PCR Cloning Research Articles

Effect of diesel leakage in circulating cooling water system on preponderant bacteria diversity and bactericidal effect of biocides

Petroleum products leakage results in adverse effect on the normal operation of a circulating cooling water system. However, relatively little research has been done to explore the effect of petroleum products leakage on circulating cooling water quality and biofilm preponderant bacteria diversity. Also, normal biocides application modes cannot fulfil the need for biofilm control. In this study, diesel oil was used as the experimental subject representing leaking petroleum products; the effect of diesel addition on biofilm preponderant bacteria diversity and the bactericidal effect of chlorine dioxide and tetradecyl dimethyl benzyl ammonium chloride (1427) was investigated. Bacterial community structures were examined by PCR-denaturing gradient gel electrophoresis and PCR cloning of 16S rDNA genes. Except for 100 mg/L diesel, increasing diesel concentration enhanced the biofilm detachment ratio compared with the control test. The microstructure of biofilm samples with 0, 300 and 900 mg/L diesel addition was observed. The species of preponderant bacteria in the biofilm sample with 300 mg/L diesel addition were more and the bacterial distribution was more uniform than those in the biofilm sample with 900 mg/L diesel addition. With ClO2 and 1427 addition, chemical oxygen demand increased, lipid phosphorus and bacterial count first decreased and then remained stable, and the bactericidal ratio first increased and then remained stable. Diesel addition variation has more obvious effect on ClO2 than 1427.

Aerobic and nitrite-dependent methane-oxidizing microorganisms in sediments of freshwater lakes on the Yunnan Plateau.

Both aerobic methane-oxidizing bacteria (MOB) and nitrite-dependent anaerobic methane oxidation (n-damo) bacteria can play an important role in mitigating the methane emission produced in anoxic sediment layers to the atmosphere. However, the environmental factors regulating the distribution of these methane-oxidizing microorganisms in lacustrine ecosystems remain essentially unclear. The present study investigated the distribution of aerobic MOB and n-damo bacteria in sediments of various freshwater lakes on the Yunnan Plateau (China). Quantitative PCR assay and clone library analysis illustrated the spatial variations in the abundances and structures of aerobic MOB and n-damo bacterial communities. Type I MOB (Methylosoma and Methylobacter) and type II MOB (Methylocystis) were detected, while type I MOB was more abundant than type II MOB. Lake sediments n-damo bacterial communities were composed of novel Methylomirabilis oxyfera-like pmoA genes. Lake sediments in the same geographic region could share a relatively similar aerobic MOB community structure. Moreover, Pearson's correlation analysis indicated that n-damo pmoA gene diversity showed a positive correlation with the ratio of organic matter to total nitrogen in lake sediment.

Symbiotic archaea in marine sponges show stability and host specificity in community structure and ammonia oxidation functionality.

Archaea associated with marine sponges are active and influence the nitrogen metabolism of sponges. However, we know little about their occurrence, specificity, and persistence. We aimed to elucidate the relative importance of host specificity and biogeographic background in shaping the symbiotic archaeal communities. We investigated these communities in sympatric sponges from the Mediterranean (Ircinia fasciculata and Ircinia oros, sampled in summer and winter) and from the Caribbean (Ircinia strobilina and Mycale laxissima). PCR cloning and sequencing of archaeal 16S rRNA and amoA genes showed that the archaeal community composition and structure were different from that in seawater and varied among sponge species. We found that the communities were dominated by ammonia-oxidizing archaea closely related to Nitrosopumilus. The community in M.laxissima differed from that in Ircinia spp., including the sympatric sponge I.strobilina; yet, geographical clusters within Ircinia spp. were observed. Whereas archaeal phylotypes in Ircinia spp. were persistent and belong to 'sponge-enriched' clusters, archaea in M.laxissima were closely related with those from diverse habitats (i.e. seawater and sediments). For all four sponge species, the expression of the archaeal amoA gene was confirmed. Our results indicate that host-specific processes, such as host ecological strategy and evolutionary history, control the sponge-archaeal communities.

Whole genome methylation profiles of myelodysplastic syndrome and its diagnostic value

To identify methylation profiles in myelodysplastic syndrome (MDS) and to provide the biomarkers for the early diagnosis and differential diagnosis of MDS. Genes were screened for hypermethylation by genome-wide DNA methylation profiles. Transcription down-regulation was determined with a gene expression microarray. Methylation-specific, real-time, and bisulfite-sequencing PCR cloning and sequencing were performed to validate selected genes in MDS cases and non-malignant hematologic diseases (controls). Diagnostic test, such as sensitivity and specificity, was used to evaluate the value of methylation patterns. A draft of methylation patterns was established and refined to 6 genes after validation in 211 patients and 60 controls. The hypermethylated genes were ABAT (97%), DAPP1 (98%), FADD (89%), LRRFIP1 (96%), PLBD1 (89%), and SMPD3 (85%). A combination of 5 or more than 5 genes showed a specificity of 95% and sensitivity of 91.4% for the diagnosis of MDS. The accuracy of diagnosis was 92.3%. We demonstrated here that the ABAT, DAPP1, FADD, LRRFIP1, PLBD1 and SMPD3 genes are hypermethylated and downregulated in MDS. The six genes could be the markers of the methylation patterns in MDS, as a noninvasive approach for the diagnosis of MDS.

P063 : IDENTIFICATION OF A NEW DQB1 AND DPB1 ALLELE WITH SBT AND GENE CLONING

Aim The objective of this study is to characterize two new HLA class II alleles that we came across during routine clinical testing. Methods HLA typing was performed by medium resolution Luminex SSO (Thermofisher), high-resolution SSP (Olerup), and SBT (Life Technologies). Long-range PCR, followed by DNA cloning and sequencing was also conducted. Results Medium resolution SSO typing revealed the MUD sample is DRB1 ∗ 13:XX, DRB3 ∗ 01:XX and DQB1 ∗ 06:XX homozygous. SBT analysis for DQB1 loci indicates it is likely DQB1 ∗ 06:03 and DQB1 ∗ 06:04 with a G/A mutation at position 599 in Exon 3, which cause an amino acid change from glycine to glutamic acid at codon 168. The confirmation of this new allele has been assured by long-range PCR covering the whole genomic region of DQB1, followed by DNA cloning and Sanger Sequencing. SBT test, covering exon 2, 3, and 4 of DPB1 region, was applied for the referred sample of a problematic DPB1 typing derived from a renal patient. SBT analysis suggested that it is DPB1 ∗ 01:01:01 and a new allele likely joined from Exon 2 of DPB1 ∗ 28:01 with Exon3 of DPB1 ∗ 05:01:02 (or similar, b/c most of the DPB1 Exon3 info are not available in IMGT). We speculate that the new DPB1 allele is very likely generated from a cross-over/conversion event within intron 2 region between DPB1 ∗ 28:01 and DPB1 ∗ 05:01:02 (or similar allele). A long-range PCR and gene cloning covering DPB1 exon 2 to 3′ UTR region has confirmed the identity of this new DPB1 allele. Conclusions We have identified a new DQB1 ∗ 06 allele with an amino acid mutation (G168E) outside the antigen recognition site and a new DPB1 ∗ 28 allele that share the same antigen recognition site as DPB1 ∗ 28:01 but with different backbones. Although these new alleles are not likely going to alter the peptide repertoire being presented to CD4+ T cells, they could be candidate allo-reaction targets in both cellular and humoral immune responses in allotransplantation.

Hunting, swimming, and worshiping: human cultural practices illuminate the blood meal sources of cave dwelling Chagas vectors (Triatoma dimidiata) in Guatemala and Belize.

Background Triatoma dimidiata, currently the major Central American vector of Trypanosoma cruzi, the parasite that causes Chagas disease, inhabits caves throughout the region. This research investigates the possibility that cave dwelling T. dimidiata might transmit the parasite to humans and links the blood meal sources of cave vectors to cultural practices that differ among locations.Methodology/Principal FindingsWe determined the blood meal sources of twenty-four T. dimidiata collected from two locations in Guatemala and one in Belize where human interactions with the caves differ. Blood meal sources were determined by cloning and sequencing PCR products amplified from DNA extracted from the vector abdomen using primers specific for the vertebrate 12S mitochondrial gene. The blood meal sources were inferred by ≥99% identity with published sequences. We found 70% of cave-collected T. dimidiata positive for human DNA. The vectors had fed on 10 additional vertebrates with a variety of relationships to humans, including companion animal (dog), food animals (pig, sheep/goat), wild animals (duck, two bat, two opossum species) and commensal animals (mouse, rat). Vectors from all locations fed on humans and commensal animals. The blood meal sources differ among locations, as well as the likelihood of feeding on dog and food animals. Vectors from one location were tested for T. cruzi infection, and 30% (3/10) tested positive, including two positive for human blood meals.Conclusions/SignificanceCave dwelling Chagas disease vectors feed on humans and commensal animals as well as dog, food animals and wild animals. Blood meal sources were related to human uses of the caves. We caution that just as T. dimidiata in caves may pose an epidemiological risk, there may be other situations where risk is thought to be minimal, but is not.

Construction of sized eukaryotic cDNA libraries using low input of total environmental metatranscriptomic RNA.

BackgroundConstruction of high quality cDNA libraries from the usually low amounts of eukaryotic mRNA extracted from environmental samples is essential in functional metatranscriptomics for the selection of functional, full-length genes encoding proteins of interest. Many of the inserts in libraries constructed by standard methods are represented by truncated cDNAs due to premature stoppage of reverse transcriptase activity and preferential cloning of short cDNAs.ResultsWe report here a simple and cost effective technique for preparation of sized eukaryotic cDNA libraries from as low as three microgram of total soil RNA dominated by ribosomal and bacterial RNA. cDNAs synthesized by a template switching approach were size-fractionated by two dimensional agarose gel electrophoresis prior to PCR amplification and cloning. Effective size selection was demonstrated by PCR amplification of conserved gene families specific of each size class. Libraries of more than one million independent inserts whose sizes ranged between one and four kb were thus produced. Up to 80% of the insert sequences were homologous to eukaryotic gene sequences present in public databases.ConclusionsA simple and cost effective technique has been developed to construct sized eukaryotic cDNA libraries from environmental samples. This technique will facilitate expression cloning of environmental eukaryotic genes and contribute to a better understanding of basic biological and/or ecological processes carried out by eukaryotic microbial communities.

Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase.

As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.

Molecular cloning and characterization of secretory and membrane-bound IgM of turbot

In recent years, increasing diseases especially bacterial diseases have brought a host of losses with the expansive cultivation of turbot (Scophthalmus maximus). In order to do more research about the immune system of turbot for better understanding the mechanism of resisting diseases, the immunoglobulin genes related to secretory and membrane-bound IgM (s-IgM and m-IgM) of turbot were cloned using homology sequences cloning and SMART RACE PCR method. The heavy chain of s-IgM cDNA is 1900 bp in length including a leader region, a variable region, four constant regions (CH1, CH2, CH3 and CH4) and a C-terminal while the cDNA of m-IgM is 1795 bp with the same leader region, variable region, three constant regions (CH1, CH2 and CH3) and two transmembrane regions (TM1 and TM2). The sequence of IgM gene was also obtained and the structure consisted of V–CH1–CH2–CH3–CH4-TM1-TM2 is similar to other fishes. The highest level of s-IgM expression was observed in spleen, followed by kidney, gills, eyes, skin of the healthy turbot whereas the same profile of m-IgM expression is found with low level. And s-IgM takes up dominant proportion of total IgM expression. Also the relative expressions of s-IgM and m-IgM were analyzed in turbot vaccinated with the live attenuated vaccine Vibrio anguillarum. Not only the transcriptions of both s-IgM and m-IgM in liver, spleen and kidney of turbot injected with V. anguillarum MVAV6203 were up-regulated but also the expressions of s-IgM and m-IgM in spleen, kidney, gut, skin and gills of bath-vaccinated turbot were increased. Comparing the ratio changes of relative expression of m-IgM and s-IgM in vaccinated turbot, we found that the proportion of m-IgM were increasing in both administration routes, which probably indicated that the increasing expression of m-IgM strengthen the phagocytic ability of B cells.

First Report of Leptosphaeria biglobosa Causing Black Leg on Raphanus sativus in Central China.

Chinese radish (Raphanus sativus) is an important vegetable grown widely in China. In 2010 to 2013, surveys for Leptosphaeria spp. on cruciferous vegetables were conducted in 17 counties in Hubei Province, China. Black leg symptoms on tuber roots and basal stems of radish were observed in Hanchuan, Jingmen, and Macheng counties. Disease incidence ranged from 2 to 25% in 10 surveyed radish fields. Five fungal isolates were obtained from diseased radish plants by surface-sterilizing radish tissue (5% NaOCl for 90 s, and then rinsed in sterilized water three times) and plating onto potato dextrose agar (PDA) plates incubated at 20°C. The isolations produced fluffy white colonies with a yellow pigment, and black-brown, globose pycnidia with pink conidial ooze formed after 10 days. Pycnidia were 150 to 200 × 80 to 100 μm. Conidia were hyaline, cylindrical, and 4 to 5 × 2 μm. The cultural and morphological characteristics of the isolates matched the description for Phoma lingam, anamorph of Leptosphaeria maculans and L. biglobosa (3). All five isolates were identified by PCR assay using the species-specific primers LbigF, LmacF, and LmacR (1), and isolate HCLB-1 was identified further by PCR cloning and analysis of the sequences coding for actin, β-tubulin, and the internal transcribed spacer (ITS) region of ribosomal DNA (3,4). Genomic DNA was extracted from mycelium of each isolate harvested from 7-day-old cultures in V8 broth using the CTAB method (5). A 444-bp DNA fragment was detected by PCR assay, suggesting that all five isolates belonged to L. biglobosa rather than L. maculans as the latter generates a 331-bp DNA fragment (1). The HCLB-1 sequences for ITS (587 bp, GenBank Accession. No. KC880981), actin (899 bp, KF307762), and β-tubulin (432 bp, KF220296) genes were 99 to 100% identical to those of L. biglobosa isolates in GenBank. All five isolates were tested for pathogenicity on R. sativus cultivars Duan Ye 13 and Qi Ye Hong. Cotyledons of 10-day-old radish seedlings and post-harvest mature roots were wounded using a sterilized needle, and 10 μl of a conidial suspension (1 × 107 conidia/ml) of each isolate was pipetted onto the wounded area on each cotyledon or root, respectively, with 12 cotyledons (= 24 wounded sites) and 1 root (= 6 wounded sites) inoculated/isolate. One wounded root and 12 wounded cotyledons inoculated with water were used as control treatments. Treated roots and seedlings were incubated at 20°C and 100% RH in the dark for 7 days, and under a 12 h light/12 h dark cycle for 12 days, respectively. While the control roots and cotyledons remained asymptomatic, the roots and cotyledons inoculated with all five test isolates formed black cankers and necrotic lesions, respectively, in the inoculated wounds. A fungus re-isolated from symptomatic roots and cotyledons resembled the original isolates in colony morphology and the 444-bp DNA fragment detected by PCR assay. No fungus was isolated from control seedlings or roots. Thus, L. biglobosa appears to be the causal agent of black leg observed on radish in Hubei, China. L. biglobosa was reported to infect wild radish (R. raphanistrum) (2). To our knowledge, this is the first report of L. biglobosa causing black leg on R. sativus.

Cloning and characterization of SnRK2 subfamily II genes from Nicotiana tabacum.

SnRK2 is a plant-specific protein kinase family involved in abiotic stress signalling. In this study, NtSnRK2.1, NtSnRK2.2, and NtSnRK2.3, were cloned from tobacco by in silico cloning and reverse transcription PCR. The three protein kinases were classed into subfamily II of the SnRK2 family using a phylogenetic tree and C-terminus analysis. Subcellular localization revealed NtSnRK2s in the nuclear and cytoplasmic compartments. Dynamic expression of NtSnRK2s in tobacco plants that were exposed to drought, salt, or cold stressors were characterised using quantitative real-time PCR. It was revealed that the three genes showed similar patterns of transcription under abiotic stress responses; there was evidence NtSnRK2s participated in abscisic acid-dependent signalling pathways. NtSnRK2.1-3 responded much faster to drought and salt than to cold stress. To investigate the role of NtSnRK2s under abiotic stresses, NtSnRK2.1 gene was over-expressed in tobacco. A stress tolerance assay showed that tobacco plants that over-expressed NtSnRK2.1 plants had greater salt tolerance. The results indicate that NtSnRK2s are involved in abiotic stress response pathways.

Identification of caleosin and two oleosin isoforms in oil bodies of pine megagametophytes

Numerous oil bodies of 0.2–2 μm occupied approximately 80% of intracellular space in mature pine (Pinus massoniana) megagametophytes. They were stably isolated and found to comprise mostly triacylglycerols as examined by thin layer chromatography analysis and confirmed by both Nile red and BODIPY stainings. Fatty acids released from the triacylglycerols of pine oil bodies were mainly unsaturated, including linoleic acid (60%), adrenic acid (12.3%) and vaccenic acid (9.7%). Proteins extracted from pine oil bodies were subjected to immunological cross-recognition, and the results showed that three proteins of 28, 16 and 14 kDa were detected by antibodies against sesame seed caleosin, sesame oleosin-L and lily pollen oleosin-P, respectively. Complete cDNA fragments encoding these three pine oil-body proteins, tentatively named caleosin, oleosin-L and oleosin-G, were obtained by PCR cloning and further confirmed by mass spectrometric analysis. Consistently, phylogenetic tree analyses showed that pine caleosin was closely-related to the caleosin of cycad megagametophyte among known caleosin sequences. While pine oleosin-L was found clustered with seed oleosin isoforms of angiosperm species, oleosin-G was distinctively grouped with the oleosin-P of lily pollen. The oleosin-G identified in pine megagametophytes seems to represent a new class of seed oleosin isoform evolutionarily close to the pollen oleosin-P.

CDR3 analysis of TCR Vβ repertoire of CD8+ T cells from chickens infected with Eimeria maxima

CD8+ T cells play a major role in the immune protection of host against the reinfection of Eimeria maxima, the most immunogenic species of eimerian parasites in chickens. To explore the dominant complementarity-determining regions 3 (CDR3) of CD8+ T cell populations induced by the infection of this parasite, sequence analysis was performed in this study for CDR3 of CD8+ T cells from E. maxima infected chickens. After 5days post the third or forth infection, intraepithelial lymphocytes were isolated from the jejunum of bird. CD3+CD8+ T cells were sorted and subjected to total RNA isolation and cDNA preparation. PCR amplification and cloning of the loci between Vβ1 and Cβ was conducted for the subsequent sequencing of CDR3 of T cell receptor (TCR). After the forth infection, 2 birds exhibited two same frequent TCR CDR3 sequences, i.e., AKQDWGTGGYSNMI and AGRVLNIQY; while the third bird showed two different frequent TCR CDR3 sequences, AKQGARGHTPLN and AKQDIEVRGPNTPLN. No frequent CDR3 sequence was detected from uninfected birds, though AGRVLNIQY was also found in two uninfected birds. Our result preliminarily demonstrates that frequent CDR3 sequences may exist in E. maxima immunized chickens, encouraging the mining of the immunodominant CD8+ T cells against E. maxima infection.

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.

Sa2059 Afferent Activation in the Proximal Colon by Enteroendocrine Mediators and Nutrients

Background: Despite the importance of gastric inhibitory polypeptide (GIP) as a physiological incretin, postprandial circulating glucose levels in GIP receptor null (GIPR-/-) and wild-type (wt) mice are similar. However, unlike wt mice, GIPR-/mice do not develop diet-induced obesity, suggesting a potential role for GIPR antagonists in the treatment of obesity. While specific GIPR antagonists, such as N-terminal truncated peptides and peptides with a Glu to Pro substitution at position 3 (Pro3-GIP), have been identified, their short circulating T1/2 has hampered their development. We hypothesize that the T1/2 of these antagonists can be increased significantly by fusing them to the Fc-fragment of IgG, a strategy that has been employed previously for this purpose. Aim: To develop long-acting antagonists to GIPR by fusion to the IgG Fc-fragment. Methods: PCR and molecular cloning were used to produce chimeric genes encoding GIP(6-42)-Fc-IgG and Pro3-GIP-Fc-IgG. After expression in HEK 293 cells, protein A agarose chromatography was used to purify protein from conditioned media. Fc-fusions were identified by Western analysis using a specific GIP antibody, and biological activity was assessed in the presence and absence of 1 nM synthetic GIP(1-42) using a GIP-specific reporter cell line. Results: Transgenic HEK 293 cells produced protein with the predicted molecular size for the Fc fusion and that possessed GIP-specific immunoreactivity. Purified Pro3-GIP-Fc-IgG induced reporter expression with an EC50 of 0.5 μM, whereas GIP(6-42)-Fc-IgG did not. Both GIP(6-42)-Fc-IgG and Pro3-GIP-Fc-IgG antagonized report gene expression induced by 1 nM GIP, with IC50 values of 4 μM and 20 μM, respectively. Summary and Conclusion: Pro3-GIP-Fc-IgG, but not GIP(6-42)-FcIgG, induced reporter gene expression, while both fusion products inhibited reporter gene expression induced by 1 nM GIP, indicating that the former is a partial agonist, while only the latter functions as an antagonist. The IC50 values for GIP(6-42)-Fc-IgG and Pro3-GIPFc-IgG were higher than the corresponding values reported for synthetic GIP(6-42) and Pro3-(GIP), indicating the possibility of partial interference of ligand binding by the Fc fragment. Nevertheless, the increase in T1/2 imparted by the fusion of N-terminal truncated GIP peptides the Fc fragment may thereby provide viable candidates for treating obesity and related disorders.

Linked production of pyroglutamate-modified proteins via self-cleavage of fusion tags with TEV protease and autonomous N-terminal cyclization with glutaminyl cyclase in vivo.

Overproduction of N-terminal pyroglutamate (pGlu)-modified proteins utilizing Escherichia coli or eukaryotic cells is a challenging work owing to the fact that the recombinant proteins need to be recovered by proteolytic removal of fusion tags to expose the N-terminal glutaminyl or glutamyl residue, which is then converted into pGlu catalyzed by the enzyme glutaminyl cyclase. Herein we describe a new method for production of N-terminal pGlu-containing proteins in vivo via intracellular self-cleavage of fusion tags by tobacco etch virus (TEV) protease and then immediate N-terminal cyclization of passenger target proteins by a bacterial glutaminyl cyclase. To combine with the sticky-end PCR cloning strategy, this design allows the gene of target proteins to be efficiently inserted into the expression vector using two unique cloning sites (i.e., SnaB I and Xho I), and the soluble and N-terminal pGlu-containing proteins are then produced in vivo. Our method has been successfully applied to the production of pGlu-modified enhanced green fluorescence protein and monocyte chemoattractant proteins. This design will facilitate the production of protein drugs and drug target proteins that possess an N-terminal pGlu residue required for their physiological activities.

Features of the RAP1A and RAP1B core promoters: recombinant approaches to testing regulatory element function in expression control (946.10)

The RAP1 genes, closely related to the RAS oncogenes, have been shown to have numerous functions, yet little is known about the expression regulation of these genes in human cells. Our lab has constructed plasmids containing putative promoter regions of human RAP1A and RAP1B, driving a gene for green fluorescent protein (GFP). We have transiently transfected these plasmids into a wide‐range of human cell types, and analyzed GFP expression via fluorescence microscopy, qRT‐PCR, and western blotting. By comparing the efficacy of each putative core promoter segment, some distinct regulatory regions have been revealed. Additionally, comparing promoter‐driven expression to the actual native expression of the RAP1B genes in specific cell types has suggested an important regulatory role for enhancer elements not included in the core promoter regions. Various sub‐cloning techniques, including inverse fusion PCR cloning (IFPC), have been employed to add putative regulatory regions to existing GFP core promoter plasmids. These should allow us to further characterize the expression control of the RAP1 genes, and to understand the cell‐type differences observed in native RAP1 expression.

Using an α-bungarotoxin binding site tag to study GABA A receptor membrane localization and trafficking.

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility(1-7). To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent α-bungarotoxin with cells expressing constructs containing an α-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the α subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity(8,9). Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors(2,10). In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP(11)) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)(12). The BBS is 3' of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Using an α-Bungarotoxin Binding Site Tag to Study GABA A Receptor Membrane Localization and Trafficking

It is increasingly evident that neurotransmitter receptors, including ionotropic GABA A receptors (GABAAR), exhibit highly dynamic trafficking and cell surface mobility1-7. To study receptor cell surface localization and endocytosis, the technique described here combines the use of fluorescent α-bungarotoxin with cells expressing constructs containing an α-bungarotoxin (Bgt) binding site (BBS). The BBS (WRYYESSLEPYPD) is based on the α subunit of the muscle nicotinic acetylcholine receptor, which binds Bgt with high affinity8,9. Incorporation of the BBS site allows surface localization and measurements of receptor insertion or removal with application of exogenous fluorescent Bgt, as previously described in the tracking of GABAA and metabotropic GABAB receptors2,10. In addition to the BBS site, we inserted a pH-sensitive GFP (pHGFP11) between amino acids 4 and 5 of the mature GABAAR subunit by standard molecular biology and PCR cloning strategies (see Figure 1)12. The BBS is 3' of the pH-sensitive GFP reporter, separated by a 13-amino acid alanine/proline linker. For trafficking studies described in this publication that are based on fixed samples, the pHGFP serves as a reporter of total tagged GABAAR subunit protein levels, allowing normalization of the Bgt labeled receptor population to total receptor population. This minimizes cell to cell Bgt staining signal variability resulting from higher or lower baseline expression of the tagged GABAAR subunits. Furthermore the pHGFP tag enables easy identification of construct expressing cells for live or fixed imaging experiments.

Integrating De Novo Transcriptome Assembly and Cloning to Obtain Chicken Ovocleidin-17 Full-Length cDNA

Efficiently obtaining full-length cDNA for a target gene is the key step for functional studies and probing genetic variations. However, almost all sequenced domestic animal genomes are not ‘finished’. Many functionally important genes are located in these gapped regions. It can be difficult to obtain full-length cDNA for which only partial amino acid/EST sequences exist. In this study we report a general pipeline to obtain full-length cDNA, and illustrate this approach for one important gene (Ovocleidin-17, OC-17) that is associated with chicken eggshell biomineralization. Chicken OC-17 is one of the best candidates to control and regulate the deposition of calcium carbonate in the calcified eggshell layer. OC-17 protein has been purified, sequenced, and has had its three-dimensional structure solved. However, researchers still cannot conduct OC-17 mRNA related studies because the mRNA sequence is unknown and the gene is absent from the current chicken genome. We used RNA-Seq to obtain the entire transcriptome of the adult hen uterus, and then conducted de novo transcriptome assembling with bioinformatics analysis to obtain candidate OC-17 transcripts. Based on this sequence, we used RACE and PCR cloning methods to successfully obtain the full-length OC-17 cDNA. Temporal and spatial OC-17 mRNA expression analyses were also performed to demonstrate that OC-17 is predominantly expressed in the adult hen uterus during the laying cycle and barely at immature developmental stages. Differential uterine expression of OC-17 was observed in hens laying eggs with weak versus strong eggshell, confirming its important role in the regulation of eggshell mineralization and providing a new tool for genetic selection for eggshell quality parameters. This study is the first one to report the full-length OC-17 cDNA sequence, and builds a foundation for OC-17 mRNA related studies. We provide a general method for biologists experiencing difficulty in obtaining candidate gene full-length cDNA sequences.