Methenyltetrahydrofolate synthetase regulates folate turnover and accumulation

Cellular folate deficiency impairs one-carbon metabolism, resulting in decreased fidelity of DNA synthesis and inhibition of numerous S-adenosylmethionine-dependent methylation reactions including protein and DNA methylation. Cellular folate concentrations are influenced by folate availability, cellular folate transport efficiency, folate polyglutamylation, and folate turnover specifically through degradation. Folate cofactors are highly susceptible to oxidative degradation in vitro with the exception of 5-formyltetrahydrofolate, which may be a storage form of folate. In this study, we determined the effects of depleting cytoplasmic 5-formyltetrahydrofolate on cellular folate concentrations and folate turnover rates in cell cultures by expressing the human methenyltetrahydrofolate synthetase cDNA in human MCF-7 cells and SH-SY5Y neuroblastoma. Cells with increased methenyltetrahydrofolate synthetase activity exhibited: 1) increased rates of folate turnover, 2) elevated generation of p-aminobenzoylglutamate in culture medium, 3) depressed cellular folate concentrations independent of medium folic acid concentrations, and 4) increased average polyglutamate chain lengths of folate cofactors. These data indicate that folate catabolism and folate polyglutamylation are competitive reactions that influence cellular folate concentrations, and that increased methenyltetrahydrofolate synthetase activity accelerates folate turnover rates, depletes cellular folate concentrations, and may account in part for tissue-specific differences in folate accumulation.     

Understanding Voltage Gating of Providencia stuartii Porins at Atomic Level

Bacterial porins are water-filled β-barrel channels that allow translocation of solutes across the outer membrane. They feature a constriction zone, contributed by the plunging of extracellular loop 3 (L3) into the channel lumen. Porins are generally in the open state, but undergo gating in response to external voltages. To date the underlying mechanism is unclear. Here we report results from molecular dynamics simulations on the two porins of Providenica stuartii, Omp-Pst1 and Omp-Pst2, which display distinct voltage sensitivities. Voltage gating was observed in Omp-Pst2, where the binding of cations in-between L3 and the barrel wall results in exposing a conserved aromatic residue in the channel lumen, thereby halting ion permeation. Comparison of Omp-Pst1 and Omp-Pst2 structures and trajectories suggests that their sensitivity to voltage is encoded in the hydrogen-bonding network anchoring L3 onto the barrel wall, as we observed that it is the strength of this network that governs the probability of cations binding behind L3. That Omp-Pst2 gating is observed only when ions flow against the electrostatic potential gradient of the channel furthermore suggests a possible role for this porin in the regulation of charge distribution across the outer membrane and bacterial homeostasis.     

Partial purification of pepsin from turkey proventriculus

In the present study, turkey proventriculus (stomach) was mixed with 10% NaCl (1:2 w/v) and extracted by centrifugation to produce crude extract. The partial purification of the extract was carried out by using Sephadex G-75 resin in gel filtration column chromatography. Crude extract (CE) and fractions obtained by gel filtration were analysed for milk-clotting activity (MCA), protein content, proteolytic activity (PA), and SDS-PAGE electrophoresis. The first 7 fractions did not have any activities; the fraction of 9, 10 and 11 had the most milk-clotting and proteolytic activities. Electrophoretic patterns proved that further purification steps should be applied for better results.     

Robust Self-Incompatibility in the Absence of a Functional ARC1 Gene in Arabidopsis thaliana

Self-incompatibility (SI) is the primary determinant of the outbreeding mode of sexual reproduction in the Brassicaceae. All Arabidopsis thaliana accessions analyzed to date carry mutations that disrupt SI functions by inactivating the SI specificity-determining S locus or SI modifier loci. S-locus genes isolated from self-incompatible close relatives of A. thaliana restore robust SI in several accessions that harbor only S-locus mutations and confer transient SI in accessions that additionally harbor mutations at modifier loci. Self-incompatible transgenic A. thaliana plants have proved to be valuable for analysis of the recognition and signaling events that underlie SI in the Brassicaceae. Here, we review results demonstrating that S-locus genes are necessary and sufficient for SI signaling and for restoration of a strong and developmentally stable SI phenotype in several accessions of A. thaliana. The data indicate that introduction of a functional E3 ligase-encoding ARC1 gene, which is deleted in all accessions that have been analyzed to date, is not required for SI signaling leading to inhibition of self pollen or for reversion of A. thaliana to its fully self-incompatible ancestral state.It is well established that specific pollen recognition in the self-incompatibility (SI) response of the Brassicaceae is determined by allele-specific interactions that occur at the stigma surface between two highly polymorphic proteins encoded in the S locus: the S-locus receptor kinase SRK and its ligand, the S-locus cysteine-rich protein SCR. Arabidopsis thaliana lacks a functional SI system and harbors nonfunctional S-locus variants that contain defective alleles of the SRK and/or SCR genes (Kusaba et al., 2001; Sherman-Broyles et al., 2007; Tang et al., 2007; Shimizu et al., 2008; Boggs et al., 2009a; Tsuchimatsu et al., 2010; Dwyer et al., 2013). Despite being highly self-fertile, A. thaliana can be made to express SI upon transformation with functional SRK-SCR gene pairs isolated from its self-incompatible close relatives (Nasrallah et al., 2002, 2004; Boggs et al., 2009a, 2009b). The first transfer of the SI trait into A. thaliana was achieved using the SRKb-SCRb gene pair isolated from the Sb locus of Arabidopsis lyrata (Kusaba et al., 2001; Nasrallah et al., 2002, 2004). Many of the subsequent studies that have been performed in the transgenic A. thaliana SRK-SCR system have used plants transformed with p548, a plasmid that we constructed by inserting the A. lyrata SRKb and SCRb genes with their 5′ and 3′ regulatory sequences into the pBIN+ binary vector (Nasrallah et al., 2004).Indriolo et al. (2014) recently used the p548 plasmid to generate SRKb-SCRb transformants and test the role of the ARM Repeat Containing1 (ARC1) gene in SI. ARC1 was originally identified as a Brassica napus protein that interacts with the SRK kinase domain in yeast (Gu et al., 1998), and it was subsequently inferred to be required for SI because downregulation of the ARC1 gene in B. napus (Stone et al., 1999) and A. lyrata (Indriolo et al., 2012), as well as overexpression of ARC1’s target, Exo70A1, in B. napus (Samuel et al., 2009), caused partial breakdown of the SI response. However, the involvement of the proposed SRK-ARC1-Exo70A1 pathway in SI has been questioned because the ARC1 gene was found to be deleted in all A. thaliana accessions analyzed to date (Kitashiba et al., 2011; Indriolo et al., 2012), including those in which the SRKb-SCRb transgenes confer a strong SI phenotype (Kitashiba et al., 2011). Additionally, overexpression of Exo70A1 did not cause weakening of the SI response in A. thaliana SRKb-SCRb plants (Kitashiba et al., 2011).Indriolo et al. (2014) reported on their characterization of the SI response in plants of the Sha and Columbia-0 (Col-0) accessions, which they either transformed with the p548 plasmid alone or cotransformed with p548 and a plasmid containing an ARC1 gene isolated from A. lyrata or B. napus. They concluded that, along with SRK and SCR, “ARC1 is the third component that is required to return A. thaliana to its ancestral self-incompatibility state.” However, this conclusion is inconsistent with results of previous studies of SI in transgenic A. thaliana SRK-SCR transformants, which have shown that several A. thaliana accessions are rendered fully self-incompatible by transformation with the p548 plasmid without the addition of a functional ARC1 gene. Contrary to Indriolo et al.’s assertion that in previous studies of A. thaliana SRK-SCR transformants, “the self-pollen rejection response was incomplete,” we reported that among 11 A. thaliana accessions tested by transformation with the p548 plasmid, five accessions (C24, Cvi-0, Hodja, Kas-2, and Sha) were converted to full SI by expression of the SRKb and SCRb genes alone (Nasrallah et al., 2004; Boggs et al., 2009a). Importantly, the SI phenotype of these self-incompatible SRKb-SCRb transformants faithfully recapitulates the SI phenotype of naturally self-incompatible Brassicaceae with respect to the four defining features of SI in this family: (1) site of pollen inhibition at the stigma surface, (2) intensity of the response, (3) developmental regulation over the course of stigma maturation, and (4) heritability. These features suggest that the inhibition of self pollen in self-incompatible A. thaliana SRK-SCR transformants is achieved via the same signaling pathway as that utilized by other self-incompatible Brassicaceae species.     

Evolution and control of imprinted FWA genes in the genus Arabidopsis

A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.     

Molecular Characterization and Evolution of Self-Incompatibility Genes in Arabidopsis thaliana: The Case of the Sc Haplotype

The switch from an outcrossing mode of mating enforced by self-incompatibility to self-fertility in the Arabidopsis thaliana lineage was associated with mutations that inactivated one or both of the two genes that comprise the self-incompatibility (SI) specificity-determining S-locus haplotype, the S-locus receptor kinase (SRK) and the S-locus cysteine-rich (SCR) genes, as well as unlinked modifier loci required for SI. All analyzed A. thaliana S-locus haplotypes belong to the SA, SB, or SC haplotypic groups. Of these three, the SC haplotype is the least well characterized. Its SRKC gene can encode a complete open-reading frame, although no functional data are available, while its SCRC sequences have not been isolated. As a result, it is not known what mutations were associated with inactivation of this haplotype. Here, we report on our analysis of the Lz-0 accession and the characterization of its highly rearranged SC haplotype. We describe the isolation of its SCRC gene as well as the subsequent isolation of SCRC sequences from other SC-containing accessions and from the A. lyrata S36 haplotype, which is the functional equivalent of the A. thaliana SC haplotype. By performing transformation experiments using chimeric SRK and SCR genes constructed with SC- and S36-derived sequences, we show that the SRKC and SCRC genes of Lz-0 and at least a few other SC-containing accessions are nonfunctional, despite SCRC encoding a functional full-length protein. We identify the probable mutations that caused the inactivation of these genes and discuss our results in the context of mechanisms of S-locus inactivation in A. thaliana.