XbaI Site Research Articles

Detection of Hemophilia A Carriers Using Intragenic Factor VIII:C DNA Polymorphisms

AbstractA DNA polymorphism for an XbaI site in intron 22 of the human factor VIII:C gene extends the utility of DNA methods for carrier detection in families segregating for hemophilia A. While the DNA polymorphism detected by a BglI site in intron 18 of the factor VIII:C gene was informative for 41% of females studied, the BglI/intron 25 polymorphism provided no additional information because of apparent linkage disequilibrium. In contrast, the XbaI intron 22 polymorphism was useful in 53% of women who were uninformative (homozygous) for either the BclI or BglI polymorphisms. Using the BclI /intron 18 and Xbal/intron 22 intragenic polymorphisms, we could provide highly accurate information for 68% of women we studied who were at risk for carriership. The carrier status of the remaining 32% could be determined utilizing the closely linked Taql/St14DNA polymorphism.

The Effect of Cell Surface Expression and Linker Sequence on the Recruitment of Arrestin to the GIP Receptor

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptor are important targets in the treatment of both type 2 diabetes mellitus (T2DM) and obesity. Originally identified for their role in desensitization, internalization and recycling of G protein-coupled receptors (GPCRs), arrestins have since been shown to act as scaffolding proteins that allow GPCRs to signal in a G protein-independent manner. While GLP-1R has been reported to interact with arrestins, this aspect of cell signaling remains controversial for GIPR. Using a (FRET)-based assay we have previously shown that yellow fluorescent protein (YFP)-labeled GIPR does not recruit arrestin. This GIPR-YFP construct contained a 10 amino acid linker between the receptor and a XbaI restriction site upstream of the YFP. This linker was not present in the modified GIPR-SYFP2 used in subsequent FRET and bioluminescence resonance energy transfer (BRET) assays. However, its removal results in the introduction of a serine residue adjacent to the end of GIPR’s C-terminal tail which could potentially be a phosphorylation site. The resulting receptor was indeed able to recruit arrestin. To find out whether the serine/arginine (SR) coded by the XbaI site was indeed the source of the problem, it was substituted with glycine/glycine (GG) by site-directed mutagenesis. This substitution abolished arrestin recruitment in the BRET assay but only significantly reduced it in the FRET assay. In addition, we show that the presence of a N-terminal FLAG epitope and influenza hemagglutinin signal peptide were also required to detect arrestin recruitment to the GIPR, most likely by increasing receptor cell surface expression. These results demonstrate how arrestin recruitment assay configuration can dramatically alter the result. This becomes relevant when drug discovery programs aim to identify ligands with “biased agonist” properties.

RS-1 enhances CRISPR-mediated targeted knock-in in bovine embryos.

Targeted knock‐in (KI) can be achieved in embryos by clustered regularly interspaced short palindromic repeats (CRISPR)‐assisted homology directed repair (HDR). However, HDR efficiency is constrained by the competition of nonhomologous end joining. The objective of this study was to explore whether CRISPR‐assisted targeted KI rates can be improved in bovine embryos by exposure to the HDR enhancer RS‐1. In vitro produced zygotes were injected with CRISPR components (300 ng/µl Cas9 messenger RNA and 100 ng/µl single guide RNA against a noncoding region) and a single‐stranded DNA (ssDNA) repair template (100 ng/µl). ssDNA template contained a 6 bp XbaI site insert, allowing targeted KI detection by restriction analysis, flanked by 50 bp homology arms. Following microinjection, zygotes were exposed to 0, 3.75, or 7.5 µM RS‐1 for 24 hr. No differences were noted between groups in terms of development or genome edition rates. However, targeted KI rates were doubled in the group exposed to 7.5 µM RS‐1 compared to the others (52.8% vs. 25% and 23.1%, for 7.5, 0, and 3.75 µM, respectively). In conclusion, transient exposure to 7.5 µM RS‐1 enhances targeted KI rates resulting in approximately half of the embryos containing the intended mutation, hence allowing direct KI generation in embryos.

Association between the estrogen receptor α gene polymorphisms rs2234693 and rs9340799 and severe and mild pre-eclampsia: a meta-analysis.

This meta-analysis was performed in order to determine the associations between the estrogen receptor α (ESR1) gene PvuII site (-397T/C, rs2234693) and XbaI site (-351A/G, rs9340799) polymorphisms with severe and mild pre-eclampsia. Eligible studies were identified by searching PubMed, Medline, Embase, China National Knowledge Infrastructure (CNKI), and WanFang databases until May 2018. The pooled odds ratio (OR) and 95% confidence interval (CI) were used to calculate the associations. Six articles (consisting of seven studies; one article was considered as two separate studies with two different subpopulations) investigated the ESR1 gene PvuII -397T/C and XbaI -351A/G polymorphisms in severe and mild pre-eclampsia patients and included controls. The pooled results indicated an increased risk of severe pre-eclampsia for the XbaI -351A/G polymorphism (OR = 1.67, 95% CI = 1.10–2.25, P=0.017 for GG compared with AA+GA; OR = 1.81, 95% CI = 1.17–2.82, P=0.008 for GG compared with GA). The GG genotype of the ESR1 XbaI polymorphism could be a genetic risk factor for severe pre-eclampsia susceptibility. However, the ESR1 gene PvuII -397T/C polymorphism was not significantly associated with the risk of severe pre-eclampsia, and there was no association between mild pre-eclampsia and the ESR1 gene PvuII -397T/C and XbaI -351A/G polymorphisms separately. The current meta-analysis indicates that the ESR1 XbaI genetic polymorphism may be associated with severe pre-eclampsia. However, there was no association of the ESR1 gene PvuII and XbaI polymorphisms with the risk of mild pre-eclampsia. Owing to the low statistical power, the results may not be sufficiently robust and this conclusion should be interpreted cautiously, which highlights the requirement for large-scale and high-quality studies in this field.

Specific cytotoxicity of epidermal groth factor variantIII oriented chimeric antigen receptor-engineered T cells on SMMC-7721

Objective To observe the specific killing effect of anti-epidermal groth factor variantⅢ (EGFRvⅢ) chimeric antigen receptor modified T cells (EGFRvⅢ/2CAR-T) on liver cancer cell SMMC-7721. Methods Expression cassette of EGFRvⅢ/2CAR (1 374 bp) was inserted into XbaⅠ and BamHⅠ sites of piggybac transposon vector PB513B-1 (PB-EGFRvⅢ/2CAR), the latter together with transposase plasmid was then transducted into human peripheral blood T cells by electroporation (1×20 ms, 840 V). The expression of EGFRvⅢ/2CAR on T cells was detected by flow cytometry. The expression of EGFRvⅢ in SMMC-7721 was detected by Western blotting. After EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721, the killing effect was detected by lactate dehydrogenase (LDH) release test and cytokine interferon (IFN)-γ secretion. Results The recombinant vector PB-EGFRvⅢ/2CAR was confirmed by XbaⅠ and BamHⅠ enzyme digestion, electrophoresis and DNA sequencing analysis. The expression of EGFRvⅢ/2CAR on T cells was detected by Flow cytometry (55.5%). Western blotting analysis showed that a protein band of approximate 145 000 molecular weight was observed in SMMC-7721 cells, which corresponds to the calculated molecular weight of EGFRvⅢ. EGFRvⅢ/2CAR-T cells were co-cultured with SMMC-7721 for 24 h, LDH release test showed that tumor specific killing was positively correlated with E∶T ratio (E∶T=1∶1, 5∶1, 10∶1, 15∶1, 20∶1); enzyme linked immunosorbent assay (ELISA) showed that cytokine IFN-γ secretion was (1 060.1±89.0) pg/ml, which was significantly higher than control group (P=0.000). Conclusion Anti-EGFRvⅢ CAR-T cells can kill specifically EGFRvⅢ+ liver cancer cell SMMC-7721, providing research basis for immunotherapy of hepatic cancer. Key words: Epidermal groth factor variantⅢ; Chimeric antigen receptor; Immunotherapy; PiggyBac transposon

Cloning of sequence of homologous crt-cluster in Streptomyces globisporus 1912-бп

Aim. The aim was to set influence to the additional homologous crt-cluster on carotenogenesis of cells of streptomycete recipient. Methods. For this purpose transformation by the hybrid plasmid pWC 9,6 was conducted. This plasmid contained the fragment of the crt-cluster sequence (9576 bp) of Crt+-mutant S. globisporus Crt4 in a Crt--recipient S. globisporus 1912-бп. To construct this hybrid plasmid, a fragment of PLR-copies of sequence of the crt-cluster of mutant S. globisporus 1912 Crt4 was cloned in the shuttle vector pWHM4 (6.6 kb). Insertion was done into unique restriction sites for endonucleases XbaI and HindIII in a polylinker of this vector. These endonucleases have not restriction sites into the crt-cluster sequence. Results. The plasmid pWC 9,6 (16.2 kb) that contains the crt-cluster sequence (9576 bp) of the Crt+-variant Crt4 of the strain S. globisporus 1912 was constructed. The plasmid successfully functions in the cells of both recipients (E. coli XL1 Blue and S. globisporus 1912-бп). It provides to them resistance to the corresponding antibiotics. The plasmid pWC 9,6 stably keeps its molecular size (16.2 kb). However, indisputable proofs of expression of the crt-clusters in transformants were not got. Conclusions. The plasmid pWC 9,6, that is able to transform and stably function in the cells of both recipient microorganisms (Streptomyces and E. coli) was constructed.
 Keywords: crt-cluster, shuttle vector, cloning, resistance, PCR.

Cloning of Lipase Gene From Thermomyces langinosus into Pichia pastoris with its Original Signal Peptide

Lipase is one of the most important industrial enzymes, which is widely used in the preparation of food additives, cosmetics, and pharmaceutical industries. In the previous study, we have cloned synthetic Thermomyces lanuginosus lipase gene into Bacillus subtilis and Escherichia coli and resulting low expression for enzyme activity. The aim of this research was to construct the Thermomyces lanuginosus lipase (TLL) gene into Pichia pastoris vector expression with TLL original signal peptide. TLL gene was amplified by PCR and contained original signal peptide and then inserted into pPICZα A between XhoI and XbaI site, and transformed into competent cell E.coli DH5α. From the transformant, two of positive recombinants were analyzed by sequencing analysis. As the result, both of two recombinant have a positive target gene which has lipase gene. The correct plasmid was linearized and then was transformed in Pichia pastoris X-33 by electroporation method. Thermomyces lanuginosus synthetic gene lipase has been successfully integrated into chromosome of P. pastoris X-33, which revealed by clear zones arund the colony on Yeast extract Peptone Dextrose Tributyrin (YPD.TB) plate with zeocin. The Thermomyces lanuginosus lipase had an open reading frame of 916bp encoding TLL of 314 amino acids with theoretical molecular mass of 35 kDa. The recombinant enzyme, Thermomyces lanuginosus lipase had optimal temperature at 80˚C and optimal pH at pH 8.

Glucose transporter 1(GLUT1) gene frequency distribution of XbaIG > T and HaeIIIT > C polymorphisms among different West Indian patients with type 2 diabetes mellitus

Glucose transporter 1 (GLUT1) which is also known as SLC2A1 is the major facilitative glucose transporter in glomerular mesangial cells. Since GLUT1 is involved in glucose uptake, the polymorphism of this GLUT1 may be an important risk factor in T2DM or in the progression of diabetes complications such as DN and CVD. A genetic association study involving West Indians patients was carried out to investigate the role of XbaI (intron 2) and HaeIII (exon 2) polymorphism in the GLUT1gene. Polymorphic XbaI and HaeIII site of GLUT1 gene were analyzed by PCR and PCR-RFLP. Real-time Quantitative PCR (qRT-PCR) of GLUT1 gene in different groups of the patient was also analyzed which shows down-regulation of mRNA transcript. The study population (n=631) consisted of 103 participants with T2DM, 102 participants with DN, 108 participants of DN without DM, 81 participants of CVD with DM, 94 patients of CVD without DM and 143 matched healthy participants. The analysis showed that the risk of developing DM, DN, and CVD in XbaI (−) carriers and HaeIII (−) carriers, when healthy individuals were considered as controls, DN shows significant value (P=0.027 and P=0.010). When DM, DN, CVD with DM and CVD without DM was compared with all groups DN and CVD with DM shows significant values with P=0.049 and 0.035 in XbaI (−) allele and P=0.040 and 0.010 in HaeIII (−) allele. GLUT1 XbaI (−) and HaeIII (−) both the allele is associated with DM, and possibly with a more severe form of the disease that can lead to development of DN and CVD.

Using Synthetic Biology methods to construct a rapid and efficient estrogen biosensor

Synthetic Biology demonstrates the integral relationship between biology, chemistry, mathematics and computer science. The field provides innovative approaches to a wide range of applications, such as bioremediation, sustainable energy production and biomedical therapies. Synthetic biology involves construction of genetic circuits when DNA sequences are modified to include standard restriction enzyme sites making the DNA sequences interchangeable and allowing for construction of a variety of circuits. New tools and devices are constantly needed to enhance the already extensive list of BioBricks housed at the Registry of Standard Biological Parts. As proof of concept we constructed a new BioBricks based on the toluene sensor built by Stiner and Haverson (AEM, 2002) in Pseudomonas. Toluene is an environmental contaminant that is the byproduct of oil contamination. Biosensors generated from BioBrick parts yield more affordable, transportable, and faster methods of pollutant detection. The tbuT cassette from Stiner and Haverson's work was digested with XbaI and SpeI sites then ligated with the GFP reporter gene and cloned into iGEM BioBrick vectors in order to create a biosensor in Escherichia coli. These BioBricks can be readily used with a variety of reporter genes to create new versions of the biosensor. We expanded our biosensor construction to create a dual functional estrogen sensitive BioBrick. The presence of estrogenic compounds (endocrine‐disruptors, EDCs) in the water supply raises concerns about human and aquatic health. Current methods for detecting estrogen contamination require expensive, time‐consuming techniques such as liquid chromatography‐mass spectrometry and high performance liquid chromatography. Previously reported estrogen biosensors required multiple cloning and transformation steps for successful detection in bacteria. Our estrogen responsive plasmid contains parts that allow for both the constitutive expression of the estrogen receptor followed by an estrogen response element toggled to a reporter gene in a single vector. When the estrogen response element is activated the reporter gene, red fluorescent protein, will produce a color change in E.coli. We will present data demonstrating the efficiency of this dual‐functional biosensor and it effectiveness for estrogenic compounds in contaminated water.Support or Funding InformationNSF grant #NSF‐GPG‐1444406, GPG: Research Collaboration: REU Pilot: Synthetic Biology Boot camp

In vitro regeneration and transient expression of recombinant sesquiterpene cyclase (SQC) in Artemisia annua L.

In the present study, we developed an efficient and reliable protocol for in vitro plant regeneration and transient gene expression in medicinal plant Artemisia annua L. For in vitro regeneration, leaf explants were cultivated in MS medium supplemented by combination of plant growth regulators including α-naphthalene-acetic acid, 6-benzyl-aminopurine, indole-3-acetic acid and 2,4-dichlorophenoxyacetic acid. The highest frequency of shoot organogenesis occurred on MS medium supplemented with 0.5mg/l NAA plus 2mg/l BA (86.6%) and MS medium supplemented with 1mg/l IAA plus 2mg/l BA (80%). Root induction was obtained on MS medium supplemented with 0.1mg/l IBA. This is a simple, reliable, rapid and high efficient regeneration system for A. annua L. in short period via adventitious shoot induction approach. Based on the sequence of a putative sesquiterpene cyclase from A. annua, 1.7kb fragment was amplified by PCR from Iranian variety of this plant after cDNA synthesis. Sesquiterpene cyclase gene was used for sub-cloning into pBI121 between XbaI and SacI sites to allow the expression of the gene driven by CaMV35S promoter. The recombinant plasmids were transferred to competent cells of Agrobacterium tumefaciens strain GV 3850 for vacuum agro-infiltration. HPLC results showed 30.09% increase in atremisinin content (0.15%) in the agroinfiltrated samples (0.67%) in comparison to the control (0.51%).

Engineered and Native Coenzyme B12-dependent Isovaleryl-CoA/Pivalyl-CoA Mutase

Adenosylcobalamin-dependent isomerases catalyze carbon skeleton rearrangements using radical chemistry. We have recently demonstrated that an isobutyryl-CoA mutase variant, IcmF, a member of this enzyme family that catalyzes the interconversion of isobutyryl-CoA and n-butyryl-CoA also catalyzes the interconversion between isovaleryl-CoA and pivalyl-CoA, albeit with low efficiency and high susceptibility to inactivation. Given the biotechnological potential of the isovaleryl-CoA/pivalyl-CoA mutase (PCM) reaction, we initially attempted to engineer IcmF to be a more proficient PCM by targeting two active site residues predicted based on sequence alignments and crystal structures, to be key to substrate selectivity. Of the eight mutants tested, the F598A mutation was the most robust, resulting in an ∼17-fold increase in the catalytic efficiency of the PCM activity and a concomitant ∼240-fold decrease in the isobutyryl-CoA mutase activity compared with wild-type IcmF. Hence, mutation of a single residue in IcmF tuned substrate specificity yielding an ∼4000-fold increase in the specificity for an unnatural substrate. However, the F598A mutant was even more susceptible to inactivation than wild-type IcmF. To circumvent this limitation, we used bioinformatics analysis to identify an authentic PCM in genomic databases. Cloning and expression of the putative AdoCbl-dependent PCM with an α2β2 heterotetrameric organization similar to that of isobutyryl-CoA mutase and a recently characterized archaeal methylmalonyl-CoA mutase, allowed demonstration of its robust PCM activity. To simplify kinetic analysis and handling, a variant PCM-F was generated in which the αβ subunits were fused into a single polypeptide via a short 11-amino acid linker. The fusion protein, PCM-F, retained high PCM activity and like PCM, was resistant to inactivation. Neither PCM nor PCM-F displayed detectable isobutyryl-CoA mutase activity, demonstrating that PCM represents a novel 5'-deoxyadenosylcobalamin-dependent acyl-CoA mutase. The newly discovered PCM and the derivative PCM-F, have potential applications in bioremediation of pivalic acid found in sludge, in stereospecific synthesis of C5 carboxylic acids and alcohols, and in the production of potential commodity and specialty chemicals.

Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2

The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2.

Overexpression and optimization of glutamate decarboxylase in Lactobacillus plantarum Taj-Apis362 for high gamma-aminobutyric acid production.

Gamma-aminobutyric acid (GABA) is an important bioactive compound biosynthesized by microorganisms through decarboxylation of glutamate by glutamate decarboxylase (GAD). In this study, a full-length GAD gene was obtained by cloning the template deoxyribonucleic acid to pTZ57R/T vector. The open reading frame of the GAD gene showed the cloned gene was composed of 1410 nucleotides and encoded a 469 amino acids protein. To improve the GABA-production, the GAD gene was cloned into pMG36e-LbGAD, and then expressed in Lactobacillus plantarum Taj-Apis362 cells. The overexpression was confirmed by SDS-PAGE and GAD activity, showing a 53 KDa protein with the enzyme activity increased by sevenfold compared with the original GAD activity. The optimal fermentation conditions for GABA production established using response surface methodology were at glutamic acid concentration of 497.973 mM, temperature 36°C, pH 5.31 and time 60 h. Under the conditions, maximum GABA concentration obtained (11.09 mM) was comparable with the predicted value by the model at 11.23 mM. To our knowledge, this is the first report of successful cloning (clone-back) and overexpression of the LbGAD gene from L. plantarum to L. plantarum cells. The recombinant Lactobacillus could be used as a starter culture for direct incorporation into a food system during fermentation for production of GABA-rich products.

Estrogen receptors 1 and 2 genotypes and age at menarche in idiopathic scoliosis

Objectives The age at menarche (AAM) is commonly in use in patients with IS as one of the maturity indicator suggesting deceleration of the growth velocity. The AAM was suggested to be related to predisposition and curve progression potential of IS. The late age at menarche was reported to be associated with higher prevalence of adolescent idiopathic scoliosis. The age at menarche is determined by both genetic and environmental factors as well as their interactions. Estrogen receptors 1 and 2 polymorphism were reported to be associated with AAM: in ESR1 XbaI and PvuII site polymorphism and in ESR2 AluI site polymorphism. The purpose of the study was to investigate associations of the ESR1 and ESR2 polymorphisms with AAM in IS patients.

Estrogen receptors genes polymorphisms and age at menarche in idiopathic scoliosis.

BackgroundThe age at menarche (AAM) is commonly in use in patients with IS as one of the maturity indicator suggesting deceleration of the growth velocity. The AAM was suggested to be related to predisposition and curve progression potential of IS. The late age at menarche was reported to be associated with higher prevalence of adolescent idiopathic scoliosis. The age at menarche is determined by both genetic and environmental factors as well as their interactions. Estrogen receptors 1 and 2 polymorphism were reported to be associated with AAM: in ESR1 XbaI and PvuII site polymorphism and in ESR2 AluI site polymorphism.The purpose of the study was to investigate associations of the ESR1 and ESR2 polymorphisms with AAM in IS patients and to evaluate association of AAM with IS severity.Methods208 females with IS Caucasian females from Central Europe underwent clinical, radiological and genetic examinations. Four SNPs were selected XbaI (A/Grs9340799) and PvuII (C/T rs2234693) in ESR1 and AluI (A/G rs4986938) and RasI (A/G rs1256049) in ESR2. Samples were analyzed with polymerase chain reaction followed by restriction fragments length polymorphism analysis (PCR-RFLP). The age of a menarche was established during personal interview with the patients and in case of children with their parents. The Cobb angle was measured.ResultsAll genotypes followed HWE. Mean AAM for patients was 154.8 ± 14.7 months (12.9 ± 1.2 years). The earliest AAM was 121 and latest 192 months. There was no statistically significant difference between AAM mean values in each genotype, for the XbaI, PvuII, AluI and RsaI site polymorphisms the p values were p = 0.7141, p = 0.9774, p = 0.7973 and p = 0.2282, respectively. Patients divided according to Cobb into mild (<30°), moderate (30°-49°) or severe (≥50°) IS revealed tendency to delay AAM: 151.9 ± 14.7; 155.2 ± 14.8 and 157.9 ± 14.0 months, respectively. There was statistical significant difference between patients with mild <30° and severe ≥50° IS, p = 0.0267.ConclusionsIn IS patients estrogen receptors polymorphisms did not show association with the AAM. Patients with severe IS form revealed delayed AAM than patients with mild IS form.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2474-15-383) contains supplementary material, which is available to authorized users.

Identification of five sea cucumber species through PCR-RFLP analysis

Sea cucumbers are traditional marine food and Chinese medicine in Asia. The rapid expansion of sea cucumber market has resulted in various problems, such as commercial fraud and mislabeling. Conventionally, sea cucumber species could be distinguished by their morphological and anatomical characteristics; however, their identification becomes difficult when they are processed. The aim of this study was to develop a new convenient method of identifying and distinguishing sea cucumber species. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of mitochondrial cytochrome oxidase I gene (COI) was used to identifing five sea cucumber species (Apostichopus japonicus, Cucumaria frondosa, Thelenota ananas, Parastichopus californicus and Actinopyga lecanora). A 692 bp fragment of COI was searched for BamHI, KpnI, PstI, XbaI and Eco31I restriction sites with DNAMAN 6.0, which were then used to PCR-RFLP analysis. These five sea cucumber species can be discriminated from mixed sea cucumbers. The developed PCR-RFLP assay will facilitate the identification of sea cucumbers, making their source tracing and quality controlling feasible.

Remodeling of Marrow Hematopoietic Stem and Progenitor Cells by Non-self ST6Gal-1 Sialyltransferase

Glycans occupy the critical cell surface interface between hematopoietic cells and their marrow niches. Typically, glycosyltransferases reside within the intracellular secretory apparatus, and each cell autonomously generates its own cell surface glycans. In this study, we report an alternate pathway to generate cell surface glycans where remotely produced glycosyltransferases remodel surfaces of target cells and for which endogenous expression of the cognate enzymes is not required. Our data show that extracellular ST6Gal-1 sialyltransferase, originating mostly from the liver and released into circulation, targets marrow hematopoietic stem and progenitor cells (HSPCs) and mediates the formation of cell surface α2,6-linked sialic acids on HSPCs as assessed by binding to the specific lectins Sambucus nigra agglutinin and Polysporus squamosus lectin and confirmed by mass spectrometry. Marrow HSPCs, operationally defined as the Lin-c-Kit+ and Lin-Sca-1+c-Kit+ populations, express negligible endogenous ST6Gal-1. Animals with reduced circulatory ST6Gal-1 have marrow Lin-Sca-1+c-Kit+ cells with reduced S. nigra agglutinin reactivity. Bone marrow chimeras demonstrated that α2,6-sialylation of HSPCs is profoundly dependent on circulatory ST6Gal-1 status of the recipients and independent of the ability of HSPCs to express endogenous ST6Gal-1. Biologically, HSPC abundance in the marrow is inversely related to circulatory ST6Gal-1 status, and this relationship is recapitulated in the bone marrow chimeras. We propose that remotely produced, rather than the endogenously expressed, ST6Gal-1 is the principal modifier of HSPC glycans for α2,6-sialic acids. In so doing, liver-produced ST6Gal-1 may be a potent systemic regulator of hematopoiesis.

A Novel Non-canonical Forkhead-associated (FHA) Domain-binding Interface Mediates the Interaction between Rad53 and Dbf4 Proteins

Forkhead-associated (FHA) and BRCA1 C-terminal (BRCT) domains are overrepresented in DNA damage and replication stress response proteins. They function primarily as phosphoepitope recognition modules but can also mediate non-canonical interactions. The latter are rare, and only a few have been studied at a molecular level. We have identified a crucial non-canonical interaction between the N-terminal FHA1 domain of the checkpoint effector kinase Rad53 and the BRCT domain of the regulatory subunit of the Dbf4-dependent kinase that is critical to suppress late origin firing and to stabilize stalled forks during replication stress. The Rad53-Dbf4 interaction is phosphorylation-independent and involves a novel non-canonical interface on the FHA1 domain. Mutations within this surface result in hypersensitivity to genotoxic stress. Importantly, this surface is not conserved in the FHA2 domain of Rad53, suggesting that the FHA domains of Rad53 gain specificity by engaging additional interaction interfaces beyond their phosphoepitope-binding site. In general, our results point to FHA domains functioning as complex logic gates rather than mere phosphoepitope-targeting modules.

25-Hydroxycholesterol Activates the Integrated Stress Response to Reprogram Transcription and Translation in Macrophages

25-Hydroxycholesterol (25OHC) is an enzymatically derived oxidation product of cholesterol that modulates lipid metabolism and immunity. 25OHC is synthesized in response to interferons and exerts broad antiviral activity by as yet poorly characterized mechanisms. To gain further insights into the basis for antiviral activity, we evaluated time-dependent responses of the macrophage lipidome and transcriptome to 25OHC treatment. In addition to altering specific aspects of cholesterol and sphingolipid metabolism, we found that 25OHC activates integrated stress response (ISR) genes and reprograms protein translation. Effects of 25OHC on ISR gene expression were independent of liver X receptors and sterol-response element-binding proteins and instead primarily resulted from activation of the GCN2/eIF2α/ATF4 branch of the ISR pathway. These studies reveal that 25OHC activates the integrated stress response, which may contribute to its antiviral activity.

Analyses of Conformational States of the Transporter Associated with Antigen Processing (TAP) Protein in a Native Cellular Membrane Environment

The transporter associated with antigen processing (TAP) plays a critical role in the MHC class I antigen presentation pathway. TAP translocates cellular peptides across the endoplasmic reticulum membrane in an ATP hydrolysis-dependent manner. We used FRET spectroscopy in permeabilized cells to delineate different conformational states of TAP in a native subcellular membrane environment. For these studies, we tagged the TAP1 and TAP2 subunits with enhanced cyan fluorescent protein and enhanced yellow fluorescent protein, respectively, C-terminally to their nucleotide binding domains (NBDs), and measured FRET efficiencies under different conditions. Our data indicate that both ATP and ADP enhance the FRET efficiencies but that neither induces a maximally closed NBD conformation. Additionally, peptide binding induces a large and significant increase in NBD proximity with a concentration dependence that is reflective of individual peptide affinities for TAP, revealing the underlying mechanism of peptide-stimulated ATPase activity of TAP. Maximal NBD closure is induced by the combination of peptide and non-hydrolysable ATP analogs. Thus, TAP1-TAP2 NBD dimers are not fully stabilized by nucleotides alone, and substrate binding plays a key role in inducing the transition state conformations of the NBD. Taken together, these findings show that at least three steps are involved in the transport of peptides across the endoplasmic reticulum membrane for antigen presentation, corresponding to three dynamically and structurally distinct conformational states of TAP. Our studies elucidate structural changes in the TAP NBD in response to nucleotides and substrate, providing new insights into the mechanism of ATP-binding cassette transporter function.