The FOXE1 gene was screened for mutations in a cohort of 34 unrelated patients with congenital hypothyroidism, 14 of whom had thyroid dysgenesis and 18 were normal (the thyroid status for 2 patients was unknown). The entire coding region of the FOXE1 gene was PCR-amplified, then analyzed using single-stranded conformational polymorphism, followed by confirmation by direct DNA sequencing. DNA sequencing analysis revealed a heterozygous A>G transition at nucleotide position 394 in one of the patients. The nucleotide transition changed asparagine to aspartate at codon 132 in the highly conserved region of the forkhead DNA binding domain of the FOXE1 gene. This mutation was not detected in a total of 104 normal healthy individuals screened. The binding ability of the mutant FOXE1 protein to the human thyroperoxidase (TPO) promoter was slightly reduced compared with the wild-type FOXE1. The mutation also caused a 5% loss of TPO transcriptional activity.
β2-Microglobulin (β(2)M) is the light chain of major histocompatibility class I (MHC I) that binds non-covalently with the α heavy chain. Both proteins attach to the antigen peptide, presenting a complex to the T cell to be destroyed via the immune mechanism.
A halo-thermophilic bacterium, Roseithermus sacchariphilus strain RA (previously known as Rhodothermaceae bacterium RA), was isolated from a hot spring in Langkawi, Malaysia. A complete genome analysis showed that the bacterium harbors 57 glycoside hydrolases (GHs), including a multi-domain xylanase (XynRA2). The full-length XynRA2 of 813 amino acids comprises a family 4_9 carbohydrate-binding module (CBM4_9), a family 10 glycoside hydrolase catalytic domain (GH10), and a C-terminal domain (CTD) for type IX secretion system (T9SS). This study aims to describe the biochemical properties of XynRA2 and the effects of CBM truncation on this xylanase. XynRA2 and its CBM-truncated variant (XynRA2ΔCBM) was expressed, purified, and characterized. The purified XynRA2 and XynRA2ΔCBM had an identical optimum temperature at 70 °C, but different optimum pHs of 8.5 and 6.0 respectively. Furthermore, XynRA2 retained 94% and 71% of activity at 4.0 M and 5.0 M NaCl respectively, whereas XynRA2ΔCBM showed a lower activity (79% and 54%). XynRA2 exhibited a turnover rate (kcat) of 24.8 s-1, but this was reduced by 40% for XynRA2ΔCBM. Both the xylanases hydrolyzed beechwood xylan predominantly into xylobiose, and oat-spelt xylan into a mixture of xylo-oligosaccharides (XOs). Collectively, this work suggested CBM4_9 of XynRA2 has a role in enzyme performance.
Novel antimicrobial agents are crucial to combat antibiotic resistance in pathogenic bacteria. Choline kinase (ChoK) in bacteria catalyzes the synthesis of phosphorylcholine, which is subsequently incorporated into the cell wall or outer membrane. In certain species of bacteria, phosphorylcholine is also used to synthesize membrane phosphatidylcholine. Numerous human ChoK inhibitors (ChoKIs) have been synthesized and tested for anticancer properties. Inhibition of S. pneumoniae ChoK by human ChoKIs showed a promising effect by distorting the cell wall and retarded the growth of this pathogen. Comparison of amino acid sequences at the catalytic sites of putative choline kinases from pathogenic bacteria and human enzymes revealed striking sequence conservation that supports the potential application of currently available ChoKIs for inhibiting bacterial enzymes. We also propose the combined use of ChoKIs and nanoparticles for targeted delivery to the pathogen while shielding the human host from any possible side effects of the inhibitors. More research should focus on the verification of putative bacterial ChoK activities and the characterization of ChoKIs with active enzymes. In conclusion, the presence of ChoK in a wide range of pathogenic bacteria and the distinct function of this enzyme has made it an attractive drug target. This review highlighted the possibility of "choking" bacterial ChoKs by using human ChoKIs.
cDNAs encoding three phospholipase A2 (PLA2) isoforms in Naja naja sputatrix were cloned and characterized. One of them encoded an acidic PLA2 (APLA) while the others encoded neutral PLA2 (NPLA-1 and NPLA-2). The specific characteristics of APLA and NPLA were attributed to mutations at nt139 and nt328 from G to C and G to A, respectively, resulting in amino acid substitutions from Asp20 and 83 in APLA to His20 and Asn83 in NPLA. Amino acid sequencing of purified protein also showed the presence of this Asp20 and His20 in APLA and NPLA, respectively. The cDNA encoding one of the PLA2 (NAJPLA-2A), when expressed in Escherichia coli, yielded a protein that exhibited PLA2 activity.
An initial study on gene cloning and characterization of unicellular green microalga Ankistrodesmus convolutus was carried out to isolate and characterize the full-length cDNA of ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit (RbcS) as a first step towards elucidating the structure of A. convolutus RbcS gene. The full-length of A. convolutus RbcS cDNA (AcRbcS) contained 28 bp of 5' untranslated region (UTR), 225 bp of 3' non-coding region, and an open reading frame of 165 amino acids consisting of a chloroplast transit peptide with 24 amino acids and a mature protein of 141 amino acids. The amino acid sequence has high identity to those of other green algae RbcS genes. The AcRbcS contained a few conserved domains including protein kinase C phosphorylation site, tyrosine kinase phosphorylation site and N-myristoylation sites. The AcRbcS was successfully expressed in Escherichia coli and a ~21 kDa of anticipated protein band was observed on SDS-PAGE. From the phylogenetic analysis of RbcS protein sequences, it was found that the RbcS of A. convolutus has closer genetic relationship with green microalgae species compared to those of green seaweed and green macroalgae species. Southern hybridization analysis revealed that the AcRbcS is a member of a small multigene family comprising of two to six members in A. convolutus genome. Under different illumination conditions, RT-PCR analysis showed that AcRbcS transcription was reduced in the dark, and drastically recovered in the light condition. Results presented in this paper established a good foundation for further study on the photosynthetic process of A. convolutus and other green algae species where little information is known on Rubisco small subunit.
Two genes that encode α-amylases from two Anoxybacillus species were cloned and expressed in Escherichia coli. The genes are 1,518 bp long and encode 506 amino acids. Both sequences are 98% similar but are distinct from other well-known α-amylases. Both of the recombinant enzymes, ASKA and ADTA, were purified using an α-CD-Sepharose column. They exhibited an optimum activity at 60°C and pH 8. Both amylases were stable at pH 6-10. At 60°C in the absence of Ca²⁺, negligible reduction in activity for up to 48 h was observed. The activity half-life at 65°C was 48 and 3 h for ASKA and ADTA, respectively. In the presence of Ca²⁺ ions, both amylases were highly stable for at least 48 h and had less than a 10% decrease in activity at 70°C. Both enzymes exhibited similar end-product profiles, and the predominant yield was maltose (69%) from starch hydrolysis. To the best of our knowledge, most α-amylases that produce high levels of maltose are active at an acidic to neutral pH. This is the first report of two thermostable, alkalitolerant recombinant α-amylases from Anoxybacillus that produce high levels of maltose and have an atypical protein sequence compared with known α-amylases.
A Bacillus sphaericus strain (205y) that produces an organic solvent-tolerant lipase was isolated in Port Dickson, Malaysia. The gene for the lipase was recovered from a genomic library and sequenced. Phylogenetic analysis was performed based on an alignment of thirteen microbial lipase sequences obtained from the NCBI database. The analysis suggested that the B. sphaericus lipase gene is a novel gene, as it is distinct from other lipase genes in Families I.4 and I.5 reported so far. Expression in Escherichia coli under the control of the lacZ promoter resulted in an eight-fold increase in enzyme activity after a 3-h induction with 1 mM IPTG. The crude enzyme thus obtained showed a slight (10%) enhancement in activity after a 30-min incubation in 25% (v/v) n-hexane at 37 degrees C, and retained 90% of its activity after a similar period in 25% (v/v) p-xylene.
Newly discovered kisspeptin (metastin), encoded by the Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of GnRH. In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125 and 115 amino acids, respectively, and its core sequence (FNLNPFGLRF, F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY, Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across nonmammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventromedial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P < 0.002), gnrh2, and gnrh3 (P < 0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased FSH-beta (2.7-fold, P < 0.05) and LH-beta (8-fold, P < 0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.
A gene (cbm71) encoding a 71,128-Da mosquitocidal protein (Cbm71) was obtained by screening a size-fractionated XbaI digest of total genomic DNA from Clostridium bifermentans subsp. malaysia CH18 with two gene-specific oligonucleotide probes. The sequence of the Cbm71 protein, as deduced from the sequence of cbm71, corresponds to that of the 66-kDa protein previously described as one of the mosquitocidal components of C. bifermentans subsp. malaysia. Cbm71 shows limited similarities with Bacillus thuringiensis delta-endotoxins, especially in the four first conserved blocks. However, Cbm71 was not immunologically related to any of the Cry toxins and thus belongs to a novel class of mosquitocidal protein. The cbm71 gene was expressed in a nontoxic strain of B. thuringiensis, and Cbm71 was produced during sporulation and secreted to the supernatant of culture. Trichloroacetic-precipitated supernatant preparations were toxic for mosquito larvae of the species Aedes aegypti, Culex pipiens, and Anopheles stephensi.
The phosphoprotein (P) gene of a heat stable Newcastle disease virus (NDV) was cloned, sequenced and expressed in Escherichia coli. SDS-PAGE analysis of the recombinant P protein (395 amino acids) and a C-terminal extension derivative (424 amino acids), gave rise to two distinct protein bands with molecular masses of approximately 53-55 and 56-58 kDa, respectively, which are approximately 26-30% heavier than those calculated from the deduced amino acid sequences. The differences in molecular mass on SDS-PAGE are thought to be attributed to the acidic nature of the P protein (pI=6.27) and also the different degrees of phosphorylation in the prokaryotic cell. Amino acid sequence comparison of the P protein among the published NDV strains showed that they were highly conserved particularly at the putative phosphorylation sites.
Immediate early response (IER) 2 gene, a member of the IER family, is a gene of unknown function which is affected by external stimuli in the brain. In the present study, the full length sequence and localization of medaka (Oryzias latipes) ier2 was investigated in the brain to understand the functions of Ier2 in the future studies. The full length sequence of medaka ier2 was identified using a 3'-, 5'- rapid amplification of cDNA ends method, and distribution in the brain was identified using in situ hybridization. The identified full length ier2 mRNA consisted of 939 nucleotides spanning along 1 exon. The deduced amino acid sequence consisted of 171 amino acid residues which contains a highly conserved sequence, nuclear localization signal. ier2 mRNA was distributed in the telencephalon, midbrain and the hypothalamus. This highly conserved primary response gene Ier2 can be used to visualize and map functionally activated neuronal circuitry in the brain of medaka.
In this study, the genome of the Plasmodium falciparum Gombak A strain was examined for the presence of a gene encoding falcipain-2, a cysteine protease, using homology-based polymerase chain reaction cloning. The nucleotide sequence obtained from the gene cloned (designated pFG1) is approximately 99% homologous to other falcipain-2 genes from different strains. Comparatively, it is 69% homologous to falcipain-3 genes. Direct cloning of the falcipain-2 gene and its resemblance to the reported corresponding mRNA transcript suggests the absence of introns in this gene. Sequence alignment and comparison revealed four amino acid differences at positions 15, 51, 59 and 414 in the falcipain-2 from P. falciparum Gombak A as compared to other falcipain-2 proteins from different strains.
Three new open reading frames were found downstream from cbm71, a toxin gene from Clostridium bifermentans malaysia (Cbm) strain CH18. The first one (91bp downstream) called cbm72, is 1857bp long and encodes a 71727-Da protein (Cbm72) with a sequence similar to that of Bacillus thuringiensis delta-endotoxins. This protein shows no significant toxicity to mosquito larvae. The two others, cbm17.1 (462bp) and cbm17.2 (459bp), are copies of the same gene encoding Cbm P18 and P16 polypeptides and located 426bp and 1022bp downstream from cbm72, respectively. They encode 17189-Da and 17451-Da proteins with sequences 44.6% similar to that of Aspergillus fumigatus hemolysin; however, they were not hemolytic in the conditions tested.
The xylanase gene from Bacillus pumilus PJ19 amplified by polymerase chain reaction (PCR) was cloned into pCRII vector and transformed into Escherichia coli strain INValphaF'. Starting from an ATG as an initiator codon, an open reading frame coding for 202 amino acids was obtained. The recombinant xylanase sequence showed a 96% homology with the xylanase sequence from B. pumilus IPO strain and had an estimated molecular weight of 22,474. Xylanase activity expressed by E. coli INValphaF' harboring the cloned gene was located primarily in the cytoplasmic fraction.
Nitric oxide associated 1 (NOA1) protein is implicated in plant disease resistance and nitric oxide (NO) biosynthesis. A full-length cDNA encoding of NOA1 protein from oil palm (Elaeis guineensis) was isolated and designated as EgNOA1. Sequence analysis suggested that EgNOA1 was a circular permutated GTPase with high similarity to the bacterial YqeH protein of the YawG/YlqF family. The gene expression of EgNOA1 and NO production in oil palm root tissues treated with Ganoderma boninense, the causal agent of basal stem rot (BSR) disease were profiled to investigate the involvement of EgNOA1 during fungal infection and association with NO biosynthesis. Real-time PCR (qPCR) analysis revealed that the transcript abundance of EgNOA1 in root tissues was increased by G. boninense treatment. NO burst in Ganoderma-treated root tissue was detected using Griess reagent, in advance of the up-regulation of the EgNOA1 transcript. This indicates that NO production was independent of EgNOA1. However, the induced expression of EgNOA1 in Ganoderma-treated root tissues implies that it might be involved in plant defense responses against pathogen infection.
Chitinases in terrestrial plants have been reported these are involved in heavy metal tolerance/detoxification. This is the first attempt to reveal chitinase gene (AcCHI I) and its function on metal detoxification in mangroves Aegiceras corniculatum. RT-PCR and RACE techniques were used to clone AcCHI I, while real-time quantitative PCR was employed to assess AcCHI I mRNA expressions in response to Cadmium (Cd). The deduced AcCHI I protein consists of 316 amino acids, including a signal peptide region, a chitin-binding domain (CBD) and a catalytic domain. Protein homology modeling was performed to identify potential features in AcCHI I. The CBD structure of AcCHI I might be critical for metal tolerance/homeostasis of the plant. Clear tissue-specific differences in AcCHI I expression were detected, with higher transcript levels detected in leaves. Results demonstrated that a short duration of Cd exposure (e.g., 3 days) promoted AcCHI I expression in roots. Upregulated expression was also detected in leaves under 10 mg/kg Cd concentration stress. The present study demonstrates that AcCHI I may play an important role in Cd tolerance/homeostasis in the plant. Further studies of the AcCHI I protein, gene overexpression, the promoter and upstream regulation will be necessary for clarifying the functions of AcCHI I.
Psychrophiles are organisms that thrive in cold environments. One of the strategies for their cold adaptation is the ability to synthesize cold-adapted enzymes. These enzymes usually display higher catalytic efficiency and thermolability at lower temperatures compared to their mesophilic and thermophilic counterparts. In this work, a psychrophilic bacterial isolate codenamed π9 was selected for the cloning of the gene encoding triose phosphate isomerase (TIM), an enzyme in the glycolytic pathway. Based on 16S rRNA gene sequence analysis, this isolate was identified as a species of the genus Pseudomonas under the P. fluorescens group. The cloning of a 816 bp fragment of TIM gene which covers the 756 bp open reading frame was achieved by a combination of degenerate and splinkerette PCRs. The partial sequence of this gene was first PCR amplified by using degenerate primers and the flanking sequences were subsequently amplified by splinkerette PCR technique. Amino acid sequence of the cloned TIM was 97% identical to TIM from Pseudomonas fluorescens and shared 51% identity with the TIM from psychrophilic Vibrio sp. This work demonstrated the use of multiple PCR techniques to clone a gene without prior knowledge of its sequence. The cloning of the TIM gene by PCR was more rapid and cost effective compared to the traditional genomic library construction and screening method. Homology model of the TIM protein in this study was generated based on Escherichia coli TIM crystal structure. The model could serve as a hypothetical TIM structure from a psychrophilic microorganism for further investigation into areas that showed deviations from the known mesophilic TIM structures.
The gene encoding for a novel cold-adapted enzyme from family II of bacterial classification (GDSL family) was cloned from the genomic DNA of Photobacterium sp. strain J15 in an Escherichia coli system, yielding a recombinant 36 kDa J15 GDSL esterase which was purified in two steps with a final yield and purification of 38.6 and 15.3 respectively. Characterization of the biochemical properties showed the J15 GDSL esterase had maximum activity at 20 °C and pH 8.0, was stable at 10 °C for 3 h and retained 50 % of its activity after a 6 h incubation at 10 °C. The enzyme was activated by Tween-20, -60 and Triton-X100 and inhibited by 1 mM Sodium dodecyl sulphate (SDS), while β-mercaptoethanol and Dithiothreitol (DTT) enhanced activity by 4.3 and 5.4 fold respectively. These results showed the J15 GDSL esterase was a novel cold-adapted enzyme from family II of lipolytic enzymes. A structural model constructed using autotransporter EstA from Pseudomonas aeruginosa as a template revealed the presence of a typical catalytic triad consisting of a serine, aspartate, and histidine which was verified with site directed mutagenesis on active serine.
Pentose phosphate pathway (PPP) composed of two functionally-connected phases, the oxidative and non-oxidative phase. Both phases catalysed by a series of enzymes. Transketolase is one of key enzymes of non-oxidative phase in which transfer two carbon units from fructose-6-phosphate to erythrose-4-phosphate and convert glyceraldehyde-3-phosphate to xylulose-5-phosphate. In plant, erythrose-4-phosphate enters the shikimate pathway which is produces many secondary metabolites such as aromatic amino acids, flavonoids, lignin. Although transketolase in plant system is important, study of this enzyme is still limited. Until to date, TKT genes had been isolated only from seven plants species, thus, the aim of present study to isolate, study the similarity and phylogeny of transketolase from sugarcane. Unlike bacteria, fungal and animal, PPP is complete in the cytosol and all enzymes are found cytosolic. However, in plant, the oxidative phase found localised in the cytosol but the sub localisation for non-oxidative phase might be restricted to plastid. Thus, this study was conducted to determine subcellular localization of sugarcane transketolase. The isolation of sugarcane TKT was done by reverse transcription polymerase chain reaction, followed by cloning into pJET1.2 vector and sequencing. This study has isolated 2,327 bp length of sugarcane TKT. The molecular phylogenetic tree analysis found that transketolase from sugarcane and Zea mays in one group. Classification analysis found that both plants showed closer relationship due to both plants in the same taxon i.e. family Poaceae. Target P 1.1 and Chloro P predicted that the compartmentation of sugarcane transketolase is localised in the chloroplast which is 85 amino acids are plant plastid target sequence. This led to conclusion that the PPP is incomplete in the cytosol of sugarcane. This study also found that the similarity sequence of sugarcane TKT closely related with the taxonomy plants.