Gene Cloning Analysis Research Articles

The effect of heavy metals on microbial community structure of a sulfidogenic consortium in anaerobic semi-continuous stirred tank reactors

The effect of heavy metals on community structure of a heavy metal tolerant sulfidogenic consortium was evaluated by using a combination of denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene and dissimilatory sulfite reductase (dsrB) gene fragments, 16S rRNA gene cloning analysis and fluorescence in situ hybridization (FISH). For this purpose, four anaerobic semi-continuous stirred tank reactors (referred as R1-R4) were run in parallel for 12weeks at heavy metal loading rates of 1.5, 3, 4.5 and 7.5mgl(-1)d(-1) each of Cu(2+), Ni(2+), Zn(2+), and Cr(6+), respectively. The abundance ratio of Desulfovibrio vulgaris detected by FISH to total cell counts was consistent with the obtained results of cloning and DGGE. This indicated that D. vulgaris was dominant in all analyzed samples and played a key role in heavy metal removal in R1, R2, and R3. In contrast, after 4weeks of operation of R4, a distinct biomass loss was observed and no positive hybridized cells were detected by specific probes for the domain Bacteria, sulfate-reducing bacteria and D. vulgris. High removal efficiencies of heavy metals were achieved in R1, R2 and R3 after 12weeks, whereas the precipitation of heavy metals in R4 was significantly decreased after 4weeks and almost not observed after 6weeks of operation. In addition, the anaerobic bacteria, such as Pertrimonas sulfuriphila, Clostridium sp., Citrobacter amalonaticus, and Klebsiella sp., identified from DGGE bands and clone library were hypothesized as heavy metal resistant bacteria at a loading rate of 1.5mgl(-1)d(-1) of Cu(2+), Ni(2+), Zn(2+), and Cr(6+.)

Gene cloning and functional analysis of yellow green leaf3 (ygl3) gene during the whole-plant growth stage in rice

Chlorophyll is an important photosynthetic pigment in the process of photosynthesis in plants and photosynthetic bacteria. Genes involved in chlorophyll biosynthesis in Arabidopsis and photosynthetic bacteria have been well documented. In rice, however, these genes have not been fully annotated. In this paper, a yellow-green leaf gene, yellow green leaf3 (ygl3) was cloned and analyzed. ygl3 encodes magnesium chelation ChlD (D) subunit, a key enzyme for chlorophyll synthesis, resulting in a yellow-green leaf phenotype in all growth stages in rice. Expression content of ygl3 is highest in the leaf blades, followed by the leaf sheaths, while there is virtually no expression of the gene in the stems and seeds. The sub-cellular structure and protein content of the photosynthetic system of the ygl3 mutant were revealed by transmission electron microscopy, BN-PAGE, and western blotting. The results show that the mutation of the ygl3 gene indirectly leads to a decrease in the protein content of the photosynthetic system and severely obstructs the formation of granum thylakoids.

Gene cloning and function analysis of cytokine-induced suppressor of cytokine signaling (SOCS) from pearl oyster Pinctada fucata

Cytokine-induced suppressor of cytokine signaling (SOCS) family acts as a negative regulator of cytokine receptor signaling to control excessive cytokine effects and inhibit a variety of signal transduction pathways, particularly the Janus kinases/signal transducers and activators of transcription (JAK/STAT) pathway. In present study, SOCS-2 homolog (PfSOCS-2) from pearl oyster Pinctada fucata was cloned and its gene has no intron. Multiple sequence alignments and phylogenetic analysis showed that PfSOCS-2 was clustered with other mollusk SOCS-2. LPS or polyI:C challenge and gene expression analysis revealed that PfSOCS-2 involved the innate immune response against bacterial and viral infections and that induction of PfSOCS-2 was varied with the different challenge stimulations. Furthermore, Dual-luciferase reporter assays showed that PfSOCS-2 involved in the regulation of vertebrate target genes containing the IFN-stimulated response element or NF-κB binding site in vitro. These results indicated that SOCS-2 from P. fucata plays a regulatory role against the stimulation.

Identification and Gene Mapping of Rice Early Senescent Leaf (e s l 3) Mutant

Leaf early senescence can directly decrease crop photosynthesis, yield and quality. Therefore, the identifications of mutants with early senescence and its gene function play key roles in crop genetic improvement. A mutant with early senescent leaf blades, named as esl3, was identified from an EMS-induced progeny in the restorer line Jinhui 10. The middle-upper leaf in mutation displayed a brown and withered phenotype from seedling stage to maturity. Correspondingly, the chlorophyll content and net photosynthetic rate declined in the abnormal leaves while enhanced in the normal green part of leaves compared with the wild type. Agronomic traits, such as panicle number, panicle length, grains per panicle, 1000-grain weight, plant height and weight of dry matter, were reduced significantly or very significantly except for seed setting rate as compared with those of wild type. Genetic analysis showed the senescence was controlled by a recessive nuclear gene. Using 391 mutation individuals in a F2 generation of Nipponbare/esl3, ESL3 was mapped between SSR marker RM19085 and InDel marker Ind05-2 on the chromosome 5 with physical distance 91 kb including 14 annotated genes. These results provide a foundation for the further gene cloning and functional analysis of ESL3 gene.

Expression and characteristic of the Cu/Zn superoxide dismutase gene from the insect parasitizing fungus Cordyceps militaris

A Cu/Zn-superoxide dismutase (SOD) gene was characterized from Cordycepes militaris by gene cloning, heterogeneous expression and function analysis. This 154-aa SOD (CmSOD) was deduced from a 465-bp gene cloned, showing 72-95 % sequence identity to Cu/Zn-SODs from other fungi. The deduced amino acid sequence of the cDNA is highly similar to Beauveria bassiana (95 %), Isaria tenuipes (94 %) and Claviceps purpurea (88 %), respectively. The SOD gene of C. militaris spin 589 bp and consisted of two introns and three exons. The CmSOD coding region sequence was inserted into plasmid pQE-30 in order to construct prokaryotic expression vector, then transformed into Escherichia coli M15 cells for expression, and a mass of rCmSOD was obtained by IPTG induction. The enzyme activity of the purified rCmSOD was approximately 714.48 U/mg after the assay. The study provided a way for in-depth research on the expression and regulation of the CmSOD, and the molecular mechanism of anti-oxidative effect in C. militaris.

Metagenomic insights into chlorination effects on microbial antibiotic resistance in drinking water

This study aimed to investigate the chlorination effects on microbial antibiotic resistance in a drinking water treatment plant. Biochemical identification, 16S rRNA gene cloning and metagenomic analysis consistently indicated that Proteobacteria were the main antibiotic resistant bacteria (ARB) dominating in the drinking water and chlorine disinfection greatly affected microbial community structure. After chlorination, higher proportion of the surviving bacteria was resistant to chloramphenicol, trimethoprim and cephalothin. Quantitative real-time PCRs revealed that sulI had the highest abundance among the antibiotic resistance genes (ARGs) detected in the drinking water, followed by tetA and tetG. Chlorination caused enrichment of ampC, aphA2, blaTEM-1, tetA, tetG, ermA and ermB, but sulI was considerably removed (p < 0.05). Metagenomic analysis confirmed that drinking water chlorination could concentrate various ARGs, as well as of plasmids, insertion sequences and integrons involved in horizontal transfer of the ARGs. Water pipeline transportation tended to reduce the abundance of most ARGs, but various ARB and ARGs were still present in the tap water, which deserves more public health concerns. The results highlighted prevalence of ARB and ARGs in chlorinated drinking water and this study might be technologically useful for detecting the ARGs in water environments.

Bacterial diversity in suspected prosthetic joint infections: an exploratory study using 16S rRNA gene analysis

Formation of biofilm is a prominent feature of prosthetic joint infections (PJIs) and constitutes a challenge to current sampling procedures and culture practices. Molecular techniques have a potential for improving diagnosis of biofilm-adapted, slow-growing and non-culturable bacteria. In this exploratory study we investigated the bacterial diversity in specimens from 22 patients clinically suspected of having PJIs. Bacteriological cultures were performed according to standard practice. A total of 55 specimens from 25 procedures ('specimen sets') were submitted to broad range 16S rRNA gene PCR, cloning, sequencing and phylogenetic analysis. More than 40 bacterial taxa within six phyla were identified in 14 specimen sets originating from 11 patients. Direct observation of biofilm was made in selected specimens by fluorescence in situ hydridization. 16S rRNA gene analysis and bacteriological cultures were concordant for 15/25 specimen sets (60%; five positive, 10 negative); additional taxa were detected in four sets by gene analysis, and discrepant results were obtained for six sets, five of which were negative on culture. Polymicrobial communities were revealed in 9/14 sets by gene analysis and 1/10 sets by culture (P<0.05). Although our study was not conclusive, these findings are consistent with a primary role of biofilm formation in PJIs.

Gene cloning and function analysis of complement B factor-2 of Apostichopus japonicus

In this study, a homologue of complement B factor (AjBf-2, GenBank ID: JN634069.1) was cloned and characterized from Apostichopus japonicus by using bioinformatics methods and molecular biotechnologies including homology cloning and RACE. The full-length cDNA of AjBf-2 was composed of 3261bp. The sequence shows 268bp in the 5′UT region, 395bp in the 3′UT region, and 2595 bp in the open reading frame. AjBf-2 gene encodes 865 amino acids. The deduced amino acids sequence and domain structure of AjBf-2 gene show significant similarity to the vertebrate Bf/C2 family protein. AjBf-2 is a mosaic protein. It has a deduced molecular mass of 96.8 kDa, with a conserved site for a D factor. AjBf-2 is composed of five short consensus repeats, a von Willebrand Factor domain, a serine protease domain and an Mg2+ binding site. It has eight consensus recognition sites for N-linked glycosylation and four cAMP- and cGMP-dependent protein kinase phosphorylation sites. Phylogenetic analysis of AjBf-2 compared with other species Bf shows that A. japonicus has a close evolutionary relationship with Strongylocentrotus purpuratus and Carcinoscorpius rotundicaud. It can be speculated that Bf in invertebrate is the ancestor of Bf in vertebrate. The result of RT-PCR shows that the AjBf-2 gene is expressed in every tested tissue of A. japonicus, and is especially high in the coelomocyte and the body wall. The expression tendency in coelomocyte and the body wall are approximately the same. After LPS induction, the expression of AjBf-2 gene peaks at 12 h in coelomocyte and 3 h in the body wall.

3-Hydroxy-3-methylglutaryl coenzyme A reductase 1 (HMG1) is highly associated with the cell division during the early stage of fruit development which determines the final fruit size in Litchi chinensis

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC: 1.1.1.34), an enzyme catalyzing the first committed step in the mevalonic acid (MVA) pathway for the biosynthesis of isoprenoids, has been reported to be involved in the fruit size determination through the regulation of early cell division. In litchi, the cell number achieved by this early cell division determines the final fruit size, but whether HMGR plays any role in this process was unknown. In this study, we set out to address this question with gene cloning and expression analysis in fruits of different pheno- or genotypes. We found that the litchi genome includes two HMGR homologues, denoted as LcHMG1 and LcHMG2. Despite 70% sequence identity at the amino acid level, they exhibited distinct expression patterns during litchi fruit development. LcHMG1 expression was highest in the early stage of fruit development, correlated with the high level of cell division. Absolute levels of LcHMG1 expression varied among fruits of different pheno- or genotypes, with expression in large-fruited types reaching higher levels for longer duration compared to that in small-fruited types. The expression patterns for LcHMG1 strongly suggest that this gene is involved in early cell division and fruit size determination in litchi. In contrast, LcHMG2 was most highly expressed in the late stage of fruit development, in association with biosynthesis of isoprenoid compounds required for later cell enlargement. These findings provided new insights on the function of HMGR genes during fruit development.

Apolipoprotein E gene cloning and tissues expression analysis inallotetraploid and its parents

As a key factor in cholesterol metabolism,Apolipoprotein E is participate in lipoprotein transport and gonadal development.To understand function of ApoE,the cDNAs of allotetraploid and their parents-red crucian carp and common carp were cloned and sequenced.The full length of allotetraploid,red crucian carp and common carp ApoE cDNAs were 1393 bp,1384 bp and 1396 bp,respectively.All the cDNAs encode 281 amino acids.Based on alignment result,ApoE of allotetraploid is more similar with common carp than red crucian carp.Meanwhile,besides characters inherited from parents,allotetraploid shows specific mutation in ApoE.By constructing phylogenetic tree using Mega software,the phylogenetic tree of ApoE was consistent with taxonomy: cyprinid fish are clustered together rather than other vertebrates.Realtime-PCR analysis indicated that the expression patterns of ApoE in allotetraploid,common carp and red crucian carp are similar.All the tissues had ApoE expression.The highest expression was in spleen.This research gave information in gene variation in the process of allotetraploid evolvement and lipid me-tabolic mechanism of fish.

Genetic analysis of morphological traits in a new, versatile, rapid-cycling Brassica rapa recombinant inbred line population

A recombinant inbred line (RIL) population was produced based on a wide cross between the rapid-cycling and self-compatible genotypes L58, a Caixin vegetable type, and R-o-18, a yellow sarson oil type. A linkage map based on 160 F7 lines was constructed using 100 Single nucleotide polymorphisms (SNPs), 130 AFLP®, 27 InDel, and 13 publicly available SSR markers. The map covers a total length of 1150 centiMorgan (cM) with an average resolution of 4.3 cM/marker. To demonstrate the versatility of this new population, 17 traits, related to plant architecture and seed characteristics, were subjected to quantitative trait loci (QTL) analysis. A total of 47 QTLs were detected, each explaining between 6 and 54% of the total phenotypic variance for the concerned trait. The genetic analysis shows that this population is a useful new tool for analyzing genetic variation for interesting traits in B. rapa, and for further exploitation of the recent availability of the B. rapa whole genome sequence for gene cloning and gene function analysis.

Genetics in Kidney Diseases

During the last two decades, developments in molecular biology and genetics have caused a revolution in medicine. Advances in gene cloning, gene mapping, and mutation analysis have contributed to an incredible amount of new information regarding the biological and pathophysiological basis for human diseases including kidney diseases. In ‘monogenic diseases”, a mutation of a single gene is sufficient to cause the disease. Conversely, in polygenic disorders, mutations of multiple genes are necessary to result in a disease. Progressive chronic kidney disease remains a major challenge in nephrology. Genome-wide expression analysis of disease processes has been pioneered in onco logy, and molecular approaches have now been included in the initial diagnosis. Although we still have difficulties in sample analyses of the kidneys, some of the molecular diagnostic approaches are now used routinely in nephrology as well. In this review, recent developments in the field of the molecular bases of kidney diseases will be addressed. (Gazi Med J 2011; 22: 118-23)

Review on Mutation in Lateral Root of Rice

Rice roots include seminal roots, adventitious roots, lateral roots and root hairs. At present, progresses in the research of rice roots have been achieved in many aspects, such as root morphology, root activity, root reaction to various environmental factors as a contribution of root growth and rice yield, the relationship between root growth and stems/leaves/flowers/rice, genetic laws of root characters, etc. However, there are very few researches on lateral root mutant. This paper reviewed progresses of the lateral root mutant of rice from the perspectives of phytomorphology to plant physiology and biochemistry to the gene mapping, consisting of mechanism of developing lateral root of rice, gene cloning and functional analysis of lateral root development, the relationship between auxin and lateral roots, agronomic traits of lateral roots mutant, structure and morphology of root hairs, gravity anomaly of root, redox metabolism and proteomics researches of the mutation in lateral root of rice.

Gene Cloning and Bioinformatics Analysis of Glycoprotein H Gene of Pseudorabies Virus Strain SL

The glycoprotein H(gH) gene homologue of pseudorabies virus wild strain SL(PRV-SL) was cloned by degenerate polymerase chain reaction (PCR) from PRV infected vero cells. Bioinformatics analysis was performed to predict the characteristics of gH, the results indicated that gH gene encoded a polypeptide, molecular mass of 71.95kda and comprising 687 amino acids. The protein had one signal peptide between 1 and 24AA, one transmembrane region 645-667AA, 27 antigenic determinants and the hydrophobicity between -2.117 and 3.178. The prediction of secondary structure showed that gH had consisted highly of alpha helix(Hh) and random coil(Cc). The phylogenetic tree showed that PRV-SL was similar to others of the Alphaherpesvirinae, and that gH had higher conservative in Alphaherpesvirinae. Cloning and analysis of gH gene laid foundation to further research and exploitation.

Progress on genetics of rice grain shape trait and its related gene mapping and cloning

Increase of crop production is the primary goal of crop breeding. Rice grain shape is a quantitative trait that is directly related to yield traits and has a close relationship with quality traits. The evaluation of grain shape is mainly grain length, grain width, grain thickness, length/width, and length/thickness. In recent years, the quantitative genetic research on rice grain shape has made a significant progress and a number of important genes associated with rice grain shape have been cloned. This paper reviews the classic genetic analysis on rice grain traits, QTL mapping, grain shape gene cloning and functional analysis, and their application in rice breeding for super high yield.

Novel Gene Discovery of Crops in China: Status, Challenging, and Perspective

Discovery of novel genes in crops is the basis to change germplasm resources from phenotypical characterization to a gene level and hence for molecular breeding. This paper reviewed progress of novel gene discovery studies in major crops, such as rice, wheat, maize, soybean, cotton, and oilseed rape in China. In last decade, Chinese scientists have achieved a number of breakthroughs on novel gene identification in crops, including: (1) Various distinctive materials for gene discovery were created, such as core collections of germplasms based on crop genetic diversity, establishment of genetic populations based on genetic resources with favorite traits, assessment of mutants derived from mutagenesis, and so on; (2) Technology and methods of gene discovery were further developed, especially the gene-based integration of various discovery technologies with combination of biometric algorithm improvement of gene/QTLs, and therefore the efficiency of gene discovery was increased; (3) Mapping genes/QTLs related to important agronomic traits of crops has become a common method for genetic studies. A number of genes/QTLs associated with disease and insect resistance, stress tolerance, good quality, nutrient use efficiency and high yield have been mapped, of which more than 500 genes have been positioned on chromosomes precisely by fine mapping; (4) Great progress in cloning and functional analysis of crop genes in China, particularly in rice, has drawn world-wide attention. More than 300 genes have been cloned in the main crops, among which more than 70 genes have been functionally validated in crops. While gene discovery in crops becomes more and more efficient, large-scale and towards utilization in the world, Chinese scientists are also making new findings in this field. However, the quality and quantity of crop gene discovery in China is still far from satisfying the needs for molecular breeding and the overall level of novel gene discovery is still behind top labs/institutions in the world. Gene discovery in different crops has developed unevenly, the number of genes discovered is not large enough and the genes found to have great value for crop improvement are few. Considering weak points in gene discovery in China and the challenges both international and from biotechnology companies, the authors provided strategies in crop gene discovery in China, with focus on speeding up gene cloning and functional analysis, emphasis on identification and evaluation of the genes with utilization potential, and research activities more orientated to the biotechnology industry.

Enhancement of GDP-Dissociation Inhibitor Gene Expression in Osteoblasts by Low-Level Laser Irradiation

Low-level laser irradiation (LLLI) stimulates bone regeneration and may be useful for bone fracture and oral implant therapy. However, the molecular mechanism of LLLI in accelerating bone formation is not well known. In order to understand the mechanism of LLLI in accelerating bone formation, we attempted to identify gene expressions enhanced by LLLI by a subtractive gene cloning and DNA microarray analysis. Osteoblastic cell MC3T3-E1 was treated with 830 nm Ga-Al-As diode laser irradiation and total mRNA was recovered and used to construct a subtractive gene library of genes with transcription enhanced by LLLI. The DNA sequences of subtracted gene was subjected to a homology search using BLASTN program in the National Center for Biotechnology Information (NCBI). Genes with mRNA levels altered by LLLI were also analysed using Affymetrix GeneChip. The mRNA levels were confirmed by RT-PCR and real-time PCR. Subtracted gene clones MCL101 indicated high homology with GDP-dissociation inhibitor beta (GDPIB) genes. Affymetrix GeneChipTM analysis was further analyzed and found that GDI alpha gene expression was also enhanced by LLLI. The increased mRNA level of GDIA and GDIB by LLLI was successfully confirmed by RT-PCR and real-time PCR. Since the up-regulation of GDIA is one of the mechanisms underlying the synergistic boneforming effect of parathyroid hormone and oestrogen. Moreover, GDIA and GDIB involve in the regulation of vesicle-mediated cellular transport and anti-apoptosis, the enhancement of GDIA and GDIB gene expressions by LLLI may be a part of mechanisms in the acceleration of bone formation by LLLI.

Development of strategy for competent cell preparation and high efficiency plasmid transformation using different methods

Current cloning technologies based on site-specific recombination are efficient, simple to use, and flexible. With the recent availability of complete genomic sequences of many organisms and plants, high-throughput and cost-efficient systems for gene cloning and functional analysis are in great demand. This study compares two different methods of preparation of competent cells using two strains of E. coli DH5α and HB101. From results the most efficient strain was found DH5α for cloning as it supports blue white screening utilizing galactosidase activity. The concentration of calcium chloride is another important factor; various concentrations of CaCl2 were tried. Optimum concentration was found to be 75 mM. However PEG also has great influence on transformation efficiency, use of 40% PEG gave the best transformation efficiency. A convenient and rapid method for the genetic transformation of E. coli with appropriate plasmid is proposed which can be utilised for high efficiency transformation in normal laboratory conditions.

Detection of t(3;5) and NPM1/MLF1 rearrangement in an elderly patient with acute myeloid leukemia: clinical and laboratory study with review of the literature

We present a novel case of acute myeloid leukemia with an NPM1/MLF1 rearrangement in a 78-year-old Korean woman. The bone marrow chromosome study showed a complex karyotype: 46,XX,t(2;13) (q13;q32),der(3)t(3;5)(q25.1;q34),der(5)del(5)(?q31q34)t(3;5),inv(9)(p11q13)c,del(20)(q11.2)[13]/49,idem,+5,+8,+der(13)t(2;13)[7]. Multiplex gene rearrangement testing, cloning, and sequencing analyses revealed an NPM1/MLF1 fusion rearrangement between exon 6 of NPM1 (ENSG00000181163) and exon 2 of MLF1 (ENSG00000178053). Although t(3;5)(q25.1;q34) or the NPM1/MLF1 rearrangement has been reported mostly as a sole karyotypic abnormality in younger patients, it should also be considered in elderly patients with complex chromosomal abnormalities in acute myeloid leukemia or myelodysplastic syndrome.

Utilization of next-generation sequencing platforms in plant genomics and genetic variant discovery

Genome-wide variant detection within a species is the primary initial step towards linking genotypic variation and phenotypes. The conversion of these genetic variants (the most prevalent of these being single-nucleotide polymorphisms or SNPs) into genetic markers is particularly important in agronomically valuable crop species to allow for cost-effective marker-assisted selection strategies, whole-genome fingerprinting, association studies, map-based gene cloning and population-based analyses. Towards these goals, an increasing number of large-scale genetic variant discovery initiatives are being undertaken in conjunction with next-generation sequencing platforms, allowing for drastically quicker and cheaper variant discovery, and leading towards a far more comprehensive view of the genome or transcriptome. This review will summarize the current status of these initiatives and will discuss the expanding role of next-generation sequencing technologies in facilitating crop improvement.