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BACKGROUND: Pig organ xenotransplantation is a potential solution for the severe organ shortage in clinic, while immunogenic genes need to be eliminated to improve the immune compatibility between humans and pigs. Current knockout strategies are mainly aimed at the genes causing hyperacute immune rejection (HAR) that occurs in the first few hours while adaptive immune reactions orchestrated by CD4 T cell thereafter also cause graft failure, in which process the MHC II molecule plays critical roles. METHODS: Thus, we generate a 4-gene (GGTA1, CMAH, ß4GalNT2, and CIITA) knockout pig by CRISPR/Cas9 and somatic cell nuclear transfer to compromise HAR and CD4 T cell reactions simultaneously. RESULTS: We successfully obtained 4KO piglets with deficiency in all alleles of genes, and at cellular and tissue levels. Additionally, the safety of our animals after gene editing was verified by using whole-genome sequencing and karyotyping. Piglets have survived for more than one year in the barrier, and also survived for more than 3 months in the conventional environment, suggesting that the piglets without MHC II can be raised in the barrier and then gradually mated in the conventional environment. CONCLUSIONS: 4KO piglets have lower immunogenicity, are safe in genomic level, and are easier to breed than the model with both MHC I and II deletion.
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The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated gene (Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12iMax, a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12iMax in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163, and MSTN via Cas12iMax in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12iMax for gene editing in livestock animals and demonstrated the potential application of Cas12iMax in the field of animal trait improvement for agricultural production.
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Sistemas CRISPR-Cas , Ganado , Animales , Bovinos , Porcinos , Ganado/genética , Edición Génica/métodos , Fenotipo , ADNRESUMEN
A novel strain with heterotrophic nitrification and aerobic denitrification was screened and identified as Klebsiella sp. TSH15 by 16S rRNA. The results demonstrated that the ammonia-N and nitrate-N removal rates were 2.99 mg/L/h and 2.53 mg/L/h under optimal conditions, respectively. The analysis of the whole genome indicated that strain TSH15 contained the key genes involved in assimilatory/dissimilatory nitrate reduction and ammonia assimilation, including nas, nar, nir, nor, glnA, gltB, gdhA, and amt. The relative expression levels of key nitrogen removal genes were further detected by RT-qPCR. The results indicated that the N metabolic pathways of strain TSH15 were the conversion of nitrate or nitrite to ammonia by assimilatory/dissimilatory nitrate reduction (NO3-âNO2-âNH4+) and further conversion of ammonia to glutamate (NH4+-N â Glutamate) by ammonia assimilation. These results indicated that the strain TSH15 had the potential to be applied to practical sewage treatment in the future.
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Amoníaco , Desnitrificación , Amoníaco/metabolismo , Nitratos/metabolismo , Klebsiella/genética , Klebsiella/metabolismo , Nitrógeno/metabolismo , ARN Ribosómico 16S , Aerobiosis , Nitrificación , Nitritos/metabolismo , Procesos Heterotróficos , Glutamatos/metabolismoRESUMEN
Understandings of the three-dimensional social behaviors of freely moving large-size mammals are valuable for both agriculture and life science, yet challenging due to occlusions in close interactions. Although existing animal pose estimation methods captured keypoint trajectories, they ignored deformable surfaces which contained geometric information essential for social interaction prediction and for dealing with the occlusions. In this study, we develop a Multi-Animal Mesh Model Alignment (MAMMAL) system based on an articulated surface mesh model. Our self-designed MAMMAL algorithms automatically enable us to align multi-view images into our mesh model and to capture 3D surface motions of multiple animals, which display better performance upon severe occlusions compared to traditional triangulation and allow complex social analysis. By utilizing MAMMAL, we are able to quantitatively analyze the locomotion, postures, animal-scene interactions, social interactions, as well as detailed tail motions of pigs. Furthermore, experiments on mouse and Beagle dogs demonstrate the generalizability of MAMMAL across different environments and mammal species.
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Imagenología Tridimensional , Captura de Movimiento , Animales , Porcinos , Ratones , Perros , Imagenología Tridimensional/métodos , Postura , Algoritmos , Movimiento (Física) , MamíferosRESUMEN
BACKGROUND: Mice with humanized livers are important models to study drug toxicology testing, development of hepatitis virus treatments, and hepatocyte transplantation therapy. However, the huge difference between mouse and human in size and anatomy limited the application of humanized mice in investigating human diseases. Therefore, it is urgent to construct humanized livers in pigs to precisely investigate hepatocyte regeneration and human hepatocyte therapy. CRISPR/Cas9 system and somatic cell cloning technology were used to generate two pig models with FAH deficiency and exhibiting severe immunodeficiency (FAH/RAG1 and FAH/RAG1/IL2RG deficiency). Human primary hepatocytes were then successfully transplanted into the FG pig model and constructed two pigs with human liver. RESULTS: The constructed FAH/RAG1/IL2RG triple-knockout pig models were characterized by chronic liver injury and severe immunodeficiency. Importantly, the FG pigs transplanted with primary human hepatocytes produced human albumin in a time dependent manner as early as 1 week after transplantation. Furthermore, the colonization of human hepatocytes was confirmed by immunochemistry staining. CONCLUSIONS: We successfully generated pig models with severe immunodeficiency that could construct human liver tissues.
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Development of thoracolumbar vertebra (TLV) and rib primordium (RP) is a common evolutionary feature across vertebrates, although whole-organism analysis of the expression dynamics of TLV- and RP-related genes has been lacking. Here, we investigated the single-cell transcriptome landscape of thoracic vertebra (TV), lumbar vertebra (LV), and RP cells from a pig embryo at 27â¯days post-fertilization (dpf) and identified six cell types with distinct gene expression signatures. In-depth dissection of the gene expression dynamics and RNA velocity revealed a coupled process of osteogenesis and angiogenesis during TLV and RP development. Further analysis of cell type-specific and strand-specific expression uncovered the extremely high level of HOXA10 3'-UTR sequence specific to osteoblasts of LV cells, which may function as anti-HOXA10-antisense by counteracting the HOXA10-antisense effect to determine TLV transition. Thus, this work provides a valuable resource for understanding embryonic osteogenesis and angiogenesis underlying vertebrate TLV and RP development at the cell type-specific resolution, which serves as a comprehensive view on the transcriptional profile of animal embryo development.
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Columna Vertebral , Transcriptoma , Animales , Costillas , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Porcinos/genéticaRESUMEN
A novel bacteria with heterotrophic nitrification and aerobic denitrification ability was obtained from a membrane bioreactor (MBR) and identified as Acinetobacter sp. TSH1. The nitrogen removal characteristics, nitrogen balance analysis, kinetic characteristics, and enhanced biological treatment in MBR of the novel isolated strain TSH1 were determined. Results showed that strain TSH1 could remove approximately 96.6% of NH4+-N, 82.9% of NO2--N and 98.7% of NO3--N in 24 h, and the corresponding maximum removal rates were 3.64 mg-N/(L·h), 1.77 mg-N/(L·h) and 3.94 mg-N/(L·h). The nitrogen balance analysis indicated that most of NH4+-N (62.6%) and NO3--N (71.9%) were transformed to gaseous nitrogen. The kinetic experiments showed that strain TSH1 had a high Km of 151.64 mg-NH4+-N/L and 203.25 mg-NO3--N/L. The enhanced biological treatment of synthetic wastewater in MBR showed that the strain TSH1 can significantly improve the nitrogen removal efficiency.
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Desnitrificación , Nitrificación , Aerobiosis , Bacterias , Reactores Biológicos , Procesos Heterotróficos , Nitritos , Nitrógeno , Aguas ResidualesRESUMEN
The study aimed to isolate a novel strain with heterotrophic nitrification and aerobic denitrification ability and evaluate the nitrogen removal characteristics. Results showed that Ochrobactrum anthropi HND19 could remove approximately 98.6% of NH4+-N (104.3 mg·L-1) and 97.6% of NO3--N (98.6 mg·L-1), and the removal rates achieved 4.28 and 4.01 mg-N/(L·h) by heterotrophic nitrification and aerobic denitrification. The optimal incubate conditions of strain HND19 were 120 rpm (shaking speed), 5 (salinity), 30 °C (temperature), 7.5 (C/N ratio) with sodium acetate as carbon resource. And the removal efficiency of the total nitrogen (TN) realized 73.4% under the optimal conditions. Functional genes (hao, napA, nirK, norB, and nosZ) involved in the nitrogen removal processes were successfully amplified from strain HND19. These findings indicate that the strain HND19 possesses great application feasibility in treating wastewater with high-intensity nitrogen.
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Nitrificación , Ochrobactrum anthropi , Aerobiosis , Desnitrificación , Procesos Heterotróficos , Nitritos , Nitrógeno , Ochrobactrum anthropi/genéticaRESUMEN
Aerobic denitrifiers have the potential to reduce nitrate in polluted water under aerobic conditions. A salt-tolerant aerobic denitrifier was newly isolated and identified as Vibrio spp. AD2 from a marine recirculating aquaculture system, in which denitrification performance was investigated via single-factor experiment, Box-Behnken experiment, and nitrogen balance analysis. Nitrate reductase genes were identified by polymerase chain reaction. Results showed that strain AD2 removed 98.9% of nitrate-nitrogen (NO3--N) with an initial concentration about 100 mg·L-1 in 48 h without nitrite-nitrogen (NO2--N) accumulation. Nitrogen balance indicated that approximately 17.5% of the initial NO3--N was utilized for bacteria synthesis themselves, 4.02% was converted to organic nitrogen, 39.8% was converted to nitrous oxide (N2O), and 31.1% was converted to nitrogen (N2). Response surface methodology experiment showed that the maximum removal of total nitrogen (TN) occurred under the condition of C/N ratio 11.5, shaking speed 127.9 rpm, and temperature 30.8 °C. Sequence amplification indicated that the denitrification genes, napA and nirS, were present in strain AD2. These results indicated that the strain AD2 has potential applications for removing NO3--N from high-salinity (3%) wastewater.
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Nitritos , Vibrio , Aerobiosis , Desnitrificación , Nitratos , Nitrificación , NitrógenoRESUMEN
This study investigated an Iron-carbon (Fe-C) micro-electrolysis method to enhance nitrogen removal of Sesuvium portulacastrum constructed wetlands (CWs) when treating mariculture effluents. The main objective was to investigate the effects of Fe-C on nitrogen purification performance and microbial characteristics of Sesuvium portulacastrum CWs. Results showed that the presence of Fe-C and Sesuvium portulacastrum could improve nitrogen removal efficiency by 20-30% and 15-30%, respectively. CWs with 33% v/v Fe-C addition performed well on nitrogen removal: TAN, 41.49 ± 13.64%; NO2--N, 13.32%; NO3--N, 60.02 ± 6.17%; TIN, 63.40 ± 12.11%. Microbial analysis revealed that Fe-C altered the microbial communities, and improved the abundance of denitrification related genera. Based on microbial enzyme activities and genes abundance, the anammox and denitrification processes were promoted by Fe-C in CWs. These findings indicate that Sesuvium portulacastrum CWs with 33% v/v Fe-C represents an effective nitrogen removal for mariculture wastewater with insufficient carbon source.
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Aizoaceae , Humedales , Carbono , Desnitrificación , Hierro , Nitrógeno , Eliminación de Residuos LíquidosRESUMEN
OBJECTIVES: Immunodeficient mice injected with human cancer cell lines have been used for human oncology studies and anti-cancer drug trials for several decades. However, rodents are not ideal species for modelling human cancer because rodents are physiologically dissimilar to humans. Therefore, anti-tumour drugs tested effective in rodents have a failure rate of 90% or higher in phase III clinical trials. Pigs are similar to humans in size, anatomy, physiology and drug metabolism rate, rendering them a desirable pre-clinical animal model for assessing anti-cancer drugs. However, xenogeneic immune rejection is a major barrier to the use of pigs as hosts for human tumours. Interleukin (IL)-2 receptor γ (IL2RG), a common signalling subunit for multiple immune cytokines including IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, is required for proper lymphoid development. MATERIALS AND METHODS: IL2RG-/Y pigs were generated by CRISPR/Cas9 technology, and examined for immunodeficiency and ability to support human oncogenesis. RESULTS: Compared to age-matched wild-type pigs, IL2RG-/Y pigs exhibited a severely impaired immune system as shown by lymphopenia, lymphoid organ atrophy, poor immunoglobulin function, and T- and NK-cell deficiency. Human melanoma Mel888 cells generated tumours in IL2RG-/Y pigs but not in wild-type littermates. The human tumours grew faster in IL2RG-/Y pigs than in nude mice. CONCLUSIONS: Our results indicate that these pigs are promising hosts for modelling human cancer in vivo, which may aid in the discovery and development of anti-cancer drugs.
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Sistemas CRISPR-Cas/genética , Subunidad gamma Común de Receptores de Interleucina/metabolismo , Neoplasias Cutáneas/patología , Animales , Animales Modificados Genéticamente/metabolismo , Línea Celular Tumoral , Modelos Animales de Enfermedad , Edición Génica , Humanos , Sistema Inmunológico/metabolismo , Subunidad gamma Común de Receptores de Interleucina/antagonistas & inhibidores , Subunidad gamma Común de Receptores de Interleucina/genética , Linfopenia/patología , Melanoma/metabolismo , Melanoma/patología , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/mortalidad , Tasa de Supervivencia , Porcinos , Porcinos Enanos , Trasplante HeterólogoRESUMEN
BACKGROUND: Xenogeneic organ transplantation has been proposed as a potential approach to fundamentally solve organ shortage problem. Xenogeneic immune responses across species is one of the major obstacles for clinic application of xeno-organ transplantation. The generation of glycoprotein galactosyltransferase α 1, 3 (GGTA1) knockout pigs has greatly contributed to the reduction of hyperacute xenograft rejection. However, severe xenograft rejection can still be induced by xenoimmune responses to the porcine major histocompatibility complex antigens swine leukocyte antigen class I and class II. METHODS: We simultaneously depleted GGTA1, ß2-microglobulin (ß2M), and major histocompatibility complex class II transactivator (CIITA) genes using clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins technology in Bamma pig fibroblast cells, which were further used to generate GGTA1ß2MCIITA triple knockout (GBC-3KO) pigs by nuclear transfer. RESULTS: The genotype of GBC-3KO pigs was confirmed by polymerase chain reaction and Sanger sequencing, and the loss of expression of α-1,3-galactose, SLA-I, and SLA-II was demonstrated by flow cytometric analysis using fluorescent-conjugated lectin from bandeiraea simplicifolia, anti-ß2-microglobulin, and swine leukocyte antigen class II DR antibodies. Furthermore, mixed lymphocyte reaction assay revealed that peripheral blood mononuclear cells from GBC-3KO pigs were significantly less effective than (WT) pig peripheral blood mononuclear cells in inducing human CD3CD4 and CD3CD8 T-cell activation and proliferation. In addition, GBC-3KO pig skin grafts showed a significantly prolonged survival in immunocompetent C57BL/6 mice, when compared with wild-type pig skin grafts. CONCLUSIONS: Taken together, these results demonstrate that elimination of GGTA1, ß2M, and CIITA genes in pigs can effectively alleviate xenogeneic immune responses and prolong pig organ survival in xenogenesis. We believe that this work will facilitate future research in xenotransplantation.
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Rechazo de Injerto/prevención & control , Xenoinjertos/inmunología , Trasplante de Órganos/métodos , Trasplante Heterólogo/métodos , Aloinjertos/provisión & distribución , Animales , Animales Modificados Genéticamente/inmunología , Sistemas CRISPR-Cas/genética , Modelos Animales de Enfermedad , Femenino , Galactosiltransferasas/genética , Galactosiltransferasas/inmunología , Técnicas de Inactivación de Genes/métodos , Genes MHC Clase II/genética , Genes MHC Clase II/inmunología , Rechazo de Injerto/inmunología , Supervivencia de Injerto/genética , Supervivencia de Injerto/inmunología , Xenoinjertos/trasplante , Humanos , Masculino , Ratones , Trasplante de Órganos/efectos adversos , Porcinos/genética , Porcinos/inmunología , Trasplante Heterólogo/efectos adversos , Microglobulina beta-2/genética , Microglobulina beta-2/inmunologíaRESUMEN
Blastocyst complementation by pluripotent stem cell (PSC) injection is believed to be the most promising method to generate xenogeneic organs. However, ethical issues prevent the study of human chimeras in the late embryonic stage of development. Primate embryonic stem cells (ESCs), which have similar pluripotency to human ESCs, are a good model for studying interspecies chimerism and organ generation. However, whether primate ESCs can be used in xenogenous grafts remains unclear. In this study, we evaluated the chimeric ability of cynomolgus monkey (Macaca fascicularis) ESCs (cmESCs) in pigs, which are excellent hosts because of their many similarities to humans. We report an optimized culture medium that enhanced the anti-apoptotic ability of cmESCs and improved the development of chimeric embryos, in which domesticated cmESCs (D-ESCs) injected into pig blastocysts differentiated into cells of all three germ layers. In addition, we obtained two neonatal interspecies chimeras, in which we observed tissue-specific D-ESC differentiation. Taken together, the results demonstrate the capability of D-ESCs to integrate and differentiate into functional cells in a porcine model, with a chimeric ratio of 0.001-0.0001 in different neonate tissues. We believe this work will facilitate future developments in xenogeneic organogenesis, bringing us one step closer to producing tissue-specific functional cells and organs in a large animal model through interspecies blastocyst complementation.
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Quimera , Células Madre Embrionarias/citología , Macaca fascicularis/embriología , Porcinos/embriología , Animales , Blastocisto/citología , Diferenciación Celular , Células Cultivadas , Quimera/embriologíaRESUMEN
Aerobic denitrification microbes have great potential to solve the problem of NO3--N accumulation in industrialized recirculating aquaculture systems (RASs). A novel salt-tolerant aerobic denitrifier was isolated from a marine recirculating aquaculture system (RAS) and identified as Halomonas alkaliphile HRL-9. Its aerobic denitrification performance in different dissolved oxygen concentrations, temperatures, and C/N ratios was studied. Investigations into nitrogen balance and nitrate reductase genes (napA and narG) were also carried out. The results showed that the optimal conditions for nitrate removal were temperature of 30 °C, a shaking speed of 150 rpm, and a C/N ratio of 10. For nitrate nitrogen (NO3--N) (initial concentration 101.8 mg·L-1), the sole nitrogen source of the growth of HRL-9, the maximum NO3--N removal efficiency reached 98.0% after 24 h and the maximum total nitrogen removal efficiency was 77.3% after 48 h. Nitrogen balance analysis showed that 21.7% of NO3--N was converted into intracellular nitrogen, 3.3% of NO3--N was converted into other nitrification products (i.e., nitrous nitrogen, ammonium nitrogen, and organic nitrogen), and 74.5% of NO3--N might be converted to gaseous products. The identification of functional genes confirmed the existence of the napA gene in strain HRL-9, but no narG gene was found. These results confirm that the aerobic denitrification strain, Halomonas alkaliphile HRL-9, which has excellent aerobic denitrification abilities, can also help us understand the microbiological mechanism and transformation pathway of aerobic denitrification in RASs.
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Biotransformación , Desnitrificación , Halomonas/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Tolerancia a la Sal , Agua de Mar/química , Aerobiosis , ChinaRESUMEN
Substantial rates of fetal loss plague all in vitro procedures involving embryo manipulations, including human-assisted reproduction, and are especially problematic for mammalian cloning where over 90% of reconstructed nuclear transfer embryos are typically lost during pregnancy. However, the epigenetic mechanism of these pregnancy failures has not been well described. Here we performed methylome and transcriptome analyses of pig induced pluripotent stem cells and associated cloned embryos, and revealed that aberrant silencing of imprinted genes, in particular the retrotransposon-derived RTL1 gene, is the principal epigenetic cause of pregnancy failure. Remarkably, restoration of RTL1 expression in pig induced pluripotent stem cells rescued fetal loss. Furthermore, in other mammals, including humans, low RTL1 levels appear to be the main epigenetic cause of pregnancy failure.
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Metilación de ADN/genética , Impresión Genómica/genética , Células Madre Pluripotentes Inducidas/citología , Complicaciones del Embarazo/genética , Proteínas Represoras/genética , Retroelementos/genética , Animales , Transferencia de Embrión/efectos adversos , Embrión de Mamíferos/citología , Femenino , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica/genética , Técnicas de Transferencia Nuclear , Embarazo , PorcinosRESUMEN
Insulin-like growth factor 2 (IGF2) is an important growth factor, which promotes growth and development in mammals during fetal and postnatal stages. Using CRISPR-Cas9 system, we generated multiple founder pigs containing 12 different mutant alleles around a regulatory element within the intron 3 of IGF2 gene. Crossing two male founders passed four mutant alleles onto F1 generation, and these mutations abolished repressor ZBED6 binding and rendered this regulatory element nonfunctional. Both founders and F1 animals showed significantly faster growth, without affecting meat quality. These results indicated that editing IGF2 intron 3-3072 site using CRISPR-Cas9 technology improved meat production in Bama pigs. This is the first demonstration that editing non-coding region can improve economic traits in livestock.
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Sistemas CRISPR-Cas , Edición Génica/métodos , Factor II del Crecimiento Similar a la Insulina/genética , Intrones , Secuencias Reguladoras de Ácidos Nucleicos , Porcinos/genética , Alelos , Crianza de Animales Domésticos/métodos , Animales , Cruzamiento/métodos , Femenino , Genotipo , Masculino , Carne/análisis , Mutación , FenotipoRESUMEN
Epigenetic modifications play significant roles in adaptive evolution. The tumor suppressor p53, well known for controlling cell fate and maintaining genomic stability, is much less known as a master gene in environmental adaptation involving methylation modifications. The blind subterranean mole rat Spalax eherenbergi superspecies in Israel consists of four species that speciated peripatrically. Remarkably, the northern Galilee species Spalax galili (2n = 52) underwent adaptive ecological sympatric speciation, caused by the sharply divergent chalk and basalt ecologies. This was demonstrated by mitochondrial and nuclear genomic evidence. Here we show that the expression patterns of the p53 regulatory pathway diversified between the abutting sympatric populations of S. galili in sharply divergent chalk-basalt ecologies. We identified higher methylation on several sites of the p53 promoter in the population living in chalk soil (chalk population). Site mutagenesis showed that methylation on these sites linked to the transcriptional repression of p53 involving Cut-Like Homeobox 1 (Cux1), paired box 4 (Pax 4), Pax 6, and activator protein 1 (AP-1). Diverse expression levels of p53 between the incipiently sympatrically speciating chalk-basalt abutting populations of S. galili selectively affected cell-cycle arrest but not apoptosis. We hypothesize that methylation modification of p53 has adaptively shifted in supervising its target genes during sympatric speciation of S. galili to cope with the contrasting environmental stresses of the abutting divergent chalk-basalt ecologies.