HindIII Site Research Articles

Milk Protein Genes Polymorphism in Indigenous and Crossbred Cattle from a private Dairy Farm in Ethiopia

Kappa casein and beta lactoglobulin genes are major milk proteins which have a direct effect on protein content in dairy cattle. Molecular-based selection through the identification of genetic polymorphism of major protein genes can be used to gain genetic improvement of milk protein yield. The objective of this study was to identify kappa casein (CSN3) and beta lactoglobulin (LGB) genes polymorphisms in indigenous and crossbreed cattle. A total of 90 whole blood samples were collected from individual animal in a private dairy farm. DNA extraction and quality assessment were done salting out procedure and gel electrophoresis, respectively. Polymerase chain reaction (PCR) was performed with gene specific primers. For genotyping, PCR products of CSN3 gene was digested with HinfI and HindIII while LGB gene was digested with HaeIII restriction enzymes. Two haplotypes A and B; three genotypes, AA, AB and BB were observed at CSN3 of HinfI site and LGB HaeIII site, but only AA and AB were observed at CSN3 of HindIII site in crossbred and indigenous cattle populations. However, at CSN3 locus, A allele was found to be more common (0.65) in indigenous cattle than the B allele (0.35), while B (0.51) allele and AB genotype (0.81) were more frequent in crossbred cattle. But, LGB B allele was higher in indigenous cattle (0.67) compared to Allele A (0.33). LGB BB genotype (0.57) is higher followed by AA genotype in indigenous cattle while both allele and genotypic frequencies are equal in crossbred cattle. Both CSN3 and LGB loci were polymorphic in studied populations. Expected heterozygosity was higher in crossbred (0.49, 0.50) than in indigenous (0.38, 0.33) cattle at CSN3 and LGB locus, respectively which might be due to breed variation. The current findings showed that both CSN3 and LGB genes could be promising diagnostic markers in selecting dairy cattle breed. However, further investigations with large sample size and association study with milk composition is required to substantiate the current result.

Efficient and Economical Targeted Insertion in Plant Genomes via Protoplast Regeneration.

Versatile genome editing can be facilitated by the insertion of DNA sequences into specific locations. Current protocols involving CRISPR and Cas proteins rely on low efficiency homology-directed repair or non-homologous end joining with modified double-stranded DNA oligonucleotides as donors. Our simple protocol eliminates the need for expensive equipment, chemical and enzymatic donor DNA modification, or plasmid construction by using polyethylene glycol-calcium to deliver non-modified single-stranded DNA oligonucleotides and CRISPR-Cas9 ribonucleoprotein into protoplasts. Plants regenerated via edited protoplasts achieved targeted insertion frequencies of up to 50% in Nicotiana benthamiana and 13.6% in rapid cycling Brassica oleracea without antibiotic selection. Using a 60 nt donor containing 27 nt in each homologous arm, 6/22 regenerated N. benthamiana plants showed targeted insertions, and one contained a precise insertion of a 6 bp HindIII site. The inserted sequences were transmitted to the next generation and invite the possibility of future exploration of versatile genome editing by targeted DNA insertion in plants.

Overexpression of the ascorbate peroxidase through enhancer-trapped pSB111 bar vector for alleviating drought stress in rice.

Rice (Oryza sativa L.) plant growth and productivity is adversely affected by various stress factors. Overexpression of drought tolerance-related genes is one of the best approaches for developing drought-resistant transgenics. Agrobacterium tumefaciens has been widely used in generating transgenic plants through plasmid vector to obtain desired characteristics and to know the specific expression profiles of genes in the plant. The enhancer trap method was developed to know the specific expression of genes at different stages of growth by entrapping the genes of an organism. In the present study, we designed a vector molecule with a feature of promoting the expression of a specific gene more than four times than its normal expression and it is useful for efficient transformation to higher plants by utilizing the trans configuration of vir genes of the plasmid A. tumefaciens, to transfer right and left sequence bordered of transferred DNA (T-DNA) into the nuclear genome of plants. We developed a binary vector consisting of 1.8-kb green fluorescent protein (GFP) cassette as a reporter gene and 1.4-kb tetramer of CaMv35S enhancer (4XEn) were cloned at HindIII site of pSB11 bar intermediate vector to tag and know the genes and their expression profiles, then mobilized into A. tumefaciens to produce a super-binary vector pSB111-bar-4XEn-GFP. The resultant construct was confirmed by polymerase chain reaction and restriction digestion methods. Finally, we discuss the role of overexpressed ascorbate peroxidase in drought stress.

Molecular cloning and characterizing of Bacillus subtilis cellulase collected from central-northern Iran forests

In the large-scale commercial bioconversion of complex lignocellulosic materials to biofuels, a cost-effective purification and production of cellulase is a huge concern. In this regard, genetically engineered strains have shown great merits for producing active cellulase. As an attempt to improve cellulase production; herein, bacterial strains with cellulolytic activity were collected from forest soils located across Mazandaran Province, in central-northern Iran. To verify cellulolytic activity, carboxymethyl cellulose (CMC) in aka cellulose gum medium was used. Afterward, in order to characterize the bacteria with most cellulolytic activity, 16S rDNA of bacterial colonies were amplified and then phylogenetic analysis was conducted. Cellulase gene was isolated from the most active bacterial strain, Bacillus subtilis B2 and then it was cloned into the bacterial expression vector pET-26b using HindIII and BamHI restriction endonuclease sites. The generated plasmid containing cellulase and expression promoter system was transformed into the BL21 (DE3) competent Escherichia coli. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis was performed to verify presence of the protein. The sequencing of cloned gene revealed 77% identity with endoglucanase of Bacillus subtilis subsp. Moreover, the molecular weight of cloned cellulase was 48 k Da as revealed by SDS-PAGE. Significance statementHerein, a newly isolated bacterial strain with high potential of cellulase production is introduced that benefits commercial bioconversion of complex lignocellulosic materials to biofuels.

Expression and Purification of a Bispecific Antibody against CD16 and Hemagglutinin Neuraminidase (HN) in E. Coli for Cancer Immunotherapy

: Immunotherapy of cancer by bispecific antibodies (bsAb) is an attractive approach for retargeting immune effector cells including natural killer (NK) cells to the tumor if the proper expression and purification of the bsAb for such applications could be addressed. Herein, we describe E. coli expression of a recombinant bsAb (bsHN-CD16) recognizing NK-CD16 and hemagglutinin neuraminidase (HN) of Newcastle Disease Virus (NDV). This bsAb might be efficient for ex vivo stimulation of NK cells via coupling to HN on the surface of the NDV-infected tumor cells. A bsAb-encoding pcDNA3.1 vector (anti-HN scFv-Fc-anti-CD16 scFv) was used as a template, and the scFv segments (after enzymatic digestion and cutting of the Fc part) were rejoined to construct the Fc-deprived bsAb (anti-HN scFv-anti-CD16 scFv; bsHN-CD16). The constructed bsHN-CD16 was inserted into the HindIII and BamHI site of the T7 promoter-based pET28a plasmid. Following restriction analyses and DNA sequencing to confirm the cloning steps, bsHN-CD16 encoding pET28a was transformed into the E. coli (Rosetta DE3 strain), induced for protein expression by IPTG, and the protein was purified under native condition by Ni/NTA column using imidazole. Analyses by SDS-PAGE and Western Blotting using Rabbit anti-human whole IgG-HRP conjugate, confirmed the expression and purification of the bsAb with the expected full size of 55 kDa and yields around 8% of the total protein. Results showed efficient production of the bsAb in E. coli for future large-scale purification.

A new molecular method for the rapid subtyping of bovine herpesvirus 1 field isolates.

Bovine herpesvirus 1 (BoHV-1) causes several clinical syndromes in cattle worldwide. There are 3 subtypes of BoHV-1: 1.1, 1.2a, and 1.2b. Several molecular methods are commonly used in the detection and characterization of BoHV-1. Among them, restriction endonuclease analysis (REA) and single-nucleotide polymorphism (SNP) analysis of the complete viral genome allow classification of BoHV-1 into different subtypes. However, developing countries need simpler and cheaper screening assays for routine testing. We designed a standard multiplex PCR followed by a REA assay allowing straightforward subclassification of all BoHV-1 isolates tested into 1.1, 1.2a, and 1.2b subtypes based on the analysis of fragment length polymorphism. Our standard multiplex PCR-REA was used to analyze 33 field strains of BoHV-1 isolated from various tissues. The assay confirmed the subtype identified previously by REA. In addition, non-polymorphic or undigested fragments were sequenced in order to confirm the mutation affecting the RE HindIII site. Our PCR-REA method is an affordable and rapid test that will subtype all BoHV-1 strains.

C synthetic gene of CryIAb-CryIAc fusion to generate resistant sugarcane to shoot or stem borer

The sugar yield lost caused by borer is about 10% or equal to 1000 kg of sugar/ha. There is no resistance trait to stem or shoot borer available in sugarcane germplasm. Genetic engineering by expressing of the Cry1Ab-Cry1Ac gene fusion is efforts to develop resistant variety to borer. A synthetic gene which consists of Rubisco gene promoter, chloroplast specific transit peptide (CTP) and the Cry1Ab-Cry1Ac was designed and assembled for a total size of 4019bp. It was inserted into pU3775CE plasmid cloning, and then into pCAMBIA5300_Ubi-tNOS plasmid vector at HindIII and KpnI sites, producing of pCAMBIA5300_RbcS::Cry1Ab- Cry1Ac. This plasmid was transforming into Agrobacterium tumefaciens and then transformed into Bulu Lawang (BL) sugarcane calli. Research aims were to identify lines produced from plant transformation molecularly and to evaluate their resistances against shoot borer. Methods applied were a DNA isolation and PCR using KAPA 2G ready mix and CryIAc-316F and CryIAc-316R primers, and plant bioassay with larva instar 1. Research progresses were 30 lines had been identified and proved containing of the cryIAc gene. Preliminary results of bioassay showed that there was variation among 10 BL Cry lines on shoots (from healthy to wilt and dry) and also on shoot borer conditions.

Konstruksi Vektor Biner Mengandung Gen Hibrid cryIB-cryIAa untuk Transformasi Agrobacterium Tanaman Padi (Oryza sativa L)

This study aimed to obtain binary vectors carrying cryIB-cryIAa hybrid gene suitable for Agrobacterium-mediated transformation of two Indonesian rice cultivars, Rajalele (Javanica) and Cisadane (Indica). Introduction of hybrid Bt toxin, encoded by cryIB-cryIAa hybrid gene with different binding receptor in the insect gut driven by maize ubiquitin promoter, was intended to reduce the possibility of the appearance of stem borer resistant to Bt toxin. A HindIII fragment containing the ubi cryIB-cryIAa hybrid gene was excised from pBKS ubi cryIB-cryIAa and cloned into the HindIII site of binary vector pCAMBIA 1301, 1303, or 1304. Restriction analysis of recombinant plasmids confirmed the insertion of the gene and demonstrated that the gene inserted in two orientations. The recombinant plasmids were transformed into electrocompetent Agrobacterium tumefaciens EHA105 by electroporation. Embryogenic calli of Rajalele and Cisadane were co-cultivated with A. tumefaciens EHA 105 harbouring binary vector pCAMBIA 1301, 1303, or 1304 containing ubi cryIB-cryIAa. GUS histochemical assay revealed that high transformation efficiency (75-100%) on both cultivars were obtained from embryogenic calli transformed with pCAMBIA 1303 or 1304.

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Precise Construction and Tuning of an Aerolysin Single-Biomolecule Interface for Single-Molecule Sensing

Relationship between porcine miR-20a and its putative target low-density lipoprotein receptor based on dual luciferase reporter gene assays.

ObjectiveMutations in low-density lipoprotein receptor (LDLR), which encodes a critical protein for cholesterol homeostasis and lipid metabolism in mammals, are involved in cardiometabolic diseases, such as familial hypercholesterolemia in pigs. Whereas microRNAs (miRNAs) can control LDLR regulation, their involvement in circulating cholesterol and lipid levels with respect to cardiometabolic diseases in pigs is unclear. We aimed to identify and analyze LDLR as a potential target gene of SSC-miR-20a.MethodsBioinformatic analysis predicted that porcine LDLR is a target of SSC-miR-20a. Wild-type and mutant LDLR 3′-untranslated region (UTR) fragments were generated by polymerase chain reaction (PCR) and cloned into the pGL3-Control vector to construct pGL3 Control LDLR wild-3′-UTR and pGL3 Control LDLR mutant-3′-UTR recombinant plasmids, respectively. An miR-20a expression plasmid was constructed by inserting the porcine pre-miR-20a-coding sequence between the HindIII and BamHI sites in pMR-mCherry, and constructs were confirmed by sequencing. HEK293T cells were co-transfected with the miR-20a expression or pMR-mCherry control plasmids and constructs harboring the corresponding 3′-UTR, and relative luciferase activity was determined. The relative expression levels of miR-20a and LDLR mRNA and their correlation in terms of expression levels in porcine liver tissue were analyzed using reverse-transcription quantitative PCR.ResultsGel electrophoresis and sequencing showed that target gene fragments were successfully cloned, and the three recombinant vectors were successfully constructed. Compared to pMR-mCherry, the miR-20a expression vector significantly inhibited wild-type LDLR-3′-UTR-driven (p<0.01), but not mutant LDLR-3′-UTR-driven (p>0.05), luciferase reporter activity. Further, miR-20a and LDLR were expressed at relatively high levels in porcine liver tissues. Pearson correlation analysis revealed that porcine liver miR-20a and LDLR levels were significantly negatively correlated (r = −0.656, p<0.05).ConclusionLDLR is a potential target of miR-20a, which might directly bind the LDLR 3′-UTR to post-transcriptionally inhibit expression. These results have implications in understanding the pathogenesis and progression of porcine cardiovascular diseases.

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.&#x0D; Keywords: crt-cluster, shuttle vector, cloning, resistance, PCR.

Heterologous expression of pikromycin biosynthetic gene cluster using Streptomyces artificial chromosome system

BackgroundHeterologous expression of biosynthetic gene clusters of natural microbial products has become an essential strategy for titer improvement and pathway engineering of various potentially-valuable natural products. A Streptomyces artificial chromosomal conjugation vector, pSBAC, was previously successfully applied for precise cloning and tandem integration of a large polyketide tautomycetin (TMC) biosynthetic gene cluster (Nah et al. in Microb Cell Fact 14(1):1, 2015), implying that this strategy could be employed to develop a custom overexpression scheme of natural product pathway clusters present in actinomycetes.ResultsTo validate the pSBAC system as a generally-applicable heterologous overexpression system for a large-sized polyketide biosynthetic gene cluster in Streptomyces, another model polyketide compound, the pikromycin biosynthetic gene cluster, was preciously cloned and heterologously expressed using the pSBAC system. A unique HindIII restriction site was precisely inserted at one of the border regions of the pikromycin biosynthetic gene cluster within the chromosome of Streptomyces venezuelae, followed by site-specific recombination of pSBAC into the flanking region of the pikromycin gene cluster. Unlike the previous cloning process, one HindIII site integration step was skipped through pSBAC modification. pPik001, a pSBAC containing the pikromycin biosynthetic gene cluster, was directly introduced into two heterologous hosts, Streptomyces lividans and Streptomyces coelicolor, resulting in the production of 10-deoxymethynolide, a major pikromycin derivative. When two entire pikromycin biosynthetic gene clusters were tandemly introduced into the S. lividans chromosome, overproduction of 10-deoxymethynolide and the presence of pikromycin, which was previously not detected, were both confirmed. Moreover, comparative qRT-PCR results confirmed that the transcription of pikromycin biosynthetic genes was significantly upregulated in S. lividans containing tandem clusters of pikromycin biosynthetic gene clusters.ConclusionsThe 60 kb pikromycin biosynthetic gene cluster was isolated in a single integration pSBAC vector. Introduction of the pikromycin biosynthetic gene cluster into the pikromycin non-producing strains resulted in higher pikromycin production. The utility of the pSBAC system as a precise cloning tool for large-sized biosynthetic gene clusters was verified through heterologous expression of the pikromycin biosynthetic gene cluster. Moreover, this pSBAC-driven heterologous expression strategy was confirmed to be an ideal approach for production of low and inconsistent natural products such as pikromycin in S. venezuelae, implying that this strategy could be employed for development of a custom overexpression scheme of natural product biosynthetic gene clusters in actinomycetes.

Cardiomyocyte-Specific Human Bcl2-Associated Anthanogene 3 P209L Expression Induces Mitochondrial Fragmentation, Bcl2-Associated Anthanogene 3 Haploinsufficiency, and Activates p38 Signaling

The Bcl2-associated anthanogene (BAG) 3 protein is a member of the BAG family of cochaperones, which supports multiple critical cellular processes, including critical structural roles supporting desmin and interactions with heat shock proteins and ubiquitin ligases intimately involved in protein quality control. The missense mutation P209L in exon 3 results in a primarily cardiac phenotype leading to skeletal muscle and cardiac complications. At least 10 other Bag3 mutations have been reported, nine resulting in a dilated cardiomyopathy for which no specific therapy is available. We generated αMHC-human Bag3 P209L transgenic mice and characterized the progressive cardiac phenotype invivo to investigate its utility in modeling human disease, understand the underlying molecular mechanisms, and identify potential therapeutic targets. We identified a progressive heart failure by echocardiography and Doppler analysis and the presence of pre-amyloid oligomers at 1 year. Paralleling the pathogenesis of neurodegenerative diseases (eg, Parkinson disease), pre-amyloid oligomers-associated alterations in cardiac mitochondrial dynamics, haploinsufficiency of wild-type BAG3, and activation of p38 signaling were identified. Unexpectedly, increased numbers of activated cardiac fibroblasts were identified in Bag3 P209L Tg+ hearts without increased fibrosis. Together, these findings point to a previously undescribed therapeutic target that may have application to mutation-induced myofibrillar myopathies as well as other common causes of heart failure that commonly harbor misfolded proteins.

Selective Inhibition of Bacterial Tryptophanyl-tRNA Synthetases by Indolmycin Is Mechanism-based

Indolmycin is a natural tryptophan analog that competes with tryptophan for binding to tryptophanyl-tRNA synthetase (TrpRS) enzymes. Bacterial and eukaryotic cytosolic TrpRSs have comparable affinities for tryptophan (Km ∼ 2 μm), and yet only bacterial TrpRSs are inhibited by indolmycin. Despite the similarity between these ligands, Bacillus stearothermophilus (Bs)TrpRS preferentially binds indolmycin ∼1500-fold more tightly than its tryptophan substrate. Kinetic characterization and crystallographic analysis of BsTrpRS allowed us to probe novel aspects of indolmycin inhibitory action. Previous work had revealed that long range coupling to residues within an allosteric region called the D1 switch of BsTrpRS positions the Mg(2+) ion in a manner that allows it to assist in transition state stabilization. The Mg(2+) ion in the inhibited complex forms significantly closer contacts with non-bridging oxygen atoms from each phosphate group of ATP and three water molecules than occur in the (presumably catalytically competent) pre-transition state (preTS) crystal structures. We propose that this altered coordination stabilizes a ground state Mg(2+)·ATP configuration, accounting for the high affinity inhibition of BsTrpRS by indolmycin. Conversely, both the ATP configuration and Mg(2+) coordination in the human cytosolic (Hc)TrpRS preTS structure differ greatly from the BsTrpRS preTS structure. The effect of these differences is that catalysis occurs via a different transition state stabilization mechanism in HcTrpRS with a yet-to-be determined role for Mg(2+). Modeling indolmycin into the tryptophan binding site points to steric hindrance and an inability to retain the interactions used for tryptophan substrate recognition as causes for the 1000-fold weaker indolmycin affinity to HcTrpRS.

Construction of a recombinant baculovirus expressing the hemaglutimin (ha) of an A/H5N1 clade 2.3.3.1b virus

Highly pathogenic avian influenza A (H5N1) virus is still a threat to public health, especially, the emergence of a new clade recently with more virulent and resistant to the vaccine developed from the previously circulating clade has raised the need for development of new vaccines. With the ultimate aim of producing influenza virus-like particles (VLPs) vaccine, in this study we created recombinant baculovirus expressing Hemagglutini (HA) from H5N1 virus clade 2.3.2.1b isolated in Quang Ngai province (DkQN37/11) – which is resistant to NIBRG14 (clade 1) currently used in Vietnam. The HA - encoding gene was amplified by PCR with a specific pair of primers, which has Kozak sequence, initation codon and BamHI site at the 5’ of the forward primer and the recognition sequence of HindIII at the 5' of the reverse primer. The PCR product was cloned into pCR2.1 and sequenced then inserted into the baculovirus vector (pBluBac4.5 / V5-His-TOPO, Invitrogen) at BamHI and HindIII sites to yield pBuBacHA. Vector pBluBacHA then co-transfected with baculovirus DNA into insect cells (Sf9) to generate recombinant baculovirus. The presence of ha gene and the expression of recombinant HA protein in the recombinant virus were detemined by PCR and Western blot using anti-HA antibody. PCR and Western blot results confirmed that we have successfully created a recombinant baculovirus expressing surface antigens of influenza A/H5N1 clade 2.3.2.1b. Our work provides essential material for further study to develop A/H5N1 VLP vaccine in Vietnam.

Expression of Recombinant pET22b-LysK-Cysteine/Histidine-Dependent Amidohydrolase/Peptidase Bacteriophage Therapeutic Protein in Escherichia coli BL21 (DE3)

ObjectivesBacteriophage-encoded endolysins are a group of enzymes that act by digesting the peptidoglycan of bacterial cell walls. LysK has been reported to lyse live staphylococcal cultures. LysK proteins containing only the cysteine/histidine-dependent amidohydrolase/peptidase (CHAP) domain has the capability to show lytic activity against live clinical staphylococcal isolates, including methicillin-resistant Staphylococcus aureus (MRSA). The aim of this study was to clone and express LysK-CHAP domain in Escherichia coli BL21 (DE3) using pET22b as a secretion vector. The pET22b plasmid was used, which encoded a pelB secretion signal under the control of the strong bacteriophage T7 promoter. MethodsThe E. coli cloning strains DH5α and BL21 (DE3) were grown at 37°C with aeration in the Luria-Bertani medium. A plasmid encoding LysK-CHAP in a pET22b backbone was constructed. The pET22b vector containing LysK-CHAP sequences were digested with NcoI and HindIII restriction enzymes. Cloning accuracy was confirmed by electrophoresis. The pET22b-LysK plasmid was used to transform the E. coli strain BL21. Isopropyl β-d-1-thiogalactopyranoside (IPTG) was added to a final concentration of 1mM to induce T7 RNA polymerase-based expression. Finally, western blot confirmed the expression of target protein. ResultsIn this study, after double digestion of pEX and pET22b vectors with HindIII and NcoI, LysK gene was cloned into two HindIII and NcoI sites in pET22b vector, and then transformed to E. coli DH5α. Cloning was confirmed with double digestion and analyzed with agarose gel. The recombinant pET22b-LysK plasmid was transformed to E. coli BL21 and the expression was induced by IPTG. The expression was confirmed by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting method. Observation of a 28.5 kDa band confirmed LysK protein expression. ConclusionIn the present study, LysK-CHAP domain was successfully cloned and expressed at the pET22b vector and E. coli BL21 (DE3).

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.

Soluble and functional expression of a recombinant enantioselective amidase from Klebsiella oxytoca KCTC 1686 in Escherichia coli and its biochemical characterization

A gene encoding an enantioselective amidase (KamH) was cloned from Klebsiella oxytoca KCTC 1686 and inserted into the EcoRI and HindIII sites of the vector pET-30a(+). When KamH with a peptide containing a His-tag and an enterokinase cleavage site was overexpressed in Escherichia coli, approximately half was found in the soluble fraction, but it lacked activity. After cleavage of the peptide by enterokinase, the enzyme activity was partly restored, reaching 420.2±33.62U/g dry cell weight (DCW). Another recombinant plasmid was constructed by inserting the KamH gene into the NdeI and EcoRI sites of pET-30a(+) to express KamH in its native form. The overexpressed amidase was found primarily in the soluble fraction and its maximum activity was 3613.4±201.68U/g DCW. This indicated that the peptide influenced not only soluble expression but also activity of KamH, perhaps by blocking the substrate-binding tunnel of KamH. Similar results were obtained with heterologously expressed amidases from Rhodococcus erythropolis MP50 and Agrobacterium tumefaciens d3. All of these amidases have an N-terminal α-helical domain. Therefore, amidases of this type may be functionally expressed in their native form. KamH hydrolyzed a range of aliphatic and aromatic amides and exhibited strict S-selectivity towards racemic amides.

Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.

Spectrum of β-Globin Gene Mutations and β-Thalassemia Haplotype Analysis among the Iranian Azeri Turkish Population

Background: A variety of mutations influencing gene transcription, translation, or mRNA processing have been identified in β-thalassemia. Several studies demonstrated a nonrandom linkage of particular RFLP haplotypes with specific β-thalassemia mutations. Linkage analysis using β-globin haplotypes is a valuable tool for indirect mutation detection. Our aim was to determine the spectrum and frequency of β-thalassemia mutations among Northwestern Iranians (Azeri Turkish population) along with determining the heterozygosity and polymorphism information content (PIC) value for seven β-globin markers. Method: We investigated spectrum of β-thalassemia mutations via ARMS-PCR technique followed by sequencing technique. Also β-globin gene cluster haplotypes was determined by PCR-RFLP technique on DNA samples of the patients and his (her) parents or siblings. Result: We detected 36 different mutations among 554 non-identical mutant chromosomes. The most frequent mutation was IVSII-1 (G>A) (23.6%), followed by IVSI-110 (G>A) (11.9%) and CD8 (-AA) (10.5%). Of the various β- globin gene cluster haplotypes, haplotypes I and IV were found to be most common among normal (21.36%) and mutant (15.76%) chromosomes, respectively. The highest observed heterozygosity (48%) was found for the HindIII Gγ and HindII 5'ψs polymorphic sites, whereas the highest expected heterozygosity (50%) was predicted for the HindIII Gγ and HindII sites, which also had the highest PIC value of 0.37. Conclusion: The current study implies mutational heterogeneity among investigated population. Haplotype study results (heterozygosity and PIC), clarifies β-globin markers usefulness for tracking mutant alleles as a complementary method to confirm the genotype in prenatal diagnosis (PND) among investigated population.