumulation of white clover mosaic virus (WClMV) in infected primary leaves. This inhibition was measured as a decrease in the accumulation of both viral mRNA and viral coat protein, especially at the early stages of infection. Salicylic acid treatment resulted in moderately increased expression of phenylalanine ammonia lyase (PAL), NPR1, PR1 and HSP70 genes that participate in resistance to pathogens in plants. In contrast, DHZ treatments did not induce significant changes in expression of these genes. The expression of the P. vulgaris alternative oxidase (AOX) gene homolog, an enzyme implicated in plant resistance to viruses, showed low constitutive expression during the first 11 days post-infection and was not affected by either SA or DHZ. It appears that, while SA induced the NPR1-PR1 pathogen defense pathway genes, both SA and DHZ may use a different pathway to induce resistance to WClMV infection in P. vulgaris plants.     

Endotoxin-induced decrease in muscle protein synthesis is associated with changes in eIF2B, eIF4E, and IGF-I

The present study examined potential mechanisms contributing to the inhibition of protein synthesis in skeletal muscle after administration of endotoxin (LPS). Rats implanted with vascular catheters were injected intravenously with a nonlethal dose of Escherichia coli LPS, and samples were collected at 4 and 24 h thereafter; pair-fed control animals were also included. The rate of muscle (gastrocnemius) protein synthesis in vivo was reduced at both time points after LPS administration. LPS did not alter tissue RNA content, but the translational efficiency was consistently reduced at both time points. To identify mechanisms responsible for regulating translation, we examined several eukaryotic initiation factors (eIFs). The content of eIF2alpha or the amount of eIF2alpha in the phosphorylated form did not change in response to LPS. eIF2B activity was decreased in muscle 4 h post-LPS but activity returned to control values by 24 h. A decrease in the relative amount of eIF2Balpha protein was not responsible for the LPS-induced reduction in eIF2B activity. LPS also markedly altered the distribution of eIF4E in muscle. Compared with control values, LPS-treated rats demonstrated 1) a transient increase in binding of the translation repressor 4E-binding protein-1 (4E-BP1) with eIF4E, 2) a transient decrease in the phosphorylated gamma-form of 4E-BP1, and 3) a sustained decrease in the amount of eIF4G associated with eIF4E. LPS also decreased insulin-like growth factor (IGF) I protein and mRNA expression in muscle at both times. A significant linear relationship existed between muscle IGF-I and the rate of protein synthesis or the amount of eIF4E bound to eIF4G. In summary, these data suggest that LPS impairs muscle protein synthesis, at least in part, by decreasing translational efficiency, resulting from an impairment in translation initiation associated with alterations in both eIF2B activity and eIF4E availability.     

Characteristics of the common bean, Phaseolus vulgaris, associated with resistance to the leafhopper Empoasca kraemeri

Experiments were conducted using the common bean, Phaseolus vulgaris, and its most important pest in Latin America, Empoasca kraemeri, in order to assess the value of insect counts and various plant characteristics as selection criteria in a plant breeding programme. The combination of insect counts with measurements of damage symptoms should make it possible to distinguish tolerance from the resistance mechanisms of antibiosis and non-preference. By this means, and by a knowledge of the plant characteristics associated with resistance, different forms of resistance may be combined in the progeny of crosses. In a comparison of two contrasting genotypes the more severely damaged genotype was the more heavily infested at early stages of plant growth, but had the lower level of infestation at later stages. The association of high early counts with high subsequent damage was not confirmed in an experiment on six genotypes, but the association of high damage with low late counts was partially confirmed. These results indicate that the level of early infestation of E. kraemeri is not consistently reflected in the subsequent levels of damage which P. vulgaris genotypes display. This suggests that these two criteria represent distinct forms of resistance which can be combined in hybrid progeny. Resistance was also associated with late maturity, indeterminate growth habit, purple flowers and black or beige seeds. However, consumer preferences and the requirements of agricultural systems place constraints upon the use of these relationships in resistance breeding.     

A series of eIF4E alleles at the Bc-3 locus are associated with recessive resistance to Clover yellow vein virus in common bean

Clover yellow vein virus (ClYVV) is capable of causing severe damage to common bean (Phaseolus vulgaris L.) production worldwide. The snap bean market class is particularly vulnerable because infection may lead to distortion and necrosis of the fresh green pods and rejection of the harvest. Three putatively independent recessive genes (cyv, desc, bc-3) have been reported to condition resistance to ClYVV; however, their allelic relationships have not been resolved. We identified, evaluated, and characterized the phenotypic and molecular genetic variation present in 21 informative common bean genotypes for resistance to ClYVV. Allelism testing phenotypes from multiple populations provided clear evidence that the three genes were a series of recessive alleles at the Bc-3 locus that condition unique potyvirus strain- and species-specific resistance spectra. Candidate gene analysis revealed complete association between the recessive resistance alleles and unique patterns of predicted amino acid substitutions in P. vulgaris eukaryotic translation initiation factor 4E (PveIF4E). This led to the discovery and characterization of two novel PveIF4E alleles associated with resistance to ClYVV, PveIF4E ³ , and PveIF4E ⁴ . We developed KASPar allele-specific SNP genotyping assays and demonstrated their ability to accurately detect and differentiate all of the PveIF4E haplotypes present in the germplasm, allelism testing, and in three separate segregating populations. The results contribute to an enhanced understanding and accessibility of the important potyvirus resistance conditioned by recessive alleles at Bc-3. The KASPar assays should be useful to further enable germplasm exploration, allelic discrimination, and marker-assisted introgression of bc-3 alleles in common bean.     

The expression of phytohemagglutinin genes in Phaseolus vulgaris is associated with organ-specific DNA methylation patterns

Using methylation sensitive restriction enzymes and DNA gel blot analyses, we have examined DNA methylation patterns at the chromosomal locus harboring the two phytohemagglutinin genes of Phaseolus vulgaris. In cotyledons, where the genes are highly expressed, several sites have become undermethylated, whereas the methylation status of other sites remains constant, both in cotyledons and in other organs. Taken together with measurements of mRNA levels, these studies provide evidence that organ-specific changes in DNA methylation patterns are associated with the organ-specific expression of nuclear genes in a dicotyledonous plant.     

A microtiter plate assay for assessing the interaction of eukaryotic initiation factor eIF4E with eIF4G and eIF4E binding protein-1

Modulation of interactions among proteins is an important mechanism for regulating both the subcellular location and the function of proteins. An example of the importance of protein-protein interaction is the reversible association of eukaryotic initiation factor eIF4E with the eIF4E binding proteins 4E-BP1 and eIF4G. When bound to 4E-BP1, eIF4E cannot bind to eIF4G to form the active eIF4F complex, an event that is required for the binding of mRNA to the ribosome. Thus, association of eIF4E with 4E-BP1 represses mRNA translation by preventing the binding of mRNA to the ribosome. Previous studies have measured the amount of 4E-BP1 or eIF4G bound to eIF4E by either affinity chromatography or immunoprecipitation of eIF4E followed by Western blot analysis for quantitation of 4E-BP1 and eIF4G. Both of these techniques have significant limitations. In the present study, we describe a microtiter plate-based assay for quantitation of the amount of 4E-BP1 and eIF4G bound to eIF4E that obviates many of the limitations of the earlier approaches. It also has the advantage that absolute amounts of the individual proteins can be easily estimated. The approach should be applicable to the study of a wide variety of protein-protein interactions.     

Helper component proteinase of the genus Potyvirus is an interaction partner of translation initiation factors eIF(iso)4E and eIF4E and contains a 4E binding motif

The multifunctional helper component proteinase (HCpro) of potyviruses (genus Potyvirus; Potyviridae) shows self-interaction and interacts with other potyviral and host plant proteins. Host proteins that are pivotal to potyvirus infection include the eukaryotic translation initiation factor eIF4E and the isoform eIF(iso)4E, which interact with viral genome-linked protein (VPg). Here we show that HCpro of Potato virus A (PVA) interacts with both eIF4E and eIF(iso)4E, with interactions with eIF(iso)4E being stronger, as judged by the data of a yeast two-hybrid system assay. A bimolecular fluorescence complementation assay on leaves of Nicotiana benthamiana showed that HCpro from three potyviruses (PVA, Potato virus Y, and Tobacco etch virus) interacted with the eIF(iso)4E and eIF4E of tobacco (Nicotiana tabacum); interactions with eIF(iso)4E and eIF4E of potato (Solanum tuberosum) were weaker. In PVA-infected cells, interactions between HCpro and tobacco eIF(iso)4E were confined to round structures that colocalized with 6K2-induced vesicles. Point mutations introduced to a 4E binding motif identified in the C-terminal region of HCpro debilitated interactions of HCpro with translation initiation factors and were detrimental to the virulence of PVA in plants. The 4E binding motif conserved in HCpro of potyviruses and HCpro-initiation factor interactions suggest new roles for HCpro and/or translation factors in the potyvirus infection cycle.     

The potyviral virus genome-linked protein VPg forms a ternary complex with the eukaryotic initiation factors eIF4E and eIF4G and reduces eIF4E affinity for a mRNA cap analogue

The virus protein linked to the genome (VPg) of plant potyviruses is a 25-kDa protein covalently attached to the genomic RNA 5' end. It was previously reported that VPg binds specifically to eIF4E, the mRNAcap-binding protein of the eukaryotic translation initiation complex. We performed a spectroscopic study of the interactions between lettuce eIF4E and VPg from lettuce mosaic virus (LMV). The cap analogue m7GDP and VPg bind to eIF4E at two distinct sites with similar affinity (K(d) = 0.3 microm). A deeper examination of the interaction pathway showed that the binding of one ligand induces a decrease in the affinity for the other by a factor of 15. GST pull-down experiments from plant extracts revealed that VPg can specifically trap eIF4G, the central component of the complex required for the initiation of protein translation. Our data suggest that eIF4G recruitment by VPg is indirectly mediated through VPg-eIF4E association. The strength of interaction between eIF4E and pep4G, the eIF4E-binding domain on eIF4G, was increased significantly by VPg. Taken together these quantitative data show that VPg is an efficient modulator of eIF4E biochemical functions.     

Fast activation of a time-dependent outward current in protoplasts derived from coats of developing Phaseolus vulgaris seeds

An outward current that appeared to activate instantaneously in response to depolarising voltage pulses at low sampling frequencies predominated in the plasma membrane of ground-parenchyma protoplasts derived from coats of developing Phaseolus vulgaris L. (cv. Redland Pioneer) seeds. However, the outward current showed time-dependent activation when higher sampling frequencies were used to measure the current. Activation of the current was best described as a double-exponential time course with the fast and slow time constants being 1 and 20 ms, respectively. The current also exhibited a rapid deactivation that followed a double-exponential time course with time constants of approximately 2 and 30 ms, respectively. “Tail-current” analysis allowed us to show that this current exhibited a low selectivity between K⁺ and Cl⁻ (P _K:Cl=1.8). Such a fast-activating current may account for some of the reports of time-independent, instantaneous currents that have been observed in plasma membranes of plant cells digitised at low sampling frequencies. Therefore, when “instantaneous” currents appear it is advisable to characterise these currents using higher sampling frequencies with correspondingly higher filtering frequency cut-offs. Received: 12 May 2000 / Accepted: 26 June 2000     

Apolipoprotein E*3-Leiden allele results from a partial gene duplication in exon 4

The apolipoprotein E3-Leiden variant has been shown to be associated with familial dysbetalipoproteinemia (FD) in a dominant manner (Havekes et al., Hum Genet 1986;73:157-163). Applying the polymerase chain reaction technique, we have cloned and sequenced relevant parts of both APOE alleles of the original proband. In exon 4 of the E*3-Leiden allele a partial gene duplication encompassing 21 nucleotides was found, leading to a tandem repeat of the codons 120-126 or 121-127. Using an E3-Leiden mutation specific oligonucleotide probe, the same mutation was found in two additional independently ascertained FD patients with an E3E3 phenotype based on isoelectric focusing. The E*3-Leiden mutation will be useful in the elucidation of the etiology of dominantly inherited forms of FD.     

The interaction of eIF4E with 4E-BP1 is an induced fit to a completely disordered protein.

4E binding protein 1 (4E-BP1) inhibits translation by binding to the initiation factor eIF4E and is mostly or completely unstructured in both free and bound states. We wished to determine whether the free protein has local structure that could be involved in eIF4E binding. Assignments were obtained using double and triple resonance NMR methods. Residues 4-10, 43-46, and 56-65 could not be assigned, primarily because of a high degree of 1H and 15N chemical shift overlap. Steady-state ¿1H¿-15N NOEs were measured for 45 residues in the assigned regions. Except for the two C-terminal residues, the NOEs were between -0.77 and - 1.14, indicating a high level of flexibility. Furthermore, the ¿1H¿-15N NOE spectrum recorded with presaturation contained no strong positive signals, making it likely that no other residues have positive or smaller negative NOEs. This implies that 4E-BP1 has no regions of local order in the absence of eIF4E. The interaction therefore appears to be an induced fit to a completely disordered protein molecule.     

A precursor of the reserve-protein, phaseolin, is transiently associated with the endoplasmic reticulum of developing Phaseolus vulgaris cotyledons

Developing cotyledons of Phaseolus vulgaris L. were labeled for 30 min with [³H] amino acids, homogenized, and the proteins fractionated on sodium dodecylsulfate (SDS) polyacrylamide gels. Fluorographs of these gels showed that the polypeptides of phaseolin, the major reserve protein of P. vulgaris, were synthesized as precursors which could be distinguished from the polypeptides of mature phaseolin by their slightly lower mobility. When extracts of cotyledons labeled for 45 min with [³H] amino acids were fractionated on isopynic sucrose gradients, radioactive phaseolin banded at the same density (1.14 g cm^-3) as the endoplasmic reticulum (ER)-marker enzyme NADH-cytochrome c reductase. Fractionation in the presence of 3 mM MgCl₂ indicated that the newly-synthesized phaseolin was associated with the rough ER. Pulse-chase experiments showed that phaseolin was transiently associated with the ER, and later accumulated in the protein bodies. Treatment of isolated ER with proteinase K showed that phaseolin polypeptides were degraded only if Triton X-100 was present, indicating that phaseolin was membrane-protected, probably enclosed within the vesicles. ER-associated phaseolin associated to an 18S form at pH 4.5 in the presence of 0.3 M NaCl and 100 mM sodium acetate. The polypeptides of ER-associated phaseolin had a slightly lower mobility on SDS-gels than polypeptides of protein body phaseolin. ER-associated phaseolin had a carbohydrate content of 6.8%, while protein body-derived phaseolin had a carbohydrate content of 6.2%. When cotyledons were labeled simultaneously with [¹⁴C] amino acids and [³H] glucosamine or with [¹⁴C] amino acids and [³H] mannose, the [³H]/[¹⁴C] ratio of ER-derived phaseolin was similar to that of protein body derived phaseolin, indicating that the faster mobility on SDS-gels was not due to the detachment of carbohydrate. Experiments in which the carbohydrate side chains were removed with endoglycosidase H, and the resulting polypeptides subjected to electrophoresis in SDS-gels showed that the differential mobility of the glycopolypeptides of phaseolin resided in their polypeptide chains.     

eIF4E is a central node of an RNA regulon that governs cellular proliferation

This study demonstrates that the eukaryotic translation initiation factor eIF4E is a critical node in an RNA regulon that impacts nearly every stage of cell cycle progression. Specifically, eIF4E coordinately promotes the messenger RNA (mRNA) export of several genes involved in the cell cycle. A common feature of these mRNAs is a structurally conserved, approximately 50-nucleotide element in the 3' untranslated region denoted as an eIF4E sensitivity element. This element is sufficient for localization of capped mRNAs to eIF4E nuclear bodies, formation of eIF4E-specific ribonucleoproteins in the nucleus, and eIF4E-dependent mRNA export. The roles of eIF4E in translation and mRNA export are distinct, as they rely on different mRNA elements. Furthermore, eIF4E-dependent mRNA export is independent of ongoing RNA or protein synthesis. Unlike the NXF1-mediated export of bulk mRNAs, eIF4E-dependent mRNA export is CRM1 dependent. Finally, the growth-suppressive promyelocytic leukemia protein (PML) inhibits this RNA regulon. These data provide novel perspectives into the proliferative and oncogenic properties of eIF4E.     

The APOE4 allele is associated with a decreased risk of retinopathy in type 2 diabetics

Background

Common polymorphisms within the apolipoprotein E (APOE) gene are suggested to be associated with the development of type 2 diabetes mellitus (T2DM), but the potential association with T2DM complications (nephropathy, neuropathy and retinopathy) remains unclear. We perform the case–control study to analyse the association between the APOE polymorphism and risk of T2DM and to analysed the potential relationship between the APOE and T2DM complications.

Methods and results

APOE variants (rs429358 and rs7412) were genotyped by TaqMan assay in T2DM patients (N?=?1274; N?=?829 with complications including retinopathy, neuropathy and nephropathy status) and with PCR–RFLP in healthy nondiabetic controls (N?=?2055). The comparison of subjects with genotypes associated with low plasma cholesterol (APOE2/E2 and APOE2/E3 carriers vs. others) did not show an association with T2DM (OR [95% CI]?=?0.88 [0.71–1.08). The differences remained insignificant after adjusting for diabetes duration, sex and BMI. Carriers of at least one APOE4 allele (rs429358) are protected against T2DM related retinopathy (OR [95% CI]?=?0.65 [0.42–0.99]. Protection against retinopathy is driven mostly by females (OR [95% CI]?=?0.50 [0.25–0.99]); and remains significant (P?=?0.044) after adjustment for diabetes duration and BMI.

Conclusion

Common APOE polymorphism was not associated with T2DM in the Czech population. Yet, APOE4 allele revealed an association with retinopathy. In particular, female T2DM patients with at least one APOE4 allele exhibit lower prevalence of retinopathy in our study subjects.