Map-based analysis of genes affecting the brittle rachis character in tetraploid wheat (Triticum turgidum L.)

The mature spike rachis of wild emmer [Triticum turgidum L. ssp. dicoccoides (Körn. ex Asch. and Graebner) Thell.] disarticulates spontaneously between each spikelet leading to the dispersion of wedge-type diaspores. By contrast, the spike rachis of domesticated emmer (Triticum turgidum L. ssp. turgidum) fails to disarticulate and remains intact until it is harvested. This major distinguishing feature between wild and domesticated emmer is controlled by two major genes, brittle rachis 2 (Br-A2) and brittle rachis 3 (Br-A3) on the short arms of chromosomes 3A and 3B, respectively. Because of their biological and agricultural importance, a map-based analysis of these genes was undertaken. Using two recombinant inbred chromosome line (RICL) populations, Br-A2, on chromosome 3A, was localized to a ~11-cM region between Xgwm2 and a cluster of linked loci (Xgwm666.1, Xbarc19, Xcfa2164, Xbarc356, and Xgwm674), whereas Br-A3, on chromosome 3B, was localized to a ~24-cM interval between Xbarc218 and Xwmc777. Comparative mapping analyses suggested that both Br-A2 and Br-A3 were present in homoeologous regions on chromosomes 3A and 3B, respectively. Furthermore, Br-A2 and Br-A3 from wheat and Btr1/Btr2 on chromosome 3H of barley (Hordeum vulgare L.) also were homoeologous suggesting that the location of major determinants of the brittle rachis trait in these species has been conserved. On the other hand, brittle rachis loci of wheat and barley, and a shattering locus on rice chromosome 1 did not appear to be orthologous. Linkage and deletion-based bin mapping comparisons suggested that Br-A2 and Br-A3 may reside in chromosomal areas where the estimated frequency of recombination was ~ 4.3 Mb/cM. These estimates indicated that the cloning of Br-A2 and Br-A3 using map-based methods would be extremely challenging.     

Natural underlying mtDNA heteroplasmy as a potential source of intra‐person hiPSC variability

Functional variability among human clones of induced pluripotent stem cells (hiPSCs) remains a limitation in assembling high‐quality biorepositories. Beyond inter‐person variability, the root cause of intra‐person variability remains unknown. Mitochondria guide the required transition from oxidative to glycolytic metabolism in nuclear reprogramming. Moreover, mitochondria have their own genome (mitochondrial DNA [mtDNA]). Herein, we performed mtDNA next‐generation sequencing (NGS) on 84 hiPSC clones derived from a cohort of 19 individuals, including mitochondrial and non‐mitochondrial patients. The analysis of mtDNA variants showed that low levels of potentially pathogenic mutations in the original fibroblasts are revealed through nuclear reprogramming, generating mutant hiPSCs with a detrimental effect in their differentiated progeny. Specifically, hiPSC‐derived cardiomyocytes with expanded mtDNA mutations non‐related with any described human disease, showed impaired mitochondrial respiration, being a potential cause of intra‐person hiPSC variability. We propose mtDNA NGS as a new selection criterion to ensure hiPSC quality for drug discovery and regenerative medicine.     

Genome‐wide SNP identification in Fraxinus linking genetic characteristics to tolerance of Agrilus planipennis

Ash (Fraxinus spp.) is one of the most widely distributed tree genera in North America. Populations of ash in the United States and Canada have been decimated by the introduced pest Agrilus planipennis (Coleoptera: Buprestidae; emerald ash borer), having negative impacts on both forest ecosystems and economic interests. The majority of trees succumb to attack by A. planipennis, but some trees have been found to be tolerant to infestation despite years of exposure. Restriction site‐associated DNA (RAD) sequencing was used to sequence ash individuals, both tolerant and susceptible to A. planipennis attack, in order to identify single nucleotide polymorphism (SNP) patterns related to tolerance and health declines. de novo SNPs were called using SAMtools and, after filtering criteria were implemented, a set of 17,807 SNPs were generated. Principal component analysis (PCA) of SNPs aligned individual trees into clusters related to geography; however, five tolerant trees clustered together despite geographic location. A subset of 32 outlier SNPs identified within this group, as well as a subset of 17 SNPs identified based on vigor rating, are potential candidates for the selection of host tolerance. Understanding the mechanisms of host tolerance through genome‐wide association has the potential to restore populations with cultivars that are able to withstand A. planipennis infestation. This study was successful in using RAD‐sequencing in order to identify SNPs that could contribute to tolerance of A. planipennis. This was a first step toward uncovering the genetic basis for host tolerance to A. planipennis. Future studies are needed to identify the functionality of the loci where these SNPs occur and how they may be related to tolerance of A. planipennis attack.     

High‐affinity cooperative Ca2+ binding by MICU1–MICU2 serves as an on–off switch for the uniporter

The mitochondrial calcium uniporter is a Ca²⁺‐activated Ca²⁺ channel that is essential for dynamic modulation of mitochondrial function in response to cellular Ca²⁺ signals. It is regulated by two paralogous EF‐hand proteins—MICU1 and MICU2, but the mechanism is unknown. Here, we demonstrate that both MICU1 and MICU2 are stabilized by Ca²⁺. We reconstitute the MICU1–MICU2 heterodimer and demonstrate that it binds Ca²⁺ cooperatively with high affinity. We discover that both MICU1 and MICU2 exhibit affinity for the mitochondria‐specific lipid cardiolipin. We determine the minimum Ca²⁺ concentration required for disinhibition of the uniporter in permeabilized cells and report a close match with the Ca²⁺‐binding affinity of MICU1–MICU2. We conclude that cooperative, high‐affinity interaction of the MICU1–MICU2 complex with Ca²⁺ serves as an on–off switch, leading to a tightly controlled channel, capable of responding directly to cytosolic Ca²⁺ signals.     

Homologous protein subunits from Escherichia coli NADH:quinone oxidoreductase can functionally replace MrpA and MrpD in Bacillus subtilis

The complex I subunits NuoL, NuoM and NuoN are homologous to two proteins, MrpA and MrpD, from one particular class of Na+/H+ antiporters. In many bacteria MrpA and MrpD are encoded by an operon comprising 6-7 conserved genes. In complex I these protein subunits are prime candidates for harboring important parts of the proton pumping machinery. Deletion of either mrpA or mrpD from the Bacillus subtilis chromosome resulted in a Na+ and pH sensitive growth phenotype. The deletion strains could be complemented in trans by their respective Mrp protein, but expression of MrpA in the B. subtilis ΔmrpD strain and vice versa did not improve growth at pH 7.4. This corroborates that the two proteins have unique specific functions. Under the same conditions NuoL could rescue B. subtilis ΔmrpA, but improved the growth of B. subtilis ΔmrpD only slightly. NuoN could restore the wild type properties of B. subtilis ΔmrpD, but had no effect on the ΔmrpA strain. Expression of NuoM did not result in any growth improvement under these conditions. This reveals that the complex I subunits NuoL, NuoM and NuoN also demonstrate functional specializations. The simplest explanation that accounts for all previous and current observations is that the five homologous proteins are single ion transporters. Presumably, MrpA transports Na+ whereas MrpD transports H+ in opposite directions, resulting in antiporter activity. This hypothesis has implications for the complex I functional mechanism, suggesting that one Na+ channel, NuoL, and two H+ channels, NuoM and NuoN, are present.     

Def1 Promotes the Degradation of Pol3 for Polymerase Exchange to Occur During DNA-Damage–Induced Mutagenesis in Saccharomyces cerevisiae

DNA damages hinder the advance of replication forks because of the inability of the replicative polymerases to synthesize across most DNA lesions. Because stalled replication forks are prone to undergo DNA breakage and recombination that can lead to chromosomal rearrangements and cell death, cells possess different mechanisms to ensure the continuity of replication on damaged templates. Specialized, translesion synthesis (TLS) polymerases can take over synthesis at DNA damage sites. TLS polymerases synthesize DNA with a high error rate and are responsible for damage-induced mutagenesis, so their activity must be strictly regulated. However, the mechanism that allows their replacement of the replicative polymerase is unknown. Here, using protein complex purification and yeast genetic tools, we identify Def1 as a key factor for damage-induced mutagenesis in yeast. In in vivo experiments we demonstrate that upon DNA damage, Def1 promotes the ubiquitylation and subsequent proteasomal degradation of Pol3, the catalytic subunit of the replicative polymerase δ, whereas Pol31 and Pol32, the other two subunits of polymerase δ, are not affected. We also show that purified Pol31 and Pol32 can form a complex with the TLS polymerase Rev1. Our results imply that TLS polymerases carry out DNA lesion bypass only after the Def1-assisted removal of Pol3 from the stalled replication fork.     

MICU1 and MICU2 play nonredundant roles in the regulation of the mitochondrial calcium uniporter

The mitochondrial uniporter is a selective Ca²⁺ channel regulated by MICU1, an EF hand‐containing protein in the organelle's intermembrane space. MICU1 physically associates with and is co‐expressed with a paralog, MICU2. To clarify the function of MICU1 and its relationship to MICU2, we used gene knockout (KO) technology. We report that HEK‐293T cells lacking MICU1 or MICU2 lose a normal threshold for Ca²⁺ intake, extending the known gating function of MICU1 to MICU2. Expression of MICU1 or MICU2 mutants lacking functional Ca²⁺‐binding sites leads to a striking loss of Ca²⁺ uptake, suggesting that MICU1/2 disinhibit the channel in response to a threshold rise in [Ca²⁺]. MICU2's activity and physical association with the pore require the presence of MICU1, though the converse is not true. We conclude that MICU1 and MICU2 are nonredundant and together set the [Ca²⁺] threshold for uniporter activity.     

Biochemical Characterization of Pathogenic Mutations in Human Mitochondrial Methionyl-tRNA Formyltransferase

N-Formylation of initiator methionyl-tRNA (Met-tRNA^Met) by methionyl-tRNA formyltransferase (MTF) is important for translation initiation in bacteria, mitochondria, and chloroplasts. Unlike all other translation systems, the metazoan mitochondrial system is unique in using a single methionine tRNA (tRNA^Met) for both initiation and elongation. A portion of Met-tRNA^Met is formylated for initiation, whereas the remainder is used for elongation. Recently, we showed that compound heterozygous mutations within the nuclear gene encoding human mitochondrial MTF (mt-MTF) significantly reduced mitochondrial translation efficiency, leading to combined oxidative phosphorylation deficiency and Leigh syndrome in two unrelated patients. Patient P1 has a stop codon mutation in one of the MTF genes and an S209L mutation in the other MTF gene. P2 has a S125L mutation in one of the MTF genes and the same S209L mutation as P1 in the other MTF gene. Here, we have investigated the effect of mutations at Ser-125 and Ser-209 on activities of human mt-MTF and of the corresponding mutations, Ala-89 or Ala-172, respectively, on activities of Escherichia coli MTF. The S125L mutant has 653-fold lower activity, whereas the S209L mutant has 36-fold lower activity. Thus, both patients depend upon residual activity of the S209L mutant to support low levels of mitochondrial protein synthesis. We discuss the implications of these and other results for whether the effect of the S209L mutation on mitochondrial translational efficiency is due to reduced activity of the mutant mt-MTF and/or reduced levels of the mutant mt-MTF.