Discovery of induced point mutations in maize genes by TILLING

Background

Going from a gene sequence to its function in the context of a whole organism requires a strategy for targeting mutations, referred to as reverse genetics. Reverse genetics is highly desirable in the modern genomics era; however, the most powerful methods are generally restricted to a few model organisms. Previously, we introduced a reverse-genetic strategy with the potential for general applicability to organisms that lack well-developed genetic tools. Our TILLING (Targeting Induced Local Lesions IN Genomes) method uses chemical mutagenesis followed by screening for single-base changes to discover induced mutations that alter protein function. TILLING was shown to be an effective reverse genetic strategy by the establishment of a high-throughput TILLING facility and the delivery of thousands of point mutations in hundreds of Arabidopsis genes to members of the plant biology community.

Results

We demonstrate that high-throughput TILLING is applicable to maize, an important crop plant with a large genome but with limited reverse-genetic resources currently available. We screened pools of DNA samples for mutations in 1-kb segments from 11 different genes, obtaining 17 independent induced mutations from a population of 750 pollen-mutagenized maize plants. One of the genes targeted was the DMT102 chromomethylase gene, for which we obtained an allelic series of three missense mutations that are predicted to be strongly deleterious.

Conclusions

Our findings indicate that TILLING is a broadly applicable and efficient reverse-genetic strategy. We are establishing a public TILLING service for maize modeled on the existing Arabidopsis TILLING Project.     

Interactions of the ectodomain of HFE with the transferrin receptor are critical for iron homeostasis in cells

Expression of wild type HFE reduces the ferritin levels of cells in culture. In this report we demonstrate that the predominant hereditary hemochromatosis mutation, C282Y(2) HFE, does not reduce ferritin expression. However, the second mutation, H63D HFE, reduces ferritin expression to a level indistinguishable from cells expressing wild type HFE. Further, two HFE cytoplasmic domain mutations engineered to disrupt potential signal transduction, S335M and Y342C, were functionally indistinguishable from wild type HFE in this assay, as was soluble HFE. These results implicate a role for the interaction of HFE with the transferrin receptor in lowering cellular ferritin levels.     

Binding to the transferrin receptor is required for endocytosis of HFE and regulation of iron homeostasis

HFE, the protein that is mutated in hereditary haemochromatosis, binds to the transferrin receptor (TfR). Here we show that wild-type HFE and TfR localize in endosomes and at the basolateral membrane of a polarized duodenal epithelial cell line, whereas the primary haemochromatosis HFE mutant, and another mutant with impaired TfR-binding ability accumulate in the ER/Golgi and at the basolateral membrane, respectively. Levels of the iron-storage protein ferritin are greatly reduced and those of TfR are slightly increased in cells expressing wild-type HFE, but not in cells expressing either mutant. Addition of an endosomal-targeting sequence derived from the human low-density lipoprotein receptor (LDLR) to the TfR-binding-impaired mutant restores its endosomal localization but not ferritin reduction or TfR elevation. Thus, binding to TfR is required for transport of HFE to endosomes and regulation of intracellular iron homeostasis, but not for basolateral surface expression of HFE.     

Transferrin receptor 2: evidence for ligand-induced stabilization and redirection to a recycling pathway

Transferrin receptor 2 (TfR2) is a homologue of transferrin receptor 1 (TfR1), the protein that delivers iron to cells through receptor-mediated endocytosis of diferric transferrin (Fe(2)Tf). TfR2 also binds Fe(2)Tf, but it seems to function primarily in the regulation of systemic iron homeostasis. In contrast to TfR1, the trafficking of TfR2 within the cell has not been extensively characterized. Previously, we showed that Fe(2)Tf increases TfR2 stability, suggesting that trafficking of TfR2 may be regulated by interaction with its ligand. In the present study, therefore, we sought to identify the mode of TfR2 degradation, to characterize TfR2 trafficking, and to determine how Fe(2)Tf stabilizes TfR2. Stabilization of TfR2 by bafilomycin implies that TfR2 traffics to the lysosome for degradation. Confocal microscopy reveals that treatment of cells with Fe(2)Tf increases the fraction of TfR2 localizing to recycling endosomes and decreases the fraction of TfR2 localizing to late endosomes. Mutational analysis of TfR2 shows that the mutation G679A, which blocks TfR2 binding to Fe(2)Tf, increases the rate of receptor turnover and prevents stabilization by Fe(2)Tf, indicating a direct role of Fe(2)Tf in TfR2 stabilization. The mutation Y23A in the cytoplasmic domain of TfR2 inhibits its internalization and degradation, implicating YQRV as an endocytic motif.     

Identification of three novel mutations in the dihydropyrimidine dehydrogenase gene associated with altered pre-mRNA splicing or protein function

Dihydropyrimidine dehydrogenase (DPD) is the initial and rate-limiting enzyme in the catabolism of the pyrimidine bases uracil and thymine, as well as of the widely used chemotherapeutic drug 5-fluorouracil (5FU). Analysis of the DPD gene ( DPYD ) in two patients presenting with complete DPD deficiency and the parents of an affected child showed the presence of three novel mutations, including one splice site mutation IVS11 + 1G-->T and the missense mutations 731A-->C (E244V) and 1651G-->A (A551T). The G-->T mutation in the invariant GT splice donor site flanking exon 11 (IVS11 + 1G-->T) created a cryptic splice site within exon 11. As a consequence, a 141-bp fragment encoding the aminoacid residues 400-446 of the primary sequence of the DPD protein was missing in the mature DPD mRNA. Analysis of the crystal structure of pig DPD suggested that the E244V mutation might interfere with the electron flow between NADPH and the pyrimidine binding site of DPD. The A551T point mutation might prevent binding of the prosthetic group FMN and affect folding of the DPD protein. The identification of these novel mutations in DPYD will allow the identification of patients with an increased risk of developing severe 5FU-associated toxicity.     

Lipooligosaccharide-independent alteration of cellular homeostasis in Neisseria meningitidis-infected epithelial cells

Neisseria meningitidis (MC) is an important cause of meningitis and septic shock. Primary loose attachment of MC to host epithelial cells is mediated by type IV pili. Lipooligosaccharide (LOS), opacity (Opa) proteins and glycolipid adhesins facilitate subsequent tight attachment. MC infection causes numerous changes in host epithelial cell homeostasis. These include cortical plaque formation, increased expression of proinflammatory cytokines and alterations in host iron homeostasis. Using both biochemical and genetic approaches, we examined the role of LOS in mediating these events. We first examined specific cellular iron homeostasis changes that occur following addition of purified MC LOS to epithelial cells. Using an MC mutant that completely lacks LOS (MC lps tbp), we examined pili-mediated attachment and cortical plaque formation in human endocervical epithelial cells (A431). We also tested whether the lack of LOS alters cellular homeostasis, including changes in the levels of host stress response factors and proinflammatory cytokines. MC lps tbp elicited the formation of cortical plaques in A431 cells. However, the plaques were less pronounced than those formed by the MC parent. Surprisingly, the proinflammatory cytokine TNF(alpha) was upregulated during infection in MC lps tbp-infected cells. Furthermore, alterations in iron homeostasis, including lower transferrin receptor 1 (TfR-1) levels, altered TfR-1 trafficking, an 'iron-starvation' gene expression profile and low iron regulatory protein (IRP) binding activity are independent of LOS. Our results demonstrate that LOS is partially involved in both the attachment to host cells and formation of cortical plaques. However, TNFalpha induction and changes in iron homeostasis observed in MC-infected epithelial cells are independent of LOS.     

Maize endosperm ADP-glucose pyrophosphorylase SHRUNKEN2 and BRITTLE2 subunit interactions

ADP-glucose pyrophosphorylase (AGP) represents a key regulatory step in polysaccharide synthesis in organisms ranging from bacteria to plants. Higher plant AGPs are complex in nature and are heterotetramers consisting of two similar but distinct subunits. How the subunits are assembled into enzymatically active polymers is not yet understood. Here, we address this issue by using naturally occurring null mutants of the Shrunken2 (Sh2) and Brittle2 (Bt2) loci of maize as well as the yeast two-hybrid expression system. In the absence of the maize endosperm large AGP subunit (SH2), the BT2 subunit remains as a monomer in the developing endosperm. In contrast, the SH2 protein, in the absence of BT2, is found in a complex of 100 kD. A direct interaction between SH2 and BT2 was proven when they were both expressed in yeast. Several motifs are essential for SH2:BT2 interaction because truncations removing the N or C terminus of either subunit eliminate SH2:BT2 interactions. Analysis of subunit interaction mutants (sim) also identified motifs essential for protein interactions.     

Bereavement after sibling death: a population‐based longitudinal case‐control study

The objective of this study was to examine mental disorders and treatment use among bereaved siblings in the general population. Siblings (N=7243) of all deceased children in the population of Manitoba, Canada who died between 1984 and 2009 were matched 1:3 to control siblings (N=21,729) who did not have a sibling die in the study period. Generalized estimating equations were used to compare the two sibling groups in the two years before and after the index child's death on physician‐diagnosed mental disorders and treatment utilization, with adjustment for confounding factors including pre‐existing mental illness. Analyses were stratified by age of the bereaved (<13 vs. 13+). Results revealed that, in the two years after the death of the child, bereaved siblings had significantly higher rates of mental disorders than control siblings, even after adjusting for pre‐existing mental illness. When comparing the effect of a child's death on younger versus older siblings, the rise in depression rates from pre‐death to post‐death was significantly higher for siblings aged under 13 (p<0.0001), increasing more than 7‐fold (adjusted relative rate, ARR=7.25, 95% CI: 3.65‐14.43). Bereaved siblings aged 13+ had substantial morbidity in the two years after the death: 25% were diagnosed with a mental disorder (vs. 17% of controls), and they had higher rates of almost all mental disorder outcomes compared to controls, including twice the rate of suicide attempts (ARR=2.01, 95% CI: 1.29‐3.12). Siblings in the bereaved cohort had higher rates of alcohol and drug use disorders already before the death of their sibling. In conclusion, the death of a child is associated with considerable mental disorder burden among surviving siblings. Pre‐existing health problems and social disadvantage do not fully account for the increase in mental disorder rates.     

Changes in health indicator gaps between First Nations and other residents of Manitoba

Background:The Truth and Reconciliation Commission of Canada has called for better reporting of health disparities between First Nations people and other Canadians to close gaps in health outcomes. We sought to evaluate changes in these disparities using indicators of health and health care use over the last 2 decades.Methods:We used linked, whole-population, administrative claims data from the Manitoba Centre for Health Policy for fiscal years 1994/95 to 1998/99 and 2012/13 to 2016/17. We measured indicators of health and health care use among registered First Nations and all other Manitobans, and compared differences between these groups over the 2 time periods.Results:Over time, the relative gap between First Nations and all other Manitobans widened by 51% (95% confidence interval [CI] 42% to 60%) for premature mortality rate. For potential years of life lost, the gap widened by 54% (95% CI 51% to 57%) among women and by 32% (95% CI 30% to 35%) among men. The absolute gap in life expectancy widened by 3.14 years (95% CI 2.92 to 3.36) among men and 3.61 years (95% CI 3.38 to 3.84) among women. Relative gaps widened by 20% (95% CI 12% to 27%) for ambulatory specialist visits, by 14% (95% CI 12% to 16%) for hospital separations and by 50% (95% CI 39% to 62%) for days spent in hospital, but narrowed by 33% (95% CI −36% to −30%) for ambulatory primary care visits, by 22% (95% CI −27% to −16%) for mammography and by 27% (95% CI −40% to −23%) for injury hospitalizations.Interpretation:Disparities between First Nations and all other Manitobans in many key indicators of health and health care use have grown larger over time. New approaches are needed to address these disparities and promote better health with and for First Nations.

Comprehensive data on the health of First Nations people in Canada are urgently needed.^1,2 This is a priority identified by the Truth and Reconciliation Commission of Canada, which highlights the striking health disparities between Indigenous and non-Indigenous populations in Canada and recognizes the disparities in their Calls to Action as “a direct result of previous Canadian government policies.”³ These health disparities are understood to be part of the continuing impact of colonization and genocidal policies aimed specifically at Indigenous people.^4,5The underlying causes of the health gap between Indigenous and non-Indigenous people, resulting from attempts by European settlers to assimilate Indigenous people into their own societies, have been detailed elsewhere.⁶ The collective trauma that Indigenous populations have experienced through colonial policies and practices include, among others, racism and marginalization in virtually every aspect of their lives; major disruptions of families and communities through forced attendance at residential schools and by the child welfare system; trauma from physical, emotional and sexual abuse, carried over into future generations; and damage to their Indigenous identity through loss of culture, language, traditions and teachings.^6,7 Other social determinants (e.g., poverty, social exclusion and poor access to clean water, quality housing, education and heath services) and the governments’ failure to address these issues also influence the health of First Nations people.^8,9A previous population-based study of First Nations’ health and health care use in Canada, published in 2002, focused on the Manitoba population, which has the highest proportion of First Nations residents among Canadian provinces.¹⁰ It identified substantial gaps in the health of First Nations and their access to health services compared with other Manitobans. Others have since reported on health disparities between Indigenous peoples and other Canadians;^11–14 however, the Manitoba data provide a unique opportunity to compare changes in health-related outcomes over nearly 2 decades. In the present study, we sought to compare indicators of health and health care use between First Nations people and other residents of Manitoba, to determine whether the gap between the 2 groups has changed over the past 18 years and establish whether any progress has been made in improving First Nations’ health.