Iranian Journal of Biotechnology
Volume:17 Issue: 3, Summer 2019

In Archaea, previous studies have revealed the presence of multiple intron-containing tRNAs and split tRNAs. The full unexpurgated analysis of archaeal tRNA genes remains a challenging task in the field of bioinformatics, because of the presence of various types of hidden tRNA genes in archaea. Here, we suggested a computational method that searched for widely separated genes encoding tRNA halves to generate suppressive variants of missing tRNAs. The exploration of tRNA genes from a genome with varying hypotheses, among all three domain of life (eukaryotes, bacteria and archaea), has been rapidly identified in different ways in the field of bioinformatics. Like eukaryotic tRNA genes, it has been established that two separated regions of the coding sequence of a tRNA gene are essential and sufficient for promotion of transcription. Our objective is to find out the two essential regions in the genome sequence which comprises two halves of the hidden tRNAs. Considering the existence of split tRNA genes widely separated throughout the genome, we developed our tRNA search algorithm to predict such separated tRNA genes by searching both a conserved terminal 5'- and 3'-motif of tRNA in agreement with the split hypothesis on the basis of cloverleaf prediction and precise insilico determination of bulge-helix-bulge secondary structure at the splice sites. By a comprehensive search for all kinds of missing tRNA genes, we have constructed hybrid tRNA genes containing one essential region from tDNA (XYZ) and the other from tDNA (ABC), both from same species in the archaea. We have also found, this type of hybrid tRNA genes are identified in the different species of the archaea (XYZ: ASN, ARG and MET; ABC: ASP,SER, ARG and PRO).These hybrid split tRNA share a common structural motif called bulge-helix-bulge (BHB) a more relaxed bulge-helix loop (BHL), at the leader exon boundary and suggested to be evolutionary interrelated. Analysis of the complete genome sequences of Metallosphaera sedula DSM 5348, Desulfurococcus kamchatkensis 1221n and Ignicoccus hospitalis KIN4/I in archaea by our algorithm revealed that a number of hybrid tRNAs are constructed from different tDNAs . Asymmetric combination of 5’ and 3’ tRNA halves may have generated the diversity of tRNA molecules. Our study of hybrid tRNA genes will provide a new molecular basis for upcoming tRNA studies.

The stress is one of main factors effects on production system. Several factors (both genetic and environmental elements) regulate immune response to stress.

In order to determine the major immune system regulatory genes underlying stress responses, a learning Bayesian network approach for those regulatory genes was applied to RNA-Seq data from a bovine leukocyte model system.

The transcriptome dataset GSE37447 was used from GEO and a Bayesian network on differentially expressed genes was learned to investigate the gene regulatory network.

Applying the method produced a strongly interconnected network with four genes (TERF2IP, PDCD10, DDX10 and CENPE) acting as nodes, suggesting these genes may be important in the transcriptome regulation program of stress response. Of these genes TERF2IP has been shown previously to regulate gene expression, act as a regulator of the nuclear factor-kappa B (NF-κB) signalling, and to activate expression of NF-κB target genes; PDCD10 encodes a conserved protein associated with cell apoptosis; DDX10 encodes a DEAD box protein and is believed to be associated with cellular growth and division; and CENPE involves unstable spindle microtubule capture at kinetochores. Together these genes are involved in DNA damage of apoptosis, RNA splicing, DNA repairing, and regulating cell division in the bovine genome. The topology of the learned Bayesian gene network indicated that the genes had a minimal interrelationship with each other. This type of structure, using the publically available computational tool, was also observed on human orthologous genes of the differentially expressed genes.

Overall, the results might be used in transcriptomic-assisted selection and design of new drug targets to treat stress-related problems in bovines.

Azo dyes are xenobiotic compounds that have bioaccumulated in the environment due to escalated industrial development. These are hazardous in nature, possessing carcinogenic and mutagenic effects on human beings.

The perspective of the present study was to isolate and to determine azo dye (Reactive Orange-16) degrading potential of marine actinobacteria isolated from sediment samples of Port Blair, India.

Actinobacteria with dye decolorization potential were isolated from sea sediment samples. The actinobacterial isolate with the highest dye decolorizing percentage was identified with the help of phenotypic, biochemical and molecular studies. The different physico-chemical parameters for dye decolorization were also optimized. The nature of decolorization by the potent isolate was determined with the help of High Performance Liquid chromatography (HPLC) and Fourier Transformed Infrared spectroscopy (FTIR) techniques. Further the toxicity of RO-16 decolorized products was investigated with the help of phytotoxcity assay.

Out of six actinobacterial isolates, VITVAMB 1 possessed the most efficient RO-16 decolorization property. It decolorized 85.6% of RO-16 (250 mg L-1) within 24hrs. Isolate VITVAMB 1 was identified to be Nocardiopsis sp. Maximum dye decolorization occurred at pH 8, temperature 35°C, 3% salt concentration and a dye concentration of 50 mg L-1.

The nature of decolorization by Nocardiopsis sp. was biodegradation. Additionally, the degraded dye metabolites were found to be less toxic than pure dye. The high decolorization potential of VITVAMB 1 and the low toxicity of its degradation products make it a prospective dye removal system. The marine origin of VITVAMB 1 also makes it an attractive source for novel azo dye reducing enzymes.

Salinity is a major environmental limiting factor, which affect agricultural production. The two Manilkara seedlings (M. roxburghiana and M. zapota) with high economic importance, could not adapt well to higher soil salinity and little is known about their proteomic mechanisms. The mechanisms responsible for the effects of salinity on the two Manilkara species leaves were examined by means of proteomic analysis. The seedlings were cultivated in a greenhouse and treated with NaCl. Leaves of control and the salt-stressed seedlings were sampled for phenol protein extraction. Proteins were separated by two-dimensional gel electrophoresis coupled with mass spectroscopy to study the change of proteins under different NaCl concentration. For M. roxburghiana leaves, 21 protein spots exhibited significant abundance variations between the control and the 6‰, 8‰ NaCl treatments, of these 13 proteins were identified. They included L-ascorbate peroxidase, chloroplast carbonic anhydrase, phosphoglycerate kinase, 5 heat-shock proteins(HSPs) which were all down-regulated; For M. zapota leaves, 35 protein spots exhibited significant abundance variations, then 24 proteins were identified, including 7 down-regulated HSPs as well as glyceraldehyde-3-phosphate dehydrogenase, Cell division protein, putative mitochondrial NAD-dependent malate dehydrogenase, ATP synthase, Rubisco large subunit-binding protein, Cytochrome c peroxidase. Based on the common identified proteins between the two M. species, our results indicated that the identificated proteins in the two Manilkara species were involved in carbohydrate metabolism, photosynthesis, defense and stress. HSPs exhibited variation strictly related to NaCl stress. The down-regulated HSPs meant the function to repair cells that have suffered damage weaken during stress process. Furthermore, except for HSP70 in M. zapota leaves, the HSPs in the two species were all small heat shock proteins (sHSPs) with molecular weights ranging from 15 to 42 kDa.

Vibrio are the main pathogenic bacteria in aquaculture. The flagellin protein C (FlaC) of Vibrio alginolyticus has good immunogenicity and the prospect of potential application in a vaccine. We aimed to evaluate the immunogenicity, protective immunity, and prokaryotic expression fermentation of V. alginolyticus FlaC protein for the vaccine in aquaculture. A molecular cloning method was used to construct the expression strain of FlaC protein, and the protein was purified with Ni-affinity chromatography. Polyclonal antiserum was prepared via mice immunized with the FlaC protein. The Western blot and enzyme-linked immunosorbent assay (ELISA) were used to check the specificity and titre of the antiserum. ELISA and pull-down assay detected the interaction between FlaC protein antiserum and Vibrio. The immune protection function of FlaC protein was detected with mice actively immunized with FlaC protein and challenged by V. alginolyticus and V. parahaemolyticus. The optimal expression conditions for FlaC protein were detected using an L9(34) orthogonal design model. The expression strain of FlaC protein was obtained successfully, and purified FlaC protein was prepared using a mice polyclonal antibody. The FlaC protein antiserum held a high specificity, and the titre was 1:3200. The antiserum directly interacted with V. alginolyticus and V. parahaemolyticus, and the FlaC protein demonstrated a significant immune protection function (50%) against V. alginolyticus infection and some immune protection function (41.66%) against V. parahaemolyticus. The optimal expression conditions for FlaC protein included a strain OD600 value of 0.8, final isopropyl-β-d-thiogalactoside (IPTG) concentration of 0.1 mmol/L, an inducing time of 8 hours, and an inducing temperature of 28°C. This study showed that the FlaC protein possesses a significant immunogenicity and immune protection effect and obtained the optimal fermentation conditions. It is expected to be a potential vaccine against V. alginolyticus and V. parahaemolyticus.

Defensin peptide isolated from plants are often heterogeneous in length, sequence and structure, but they are mostly small, cationic and amphipathic. Plant defensins exhibit broad-spectrum antibacterial and antifungal activities against Gram-positive and Gram-negative bacteria, fungi and etc. Plant defensins also play an important role in innate immunity, such as heavy metal and some abiotic stresses tolerance. In this paper, in vitro broad-spectrum activities, antimicrobial and heavy metal absorption, of a recombinant plant defensin were studied. SDmod gene, a modified plant defensin gene, was cloned in pBISN1-IN (EU886197) plasmid, recombinant protein was produced by transient expression via Agroinfiltration method in common bean. The recombinant protein was tested for antibacterial activity against Gram-negative, Gram-positive bacteria and Fusarium sp. the effects of different treatments on heavy metal zinc absorption by this peptide were tested. We confirmed the antibacterial activities of this peptide against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Bacillus cereus) bacteria, and antifungal activities of this peptide against Fusarium spp. (Fusarium oxysporum and Fusarium solani). High metal absorption coefficient for this peptide was also observed. Out of six actinobacterial isolates, VITVAMB 1 possessed the most efficient RO-16 decolorization property. It decolorized 85.6% of RO-16 (250 mg L-1) within 24hrs. Isolate VITVAMB 1 was identified to be Nocardiopsis sp. Maximum dye decolorization occurred at pH 8, temperature 35oC, 3% salt concentration and a dye concentration of 50 mg L-1. Results suggesting that modified defensin peptide facilitates a broader range of defense activities. dedefensins are an important part of the innate immune system in eukaryotes. These molecules have multidimensional properties that making them promising agents for therapeutic drugs.

In plant breeding program to produce hybrid varieties, pair of male sterile and restorer fertility lines are required. Differentiation of lines possessing restorer fertility allele from the lines lacking it remove the need for the progeny test, and thus reducing the time and the cost in the hybrid production procedure. Canola breeding program in Iran has concentrated toward production of domestic hybrid varieties, however, it suffers from lack of molecular information in restore fertility status of lines, and therefore it needs time and tedious activities. To design gene-based markers for distinguishing R-, A- lines and hybrids in sunflower breeding programs. Aligning sequences of locus responsible for male sterility and that of male fertility resulted in finding differences in the loci, which used to define two set of suitable primer pairs. Genomic DNA from 25 R-lines (23 inbred lines and two commercial lines), 9 A-lines (7 inbred lines and two commercial lines), one B-line and two commercial hybrids were extracted and used in PCR as template. Using one-primer pairs, a band of nearly 1500 bp was amplified in restorer lines but not in A-, B- lines. Another primer pair used to distinguish hybrids (heterozygout) from restorer lines. Results of the report is predicted to be used in canola breeding for hybrid production. Although the molecular bases for the male sterility and fertility restoration in rapeseed is not published, taking advantages of gene-based markers, make rapeseed breeding program more efficient regarding time and costs.

The chaperone activity of Mycobacterium tuberculosis Acr is an important function that helps to prevent misfolding of protein substrates inside the host, especially in conditions of hypoxia. The aim of this study was to establish the correlation of structure and function of recombinant Acr proteins both before and after gel filtration chromatography. The aim was also to find the oligomeric conformation of these samples and use this information to explain differences in activity M. tuberculosis acr gene was cloned with an N-terminal His-tag in pET28a and expressed with IPTG induction in BL2 (DE3) competent Escherichia coli. The activity of a recombinant Acr without gel filtration was checked by preventing thermal aggregation of citrate synthase at 45°C and the chaperone activity against insulin B chain aggregation at 60°C and 37°C. On further purification using gel filtration chromatography, the protein was again tested for chaperone activity using insulin as substrate at 37°C with two types of samples without and with gel filtration designated A and B respectively. The effects of pre–heat treatment at 60 °C on chaperone activity of both A and B samples were studied by performing the chaperone assay at 37°C. The level of expression was 40 to 50 mg /l. The protein was expressed in a soluble form at 37°C and subsequently purified by a 3 step gradient of imidazole using Ni-NTA resin. Gel filtration chromatography showed recombinant Acr to be a mixture of 9 to 15-mers, whereas Native-PAGE analysis showed a large proportion of 5 and 7 mers in the non gel-filtered sample, while non gel –filtered samples showed more proportions of higher size oligomers. The chaperone activity of non gel-filtered (A) samples was less than gel-filtered (B) samples at 37°C with 24 µM required of A for complete inhibition as compared to 6 µM of B. The chaperone activity of non gel–filtered samples at 60°C showed complete inhibition of activity at a concentration of 44 µM. Molecular interaction studies showed influence of size of oligomers on molecular coverage of insulin B chain. Pre-heat treatment improved the activity only after the gel filtration. The larger proportion of monomers in the non gel-filtered sample could explain the difference in activity as compared to the gel-filtered samples in terms of molecular interaction with insulin. Increased oligomer size favorably affected secondary structure, a finding not reported so far, and warranting further investigation. A molecular level interaction of inhibition was predicted using Avogadro number of molecules and oligomer size. The difference in activity after pre–heat treatment seemed to indicate an important role for oligomerization.

Using Brucella abortus Strain 19 (S19) to control bovine brucellosis is restricted due to induce antibodies to the O-side chain of the smooth lipopolysaccharide (LPS) which may be difficult to differentiate vaccinated and infected animals. Furthermore, it is virulent for humans and can induce abortion to cattle. The aim of this study was to employ gene knockout B. abortus S19 for the first time to eliminate diagnostic defects and obtain the attenuated mutant strain. The wbkA gene, which is one of the LPS O-chain coding genes, was knocked out in vaccinal Brucella abortus S19. The proliferative response and immunoglobulin M production were analyzed in wbkA deletion strain-infected BALB/c mice. The loss of wbkA gene function resulted in induction of the splenocyte proliferative response in mice infected by the mutant S19 strain compare to those induced by parental S19 and RB51 strains. Moreover, wbkA mutant did not induce any IgM antibody response using the enzyme-linked immunosorbent assay. As a result, the new mutant S19 strain had deficiency in its LPS O-chain structure, besides cannot induce IgM response then, reduce mistakes to discriminate between vaccinated and infected animal, and also can be considered as a new vaccine candidate.

DNA markers are inevitable tools of human identification in forensic science. Single Nucleotide Polymorphisms (SNPs) are one category of these markers which is concerned to use especially in the case of degraded DNA because of their short amplicons. Detection of highly informative SNPs by the criteria is the essential step to develop a useful panel of SNP markers. The purpose of this work is to get high informative SNPs for human identification in Persian ethnic of the Iranian population. Genotype and allele frequencies of 10 SNPs from the SNPforID browser were determined by a PCR-RFLP method on 100 samples that was taken from 100 unrelated Persian people. These ten SNPs were in Hardy-Weinberg equilibrium (P value > 0.1) except rs1355366 (P value = 0.02) and Heterozygosity of seven SNPs is greater than 0.45 but minor allele frequency of only four SNPs is more than 0.45. According to criteria only three SNPs rs1454361, rs2111980 and rs2107612 can pass all standards and are highly informative in population for forensic uses. Our data showed that the CPI (Combined probability of Identity) and CPE (Combined Power of Exclusion) for ten SNPs are 1.13 E-04 and 0.809 respectively. It was also showed based on the criteria only three SNPs (rs2107612, rs1454361 and rs2111980) are highly informative in Persian population. If we can find 39 SNPs with PE and PI close to PE and PI of these three SNPs (rs2107612, rs1454361 and rs2111980), we will be able to use of these 39 SNPs in human identification with sufficient power of discrimination.