End-3 Genes Research Articles

New biomarkers underlying acetic acid tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii

Evolutionary engineering experiments, in combination with omics technologies, revealed genetic markers underpinning the molecular mechanisms behind acetic acid stress tolerance in the probiotic yeast Saccharomyces cerevisiae var. boulardii. Here, compared to the ancestral Ent strain, evolved yeast strains could quickly adapt to high acetic acid levels (7 g/L) and displayed a shorter lag phase of growth. Bioinformatic-aided whole-genome sequencing identified genetic changes associated with enhanced strain robustness to acetic acid: a duplicated sequence in the essential endocytotic PAN1 gene, mutations in a cell wall mannoprotein (dan4Thr192del), a lipid and fatty acid transcription factor (oaf1Ser57Pro) and a thiamine biosynthetic enzyme (thi13Thr332Ala). Induction of PAN1 and its associated endocytic complex SLA1 and END3 genes was observed following acetic acid treatment in the evolved-resistant strain when compared to the ancestral strain. Genome-wide transcriptomic analysis of the evolved Ent acid-resistant strain (Ent ev16) also revealed a dramatic rewiring of gene expression among genes associated with cellular transport, metabolism, oxidative stress response, biosynthesis/organization of the cell wall, and cell membrane. Some evolved strains also displayed better growth at high acetic acid concentrations and exhibited adaptive metabolic profiles with altered levels of secreted ethanol (4.0–6.4% decrease), glycerol (31.4–78.5% increase), and acetic acid (53.0–60.3% increase) when compared to the ancestral strain. Overall, duplication/mutations and transcriptional alterations are key mechanisms driving improved acetic acid tolerance in probiotic strains. We successfully used adaptive evolutionary engineering to rapidly and effectively elucidate the molecular mechanisms behind important industrial traits to obtain robust probiotic yeast strains for myriad biotechnological applications.Key points•Acetic acid adaptation of evolutionary engineered robust probiotic yeast S. boulardii•Enterol ev16 with altered genetic and transcriptomic profiles survives in up to 7 g/L acetic acid•Improved acetic acid tolerance of S. boulardii ev16 with mutated PAN1, DAN4, OAF1, and THI13 genesGraphical

Analysis of genetic characteristics in two Chinese children of type Ⅱ Waardenburg syndrome

Objective: To screen and analyze the mutations of MITF gene in two children of type Ⅱ Waardenburg syndrome (WS2) from different families in Yunnan,China,and to explore the possible molecular pathogenesis. Methods: With informed consent, medical history collection, physical examinations, audiological evaluation, and high resolution computer tomography (HRCT) scan of temporal bone were performed on the two WS2 probands and their family members. Genomic DNA was extracted from peripheral blood of all individuals. The coding regions including all exons, part of introns and promoters of MITF, PAX3, SOX10, SNAI2, END3, ENDRB, and KITLG genes were sequenced by high-throughput sequencing. According to the results of high-throughput sequencing, pathogenic mutations detected in the probands and their parents were verified by Sanger sequencing. Results: The proband 1 carried c.641_643delGAA mutation in the 7th exon of MITF gene, which was a frame-shift mutation resulting in an amino acid change of p.214delR. It was a de novo mutation as the parents of proband 1 showed no variation on this site. The proband 2 carried heterozygous loss of the large fragment ranging from exon 1 to exon 9 of MITF gene, which defected the function of MITF protein. Conclusion: Genetic examinations provide important evidence for diagnosis of Waardenburg syndrome. Heterozygous mutation c.641_643delGAA and heterozygous loss of the large fragment ranging from exon 1 to exon 9 of MITF gene might be the molecular pathogenesis of the two WS2 probands in this study.

Studies on genes expression pattern of antioxidant enzymes and enzymes involved into the genetic information implementation in E.coli cells due to the antibiotic resistance against apramycin and cefatoxime

The emergence of antibiotic-resistant bacteria is considered a serious problem. The resistance of bacteria against antimicrobial substances becomes important in the repair systems for damage to DNA and RNA molecules. The role of the antioxidant system in the development of bacterial resistance against antibiotics is not yet practically studied. The article studied the expression regulation of the genes of antioxidant enzymes and enzymes involved in the genetic information in E. coli cells with the antibiotic resistance against apramycin and cefatoxime. The study was conducted on bacterial cells resistant against these two antibiotics. The genes blaOXA-1, blaSHV, blaTEM, mdtK, aadA1, aadA2, sat, strA, blaCTX, blaPER-2, tnpA, tnpR, intC1 and intC1c were identified in bacterial cell case. This indicates the presence of plasmids in bacteria with these genes, which provide bacterial resistance to apramycin and cefatoxime. It was established that during the formation of cefotaxime resistance, there was a sharp increase in the expression of the Cu, Zn superoxide dismutase gene: in comparison with the control group, the representation of its transcripts increased 141.04 times for cefotoxime and 155.42 times for apramycin. It has been established that during the formation of resistance to the studied antibiotics in E. coli, an increase in the expression of the end4 and end3 genes is observed. There is tendency toward an increase in the number of transcripts of the pol3E gene observed in the formation of resistance against cefotaxime and apromycin.

Partially compromised specification causes stochastic effects on gut development in C. elegans

The C. elegans gut descends from the E progenitor cell through a series of stereotyped cell divisions and morphogenetic events. Effects of perturbations of upstream cell specification on downstream organogenesis have not been extensively investigated. Here we have assembled an allelic series of strains that variably compromise specification of E by perturbing the activation of the gut-specifying end-1 and end-3 genes. Using a marker that allows identification of all E descendants regardless of fate, superimposed with markers that identify cells that have adopted a gut fate, we have examined the fate of E lineage descendants among hundreds of embryos. We find that when specification is partially compromised, the E lineage undergoes hyperplasia accompanied by stochastic and variable specification of gut fate among the E descendants. As anticipated by prior work, the activation of the gut differentiation factor elt-2 becomes delayed in these strains, although ultimate protein levels of a translational ELT-2::GFP reporter resemble those of the wild type. By comparing these effects among the various specification mutants, we find that the stronger the defect in specification (i.e. the fewer number of embryos specifying gut), the stronger the defects in the E lineage and delay in activation of elt-2. Despite the changes in the E lineage in these strains, we find that supernumerary E descendants that adopt a gut fate are accommodated into a relatively normal-looking intestine. Hence, upstream perturbation of specification dramatically affects the E lineage, but as long as sufficient descendants adopt a gut fate, organogenesis overcomes these effects to form a relatively normal intestine.

Blocking endocytotic mechanisms to improve heterologous protein titers in Saccharomyces cerevisiae.

Saccharomyces cerevisiae is a useful platform for protein production of biopharmaceuticals and industrial enzymes. To date, substantial effort has focused on alleviating several bottlenecks in expression and the secretory pathway. Recently, it has been shown that highly active endocytosis could decrease the overall protein titer in the supernatant. In this study, we block endocytosis and trafficking to the vacuole using a modified TEV Protease-Mediated Induction of Protein Instability (mTIPI) system to disrupt the endocytotic and vacuolar complexes. We report that conditional knock-down of endocytosis gene Rvs161 improved the concentration of α-amylase in supernatant of S. cerevisiae cultures by 63.7% compared to controls. By adaptive evolution, we obtained knock-down mutants in Rvs161 and End3 genes with 2-fold and 3-fold α-amylase concentrations compared to controls that were not evolved. Our study demonstrates that genetic blocking of endocytotic mechanisms can improve heterologous protein production in S. cerevisiae. This result is likely generalizable to other eukaryotic secretion hosts.

Roles of the Wnt effector POP-1/TCF in the C. elegans endomesoderm specification gene network

In C. elegans the 4-cell stage blastomere EMS is an endomesodermal precursor. Its anterior daughter, MS, makes primarily mesodermal cells, while its posterior daughter E generates the entire intestine. The gene regulatory network underlying specification of MS and E has been the subject of study for more than 15 years. A key component of the specification of the two cells is the involvement of the Wnt/β-catenin asymmetry pathway, which through its nuclear effector POP-1, specifies MS and E as different from each other. Loss of pop-1 function results in the mis-specification of MS as an E-like cell, because POP-1 directly represses the end-1 and end-3 genes in MS, which would otherwise promote an endoderm fate. A long-standing question has been whether POP-1 plays a role in specifying MS fate beyond repression of endoderm fate. This question has been difficult to ask because the only chromosomal lesions that remove both end-1 and end-3 are large deletions removing hundreds of genes. Here, we report the construction of bona fide end-1 end-3 double mutants. In embryos lacking activity of end-1, end-3 and pop-1 together, we find that MS fate is partially restored, while E expresses early markers of MS fate and adopts characteristics of both MS and C. Our results suggest that POP-1 is not critical for MS specification beyond repression of endoderm specification, and reveal that Wnt-modified POP-1 and END-1/3 further reinforce E specification by repressing MS fate in E. By comparison, a previous work suggested that in the related nematode C. briggsae, Cb-POP-1 is not required to repress endoderm specification in MS, in direct contrast with Ce-POP-1, but is critical for repression of MS fate in E. The findings reported here shed new light on the flexibility of combinatorial control mechanisms in endomesoderm specification in Caenorhabditis.

Maternal deployment of the embryonic SKN-1 → MED-1,2 cell specification pathway in C. elegans

We have previously shown that the MED-1,2 divergent GATA factors act apparently zygotically to specify the fates of the MS (mesoderm) and E (endoderm) sister cells, born at the 7-cell stage of C. elegans embryogenesis. In the E cell, MED-1,2 activate transcription of the endoderm-promoting end-1 and end-3 genes. We demonstrate by in situ hybridization that med transcripts accumulate both in the EMS cell and in the maternal germline in a SKN-1-dependent manner. Removal of zygotic med function alone results in a weakly impenetrant loss of endoderm. However, med-1,2(−) embryos made by mothers in which germline med transcripts have been abrogated by transgene cosuppression fail to make endoderm 50% of the time, similar to the phenotype seen by RNAi. We also find that reduction of Med or End activity results in aberrant numbers of intestinal cells in embryos that make endoderm. We further show that regulation of the paralogous end-1 and end-3 genes consists of both shared and distinct inputs, and that END-3 activates end-1 expression. Our data thus reveal three new properties of C. elegans endoderm specification: both maternal and zygotic activities of the med genes act to specify endoderm, defects in endoderm specification also result in defects in gut cell number, and activation of the paralogous end-1 and end-3 genes differs qualitatively in the relative contributions of their upstream regulators.

The Wnt effector POP-1 and the PAL-1/Caudal homeoprotein collaborate with SKN-1 to activate C. elegans endoderm development

POP-1, a Tcf/Lef-1-like target of the convergent Wnt and MAP kinase (MAPK) signaling pathways, functions throughout Caenorhabditis elegans development to generate unequal daughters during asymmetric cell divisions. A particularly prominent such asymmetric division occurs when the EMS blastomere divides to produce MS, a mesoderm precursor, and E, the sole endoderm progenitor. POP-1 allows mesoderm development in the MS lineage by repressing the endoderm-promoting end-1 and end-3 genes. This repression is relieved in the E lineage by Wnt/MAPK signaling, which results in phosphorylation and export of POP-1 from the E nucleus. Here, we report that, in addition to repressing E development in MS, POP-1 also functions positively in endoderm development, in conjunction with the well-characterized endoderm-promoting SKN-1→MED regulatory cascade. While removal of POP-1 alone results in derepression of endoderm development in the MS lineage, mutations in several genes that result in impenetrant loss of endoderm are strongly enhanced by loss of pop-1 function. A Lef-1-like binding site is essential for activation of an end-1 promoter fusion, suggesting that POP-1 may act directly on end-1. Thus, POP-1 may generate developmental asymmetry during many cell divisions in C. elegans by reiteratively switching from repressive and activating states. Furthermore, we report that the Caudal-like homeodomain protein PAL-1, whose role in early embryogenesis was thought to be exclusive specification of mesectodermal development in the lineage of the C blastomere, can act with POP-1 to activate endoderm specification in the absence of the SKN-1→MED transcriptional input, accounting for the impenetrance of mutants lacking SKN-1 or MED-1,2 activity. We conclude that the combined action of several separate transcriptional regulatory inputs, including SKN-1, the MEDs, PAL-1, and the Wnt/MAPK-activated form of POP-1, are responsible for activating end gene transcription and endoderm development.

Dynamics of a Developmental Switch: Recursive Intracellular and Intranuclear Redistribution of Caenorhabditis elegans POP-1 Parallels Wnt-Inhibited Transcriptional Repression

POP-1, a Tcf/Lef factor, functions throughout Caenorhabditis elegans development as a Wnt-dependent reiterative switch to generate nonequivalent sister cells that are born by anterior–posterior cell divisions. We have observed the interaction between POP-1 and a target gene that it represses as it responds to Wnt signaling. Dynamic observations in living embryos reveal that POP-1 undergoes Wnt-dependent nucleocytoplasmic redistribution immediately following cytokinesis, explaining the differential nuclear POP-1 levels in nonequivalent sister cells. In unsignaled (anterior) but not Wnt-signaled (posterior) sister cells, POP-1 progressively coalesces into subnuclear domains during interphase, coincident with its action as a repressor. While the asymmetric distribution of POP-1 in nonequivalent sisters apparently requires a 124-amino-acid internal domain, neither the HMG box nor β-catenin interaction domains are required. We find that a transcriptional activator, MED-1, associates in vivo with the end-1 and end-3 target genes in the mesoderm (anterior sister) and in the endoderm (posterior sister) following the asymmetric cell division that subdivides the mesendoderm. However, in the anterior sister, binding of POP-1 to the end-1 and end-3 genes blocks their expression. In vivo, binding of POP-1 to the end-1 and end-3 targets (in the posterior sister) is blocked by Wnt/MAPK signaling. Thus, a Tcf/Lef factor represses transactivation of genes in an unsignaled daughter cell by abrogating the function of a bound activator.

The sequence of a 17,933 bp segment of Saccharomyces cerevisiae chromosome XIV contains the RHO2, TOP2, MKT1 and END3 genes and five new open reading frames.

We report the DNA sequence of a 17,933 bp fragment from the left arm of chromosome XIV of Saccharomyces cerevisiae. Analysis of the sequence reveals the presence of ten open reading frames (ORFs) larger than 100 codons. Four of these were previously identified as genes RHO2, TOP2, MKT1 and END3. Additionally, the NH2 end coding region of PMS1 is found in the 3' end of the sequence. No significant homology to any known protein has been found for the other five ORFs.