Allele | bingo | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Mutation Type | missense | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Chromosome | 15 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Coordinate | 78,449,168 bp (GRCm39) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Base Change | T ⇒ C (forward strand) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gene | Rac2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gene Name | Rac family small GTPase 2 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Chromosomal Location | 78,443,369-78,456,983 bp (-) (GRCm39) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MGI Phenotype |
FUNCTION: [Summary is not available for the mouse gene. This summary is for the human ortholog.] This gene encodes a member of the Ras superfamily of small guanosine triphosphate (GTP)-metabolizing proteins. The encoded protein localizes to the plasma membrane, where it regulates diverse processes, such as secretion, phagocytosis, and cell polarization. Activity of this protein is also involved in the generation of reactive oxygen species. Mutations in this gene are associated with neutrophil immunodeficiency syndrome. There is a pseudogene for this gene on chromosome 6. [provided by RefSeq, Jul 2013] PHENOTYPE: Homozygotes for a targeted null mutation exhibit peripheral blood lymphocytosis, reductions in peritoneal B-1a lymphocytes, marginal zone lymphocytes, and IgM-secreting plasma cells, decreased levels of serum IgM and IgA, and abnormal T cell migration. [provided by MGI curators] |
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Accession Number | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Mapped | Yes | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Amino Acid Change | Threonine changed to Alanine | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Institutional Source | Beutler Lab | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Gene Model | predicted gene model for protein(s): [ENSMUSP00000036384] [ENSMUSP00000154826] | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
AlphaFold | Q05144 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
SMART Domains |
Protein: ENSMUSP00000036384 Gene: ENSMUSG00000033220 AA Change: T115A
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Predicted Effect | probably damaging
PolyPhen 2 Score 1.000 (Sensitivity: 0.00; Specificity: 1.00) |
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Predicted Effect | probably benign | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Predicted Effect | unknown | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Meta Mutation Damage Score | 0.9533 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Is this an essential gene? | Non Essential (E-score: 0.000) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Phenotypic Category | Autosomal Recessive | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Candidate Explorer Status | loading ... | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Single pedigree Linkage Analysis Data |
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Penetrance | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Alleles Listed at MGI | All mutations/alleles(4) : Gene trapped(2) Radiation induced(1) Targeted(1) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Lab Alleles |
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Mode of Inheritance | Autosomal Recessive | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Local Stock | Live Mice | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
MMRRC Submission | 038186-MU | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Last Updated | 2019-09-04 9:48 PM by Anne Murray | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Record Created | 2014-06-16 10:33 PM by Kuan-Wen Wang | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Record Posted | 2015-04-20 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Phenotypic Description |
The bingo phenotype was identified among G3 mice of the pedigree R0627, some of which showed a diminished T-independent antibody response to 4-hydroxy-3-nitrophenylacetyl-Ficoll (NP-Ficoll; Figure 1). |
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Nature of Mutation |
Whole exome HiSeq sequencing of the G1 grandsire identified 105 mutations. The diminished T-independent antibody response to NP-Ficoll was linked by continuous variable mapping to a mutation in Rac2: an A to G transition at base pair 78,564,968 (v38) on chromosome 15, or base pair 7,816 in the GenBank genomic region NC_000081 encoding Rac2. Linkage was found with a recessive model of inheritance, wherein 7 variant homozygotes departed phenotypically from 10 homozygous reference mice and 19 heterozygous mice with a P value of 9.038 x 10-6 (Figure 2). The mutation corresponds to residue 481 in the mRNA sequence NM_009008 within exon 5 of 7 total exons.
The mutated nucleotide is indicated in red. The mutation results in a threonine (T) to alanine (A) substitution at position 115 (T115A) in the Rac2 (Ras-related C3 botulinum toxin substrate 2) protein, and is strongly predicted by Polyphen-2 to cause loss of function (probably damaging; score = 1.00) (1). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Illustration of Mutations in Gene & Protein |
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Protein Prediction |
Rac2 is a member of the Rac subfamily of Rho guanosine triphosphatases (Rho GTPases). Rho GTPases have several conserved domains including five GTP binding and hydrolysis domains (G-boxes; G1-G5), two switch regions (switch I and II), a polybasic domain, and a prenylation site [Figure 3; (2)]. G-boxes function in GDP binding and exhibit GTPase activity (3). In Rac2, these regions correspond to amino acids 10-17 (G1), Thr35 (G2), 57-61 (G3), and 115-118 (G4), and 157-160 (G5). The Rac proteins each have two highly conserved switch regions, switch I (amino acids 27-40) and switch II (amino acids 56-71), situated on either side of the bound nucleotide [Figure 3 and 4; PDB:2W2X; (4)]. Both switch regions are sites of interactions between the Rac proteins and guanine nucleotide exchange factors (GEFs) and guanine nucleotide-dissociation inhibitors (GDIs) as well as with downstream protein targets (5). The main difference between the Rac family members is at the C-terminal polybasic tail. Rac1 has a stretch of basic amino acids, while Rac2 has several nonbasic residues that interrupt the region. The polybasic region of Rac2 (RQQKRP; amino acids 183-188) is required for its function as a regulator of NAPDH oxidase (see the Background section) (5;6). In addition, the polybasic domains of Rac1 and Rac2 regulate binding to effector proteins such as PAK1, phosphatidylinositol 5-kinase, and the adaptor Crk (7;8). Lipid prenylation at the C-terminus (Cys-A/S-L-L-COOH) and the polybasic domain regulate the localization of Rho GTPases to the membrane and facilitate protein-protein interactions (5;6;9;10). After prenylation, the C-terminal tripeptide (A/SLL) is preotylically removed and the new C-terminus is methylated (6;11). The bingo mutation results in a threonine (T) to alanine (A) substitution at position 115 (T115A) within the G4 G-box. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Expression/Localization | Rac2 is expressed solely in hematopoietic cells (9;12;13). Rac2 is localized to the cytoplasm and perinuclear pool, but upon cell activation, Rac2 is translocated to the plasma membrane (6). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Background |
The Rho-like GTPases comprise several proteins including Rho, Rac1/2/3, Cdc42, RhoD, RhoG, RhoE, and TC10 [reviewed in (14)]. The Rho GTPases integrate receptor-mediated signals through binding to effectors and regulators of the actin cytoskeleton and affect multiple cellular activities including cell morphology, polarity, migration, proliferation, apoptosis, phagocytosis, cytokinesis, adhesion, vesicular transport, and transcription. The Rho GTPases are active when bound to GTP and are inactive in their GDP-bound form [Figure 5; reviewed in (15;16)]. Regulation of Rho GTPase activity is complex and involves GEFs that promote the exchange of GDP for GTP, GTPase-activating proteins (GAPs) that enhance the GTPase activity of Rho proteins, and GDIs that sequester Rho GTPases in a GDP-bound state (17). Rac2 (and the other Rac proteins) function in actin polymerization resulting in lamellopodial extension and membrane ruffling, directed migration, chemotaxis, and superoxide (O2−) production in phagocytic cells as well as cytoskeleton organization in red blood cells and osteoclasts [(18-23)]. The Rac proteins regulate leukocyte migration by transducing signals from cell surface receptors (e.g., the Fcγ receptor, formylmethionyl-leucyl-phenylalanine (fMLP) receptor, and β2 integrins) to the actin and microtubule cytoskeletons through cytoplasmic effectors (e.g., tyrosine kinases, scaffolding/adapter proteins, nucleotide exchange proteins, and phosphatases) upon binding of GTP (24). Rac2 dually regulates phospholipase D (PLD) activity (25). PLD is an enzyme at the plasma membrane that catalyzes the hydrolysis of phosphatidylcholine to phosphatidic acid, which regulates cytoskeleton dynamics, calcium mobilization, secretion, superoxide production, endocytosis, exocytosis, vesicle trafficking, glucose transport, mitogenesis, and cell survival (26). Rac2-mediated inhibitory responses on PLD can be reversed in the presence of phosphatidylinositol 4,5-bisphosphate (PIP2), indicating a connection between Rac2-PIP2-PLD that regulates cell migration (25). Rac2-associated cell type-specific functions are described in more detail, below.
Neutrophils In neutrophils, Rac2 is essential for F-actin polymerization, L-selectin (see the record for dim_sum)-mediated adhesion, primary granule release of the mediators myeloperoxidase and elastase (see the record for Ruo) in response to cytochalasin B/fMLP (CB/fMLP) and CB/leukotriene B4 (CB/LTB4), the formation of neutrophil extracellular traps (NETs) that bind invading pathogens through the regulation of nitric oxide and reactive oxygen species, chemotaxis in response to fMLP, complement C5a, or LTB4, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase function [Figure 6; (21;27-29)]. NADPH oxidase is a multiprotein phagocytic oxidase complex that mediates the production of reactive oxygen species in response to growth factors or inflammatory cytokines. The NADPH is assembled from a membrane-spanning flavo-cytochrome b558 (cyt b; composed of both gp91phox and p22phox) and four cytosolic factors (p47phox, p67phox, p40phox, and Rac2). The cytosolic factors translocate to the cyt b to generate an active enzyme. Upon neutrophil activation, Rac2 is activated by a membrane-associated GEF and subsequently assembles into a membrane-localized NADPH oxidase through a mechanism coordinated with the translocation of the p47phox–p67phox complex (30;31). Rac2 acts independently of p67phox to regulate initial transfer of electrons from NADPH to the to cyt b-associated flavin adenine dinucleotide (FAD). However, Rac2 binding to p67phox is necessary for the subsequent electron transfer from FAD to molecular oxygen to form superoxide [(32); reviewed in (33)]. Rac2 -/- mice have delayed wound closure to defects in Rac2-dependent NAPDH oxidase activity during wound healing (34). Macrophages Rac2 is a component of the myeloid α4β1- and αv-directed signalosome in macrophages through a myeloid-specific association with the nonreceptor protein tyrosine kinase Syk (see the record for poppy) (35). Rac2-deficient macrophages also exhibit a reduction in superoxide production and phagocytosis after phorbol ester (PMA), fMLP, or FcγR stimulation (36). Yamauchi et al. found that the cell morphology and actin responses in the Rac2-deficient macrophages was similar to those in wild-type macrophages (36). In contrast, Wheeler et al. determined that loss of Rac2 expression resulted in reduced levels of polymerized actin and podosome formation (24). Loss of Rac2 expression had little effect on macrophage migration speed (24). Mast cells Rac2 is required for the migration, degranulation, integrin-mediated adhesion to fibronectin, and growth-dependent survival of mast cells (22). The deficiency of mast cell growth upon loss of Rac2 expression correlated with increased apoptosis after growth factor simulation and a concomitant reduction in Akt activation (22). Also, loss of Rac2 expression resulted in increased expression of the proapoptotic protein BAD and decreased expression of the antiapoptotic protein Bcl-XL (22). In mast cells, Rac2 is required for stem cell factor-induced activation of JNKs and the c-Jun pathway to regulate the expression of 38 genes including those of several mast cell proteases [e.g., mouse mast cell protease 7 (MMCP-7)] (37).
T cells Rac1 and Rac2 have redundant roles at several stages of T cell development through the regulation of survival and proliferation signals (38). Rac1 and Rac2 are both required for optimum T cell activation downstream of the T cell receptor and costimulatory proteins CD28 and CD5 (39-41). Antisense-mediated knockdown of Rac2 in Jurkat cells results in reduced actin polymerization triggered by L-selectin (42). Rac1 and Rac2 mediate the initial interaction between mature dendritic cells and naïve T cells by controlling the formation of dendrites from the mature dendritic cells, controlling the polarized short-range migration of dendritic cells toward T cells, and T cell priming (43). Rac2 is essential for T cell activation in response to anti-CD3 and T cell receptor-specific antigens; costimulation with anti-CD28 or the addition of IL-2 partially compensates for the defects observed [Figure 7; (44)]. Rac2 is a substrate of Vav GEF activity and is proposed to relay Vav signaling during T cell activation (44). Rac2-/- T cells exhibited reduced actin polymerization upon TCR cross-linking or antigen stimulation as well as reduced phosphorylation of ERK1/2 and p38. Calcium flux was also reduced upon antigen stimulation (44). Rac2 is required for normal IFN-γ production by activated CD4+ T Helper 1 (TH1) cells through the activation of the NF-κB and p38 pathways (45). In Rac2-/- mice, there is a 90% increase in peripheral blood CD4+ and CD8+ T-lymphocyte numbers compared to wild-type mice (46). The proportions and numbers of double-negative (CD4-CD8-), double-positive (CD4+CD8+), and single-positive (CD4+CD8-, CD4-CD8+) T lymphocytes in the thymus were similar between wild-type and Rac2-/- mice (46). In the spleen, CD8+ and CD4+ T-lymphocyte numbers were increased in the Rac2 -/- mice compared to wild-type mice (46). Lymph node T lymphocyte (Thy1+, CD4+, CD8+) chemotaxis was reduced in Rac2-/- mice (46). In addition, filamentous actin generation in T lymphocytes in response to chemoattractants was reduced in the Rac2 -/- mice compared to wild-type mice (46). B cells Rac2 is required for B cell development as well as for either B cell receptor (BCR) signal transduction and subsequent calcium mobilization or in determining the efficiency of BCR ligation (47;48). Rac2 is required for efficient signaling after the co-ligation of CD19 and BCR. Rac2-deficient mice exhibit a 30% reduction in B cell numbers due mainly be a reduced number of recirculating B lymphocytes in the bone marrow (47). In the peripheral blood, Rac2-/- mice had an increase in total leukocyte number including both B and T cells (47). B cell numbers were reduced in the spleen due to a loss of mature and/or marginal zone B cells (47). The levels of IgG1 and IgG2b were increased in the serum of Rac2-/- mice, while the levels of serum IgM and IgA were reduced (47). Rac2-/- lymphocytes were able to class switch to IgG1 in response to a T-dependent stimulus. After immunization with the T-dependent antigen DNP-dextran, TNP-specific IgM was reduced in the Rac2-/- mice compared to wild-type mice; the production of TNP-specific IgG3 was comparable between the Rac2-/- and wild-type mice. Dendritic cells Loss of Rac2 expression in CD8+ DC cells resulted in loss of phagosomal ROS production and reduced efficiency of antigen cross-presentation to CD8+ T cells to subsequently initiate cytotoxic immune responses (49). The phenotype of the Rac2-deficient CD8+ DC cells resembled that of CD8- DC cells in that they had reduced ROS production and increased acidification. Osteoclasts Rac1 and Rac2 have overlapping functions in the regulation of preosteoclast motility (50;51). Combined deletion of both Rac1 and Rac2 results in arrested bone resorption and severe osteopetrosis through the dysregulation of the osteoclast cytoskeleton (50). In mature osteoclasts, Rac2 has a nonredundant function in chemotaxis, resorptive activity, integrin-mediated actin remodeling, and motility (20). Osteoclasts and preosteoclasts from Rac2 -/- mice exhibited a reduced rate of bone resorption and reduced chemotaxis in response to colony stimulating factor 1 (CSF1) (20;51). In addition, male Rac2 -/- mice have increased trabecular bone mass when compared to wild-type mice (20;51). Endothelial cells Rac2 is required for avβ3, α4β1 and α5β1 integrin-associated migration and for the control of angiogenesis (52). During endothelial cell migration via the avβ3 and α4β1 integrins, Syk mediates the activation of Rac2 (52). Rac2 is also required for the aortic ring endothelial outgrowth response and neovascularization of the hindlimb after ischemic injury (52). Human conditions Mutations in RAC2 are linked to neutrophil (alternatively, phagocytic) immunodeficiency syndrome [NIS; OMIM: #608203; (53-55)] and decreased numbers of peripheral T and B cells. Patients with NIS have severe, recurrent infections, poor wound healing, and exhibit reduced neutrophil migration, azurophilic granule secretion, and superoxide production (53-55). | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Putative Mechanism | Rac2-/- mice exhibit a normal frequency of peripheral T cells, but exhibit leukocytosis due to an increased frequency of mature neutrophils in the peripheral blood (21). Rac2 -/- neutrophils (21), B cells (47) and T cells (46) show reduced migration and F-actin polymerization in response to chemokines, although chemotaxis is not completely abolished. Consistent with these observations, recruitment of Rac2-/- neutrophils to sites of inflammation is impaired. The incomplete block in chemotaxis most likely reflects the continued presence of Rac1 in these cell types. Rac2-/- mice also display a lack of peritoneal B-1 and MZB cells, as well as abnormal T-independent and T-dependent antibody responses (47). The reduction in T-independent antibody responses observed in the bingo mice indicates loss of Rac2 function. A change in neutrophil frequency in the peripheral blood was not observed, indicating that some Rac2 function remains or that Rac1 may be compensating for the loss of Rac2 function and/or expression. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Primers |
PCR Primer bingo_pcr_F: GGACCAAGTCATTTTACCCAGGCAC bingo_pcr_R: AGGGTCACTAGTACCACACAGGAC Sequencing Primer bingo_seq_F: tgacagagaaacacaggcatag bingo_seq_R: ACAGTGAGGGTTCCTCCTG |
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Genotyping | PCR program 1) 94°C 2:00 The following sequence of 731 nucleotides is amplified (chromosome 15, - strand): 1 agggtcacta gtaccacaca ggaccaatga gaggagagtc tccttgggct gtgtgtgtgt Primer binding sites are underlined and the sequencing primers are highlighted; the mutated nucleotide is shown in red. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
References |
15. Jaffe, A. B., and Hall, A. (2005) Rho GTPases: Biochemistry and Biology. Annu Rev Cell Dev Biol. 21, 247-269.
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Science Writers | Anne Murray | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Illustrators | Peter Jurek, Katherine Timer | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Authors | Bruce Beutler, Jin Huk Choi, Kuan-Wen Wang, Ming Zeng |